CMMB 343 Lab Manual 2018

Name: ____________________ TA Name and Email:

Lab Section: __________

_____________________________

Lab Partners’ Name and Contact Info: ________________________________________ _______________________________________ _______________________________________

Cellular, Molecular and Microbial Biology 343

Microbiology Laboratory Manual

2018 Department of Biological Sciences The University of Calgary W. Huddleston F. Cusack Copyright © 2018 The University of Calgary

ii

Table of Contents Laboratory Schedule .........................................................................................................

v

Laboratory Policies ...........................................................................................................

vi

Emergency and Safety Information ................................................................................

ix

Preface and Introduction to CMMB 343 Laboratories ..................................................

xiii

Laboratory 1 - The Microbiology Laboratory ............................................................... Laboratory 2 - Laboratory Techniques ...........................................................................

1 5

Laboratory 3 - Microbial Morphology andin vitroCulture ..........................................

9

Laboratory 4 - Effect of Environment on Bacterial Growth ...........................................

15

Laboratory 5 - Effect of Chemical Agents on Bacterial Growth ...................................

21

Class Presentations ..........................................................................................................

27

Lab Exam 1 ................................................................................................................

29

Term Project - Identication of Bacterial Unknowns ......................................................

31

Laboratory 6 - Microbial Metabolism ............................................................................

35

Laboratory 7 - Transposon Mutagenesis........................................................................

39

Laboratory 8 - Effect of Radiation on Viral Growth ....................................................

41

Lab Exam 2 ................................................................................................................

45

Appendix 1- Laboratory Exercises ....................................................................................

49

Appendix 2 - Microscopy ...............................................................................................

51

Appendix 3 - Media and Methods Used for Cultivating and Isolating Bacteria .............

53

Appendix 4 - Review of Microbiological Techniques .....................................................

59

Appendix 5 - Bacterial Culture Media ............................................................................

71

Appendix 6 - WHMIS .....................................................................................................

77

Appendix 7 - Biohazard Safety Level Designations .......................................................

79

Appendix 8 - Image Reference Sheet ........................................................................

81

Appendix 9 - TA Evaluation Form ..................................................................................

83

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iv

LABORATORY SCHEDULE B01/B03/B05/B07 - EEEL 369 B02/B04/B06/B08 - EEEL 303 WARNING: this course requires time in the laboratory outside of scheduled class time (see page xiv) Date

January 9/11 January 16/18 January 23/25

Exercise

Assignment

LABORATORY 1 Exercise 1 due January 16/18 Complete Laboratory Quiz 1before your next scheduled lab LABORATORY 2

Exercise 2 due January 19/22

LABORATORY 3

Exercise 3 due January 26/29

Complete Laboratory Quiz 2before your next scheduled lab Jan. 30/Feb. 1

Exercise 4 due February 2/5

LABORATORY 4

Complete Laboratory Quiz 3before your next scheduled lab February 6/8

LABORATORY 5

Ames Test Exercise due February 6/8 Exercise 5 due February 9/12

February 13/15

CLASS PRESENTATIONS

February 20/22

NO LABS (READING WEEK)

Feb 27/March 1

MIDTERM LAB EXAM BACTERIAL UNKNOWN

March 6/8

LABORATORY 6 BACTERIAL UNKNOWN

Winogradsky Exercise due March 2/5 Unknown Exercise due March 2/5 Exercise 6 due March 9/12

Complete Laboratory Quiz 4before your next scheduled lab March 13/15


Exercise 7 due March 20/22

Complete Laboratory Quiz 5before your next scheduled lab March 20/22


March 27/29

NO LABS

April 3/5

FINAL LAB EXAM

Exercise 8 due March 27/29 Journal due March 20/22

v

LABORATORY POLICIES Composition of Laboratory Mark: Component:

Weight:

Submissions

10 %

Quizzes

% 5

Presentation

5%

UnknownJournal

4%

MidtermLabExam*

8%

Final Lab Exam*

8%

Total**

Both individual and group work will be used to evaluate your understanding of concepts and techniques. Lab exam questions will be short answer and will cover both practical and theoretical aspects of the laboratories. * You must complete this course component in order to pass the course ** You must pass (>50%) the lab component in order to pass the course

40 %

The Final Lab Exam will take place in the s cheduled laboratory the week of April 2

Re-evaluation of Laboratory Work The course policy on re-evaluation of student work is that students must rst take the time to read over their TA’s comments; for this reason, requests for re-evaluation cannot be made until the day after graded work has been returned. If you would like a piece of term work re-evaluated, prepare a written summary of your concerns related to the grading of your work using the ReEvaluation Request Form available on D2L, andmake an appointment to meet with your TA. This re-evaluation must be requested within 15 DAYS of the date when the graded work was srcinally handed back or when your mark was posted . If you still have concerns, you should submit the work to the LabCoordinator for an independent reappraisal. Seethe University of Calgary Calendar for more information on re-evaluation of term work (http://www.ucalgary.ca/ pubs/calendar/current/i-2.html).

Policy on Late Assignments Assignments are due at12 pm in the submission box (outside EEEL 369) on the day indicatedon the laboratory schedule (see page v) unless indicated otherwise. Late work willnot be accepted and will be graded as a zero, unless you provide the Lab Coordinator with documentation to support a valid reason for handing in an assignment late. Valid reasons (and documentation ) include: • personal illness (doctor’s note on the University of Calgary‘s ofcial stationery available at: http://www.ucalgary.ca/UofC/departments/UHS/PDFs/deferred_exam_form.pdf);

• family emergency (doctor’s note, death notice,etc., on ofcial stationery); • professional school interview (letter of invitation); • team absences (coach’s letter) Once marked assignments have been returned to students, late assignments will not be accepted for any reason.

vi

Academic Misconduct Common forms of academic misconduct are cheating and plagiarism.Cheating is receiving or giving information on an assignment, exam or quiz.Plagiarism is the inclusion of someone else’s ideas or information in a written or oral assignment without giving credit or acknowledging the source. This is considered to be the theft of another’s ideas. Copying all or part of someone else’s written assignment is one example of plagiarism. In addition, plagiarism includes word for word copying of text from a textbook, a journal article or a document posted on the world wide web, even if the source is referenced. Cooperation in the carrying out of laboratory experiments is often necessary and both formal and informal discussions on the interpretation of data is encouraged. HOWEVER, lab reports and other assignments must be written in your own words (i.e. produced independently). There are instances in which students are instructed to prepare joint reports. When this is specically requested, this is, of course, acceptable. Penalties for academic misconduct range from reduced or failing grades on a course component to expulsion from the University.See the University Calendar (http://www.ucalgary.ca/pubs/ calendar/current/k.html) for more information.

Attendance Attendance is required at all laboratory classes. You may attend only the lab section in which you are registered, due to space and equipment limitations. If you miss a lab, you must contact the Lab Coordinator within 48 hours of the absence. The documentation must explicitly state the day(s) missed, the reason for the absence, and when you are able to return to classes. Please note that lab exercises run for one week at a time and are not repeated in subsequent weeks, so if you miss an entire week of classes, you will not be able to make up that lab. You are still responsible for all the material covered in that lab. Assignments will not be accepted from a student who was absent from the lab in which the data were collected, unless you have a validexcuse and supporting documentation. If you miss a lab in which an assignment was due or an exam was written and have a valid excuse, you must present the Lab Coordinator with supporting documentation. Students who miss a substantial number of labs may not be permitted to write the lab na l exam. Depending on the circumstances, if you cannot make up the lab, you may either be given an excused absence for the assignment, or other arrangements will be made.

Required Laboratory Components Attendance is required at all laboratory classes. Students who miss a substantial number of labs will not be permitted to ta ke the lab exams. You must take the lab exams in order to pass the course. Students who do not show competency with the laboratory material will not receive a passing grade in the course. You must pass (>50%) the laboratory component of the course in order to pass the course.

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viii

Emergency and Safety Information Emergency Numbers Campus Security

220-5333

Safety Ofce

220-6345

University Health Services

220-5765/8

Poison Control Center

403-670-1414

Emergency Procedures Fire*

• Pull nearest re alarm.

• Phone 220-5333 (24 hour Campus Security number) • Give name, telephone number, location and nature of re. • Use re extinguisher if small re. • If possible, students must turn off all sources of heat, gas or open ames, e.g. hot plates, Bunsen burners.

• All personnel must leave the lab; ensure lab doors are closed. • Exit the building via closest emergency exit. • Meet re ghters outside door closest to re alarm panel and inform them of relocation and hazards. Serious Personal Injury*

• Do not move seriously injured persons unlessthere is a danger to their safety, taking precautions for your own safety. • Phone Campus Security: 220- 5333 (24 hours) (give name,telephone number,location and type of injury). If possible have someone go to main entrance and ag down ambulance. Minor Personal Injury*

• •

Telephone Campus Security: 220-5333

•

Phone University Health Services: 220-5765/5768

Make injured person comfortable.

* Notify one of the laboratory coordinators as soon as possible after the incident. Building Damage, Floods, Utility Failure*

• Inform the coordinator ofthe teaching laboratories andtelephone the Safety Ofce at

220-6345 and describe the problem. • After 4:30 phone Campus Security at 220-53 33. Give your name, telephone number, location and describe the problem.

ix

Guidelines for Safety Procedures in Teaching Laboratories in the Department of Biological Sciences Objectives:

Students in laboratories in the Biological Sciences should be aware that there are risks of personal injury through accidents, e.g. re or explosion, exposure to biohazardous materials, corrosive chemicals, fumes, cuts. These guidelines are meant to minimize the risk of injury by emphasizing safety precautions and to clarify emergency procedures in the event of an accident. Emergency Equipment:

You should know the locations of thefollowing equipment; your instructor willinform you of at the rst lab: • Eyewash facilities and explanation of operation

• Closest emergency exit

• Fire extinguisher and explanation of use.

• Closest re alarm and pull station.

• Safety showers and explanation of operation.

• First aid kit.

• Closest emergency telephone and emergency phone #s. General Safety Regulations:

1) Eating, drinking and smoking are prohibited in all laboratories. Cell phones are not allowed in the laboratories. 2) Always wash your hands prior to leaving the laboratory. 3) Laboratory Coats and Safety Glasses (splash-proof glasses recommended) andappropriate attire are required for all laboratories unless otherwise indicated by the coordinator of the course. 4) Students are not allowed to work in a laboratory unless a lab instructor is present. 5) Before starting an experiment, establish a plan of action and list of required materials. Compile all materials before starting. Confusion can cause accidents. 6) Report equipment problems to the labinstructor immediately. 7) Report all spills to the lab instructor immediately. 8) Long hair must be tied back to keep from being caught in equipment, Bunsen burners, chemicals, etc. 9) It is recommended that contact lenses not be worn in laboratories. Use chemical splash goggles over normal eyeglasses. 10) Enthusiasm is encouraged but pranksand horseplay can be hazardous.

x

Spills: Minor spill of SOLUTION/CHEMICAL: Put on gloves and wipe up the spill using paper towels

and a sponge as indicated by the lab instructor. Major spill of ACID/BASE/TOXIN: Immediately contact your lab instructor. DO NOT TOUCH!

Radioactive material: Small volume/little activity: Contact your labinstructor. While wearing lab coats/gloves, clean up the uid and deposit all materials in the solid waste container. Large volume/unsure of total activity: Contact your lab instructor and the Safety Ofce will be

called. In EITHER SITUATION, contact the coordinator of the teaching laboratories and notify them of the time, location and person(s) involved in the accident. All spills of radioactive material on individuals or on lab coats shall be reported to the Safety Ofce as stated in the Radiation Policies and Procedure Manual.

Blood/Bacterial/Fungal/Virus spills: If you spill a container of a biohazardous material, immediately tell your lab instructor and students around the spill area. If necessary, remove any contaminated protective and personal clothing. Prevent anyone from going near the spill. Wash your hands and face. The lab instructor and laboratory coordinator are required to supervise the clean up at this point.

Health concerns: 1)

Allergies:

Students with allergies (chemical, plant, animal or antibiotic) should inform the laboratory coordinator if the allergy may be relevant to any laboratory exercises in the course. The student should then receive a note from his/her doctor to indicate if the student should be allowed to carry out the experiment(s) using appropriate precautions arranged through the Safety Ofce or if the student should not be present during the experiment. 2)

Pregnancy

Some chemicals used in laboratory teaching are teratogens, which cause fetal deformities. It is important that students who are pregnant inform the laboratory coordinator so that the appropriate precautions are taken in consultation with the Safety Ofce. 3)

Other health concerns:

a) Animal Bites must be reported to the lab instructor immediately, the student will then be taken to Health Services for a tetanus shot. b) The student should make the laboratory coordinator aware of any serious healthconditions which may be exacerbated by work in the laboratory.

xi

Disposal of Wastes Most waste produced by laboratories cannot be disposed of in the sink or regular garbage cans. Please pay close attention tothe following procedures. Radioactive liquid: All radioactive LIQUID must be poured into a radioactive liquid waste container from the Safety Ofce. Do not pour anything down the designated sink without rst check ing with your lab instructor or the laboratory coordinator. All SOLID radioactive waste such as pipette tips, gloves, paper towels, microfuge tubes, etc., is placed n i the solid radioactive waste container (Safety Ofce). Yellow Container All sharps: needles, scalpels, syringes, razor blades and other sharp items. Broken glassware and materials such as pipette tips, microtitre plates, gloves, paper towels CONTAMINATED with BLOOD, BODY FLUIDS or other BIOHAZARDOUS MATERIAL.

Blue Container Clean Lab Glassware: broken glass, Pasteur pipettes, broken pipettes Clean Plastic Labware NO CHEMICAL, BIOLOGICAL , OR RADIOACTIVEMATERIALS

NO CHEMICALOR RADIOACTIVE MATERIALS

Petri plates: Place in clear plastic biohazard bags for autoclaving. Bacterial or fungal liquid: Tubes and asks containing liquid cultures are placed in marked trays

or on carts for autoclaving. Viruses: Pour liquid and place materials into a bleach solution. (This is followed by exposure to UV light and autoclaving before disposal). Animal Tissues/Carcasses: Place in plastic bags for disposal through the Animal Resources

Center (Main Campus). Liquid Chemicals/Fixatives: Place in 20 L containers provided by the Safety Ofce. Other la belled screw-cap bottles may be provided to keep certain chemicals separate. Solid Chemicals: Place in containers for disposal by Safety Ofce. In case of any uncertainty in waste disposal please contact the lab instructor

xii

Preface Welcome to CMMB 343! This course is intended for students wishing to major in the Cellular, Molecular and Microbial Biology Program in the Department of Biological Sciences. The study of microorganisms is a dynamic and fast-paced eld, with advances and discoveries occurring almost every day. This course focuses on the bacteria, but consideration will be made for the archaea, eukaryotic microorganisms and viruses. Microbiology is a relevant science that impacts our daily lives in numerous ways. By performing the exercises in this manual you will develop an appreciation of the variability of microbes that are involved in areas such as microbial genetics, ecology, medicine and biotechnology. It is hoped that the scope of the experiments in this laboratory manual challenge, interest and inspire you to further your studies in this interesting eld. INTRODUCTION TO THE CMMB 343 LABORATORY

A. Objectives of the Laboratory The main purpose of laboratory manipulations is to put into practice concepts you are learning in your lectures, textbooks and the scientic literature. It is in the laboratory where the tools and techniques necessary to develop new ndings and ultimately new concepts are incorporated. Science, by nature, is a product of laboratory effort. Therefore one can say that real science is found in the laboratory. The overall objectives of the CMMB 343 laboratories are to: 1. reinforce aseptic technique and basic microbiological methodology 2. provide practical information on heterotrophic, chemotrophic and phototrophic microorganisms 3. provide information that demonstrates the importance of microorganisms in the environment, in the food industry and in medicine A primary feature of the microbiology laboratory is that living organisms are employed as part of the experiment. Most of the microorganisms are harmless. However, whether they are nonpathogenic or pathogenic, the microorganisms are treated with the same respect to ensure that personal safety in the laboratory is maintained. Careful attention to technique is essential at all times. Your attitude to laboratory study is an important factor in determining how well the session will go. If you are interested in learning and willing to put in both time and effort, it will be a most pleasant and rewarding experience.

xiii

B. Open Laboratory Due to the nature of microbial growth, it is necessary to incubate the cultures prepared in the labs for 24 - 48 hours at the appropriate temperature to allow time for the microorganisms to grow. As a result, it will be necess ary for you to return to the lab the day following the scheduled lab to observe your results.

The lab submissions following each exercise are scheduled to be handed in with enough time to properly observe the results. References will be available in the open labs and on reserve in the library during the week. Use these resources to help you answer the submission questions and interpret your results. OPEN LAB SCHEDULE

Monday: 0900-1200 Wednesday: 0900-1500 Friday: 0900-1500 If your class schedule conicts with the open lab schedule, contact Mr. Huddleston ([email protected]) to make alternate arrangements.

Laboratory Dress Code You can’t put a covering over the entire surface of the Earth, so instead, wear sandals -Buddhist wisdom Throughout this term, you will be working directly with microorganisms. Although the organisms used in this course have been approved for use in an undergraduate laboratory, care must be taken to ensure safe handling practices. We will therefore treat all organisms as if they were classied a s Biosafety Level 2 (BSL2 - see Appendix 7). We will be maintaining a strict regulation of dress code in the lab that is appropriate for working with BSL2 organisms. In addition to using aseptic technique when working in the laboratory, your dress must meet the following guidelines at all times: 1.

No open-toed footwear (e.g. sandals, etc.)

2.

A laboratory jacket must be worn properly at all times

3.

No exposed skin is allowed that is not covered by the laboratory jacket (e.g. no shorts, skirts, etc.)

4.

Long hair must be tied back and tucked into the laboratory jacket

Failure to meet these criteria will result in denied admittance to both the scheduled and open laboratory xiv

Laboratory 1 Objectives:

1.

To become familiar with the microbiology laboratory environment

Recommended Laboratory Resources:

Madigan, MT, KS Bender, DH Buckley, WM Sattley and DA Stahl. 2018. Brock: Biology of Microorganisms, 15th edition. Pearson Education, Inc., New York. pp. 2; 18 Lab Manual Appendices 1, 4, and 7 A.

Ubiquity of Microorganisms

Unless special precautions are taken to exclude them from an environment, microorganisms are always present (ubiquitous) in the air, on surfaces, and on your skin, clothing, hair,etc. Microbiologists must be acutely aware of the ubiquity of microorganisms because their presence is a potential source of contamination. Aseptic (sterile) technique is essential in order to protect you (and your colleagues!) from the organisms with which you are working and to prevent contamination of the cultures you are studying.

Importance of Aseptic Technique To demonstrate the importance of thorough hand-washing and workspace sterilization, you will inoculate nutrient agar plates before and after the use of soap and disinfectant. Your TA will demonstrate how to handle agar plates and how to work aseptically in order to minimize the risk of contamination.

Procedure: (work individually) 1. Hand-washing

1. Obtain 1 nutrient agar (NA) plate from the side bench. 2. Ensure that you keep the lid on the plate until you are ready for inoculation. 3. Without washing your hands, remove the lid and gently roll each nger and thumb from one hand across the surface of the agar of the plate - pressgently so you do not gouge the agar. Replace the lid as soon as you are nished. 4. Label the plate properly (page 63)as demonstrated by your TA. 5. For now, place the plate on your bench where it will not be disturbed. 2. Environmental survey

1. Obtain 1 nutrient agar(NA) plate from the side bench. Use a marker to “divide” the platein half as demonstrated by the TA. 2. Swab a surface (e.g. oor, bench, shoe, etc.) of your choice with a moist applicator and gently wipe it across the surface of the agar on one side of the plate. Ensure that the lid is only removed during the inoculation of the plate. 3. Label the plate properly (page 63) and place it with your hand-washing plate. 1

Aseptic Technique and Laboratory Standard Operating Procedures Read these guidelines carefully. They must be strictly adhered to so theybecome routine. Remember, the microbiology laboratory is considered a BIOHAZARDOUS area. 1. Thoroughly read each day’s experiment(s) before coming tothe laboratory. When you are prepared, the lab runs more efciently and accurately. As Pasteur stated, “chance favours the prepared mind”. 2. Laboratory coats must always be worn properly. Do not come to the laboratory with long loose hair, loose clothing or open-toed footwear. Avoid the use of hair spray before coming to the lab. 3. Do not bring anything that is not required (coats, bags,cell phones,etc.) into the laboratory. Smoking, eating, or drinking are not permitted. Do not place anything (pencils, ngers, etc.) in your mouth. 4. Place your lab manual to the side of your work area. Work over the lab bench - not over your book.

5. Clean the bench top properly with disinfectant before and after your laboratory work. 6. Wash your hands properly with soap and water before and after your laboratory work. 7. Know the location of the re extinguisher, eye wash, shower and rst-aid kit.

8. Lab benches must be completely cleared andreset after each laboratory session.E.g. microscopes are cleaned and stored properly, inoculating loops are placed in their correct holders, etc. 9. It will often be necessary to incubate cultures overnight. Culturesshould always be labelled properly (Ap4-5). Instructions will be provided regarding where to place each culture and for how long each culture needs incubation. Any plates left on the student bench will be discarded. 10. Any spillage of culture must be cleaned up immediately by covering with paper towel and pouring disinfectant onto the covered spill. Wait 5 minutes before wiping. Immediately report any spill. 11. Always use the appropriate equipment properly when handling chemicals, reagents or bacterial cultures. Instructions will be given for each exercise as to which piece of equipment to use and how to use it. 12. Used micropipette tips must be ejected directly into the red biohazard disposal bag located on the student bench. Used tips areNEVER to be placed on the bench. 13. Used glass pipettes must be placed immediately in the pipette bucket located on the side bench. Used pipettes are NEVER to be placed on the bench. 14. Agar plates to be discarded are placed in theautoclave bucket at the front of the lab. Culture tubes to be discarded are placed in thetest tube rack at the front of the lab. Microscope slides to be discarded are placed in the used slide container at the front of the lab. 15. All broken, but sterile glassware and plastic is placed in theblue bucket at the front of the lab. 16. All broken, contaminated glassware and plastic is placed in theyellow bucket at the front of the lab. 17. Never remove cultures or any other materials or equipment from the laboratory. IF YOU ARE UNSURE OF ANY PROCEDURES, USE OF EQUIPMENT, OR TECHNIQUES, ASK THE LAB INSTRUCTOR FOR HELP

2

LEVEL II LABORATORY:

Assume that you are always working with BSL II microorganisms in the laboratory (page 79). Although all organisms have been approved for use in the laboratory, proper microbiology techniques should be used in all experiments. Now that your TA has discussed lab safety and aseptic technique, you will follow these guidelines and inoculate a second set of hand-washing and environmental survey plates.

Procedure: (work individually) 1. Hand-washing

1. Obtain 1 nutrient agar (NA) plate from the side bench. 2. Wash your hands and then use aseptic technique to inoculate the plate by gently rolling each nger and thumb from one hand across the surface of the agar of the plate - press gently so you do not gouge the agar. Use the same hand you used to inoculate the rst agar plate. 3. Label the plate properly, stack both hand-washingplates together and place them in the correct box on the side bench. They will be incubated and returned to you next week. 2. Environmental survey

1. Use the environmental survey nutrient agar(NA) plate you inoculated earlier. Remember, you divided the plate in half. 2. Use the supplied detergent to clean the surface you sampled earlier. 4. Use a new moist applicator to swab thesurface again to inoculate thesecond half of the agar plate. 5. Place it in the correct box on the side bench. It will beincubated and returned to younext week.

B.

Lab Quiz 1 You must complete Lab Quiz 1 (posted on D2L)before your next scheduled lab.

Checklist for Laboratory 1: q

Each student should inoculate two hand-washing plates andone environmental survey plate

q

Each student should complete Lab Quiz 1before their next scheduled lab

3

4

NAME: _____________________________

EXERCISE 1 SUBMISSION

LAB SECTION: ______________________ SCORE: __________

A.


1. Note that part of the grade for this submission is from theassessment of the threeplates you submit. Ensure you have labelled and stacked them properly as demonstrated by your TA.4()

2. Record your observations ofthe two hand washing plates and the environmental survey plate. Notethe relative abundance and diversity of organisms on the plates. 4( ) Hand-washing plates Before soap

After soap

Environmental survey plate Before detergent

After detergent


1.

To gain competence with the use of common laboratory techniques used in microbiology

2.

To describe the colony and cellular morphology characteristics of microorganisms


Madigan, MT, KS Bender, DH Buckley, WM Sattley and DA Stahl. 2018. Brock: Biology of Microorganisms, 15th edition. Pearson Education, Inc., New York. pp. 11-14; 35-36; 4245; 74-75; 144-150. Lab Manual Appendices 3 and 4 A.


Importance of aseptic technique Procedure: (work individually) 1. Collect the two hand washing plates and one environmental survey plate youprepared in Lab 1. 2. Compare the abundance and diversity of bacteria on each plate. Record your observations on the Exercise 1 submission sheet. 3. Discard the plates properly in the autoclave bucket at the front of the lab. 4. Submit the Exercise 1 submission sheetbefore you leave the lab.

B.

Nutrition and Culture of Microorganisms

In order for microorganisms to grow and divide, they must be able to make the complex macromolecules needed for life. Organisms make these macromolecules by obtaining nutrients from the environment, transforming them into the precursor molecules they need, and than building the specic polymers they require. Microbial nutrition involves nutrient acquisition, enzymatic transformation and polymerization of these complex macromolecules. Due to the diversity of microbial life, the amounts and types of nutrients required for life can vary greatly macronutrients (nutrients required depending on the species. However, all organisms must acquire a source of in large amounts) such as: oxygen (H2O, O2, etc.), carbon (CO2, glucose, etc.), nitrogen (NH3, NO3-, etc.), and minerals (P, S, K, Mg). Organisms also requiremicronutrients (nutrients required in trace amounts) such as: metal ions (Fe, Cu, Ni,etc.) and organic growth factors such as vitamins (thiamine, biotin,etc.).

5

Culture Media for Bacteria To study microorganisms in the laboratory, we must be able toculture them. Microbiologists refer to the nutrient solutions used to culture organisms asmedia (singular: medium). Chemically dened media consist of a basal (basic) salt solution, which supplies the inorganic requirements of the organism, and organic components that provide a source of carbon and energy. The exact chemical composition of each componentis known. Complex media contain substances that are rich in both inorganic and organic nutrients such as meat or vegetable infusions, blood and hormones. The exact chemical composition of the media is not known. Enriched mediaorganisms. are loadedMinimal with nutrients macromolecular monomers to promote the growth of even the fastidious mediaand most contain only the most basic nutrients.

Microorganisms are ubiquitous, therefore culture media must be sterilized in a giant pressure cooker called an autoclave. The media is prepared and autoclaved at 15 pounds pressure (psi) at a temperature of 121°C. petri dishes or test tubes using aseptic After autoclaving, the media is cooled (~50°C) and poured into sterile technique. Media may be prepared as a liquid b ( roth), or have agar added to provide a semisolid support for growth. Agar liquees at high temperatures (100°C) and chemically dened or complex media can be added. As media containing agar continues to cool, it solidies (42°C) forming a rm transparent gel. Broth or agar media can then beinoculated with a sample and incubated under the desired environmental conditions. On agar media, microbial cells are immobilized, allowing them to grow in aggregates of cells that eventually form visiblecolonies. In broth, microbial cells grow in solution in aplanktonic (free-living), unattached phase.

Colony Morphology of Bacteria Robert Koch was the rst to notice that different species of bacteria could be distinguished from one another when grown on agar media. Today we know that individual microbial cells placed on agar media grow and divide, eventually forming a mass of cells visible with the naked eye. These masses of cellscolonies ( ) have physical characteristics that differ depending on the species of bacteria growing on the agar. We refer to these characteristics as the organism’scolony morphology (page 62). For many years, colony morphology was a major distinguishing feature used by microbiologists to isolate and describe species of bacteria and is still used extensively today. When only one colony morphology (and therefore only one microbial species) is present on an agar plate, we say that we have apure culture.

Procedure: (work individually) 1.

Observations of microbial cultures (may be completed in the open lab)

1. Observe the sample agar plates on the side bench that demonstrate various colonymorphologies. These can be used to help you recognize the morphologies on your plates. 2. Observe the streak and spread plates on the side bench that demonstrate commonly observed errors. These can help you identify how to improve your technique.

6

2.

Streak plate method to obtain single colonies (page 62):

1. Obtain one stock plate, oneNA plate and one minimal glucose (MG) plate fromthe side bench. 2. Observe the TA demonstration for preparinga streak plate. 3. Prepare a complex media streak plate on the NA plate. 4. Prepare a chemically-dened media streak plate on the MG plate.

5. Label both plates properly andplace them in the correct box on the side bench. Return the stockplate to the side bench. 6. In the open lab, collect your plates from the box on the side bench, note the pattern of growth, conrm the presence of single colonies and record thecolony morphology for each plate on the Exercise 2 submission sheet. 7. Submit your best streak plate to the correct box on the side bench anddiscard the other plate properly. 3.

Spread plate method to enumerate bacteria (pages 64-66)

1. Obtain one stock broth culture tube and three NA plates from the side bench. 2. Compare your broth culture tube with the turbidity standards onthe side bench and record your estimate of the cellular concentration on the Exercise 2 submission sheet. 3. Observe the TA demonstration for preparing serial dilutions anda spread plate. 4. Prepare seven serial 10-fold dilutions (10-1 to 10-7) of the stock broth culture. -5

-6

-7

5. Prepare a spread plate for the 10 , 10 , and 10 dilutions on separate NA plates. 6. Label the plates properly and place in the correct box on the side bench. Return the stock broth culture tube to the side bench. 7. In the open lab, collect your plates, determine which one has between 30 and 300 colonies (page 66), calculate the concentration of the srcinal stock broth culture and record your answer on the Exercise 2 submission sheet. 8. Discard the plates properly.

7

C.

Microscopy and Staining

Your TA will review the parts, function and proper handling of the microscope (Appendix 2). Make sure you have working knowledge of the microscope before you proceed. Gloves will be available to reduce the risk of staining your hands.

Cellular Morphology of Bacteria Under a light microscope, most bacterial cells do not provide enough contrast to be detected without the aid of stains. Once stained, microbial cells can only be visualized under the oil-immersion lens at the very limits of a light microscope’s resolving ability. We refer to the characteristics observed under a microscope as the organism’s cellular morphology. Many techniques have been developed that stain specic structures such that we can observe a variety of physiological characteristics of bacteria. Differential stains can be used to highlight differences between types of bacteria because they do not stain all cells the same. The major features recorded when describing cellular morphology are listed on page 60.

Procedure: (work individually) Gram stain preparation and observation (pages 59-60):

1.

1. Obtain a stock plate from the side bench 2. Your TA will demonstrate how to prepare abacterial smear and how to carry out theGram stain. 3. Focus on a patch of cells under the 10x objective and then follow the procedure forusing the oil immersion lens (page 52). 4. Use the space on the Exercise 2 submission sheet to record thecellular morphology and prepare a diagram of a representative microbial cell. 5. Look at your colleague’s slides: some of the samples are Gram-positive and someare Gram-negative. You should be able to identify both of these Gram reaction morphologies. 6. Discard the slide properly in the slide bucket at the front of the lab. Return the stock plate to the side bench. Maintaining the microscope:

2.

1. Clean the lenses and put the microscope away properly as demonstrated byyour TA.

D.

Class Presentation

You (in a group of students) will give a 20 minute class presentation in the lab the week of February 12. Your TA willassign a topic before you leave the lab this week. More information can be found on page 27 and will also be provided by your TA in the lab.


Each student complete and submit the Exercise 1 submission sheet

q

Each student should prepare a NA and MG streak plate

q q

Each student should prepare 3 NA spread plates Each student should prepare a Gram stain and diagram

q

Each student should record their results in the open lab and discard their plates properly

q

Each student should submit the Exercise 2 submissionbefore noon on January 19/22

q

Each student group should start preparing for the class presentation 8

NAME: _____________________________



Nutrition and Culture of Microorganisms 1. Note that part of the grade for this submission is from the assessment ofthe streak plate you submit. (2) 2. Record the colony morphology ofthe organism from the NA plate and the MG plate. (2) MG

3. Using the turbidity standards, whatdo you estimate the cellular concentration ofyour unknown broth culture tube to be? (1)

4. What is the cellular concentration of yourunknown broth culture tube based on the spread plate method of enumeration (page 66)? Show your calculation and use proper scientic notation. (3)

5. Use the space below to prepare a diagram of a representative microbial cell fromyour Gram stain (page 61). Ensure your caption indicates the cellular morphology of the organism.5()

Laboratory 3 Objectives: 1.

To identify an organism by growth and morphology on selective and differential media

2.

To use staining techniques to describe the cellular morphology characteristics of microorganisms

3.

To identify an organism based on differential staining of morphological characteristics

4.

To prepare a Winogradsky column for weekly observations of metabolic diversity and ecosystem stratication


Madigan, MT, KS Bender, DH Buckley, WM Sattley and DA Stahl. 2018. Brock: Biology of Microorganisms, 15th edition. Pearson Education, Inc., New York. pp. 24; 48; 53; 56-57; 413-315; 585-588. Lab Manual Appendices 2, 3 and 4 A.

Use of selective and differential media

Compounds can be added to dened or complex media that help select for or differentiate between species of bacteria in a mixed culture.Selective media contain compounds that selectively enrich and/or selectively repress the growth of certain organisms while not affecting the growth of others. In selective enrichment, the nutritional or environmental conditions are controlled to favor the growth of a sought-after species. Selective repression involves inhibiting the growth of interfering species while permitting the growth of a sought-after species.

Some selective media are also differential.Differential media contain an indicator that differentiates the occurrence of specic chemical reactions such that groups of bacteria can be differentiated from each other based on their ability to carry out that reaction (page 57).

Procedure: (work individually) You will be given a pure broth culture containing anyone unknown bacterial species: A, B, C or D.

Record your unknown code on the Exercise 3 Submission sheet 1. Prepare a streak plate on Brain-Heart Infusion (BHI), PhenylethylAlcohol (PEA), Desoxycholate (DOC), and Mannitol Salts (MSA) agar plates. 2. Label each plate properly and place them in the correct box on the side bench. 3. Your TA will demonstrate how to use the DifcoTM & BBLTM Manual. It contains detailed information TM about the media used in many microbiological applications. Using the Difco & BBLTM Manual and Appendix 3, list, in point form, on the Exercise 3 submission sheet, the ingredient(s) in each medium which make that medium either selective, differential or both. For the selective ingredients, indicate which organisms are either inhibited or enhanced and explain why. For the differential ingredients, explain how they are differential. Your TA will work through an example with you. A link to the DifcoTM & BBLTM Manual is posted on D2L. 4. In the open lab, note the presence of growth and record the colony morphology on the Exercise 3 submission sheet. Discard the plates properly.

9

B.

Microscopy and Differential Staining

Last week you reviewed the parts, function and proper handling of the microscope (Appendix 2). Make sure you have working knowledge of the microscope before you proceed. Your TA will demonstrate how to prepare the acid-fast, capsule, endospore and agella stains (pages 59-60).

Procedure: (work in a group of 4) You will be given a pure agar streak plate containing anyone unknown bacterial species:a, b, g, d.

Record your unknown code on the Exercise 3 Submission sheet 1. Look at the demonstration Gram, acid-fast,capsule, endospore, andagella stains of the positive controls located on the side bench. Use these as a guide to help you interpret the results of your stains.

Ensure you can explain how each stain works and how to interpret the results for each. 2. Prepare a Gram, acid-fast, capsule and endospore stain (i.e. one stain per student) of your unknown organism and record the results on the Exercise 3 submission sheet. 3. Use the chart (page 12) to identify your unknown. 4. Discard all slides properly and return the unknown agar plate to the side bench.

C.

Winogradsky Column

In the 1880s, Sergei Winogradsky devised a procedure to study soil microorganisms. The Winogradsky column models an anaerobic ecosystem that can be used for the isolation of bacteria and archaea and is an effective means to study some of the most numerous organisms on Earth. These microorganisms show extreme metabolic diversity, including oxygenic and anoxygenic photoautotrophy, chemolithoautotrophy, and photoheterotrophy. They are also descendants of the rst organisms on the planet, with a history that goes back 4 billion years. In this exercise, you will use an environmental slurry (a mixture of soil and pond water from Calgary) to observe and study some of the different types of metabolism illustrated by this diverse group of microbes. Each student bench will prepare four Winogradsky columns: a control column (A), a column containing added carbon (B), a column that contains added sulfur (C), and a column that contains both added carbon and sulfur (D). The columns will be incubated for several weeks and you will make a series of observations as the microorganisms develop within the column.

Procedure: (Please work as a lab bench) Each lab bench will prepare four Winogradsky columns, one of each treatment: A: control

B: +carbon

C: +sulfur

D: +carbon/+sulfur

Work with a partner to prepareone of the above Winogradsky columns 1. Label a Winogradsky column tubewith your initials, lab section, and thetreatment (A, B, C or D) Mark the tube 10 cm and 13 cm up from the base of the tube as demonstrated by your TA 2. Fill your column with the appropriate soil (A, B, Cor D) up to the 10 cm line 3. Gently tap the tube to remove as much air from the slurry as possible without compacting thesoil 4. Add water to the tube until it has been lled to the 13 cm line

10

5. Place the lid on the tube and turn until it is halfway closed. Do NOT tighten! 6. Use the space below to record your initial observations ofall four columns. This should include the colour of each layer, changes to the sediment, development of layers within the sediment and water, changes in the thickness of sediment layers, and differences among the columns and anything else that becomes evident (don’t forget that smell is a great sense!) You may take a photograph of your columns as long as you maintain aseptic technique. 7. Place your column in the appropriate rack on the light cart 8. Clean up any mess that you made while assembling the column.Your entire bench willbe penalized if your workspace is not cleaned up properly. Winogradsky Column Observations (each pair should observe all four columns)

You will makeweekly observations of all four columns throughout this5 week exercise, so you will need to prepare a table to organize your work. You may use the space on page 13 for your table (or using your own resources). Make sure you include your Week 0 observations! You should also start thinking about the questions on page 14 as the Winogradsky columns develop over the course of the exercise. Suggested Resources (links provided on D2L)

1. Microbial Life Educational Resources-Winogradsky Column. http://serc.carleton.edu/microbelife/topics/special_collections/winogradsky.html 2. The Winogradsky Column. http://www.sumanasinc.com/webcontent/animations/content/winogradsky.html Note: This exercise has been adapted from a Howard Hughes Medical Institute Laboratory exercise with many thanks. 11

Table 3.1: Table of Staining Results for Known Organisms Gram

Cell

Cell

Acid-Fast

Capsule

Spore

Flagella

Stain

Morphology

Arrangement

Stain

Stain

Stain

Stain

Bacillus coli

-

rod

single cells

-

+

+

+

Actinobacterium aureus

+

rod

single cells

-

+

+

+

Lactobacter caseus

-

rod

single cells, pairs or short chains

Seratia pneumoniae

+

rod

pairs chains or

Micrococcus citrus

+

coccus

pairs or tetrads

Pseudomonas catarrhalis

weak+

Organism

Mycobacterium

rod

-

-

+

+

+

single cells or in dense clusters

--

-

-

-

-

-

+---

-

coccus

pairs

-

+

-

+

coccus

single cells, pairs, tetrads or clusters

-

+

--

-

shmegmatis Escherichia boydii

D.

Lab Quiz 2

You must complete Lab Quiz 2 (posted on D2L)before your next scheduled lab.


Each student should prepare 3 selective/differential streak plates of their unknown (A, B, C, or D)

q

Each student group should complete the differential stains (4) on their unknowna, ( b, g, or d)

q

Each student pair should prepare a Winogradsky column

q


q

Each student should submit the Exercise 3 submissionbefore noon on January 26/29

q


12

s n o it a v re s b O n m u l o C y k s d a r g o in W

13

Winogradsky Column Questions 1. How do your columns differ? How are they the same? Explain the differences.

2. Did you observe changes in the control column? If so, explain why they occurred.

3. Winogradsky columns form oxygen concentration gradients. Predict thedistribution of oxygen throughout the column (sediment, water, air).

4. Winogradsky columns form sulde concentration gradients aswell. In the columns that contain egg yolk, predict how sulde will be distributed throughout the column (sediment, water, air).

5. Desulfovibrio are an example of bacteria that grow in Winogradsky columns. Whattype of metabolism do they carry out? What substrates are required? What products are formed? Where in the column would you expect to nd them?

6. Winogradsky Chromatiaceae, Rhodospirillaceae, Chlorobacteriaceae are examples of substrates bacteria that in columns. What type ofand metabolism do they carry out? What aregrow required? What products are formed? Where in the column would you expect to nd each of them?

7. Cyanobacteria and algaeare examples of organisms that grow in Winogradsky columns. Whattype of metabolism do they carry out? What substrates are required? What products are formed? Where in the column would you expect to nd each of them?

8. Explain how Winogradsky columns illustrate the diversity ofmicroorganisms found onEarth today in terms of the diversity of niches they occupy.

9. Explain what the Winogradsky columns illustrateabout life on early Earth.

14

NAME: _____________________________


SCORE: __________

A.

LAB SECTION: ______________________

Use of Selective and Differential Media SELECTIVE/DIFFERENTIAL PLATES UNKNOWN CODE: _________

1. For the following media, use the DifcoTM & BBLTM Manual to: (8) a. Indicate the selective and/or differentialcomponents and explain how each component is selective and/ or differential b. Indicate which component provides a source of macronutrients

Selective:

Phenylethyl Alcohol (PEA) agar

Differential:

Macronutrients:

Desoxycholate (DOC) agar Selective:

Differential:

Macronutrients:

Mannitol Salts (MSA) agar Selective:

Differential:

Macronutrients:

2. Record the colony morphology (if available) ofyour unknown organism on each of the 4 plates. (4) Brain-Heart Infusion (BHI) agar

Phenylethyl Alcohol (DOC) agar

Desoxycholate (DOC) agar

Mannitol Salts (MSA) agar

3a. Identication of unknown organism: ________________________________________________1)( 3b. Provide a rationale for your identication. 3()

B.

Microscopy and Differential Staining Differential Staining Unknown Code: __________

4a. Record the results of the Gram, acid-fast, capsule, and endospore stains for your group’s unknown. 4)(

4b. Identication of unknown organism: _______________________________________________1)(

5. What is one advantage, to your unknown organism, of possessing the morphological property above? 1()


1. To become familiar with the effects of various environmental parameters on the growth of microorganisms 2. To observe the effect of heat on bacterial cell viability in spore-forming and non-sporeforming organisms 3. To screen potentially carcinogenic chemicals using the Ames test Recommended Laboratory Resources:

Madigan, MT, KS Bender, DH Buckley, WM Sattley and DA Stahl. 2018. Brock: Biology of Microorganisms, 15th edition. Pearson Education, Inc., New York. pp. 53-56; 152-159; 310-313. A.

Factors Affecting the Growth of Bacteria

We have considered the nutrient requirements of microorganisms grown in culture and the ability to selectively culture organisms based on the nutrient composition of media. However, microbial growth is also inuenced by the physical and chemical characteristics of the environment. Understanding the environmental requirements of an organism helps us understand the diversity of life on Earth and allows us to study a greater range of organisms in the laboratory. 1.

The Effect of Temperature

Temperature has one of the greatest inuences on the growth of microorganisms since all the processes of growth are dependent on chemical reactions that are affected by temperature. Growth occurs at temperatures that range between a minimum, the temperature below which growth cannot occur, an optimum at which they grow most rapidly, and a maximum above which the organism cannot grow. We refer to these three temperatures as the cardinal temperatures.

The cardinal temperatures are unique to each organism and allow us to classify microbes into four groups dened by the range of temperatures between their minimum and maximum growth requirements. Research Applications

A clear understanding of the cardinal temperatures of an organism is essential for expression of recombinant proteins. The temperature a bacterial culture is grown at can have a signicant impact on the expression of soluble recombinant protein. When a recombinant protein is expressed at high levels in E. coli, for example, the protein may end up in an insoluble protein fraction, called inclusion bodies. These bodies contain misfolded, inactive proteins that are targeted for degradation by intracellular mechanisms. Thus, if the recombinant protein ends up in inclusion bodies, the protein is difcult to purify and is often unusable for further experiments. The optimum temperature for the expression of each recombinant protein must be determined experimentally and must also t within the temperature range at which the host organism will grow. 15

Procedure: (work in a group of 4) 1. Divide 4 BHI agar plates into quadrants. Label one plate 15°C, the second 25°C, the third 37°C and the fourth 56°C. 2. Collect one broth culture tube of each of the four organisms on the side bench. 3. Aseptically spread a loopful of each culture (remember to vortex each tube well before using) within one quadrant of each plate (you arenot interested in obtaining single colonies!). 4. Place each properly labeled plate in the appropriate box (i.e. there are four boxes!) on the side bench and incubate for 24 hours. 5. In the open lab, rank the relative abundance of growth for each organism on each plate on the Exercise 4 submission sheet. The TA can help you if you require assistance. 2.

The Effect of Hydrogen-Ion Concentration

The hydrogen ion concentration (measured by pH) of an organism’s environment affects the growth of most bacteria. The pH range of an organism is the difference between the maximum and minimum pH at which the organism will grow. The optimum pH is the pH at which the organism grows best. It is important to note that these pH ranges refer to the organism’s external environment. The internal pH of most organisms can only tolerate very minor adjustments before life is no longer possible.

Procedure: (work in a group of 4) 1. Each pair of students needs to aseptically prepareone set of the following solutions using micropipettes (as demonstrated by your TA). Tube #

nutrient broth

0.2 M K2HPO4

0.1 M citric acidtotal

pH

1

8 ml

0.10 ml (100 µl)

1.90 ml (2 x 950 µl)

10 ml

2.8

2

8 ml

0.30 ml (300 µl)

1.70 ml (2 x 850 µl)

10 ml

3.6

3

8 ml

0.60 ml (600 µl)

1.40 ml (2 x 700 µl)

10 ml

4.4

8 ml

1.00 ml (1000 µl)

1.00 ml (1000 µl)

10 ml

5.2

5

8 ml

1.30 ml (2 x 650 µl)

0.70 ml (700 µl)

10 ml

6.0

6

8 ml

1.50 ml (2 x 750 µl)

0.50 ml (500 µl)

10 ml

6.8

7

8 ml

1.90 ml (2 x 950 µl)

0.10 ml (100 µl)

10 ml

7.6

0.2 M boric acid

0.2 M NaOH

8 ml

1.30 ml (2 x 650 µl)

0.70 ml (700 µl)

10 ml

8.4

9

8 ml

1.00 ml (1000 µl)

1.00 ml (1000 µl)

10 ml

9.2

10

8 ml

0.20 ml (200 µl)

1.80 ml (2 x 900 µl)

10 ml

10.0

4

8

16

2. One pair will aseptically inoculate 1 set of tubes with 100 µl from a broth culture of organism A. One pair will aseptically inoculate theother set of tubes with 100 µl from a broth culture of organism B. 3. Place the properly labeled tubes in the appropriate box on the side bench and incubate for24 hours. 4. In the open lab, gently resuspend the cells that may have settled to the bottom of each tube. Rank the relative turbidity for each tube on the Exercise 4 submission sheet. The TA can help you if you require assistance. Tubes 1-3 may form a precipitate because of the strong acidity of the solution. Be sure to compare your tubes with the control tubes which will be available in the open lab. 3.

The Effect of Heavy Metals

Heavy metals (Cu, Ag, Hg,etc.) are capable of exerting a lethal effect upon bacteria at high enough concentrations even though some may be required in trace amounts. Resistance to heavy metals is facilitated by plasmids that enable bacteria to pump the ions out of the cell or alter the oxidation state of the ion. Procedure (work in a group of 4)

1. Remove a tube of molten NA from the water bath and inoculate with 100µl ofE. coli broth culture. Gently rotate the tube between the palms to obtain a uniform distribution of organisms and then pour the agar into the empty sterile petri dish. Allow the agar to harden. 2. Using sterile forceps, dipa sterile paper disc into a solution of 1% silver nitrate. Place thedisc in the centre of the plate. 3. Place the properly labelled plate in the appropriate box on the side bench and incubate for24 hours. 4. In the open lab, observe the growth pattern of the organism. Measure the diameter of the inhibition zone and record your results on the Exercise 4 submission sheet.

B.

Effect of Spore Formation on Culture Viability

Early microbiologists noted that at temperatures above the maximum cardinal temperature, most microbial cells are killed. Consequently, the use of heat (autoclaving, boiling, and aming) became (and still is) a standard part of aseptic technique used to kill microorganisms to sterilize environments. However, it was also noted that heat was not always an effective means of destroying all microbial life. Ferdinand Cohn studied heat-resistant bacteria and eventually characterized the genus Bacillus. It was Cohn who rst discovered and described endospore formation and brought to light why some methods of sterilization were not always successful. Endospores are differentiated bacterial cells that form due to changes in the nutritional and physical environment of the organism. The highly resistant nature of endospores allow spore forming bacteria to survive extremes of heat, dessication (drying), UV radiation, pH, and chemical disinfectants compared with vegetative (undifferentiated) cells. Endospores can remain dormant for long periods of time - until environmental conditions become favorable again - and will then germinate into a fully viable vegetative cell. Endospore formation is limited to a few microbial species, but forms part of the normal life cycle of the sporeforming bacteria. The location of the endospore in the cell is usually characteristic of the species. For example, the location and shape of Bacillus endospores are different from the location and shape ofClostridium endospores. Therefore, the presence or absence of endospores and the description of the endospore is useful to a microbiologist as an aid in identication. 17

Procedure: (work in pairs) 1. Collect a mixed broth culture tube from the side bench. Vortex the tube well to resuspend the culture. 2. Prepare 2 streak plates on BHI agar for single colony isolation. Place each properly labeled plate in the appropriate box on the side bench and incubate for24 hours (one at 37°C and one at 55°C). 3. Place the broth culture inthe 80°C water bath for 10 minutes. Prepare astreak plate on BHI agar for single colony isolation. Place the properly labeled plate in the appropriate box on the side bench and incubate for 24 hours at 37°C. 4. Place the broth culture inthe 100°C water bath for 15 minutes. Prepare astreak plate on BHI agar for single colony isolation. Place the properly labeled plate in the appropriate box on the side bench and incubate for 24 hours at 37°C. 5. In the open lab, note the presence of growth on each plate and observe the demo Gram stains. Record the colony and cellular morphologies on the Exercise 4 submission sheet.

C.

The Use of Bacteria to Screen for Carcinogenic Chemicals Using the Ames Test

The conventional way todetermine whether or nota chemical substance is carcinogenic is to inject the material into animals and look for the development of tumours. If tumours develop, it is presumed that the substance can cause cancer. Although this method works well, it is costly and time consuming. The fact that carcinogenic compounds induce increased rates of mutation in bacteria has led to the use of bacteria for screening chemical compounds for possible carcinogenesis. The Ames Test, developed by Dr. Bruce Ames at the University of California, Berkley, has been widely used for this purpose. The correlation between carcinogenesis andmutagenicity is between 85% and 90%. Scientists are now using the Ames test to screen many chemicals quickly and inexpensively to determine which are mutagenic and therefore, potentially carcinogenic. back mutations The standard way to test chemicals for mutagenesis has been to measure the rate of (reversions) in strains of auxotrophic bacteria. In the Ames test, a strain ofSalmonella entericathat is auxotrophic for the amino acid histidine and lacks DNA repair enzymes (to prevent the correction of DNA injury) is exposed to a chemical agent. After chemical exposure and incubation on histidine-decient medium, the rate of reversion toprototrophy is determined by counting the number of colonies that are seen on the histidine-decient medium. Each colony represents a his —> his+ revertant. A positive result, indicating mutagenicity, is obtained when an obvious increase in the number of colonies is evident when compared to the number of spontaneous revertants in the negative control.

Keep in mind, when making your observations and conclusions, that this is a simplied version of the Ames test. Only one tester strain ofSalmonella enterica is being used in this experiment. Normally, several other strains would be used in order to accommodate different kinds of chemical compounds. While one chemical agent may be mutagenic on onetester strain, it may produce a negative result on another strain.Also, the test chemical agents are normally treated with a mammalian liver enzyme preparation. There is evidence that liver enzymes (mixed-function oxygenases) activate many carcinogenic chemical agents once they are in the body that are not active otherwise. This reagent will not be used in this experiment since the experiment will work well without it.

18

Procedure: (work in pairs) The rst three steps must be performed very quickly, before the top agar cools (about 20 seconds).

1. Pipette 100 l ofSalmonella mutant TA1538 into 1 tube of soft agar (from the hot water bath on the side bench).

2. Gently mix the contents of the tube by rotating between the palmsof both hands -do not shake! 3. Pour the contents of the tube over a plate of minimal glucose (MG)agar. Gently swirl the plate around to spread the agar evenly over the entire surface. Allow to harden. 4. Repeat the steps 1-3 twice more using two new soft agar tubes and MG agar plates. 5. Label the rst plate “negative control”. Dip asterile disc, using sterileforceps, into sterile water. Drain any excess water by touching to the side of the container. Place the disc in the centre of the plate and press gently so it will adhere to the surface of the agar.

6. Label the second plate “positive control”.Aseptically moisten a disc in a sodium azide solution, place the disc in the centre of the plate and press gently so it will adhere to the surface of the agar. Sodium azide is a carcinogenic substance and should be handled with extreme care

7. Label the third plate “unknown”. Usingthe same technique as above, moisten a disc in an unknown solution, place the disc in the centre of the plate, and press gently so it will adhere to the surface of the agar. 8. Place the plates in the appropriate box on the side bench. The plates will be incubated for72 hours and stored in the refrigerator. They will be returned in Lab 5.

D.

Lab Quiz 3 You must complete Lab Quiz 3before your next scheduled lab.


Each student group should inoculate 4 temperature plates, 20 pH tubes and 1 heavy metal plate

q

Each student pair should inoculate 4 endospore BHI plates

q

Each student pair should inoculate 3 Ames test plates

q

Each student should record their results in the open lab and discard their plates and tubes properly

q

Each student should submit the Exercise 4 submissionbefore noon on February 2/5

q


19

20

NAME: __________________________


LAB SECTION: ___________________ SCORE: __________

A.

Factors Affecting the Growth of Bacteria

1. What are the four classes used to distinguish microorganismsin relation to their temperature optima? Indicate the approximateoptimum temperatures for each class. (4)

2. Indicate the relative abundanceof growth (ranked from 0 to 3) of each organism on the temperature plates. Use the standards on the side bench (and the TA!) to help you gauge the level of growth. Classify each of the organisms into the appropriate temperature class.4() 1

2

3

4

15°C 25°C 37°C

56°C Classication: 3. Briey explain why theoptimum cardinal temperature of anorganism is closer to the maximum cardinal temperature than to the minimum cardinal temperature.2()

4. What is one molecular adaptation that alloworganisms that grow in hot environments (greater than 45°C) to survive? (1)

5. What three classes areused to distinguish microorganisms in relation totheir pH tolerances? 3( )

6. Use the table below to indicate the relative turbidity (ranked from 0to 3) for each of the pH tubes. Use the standards on the side bench (and the TA!) to help you gauge the level of growth. Classify the organism into the appropriate pH class. 4 () A Tube 1

pH 2.8

2

B Turbidity

Tube 1

pH 2.8

3.6

2

3.6

3

4.4

3

4.4

4

5.2

4

5.2

5

6.0

5

6.0

6

6.8

6

6.8

7

7.6

7

7.6

8

8.4

8

8.4

9

9.2

9

9.2

10

10.0

10

10.0

Classication

Turbidity

Classication

7. Explain how microorganismsmaintain a constant internal pH despite changes in the external pH. (2)

8. What is the zone of inhibition (diameter) around the heavymetal disc? Based on your results, is the organism resistant or susceptible to silver? 2()

9. How does silver inhibit microbial growth? (2)

B.

Effect of Spore Formation on Culture Viability

10. In the space below, note the presence of growth and record the colony and cellular morphology (if available) of any organisms that grow on the four BHI streak plates.4()

11. Explain your results, indicating the purpose of each plate and what information they told you about the organisms in the mixed culture. (4)


1.

To determine the phenol coefcient of a test disinfectant

2.

To evaluate the effectiveness of samples of commercially prepared antibacterial agents

3.

To observe the effect of several antibiotics using the Kirby-Bauer disk diffusion method


Madigan, MT, KS Bender, DH Buckley, WM Sattley and DA Stahl. 2018. Brock: Biology of Microorganisms, 15th edition. Pearson Education, Inc., New York. pp. 167-170; 839840; 852-858. A.

The Effect of Chemical Agents on Bacterial Growth

Antimicrobial substances are eithercidal (bacteriocidal, fungicidal,etc.), concerned with the killing of microorganisms; or static (bacteriostatic, etc.), growth-inhibiting.Antiseptics are cidal or static against Disinfectants are more potent cidal agents microorganisms, but are still safe enough for use on living tissues. that destroy nearly all microorganisms, but can be applied only to inanimate material because of their toxicity. The effectiveness of a chemical agent is measured by determining theminimum inhibitory concentration (MIC), which is the lowest concentration of agent that completely inhibits the growth of the test organism.

1.

Phenol coefcient

In the 1800s Joseph Lister introduced dilute phenol, formerly called “carbolic acid,” as a disinfectant for surgical dressings. Because his disinfection procedure dramatically reduced postsurgical infection and death, other surgeons soon accepted it. Phenol, in various dilutions was the rst disinfectant and antiseptic to gain favour in western medicine. For this reason, other solutions are now compared with it. The phenol coefcient is the ratio of the test agent’s MIC to the MIC of phenol. The test agent and phenol are tested against the same organism under the same conditions in order to generate the MICs. If the coefcient is greater than 1, the test disinfectant is better than phenol; if the coefcient is less than 1, then it is not as effective as phenol. Phenol coefcient =

reciprocal dilution of test disinfectant that kills at 10 minutes but not at 5 minutes reciprocal dilution of phenol that kills at10 minutes but not at 5 minutes

Research Applications

E. coli is one of the primary workhorses of modern molecular biology. It is used routinely for producing copies of cloned genes and for the production of recombinant heterologous proteins (produced from a cloned gene from a different species or organism).E. coli has been used for production of human insulin, bioremediation and industrial scale fermentative production of specic proteins. In the lab today, you will use E. coli to test the effectiveness of chemical agents on microbial growth and survival.

21

Procedure: (work in pairs) 1. One partner will use the 5% phenol and follow steps2a through 5a. The other partner will use the test disinfectant (1% Savlon) and follow steps2b through 5b. USE THIS PROCEDURE IF YOU ARE USING PHENOL

2a. Divide 2 BHI plates into quadrants and label with the appropriate duration (5 or 10 minutes), disinfectant (phenol), and the 4 dilutions (see below). 3a. Label 4 sterile 16 mm test tubes “1”, “2”, “3”, and “4”. Use a micropipette and sterile tip to dispense the following into the sterile tubes. PipetteE. coli last, at 1 minute intervals and immediately vortex the tubes. TUBE

1

distilled water 5% phenol nal dilution of phenol E. coli

3.5 ml 1000 µl 1/100

500 µl

2

3

4

3.6 ml 900 µl 1/110

3.7 ml 800 µl 1/125

3.8 ml 700 µl 1/150

500 µl

500 µl

500 µl

4a. Exactly 5 minutes after the addition of E. coli to the tubes, use a sterile loop to spread the appropriate quadrant of the 5 minute BHI plate. 5a. After a second interval of 5 minutes , repeat step 4a on the 10 minute BHI plate. USE THIS PROCEDURE IF YOU ARE USING SAVLON

2b. Divide 2 BHI plates into quadrants and label with the appropriate duration (5 or 10 minutes), disinfectant (Savlon), and the 4 dilutions (see below). 3b. Label 4 sterile 16 mm test tubes “1”, “2”, “3”, and “4”. Use a micropipette and sterile tip to dispense the following into the sterile tubes. PipetteE. coli last, at 1 minute intervals and immediately vortex the tubes. TUBE distilled water 1% Savlon nal dilution of Savlon E. coli

500 µl

1

2

3

4.2 ml 300 µl 1/1500

4.4 ml 100 µl 1/5000 500 µl

4

4.45 ml 50 µl 1/10,000 500 µl

4.49 ml 10 µl 1/50,000 500 µl

4b. Exactly 5 minutes after the addition of E. coli to the tubes, use a sterile loop to spread the appropriate quadrant of the 5 minute BHI plate. 5b. After a second interval of 5 minutes , repeat step 4b on the 10 minute BHI plate. -------------------------------6. Place all four plates in the appropriate box on the side bench and incubate for24 hours. 7. In the open lab, determine the MIC of phenol and Savlon that killsE. coli at 10 minutes but not at 5 minutes and calculate the phenol coefcient. Record your results on the Exercise 5 submission sheet.

22

2.

Evaluation of commercially prepared antibacterial agents

Many commercial preparations claim to be antibacterial. The purpose of this investigation is to make a limited evaluation of these claims and to compare the effectiveness of competitive products.

Procedure: (work in pairs) 1. One partner will use a Gram-positive organism andthe other partner will use a Gram-negative organism. 2. Prepare a microbial lawn by placing 100 µl ofyour organism onto a BHI plate. Spread the culture evenly over the plate using a glass spreader sterilized in alcohol and amed as demonstrated by your TA. When dry, divide the plate into quadrants and label with the organism and names of the test solutions. 3. Obtain a sample of the 4 antiseptic/disinfectant test solutionsfrom the side bench. 4. Using sterile forceps (see TA demonstration), dip a lter paper disc intoa test solution and place it on one of the quadrants on the BHI plate. With the tip of the forceps, touch the surface of the disc to improve its contact with, and adhesion to, the medium. Be sure to sterilize the forceps before picking up the next disc.

5. Place both plates in the appropriate box on the side bench and incubate for 24 hours. 6. In the open lab, observe and measure thezone of inhibition (i.e. diameter in mm) around the discs for each organism and record your results on the Exercise 5 submission sheet.

B.

The Effect of Chemotherapeutic Agents on Bacterial Growth

The previous exercise dealt with chemicals that control the growth of microorganisms outside the human body, but most of them are too toxic to be taken internally. Chemotherapeutic agents are antimicrobial compounds that can be administered internally and are important in human and veterinary medicine. Paul Ehrlich began to look for chemotherapeutic agents that had selective toxicity: the ability to kill or inhibit microorganisms, but have no adverse effects in the host. Antibiotics are compounds naturally produced by bacteria and fungi that kill or inhibit other microorganisms and provide a competitive advantage for the producing organism. Most antibiotics are produced by the two bacterial genera Streptomyces and Bacillus and the fungal genus Penicillium. Synthetic agents are compounds developed in laboratories that have antimicrobial action and also display selective toxicity. Antibiotics that have been structurally modied in the laboratory to improve activity and minimize toxicity are referred to as semisynthetic antibiotics. Different chemotherapeutic agents have a wide range of effects on different microbes. Some agents are broad spectrum: effective against a wide range of microorganisms (Gram-positive, Gram-negative, fungi, etc.). Others have a narrow spectrum of activity: only a few species are killed or inhibited by these agents.

23

1.

Kirby-Bauer antimicrobial susceptibility test

The Kirby-Bauer antibiotic disc diffusion test is a rapid, inexpensive simple test used in diagnostic laboratories to determine the effectiveness of an antibiotic on a particular strain of bacteria. The procedure is designed to evaluate one variable, the sensitivity (susceptibility) of a pathogen to assorted antibiotics, all other variables are held constant. Variables that can alter results of an antibiotic disc diffusion test include concentration and rate of diffusion of the antibiotic in each disc, density of bacterial growth, thickness of medium, andtemperature and length of incubation. The discs are prepared commercially by adding known amounts of the antibiotic to the disc. During incubation, the antibiotic diffuses from the lter paper into the agar, the further it gets from the lter paper, the weaker the concentration of the antibiotic.

Procedure: (work in pairs) 1. Prepare a lawn of a Gram-positive organism anda Gram-negative organism on different BHI plates. 2. Obtain the 3 antibiotics from the sidebench and aseptically place the discsevenly around both BHI plates. The discs must not be closer than 3 cm from each other and not closer than 2 cm from the edge of the plate. Ensure that the disc has made rm contact with the agar. Label both plates appropriately so that a comparison between the effectiveness of the antibiotics on Gram-positive and Gram-negative organisms can be made. 3. Place the plates in the appropriate box on the side bench and incubate for24 hours. 4. In the open lab, observe and measure the zone of inhibition (in mm) around the discs. Use the standards to determine the susceptibility of each organism to the antibiotics. Record your results on the Exercise 5 submission sheet.

C.

Ames Test Results

Collect the three Ames test plates you prepared in Lab 4. Record your results on the Ames Test submission sheet, which is due before you leave the lab. a) Count the number of colonies present on each plate. Ignore the barely visible background lawnof bacterial growth. b) Determine and record the number of chemically induced mutations by subtracting thenumber of colonies on the negative control plate, representative of spontaneous mutations, from the number of colonies on: i.

The positive control plate, and

ii. The unknown test plate c) Determine and record the relative mutagenicity ofthe test compound on the basis of the number of induced mutations: If below 10, (-); if greater than 10 (1+); if greater than 100 (2+); and if greater than 500 (3+). The TA can help you with this classication.

24


Each student pair should inoculate 4 phenol coefcient plates

q

Each student pair should inoculate 2 antiseptic/disinfectant BHI plates

q

Each student pair should inoculate 2 Kirby-Bauer plates

q

Each student should complete and submit the Ames Test submission

q


q

Each student should submit the Exercise 5 submissionbefore 1500 (3:00pm) on February 9/12

25

26

NAME: _____________________________



A.

The Effect of Chemical Agents on Bacterial Growth

1a. Calculate the phenol coefcient of Savlon. Show your calculations.3()

MIC of phenol that inhibits growth at 10 minutes, but not at 5 minutes:

MIC of Savlon inibits growth at 10 minutes, but not at 5 minutes:

Phenol coefcient of Savlon:

1b. Based on your results, which is the better disinfectant: 5% phenol or 1% Savlon? Explain.1)(

2a. Complete the table below indicating the zone of inhibition on the bacterial lawn of the Gram-positive organism and the Gram-negative organism for each of the antiseptics/disinfectants you used.4)( Name of Agent

Antiseptic OR Disinfectant?

Zone of inhibition (mm) for Grampositive

Zone of inhibition (mm) for Gramnegative

2b. Based on your results, are there any differences in the effectiveness of antiseptics or disinfectants, or in their effectiveness against Gram-positive organisms or Gram-negative organisms?2)(

B.

The Effect of Chemotherapeutic Agents on Bacterial Growth

3a. Complete the table below indicating the zone of inhibition on the bacterial lawn of Staphylococcus for each Complete the table below indicating the zone of inhibition on the bacterial lawn of the Gram-positive and Gram-negative organisms for each of the antibiotic discs you used. Indicate whether the organism is susceptible (S), resistant (R), or intermediate (I) to the agent.3() Agent Organism

Zone (mm)

S/R/I

Zone (mm)

S/R/I

Zone (mm)

S/R/I

Gram-positive Gram-negative

3b. Based on your results, classify the antibiotics that you used as either broad or narrow spectrum.1)(

NAME: _____________________________

AMES TEST SUBMISSION


Screening for Carcinogenic Chemicals Using the Ames Test 1. Complete the table below indicating the number of colonies on the three Ames test plates. Determine the number of induced mutations for each plate and rank the relative mutagenicity for each plate.4)( Test

Numberofcolonies

Number of induced

Degree of mutagenicity

mutations Negative control Positive control Unknown

2. The MG plates you used for this test contained trace amounts of histidine, which mayhave resulted in a faint background lawn of growth on the plates. Why was histidine added, why did the background growth not develop into a complete lawn, and why did some colonies develop fully?4()

3. Why are the positive and negative controls important for theAmes test? (2)

Class Presentations Now that you are well on your way to becoming a world-renowned microbiologist, it is time to share your knowledge and familiarity of everything microbial with the world! You (in a group of students) will be assigned either a microorganism, a microbiologist, or an everyday/global problem with a microbial solution, and give a short presentation to your colleagues in lab the week of February 12. Your presentation should outline any pertinent information relevant to your topic with a focus on the properties of the microorganism involved, major scientic contribution of the biologist, and any other relevant practical information you discover. The presentation must be between 15 and 20 minutes in duration. You will be graded on your oral presentation by your TA and will be expected to answer any questions that arise. There will be a small whiteboard, digital projector and a PC (with Microsoft PowerPoint) available for your use. You must bring your presentation on a ash drive or bring your own computer (with appropriate cables for connecting to the projector). Computer/le issues are NOT a valid reason for not being able to complete your presentation. The 20-minute time limit includes time for set-up, presenting, answering questions and wrap-up.

In addition to giving the presentation, you will be expected to participate in the presentation of your peers by paying attention and asking questions. There will be questions on the lab midterm exam pertaining to the presentations. Any student identied as being disrespectful during a colleague’s presentation will receive a zero for this component of the course.

Presentation topics will be assigned the week of January 15. You must present the topic assigned by your TA and you must provide your classmates with an introduction to the topic and current research being carried out for that area. You must present as thorough a review of the material as possible without exceeding the time limit.

27

EXPECTATIONS FOR ORAL PRESENTATIONS Your presentation will be graded on 4 main criteria. Make sure you consider these criteria as you plan and practice your presentation. There will also be a peer evaluation mark. Your presentation should provide a suitable introduction for your colleagues and outline any current and relevant information regarding your topic. Have fun and make sure you ask your TA if you have any questions. Grading Components: 1. Information (3% of course grade) Content (7 marks):

Your group will be graded on how well you introduce your topic, how well you use appropriate terminology, how well you demonstrate understanding of the material and how comfortable you are presenting the information. Answering Questions (2 marks):

Your group will be graded on your ability to understand and answer simple questions based on your presentation. 2. Presentation (0.5% of course grade) Presentation Skills (7 marks):

Each individual will be graded on their speaking skills, timing, condence, organization, and

engagement with the audience. 3. Citations (0.5% of course grade) Literature Cited (2 marks):

Your group will be graded on your use of in-text citations and the literature-cited section that you hand in prior to beginning your presentation. 4. Collaboration (1% of course grade) Peer Evaluation (7 marks):

Each member of your group will assess the other members on their participation and cooperation within the group dynamic. This is a required component of the presentation - if you do not submit a peer evaluation form (posted on D2L), you will get a zero on all Presentation components (5% of course grade).

28

Lab Exam 1 and Inoculations Lab Exam 1 You will write Lab Exam 1 in your scheduled section on February 27/March 1. The exam will cover material learned in Exercises 1-5 (plus the presentations) and will be a mix of factual recall, practical and application questions. The exam will be 2 hours in length and consist of ~10 short answer/practical quesions. You may use a pen or pencil and a non-programmable calculator. You maynot bring personal electronic devices (PDAs, MP3s, cell phones, etc.) What should you study? Terminology - know how to use the proper terms at the proper times.I.e. a bacterial cell is cocci, not

circular Procedures and theory - know how to do a Gram stain, a streak plate,etc. and how to classify an organism

based on test results. Results and calculations - know how to interpret any of the tests we have done, how to determine phenol coefcients, etc. Conclusions and study questions - know how to draw conclusions from results. Study the questions in the

lab manual and your submissions. Experimental design - understand how/why an experimental procedure works and be able to design an experiment to answer a question Aseptic technique - you should follow proper safety guidelines and know how to maintain a safe, aseptic

work environment What should you NOT study? Quantities and lists - you will not be asked how many grams of agar need to be added to 200ml water or list the ingredients of BHI agar,etc. Specic results - you will not be asked to recall the specic results of the hand-washing plates, etc.

Preparation for Exercise 6 Homolactic Fermentation Procedure:(work in pairs) 1. Using the sample provided, inoculate one tubeof litmus milk medium with a loop of yogurt. 2. Place the properly labeled tube in the appropriate box on the side bench and incubate forone week.

29

Winogradsky Columns Procedure: (work individually): 1.

Make your nal observations of the four Winogradsky columns.

2.

Submit your observation chart (page 13 or the one you generated)before noon on March 2/5

Preparation for Bacterial Unknown Project Procedure: (work individually) 1. Collect two pure unknown culture plates from the side bench and record your unknown number on page 29 and on the Unknown submission sheet. 2. Prepare streak platesfor single colony isolation andincubate the plates according to the culture conditions “hint” provided by the TA. 3. Return the srcinal plates to your TA. Before next week’s lab: 1. Maintain a pure culture of each organism (if you need to replace any of your plates throughout this project, you will be penalized on your journal grade). 2. Perform a Gram stain on each unknown fromyour plates. 3. Complete the Unknown submissionbefore noon on March 2/5

30

Term Project Identication of Bacterial Unknowns Objectives:

1. To identify two unknown organisms using classic bacteriological techniques and a dichotomous key

Identication of Heterotrophic Bacteria Heterotrophic bacteria have been more extensively studied than the autotrophic bacteria since all animal and plant pathogens are heterotrophic and they form the greater part of our immediate environment. Since heterotrophic bacteria require an organic form of carbon, they are most easily cultivated on complex media such as nutrient agar or brain heart infusion agar or broth. Under normal conditions, most environmental samples are a heterogeneous mixture of microbial species that rst need to be separated before individual organisms can be identied. In order to separate and identify organisms in a mixed culture the streak plate technique may be used. Once pure cultures have been prepared, then differential media and a variety of biochemical and staining tests are employed in order to identify the organisms. A bacteriological dichotomous key simplies the procedure since only certain characteristics are specic to a particular genus or species.

Isolation and Identication of an Unknown Bacterial Species Last week, you received two pure culture plates containing unknown bacterial species.

Unknown # ___________________ Make sure you collect the Unknown submission sheet and the pure culture streak plates of each organism. Remember, you will be responsible forproperly maintaining both of your organisms. You will need to plate, incubate and refrigerate your cultures until you have successfully identied both of them. Over the next few weeks (in the lab and open lab), you will collect data about the environmental, physiological, enzymatic and nutritional characteristics of the unknowns and make tentative identications using classic taxonomic methods. To assist you in this effort, you were given culture hints and you will have access to a dichotomous key (posted on D2L and in the lab). Your TA will give a brief description on its use. As you collect information about your unknowns, you need to rationalize the important steps you take during the identication process, provide a ow chart indicating how you moved through the dichotomous key, record the results of the tests that you perform, illustrate your understanding of each test and what information each test provides about your organism, and answer a discussion question once you have identied both organisms. To facilitate this, you will maintain an “Unknown Journal”.

31

UNKNOWN JOURNAL You may use any format you feel is appropriate for your journale.g ( . a hard-cover lab notebook, loose leaf, computer document, etc.). To properly record and present the information you collect, please organize your journal following these guidelines: 1. Prepare a Table of Contents directing the reader to specic information

E.g. Results of Gram Stain – page 4

2. Number the pages of your journal (so that your TOC is useful!) 3. Prepare a ow chart In order to increase efciency and decrease waste, indicate how you used the dichotomous key to complete your analysis, characterization and identication ofboth organisms.

4. Prepare Headings for each result Please make these as detailed as possible.E.g. Growth on Mannitol Salt Agar. 5. Record your results accurately and completely Your TA may not see the results that you obtain, so you must record a description of how your organisms responded to each test.I.e. the colour, extent of growth,etc. 6. Write (discuss) your results In a few sentences, explain the rational for performing each test and explain the interpretation of each result, indicating what the results for each test tells you about your organisms. I.e. what you learned about the physiology and biochemistry of your organisms. 7. Identify your unknown Make sure you rationalize the identity of both unknown organisms. 8. Complete the discussion question Once you know the identity of your unknown organisms, a discussion question for “Organism A” will be made available to you. Provide a complete, accurate and concise answer to the question in your journal. Ensure you use CSE citation rules for both the in-text citations and Literature Cited (see page 49) You will submit your journal the week of March 19. Your TA will assess the format of your journal, the results and appropriateness of the tests that you performed, the identication of your unknowns, and the nal discussion. WARNING: You are responsible for the proper maintenance of your unknowns. You will need to keep a pure culture that is fresh (less than 1 week old) before you inoculate each test. If you fail to maintain your culture (contamination, desiccation,etc.) or if you fail to label, store or destroy your media and tests properly, you will receive a penalty on your journal.

After you have completed your identications, you need todestroy all plates and tests.

32

PROTOCOL FOR HANDLING UNKNOWNS 1. The identication of theorganism is to be determined using the mediaand resources providedin the lab. All of the media required will be in the shelves along the side bench. If you cannot nd the media you require, ask a TA or technician for assistance. Refer to Appendices 4 and 5 for a review of staining procedures and biochemical tests. The medium you use must be applicable to the identication of your organism. Media are not to be randomly inoculated in the hope of achieving successful identication of the unknown. Remember, you must present a rationale for the use of each test in your journal. Use of the dichotomous key will facilitate your procedure and save valuable time.

2.

Ensure that you are maintaining a fresh culture of your organisms throughout this exercise. You will need to use single, isolated colonies from a fresh (less than 1 week old) plate to inoculate the tests and media. The TAs will be looking at your test results and source of inoculum regularly throughout this exercise. Make sure you areproperly disposing old plates and tests when they are no longer required.

3.

Ensure that you are recording all of your results in your unknown journal and that it is kept up to date regularly. This will help make sure that you do not forget anything and will spread the written component of this exercise over a manageable amount of time.

4.

Use the dichotomous key and references that are available in the library or in the lab. Some references that may be used to facilitate identication of the organisms are: a. Bergey’s Manual of Determinative Bacteriology orBergey’s Manual of Systematic Bacteriology, Vol. 1 & 2. b. DifcoTM & BBLTM Manual - lists all the media made by Difco with a discussion of the media including the purpose and application of each kind of medium.

5.

TIDY your working space before you leave the lab. For example:

a. Rell staining bottles as necessary from the supply bottles on the shelf above the side bench b. Put your microscope away properly c. Put the stool back under the bench before you leave If your work area is not properly maintained, you will receive a penalty on your journal

6.

If you have any major problems with your unknowns, talk to your TA right away. The journal will be due the week of March 19

33

34

UNKNOWN SUBMISSION

NAME: _____________________________ LAB SECTION: ______________________ UNKNOWN # ________________ SCORE: ________________

1. What is the colony morphology of your two unknown organisms?(4)

2. Submit a pure culture streak plate of both organisms. (4)

3. What is the cellular morphology ofyour two unknown organisms? Makesure the cellular morphology for each organism is matched to the appropriate colony morphology from Question 1.4)(

4. Use the space below to diagram both of the unknown organisms(page 61). Make sure thediagram for each organism is matched to the appropriate morphology from Questions 1 and 3.6()


1. To become familiar with the effects and consequences of oxygen on microbial growth and metabolism 2. To become familiar with fermentation pathways common tomany microorganisms 3. To become familiar with respiratory pathways common to many microorganisms Recommended Laboratory Resources:

Madigan, MT, KS Bender, DH Buckley, WM Sattley and DA Stahl. 2018. Brock: Biology of Microorganisms, 15th edition. Pearson Education, Inc., New York. pp. 85-95; 160-163; 419-421; 434-438. Lab Manual Appendices 3, 4, and 5 *

Collect a broth culture tube and a streak plate of one of the unknown organisms (A, B, C, D, E or F) from the side bench. Remember to vortex the tube well before each use. Record the unknown code on the Exercise 6 submission sheet. You will need these cultures for parts A and B below.

A.

Oxygen as a Nutrient

Although oxygen is a major component of macromolecules and is thus needed for life, molecular oxygen (O ) is 2 never used as a source of these atoms. The oxygen found in these compounds is derived from water and organic molecules. Molecular oxygen is normally only required as the nal electron acceptor in the electron transport chain of respiring organisms and it is in this sense that we are considering oxygen a required nutrient. 1.

Oxygen tolerance

Organisms that respire have to remove toxic oxygen by-products (O , H2O2, OH.) that form during the 2 reduction of O2 to H2O. If not removed, organic cellular components are oxidized and the cell dies. Not all microorganisms require the same levels of oxygen for metabolism, and some organisms cannot tolerate any oxygen at all.

Procedure: (work individually) 1. Record the colony morphology of your unknown organism on the Exercise 6 submission sheet. 2. Prepare one tube of each of the following media for the unknown culture (A, B, C, D, E, or F). i.

BHI slant - aseptically inoculate and spread a loopful across the surface of the agar.

ii. Deep shake culture (page 55) - aseptically inoculate 200

l into a tube of molten tryptose agar. Mix

gently by rotating the tube between the palms of your hands.Allow to harden in an upright position. below iii. Thioglycollate medium (page 55) - aseptically inoculate 200 l into a thioglycollate broth tube the upper, pink layer. Handle very carefully so the oxygenated layer is not disturbed. 3. Place both properly labeled tubes in the appropriate box on the side bench and incubate for24 hours. 4. In the open lab, record your observations and interpretations on the Exercise 6 submission sheet. 35

2.

Respiratory enzymes

The respiratory electron transport chain is a sequence of enzymes and electron carriers responsible for transferring reducing equivalents from substrates to molecular oxygen. Oxygen acts as the nal electron/ hydrogen acceptor in the respiratory chain producing either water or hydrogen peroxide, depending upon the species and its enzyme system. Two enzymes which are part of the respiratory chain are cytochrome c oxidase and catalase.

Procedure: (work individually) 1. Use the agar streak plate of the unknown organism (A, B, C, D, E, or F) to completeeach of the following tests. i.

Oxidase test (page 67) - Use an oxidase stick (hold the red end) to pick off a small mass of cells (with the brown end) from a single colony. Examine the stick over a 3 minute period.

ii. Catalase test (page 67) - Add two drops of hydrogen peroxide to a colony on the agar plate. 2. Record the results and interpretations on the Exercise 6 submission sheet.

B.

Utilization of Glucose (Fermentation or Respiration?)

Glucose, because of its wide distribution and importance in animal metabolism, has served as the initial substrate for most studies of product formation and mechanism of carbohydrate metabolism in microorganisms. Furthermore, glucose is assigned a major role in reactions leading from polysaccharides and as a substrate for phosphorylation prior to rearrangement and cleavage in fermentation and oxidation pathways. Glucose may be utilized in a variety of ways. It may be respired aerobically when oxygen is present or it may be anaerobically fermented. Facultative organisms may respire or ferment glucose depending upon the presence or absence of oxygen. 1.

Hugh and Leifson (H&L) glucose media (page 67)

Procedure: (work individually) 1. Aseptically stab inoculate 2 tubes ofH&L glucose from an isolated colonyon the agar streak plate (A, B, C, D, E, or F). Gently overlay one tube with a layer of mineral oil as demonstrated by your TA. 2. Place both properly labeled tubes in the appropriate box on the side bench and incubate for24 hours. 3. In the open lab, record your observations and interpretations on the Exercise 6 submission sheet.

36

C.

Fermentation Pathways

Heterotrophic microorganisms may break down carbohydrates to obtain energy by fermentation. Fermentation has four basic features: (1) it occurs in the absence of oxygen; (2) energy producing electron transport is absent; (3) it is much less energy efcient than respiration; and (4) metabolic intermediates or fermentation end products are produced. Fermentation of glucose occurs in two stages. The rst involves the splitting of glucose and the removal of two pairs of hydrogen atoms, resulting in the formation of carbon compounds more oxidized than glucosei.e. ( pyruvate). In the second, or reductive part of fermentation, the oxidized compounds are reduced, recycling the hydrogen atoms removed in the rst stage. Since an oxidation cannot proceed without an equivalent reduction, the number of hydrogen atoms removed in the rst part of a fermentation is always equal to the number used in the second part.

The two major used for bacterial fermentations areInthe Embden-Meyerhof-Parnas (glycolysis) andpathways the hexose monophosphate (HMP) scheme. either pathway, pyruvic acid is(EMP) alwaysscheme one of the compounds formed in the rst stage of bacterial fermentation. The end products characteristic of the various bacterial fermentations are derived from the pyruvic acid and the hydrogen atoms produced in the rst (or oxidation) stage of fermentation. Among the bacteria, several different pathways for the fermentation of carbohydrates have been identied. Each is associated with a specic set of end products and each is characteristic of a particular species. 1.

Alcohol fermentation

Bacteria (i.e. Streptococcus sp.and some Lactobacillus sp.) rst oxidize a carbohydrate (glucose, lactose,etc.) to pyruvic acid via the Embden-Meyerhof-Parnas (EMP) pathway. The pyruvic acid is then reduced almost entirely to lactic acid. Last week, you inoculated a litmus milk tube with yogurt to observe the homolactic fermentation of milk. Follow the procedure below to complete this observation.

Procedure: (work in pairs) 1. Observe the changes in the litmus milk tube (page 68) and record your results and interpretations on the Exercise 6 submission sheet. 2.

Mixed acid or Butanediol fermentation (page 69)

Bacteria (i.e. Escherichia coli) rst oxidize a carbohydrate (glucose, lactose,etc.) to pyruvic acid via the Embden-Meyerhof-Parnas (EMP) pathway. The pyruvic acid is then reduced to a variety of acids (lactic acid, acetic acid, succinic acid,etc.), which accumulate in the media. Bacteria (i.e. Enterobacter aerogenes) rst oxidize a carbohydrate (glucose, lactose,etc.) to pyruvic acid via the Embden-Meyerhof-Parnas (EMP) pathway. The pyruvic acid is then reduced to a variety of acids, which are then converted to acetoin and 2,3-butanediol, which accumulate in the media.

Procedure: (work in pairs) 1. Aseptically inoculate twosterile MR-VP broth tubes with E1 and two more with E3. 2. Place the properly labeled tubes inthe appropriate box on the side bench and incubate for24 hours. 3. In the open lab, collect the tubes and vortex each tube well. Carry out the Methyl Red test on one set of tubes and the Voges-Proskauer test on the other set. Record the results and interpretations on the Exercise 6 submission sheet. 37

D.

Anaerobic Respiration

Respiration may occur with a nal electron acceptor other than oxygen, which results in the reduction of sulfates, nitrates, CO2, ferric iron, or several organic molecules such as fumarate. 1.

Nitrate respiration

Nitrates may be produced in the soil by nitrifying bacteria through the oxidation of ammonia. Alternatively, they may be applied to the soil in the form of commercial fertilizer. Nitrates are very soluble and if not promptly assimilated by plants, may be lost from the soil either by leaching or by microbial reduction. Many bacteria, as well as plants and some fungi, are capable of reducing nitrates to ammonia and subsequently incorporating ammonia into amino acids and proteins. When NO is reduced for use as a nutrient source in 3 this way, it is assimilative nitrate reduction . This occur anaerobically as ammonia well as under aerobic conditions. In called general, assimilative nitrate reductases aremay soluble proteins which are repressed. Dissimilative nitrate reduction is restricted to bacteria and occurs when NO is used as an electron acceptor 3 in energy metabolism (anaerobic respiration). Dissimilative nitrate reductases are membrane bound proteins which are repressed by O2 and synthesized under anaerobic conditions only. These bacteria are able to use nitrate instead of oxygen as the nal electron acceptor in the electron transport chain.

The ability to reduce nitrate to nitrite in the absence of oxygen is possessed by many bacteria and the process is called nitrate reduction: NO3- + 2H+

anaerobic

> NO2- + H2O

However, the ability to reduce nitrate beyond nitrite is limited to a relatively small number of genera. The formation of N2O, NO or N2 is called denitrication: NO 3

anaerobic

> NO -

anaerobic

2

> N (NO or N O) 2

2

Since these products are all gaseous, they can be easily lost into the atmosphere. This process is the main means by which gaseous N2 is formed biologically, and since N2 is much less readily available to organisms than nitrate as a source of nitrogen, denitrication is considered a detrimental process.

Procedure: (work in pairs) 1. Aseptically inoculate twosterile nitrate reduction brothtubes (page 71) with E3 and P9 as demonstrated by your TA. Watch out for the Durham vial! 2. Place the properly labeled tubes in the appropriate box on the side bench and incubate for24 hours. 3. In the open lab, collect the tubes and record the results on the Exercise 6 submission sheet.

E.

Lab Quiz 4

You must complete Lab Quiz 4 before your next scheduled lab

38


SCORE: __________

A.

Oxygen as a Nutrient

NAME: _____________________________ LAB SECTION: _________________

Unknown Code: ____

1. Record your observations and interpretation of eachmedia inoculation or the results of each of the tests you performed on the unknown organism. 6( ) BHI plate (colony morphology):

BHI Slant:

Deep Shake tube:

Thioglycollate broth:

Oxidase test:

Catalase test:

B. Utilization of Glucose (Fermentation or Respiration?) 2. Record your observations and interpretation of theH&L glucose test you performed on the unknown organism. (2)

3. Based on the results (question 1),to which oxygen requirement class doesthe organism belong? What do you know about the metabolic capabilities of the organism? Be specic.4()

4. What was the purpose of the H&Lglucose test tube that youcovered with mineral oil? Explain.2()

5. What were the results (and signicance) ofthe litmus milk test for the yogurt sample? 2( )

6. Record your observations andinterpretation of the MR-VPmedia for both organisms inoculated. 4( )

7. Why can you read the results of the methyl red test immediately after addingthe methyl red reagent, but you have to wait for the results of the Voges-Proskauer test?2()

8. Record your observations and interpretation of thenitrate reduction test forboth of the organisms tested. (3) Gas Organism

Y/N

Interpretation

E3

P9

9. Explain why gas production suggests, but does not conrm, nitrate reduction. (2)


1. To introduce transposons and techniques for generating transposon mutants Recommended Laboratory Resources:

Madigan, MT, KS Bender, DH Buckley, WM Sattley and DA Stahl. 2018. Brock: Biology of Microorganisms, 15th edition. Pearson Education, Inc., New York. pp. 307-308, 314-315, 320-321, 325-328.

Transposon Mutagenesis Creation of mutations and subsequent genetic mapping can elucidate the identity, relative size, number and organization of genes involved in a physiological process. Three general treatments can be used to mutagenize microorganisms: electromagnetic radiation, chemical mutagens and transposons. A transposon (Tn) is a genetic element which can move from one site on a DNA molecule to another site either on the same or on a different DNA molecule. When the insertion site of a Tn is within a gene, the linear continuity of that gene is disrupted,i.e., the Tn insertion is mutagenic. As well as encoding transposition genes, Tn often encode for genes conferring resistance to antibiotics. A modied transposable element,Tn5, encodes for resistance to the antibioticgentamicin and will be used to illustrate the use of Tn5 as a mutagen usingEscherichia and Rhizobium Research Applications

Scientists have taken advantage of the unique ability of transposons to jump from site to site in a genome, using them to create large mutant collections. During cell division, transposons can become activated and jump randomly in a genome. In doing so, the transposon has a chance of inserting into a region that disrupts the expression of a gene, knocking the gene out in the process. Using these single knockouts, researchers are able to study the effects of losing the function of specic genes on an organism. However, transforming genes into a host organism is inefcient. Often only a small percentage of the host cells successfully incorporate the target DNA (e.g. plasmid). To identify (select) the transformed cells, antibiotic resistance genes are included in the plasmid, allowing transformants to grow in media containing the selected antibiotic. Untransformed cells do not survive this treatment.

Plasmid Vector Wild-type Rhizobium VF39 (recipient strain) will be mutagenized by conjugation with anE. coli S17-1 (donor) strain that harbours a plasmid pSUP102::Tn5-B22 ( ) that carries the transposon,Tn5-B22. The pSUP102::Tn5-B22 and the E. coli mutagenesis protocol relies upon the special properties of the plasmid, strain that harbours it.

39

Plasmid pSUP102::Tn5-B22 contains three important properties: 1. A modied Tn5 transposonthat has had the normal antibiotic resistance geneof Tn5 (kanamycin) replaced with a gene coding for antibiotic resistance togentamicin.

2. An srcin of transfer . This sequence of DNA is recognized by the conjugation apparatus and will allow transfer of the plasmid during conjugation between two cells. 3. An E. coli specic srcin of replication . This plasmid is unable toreplicate in Rhizobium and therefore will be lost as the Rhizobium replicates. This makes the plasmid a “suicide vector”. The E. coli strain contains the conjugal transfer genes on the chromosome. It is capable of taking any plasmid that has an srcin of transfer and mobilizing it to a suitable recipient. In this experiment, the plasmid pSUP102::Tn5-B22 from theE. coli strain will be mobilized by conjugation to aRhizobium wild type stain that has a chromosomal gene coding for antibiotic resistance to streptomycin. After conjugation, both the donor and recipient cells will be plated on TY +streptomycin + gentamicin (TY + Sm + Gn) plates.

Procedure: (work in pairs) 1. Pipette 1.0 ml of E. coli S17-1 pSUP102::Tn5-B22 (donor cells) into a sterile centrifuge tube. Pipette 2.0 ml of Rhizobium (recipient cells) to the same centrifuge tube. 2. Centrifuge at 4,000 rpmfor 10 minutes and decant the supernatant very carefully by pouring it into a waste beaker. Resuspend the pellet in 200 l of sterile TY broth. Mix well on the vortex. 3. Place 3 sterile nitrocellulose lters evenly around thesurface of a dry TY plate (as demonstrated byyour TA). The lters must not be closer than 3 cm from each other and not closer than 2 cm from the edge of the plate. Ensure the lters make rm contact with the agar.

4. Using a micropipette with a sterile yellow tip attached, aliquot 40 l of resuspended cells onto 1 nitrocellulose lter. If necessary, spread the cells around on the lter using the tip. Do the same with the remaining two lters. Allow the spots to dry on the lters at room temperature, place the properly labeled plate in the appropriate box on the side bench and incubate for24 hours.

In the open lab: 5. Aseptically pipette 1000 lof sterile distilled water into3 sterile test tubes. Use sterile forcepsto carefully pick up each lter and place each into a different test tube. You may have to push the lter gently into the water using a sterile glass rod. Mix very well on the vortex to wash the cells from the lter into the water. 6. Pipette 100 l from eachtube onto separate TY+ Sm + Gn plates. Spread each plate with asterile glass spreader. Place the properly labeled plates in the appropriate box on the side bench. They will be incubated for 5 days and returned in Lab 8.

Lab Quiz 5 You must complete Lab Quiz 5before your next scheduled lab (Lab 8).

40

Laboratory 8 Objectives: 1. To explain the effect of UVradiation on the replication of T4 and explore the useof sunscreens through UVinduced mutation in T4 2. To perform a plaque assay with bacteriophage T4 andits host cellE. coli


Madigan, MT, KS Bender, DH Buckley, WM Sattley and DA Stahl. 2018. Brock: Biology of Microorganisms, 15th edition. Pearson Education, Inc., New York. pp. 166; 224; 227229; 312.

A.

Effect of Radiation on Viral Growth

Bacteriophage In 1917, F. d’Herelle, a Canadian microbiologist working in Paris and F. Twort, a British scientist, discovered bacterial viruses, which they calledbacteriophage, meaning “to eat bacteria”. All viruses are considered to be noncellular infectious entities having either a DNA or RNA genome. They replicate only in living cells, using the cell’s metabolic machinery to synthesize copies of themselves for transfer of their own genomes to new hosts. Although a virus contains nucleic acid hereditary material and is capable of reproducing itself, it has none of the other attributes of a living organism. The virus used for this experiment is anE. coli T4 bacteriophage. It is a classical virulent virus that can go through the lytic cycle but not the lysogenic cycle. It is one of largest of the bacterial viruses. The genome is composed of one, linear, double stranded DNA molecule with a total size of 169 kbp (kilo base pairs).

The Effect of UV Radiation on DNA Replication Biologically signicant radiation, such as that used for photosynthesis, has wavelengths in the visible range of the electromagnetic spectrum from 380-750 nm. Ultraviolet light is just below this visible range of the spectrum and is divided into three types: UVA, UVB, and UVC. Known as the “tanning spectrum”, UVA wavelengths are the longest. This energy causes increased melanin production by melanocytes found just below the epidermis of the skin. Although still used in tanning salons, UVA radiation is now known to penetrate the skin causing the skin to age 40 times faster than normal. As well, UVA light is associated with cataract formation and with malignant melanomas, the most deadly form of skin cancer. UVB radiation, the “burning spectrum”, is more energetic and is associated with sunburn and the most common form of cancer, basal cell carcinoma. UVC radiation comprises cosmic rays which rarely reach the surface of Earth.

UV light is a powerfulmutagen. It is now well established that the damage done to DNA due to UV light is the induction of pyrimidine dimers e.g. ( thymine dimers). This occurs when two adjacent pyrimidine residues along the DNA backbone become “fused” to each other rendering them unable to hydrogen bond with the complementary base on the opposite DNA strand. Other changes to DNA structure which can occur as a result of exposure to UV light are strand breakages, duplications, deletions, inversions and translocations. Any one of these alterations in DNA, if not repaired, will ultimately disrupt DNA replication and protein synthesis. 41

The Role of Sunscreen The term sunscreen refers to anything that blocks UV radiation. The rst sunscreen lotions contained the chemical para-amino-benzoic acid (PABA), which absorbs and blocks UVB radiation. Many people have developed sensitivities to PABA and it is not widely used anymore. Current sunscreens are combinations of chemicals that block a range of UV wavelengths. These chemicals include PABA-esters and cinnamates which block UVB, as well as Parsol 1789 and benzophenones which block UVA. SPF (sun protection factor) is a means of rating the effectiveness of a sunscreen based on numerical classication of skin sensitivities to sun exposure i.e. ( how effectively the cream blocks UVB radiation).

Skin types range from 1 (white skin/freckles) to 6 (brown/black skin). Redness occurs in 10-20 minutes in type 1 and 40-75 minutes in Type 6 depending on the latitude where exposure occurs. By multiplying the SPF by the amount of time it takes to cause your skin to burn, you can calculate how much protection is offered by the sunscreen. For instance, if your skin burns in 15 minutes, and you apply a sunscreen with an SPF of 8, it will take 120 minutes for your skin to start to burn (8 x 15 minutes). To illustrate the effect of UV light on DNA, a model system using a T4 bacteriophage and its host bacterium E. coli C will be used.The UV source will be a germicidal tube with a wavelength of 264 nm. On exposure of phage to UV light, the types of DNA damage described earlier occur. This damage will ultimately affect the phage’s ability to propagate successfully within the host cell and cause cell lysis.Therefore, UV damage can be indirectly visualized by observing the number of plaques produced from UV exposed phage and comparing this number with plaques produced by phage which have not been exposed to UV light. In this exercise, the effectiveness of sunscreen with various degrees of protection factors will be tested. However, instead of risking damage to human skin, the DNA of T4 phage will be exposed to the UV light. So, relax, think about a hot day at the beach and get ready to “catch a few rays”.

The Plaque Assay To determine titer (concentration) throughout this experiment, samples will be taken at intervals after infection and aphage plaque assay will be performed. When a population of host bacteria are grown as a lawn on an agar plate, individual cells infected with a virus will lyse, exposing adjacent cells to virions. Over time, this will create an area of clearing on the agar plate called aplaque. It can be assumed that this area was cleared of host cells by progeny of one srcinal virion and represents aplaque-forming unit (PFU). By counting the number of plaques on an agar plate, you are counting the number of virions (or infected host cells) present in the culture at the time the plate was made. Viral titer has the units PFU/ml.

Note: This exercise has been adapted from a University of Lethbridge Biology Laboratory Manual with many thanks to Ms. Laurie Pacarynuk. 42

Procedure: (work in pairs) 1. Prepare a cardboard template by spreading a piece of plastic wrap across the circular opening in the cardboard frame and taping the wrap to the cardboard, pulling the wrap tight as you do so. 2. Your TA will assign you one sample of sunscreen with an unknownSPF. *

Controls will be prepared for you. For the rst control, no sunscreen will be applied to the plastic wrap. The phage will be UV treated and samples assayed in an identical manner to the phage tested with the lotions. For the second control, the phage will not be treated with sunscreen or UV but samples will be assayed in the same manner as the procedure indicates.

3. Weigh 0.2 g of the sunscreen assigned to youand rub the lotion over the plastic wrap to create athin lm over the template. There should not be any visible white streaks on the plastic wrap. Use a small piece of tape to label your template with the name of your sample sunscreen. 4. Obtain 6 sterile 13 mm test tubes and 6 H-agar plates. Label thetubes and plates with your name and the time intervals of 0, 5, 10, 15, 20, and 30 minutes. 5. Aliquot 100 µl of E. coli (108 CFU/ml) to each of the 6 labelled test tubes. 6. Aliquot 2.0 ml of T4 bacteriophage (103 PFU/ml) to an empty sterile petri dish. *Caution: UV light is damaging to the naked eye and exposed skin. Always cover bare skin and view through safety glasses which absorb harmful wavelengths*

7. When you are ready to start the assay: a. Aseptically aliquot 100

l ofphage from the petri dish to the test tube labelled “0”.

b. Place the petri dish inthe UV hood (without the lid) assoon as you have removed your time0 sample. Place your cardboard template over the petri dish. c. Quickly vortex the phage/E. coli suspension and incubate for 15 minutes at room temperature. For the best results, keep your 15 minute incubations consistent! You may want to keep track of the time you added the phage to theE. coli and the time when you need to add the overlay (terminate incubation). 8. Aliquot 100 l of phage from your petri dish to the appropriately labelled tube of E. coli, vortex and incubate for 15 minutes at each sample time (5, 10, 15, 20 and 30 minutes). 9. Upon completion of the 15 minute incubation for each tube: a. Quickly remove a tube of soft overlay agar from the 55°C water bath and pour into the appropriately labelled test tube containing the phage/E. coli suspension. b. Mix well by rotating the tube between the palms of your hands, immediately pour overan H-agar plate, and tip the plate to spread the overlay agar over the surface. Allow to harden. 10. Place the 6 plates in the appropriate box on the side bench to be incubated for 24 hours at 37°C. Remove the plastic wrap and all tape from your template and discard the plastic wrap. Do not discard the template (replace on the side bench). 11. In the open lab, count the plaques on each plate as demonstrated by your TA. Recordyour results in the appropriate column in the tableon the class record sheet posted in the open lab.Results must be recorded by noon on March 23/26. Submit your plates to the correct box on the side bench

12. The class results (and the controls) will be posted on D2L. The Exercise 8 submission is due, with your Unknown Journal, on March 27/29.

43

B.

Transposon Mutagenesis

1. Controls have been prepared for you. Donor cells ( E. coli S17-1) were plated alone on a TY + Sm + Gn plate. Record the number of cells that grew on this plate on the Exercise 7 submission sheet (Question 1). Recipient cells (wild-type Rhizobium VF39) were plated alone on a TY + Sm + Gn plate. Record the number of cells that grew on this plate on the Exercise 7 submission sheet (Question 2). Recipient cells (wild-type Rhizobium VF39) were also plated alone on a TY + Sm plate. Record the number of cells that grew on this plate on the Exercise 7 submission sheet (Question 3). 2. Collect the three TY + Sm + Gn plates you prepared in Lab 7. Count the number of streptomycin- and gentamicin-resistant colonies observed on the three plates. Record the average number of transposedRhizobium cells that cam from the nitrocellulose lters on the Exercise 7 submission sheet (Question 4). 3. To determine the frequency of transposition, weneed to know the number ofRhizobium cells that underwent both conjugation and transpositionfrom the srcinal culture . From step 2 above, we know the average number of cells that successfully completedboth events. Determine the number of transposed cells in the 200 l suspension (step 2 of the procedure) and record the answer on the Exercise 7 submission sheet (Question 5).

4. The Rhizobium cells in the 200 l suspension were from a pellet of cells that, before centrifugation, were suspended in 2 ml of broth (we can ignore the 1 ml of broth that contained the E. coli). Determine the number of transposed cells that would have come from 1 ml of the srcinal Rhizobium culture (step 1 of the procedure) and record the answer on the Exercise 7 submission sheet (Question 6). 5. Determine the frequency oftransposition. This is denedas the number of transposon mutants per ml of srcinal culture divided by the total number of viable Rhizobium in the srcinal culture. Record the answer on the Exercise 7 submission sheet (Question 7).

The Exercise 7 submission sheet isdue at the end of your scheduled lab (March 20/22). Submit your plates to the correct box on the side bench

44

NAME: _____________________________



A.

Plasmid Vector

1. Note that part of the grade for this submission is from the assessment of your results (plates). 2() 2. How many E. coli S17-1 cells grew on the TY + Sm + Gn plate? Explain this result.2()

3. How many wild-type Rhizobium VF39 grew on the TY + Sm + Gn plate? Explain this result. 2()

4. How many wild-type Rhizobium VF39 grew on the TY + Sm plate? Explain this result. 2()

5. What was the average number of transposedRhizobium on the nitrocellulose lters? (1)

6. What would be the number of transposed Rhizobium cells in the 200 for full marks. (2)

l suspension? Show your calculations

7. What would be the number of transposed Rhizobium cells that would have come from 1 ml of the srcinal Rhizobium VF39 culture? Show your calculations for full marks. 2()

8. What was the frequency of transposition? What does this number tell you?(2)

NAME: _____________________________



The Effect of UV Radiation on DNA Replication 1. Note that part of the grade for this submission is from the assessment of your results (plates). 2() 2. Record the class results (posted on D2L) in the following table. 3( ) Number of Plaques ABCD

No sunscreen (Control 1)

No sunscreen No UV (Control 2)

0 min 5 min 10 min 15 min 20 min 30 min 3. Prepare one gure (Page 50) showing the effect that length of exposure to UV radiation has on the titer of T4 for each of the sunscreens tested. Label the gure with as much information as you can determine from your results (i.e. assign an SPF value to each sunscreen).Attach the gure to your submission . (10) 4. Explain why a germicidal tube of the 264 nm is used in this experiment. 2( )

5. Explain the results ofthis experiment. Make sureyou refer to the data, controls andthe gure in your discussion. (5)

Lab Exam 2 You will write Lab Exam 2 in your scheduled section on April 3/5. The exam will cover material presented throughout the term and will be a mix of factual recall, practical and application questions. The exam will be 2 hours in length and consist of short answer/practical questions. You may use a pen or pencil, ruler and a nonprogrammable calculator. You may not bring personal electronic devices (PDAs, MP3s, cell phones,etc.) What should you study? Terminology - know how to use the proper terms at the proper times.I.e. a bacterial cell is cocci, not

circular Procedures and theory - know how to do a Gram stain, a streak plate,etc. and how to perform an experiment to nd information about an organism based on test results. Results and calculations - know how to interpret any of the tests we have done, how to determine phenol coefcients, frequency of transposition,etc. Conclusions and study questions - know how to draw conclusions from results. Study the questions in the

lab manual and your submissions. Experimental design - understand how/why an experimental procedure works and be able to design an experiment to answer a question Aseptic technique - you should follow proper safety guidelines and know how to maintain a safe, aseptic

work environment What should you NOT study? Quantities and lists - you will not be asked how many grams of agar need to be added to 1000 ml water or list the ingredients of DOC agar, or to reproduce thespecic procedure for transposingE. coli with Rhizobium, etc. Specic results - you will not be asked to recall the specic results of the endospore stain for unknown a, etc. Unknowns - you will not be asked anyspecic questions about your unknowns Pathways - you will not be asked to illustrate the dissimilative nitrate reduction pathway of E. coli, etc.

45

46

Appendices

Appendix 1: Laboratory Exercises For the laboratory component of CMMB 343, no formal lab reports will be submitted for grading. Instead, you will be required to complete: 1. Exercise submission sheets, which are located at the end of each exercise and handed in for grading. On the submission sheets, the results of the experiments will be recorded and questions answered and/or discussion included. 2. Online (D2L) quizzes to be completed between labs. 3. A journal (format to be discussed with your TA) detailing the information you collectabout an unknown organism that you will identify. cannot be submitted for You may complete your submissions in pen or pencil, but exercises completed in pencil regrading.

A list of the literature you have cited in answering the questions or discussing your results does not need to be included (except for the journal - see page 32). Generally, the resources required to answer the questions will be found within your textbook - additional references are available in the library. When a list of references is required, you must be follow CSE citation rules. The references are alphabetized according to the last name of the rst author of each work, followed by the date of publication. The title of the paper and the journal, the volume of the journal, and the pages cited, follow in that order. If a book is being referenced, the edition, publisher and place of publication is included. In-text citations will not be necessary, unless specically called for. The use of general interest websites, wikipedia, and dictionaries not is acceptable, if you start with these sites, ensure you go to the srcinal source that informed these sites. Example: Brock, TD and Madigan, MT. 1991. Biology of Microorganisms, 6th edition. Prentice Hall Inc., New Jersey. Pages 87-99. Lechevalier, H 1986. Nocardioforms. In Bergey’s Manual of Systematic Bacteriology. Vol. 2. Edited by P.H. Sneath, N.S. Mair, M.E. Sharpe, and J.G. Holt. Williams and Wilkens Co., Baltimore, MD. p. 14581484. Morrin, M and Ward OP. 1989. Investigation of cell wall composition of different mycelial forms of Rhizopus arrhizus. Mycology Research. 93: 524-528. Feel encouraged to work on the results, questions and discussions with colleagues in the course, but remember that writing the nal version of the lab submission is anindividual effort. Using or copying another student’s lab submission and handing it in as your own is plagiarism.If this situation occurs the students will be referred to the Department Head for disciplinary action . It is suggested that you keep a lab note book for notes and preliminary results. Remember, two lab exams will be written during the term (see the schedule on page v). For the majority of the exercises, it will be necessary toreturn to the lab the day after your scheduled lab to make observations of your results. In these cases the technician will remove the plates and tubes from the incubator and place them in the lab. Discard, in the appropriate place, all your plates and tubes after you have made your observations, unless directed otherwise. 49

Figures Figures must be completed on appropriate paper •

Computer-generated gures are acceptable, as long as they meet the format guidelines that follow

• Graph paper is always acceptable Figures must show what experiment was performed and the results obtained •

Figures are self-contained, which means someone looking only at the gure should understand the experiment without seeing the procedure or any other results

• Figures include all related treatments and controls Figures must have an appropriate caption •

Captions are placed below the graph and are labelled (“Figure 1”, etc.)

• Captions must indicate the variables involved, the names of all organisms, chemicals or molecules used, and all appropriate statistical information (sample size, etc.) Figures must have properly formatted and labelled axes •

Independent variable is plotted on the x-axis; dependent variable is plotted on the y-axis

• Axes start at “0”, and scale must be continuous and appropriate (arithmetic, logarithmic, etc.), such that there isn’t a lot of empty space. You may use breaks if appropriate (i.e. they do not affect the trend) • Axes are labelled appropriately and units are clearly indicated (if the variable has units) Figures displaying multiple treatments must clearly delineate each treatment •

Each line of best t must use a distinguishable colour and/or style (solid, dashed, etc.)

• A legend must demarcate each line Figures requiring labels must be clear and neat •

Labels appear close to the area being identied

•

A bar clearly indicates the area being identied

50

Appendix 2: Microscopy The care and use of the brighteld microscope: This procedure applies to the Olympus microscope (model CX31) that will be used in the laboratory: A. Handling the microscope

1. Microscopes are calibratedinstruments - handle with care and avoid sudden or severe impact. 2. Carry the microscope upright with one hand under thebase and the other hand holding the recessed handle on the rear of thearm. Never hold by the stage, nosepiece, or eyepiece. 3. Always lift the microscope to move it.Never slide the microscope across a work surface. 4. Position the microscope sothat you can sit comfortably at your stool when observing a specimen. B. Preparation before use

1. Remove the dust cover and place it in the cupboard (not on the bench) until you are nished. 2. Ensure the main switch is set to “O” (off) and the light intensity control knob is set to “1”. 3. Plug the power cord into the nearest electrical outlet. Keep theexcess cord neat and behind the microscope to prevent pulls and tangles. 4. Ensure: a. the 4x (red) objective is engaged b. the stage is in its lowest position (rotate the course adjustment knobtoward you) c. the condenser lens is in the highest position (rotatethe condenser height adjustment knob) d. the aperature iris diaphragm is closed (slide the adjustment leverto the right as far as it will go) e. the eld iris diaphragm is open(turn the adjustment ringclockwise as far as it will go) C. Placing the specimen

1. Always place the specimen with great care to avoid damaging the microscope or breaking theglass slide 2. Open the curved nger on the specimen holder and place the slide on the stage from the front

3. Gently release the spring-loaded curved nger until it contacts the slide. 4. To adjust the position ofthe specimen, turn the upper(Y-axis) and lower (X-axis) stagecontrol knobs. Do not adjust the stage or specimen holder directly. The specimen should be centered over the condenser lens. D. Observing the specimen

1. Set the main switch to “|” (on) and adjust the light intensity knob to “4”. You may adjust the intensity knob to make the illumination brighter or darker as needed. 2. Rotate the revolving nosepiece to engage the 10x (yellow) objective. 3. While looking throughthe eyepieces, adjustthe interpupillar distance untilthe left and right elds of view coincide completely.Don’t pinch your ngers!

4. Focus on the specimen by rotating the coarse adjustment knob (rotate awayfrom you). Rack the stage up slowly until the specimen becomes visible. Watch to ensure the objective lens does not come into contact with the slide. Improve the focus with the ne adjustment knob (rotate away from you) until the image is fully resolved. 5. You may move the specimen by gently adjusting the X- andY-axis knobs. 51

E. Adjusting the diopter

1. Looking through the right eyepiece with your right eye, bring the image into focus. 2. Looking through the left eyepiece with the left eye, turn the diopter adjustment ring to focus the image. 3. This will reduce eye strain as you manipulate many specimens. F. Adjusting Magnication

1. The objective lenses are parfocal. Once the 10x objective is focused, the 40x or 100x objective can be engaged and focused with only slight manipulation of the ne adjustment knob. 2. As you adjust magnication, you mayneed to adjust the light intensity, eld iris diaphragm, and aperture iris diaphragm to maximize focus and resolution. G. Using the Immersion Objective

1. Focus on the specimen with the 10x objective engaged. 2. 3. 4. 5.

Rotate the revolving nosepiece to engage the 4x objective. Carefully add 1 drop of immersion oil where the light from the condenser is shining through the slide. Engage the 100x objective (directly from 4x)and adjust the focus using the ne adjustment knob. When nished, CLEAN THE OIL FROM THE OBJECTIVE WITH LENS PAPER THAT HAS BEEN MOISTENED WITH CLEANING SOLUTION. WIPE DRY WITH LENS PAPER. *NEVER LEAVE OIL ON THE OBJECTIVES OR ON THE STAGE OF A MICROSCOPE*

H. Maintaining the microscope

1. Remove the slide and dispose properly! 2. Clean the objectives with cleaning solution, engagethe 4x objective, lower the stagecompletely, set the light intensity to “1”, turn the power switch to O “ ” (off), wrap the cord around the microscope, replace dust cover and place in the correct cupboard. 3. Report any malfunction or accident immediately. Interpupular distance adjustment Diopter adjustment ring

Arm

with curved nger

height adjustment knob

lens Field iris diaphragm adjustment ring

Base (X- Y-axis knob)

52

Appendix 3: Media and Methods Used For Isolating and Cultivating Bacteria A. General preparation of media Bacteria can rarely be identied only from a study of their cellular morphology and it is necessary therefore to obtain pure cultures of these organisms growing in the laboratory. Bacteria display many variations for the major nutritional requirements and so the utilizable sources of energy also vary. However, media for the cultivation of bacteria must contain a) water , b) a source of energy, usually in the form of a carbon compound, c) essential elements including N, P , and S, d) metallic elements such as K, Mg, Ca, Fe, Mn, Cu, and Zn in varying proportions, and e) organic nutrients. These are known as growth factors, compounds that an organism requires as a precursor of its organic cell material but which it cannot synthesize from simpler carbon sources. They include: i) amino acids ii) purines and pyrimidines and iii) vitamins. In prepared media, chemicals of dened composition are to be preferred to those of undened composition. Since the nutritional patterns of many bacteria are not sufciently well known, it is often necessary to add substances of complex organic compositione.g. peptone. In addition, media are often solidied with agar, a complex polysaccharide extracted from red algae. It is essential therefore, to use the same source of these materials for any comparative experiments. Most chemicals should be dissolved in distilled water. Tap water may contain toxic elements, particularly copper.

Most bacteria can only grow within a restricted pH range. The reaction of the medium must be adjusted so that the nal pH, after sterilization is between 6.8 and 7.2, unless a different reaction is needed for some special purpose. Buffers, which prevent large changes in pH, are often required to facilitate growth. This is particularly true of media composed of simple compounds or in which acid-producing bacteria are cultivated. Mixtures of sodium and potassium phosphates are often employed. In complex media, buffering is provided by the peptides and amino acids. To determine pH changes during growth, indicators may be included in the medium. Where critical experiments are being performed it is often advisable to add the indicator to a sample tube only as a control, and to a small quantity of the actual culture uid at theend of the experiment. Otherwise, it is possible that the indicator or the alcohol it is dissolved in might act as a substrate for bacterial growth.

B. Types of media used for cultivating bacteria The nutritional classication of organisms is based on three parameters: the energy source, the principal carbon source and the source of reducing power. With respect to energy source, phototrophs are photosynthetic organisms that use light as their energy source and chemotrophs are organisms 2 that depend Heterotrophs on a chemicaldepend energyon source. Organisms to use as a principal carbon are autotrophs. an organic carbonable source. ToCO designate the source of source reducing power, the term lithotroph or organotroph is applied. Lithotrophs use inorganic compounds as their source of reducing power, and organotrophs use organic compounds as their source of reducing power.

53

To summarize: energy source

source of carbon source

photoautotroph (photolithotroph)

light

CO2

photoheterotroph (photoorganotroph)

light

organic

organic

chemoautotroph (chemolithotroph)*

chemical oxidation of reduced inorganic compounds , NO2- and H2 3

CO2

inorganic

chemical

organic

organic

e.g. NH chemoheterotroph (chemoorganotroph)

reducing power inorganic oxidizable substrate

*All chemoautotrophs are chemolithotrophs, but not all lithotrophs are autotrophic. For example, the methylotrophic bacteria can use organic carbon as their carbon source* 1. Media for autotrophic bacteria Autotrophic bacteria are cultivated in solutions of mineral salts without organic additives (except in those species requiring growth factors). The media will be variable depending on whether the organism to be grown is a phototroph or a chemotroph. 2. Media for heterotrophic bacteria Many different types of media have been perfected for the cultivation of heterotrophic microorganisms depending on their metabolic processes and nutritional requirements. Those organisms with complex requirements are said to be nutritionally exacting and include several of the pathogenic organisms and certain soil species. They are usually grown on complex natural media while the less exacting types (and the autotrophs) are grown on synthetic media of known composition. TM Recipes for various types of media can be found in the Difco & BBLTM Manual (http://www.bd.com/ ds/technicalCenter/documents.asp).

C. The cultivation of anaerobic bacteria

Many species of bacteria are facultative aerobes,i.e. they can grow under aerobic or anaerobic conditions, the latter ability being dependent upon the presence of some substance that can be utilized as a hydrogen acceptor by the species concerned. Generally, facultative organisms prefer to grow aerobically rather than anaerobically. Aerobically, a larger amount of ATP and a larger amount of all mass is produced. Some bacteria are obligate aerobes, unable to use anything but oxygen as a nal electron acceptor. Aerotolerant anaerobic organisms are fermentative organisms that are able to grow in the presence of oxygen. Others are obligate anaerobes which cannot use oxygen as an electron acceptor. A few bacteria are somewhat intermediate, growing best in low oxygen tensions. These are called microaerophilic bacteria.

54

Anaerobic forms occur in several families of bacteria such asBacteroidaceae and Peptococcaceae. Other anaerobic organisms include the GenusClostridium, an endospore-forming organism, certain autotrophic bacteriae.g. the purple sulphur bacteria, and some other soil or water bacteria e.g. Desulfovibrio. During growth aerobic bacteria tend to utilize all available oxygen and so reduce the medium. Thus, in mixed cultures the oxidation-reduction potential (Eh) of the medium may become low enough to allow anaerobes to develop. 1. Methods involving removal of oxygen: a. Stab and shake cultures

Many anaerobes can grow in deep stab or shake cultures in glucose agar. The method is particularly useful for microaerophilic species. A seal of liquid parafn or vaseline is sometimes advocated to help maintain anaerobic conditions,e.g. Hugh and Leifson fermentation test. This method is not entirely reliable and some evidence has been collected showing that oxygen diffuses through parafn quite quickly. b. Anaerobe jar culture

If surface cultures are required, the plates or slopes should be placed in an anaerobe jar e.g. the McIntosh and Fildes jar. This is a heavy metal or glass jar with a lid that can be clamped rmly down to form an effective seal. Inlet and outlet valves allow the air in the jar to be replaced with hydrogen, after slow and careful evacuation of most of the oxygen. When this has been done, the valves are closed and a current is passed through a small heating coil packed with palladinized asbestos and enclosed in a wire gauze cage, attached to the inside of the lid. The gently heated palladium catalyses the combination of any remaining traces of oxygen with the hydrogen. A tube of methylene blue agar tted to the jar acts as an indicator. Recently, many adaptations of this jar have been introduced. The one used presently is a commercially prepared envelope by Merck. The envelope called an Anaerocult A is placed in the jar. The envelope incorporates a system which allows for the chemical binding of oxygen which creates an oxygen-free milieu and a CO2 atmosphere simply by adding 35 ml of water to the contents. To assure that anaerobic conditions are provided, a disposable anaerobic indicator, methylene blue, is included in the jar. c. Thioglycollate medium

This is prepared by adding 0.1% (up to 0.4%) thioglycollic acid to nutrient broth before adjusting the pH. 1% glucose must also be added. Although the medium may be solidied with agar, it is more usual to use a semi-solid medium,i.e. 0.5% agar. The increased viscosity of the medium prevents the distribution of oxygen (dissolved at the exposed surface), by convection currents. Resazurin is added to act as an indicator of reducing conditions. It is colourless when reducedi.e. ( between Eh + 0.12 and 0.3 volts, which will allow most anaerobes to develop). A pink layer at the surface shows the depth to which oxygen has diffused into the medium. Inocula should be introduced carefully by means of a ne pipette, at the bottom of the tube.

55

D. Methods of isolating bacteria:

1. Isolation from contaminated cultures Progressive dilution is the basis of isolation techniques. This can be applied to suspensions containing two or more bacterial species as follows: a. Poured plate method

Melt and cool to 45°C two tubes of an agar medium. Inoculate one tube with one loopful of the suspension and mix well by rotating it between the hands. Flame the loop thoroughly and transfer one loopful of the mixture from the rst tube to the second. Mix again. Pour the contents of both tubes into sterile Petri dishes and allow to set. Take care that the agar does not set before it is poured. The second plate should show well separated colonies after incubation. Distinct colonies can then be picked off and examined. If the suspension is very heavy initial dilution in sterile saline may have to be made. b. The streak plate method

This is a quicker, though less reliable method. Prepare a plate of solid medium. Dry the plates at 50°C for 30 minutes before use. Prepare a streak plate for single colony isolates using the method on pages 62-63. After incubation, well separated colonies should be found along the streak marks. Before deciding that a culture is pure by either method, colonies should be picked off, grown and then reseparated, until all colonies are the same. Morphological variants can upset strict application of this principle. Staining can be used as a check on the purity of the nal isolations. 2. Isolation from natural sources all live organisms in a given The objective of many viable counting techniques is to estimate the number of sample of material. To do this, a medium satisfying the nutritional requirements of as many of the bacteria in the sample as possible, is required. Thus, in soil bacteriology, soil extract media have been developed as most resembling an ideal non-selective medium. Other types of viable counts and all isolation techniques embody the reverse principles. Here, it is necessary to pick out and encourage one type of organism and to prevent or depress the development of other types. In natural habitats, the organisms which one is isolating may be present only in small numbers. The rst step in such cases is thus to obtain an enrichment culture by one or more of three methods:

a) by using selective media b) by using selective conditions of incubation c) by selective pretreatment of the material Several generations of sub-cultures on liquid, or solid media may be necessary, but the nal step will consist

of plating out the organisms by one of the methods described above.

56

a. The use of selective media

A completely selective medium, allowing the growth of only a single species, is not attainable in practice, but media can be obtained which will discourage all but the required species. Some selective media are alsodifferential. Certain organisms, when grown on them, exhibit distinctive biochemical or morphological characters which enable them to be recognized easily. This may be very important from the medical point of view, where the range of species concerned is small. Where a large range of species is involved,e.g. in soil, differential media cannot be used to separate taxonomic groups, but are useful to distinguish groups concerned in some biochemical process, e.g. cellulose decomposition. Selectivity may be achieved in three ways: i) Addition of substances to a medium

A classic example of selective media is MacConkey’s bile-salt lactose broth. Bile salts discourage the growth of most bacteria except those of intestinal srcin. The presence of 1% lactose and a pH indicator make the medium differential as well. Coliform organisms ferment the lactose to produce acid and gas from the medium indicating fecal pollution in water and milk, etc. The same medium solidied with agar is used to isolate the pathogenicSalmonella/Shigella group of bacteria. While the coliforms produce acid and therefore a color change, theSalmonella and Shigella group are not able to ferment lactose and no color change occurs. Other examples of substances added to media to make them selective are: potassium selenite (0.4%) sodium azide

inhibits coliforms inhibits the cytochrome oxidase enzyme in the electron transport chain where the transport is coupled with ATP synthesis 0.025% used to isolate fecal streptococci

potassium tellurite (0.002%) agar to isolate crystal violet

inhibits coliforms and pseudomonads used in blood Corynebacterium diphtheriae

a triphenylmethane dye that exerts its inhibitory effect by interfering with cellular oxidase processes inhibits Gram-positive bacteria at very low concentrations (0.01%) Used for isolating Gram-negatives since they are affected by much higher concentrations.

rose bengal (0.006%) actidione

discourages all bacterial growth inhibits yeasts and fungi, 0.0005% used to enumerate bacteria in mixed ora

nystatin

inhibits yeasts and fungi

antibiotics

e.g. penicillin (1000 units/ml)

57

ii) Alteration of the pH of a medium

Organisms tolerating a high pH,e.g. Vibrio and Pseudomonas sp., can be encouraged by adjusting the reaction to pH 9-10,e.g. Dieudonne’s blood-alkali agar for isolatingVibrio cholerae. Acid tolerant bacteria can be more readily isolated if the pH is reduced to 5 or lower. Thus, Lactobacilli are isolated on media containing 0.5% acetic acid. After isolation, the organisms should be grown on normal media. iii) Omission of substance

Media without organic substances are used to isolate and grow autotrophic microorganisms. Several transfers are usually needed. Although the rst cultures show much growth, some organic matter is inevitably introduced with the inoculum causing contamination. Mannitol phosphate media used for nitrogen xing organisms is an example of this method of selection. b. The use of selective incubation

Selective incubation of cultures can select quite different parts of the microbial population. i) Temperature

Microorganisms have more or less denite temperature requirements, with maximum temperatures above which they fail to grow and minimum temperatures below which they fail to grow. These limits vary depending on cultural conditions, but are sufciently precise to be used in isolation procedures.

The temperature range of a given microorganism is relatively easy to establish. One culture of a given species, uniformly inoculated, should be incubated over a range of temperatures,e.g. 50°C, 45°C, 37°C, 25°C, 18°C, 10°C, 2°C. Tubes incubated at high temperatures must be enclosed in polythene bags to prevent excessive desiccation. The amount of growth occurring after one day, two days or two weeks incubation should be noted visually and recorded as a scale from zero to three i.e. 0, 1, 2, or 3. The method may be made more precise by employing stringent inoculation procedures and using an spectrophotometer to assess the turbidity of a suspension. The results obtained from this experiment will vary,thermophiles growing at 45°C-50°C and above, mesophiles rarely growing above 40°C, body organisms having an optimum at 37°C, soil organisms growing at about 10°C-25°C. Organisms growing at low temperatures are usually termed psychrophiles. These temperature distinctions are made for convenience and the precise numbers should not be taken as absolute. Thermophiles occur widely in soil and are important in the production of silage and compost, and in the canning and sugar-rening industries. To isolate such organisms from soil, a moist sample should be incubated at 45°C-60°C or higher for three days. A suspension of this material should then be made in sterile Ringer’s solution and heated in a water bath at 100°C for twenty minutes. Add l ml of this suspension to 15 ml of nutrient agar melted andcooled to 45°C. Pour into a sterile petri dish. Incubate at 45°C-60°C with the dishes in a polythene bag to prevent drying. Thermophiles are thermoduric. However, a similar sample should be incubated at 25°C to demonstrate that some thermoduric organisms are not thermophiles. The distinction between mesophiles and thermophiles may be imprecise. Extreme thermophiles have been found in hot springs such temperatures at the boiling point, in steam vents (fumaroles) where temperatures may reach 150°C - 500°C and in geothermal vents at the bottom of the ocean which have temperatures of 350°C and higher. ii) Aeration

Bacteria have a denite relationship to aeration conditions.Some will only grow with asupply of oxygen, the obligate aerobes. Others will only grow in the absence of oxygen, the obligate anaerobes. The great bulk of organisms will grow in either state to a certain degree. These are known as facultative.

58

Appendix 4: Review of microbiological techniques I. Preparation of bacterial smears for staining

To make a good lm for staining, obtain a clean grease-free slide. Draw a circle on it approximately 2 cm in diameter with a grease pencil.Turn the slide over.

If the inoculum is from solid (agar) media, add a drop of water within the circle. With a sterile loop or stick, remove a very small quantity of surface growth from the agar, mix this with the water to make a homogenous suspension of the cells, and allow toair dry. If thethe slide is prepared from a broth culture, use a sterile loop or swab to place a drop of the broth culture onto slide (do not add water). A loop must be amed again before replacing it in the holder; a swab can be discarded in the autoclave garbage. When dry, the lm should be onlyfaintly visible; a thick opaque lm where the bacteria are piled on top of one another is useless.

During the staining process, cells can be washed away, leaving very few cells for observation. To prevent this from happening, the cells must bexed to the slide using one of the following methods: Heat Fixing: pass the end of the slide with the smear through a Bunsen ame several times. Do not hold the slide in the ame: if the slide is over-heated, the bacteria will become distorted, making it hard to observe

under the microscope. Methanol Fixing: add a drop of methanol to the thoroughly dried smear. Allow the methanol to air dry. II. Staining procedures:

A. Acid-fast stain (Kinyoun method) 1. Prepare a bacterial smear as outlined inI above. 2. Flood the smear with Kinyoun carbolfuchsin for 10 minutes. 3. Rinse the slide with water to remove excess stain. 4. Decolourize with acid-alcoholuntil the run-off is clear. Rinse with water. 5. Counterstain with Brilliant Green for1 minute. Rinse with water, blot dry and observe. ACID FAST ORGANISMS ARE RED; OTHERS ARE BLUE/GREEN

B. Capsule stain (does not require a bacterial smear) 1. Aseptically place a loop ofculture in a drop of Congo Red on one end of a glass slide. 2. Using the end of a second slide, draw the Congo Redto the opposite end of the slide. The Congo Red should form a thin translucent layer on the slide. Dispose of the second slide appropriately. 3. Do not heat x, but allow to air dry. 4. Flood the slide with Maneval’s Stain for1 minute. Rinse gently. Air dry and observe. CELLS ARE RED, CAPSULES ARE CLEAR, AND THE BACKGROUND IS BLUE. DIFFERENTIATE BETWEEN BACTERIAL CELLS AND ARTIFACTS ON THE SLIDE. WHEN THE EDGE OF THE CAPSULE IS IN FOCUS, THE RED CELLS SHOULD BE VISIBLE.

59

C. Endospore stain 1. Generally, the organism is grown on a sporulation agarplate for 48 hours before doing an endospore stain. 2. Prepare a bacterial smear as outlined inI above. 3. Place the slide on a heating rack in the fume hood. Coverthe smear with a small piece of paper towel and ood the smear with Malachite Green. Steam gently for 5 minutes, keeping the paper moist with stain. 4. Remove the paper towel with forceps and rinse the slide with water to remove excess stain. 5. Counterstain with Safraninfor 1 minute. Rinse with water, blot dry and observe. The endospores will stain green and the rest of the cell pink

D. Gram stain 1. Prepare a bacterial smear as outlined inI above. 2. Flood the smear with Crystal Violet for 1 minute. Rinsewith water. Remove excess water bytapping slide gently on the staining rack. 3. Flood the smear with Lugol’s iodine for 1 minute. Rinse with water. Remove excess water bytapping slide gently on the staining rack. 4. Decolourize with 95% ethanol. Drip alcohol downthe slide for approximately 10 seconds andrinse with water. Alcohol and water are alternated until the drippings are colourless. Remove the water after the last rinse. 5. Counterstain with Safraninfor 1 minute. Rinse with water, blot dry and observe. Gram-positive organisms stain blue (purple) and Gram-negative organisms stain pink (red). III. Cellular morphology

Cellular morphology is generally obtained from a Gram-stained slide. The cellular morphology includes: size diameter in length in

as determined by calibration of the microscope mm (cocci) mm (bacilli)

shape

cocci (spherical), bacilli (rod)

arrangement of cells

random, pairs, tetrads, chains, clusters

Gram reaction

positive, negative

A diagram drawn to scale usually accompanies the cellular morphology

60

A. Diagrams of Cells: 1. Make all diagrams with pencil. Diagrams areto be prepared on blank white paper. 2. Draw what you observe through the microscope ratherthan your interpretation orwhat you expect to see. 3. Guidelines for scientic diagrams:

a) Diagrams should be large. Usually only one representative cell needsto be drawn in detail, other cells are drawn in rough to represent the surrounding medium. b) Do not shade. c) Do not write notes on the diagrams. d) Label identiable structures. Aruler is used to draw a straight line horizontally from thestructures to the label (usually the right side).

4. All diagrams must be labelled, below the diagram, with: a) Title of the diagram. b) Total magnication of the specimen. Multiply the power of theoculars by the power of the objective used. For example, under the 10x objective, the total magnication is 100x since the oculars also magnify 10x. c) All diagrams must be drawn to scale. To demonstrate the magnication of thedrawing, divide the size of cell as drawn by the size of cell as measured on the slide. Remember scale! 5. The diagram should bedrawn in proportion sothat one measured dimension is sufcient whendrawing to scale. Use a size bar to indicate the actual size of the cell. See Figure Ap4.1 below: Your microscope was calibrated so that under the 100x objective, 1 ocular division = 1.0 micrometer, and the rod shown measures 5 ocular divisions in length, the length of the rod is therefore 1.0 micrometers x 5 = 5 micrometers. The length of the rod that has been drawn is 73 mm, so:

5 µm

Cell Wall

Figure Ap4.1: Bacilli bacterial cell Total Magnication: 1000x Magnication of drawing: 73 000 µm / 5 µm = 14 600x

61

IV. Colony morphology

The following characteristics are those most commonly used to describe colony morphology. A single colony is observed when describing colony morphology. 1. Shape:

2. Surface: smooth moist

rough dry

mucoid powdery

3. Elevation:

4. Size: in mm, as measured with a ruler 5. Pigment: cream, white or beige coloured organisms are usually considered to be non-pigmented. Pigments may be purple, red, pink, yellow, brown, blue, grey,etc. Water soluble pigments diffuse into the media. 6. Opacity: Transparent (can see through) or opaque. V. Streak plate:

If a colony morphology is to be obtained from a plate culture, the agar plate must be streaked in such a manner so as to obtain single colonies of the bacteria. See Figure 5.12 in your text and Figure Ap4.2 on page 63. 1. A loop/swab of liquid culture, or asmall amount (1 colony)of bacterial growth from aplate culture, is transferred aseptically to a sterile plate and spread across the surface of one quadrant of the plate in a zigzag pattern. 2. The loop is amed to re-sterilize the surface and cooledby touching the loop to the surface of the agar near one edge of the plate. This will cause the agar to sputter, so make sure your plate is close to your bunsen ame to reduce the spread of aerosols. To avoid aerosols, sterile sticks can be used and discarded between each set of streaks. This will be demonstrated by your TA.

3. NOTE: The loop/stick is not to be reintroduced into the broth or plate culture used for the rst streak. Make a second set of zigzags on a second quadrant of the plate, being sure to pass through part of the rst set of streaks to pick up inoculum from the rst quadrant. This will dilute the number of cells spread in the second streak compared to the rst streak.

4. Flame the loop, cool in the agar, and make a third set of zigzag streaks. Repeat for a4th set. After incubation, fewer and fewer bacterial cells should appear in successive streaks resulting in isolated colonies in the area of the 4th set of streaks. 62

Figure Ap4.2: How to do a steak plate for single colony isolation VI. Labeling of Cultures: date, your initials, lab section, and Individual tube and plate cultures should be labelled legibly with the specimen information. The plates are labelled along the outside edge on the bottom only (agar side) since the lids may get mixed up. Plates should be incubated agar side up so any condensation will fall on the lid. Tubes are labelled on the test tube, NOT on the lid. VII. Incubation of Cultures:

Cultures should be incubated at the temperature most favorable to growth or the specic activity being studied. Human pathogens and commensal species grow best at body temperature,i.e. 37°C. Soil organisms and plant pathogens are normally incubated at 20-25°C. The optimum temperature is that temperature at which the growth rate is maximal for a particular organism. Cultures will usually grow at their optimum temperature in 2448 hours. Some specic tests may take 48-96 hours of incubation to reach maximum growth. VIII. Pipetting using sterile technique:

Whenever pipetting is done in the microbiology laboratory, strict aseptic technique must be used. In CMMB 343, glass (or plastic disposable) pipettes will be used to pipette large volumes and micropipettes will be used to dispense smaller volumes. Pipetting with a sterile glass (or disposable plastic) pipette and a propipette

The can of pipettes has been sterilized and all the pipettes must be kept sterile. Take the can of pipettes to the work bench. Remove the top of the can, ame the opening and remove one pipette, making sure that only the top of the pipette is touched. Reame the opening and replace the top. Carefully place the propipette on the pipette without contaminating the lower 3/4 of the pipette. When the pipetting is completed, remove the propipette carefully and place the used pipette in the pipette bucket.

63

Pipetting using a micropipette

These micropipettes are very expensive and very delicate. Please handle them carefully and do not exceed the maximum or minimum volumes. When not in use, the micropipettes are kept in the micropipette stands. Micropipettes consist of a barrel (that houses a spring-loaded piston) attached to a button that is depressed with your thumb to draw up and expel liquid. Micropipettes must be used in conjunction with plastic tips that have been sterilized in special boxes. A tip must be placed securely on the barrel of the pipette so that there is an air-tight seal. If it is too loose, the tip will leak and not transfer an accurate volume. Since the tip is the only sterile part of the instrument, only allow the tip to come into contact with the bacterial culture, not the barrel. These tips are disposable and a clean tip should be used every time a solution is measured. Discard used tips into the disposal bags on your bench. 1. Eppendorf micropipette

Figure Ap4.3: Eppendorf micropipette a) The control button on top of the pipette has three positions: At Rest

-

Stop 1

-

Stop 2

-

The position of the button when it is not being used This measures the volume selected in the window. Depressing the button from the “At Rest” position to “Stop 1” measures the volume selected in the window. This blows out the last drops when expelling liquid from the tip.

b) To set the volume required: i) Turn the setting ring. Note the “-” and “+” directions and turn accordingly. For theblue-coded micropipettes, never turn below 100 l (shown as 0100 from top to bottom) or above 1000 l (shown as 1000 from top to bottom). Theyellow-coded micropipettes have a range between 10 l and 100 l. 64

* Remember to use aseptic technique *

c) To measure a required volume of liquid: i) Securely attach a micropipette tip from asterile tip box. Keep the lid of the box closed as much as possible to maintain sterility ii) Press the control button down to the “First Stop” and hold. iii) Hold micropipette vertically and immerse tip approximately 3 mm into the liquid. iv) Allow control button to glide backSLOWLY to the “At Rest” position. Never allow it to snap back. v) Slide tip out of the liquid along the inside of the vessel toremove any remaining droplets. The volume in the tip should now be the volume shown in the window. d) To transfer this volume of liquid: i) Place the tip in the receiving vessel, holding thetip at an angle against the inside of the vessel. ii) Press the control button slowly down to the “First Stop” to dispense the liquid. iii) Press the control button down to the “Second Stop” (blow-out) to empty the tip completely. iv) Hold down the control button. Slide tip out along the inside of thevessel. v) Let the control button glide backslowly to the “At Rest” position. vi) To eject the tip after the liquid has beendispensed, hold the micropipette over the disposal bag and depress the eject button. This will shoot the tip off the end of the micropepette into the disposal bag. Be careful to aim correctly.

Figure Ap4.4: Technique of holding micropipette, test tube and cap Figure 5.12 in your text shows a technique to aseptically retrieve a loop of broth culture from a tube. The technique to use a micropipette to aliquot liquid between vessels is similar. 65

IX. Serial ten-fold dilutions

It is sometimes necessary to determine the concentration of the organism with which you are working. Working concentrations are often very high, which makes counting difcult, so the stock solution must rst be diluted before plating and subsequent counting. A series of 10-fold dilutions is typically made, and a sample from each dilution is then plated and incubated. It is assumed that each colony on the resulting plates srcinated from a single organism from the dilution, so we call each colony a colony forming unit (CFU) and we measure bacterial concentration as the number of CFUs per ml of srcinal culture. The dilution that produces a colony count between 30 and 300 CFU/ml is normally chosen to perform the calculation. It is not considered statistically reliable to count from a plate containing more than 300 colonies and a plate with less than 30 colonies. Thus, only one set of plates will normally be counted if serial ten-fold dilutions are used. Figure Ap4.5 below shows how to perform serial 10-fold dilutions. Stock Culture Solution

100 µl

1µ 00l

Dilution 1µ 00l 100 µl 1µ 00l

1µ 00l

900 µl sterile broth 1/10 10-1

1/100 10-2

1/1000 10-3

1/10 000 10-4

1/100 000 1/1 000 000 10-5 10-6

Figure Ap4.5: Serial dilutions (refer to Figure 5.15 in your text) X. The dilution-plate method of viable counts

a) Serial ten-fold dilutions are prepared. 1000 l ofeach dilution is mixed with 15or 20 ml of an agar medium, previously melted and cooled to 45°C and poured into separate sterile petri dishes and even distribution is ensured. When set, the plates are incubated at the required temperature. The appropriate plate is counted and the concentration is determined in CFU/ml. b) Alternatively, 100 l of each dilution is added to a separate agar plate and thedilution is spread with a sterile glass spreader. When dry, the plates are incubated at the required temperature. The appropriate plate is counted and the concentration is determined in CFU/ml. -4 -3 For example: If you count 185 colonies on the 10 dilution plate (theoretically 1850 on the 10 and 18.5 on the -5 4 10 plates), you need to multiply by 10 (you plated 100 l onto the plate) and multiply by 10 (the inverse of the 7 dilution) to determine the concentration of the stock culture solution: 1.85 x 10 CFU/ml.

66

XI. Biochemical tests 1. Oxidase test

Cytochrome c is a major enzyme involved in many metabolic electron transport systems and becomes reduced when it oxidizes the cytochrome bc1 complex. The electrons are then passed to Complex IV, which, in turn, reduces O2 to H2O. If present, cytochrome c will also oxidize the articial reagent dimethylpphenylenediamine, which changes from colourless (reduced) to blue (oxidized). 2. Catalase test

Flavoprotein (FMN) is the rst enzyme inmany metabolic electron transport systems and becomes reduced when it oxidizes NADH. FMN normally passes electrons onto other proteins within Complex I, but can reduce oxygen directly. When this occurs, toxic oxygen radicals like hydrogen peroxide (H 0 ) and superoxide radical 2 2

(O2-) are produced. The enzyme catalase functions by converting hydrogen peroxide to water and oxygen gas (02). When hydrogen peroxide is added directly to cells that produce catalase, oxygen bubbles form instantly. 3. Hugh and Leifson (H&L) test

Utilization of a carbohydrate (glucose, lactose,etc.) results in the production of acidic end products or intermediates. Excretion of these acidic compounds into the medium results in the pH indicator, bromthymol blue, changing from green to yellow. This is considered apositive result for carbohydrate utilization. The location of the colour change, from green to yellow is also important. If the organism is can only ferment the carbohydrate, or both respire and ferment the carbohydrate, then the colour change will occur in both the sealed (mineral oil) and unsealed media. If the organism can respire only, then the colour change will occur in the unsealed media only. Also note that each tube will contain an oxygen gradient, with the aerobic zone near the top of the tube and the anoxic zone near the bottom of the tube. If the organism cannot utilize the carbohydrate, but can grow in the medium utilizing peptones, the organism will produce alkaline end products. Excretion of these basic compounds into the medium results in the bromthymol blue changing from green to blue at the top of the medium. This is negative a result for carbohydrate utilization. 3. Phenol red test

Utilization of a carbohydrate (glucose, lactose,etc.) results in the production of acidic end products or intermediates. Excretion of these acidic compounds into the medium results in the pH indicator, phenol red, changing from red to yellow. This is considered apositive result for carbohydrate utilization. When this medium is prepared as a broth, gas production can be detected by placing an inverted Durham vial in the tube. If the organism produces gas as a result of fermentation, the medium turns yellow and a bubble forms in the Durham vial. When the medium is prepared as a gel (with agar), gas production can be detected when cracks appear in the agar. 4. Citrate test

Simmons citrate medium is a mineral medium with 0.3% sodium citrate as the sole carbon source. The indicator bromthymol blue is incorporated into the medium. It is green at pH 6.8 and blue at pH 7.6. The organism is streaked onto the surface of the slant and incubated. Organisms able to utilize the citrate grow on the surface of the medium. Due to oxidative formation of sodium carbonate, the medium becomes alkaline and changes colour from green to blue.

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5. Coagulase test

The organism being tested is inoculated into a tube of0.5 ml blood plasma which is prepared fresh daily . The tube must be incubated for at least 6 - 8 hours and at longest 24 hours. A solid clot indicates a positive result. The organism produces the enzyme coagulase which coagulates the blood plasma to form a clot. 6. H2S test

Hydrogen sulde may be produced from sulphur containing amino acids, e.g. cysteine, present in peptone. The organism is inoculated into a tube of peptone water and a piece of lead acetate paper is suspended into the tube (not touching the liquid) by the cap. After overnight incubation, if the organism breaks down cysteine and produces H2S, the lead acetate paper turns black due to the formation of lead sulde. A negative result is indicated by the lead acetate paper remaining white. 7. Indole test

Indole is produced by some bacteria upon degradation of the amino acid tryptophan. The organism is inoculated into a tube of peptone water and incubated overnight. Several drops of Kovac’s reagent are added after incubation. If the organism breaks down tryptophan to produce indole, the indole and the amyl alcohol present in the Kovac’s reagent produce a cherry red colour at the interface of the medium and the Kovac’s reagent. A negative result is indicated by a brown colour. 8. Litmus milk

Litmus milk is composed of skim milk with sufcient litmus to produce a lilac colour. There is a small amount of glucose present in the milk and a larger amount of lactose. Casein is the main protein in milk and gives the milk its opacity. Reactions occur due to the fermentation of the carbohydrates or utilization of the proteins. The pH indicator, litmus, changes colour as a result of the reactions that occur. The organism is inoculated into the medium and incubated for 2 - 5 days.

Litmus indicates changes in the pH of the media and also in the oxidation-reduction state of the media: a)

acid production

litmus turns pink indicates lactose (glucose) fermentation with acidic end products

b)

alkaline reaction

litmus turns purple/blue partial casein digestion with alkaline end products (ammonia)

c)

reduction

litmus turns white litmus reduction by a fermentation reductase enzyme (anaerobic)

d)

coagulation

the medium solidies

acid reaction precipitates casein gas production can create ssures in the clot (coagulated casein)

e)

peptonization

the medium loses its opacity (sometimes white, pink or brown on top) Casein is digested leaving whey (straw colour)

9. Mannitol yeast extract congo red agar

The organism is streaked onto a plate of mannitol yeast extract congo red and incubated for 2-5 days. The organism grows on the mannitol yeast extract agar. If it can absorb the congo red, the colonies are red. A negative result is indicated by growth of translucent colonies on the agar. 68

10. Methyl Red - Voges Proskauer (MR-VP) test

The Methyl Red (MR) test and the Voges-Proskauer (VP) test determines two possible end products of the fermentation of glucose. The organism is inoculated into two tubes of MR-VP broth and incubated overnight. In the MR test, the organism ferments the glucose in the medium to the end products acetic, lactic and succinic acids, and ethanol, CO2 and H2. The high concentration of organic acids overcomes the phosphate buffer in the medium and the pH becomes very low. Several drops of methyl red are added: a red colour indicates mixed acid fermentation (a positive result). A yellow (or orange) colour indicates that little acid has been produced by the organism (a negative result). In the VP test, the organism ferments the glucose in the medium to pyruvate, which is then converted to acetoin and 2, 3-butanediol. 15 drops ofa-naphthol and 5 drops of 40% KOH are added and the tube is vortexed gently to expose the medium to oxygen in order to oxidize the acetoin (if present) to diacetyl. The diacetyl then reacts with peptones, which results in a red pigment (a positive result). If there is no acetoin present, the reagents will turn the medium a brown (copper) colour. 11. Motility

A stab is used to inoculate the organism into the TTC motility medium. Stab carefully to obtain only a single stab line and incubate overnight. The triphenyl tetrazolium chloride is reduced when broken down by the organism and turns red. Therefore, the medium turns red where it is inoculated. If the organism is a facultative organism and motile it swims throughout the medium and the whole tube becomes red. If the organism is an aerobic organism, the stab line turns red and the entire top of the medium turns red where the organism swims in the presence of oxygen. 12. Ornithine decarboxylase test

Some amino acids can be utilized as a carbon source after decarboxylation. In this process, the carboxyl group is converted to CO2 and the amino acid is converted to an amine in the presence of the coenzyme pyridoxal phosphate. The production of the amine results in a rise in pH which changes the colour of the indicator in the media used. The amino decarboxylases areof lysine, arginine andoccur ornithine. will the amino ornithine which is notacid found as aconstituent protein but does in theThis free test form. Theuse medium usedacid is Møller’s amino acid medium. It incorporates basal salts, glucose, the indicator bromcresol purple, as well as 1% Lornithine. A tube of ornithine decarboxylase TEST medium and a tube of decarboxylase CONTROL medium is inoculated with the organism, and approximately 1 ml ofsterile mineral oil is poured into each tube. The tubes are incubated overnight. The indicator, bromocresol purple is yellow at pH 5.2 and purple at pH 6.8. If the organism can utilize the glucose in the control tube, the medium turns yellow because of acid production. If the organism can utilize the glucose and the ornithine in the test tube, the medium rst becomes yellow because of acid production from glucose, later if decarboxylation occurs, the medium turns alkaline and back to purple. A positive result is indicated by a yellow control tube and a purple test tube. A negative result is indicated by a yellow control and a yellow test tube. 13. Pigment Solubility Tests

The pigment produced by some microorganisms can be either a water soluble pigment or organic solvent soluble. To determine water solubility, observe the NA or BHI agar on which the organism is grown. Waterinsoluble pigments remain conned within the cells; water-soluble pigments dissolve into the surrounding medium. To determine organic solvent solubility, add 10 drops of alcohol: acetone (1:1) mixture to a test tube. Add a loop of the cells grown on a NA or BHI agar plate. If the pigment is soluble, the liquid will be tinted the colour of the pigment and will be transparent. If the pigment is insoluble, the liquid may appear to be tinted due to the suspended cells, but the suspension will be opaque. 69

14. Urea test

A solution of urea is added aseptically to a sterile cooled basal medium in which phenol red is added as a pH indicator. Phenol red is red at pH 8.0 and yellow at pH 6.6. The organism is inoculated into the medium and incubated overnight. If the organism possesses the enzyme urease, it will break down the urea, ammonia is produced and a red colour results. A negative result is indicated by the medium remaining yellow.

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Appendix 5: Bacterial Culture Media The following media is prepared from Difco dehydrated culture media: 1. Blood agar base + 5% debrinated whole blood

2. Brewer thioglycollate gelatin media 3. MacConkey agar 4. Mannitol Salt agar 5. Phenol Red Broth base + millipore ltered lactose, sucrose, maltose, xylose or sorbitol added to a concentration of 1% after autoclaving.

6. SF agar 7. SIM media 8. Mannitol nitrogen free agar MnSO4•4H20 dH20 CaCO3 NaCl MgSO4 K2HPO4 Mannitol (sugar) FeCl3•6H2O

trace 1.0 L 1.0 g 0.2 g 0.2 g 0.5 g 10 g granule

agar (1.5%) salt molybdenum

15 g trace

To remove the precipitate which forms on autoclaving, autoclave for 5 minutes at 15 lb.Cool. Filter through 3 pieces of Whatman #1 in a Buchner or if necessary use a sintered glass funnel with suction. Distribute in asks. Re-autoclave - 15 minutes at 15 lb. 9. Carbon free agar Prepare minimal glucose agar, but omit glucose and do not amend with any amino acids. 10. Nitrate reduction broth beef extract peptone KNO3 dH20

1.2 g 2.0 g 0.4 g 400 ml

Distribute 10 ml in large test tube. Add Durham vials upside down for trapping nitrogenous gases.

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11. Butlin’s medium K2HPO4 NH4Cl Na2SO4 CaCl2•6H20 MgSO4•7H2O yeast extract FeSO4 dH20 Na lactate

0.5 g 1.0 g 2.0 g 0.1 g 1.0 g 1.0 g 0.002 g 1000 ml 3.5 ml of 60% solution

Adjust pH to 7.5 before autoclaving. Fill screw cap test tubes approximately 5/6full. An iron nail (sterile) may be added to the tube, however FeSO4 in the media provides the iron required for formation of FeS. 12. Rogosa SL agar Bacto Tryptose yeast extract Dextrose Arabinose Saccharose (sucrose) Sodium acetate Ammonium citrate KH2PO4 6.0 g magnesium sulfate manganous sulfate ferrous sulfate Tween 80 (sorbitan monooleate liquid) agar dH20

10.0 g 5.0 g 10.0 g 5.0 g 5.0 g 15.0 g 2.0 g 0.57 g 0.12 g 0.03 g 1.0 g 15.0 g 1000 ml

Heat to dissolve. Add 1.32 ml glacial acetic acid/liter. Mix and then heat to 90 - 100°C for 3 minutes. Do not autoclave. Distribute in tubes or Petri plates. 13. Sucrose agar Tryptone yeast extract K2HPO4 ammonium citrate sucrose agar dH20

1.0 g 0.5 g 0.5 g 0.5 gg 5.0 1.5 g 100 ml

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14. Minimal glucose agar (MG) 2X salts

K2HPO4 KH2PO4 (NH4)2SO4 MgSO4•7H20 dH20

21.0 g 9.0 g 2.0 g 0.2 g 1000 ml

2X agar

Bacto agar dH20

6.0 g 200 ml

Prepare salts in 400 ml bottles. Make 200 ml of salts/bottle. Prepare agar and distribute into 200ml quantities. Autoclave separately. Add 2 ml of 40% glucose (volumetric) to the cooled salts. Pour hot agar into salts, mix and pour plates. 15. Amino acids Add the following amounts of millipore ltered a.a. to 200 ml of cooled 2X salts.

1-leucine threonine thiamine tryptophan

1.2% solution 1.2% solution 1.0% solution 1.2% solution

1 ml 1 ml 0.125 ml 1 ml

16. Nitrate reduction broth Peptone NaCl KNO3 dH20

1.0 g 0.5 g 1.0 g 100 ml

17. Stevenson’s medium Inorganic Compound (NH4)2SO4 NaNO2 MgSO4•7H20 FeSO4•7H20 NaCl

MgCO3 Na2CO3 K2HPO4 dH20

Ammonium Medium 2.0 g

Nitrite Medium -

0.5 g 0.1 g 5.0 g

1.0 g 0.5 g 0.1 g

0.3 g

1.0 g

1.0 g

0.3 g 1.0 g

1000 ml 73

1000 ml

18. Peptone Yeast Extract agar Ferric ortho Phosphate Yeast Extract Peptone

0.01 g 1.0 g 5.0 g

dH20 agar

1.0 L 15.0 g

19. Starkey’s medium (NH4)2SO4 KH2PO4 MgSO4•7H20

0.3 g

CaCl 3.0 g 0.5 g

2

Ferric sulfate dH20

0.25 g 2.0 ml of a 0.5% solution 1000 ml

Mix well, check pH. Should be at pH 4.5. Autoclave. Add 1 g of sulfur that has been sterilized by steam for 30 minutes on 3 consecutive days. Sulfur is sieved (20 mesh or less) and blown or gently poured onto surface of media. Positive growth is indicated by pH changing to 1.0, increased turbidity, and sulfur granule precipitation. 20. Tryptic Soy Broth tryptone polypeptone

15.0 g 5.0 g

NaCl dH20

5.0 g 1000 ml

20.0 g 1.0 g 5.0 g

agar dH20

15.0 g 1000 ml

10.0 g

CaCO3

21. Tryptose dextrose agar Tryptose Dextrose NaCl Dissolve in steamer. Autoclave. 22. Yeast extract - mannitol aga r (YMA) mannitol K HPO MgSO4•7H20 NaCl 2

4

0.5 g

0.2 g 0.1 g

yeast extract agar dH20

4.0 g 0.4 g

15.0 g 1000 ml

Adjust pH to 6.8 - 7.0. Before autoclaving, add 2.5 ml of a 1% congo red solution 23. Winogradsky column In the 1880s, Sergei Winogradsky devised a medium and procedure to study the development of soil microorganisms in vitro Mix together 1 teaspoon of nely chopped grass and 2 tablespoons mud Add 1 teaspoon of Plaster of Paris and mix until a thick, gummy consistency is achieved Use a glass rod to tightly pack the mixture into the bottom of a glass tube When the column is half full of the mixture, add bog water, leaving one inch of space at the top of the tube. Screw the cap on loosely and incubate under a light rack at room temperature for 2-3 days Remove any air bubbles trapped within the column, top up with bog water, screw the cap on tightly and observe the development of the miniature ecosystem

24. Rhizobium enrichment culture Aseptically add 4-6 clover seeds to the surface of 2 water agar slants Once the clover has germinated, inoculate one of the tubes withRhizobium, cap loosely and observe the development of the endosymbiotic relationship between the two organisms in the inoculated tube 74

Reagents and Stains 1. Arsenomolybdate Reagent ammonium molybdate dH20

25.0 g 450 ml

Add 21 ml concentrated H2SO4 Dissolve 3 g sodium arsenate in 25 ml dH20. Add to the above solution. Put in a brown bottle and incubate 48hours at 37°C. Should be yellow with no green tint. 2. Benedict’s reagent for Glucose copper sulfate sodium citrate sodium carbonate, anhydrous (or 100 g Na2CO3•10H20)

8.65 g 86.5 g 50.0 g

Dissolve citrate and carbonate in approximately 300 ml ofwater, heat and lter, Dissolve CuSO4 in 50 ml of water, heat and pour slowly, stirring constantly into rst solution. Cool and add water to bring volume to 500 ml.

3. Kovac’s reagent for Indole Dissolve 5 g p-dimethylaminobenzaldehyde in 7.5 ml amyl alcohol.Add 2.5 ml hydrochloric acid slowly, in a fume cabinet. 4. Nelson’s reagent for Glucose Reagent A

Na2(anhydrous) CO3 NaK tartrate Na(anhydrous) SO4 2 dH20

12.5 g 12.5 g 100.0 g 350 ml

Pour into a graduated cylinder and dilute to 500 ml. Reagent B

CuSO4•5H20 dH20 concentrated H2SO4

7.5 g 50 ml 1 drop

To 100 ml of Solution A, add 4 ml of Solution B just before using.

75

5. Nessler’s reagent for Ammonia Dissolve 10 g sodium hydroxide in 100 ml dH20 (1) To 15 ml of dH20 add 15 ml of Nessler’s stock reagent. (2) Add solution (2) to 70 ml of solution (1). Do not use if precipitation occurs. 6. Nitrate reagents A. Diphenylamine reagent

diphenylamine Hconcentrated SO4 2 dH20 HCl concentrated

0.7 g 60 ml 28.8 ml 11.3 ml

Dissolve diphenylamine in H2SO4 and add dH20. Cool and add HCl. Allow to stand overnight. B. concentrated H2SO4

N.B. do not use if black precipitation occurs (or if the solution becomes black) 7. Nitrite reagents A. Grieses reagent A

glacial acetic acid dH2O dimethyl naphthylamine

29.4 ml 70.6 ml 0.25 g

Add acid to water slowly to prevent boiling B. Grieses reagent B

glacial acetic acid dH2O sulfanilic acid

29.4 ml 70.6 ml 0.8 g

Add acid to water Store in separate containers. Just before use mix equal quantities ofA and B. Should be a pale pink-purple colour. 8. Phenolphthalein solution phenolphthalein 1.0 g 95% ethanol 100 ml pH ranges 8.3 - 10.0. Colourless in the acidic range; red when alkaline 9. Sudan Black B stain 0.3 g sudan black B in 70% alcohol. Allow to stand overnight before using.

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Appendix 6: WHMIS WHMIS stands for Workplace Hazardous Materials Information System. It is a nationwide system to provide information on hazardous materials used in the workplace. Exposure to hazardous materials can cause or contribute to a variety of health effects such as irritation, burns, sensitization, heart ailments, cancer, kidney and lung damage. Some materials may also be safety hazards that can contribute to res, explosions and other accidents if improperly stored or handled. WHMIS is an information system which provides the following: Labels on hazardous materials and their container that alert employers and workers to the danger of the product and basic safety precautions. Materials Safety Data Sheets (MSDS) - technical bulletins which provide detailed hazard and

precautionary information on the product. Please see your TA or the Lab Coordinator if you would like further information on any of the controlled products that are being used in the laboratory.

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Appendix 8: Image Reference Sheet CMMB 343 Laboratory Manual 2018

Page 52 Microscope image from Olympus CX31 compound light microscope user manual www.olympus-lifescience.com Olympus Scientic Solutions Americas Corp., Waltham MA Page 61 Image drawn in-house Page 62 Images drawn in-house Page 63 Image drawn in-house Page 64 Image drawn in-house Page 65 Image drawn in-house Page 66 Image Madigan, Bender, Buckley, Sattley and Stahl. 2018. Brock: Biology of Microorganisms, 15th Edition.from Pearson Education Inc., San Francisco. p. 150. Page 79 Document from Centers for Disease Control and Prevention. Updated March 13, 2015. Biosafety in Microbiological and Biomedical Laboratories, 5th Edition. http://www.cdc.gov/biosafety/publications/bmbl5/. Accessed December 4, 2017.

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Notes

Notes

Notes

Notes

CMMB 343 Lab Manual 2018

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