Biology 1 Lab Manual 201405

Table of Contents: FHSB 1214 FHSC 1214 Biology I Cell Biology Introduction Practical 1 Cell Biology Studies I Practical 2 Cell Biology Studies II Practical 3 Cell Biology Studies III

Practical 4 Cell Biology Studies IV Practical 7 Cell Biology Studies VII

Practical 5 Cell Biology Studies V Practical 6 Cell Biology Studies VI Practical 8 Cell Biology Studies VIII Practical 9 Cell Biology Studies IX

Practical 1 Biological molecules I Practical 2 Biological molecules II Practical 3 Enzyme studies I (Experiment 1)

Experiment Description

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Exercise 1: Writing of Lab Reports Exercise 2: Notes on Biological Drawings Identification of Biochemical in Their Pure Form

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Investigation of Action of Saliva and HCl in Two Carbohydrate Solutions

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Investigation of the Effects of Catalase Concentration on Hydrogen Peroxide

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Synthesis of Starch Using an Enzyme Extracted from Potato Tuber

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Investigation of the Enzymatic Effects of Materials on Hydrogen Peroxide

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43 49 50 52

Practical 6 Cell studies II Practical 7 Cell studies III

Exercise 1: The Microscope Microscope and Its Uses Exercise 2: On-site Assessment Exercise 3: Preparation of Wet Mount Exercise 4: Preparation of Microscopic Slides Exercise 5: Measurement with a Microscope Exercise 6: Observation of Starch Grains (Additional practice tasks if time permits) Exercise 7: Observation of Hair (Additional practice tasks if time permits) Extraction of Cell Organelles by Differential Centrifugation Determination of Solute Potential of Potato Cell Sap

Practical 8 Cell studies IV

Effects of Various Treatments on Pieces of Stained Potato Cells

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Practical 9 Energetics I

Respiration Respirati on of Germinating Beans

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Exercise 1: Microscopic Examination of Cells at Various Stages of Plant Mitosis Exercise 2: Reproductive Tissues in Plants (Histology of Plant - Lily Reproductive Structures) [Meiosis]

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Optional: Optional : Practical 3 Enzyme studies I (Experiment 2) Practical 4 Enzyme studies II Practical 5 Cell studies I

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Lab manual version 4_201405 Biology I & Fundamentals of Cell Biology

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FHSB 1214 Biology I Practical 10 Cell Biology Studies X -

FHSC 1214 Cell Biology -

Practical 10 Energetics Energetics II

Experiment Description

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Exercise 1: Mitosis and Meiosis Modelling Exercise 2: DNA Replication Modelling

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Respiration Respirati on of Yeast

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Appendix : Case study


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E-lab manuals: towards saving the earth More and more people all over the world and in Malaysia are getting involved in saving the planet – – electric cars, wind and solar energy, and paper-less projects. What can YO U do? do? Beginning January 2011, CFS PJ Biology has piloted test e-lab manuals in an attempt to join global conservation efforts to save the planet by saving trees. Less paper means more trees will be left standing to absorb carbon dioxide and reduce the greenhouse effect. Don’t Don’t you & your future loved ones deserve to enjoy a cooler planet? Another advantage is that full-colour biology pictures will be accessible to students for the first time. Important rules on tests and lab assignments Summary:  No MCs or any valid reasons accepted for late/missed assignments and tests.  It is the student’s responsibi lity to submit another assignment in-lieu or sit for a replacement test when announced (on different topic).  The lecturer will NOT remind students to submit late/missed late/miss ed assignments nor attend replacement tests. (Treating you as adults.) Details:  Unacceptable: MCs, valid reasons (chicken pox, met with with an accident, menstrual cramps, stomach ache, dog died etc, non-valid reasons (forgetfulness; lateness; server/ IT problems). This is to be fair to everyone as fake MCs and liars are present in Malaysia.  If a student student fails to submit an assignment or misses a test, the lecturer lecturer will NOT NOT remind you to submit a new assignment nor to sit for a replacement test. The replacement test will be announced to everyone in general and not to individual absentees. Those who are supposed to attend must turn up and will not be reminded. It will be conducted at the end of the semester on a different topic (usually more difficult) when all students are so busy with tests and assignments.  It is the responsibility responsibili ty of the student to choose one other assignment to be submitted later in the semester when all students are so busy with tests and assignments.  If a submission is done online, a minimum of of 7 days are given to submit your assignment. As such, n o excuses will be entertained if there’s a server/ IT failure or technical problems with your UTAR account. Hence, you have an option to submit your assignment on day 1 to be safe, or on day 7 to be stupid. If you’re late, a link a link for ‘Late submissions’ is available if you don’t mind getting getting a 50% discount, or you may choose to submit another assignment which the lecturer will n o t bother to remind you about. I acknowledge reading the above & agree to be bound by terms therein. Your signature: signature:


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How YOU can do well in BIOLOGY Follow the 4A’s and you can expect A’s.

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ttitude  Attend ALL lectures, tutorials and practicals on time without fail.  Be attentive in class and revise your notes after class while the topic is still fresh in your mind. Why waste time re-reading 2-3 months later?  Do your assignments faithfully as they carry marks for the finals.  Come prepared for lessons (i.e. read up beforehand).  Read up beforehand before attending lectures so that you won’t be lost and wasted hours of your life week after week.  Why stress yourself out if you can avoid it? Do NOT count on last minute revision for tests and examinations, as it will be too late to catch up and seek help in areas where you may find confusing or unclear of.  Why panic before exams because you can’t find this or that? Keep separate files for lecture, tutorial and practical. File up the respective notes systematically so that you do not lose them along the semester.  Do you expect the lecturer/ tutor to be available all the time to answer your questions? It is YOUR responsibility to take the initiative to clear your doubts or satisfy your curiosity to understand certain scientific phenomena by reading up on the relevant topics.

Based on a true story… A professor at the National University of Singapore recounts how on one occasion a student consulted him days before the exam. Student: Prof, could you explain this page to me please? Professor: What don’t you understand about this page? Student: EVERYTHING. Professor: But I already went through this during lecture. Student: Oh, I didn’t attend most of the lectures actually. As for the next page, could you explain this page to me please? ... and this page too… and that too… Prof: I’m sorry, I can’t help you. Student: (Hmmmph, HE’S so selfish. Hey, I paid to study here!) What do YOU think?

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If the student failed, whose fault was it? Was this student clever in skipping lectures? Was it fair for the student to make demands on the lecturer’s precious time to answer his questions? How would the student have benefited himself if he looked up books and other sources of information for himself first?

 ttendance for lectures, tutorials and practicals  Lectures, tutorials and practicals carry marks that count towards your finals.   You are expected to be present at ALL lectures, tutorials and practicals.  Absence from any lesson must be accompanied by a photocopy of your medical certificate presented to your lecturer/ tutor at your next meeting.  If you know in advance that you will not be able to attend the practical for a particular week, you are expected to inform your tutor latest by the Friday before the affected week.


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ssignments  Use proper A4 foolscap for all handwritten assignments.  Write neatly and legibly in blue or black ink. Your tutor reserves the absolute right to reject your assignment and ask you to re-do the assignment should he/she consider it to be below the expected quality.  Submit your assignment on time. Late submissions may entail mark deduction or not be graded at all.

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ssessments  ALL academic tests and examinations help prepare you better for the finals.  As such, to sit for them all is not only compulsory, but beneficial. After sitting for one, you’ll just want to sit for another, and another, and another…  Absence from tests and examinations MUST be covered by a medical certificate, or will be considered to have failed the tests.


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Introduction Exercise 1 Writing of Lab Reports

W

hy should I bother writing lab repo rts in the correct way?” The Foundation Programme is designed to prepare you for undergraduate studies at UTAR which will require the writing of lab reports all years generally. At the end of your third year, you may have an opportunity to work on scientific projects which will culminate in an official scientific report. Depending on the quality of your report, the golden chance remains of publishing your report in a scientific journal. Such recognition may open doors of opportunity (e.g., strengthen application for scholarships and further studies etc.). Science professors are evaluated in most parts of the world by the papers they write.

Format of a lab report Your lab report should be preceded by a cover page which contains the following:  Name  Partner ’s name  Group  Date  Program  Unit code  Unit description  Year and semester of study  Title of lab report  Lecturer’s name


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Example:

Your lab report should contain the following sections:  Title  Objective  Apparatus, materials and methods (if your assignment is submitted online, this

step may be omitted)  Observations and/or results with discussion  Conclusion The following guidelines on report writing are those required by the actual internationallyrecognized scientific community. The text in quotation marks in the following section is taken from Warren D. Dolphin of Iowa State University. Credit has been given to the author by citing the source. This is good practice as opposed to plagiarism, in which copied material is claimed as the possession of the copyist.

1 Apparatus, materials and methods “As the name implies, the materials and methods used in the experiments should be reported in this section. The difficulty in writing this section is to provide enough detail for the reader to understand the experiment without overwhelming him or her. When procedures from a lab book or another report are followed exactly, simply cite the work, noting that details can be found in that particular source. However, it is still necessary to describe special pieces of equipment and the general theory of the assays used. This can usually be done in a short paragraph, possibly along with a drawing of the


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experimental apparatus. Generally, this section attempts to answer the following questions: 1. What materials were used? 2. How were they used? 3. Where and when was the work done? (This question is most important in field studies.)”

2 Observations and/or results with discussion Results “The results section should summarize the data from the experiments without discussi ng their implications. The data should be organized into tables, figures, graphs, photographs, and so on. But data included in a table should not be duplicated in a figure or graph. All figures and tables should have descriptive titles and should include a legend explaining any symbols, abbreviations, or special methods used. Figures and tables should be numbered separately and should be referred to in the text by number, for example:  Figure 1 shows that the activity decreased after five minutes.  The activity decreased after five minutes (fig. 1). Figures and tables should be self-explanatory; that is, the reader should be able to understand them without referring to the text. All columns and rows in tables and axes in figures should be labelled. This section of your report should concentrate on general trends and differences and not on trivial details. Many authors organize and write the results section before the rest of the report.”

2.1 Recording Qualitative Data Qualitative experiments include those that require observations of non-quantifiable data such as observations of colour, slides and whole specimens. Below are guidelines on reporting a segment of qualitative experiments. Liquid in con tainer:

Be careful to distinguish accurately among solution, suspension, emulsion etc. Often, “mixture” is a safe descriptive term to employ. It is your responsibility to look up the definitions as studied in secondary school.  

KI solution was added to the starch suspension emulsion of lipid droplets in water

A m o u n t o f l i g h t p e n et r a ti n g s o l u t i o n

Be careful to distinguish accurately among transparent, translucent and opaque. It is your responsibility to look up the definitions as studied in secondary school.


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Colour

Some descriptions of colour are unacceptable as they are ambiguous.  Light/pale brown, instead of beige  Murky/ cloudy white, instead of milky If there’s a change in colour ation, you may choose to report as follows. 

The initial blue colouration of the mixture turns green, then yellow and may finally appear brick red.

If the transition cannot be easily seen, at least state the initial and final colours. If there is no change, one must state the colour (e.g., “ it remained blue”). It is incomplete to only report “there was no colour change” wi thout at least recording the initial colour. Precipitate

One should comment on the precipitate colour and relative quantity. To do so, the mixture must be left to settle.  

Colour of precipitate - green, yellow, brick red precipitate Amount of precipitate - a little, moderate amount, abundant

Example: When describing observations involving Ben edict’s test, one should report that w hen one shakes the test tube containing Benedict’s solution and precipitate, the entire mixture will take the colour of the precipitate. This colour upon shaking is recorded and also the amount of light penetrating solution (transparent/ translucent/ opaque). 

Moderate amount of brick red precipitate suspended in solution, which bore a tinge of blue. Solution wa s opaque.”

Note: Particles cannot be regarded as precipitate. (e .g. groundnut particles in water.) 2.2 Recording Quantitative Data Quantitative experiments include those that require observations of quantifiable data such as time, quantity, weight, etc. T a b u l a ti o n a n d g r a p h i n g

There are two categories of data normally used in reporting quantitative results – raw data and processed data. Raw data refers to the readings obtained from measurements (e.g., length, weight, height, quantity, etc.). The table must be accompanied by the following features:  Informative table title  Gridlines  Columns/ rows with appropriate headings and units ( units and calculations

should not be in the table body )


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 All processed data related to and required for plotting graph must be shown in

the table. E.g. Averages, rate of yeast respiration in terms of no. of bubbles formed per minute. Precision and d ecimal places:

One must express data according to the precision afforded by the instrument. E.g., if the instrument can weigh an item as light as 0.1 g, then do not record it as ‘0.10 g’, so as to correctly reflect the precision of the instrument. Note that the decimal places in the table must be the same for the same unit of measurement, and reflect the precision of the instrument. If a measurement unit is converted to percentage or any other unit, one is not bound by the precision of the instrument. However, the recording should maintain a consistent and reasonable use of the number of decimals (e.g., avoid too many decimals ’88.8888888 %’). No te that the table and graph below feature such consistency of decimal places. Precision of processed data can be presented in the following manner: 

Averages calculated need not follow the decimal places of the raw data.

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Processed data involving summation and/ or subtraction should follow decimal places of the raw data.

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Decimals arising from processed data involving multiplication and/ or division should be reasonable (e.g., not unnecessarily long).

Sample table: Title: Mass of precipitate of standards at various concentrations of glucose solutions.

Glucose concentration (%) 4 2 1 0.5 0.1

Reading 1 0.1 8.2 5.2 2.3 0.4

Precipitate mass (g) Reading 2 Reading 3 18.6 9.3 4.5 1.8 0.3

18.4 9.0 4.8 2.1 0.4

Average 18.7 8.8 4.8 2.1 0.4

Graph

Plot a graph that will show the trend of the investigation. Include the following in the plotting of graph: 

Informative title

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x-axis : labelled, including units (independent variables)

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y-axis : labelled, including units (dependent variables)

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appropriate scale used

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points plotted


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Shape of graph can only be drawn using pencil, blue and black ink pen 

points plotted according to table of data

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best fit line/ curve

Sample graph: Average mass of precipitate of standards at various concentrations of glucose solutions 20

Ave. precipitate mass (g)

18 16 14 12 10 8 6 4 2 0 0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Concentration of glucose solution ( %)

Note: The line of the plot does not go beyond the concentrations used (no extrapolation of points). Hence, one should not extrapolate otherwise it is a claim that a certain y value is predicted for a certain concentration.

Avoid clashing headings with clashing units (e.g., headings with two different units but both have gram in their units – gram eggs vs. gram nutrients per gram plain feed) Amount of nutrients (g/ g plain feed) Mean

0.30 78.0

Mass of eggs laid in a week (g) 0.25 0.20 0.15 0.10 74.0

69.3

62.7

59. 7

0.00 58.0

2.3 What if I don’t obtain desired results? For the purpose of your UTAR lab report, if you don’t obtain the desired results, just record them as they are. By right, you should repeat it – however, you may be constrained by a limited amount of supplied solutions in the UTAR lab and time. Hence, if your repeats involve consuming more solutions, please ask your tutor first. You may put a footnote concerning the expected results. In your discussion, be sure to explain the possible reasons for the anomaly.


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3 Discussion “This sec tion should not just be a restatement of the results but should emphasize interpretation of the data, relating them to existing theory and knowledge. Speculation is appropriate, if it is so identified.” “ Suggestions for the improvement of techniques or experimental design may also be included here” . “ In writing this section, you should explain the logic that allows you to accept or reject your original hypotheses. You should also be able to suggest future experiments that might clarify areas of doubt in y our results.”

3.1 General Comments on Style 1.

All scientific names (genus and species) must be italicized. Underlining indicates italics in a typed paper.

2.

Use the metric system of measurements. Abbreviations of units are used without a following period.

3.

Be aware that the word data is plural while datum is singular. This affects the choice of a correct verb. The word species is used both as a singular and as a plural.

4.

Numbers should be written as numerals when they are greater than ten or when they are associated with measurements  6 mm or 2 g  two explanations of six factors.

When one list includes numbers over and under ten, all numbers in the list may be expressed as numerals; for example,  17 sunfish, 13 bass, and 2 trout.

Never start a sentence with numerals. Spell all numbers beginning sentences. 5.

Be sure to divide paragraphs correctly and to use starting and ending sentences that indicate the purpose of the paragraph. A report or a section of a report should not be one long paragraph.

6.

Every sentence must have a subject and a verb.

7.

Avoid using the first person, I or we, in writing. Keep your writing impersonal, in the third person. Instead of saying, " We weighed the frogs and put them in a glass jar, " write, "The frogs were weighed and put in a glass jar. "

8.

Avoid the use of slang and the overuse of contractions.


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9.

Be consistent in the use of tense throughout a paragraph--do not switch between past and present. It is best to use past tense.

10.

Be sure that pronouns refer to antecedents. For example, in the statement, "Sometimes cecropia caterpillars are in cherry trees but they are hard to find ." Does "they" refer to caterpillars or trees? After writing a report, read it over, watching especially for lack of precision and for ambiguity. Each sentence should present a clear message. The following examples illustrate lack of precision:  "The sample was incubated in mixture A minus B plus C."

Does the mixture lack both B and C or lack B and contain C?  "Protection against Carcinogenesis by Antioxidants "

The title leaves the reader wondering whether antioxidants protect from or cause cancer. The only way to prevent such errors is to read and think about what you write. Learn to reread and edit your work. Identify trends/ patterns by in words the trend shown in the graph. Remember to make reference to the values shown on the graph. Explain all the observations or trend obtained during the investigation. o

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 As temperature increases from 25 C to 50 C, rate of yeast respiration/ mean

number of bubbles formed per 3 mins. increases proportionately/ linearly from 7 to 28. In summary, the discussion should be correctly applying the theoretical concept involved in the experiment.

4 Conclusion State the general trend obtained through the investigation and provides a concise conclusion about the investigation.

5 Literature Cited This section lists all articles or books cited in your report. It is not the same as a bibliography, which simply lists references regardless of whether they were cited in the paper. The listing should be alphabetized by the last names of the authors. Different journals require different formats for citing literature. For articles:

Fox, J.W. 1988. Nest-building behavior of the catbird, Dumetella carolinensis. Journal of Ecology 47: 113-17. Lab manual version 4_201405 Biology I & Fundamentals of Cell Biology

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For Books:

Bird, W.Z. 1990. Ecological aspects of fox reproduction . Berlin: Guttenberg Press. For chapters in book s:

Smith, C.J. 1989. Basal cell carcinomas. In Histological aspects of cancer , ed. C.D. Wilfred, pp. 278-91. Boston: Medical Press. When citing references in the text, do not use footnotes; instead, refer to articles by the author's name and the date the paper was published.  

Fox in 1988 investigated the hormones on the nest-building behavior of catbirds. Hormones are known to influence the nest-building behavior of catbirds (Fox, 1988).

When citing papers that have two authors, both names must be listed. When three or more authors are involved, the Latin et al. (et alia) meaning "and others" may be used. A paper by Smith, Lynch, Merrill, and Beam published in 1989 would be cited in the text as: Smith et al. (1989) have shown that... This short form is for text use only. In the Literature Cited, all names would be listed, usually last name preceding initials.


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Introduction Exercise 2 Notes on Biological Drawings ______________________________________________________________________ Drawings are an aid to precise observations and for this reason they are an important part of laboratory work. In the practical examination, the examiner will have only your written recordings and drawings to assess you. Therefore full recordings and neatly labelled drawings are of great importance. Keep the following points in mind when making drawings: 1. Use a s h a r p , p o i n t e d H B / 2 B p e n c i l . 2. Drawings should be as l a r g e as possible and made to f i t i n t o t h e s p a c e a v a i l ab l e . 3. Attention must be given to the g e n e r al s h a p e an d p r o p o r t i o n of the specimen. First consider what you want to show. Then plan your drawing so that various parts are in proportion and fit on the page. Small marks indicating the length and breadth of the drawing are of great help in planning, and a faint outline can be rapidly drawn to show the relative positions of the parts. 4. The final outline should be drawn with c l e a n f i r m l i n e s (not sketchy broken lines). Details should be put in clearly with a sharp pencil. If important details are too small to be shown in proportion, they can be shown in an enlarged drawing on the side. 5. Drawings should be accurate records of your observations. The biologist is not expected to be an artist, but to become, in some degree, a draughtsman. Clear and accurate line drawings are needed. 6. S h a d i n g a n d c o l o u r i n g s h o u l d b e av o i d e d . It should be possible to make the drawing perfectly clear by the judicious use of thick and thin pencil lines and careful cross-hatching. Get into the habit of making your drawings large and clear. 7. As important as the drawing is the l a b e l l i n g . This should be done neatly i n p e n c i l and the l e t t er s p r i n t e d . Each label should be connected to the appropriate part of the drawing by a clear guideline without arrowheads. Do not label too close to the drawings, and never write on the drawing itself. Always make sure that each drawing is fully labelled before you leave it. Guidelines to the labels must be drawn with pencil and ruler and never crossing one another. 8. Each drawing must always have a title. The title should specify whether it is a transverse section, s longitudinal section, whole mount, etc. 9. M a g n i f i c a t i o n o f d r aw i n g can be stated if necessary. Calculate using the formula, Magnification = size of drawing___ Actual size of specimen 10. P l a n d i a g r a m s of microscopic sections s h o u l d n o t i n c l u d e a n y c el l s t r u c t u r e . They are outline drawings showing relative amounts and distribution of various tissues. 11. In making h i g h - p o w e r d e t ai l e d d r a w i n g s , repeated features need not all be drawn but only a s m a l l r e p r e s en t a t i v e portion showing a few large accurate cells (3 or 4 adjacent cells) of each type must be indicated. 12. It is sometimes appropriate, particularly when drawing live specimens, to make succinct notes to the labels. These are called a n n o t a t e d d r a w i n g s , which are particularly valuable as they combine a record of structure with functional observations. Annotations must be beneath the labels.


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Example of an annotated drawing Title: Detailed drawing of a Hydrilla leaf cell Magnification power: 10x. 40x Chloroplast (moves in cytoplasm, site of photosynthesis; stained brownish with iodine solution) Cytoplasm (granulated; found at periphery of cell; stained light yellow with iodine solution)

Cell vacuole (contains salt and sugar solution; bound by membrane tonoplast; colourless)

Cell wall (made of cellulose, stained yellow)

Nucleus (doubled membrane organelle embedded in the cytoplasm; control center of the cell; stained orange brown with iodine solution.)

Note: The distinction between a plan diagram and a drawing: A drawing is an e x a c t a n d a c c u r a t e representation of an object, unlike a diagram which is a s i m p l i f i e d o u t l i n e . Warning: M e m o r i z e d t ex t b o o k d r a w i n g s o r d i a g r a m s , b e ar i n g l i t t l e l i k e n e s s t o t h e s p e c i m e n s o r o b s e r v a t i o n s w i l l n o t b e a w a r d ed m a r k s .


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A tissue map or plan diagram refers to a generalized outline of the tissue regions of a specimen. If such is requested, no detailed drawings of individual cells are required. The illustration below is a cross section of a leaf. The items in the square box are detailed drawings of cells whereas those of the vascular bundle reflect a tissue map or plan diagram.

Is the picture below a drawing or diagram?


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General Marking Scheme It is your responsibility to read the guidelines in the introduction to this lab manual. In the past many clever students lost marks simply because they failed to look up instructions already provided. Would you like to be as clever as them? This is an example of what a marking scheme may look like for: General instructions for students:  One to two slides may be drawn [please consult your lecturer]. If two drawings are to be done, each student is allowed only 30 mins per slide for drawings to be done in the 1st half.  The second half of the practical wi ll be used to assess students’ microscope and / or identification skills on one type of slide. Students are required to identify at least any three structures which they choose.  For online submissions, students are required to upload pictures of their drawings (more instructions on WBLE).  In order not to lose marks unnecessarily, please ensure that you comply with the instructions on writing lab reports at the beginning of this manual, including your particulars (e.g., name, group, etc.) as stated in the instructions.


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Checklist for biological drawings This section may comprise 10 marks out of 20 marks. Any mistake will result in subtraction of 1 mark.

Items 1. Appropriate & comprehensive (detailed and complete) title 2. Written indication of objective used (multiplied by __x ocular lens) 3. Requirement for detailed drawing or plan diagram instruction adhered to

Please read full version for your personal success Remarks from Lab Introduction “The title should specify whether it is a transverse section, s longitudinal section, whole mount, etc.”

“A tissue map or p l a n d i a g r a m refers to a generalized outline of the tissue regions of a specimen. If such is requested, no detailed drawings of individual cells are required.”

4. Correctly labelled items 5. Annotations if requested

6. Drawing is as what is seen under microscope (i.e., not textbook-perfect picture) 7. Magnification if requested

“…annotated drawings, which are particularly valuable as they combine a record of structure with functional observations.” “M emorized textbook drawings or diagrams, bearing little likeness to the specimens or observations will not be awarded marks .” “ M a g n i f i c a t i o n = __ _s ize o f d r aw i n g

8. Method of calculation if requested 9. Early submission up to tutor to delete marks according to lateness 10. Overall impression of drawings (e.g., neatness) a. Satisfactory b. Quite good c. Good d. Very good e. Excellent


”

Actual size of specimen

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General guidelines for biological photo(s)

Items 1. Title - Appropriate & comprehensive (detailed and complete) 2. Magnification - Written indication of objective used (multiplied by __x ocular lens) 3. Label - Correctly labelled items

please read full version for your personal success Remarks from lab introduction Specify whether it is a transverse section/cross section, longitudinal section, whole mount, etc.”

10x. 4x or 40x / 10x. 10x or 100x

Label box should not overlap the photo. Label line should point to the specific area. Label line should not overlap each other.

4. Annotations if requested

Include the structure and function Annotation should be beneath the label.

5. Images and tidiness

Photos taken must be clear.

6. Late submission - up to tutor to delete marks according to lateness

General instructions for lecturers: Two areas of assessment: 1) Biological drawings 1st hour allocated for drawing Kindly instruct students to draw slides according to availability (as supplied). The manual provides options to lecturers to request 1 detailed drawing or 2 plan diagrams. Note: if students are to submit drawing on the spot, 1 drawing should suffice. Recommended time allocation: Briefing (5-10 mins) + Slide familiarisation & reading (30 mins) + drawing (30 min if 1 detailed drawing; longer if 2 plan diagrams required) 2) On-site assessment 2nd hour allocated for on-site assessment. On-the-spot identification of 3 structures (10 marks); 2-3 students assessed within 3 minutes. Drawing (10 marks) Recommended time allocation: On-site assessment (50 min).

General instructions for lab reports:  Prepare your answers on your own A4 sheets of paper.  You are not required to re-write questions.


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Practical 1 (FHSB 1214 Biology I & FHSC 1214 Cell Biology) Identification of Biochemical in Their Pure Form ______________________________________________________________________ Objective: To identify the components of the solution in its pure form with various food tests and state the justifications. Important notice: Any heating that has to be done in the following tests should be carried out in a water bath at 95 oC. Direct heating of test-tubes should not be taken place. Apparatus & Equipments: Test tubes Water bath, 95 oC Test tube holder Materials:  Iodine  1 M hydrochloric acid  Sudan III  Starch solution  Corn oil  Egg albumin  1% copper sulphate solution  DCPIP (dichlorophenolindophenol) solution  Ascorbic acid (or vitamin C tablet, or lemon juice)

Test tube rack Spatula

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  

1% sucrose solution (Analar sucrose must be used to avoid contamination with a reducing sugar Benedict’s reagent 1 M sodium hydroxide (or potassium hydroxide or sodium hydrogen carbonate) Millon’s reagent 1% glucose solution Absolute ethanol

Introduction The nutrients in the food you eat supply your body with energy for growth and repair. These principle substances include carbohydrates, proteins, fats, minerals and vitamins. We can test for the presence of these important compounds in food by using chemical reagents that react in predictable ways in the presence of these nutrients. Please refer to the notes given above on:  How to record qualitative data. (Marks will be awarded based on proper recording.)  What to do if you don’t obtain the desired results.


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Flowchart Students will be allowed to proceed with the experiment only if they have come into the laboratory with a flowchart of the day’s experiment. Procedures: The following tests are to be done in pairs unless otherwise specified.

Part 1: Identification of Carbohydrates Test for reducing su gars

The reducing sugars include all monosaccharide, such as glucose and fructose, and some disaccharides, such as maltose and lactose, use 0.1 – 1% sugar solutions. Common tests for reducing sugars include Benedict’s test (described below) and Fehling’s test (not done here). See ‘basis of test’ below for explanation of the following reaction:

Benedict’s test for reducing sugars: Procedure*

Basis of test

Observation

Benedict’s solution contains copper sulphate. Reducing sugars reduce soluble alkaline blue copper sulphate containing copper (II) ions, Cu2+ to insoluble red-brown copper oxide containing copper (I). The latter is seen as a precipitate.

[Note: report after shaking and after contents settle down; see introduction pg. 9]

R e d u c i n g s u g a r t es t

Add 2 cm3 of any o n e solution of the reducing sugar provided to testtube. Add an equal volume of Benedict’s solution. Using a test-tube holder, shake and heat at a high temperature for one minute (a water bath is provided), shaking continuously to minimize spitting.

*: Please do NO T remove measuring cylinder or any other item from the stations provided. Observe and report characteristics of tube contents before and after precipitate settles to bottom of tube, taking note of liquid, colour and precipitate. T e s t f o r n o n - r ed u c i n g s u g a r s

The most common non-reducing sugar is sucrose, a disaccharide. If reducing sugars have been shown to be absent (negative result in above test), a brick-red precipitate in Lab manual version 4_201405 Biology I & Fundamentals of Cell Biology

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the test below indicates the presence of a non-reducing sugar. If reducing sugars have been shown to be present, a heavier precipitate will be observed in the following test than with the reducing test if non-reducing sugar is also present. The proper procedure for testing for an unknown carbohydrate sample for non-reducing sugars involves: 

First test for reducing sugar s: Benedict’s test on the unknown fresh sample Why is this step necessary? What results will one get which will cause this step to be called a ‘negative test’?



Second test for reducing sugars: Benedict’s test on the acid -hydrolysed unknown sample What results will one get which will cause this step to be called a ‘positive test’?

Procedure*

Basis of test

Observation

Non-reducing sugar test

Add 2 cm3 of fresh A polysaccharide or sucrose solution to a test disaccharide can be tube. Add 1 cm 3 O.1 M hydrolyzed to smaller hydrochloric acid. Using a component constituents test-tube holder, heat at a by boiling with O.1 M high temperature for one hydrochloric acid. minute. Sucrose is hydrolyzed to Carefully neutralize with glucose and fructose, 3 equal volume (1 cm ) of 1 both of which are M sodium hydroxide or reducing sugars and give sodium hydrogen the reducing sugar result carbonate or potassium with the Benedict’s test. hydroxide. (Care is required because effervescence occurs.) Finally, add an equal volume (4 cm 3 ) of Benedict’s solution to the acid-hydrolysed sugar solution. Using a test-tube holder, shake continuously to minimize spitting when heating at a high temperature for one minute (a water bath is provided).


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Addit ional Information

The mixture is likely to bump violently during heating and extra care should therefore be taken. The test is semi-quantitative , that is, a rough estimation of the amount of reducing sugar present will be possible. The final precipitate will appear green to yellow to orange to red-brown with increasing amounts to reducing sugar. The initial yellow colour blends with the blue of the copper sulphate solution to give the green colouration. Is the precipitate that of reducing sugar or copper oxide? *: Please do NOT remove measuring cylinder or any other item from the stations provided. Observe and report characteristics of tube contents before and after precipitate settles to bottom of tube, taking note of liquid, colour and precipitate.

Test for starch

Starch is only slightly soluble in water, in which it forms a colloidal suspension. It can be tested as a mainly solid in suspension. Procedure*

Basis of test

Observation

Iodin e test

***Note: The starch prepared for you is already cooked starch.

A polyiodide complex is formed with starch.

Add a few drops of 1% cooked starch solution on a white tile. Add a few drops of I2/KI solution (iodine). Be sure to mix them together on the tile with an object such as your pen cover. *: Please do NO T remove measuring cylinder or any other item from the stations provided.


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Part 2: Identification of Lipids Lipids include oils (such as corn oil and olive oil), fats and waxes. Procedure*

Basis of test

Observation

Sudan III

Sudan lll is a red dye. Add 2 cm3 of oil to 2 cm 3 of distilled water in a testtube. Add a few drops of Sudan III and shake.

Fat globules are stained red and are less dense than water.

E m u l s i o n t es t

Add 2 cm3 fat or oil to a Test-tube containing 2 cm 3 of absolute ethanol. Dissolve the lipid by shaking vigorously. Add an 4 cm3 volume of cold (or tap) water.

Lipids are immiscible with water. Adding water to a solution of the lipid in alcohol results in an emulsion if tiny lipid droplets in the water which reflect light and give a white, translucent appearance.

[***Note: report after shaking and after contents settle down]

*: Please do NO T remove measuring cylinder or any other item from the stations provided.


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Part 3: Identification of Proteins A suitable protein for these tests is egg albumen. Procedure*

Basis of test

Observation

Millon’s Test

Add 2 cm3 protein (albumin) solution or suspension to a test-tube. Add 1 cm3 Millon’s reagent. Using a test-tube holder, heat at a high temperature for one minute (a water bath is provided). Millon’s reagent is poisonous: be extremely careful!

Millon’s reagent contains mercury acidified with nitric acid, giving mercury (II) nitrate and nitrite. The amino acid tyrosine contains a phenol group which reacts to give a red mercury (II) complex. This is a reaction given by all phenolics and is not specific for proteins. Protein usually coagulates on boiling. Thus appearing solid. The only common protein lacking tyrosine likely to be used is gelatin.

Biuret Test

Add 2 cm3 (albumin) protein solution to a test tube. Add an equal volume of 5% sodium hydroxide (or potassium hydroxide) solution and mix. Add 2 drops of 1% copper sulphate solution and mix. No heating is required.

A test for peptide bonds. In the presence of dilute copper sulphate in alkaline solution, nitrogen atoms in the peptide chain form a purple complex with copper (II) ions, Cu2+. Biuret is a compound derived from urea which also contains the – CONH – group and gives a positive result.



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Part 4: Identification of Vitamin C (ascorbic acid) ***Note: If more than 5 drops of ascorbic acid are required to turn DCPIP to colourless, please dilute the latter significantly. This test can be conducted on a quantitative basis if required, in which case the volumes given below must be measured accurately. A suitable source of vitamin C is a 50/50 mix of fresh orange or lemon juice with distilled water. Vitamin C tablets may also be purchased. Procedure*

Basis of test

Observation

DCPIP test

Using 0.1% ascorbic acid solution as a standard. Add 1 cm3 of DCPIP solution to a test-tube.

DCPIP is a blue dye which is reduced to a colourless compound by ascorbic acid, a strong reducing agent.

***Add the 0.1% ascorbic acid to the DCPIP drop by drop until it becomes approximately colourless (or by stirring gently if you’re provided with a syringe needle/ glass rod). Note the no. of drop(s) of ascorbic acid solution used. Addit ional Information

Shaking the solution would result in oxidation of the ascorbic acid by oxygen in the air. The effects of shaking and of boiling could be investigated.



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Assignments Please check with your tutor which option is required for you. Option 1: (please refer to WBLE/ Turnitin for instructions which may incorporate other options below) Option 2: Skills Based Assessment: Tabulation of qualitative data 1. Tabulate your observations above for each biochemical food test executed, according to the guidelines provide in the introduction on writing lab reports. Note: The table in the lab manual for this task is not presented correctly. 2. Wrong results are alright for this experiment. 3. No need to write procedure, basis of test, discussion or conclusion. 4. You may choose to construct one or more tables. 5. For tests involving carbohydrates, observe and report characteristics of tube contents before and after precipitate settles to bottom of tube, taking note of liquid, colour and precipitate as above. o Liquid mixture, solution, suspension, emulsion?   transparent, translucent, opaque? o Colour state initial and final colours?  o Precipitate (if any) colour of precipitate?   amount of precipitate? Better understanding of terms:  Mixture  Solution  Suspension

Option 3: Skills Based Assessment: Critical thinking/ Discussion 1. How could you determine the concentration of ascorbic acid in an unknown sample? 2. You are provided with three sugar solutions. First one contains glucose, second one is a mixture of glucose and sucrose, and lastly is sucrose solution. (a) How could you identify each solution? (b) Supposing that the apparatus were available, and time permitted, briefly discuss any further experiments you could perform to confirm your results. 3. After carrying out Benedict’s test, a student concludes that the obtained positive results prove that glucose is present. True or false? Provide a reason. 4. After carrying out Benedict’s test, a student identifies the coloured precipitate as reducing sugar. True or false? Provide a reason. 5. A student pours Benedict’s solution into a tube containing a carbohydrate. No colour change is obtained. The student concludes that the carbohydrate is not a reducing sugar. True or false? Provide a reason. Lab manual version 4_201405 Biology I & Fundamentals of Cell Biology

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6. A student adds acid to a solution of sucrose followed by neutralization and, finally, Benedict’s test. A negative (instead of positive) result is obtained. Explain why. 7. Why does sucrose yield positive results after carrying out the non-reducing sugar test? What are the components of sucrose?

For those who have done Sem 3 3. How would you make 100 cm 3 of a 10% glucose solution? 4. Starting with stock solutions of 10% glucose and 2% sucrose how would you make 100 cm 3 of a mixture of final concentration 1% sucrose and 1% glucose?


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Practical 2 (FHSB 1214 Biology I & FHSC 1214 Cell Biology) Investigation of Action of Saliva and 3 M Hydrochloric Acid in Two Carbohydrate Solutions ______________________________________________________________________ Objective: Students are expected to state the objective of this experiment . Apparatus & Equipments: Boiling tubes Pipette filler Water bath, 37-40 oC Beaker

Metal test tube racks Graduated glass pipette, 10ml Water bath, ~90-95 oC Pasteur pipette

Materials: Carbohydrate solution A Carbohydrate solution B Benedict’s solution 3 M Hydrochloric acid 3 M Sodium hydroxide (or potassium hydroxide) Flowchart Students will be allowed to proceed with the experiment only if they have come into the laboratory with a flowchart of the day’s experiment. Procedures: This experiment is to be done in pairs. To avoid congestion, each pair should collect the following before beginning the experiment:  8 ml NaOH  16 ml Benedict’s Solution  2ml Solution A  42ml Solution B  2ml HCl  1 pipette and 1 rubber teat (to be washed with distilled water each time before reuse)  5 ml measuring cylinder (to be washed with distilled water each time before reuse)  Metal test tube racks (not wooden) Overview Please see tables 1 & 2 on the next page to get a rough idea of what is required in the experiment. Can you identify in the instructions that follow, how the tubes are to be placed under various temperatures and time periods? Carry out your investigation as follows. 1.

Prepare two test tubes containing 2 ml solution A and 2 ml solution B respectively. Add 2 ml Benedict’s solution to each test tube. Heat both tu bes together in the hotter (~90-95 oC) water bath for two minutes. Record the results in table 1.

2.

Pipette 10 ml solution B into each of four test-tubes and, label the tubes 1, 2, 3 and 4 respectively with labelling paper (or masking tape) near mouth of tube. Write the initials of your group name or individuals.


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3.

Place tubes 1 and 2 in a water bath of ~37 o, and tubes 3 and 4 in a water bath of ~95oC (It doesn’t matter how long you put it in at this stage as no saliv a or HCl have been added yet).

4.

Salivate into a separate test-tube till it reaches a height of about 1 cm - 1.5 cm. Dilute the saliva with an approximately equal volume of distilled water.

5.

Ensure that the following two steps (5 and 6) adding of saliva or HCL into the respective tubes (mentioned in the next sentence and below) is done approximately at the same time. (Why is this necessary?)

Note: for the following, please ensure that the respective tube into which saliva is going to be dropped does NOT leave the water bath (especially 95 oC) for more than 30 seconds! (Why is this necessary?) 6.

Use a 5 ml measuring cylinder to measure out 2 ml of the diluted saliva prepared in (3) and pipette 1 ml each into tubes 1 and 4. Shake the contents of the tubes well to ensure thorough mixing.

7.

Use a measuring cylinder to measure out 2 ml HCl and pipette 1 ml each into tubes 2 (already in water bath of ~37 oC) and 3. Place tubes 3 and 4 in a water bath set at 95 oC. Let tubes 1, 2 (already in water bath of ~37 oC), 3 & 4 (recently in water bath of ~95oC) incubate at their respective temperatures (see Table 2) for 35 minutes from this moment.

8.

Label 4 more new tubes (either test tubes or boiling tubes) as follows: 1’, 2’, 3’ and 4’. After 5 minutes of incubation of tubes labelled 1 to 4 prepared previously, pou r out about one-third of the total volume of the contents from all these tubes into the respective newly labelled test tubes (e.g., 1 into 1’, 2 into 2’ etc.). Ensure that the volume in each of the tubes 1’ -4’ is approximately the same (why is this important?). Straightaway, place back the original tubes (labelled 1-4) back into the respective temperatures of incubation.

9.

Neutralize the acid in each of tube labelled 2’ and 3’ with 2ml of sodium hydroxide (or potassium hydroxide) (each). Shake each tube (2’ and 3’) to ensure uniform mixing.

10.

Remove 1ml of the solution from each tube (1’ to 4’) into new tubes and label appropriately as you wish as long as you don’t get confused . To carry out Benedict’s test, add an equal volume of Benedict’s solution (1 ml) f or each tube. Using a test-tube holder, shake and heat at a high temperature for one minute (use the hotter water bath provided), shaking continuously to minimize spitting. Record your observations in table 2.

11.

Wash the test tubes 1’ to 4’. After 35 minutes of incubating tubes 1 to 4, pour out about one-third of the total volume from test samples from all the tubes into the respective tubes labelled 1’ - 4’.

12.

Neutralize the acid in each test tube labelled 2’ and 3’ with 1ml of sodium hydroxide (or potassium hydroxide). (Why is neutralization necessary?) Remove 1ml of solution from each tube 1’ to 4’ and carry out Benedict’s test with an equal


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volume of Benedict’s solution (1 ml) for each tube. Remember to heat your sample (please see previous. Record your observations in table 2. 13.

Add a few drops of fresh solution A and B separately spaced on a white tile. On each solution, add 1-2 drops of I 2/KI solution (iodine). Be sure to mix them together on the tile with an object such as your pen cover. Record your observations in the table 1.

Note: no penalization for unexpected results. Please refer to Practical 1 Exercise 1 “Writing lab report”.

Table 1: (title) Observations

Conclusions

Benedict’s test: Solution A Iodine test: Benedict’s test: Solution B Iodine test:

Table 2: (title) Benedict’s Test—Colour Observation Tube

Contents

Temp (°C)

1

10 ml solution B 1 ml saliva

37

2

10 ml solution B 1 ml 3 M HCl

37

3

10 ml solution B 1 ml 3 M HCl

95

4

10 ml solution B 1 ml saliva

95


After 5th min (from tubes 1 – 4 into 1’ – 4’)

After 35th min (from tubes 1 – 4 into 1’ – 4’)

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Guidelines Observations For Benedict’s test and Iodine tests , please follow lab manual guidelines for students on writing lab report on the following: o Liquid mixture, solution, suspension, emulsion?   transparent, translucent, opaque? o Colour  state initial and final colours? o Precipitate colour of precipitate?  amount of precipitate?  Conclusions  Absence/presence of what type of carbohydrate?

Results and Discussions: 1. The results and discussion sections of your report should not exceed 2 pages. 2. Ensure that the guidelines for constructing tables and recording results for this experiment are adhered to (see introduction). 3. If you’re required to wri te a discussion straight-to-the-point, follow the numbering below (please check with your lab tutor). If your report is full-length, write your discussion in prose form (please check with your lab tutor). Theory to apply: Refer to relevant information from lecture topics on biological molecules and enzymes. Discussion should contain: 1) Name of enzyme involved 2) Specific action(s) of enzyme involved 3) 4) Effect of HCl on substances (e.g., Solution B) 5) Effect of temperature on substances (e.g., Solution B, saliva content) 6) Product: a. Identification (make suggestion(s)/ educated guesses) b. Structure (e.g., chemical classification etc.) 7) Basis of test() used 8) Which carbohydrate is more complex, A or B? Give a reason. 9) Conclusion: Summary of results


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Practical 3 (FHSB 1214 Biology I & FHSC 1214 Cell Biology) Investigation of the effects of catalase concentration on hydrogen peroxide

Objective: To investigate the effects of different catalase concentration on the decomposition of hydrogen peroxide. Apparatus and Materials: 5 test or boiling tubes Scalpel/ pen knife 3 1 beaker (500cm ) White tile 3 1 beaker (250cm ) Mortar and Pestle 1 Retort Stand (optional) Weighing boat 1 rubber bung with delivery tube 4 filter funnel and filter paper (optional) 4 test tubes or plastic vials (if provided) Potato 1% hydrogen peroxide solution Hand-held pipette * *C a u t i o n : Hydrogen peroxide is form ed continu ously as a by-produ ct of c h e m i c a l r e a c t i o n s i n l i v i n g c e l l s ; i t i s a v er y t o x i c ( p o i s o n o u s ) s u b s t a n c e .

Note to lecturer: This experiment may be done together with Experiment 2 if the lab session is 3 h long. Introductory instructions:  You may perform this experiment in groups of 3-5. Introduction: Enzymes are proteinaceous molecules that speed up chemical reactions within living systems. In this experiment, the effect of catalase on hydrogen peroxide is investigated. Catalase is an enzyme present in the cells of plants, animals and aerobic (oxygen requiring) bacteria. It promotes the conversion of hydrogen peroxide, a powerful and potentially harmful oxidizing agent, to water and molecular oxygen. 2H2O2 + catalase → 2H 2O + O2 Warning: H2O2 is corrosive. For the person handling, please wear gloves. Flowchart Students will be allowed to proceed with the experiment only if they have come into the laboratory with a flowchart of the day’s experiment. Procedures: 1. Optional: Set up an electric water bath at 37 oC. (If this is not provided, it’s ok.) 2. Depending on the size of the rubber bung holding the delivery tube, select either one boiling or test tube and label it as tube A. 3. From the potato sample given, cut (with a pen knife/ knife/ scalpel) and weigh 5g of potato using a weighing boat so as not to dirty the weighing balance.


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4. Cut the potato samples into smaller pieces (the smaller the easier for you to mash) and mash the potato sample using the mortar and pestle. Note: don’t spend too much time on this – it doesn’t have to be KFC mashed potato quality! Add 6 cm 3 of distilled water to the potato samples after the mashing process. 5. You can do two things: (i) separate the solid mashed potato from the liquid in any way you choose and pouring the liquid into a test tube; or (ii) by filtering the mashed potato sample (with filter paper and funnel) and collect the filtrate in a test tube or plastic vial (if provided) [using filter paper and funnel is more time-consuming]. 6. Fill an empty test tube with tap water (see picture below). 7. Add 5cm 3 of hydrogen peroxide into Tube A using the hand-held pipette provided. 8. Draw 1cm3 of the filtrate from the mashed potato samples and add to Tube A. Immediately close the test tubes with the rubber bung. Seal the end of the delivery tube furthest away from the bung with parafilm. 9. Set up the apparatus as shown below (if retort stand is provided; if not just use each other’s hands). Note: You need neither the water bath n o r retort stand.

10. Remove the parafilm and immediately immerse the tube in the water bath quickly (use a beaker for this and pour into it water from an electric water bath) and start your watch. Count the number of gas bubbles produced for 2 minutes and record it. After you finish, return the water you took back to the electric water bath. [Note: water can maintain the heat in it for quite some time.] 11. Optional, depends on time available: To get a 2 nd measurement, dispose the contents of tube A. Repeat step 7 to 10. After you finish, return the water you took back to the electric water bath. 12. Repeat Step 2 to 11 but with 10g of potato, then 15g and finally 20g (optional, depends on time available).


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13. Record the data in table 1. Your class is to record their data on the whiteboard. Calculate the averages in order to plot graphs. Table 1: (title)

st

5g 2nd

(What heading should you write here?) 10g 15g *20g rd st nd rd st nd rd st *3 1 2 *3 1 2 *3 *1 *2nd *3rd

Number of 1 Attempt Number of gas bubbles produced *Optional, depends on time available.

After the experiment Please dispose of potato pieces, masking tape, parafilm etc. into the dustbins provided. Please clean the sink, removing any potato pieces, masking tape, parafilm etc. Assignments Please check with your tutor which option is required for you. Option 1: (please refer to WBLE/ Turnitin for instructions which may incorporate other options below) Option 2: Skills-Based Assessment: Tabulation of quantitative data (Table 1) Option 3: Skills-Based Assessment: Graphing of quantitative data Present your graph (pasted from Excel) of the average number of bubbles produced against potato samples used. Use a best fit curve. To get full marks, please observe the guidelines given on pp6-7 as well. No need to write procedure, draw table, write a discussion or conclusion. Option 4A: Skills-Based Assessment: Discussion Data provided to students to discuss Write your discussion in prose form and without numbering. Excluding your cover page, your discussion and conclusion should NOT exceed ONE A4 page of Word document (standard/ default size). Anything in excess will NOT be graded. • Font Arial, size 11. • Margins: 1 inch from top, bottom, left and right (no need to change if you’re using the standard/ default size when MS Word opens). • Theory to apply: Refer to relevant information from lecture topics which may or may not have been covered yet. *Option 4B: Skills-Based Assessment: Discussion Students use own data to discuss From the data you have collected in the practical, account fully for the results which you have obtained. Discuss any anomalous data/ results that you might have. Explain the trend or pattern of the graph.


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Practical 3 Experiment 2 (FHSC 1214 Cell Biology ONLY) Synthesis of Starch Using an Enzyme Extracted from a Potato Tuber ______________________________________________________________________ Objective: To find out which compounds the enzyme in the potato can act on to produce starch (investigate three possible substrates: glucose, maltose and glucose-1-phosphate). Apparatus and Materials: Centrifuge and centrifuge tubes Test tube rack Pestle and mortar Knife Labelling paper (or masking tape) Test tubes Pipette White tile

Glucose-1-phosphate (2%) Glucose solution (2%) Maltose solution (2%) Iodine solution Potato tuber

Procedures: Introductory instructions:  Create a flowchart before you enter the lab in order to understand the steps in this experiment. Show this to your tutor before starting the experiment.  You may perform this experiment in pairs.  Take 5 ml iodine only when ready to begin the reaction.  Groups may have to take turns to centrifuge, depending on the number of groups and holders in the centrifuge.  NOTE: After carrying out steps 1 to 2, proceed to Experiment 2. Return to Experiment 1 only during the waiting periods of Experiment 2. A. Extracting the enzyme from potato tissue 1. Peel a medium-sized half potato. Cut half of it into small cubes on a white tile (the smaller the easier for you to grind). Grind a few pieces of potato cubes in a pestle and mortar with 20cm 3 of water. 2. Separate the aqueous part of the extract from the solid as best as possible. You can do this by pouring it out while restraining the solids with your fingers or an appropriate instrument. Divide the aqueous part of the extract into two equal portions and pour them into two centrifuge tubes. As far as possible, avoid letting sand and solid matter to get into the tubes. 3. Spin the extracts in a centrifuge for ten minutes at 5000 rpm so that the starch, cell walls and other solid matter will settle at the bottom of the centrifuge tubes. The starch-free liquid above the deposit, or supernatant, should contain the enzyme. 4. Using a teat pipette, carefully, without disturbing the deposit beneath, withdraw as much the clear enzyme solution as possible from the centrifuge tube. 5. To check whether this enzyme solution is starch-free, transfer a few drops of it into a test tube and add 2 drops of iodine solution onto it. If a blue colour appears, then the potato extract would need to be centrifuged again.


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B. Attempting starch synthesis 6. Label three clean test tubes G, M and G1P, respectively. Use a separate teat pipette and measuring cylinder in each case to place 3 cm 3 of glucose solution in the G tube, 3 cm 3 of maltose solution in the M tube, and 3cm 3 of glucose-1-phosphate solution in the G1P tube. 7. To synthesise starch, pour 2 cm 3 of the enzyme solution (the liquid or supernatant you obtained after centrifuging above) into the substrate tube (G, M and G1P), mix well and note the time. 8. For each substrate, place 15 discrete drops of iodine solutions on clearly labelled piece of white tile. 9. After one minute of the reaction use a teat pipette to place one drop of enzymesubstrate solution onto one existing drop of iodine solution on the white tile. Stir with a suitable object (e.g. woodsplint or tooth pick) and record the colour produced. Repeat at intervals of 1 minute over 15 minutes, all the three tubes simultaneously. Assignments Please check with your tutor which option is required for you. Option 1: (please refer to WBLE/ Turnitin for instructions which may incorporate other options below) Option 2: Skills-Based Assessment: Discussion Discuss the following questions: 1. Draw the structural formula of the substrates. What features of the starchsynthesizing substrate molecule might have been recognized by the starchsynthesizing enzyme? 2. The synthesis of polymers such as starch requires metabolic energy. What was the energy source in the successful reaction? 3. The enzyme isolated from potatoes is known as starch phosphorylase. In the intact potato tuber it is also used to break down starch. How did conditions in the test tube favor starch synthesis? In what circumstances does the enzyme bring about starch synthesis in a potato? 4. In plant leaves, starch accumulates in chloroplasts. The synthesis of starch requires ATP. Where do you think this ATP comes from?


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Practical 4 (FHSB 1214 Biology I & FHSC 1214 Cell Biology) Investigation of the Enzymatic Effects of Materials on Hydrogen Peroxide Solution

Objective: To investigates the enzymatic effect of various materials in the hydrogen peroxide solution. Apparatus & Equipment: Beaker Test tubes o Either: water bath (95 C) or Bunsen burner Materials: Fresh Liver Manganese dioxide Wood splints

Potato cubes Hydrogen peroxide**

* *C a u t i o n : Hydrogen peroxide is form ed continu ously as a by-prod uct of chemical reactions in living cells; it is a very toxic (poisonou s) substance.

Flowchart Students will be allowed to proceed with the experiment only if they have come into the laboratory with a flowchart of the day’s experiment. Procedures: Create a flowchart before you enter the lab in order to understand the steps in this experiment. Show this to your tutor before starting the experiment. Wear gloves when handling liver tissue, so as not to be contaminated by any pathogen associated with the liver tissue used. Please stick to using one pair of gloves per person to prevent wastage. [Note: using boiling tubes may provide better results.] 1. Label six fresh empty test or boiling tubes 1, 2, 3, 4, 5, 6 and stand them in a rack. 2. Using a razor blade, cut the provided liver into several pieces of roughly 0.8 cm x 0.8 cm x 0.5 cm. 3. Place one piece of liver into tube 1. 4. Boil 100 cm 3 of water in a beaker. (If you’re using a water bath set at 95 oC, this step is not necessary). 5. Place the second piece of liver into the bottom of tube 2. Using a wooden splint, gently spread the liver, without mashing it, over as wide an area as possible of the bottom of the test or boiling tube. Place tube 2 in the boiling or water bath (95 oC) for about five minutes. 6. Using the weighing balance, measure out two 0.5 g portions of manganese dioxide powder each onto a weighing boat. Pour each portion into tube 5 and tube 6. Lab manual version 4_201405 Biology I & Fundamentals of Cell Biology

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7. Put tube 6 in the beaker of boiling water or water bath (95 oC) for five minutes. 8. After five minutes let cool tube 2 and 6. 9. *Now put the third piece of liver into test or boiling tube 3. With the wooden stick provided, mash it gently into a pulp. 10. Now put the third piece of liver onto a white tile. With a mortar and pestle, mash it gently into a pulp. Scoop the pulp into tube 3. 11. Cut potato cubes of roughly 0.8 cm x 0.8 cm x 0.5 cm. Place one cube into a tube 4. 12. Prepare another six fresh empty test or boiling tubes and stand them in a rack. Put 5 cm3 of hydrogen peroxide into each of them. 13. Next, quickly add hydrogen peroxide into the test or boiling tubes 1, 2, 3, 4, 5, and 6. If needed, you may push down some materials with one end of the wood splints provided. **Step 12 and 13 are to be done quickly. 14. Using the parafilm provided, stretch it quickly seal the mouth of the test or boiling tubes by stretching the film over it. In order to prevent the parafilm from being displaced if a lot of gas is produced, secure the parafilm covering the side of the test or boiling tube with masking tape. 15. Leave for 20 minutes or till when you see quite a lot of gas being produced in some test or boiling tubes as evidenced by the bulging of parafilm from the test or boiling tube mouths. 16. Once enough gas has accumulated in some test or boiling tubes, insert a glowing splint (flame extinguished but glow remains) into each tube one at a time by just penetrating the parafilm with it. You may use the same splint. Why is it important to test each test or boiling tube at least without too much difference in the duration of sealing among the tubes? 17. Record all your observations in the table. Record your observations on each tube immediately after the reaction has started. [Note: be sure to use the following terms correctly: glowing splint glowed brighter, flame rekindled, effervescence (bubbles) observed, reference to sound, etc.]


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Table 1: (title) Test Tube 1 2 3

Contents with 5 cm 3 hydrogen peroxide Fresh liver Boiled liver (cooled) Pulped liver

4

Potato cubes

5

Manganese dioxide (untreated)

6

Observations before and after using wood splint

Boiled manganese dioxide (cooled after heating)

Washing up Thoroughly wash and scrubbed all apparatus containing liver pieces with detergent or Dettol solution provided to rid it of unpleasant odours.


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Assignments Please check with your tutor which option is required for you. Option 1: (please refer to WBLE/Turnitin for instructions which may incorporate other options below) Option 2: Skills-Based Assessment: Tabulation of qualitative data Tabulate the results you obtained (unexpected results accepted). The results table should not exceed 1 page. Option 3: Skills-Based Assessment: Discussion Write your discussion in prose form and without numbering. Excluding your cover page, your discussion and conclusion should NOT exceed ONE A4 page of Word document (standard/ default size). Anything in excess will NOT be graded. • Font Arial, size 11. • Margins: 1 inch from top, bottom, left and right (no need to change if you’re using the standard/ default size when MS Word opens). • Theory to apply: Refer to relevant information from lecture topics which may or may not have been covered yet. Please be sure to address the following: 1. What is the equation of the reaction observed? 2. What plant or animal organelle is involved? 3. What effect does pulping the liver have upon the reaction? Account for this. 4. What effect does boiling the liver have upon the reaction? Account for this (include reference to enzyme structure (bonds, molecular motion, shape, active site). 5. What were the differences between the reactions with fresh liver and with fresh potato cubes? Account for these differences (include reference to enzyme structure (bonds, molecular motion, shape, active site) 6. What were the differences between the effects on the reaction of boiling the liver and heating the manganese dioxide? Account for these differences (include reference to susceptibility (sensitivity) to heat, enzyme shape, bonds etc).


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Practical 5 (FHSC 1214 Cell Biology); Practical 7 (FHSB 1214 Biology I) Exercise 1 The Microscope and Its Uses

Microscope usage (revision) Note to lecturer: Before any microscope work (viewing of histological slides) commences, please ensure students have gone through this introductory session. Objective: To study the uses of microscope and its maintenances. To learn microscopic techniques such as focus the object with correct illumination under different power of magnifications. Introduction: The microscope is a basic tool of the biologist. It is a valuable precision optical instrument easily damaged by careless usage. It is very important for the student to become familiar with the parts of the microscope and the procedures in the handling of it. Treat your microscope well and it will serve you well. Apparatus and Materials: Binocular Microscope Microscope slide Plastic millimeter ruler

Cover slips Newspaper (1 page) Wash bottle

Setting up the Microscope: The microscope when not in use is usually kept in a case. Remove it by grasping the handle arm while placing one hand under the base. Set it down gently on the laboratory table and at a reasonable distance from the table edge. Always keep the microscope upright in the vertical position and never touch any of the lens surfaces with the fingers since it will deposit a thin film of oil on the glass.


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Parts of the Microscope:

Component Arm Base Body tube Eyepiece or ocular lenses

Revolving nosepiece Objective lenses

Function For lifting and carrying the microscope. To provide stability. To house the lenses. This is a set of lenses that rests loosely at the top end of the body tube. It is obvious that if the microscope is tilted while being carried, the lens may fall out and be ruined. The magnification of the eyepiece (given as 10X) is printed on the metal part of the ocular. Located at the lower end of the body tube, it carries 3 objectives of different lengths. Rotating this part changes the magnification of the objectives. They are of different magnifications with the following visible properties: Objectives Magnification Length Lens opening Scanning lens 4x Shortest Widest Low power lens 10x short wide High power lens 60x longest narrowest


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Component Focusing adjustments

Stage Mechanical stage Specimen holder Vertical feed knob Horizontal feed knob Condenser Built-in light source Brightness adjustment knob Main switch

Function These comprise two knobs located on either side of the microscope which are used to change the distance between the object being viewed and the objective lens. Changing the distance determines the focus. For the object to be viewed in focus under high magnification, the lens must be much closer to the object than when it is under low magnification. Coarse adjustment Made by the large knob beside the body tube for focusing under low power magnification. Fine adjustment Made by the small knob, which is for focusing under high power magnification and accurate focusing. Precautions when using the focusing adjustments:  Turn both adjustment knobs at the same time. Do not overturn the adjustment knobs (i.e. do not force them to  go beyond their limits)  Do not use the coarse adjustment knobs when focussing under the 60x objective lens. This is the platform for slides and specimens to be viewed under the microscope. This movable portion of the stage is attached to the specimen holder and allows the slide to be moved in different directions to facilitate viewing. This holds the glass slide in place. Rotating this moves the glass slide in the vertical direction. This moves the glass slide in the horizontal direction. Located just beneath the stage of the microscope, it incorporates a lens which collects light on the stage to bear on the object. This is situated below the iris-diaphragm to provide light for illuminating the object. It can be switched on or off. This provides adjustment to the illumination brightness.

This ensures that power is turned on or off.


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Preliminaries before Use: 1. Use the coarse adjustment to raise the body tube so that the objective can clear the stage when the revolving nosepiece is turned. 2. Turn the nosepiece until the scanning objective is in-line with the eyepiece. You should hear a soft click or else feel a distinct falling into place as the objective moves into position. If not, the field of view is totally dark or an illuminated crescent instead of a complete circle. 3. Turn the diaphragm to its largest opening. 4. Look into the eyepiece and make a final adjustment to the light adjustment knob so that the field of view (i.e., the lit circle which you see) is evenly illuminated. Any glare should be removed by adjusting the diaphragm. 5. Should either of the lenses appear dirty, wipe it gently with a piece of special lens paper. Use a circular motion with very light finger pressure. You should never use any other type of paper or cloth. Discard the lens paper after use. 6. The microscope is now ready for use. 7. If you’re using a binocular compound light microscope like the diagram above , position it so that the stage faces you. 8. Connect the microscope to the power supply and turn on the built-in light. 9. Ensure that the microscope stage is at its lowest position. This will prevent breaking of slides and lenses by mistake when adjusting the objectives by moving the stage with the coarse adjustment knob.


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Using a higher power objective 1. Great care must be taken when using higher power objectives. DO NOT focus the high power objectives with the coarse adjustment knob. 2. Most microscopes have parfocal objectives. This means that if one switches from viewing a specimen in sharp focus under a lower power objective to a higher one, the object should automatically come approximately into focus. Only some slight further focussing with the fine adjustment knob is required to see the specimen clearly. Therefore, if you’re using the higher power objectives, do not use the coarse knob to refine focus or you’ll risk breaking the slide and lenses. I f t h e o b j e c t i v e s a r e n o t p a r f o c a l , adjust the stage such that it is about 1cm from

the low-power objective. Change to the high-power objective and then adjust the stage with the coarse adjustment knob until it is about 1mm away from the objective. This is determined by looking from the side of the microscope. Using the fine adjustment knob and looking through the eyepiece now, slowly bring the object into focus. Repeat the procedure carefully if the first attempt at finding an object under highpower magnification is unsuccessful. 3. When changing from one objective to another, you will hear a ‘click’ when the objective is set in position. 4. You are now ready to switch from the scanning objective to a higher power objective after obtaining a sharp focus of the object. 5. If required, adjust the fine adjustment knob to see the specimen clearly. 6. If you’re going to swit ch to the next higher power objective, look from the side of the microscope and move the revolving nosepiece slowly till that higher power objective clicks into position. Be careful that it does not touch the slide (normally it shouldn’t unless the specimen is too thick and also covered by a thin cover slip). 7. Take care that the lower end of the high power objective does not touch the cover slip. If this happens, you must repeat the whole procedure focusing again, starting with the scanning objective. Trouble-shooting Below are some common problems associated with not being able to find and focus on an object under high-power magnification.     

Is the objective lens in position? Is the cover slip on the slide facing upwards? Is the object in the centre of the stage? Are the lenses clean and free from dirt and moisture? Is the condenser adjusted and focused?


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Oil Immersion: If your microscope comes with a 100 x objective, please do NOT use it. Used the improper way, it will break. If you require a particularly high magnification, immersion oil may be used. Fluid with the same refractive index as the objective lens is placed between a special objective lens and the cover slip so that it touches both. The fluid permits a larger cone of light rays to enter the objective from the specimen, and this increases the resolving power obtainable. Microscope Care: Like all laboratory instruments, the microscope needs proper care for best service. Observe the following: 1. Turn the resolving nosepiece until the scanning objective is in position. 2. Adjust the boy tube so that the lower end of the objective is about 1 cm above the stage. 3. Ensure that the stage surface is clean and dry. 4. Return the microscope in an upright position to its storage case.

Activity: Manipulation Skill practice task Note to lecturer: this activity may be graded. Any mistake will result in subtraction of 1 mark. Microscope manipulation checklist

Observed Yes No

Skill: Manipulation 1. Position compound light microscope so that the stage faces you. 2. Ensure that the microscope stage is at its lowest position. 3. Position the specimen holder such that it is roughly in the middle of the stage and not at either left or right extremes. 4. Secure the slide in position correctly with the specimen holder 5. Ensure that the scanning objective is first employed. 6. Ensure that the field of view is a complete circle and not totally dark or an illuminated crescent. 7. Both eyes open and used to look through the eyepieces. 8. Adjust the brightness adjustment knob to give the right amount of light for viewing the object details clearly (i.e., instead of either too dark or too bright, obscuring the object’s finer details). 9. Focus on image accurately and sharply by using the coarse and fine adjustment knobs. 10. When using the next higher power objective, look from the side of the microscope to ensure that it does not touch the slide. 11. When using higher power objectives (e.g., 40 X onwards), only the fine adjustment knob is used (i.e., not the coarse adjustment knob).


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Practical 5 (FHSC 1214 Cell Biology); Practical 7 (FHSB 1214 Biology I) Exercise 2 On-site Assessment

Each student will be assessed on-the-spot identification of 3 structures within certain minutes (10 marks) (The duration will be decided by the tutor). This section may comprise 10 marks out of 20 marks. Any mistake will result in subtraction of 1 mark. Checklist for on-site slide structure identification Observed Yes

No

Skill: Manipulation 12. Position compound light microscope so that the stage faces you and ensure that the microscope stage is at its lowest position. 13. Position the specimen holder such that it is roughly in the middle of the stage and not at either left or right extremes. 14. Ensure that the scanning objective is first employed. 15. Ensure that the field of view is a complete circle and not totally dark or an illuminated crescent. 16. Both eyes open and used to look through the eyepieces. 17. Adjust the brightness adjustment knob to give the right amount of light for viewing the object details clearly (i.e., instead of either too dark or too bright, obscuring the object’s finer details). 18. When using the next higher power objective, look from the side of the microscope to ensure that it does not touch the slide. 19. When using higher power objectives (e.g., 40 X onwards), only the fine adjustment knob is used (i.e., not the coarse adjustment knob). 20. Focus on image accurately and sharply by using the coarse and fine adjustment knobs. Skill: Identification 21. Able to name the specimen from the slide or identify two - three structures from the slide. Total marks


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Practical 5 (FHSC 1214 Cell Biology); Practical 7 (FHSB 1214 Biology I) Exercise 3 Preparation of Wet Mount

Materials for microscopic examination are usually placed on the glass slide of standard size, the microscope slide. In most cases, the materials are then covered by small thin piece of glass, the cover slip. Both microscope slide and cover slip should be very clean before use. Cleaning microscope slides Hol d t he m icr osc ope sli de by t he edg es between the index flinger and the thumb and dip in water. Then wipe dry using a soft tissue or a clean piece of cloth. Dirty handkerchiefs will n o t do. Cleaning cover slips Cover slips are very fragile and need careful handling. Hold a cover slip by the edges between the index finger and the thumb and then dip in water. To wipe dry insert the cover slip into the fold of a piece of clean cloth or lens paper and apply gentle pressure between the finger and thumb to both surfaces at the same time. Use a gentle circular wiping mo tion for of effective cleaning.

REMEMBER Always handle glass slides and cover slips by their edges, never by their flat surfaces.

Focusing the Microscope - ‘e’ slide 1. Prepare a microscope slide to view the letter “e”. Using a pair of scissors, cut a piece of newspaper about 3 mm square that includes a tiny sharp-lined letter (an “R” or "e" is best). 2. Place this square piece of newspaper in the centre of the slide with the print ed side up. 3. Add one or two drops of water onto the newspaper using a dropper. The water should be sufficient for the newspaper to absorb and still leave some remaining around it. 4. Pl ace the cover slip carefully over the newspaper. If this is done properly, the remaining water should spread out evenly with minimum formation of air bubble between cover slip and slide. This may take bit of practice. One effective method is to hold the cover slip about 45º to the slide, let it slip down the slide till the lower edge touches the water, and then slowly lower the cover slip down onto the slide. Use a mounted needle if necessary. Lab manual version 4_201405 Biology I & Fundamentals of Cell Biology

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Some air-bubbles may still be trapped even after the most careful preparation. If so, gentle tapping of the cover slip with a pencil point may help remove them. Anyway, a few bubbles should not hinder most obser vations to be made. 5. Make a drawing of the image under 4x magnification.

How to Mount an Object for Microscopic Examination Carry out the observations as follows: 1. Compare the position of image as seen through the eyepiece with that of the printed letter as seen with the unaided eye. Does the image appear to be reversed, i.e. as it would appear if seen in a mirror? 2. Slowly move the slide from left to right, observe and describe the way the image moves. Repeat right to left. 3. Move the slide away from yourself and describe observe the movement of the image again.


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Practical 5 (FHSC 1214 Cell Biology); Practical 7 (FHSB 1214 Biology I) Exercise 4 Preparation of Microscopic Slides ______________________________________________________________________ Objective: To study the microscopic structure of biological samples and to learn the preparation of biological samples for microscopic study purposes. Introduction: Examination of biological materials under the microscope will usually entail long periods of looking into the eyepiece. It is useful to develop the habit of keeping both eyes open and relaxed , as though you were looking at a distant object. (The final image is theoretical focused at infinity as any book on optical instruments will tell us). This will cut out eye-strain caused by continual forcing of one eye to remain closed. Apparatus and Equipments: Binocular Microscope Microscope slide Plastic millimeter ruler Forcep

Cover slips Soft tissue papers (lens cleaner) Wash bottle

Materials: Potato Hair Safranin

Onion Iodine

Observation of Onion Cells: The onion scale leaf (see figure below) has generally two major surfaces – an outer surface which faces the exterior and an inner surface which faces the interior of the onion. The outer surface may have pigmented portions of its outer epidermis while the inner surface may not.

(Mackean, D. G., 1973. Introduction to biology, pg 25.) Lab manual version 4_201405 Biology I & Fundamentals of Cell Biology

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Part 1 - Preparation of microscopic slides 1. Cut an onion bulb into quarters. Remove one of its fleshy scale leaves. 2. Bend the onion scale leaf towards the outer epidermis until it breaks on the upper surface. 3. Although broken, there is some thin tissue layer of the inner epidermis still intact. It appears as a transparent paper-thin skin with a ragged edge along the broken edge of the leaf. 4. With your fingers, pull the inner epidermis gently away from the scale leaf. 5. You may use forceps, scissors or a scalpel (used against a white tile) to pull out 2 small-sized portions of the inner epidermis (roughly 5 mm X 5 mm). Make sure that it is not too big so as to reach the width of the cover slip or of such a size that it rolls up easily.

6. Using a dropper, place 1-2 drops of water on the slide and place the epidermis on the water. Make sure that there is some water covering the specimen and surrounding it. 7. If there are any bubbles, try to get rid of them by pricking them with a mounting needle provided. Why are bubbles undesirable?


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8. Referring to the diagram below, hold a cover slip at about 45  to the slide and lower it so that one edge touches the water droplet.

9. Slowly lower the cover slip onto the slide using the points of a pair of fine forceps, pencil or a mounted needle. If this is done properly, the remaining water should spread out evenly with minimum formation of air bubble between cover slip and slide. Some air-bubbles may still be trapped even after the most careful preparation. If so, gentle tapping of the cover slip with a pencil point may help remove them. Anyway, a few bubbles should not hinder most obser vations to be made. 10. Remove excess water from on top or around the cover slip with a piece of tissue paper provided – be careful not to absorb all the water from under the cover slip. Removal of excess water ensures that when the slide is viewed under the microscope, water will not spill onto the stage. 11. The mounting of a specimen on a slide with solution is called a wet mount. Avoid tilting the microscope when using a wet mount.

Part 2 - Viewing the slides 1. Place the slide carefully on the stage and position such that the specimen is in the centre of the hole in the stage and also in the middle of the ‘circle’ of light emanating from the lamp through the stage hole. 2. Ensure that the scanning objective is in place by moving the revolving nosepiece. The revolving nosepiece is in correct position if the objective lens is felt to ‘click’ to fit an unseen internal groove that aligns the lens’s field of view with the eyepieces. If not, the field of view is totally dark or an illuminated crescent instead of a complete circle. 3. If the bifocal compound light microscope you’re using has eyepiece lenses which can be slid horizontally, slide the eyepieces to the maximum length away from each other first. Place your head just above the eyepieces. Slowly, slide the eyepieces towards each other horizontally so that they fit the position of the eyes on your head. If the eyepieces are in correct position, you should be able to observe only one illuminated circular field of view. If not, you’ll see two overlapping illuminated circles.


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4. Adjust the brightness adjustment knob to give the right amount of light for viewing the object clearly. Some materials are best viewed in dim light, others in bright light. A common cause of poor definition of (i.e., not being able to see details clearly) the image is that the object is over-illuminated. Best definition is often obtained by cutting down the amount of light and not increasing it. If the condenser is not adjusted and focused, the specimen may appear too dark or too bright, obscuring the object’s finer details. 5. Looking down the eyepiece, slowly adjust the position of stage with the coarse adjustment knob until the object comes into focus. Focus accurately by using the fine adjustment knob. 6. Keep both eyes open when viewing through the eyepiece. Get accustomed to using both eyes otherwise this will strain your eye or give you a headache over time. 7. If the details of the specimen are not clear, adjust the brightness adjustment knob and/or condenser. 8. Once the object is in sharp focus, it’s time to view it at higher magnification (i.e., the 10X objective). 9. Never to lower the body tube while looking into the eyepiece and using the coarse adjustment. If for some reason you miss the image, look up and repeat the whole procedure of focusing. 10. For viewing under every objective lens, get a sharp focus first. Use the fine adjustment to sharpen the focus of the specimen. 11. Count the number of cells you see at 10X magnification. Notes: The lines that form the network between individual cells are non-living cell walls made up chiefly of cellulose. This cell wall is the outermost part of the cell and immediately surrounds the cell membrane, also called plasma membrane, which in turn enclose the cytoplasm. The central part of most plant cells is taken up by a vacuole filled with a fluid made up mostly of water and various salts. The nucleus appears as a dense body in the translucent cytoplasm. 12. Turn again to the scanning objective and remove the slide from the stage.

Staining with iodine

13. Stain the grains by the technique of irrigation. This is done by placing a drop of iodine at one edge of cover slip. A small piece of filter paper is brought into contract with the water at the opposite edge of the cover slip. As water is absorbed the iodine from the other side will be drawn under the cover slip. Continue this until the iodine is drawn halfway across the space beneath the cover slip. The iodine will then slowly spread throughout the mount.


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Tissue paper

The Technique of Irrigation 14. Examine first under low power (begin with the scanning objective first) and then under high power. Answer these questions: 1. What are the effects of the iodine stain on the cells? 2. Can you observe any changes in the cells? If so, describe them. 3. Are there starch grains in the cells? 4. How can you identify the starch grains if they are present? Staining with safranin

15. Prepare another slide of the onion epidermis. This time add a drop of safranin onto the epidermis instead of distilled water. Allow the stain to take for 10 minutes before drawing it off with tissue paper. Use the irrigation technique to dilute and wash off the excess free stain. Finally put on a cover slip. 16. Examine first under low power (begin with the scanning objective first) and then under high power. Answer these questions: 1. What are the effects of the safranin stain on the cells? 2. How is this preparation different from the previous one observed in step 14? Add to your drawing any additional details you may observe with this second preparation.


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Assignments Please check with your tutor which option is required for you. Option 1: On-site assessment  This option may be used for stained (iodine or safranin) cells.  Students will be graded based on microscope skills stated in ‘ Activity: Activity: Manipulation Skill practice task ’ under ‘Exercise 1 The Microscope and Its Uses’ and ‘Exercise 2 On-site Assessment ’.  For ‘Exercise 2 On-site Assessment ’, students have to identify at least TWO structures - cell wall, nucleus or starch grains (if visible) in under 1.5 minutes .  There should be minimal folding of epidermal tissue.  Mark allocation: 5 marks.

(Minus marks for any violation of microscope usage or unsatisfactory specimen preparation) Option 2: Drawing – 6 cells,  For specimens stained with either iodine or safranin, make a drawing of 4 – 6 each 2 – 2 – 3 3 cm long. Include only the details you can observe in your preparation. TWO structures: cell wall, nucleus or starch grains (if visible).  Label at least TWO structures:  Students will be graded based on ‘ Introduction Exercise 2: Notes on biological drawings’ above.


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Practical 5 (FHSC 1214 Cell Biology); Practical 7 (FHSB 1214 Biology I) Exercise 5 Measurement with a Microscope

Quantitative observations under the microscope – – making microscopic measurements A more exacting exacting type of observation observation introduces introduces the idea of amount amount and is called quantitative. How big? How fast? Measuring instruments such as rulers, balances and thermometers must be used to extend the range and accuracy of the senses. The microscope can be used for the dual purpose of measuring very small objects (too small to be measured with an ordinary ruler) and observing them accurately. Magnification When looking through the microscope, it is important to know how much the object being observed is magnified. To find the degree of magnification, multiply the magnification on the objective being used by that on the eyepiece. For example, if the magnification magnificatio n of the eyepiece is 10x, and that of the objective is 40x, then the total magnification is 10x40, or 400x. Objects viewed under the microscope will be seen in three-dimensions. three- dimensions. For a given object, the distance that allows it to be in focus should thus be investigated using the fine adjustment knob. This distance is the focal depth of of the object being observed. The focal depth decreases with increasing magnification. Determining the diameter of the field of view by means of a stage graticule/ micrometer or an eye-piece graticule/ micrometer A graticule / micrometer is a ‘micro‘micro -ruler’. It is a piece of of plastic or glass slide which is finely calibrated to 0.1mm or even smaller (0.01mm). It is used for measuring microscopic microscopi c structures. There are two types of graticule / micrometer: 1.

Stage graticule/ micrometer This is placed on the stage over the specimen slide. When viewed under the microscope, the finely calibrated division marks can be seen clearly and are magnified according to the magnification magnificat ion of the two lenses used. Since it is more finely calibrated than a ruler, it more accurately measures the size of a specimen under the microscope.

2.

Ocular/ Eyepiece graticule/ micrometer This is placed in the body tube together with the eyepiece lens. It is also used to measure microscopic structures under the microscope. However, it is magnified differently from that of an object on the stage. The object on the stage is magnified by both the objective lens and the eyepiece lens, while the eyepiece graticule is only magnified by the eyepiece lens. Thus, there is a need to further calibrate the scale when different objective lens is used.


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Calibrating the stage graticule/ micrometer Each graticule/ micrometer, whether it is the stage type or the eyepiece type; must be calibrated before it can be used. (i)

Place a stage micrometer on the microscope stage, and using the lowest magnification (4X), focus on the grid of the stage micrometer.

(ii)

Rotate the ocular micrometer by turning the appropriate eyepiece. Move the stage until you superimpose the lines of the ocular micrometer upon those of the stage micrometer. With the lines of the two micrometers coinciding at one end of the field (align ( align the start (0 mm) of divisions on the stage graticule with the start (0mm) of divisions on the eyepiece graticule ).(Figure 1)

(iii)

Count the spaces of each micrometer micromete r to a point at which the lines of the micrometers coincide again (Figure 2) (i.e. find the division /scale of eyepiece graticule which is parallel with a particular division/scale of the stage micrometer). micrometer). Since each division of the stage micrometer measures 10 micrometers (m)/0.01mm, and since you know how many ocular divisions are equivalent to one stage division, you can now calculate the number of micrometers in each space of the ocular scale. *Alternatively: Suppose the full length of the ocular / eyepiece graticule covered 250 divisions of the stage micrometer. Then the full length of the ocular/eyepiece graticule is equivalent to (250 x 0.01mm) = 2.5mm long. For an ocular/eyepiece graticule with 100 divisions, each division will measure 25μm at the stage for this magnification.

(iv)

Record your calculations as shown below. (Using the lowest magnification (4X objective / total magnification 40x) _____number _____number of divisions on eyepiece eyepiece graticule corresponds corresponds to_____divisions to_____divisions of stage micrometer. Value for each division of the stage micrometer = __________mm = __________ m Thus value/size for each division of eyepiece micrometer = ____________ m (give your answer in  m) m)

(v)

Repeat your measurements for 10x, 40x/60x, and 100 x objectives.

(vi)

Fill in your calibrated values for 1 ocular / eyepiece graticule scale at various magnifications in Table 1. 1.


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Figures 1 & 2 illustrate the calibration of the eye-piece/ ocular graticule/ micrometer.

Figure 1 Note: the display of scales of stage and ocular micrometers shown here may be different from the actual ones provided in your laboratory


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Figure 2

Table 1 Value of Eyepiece Graticule / Micrometer Division for Various Objective Magnifications 1

Objectives magnification 4X

*Calibrated values of eye piece graticule 1 eyepiece graticule unit = ___________ mm = __________ μm

10X

1 eyepiece graticule unit = ___________ mm = __________ μm

3

40X/60x

1 eyepiece graticule unit = __________ mm = __________ μm

4

100x

1 eyepiece graticule unit = __________ mm = __________ μm

*Note: Please keep these calculations for your on-site assessments (see below). Note: all values to have precision up to 4 decimal places for mm and 2 decimal places for m)


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Application exercise Option 1: Mock on-site-assessment     

Use any given specimen slide provided which may not be used for the on-site assessment or assignment this semester (e.g., skin, trachea etc.). In 2 minutes, identify any three structures of your choice. You will be assessed according to the ‘ Microscope manipulation checklist ’ under ‘Activity: Manipulation Skill practice task ’ above. For the final structure, determine the number of eyepiece divisions spanning the size (e.g., length, breadth, diameter etc.) of the structure. Multiply by the correct factor according to the corresponding lens (see ‘Table 1: value of eyepiece graticule’ above) to obtain the actual size of the structure in micrometers.

Option 2: Biological drawing with scale bar Name of specimen: Magnification power: Draw your specimen and show the measurement/size of the specimen in m using the scale bar as shown in the figure below (refer to the eyepiece micrometer and convert the length into m according to the objective lens used):


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http://www.mun.ca/biology/Help_centre/1001_2_tutorialpages/Measuring_scope.html http://www.nature.com/nature/journal/v474/n7349/full/nature09974.html


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Practical 5 (FHSC 1214 Cell Biology); Practical 7 (FHSB 1214 Biology I) Exercise 6 Observation of Starch Grains (Additional practice tasks if time permits)

1. Place a clean glass slide on the white white tile. Place a small piece of potato in the centre of the slide and rub the piece of the potato in a circular pattern to distribute the potato juice in an even layer. layer. Discard the piece of of potato. potato. 2. Add a drop of water and then a clean cover slip to the slide. Take the usual precaution of avoiding air-bubbles. 3. Examine the preparation under low power (begin with the scanning objective first). The starch grains in the mount can be more readily observed if sized of the opening in the iris diaphragm is decreased. This will increase the contrast between the starch grains and the surrounding water. 4. Move the slide on the stage until you locate a field in which the grains are well separated. Examine them under low or high power. Make a drawing of 4 – 6 – 6 starch grains to illustrate their typical shape with any other observable details. 5. After completing completing your drawings, turn again to the scanning objective. objective. Remove the slide from the stage and place it on a white tile. Stain the grains with iodine using the technique of irrigation earlier mentioned. 6. Examine various aspects of the iodine-stained iodine-st ained mount first under the scanning objective and then under low and high power. Note the effects of different iodine concentration on the starch grains. Draw 4 – 6 – 6 typical starch grains to illustrate their shape and structure. Answer these questions: 1. What observable changes may be seen in the starch grains exposed to relatively relativel y high iodine concentration? 2. What observable differences are there between these starch grains when compared to those exposed to lower iodine concentration? 3. Can the internal grain structure better observe in strained grains or unstained ones? 7. Prepare another slide of starch as outlined in step no. 1 but do not add the cover slip yet. The grains are stained first by adding a drop of iodine onto them and the slide gently rotated by tilting to-and- fro so that the whole area of grains is evenly covered by iodine. Excess stain is drained off before a cover slip is added. Examine this preparation carefully.

Skills-Based Assessment: Discussion Biological materials are often stained before examination under a microscope. Based on your experience in this exercise suggest reasons for such use of stains.


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Practical 5 (FHSC 1214 Cell Biology); Practical 7 (FHSB 1214 Biology I) Exercise 7 Observation of Hair (Additional practice tasks if time permits)

1. Mount a small portion of your own hair in a drop of water on a slide. Add a cover slip, taking the usual precautions not to trap air beneath it. 2. Adjust the diaphragm of the microscope to its largest opening and bring the hair into sharp focus under low power (begin with the scanning objective first). Reduce light gradually by progressively closing the diaphragm. In this way, determine the diaphragm setting that provides the clearest image of the hair. As you further examine the hair, shift the focus by slowly turning the fine adjustment back and forth. 3. Move the hair to the centre of the scanning field and shift to higher power magnifications. Note any changes in the brightness of the field of view. Bring the hair image into the sharpest possible focus and examine carefully. Answer these questions: 1. While shifting the focus with the fine adjustment, what changes in the image can be observed? Explain why these changes take place. 2. Does higher-power magnification allows greater detail to be seen? 3. Is the depth of focus as great with higher power as with low power? 4. Is the resolving power increased or or decreased when magnification magnificatio n is increased? Using the procedure outlined in Exercise 2, estimate the width of the hair. State his measurement in millimeters as well as in micrometers. 4. As an interesting interesti ng corollary of this exercise you could examine hair from different members of the class and try to determine differences between fine and coarse hair, curly and straight hair, and between hair of different shades or colours. The differences and similarities could be represented by diagrams based on your observations 5. The exercise in step 4 above can be further extended by using hair from different animals, e.g. dog, cat, mouse, rabbit, guinea pig, etc.


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Practical 6 (FHSC 1214 Cell Biology ONLY) Extraction of Cell Organelles by Differential Centrifugation ___________________________________________________________________ Objective: To show how organelles can be purified from homogenated liver tissue by differential centrifugation. Introduction: Cell fractionation A. Homogenization Cells or tissues are ground up/ blended in such a way that its consistency is even. This is to destroy the cell membrane so that the cytoplasmic components flow out. B. Centrifugation Principle: Different cell components are of a certain size and density, and descend to the bottom of the centrifuge tube at different speeds. The faster the rotation of the centrifuge, the smaller the particles is sediment. Components can be separated from larger to smaller ones based by using a series of increasing speeds. This is called differential centrifugation. A cell component can be designated 70S. S is ‘Svedberg’ unit or sedimentation coefficient. It refers to how fast a substance /particle sediments in an ultracentrifuge, based on its size and shape. The greater the S number, the greater the rate of sedimentation. The process of differential centrifugation is based on the fact that organelles have differences in size, shape and density. As a result, the effect of gravity on each is different. We can use this principle to separate an organelle from a homogenous solution of particles by artificially controlling the gravity of a solution. This is done by putting the solution in a variable speed centrifuge and rotating them at a high rate of speed. This creates a force that can be much greater than the force of gravity, and particles that would normally stay in solution will fall out and form a pellet at the bottom of the tube. The relative centrifugal force can be calculated by the following equation: R.C.F. = 1.119 x 10 -5 (rpm2) r Where rpm is the revolutions per minute of the rotor and r is the distance (in cm) of the particle from the axis of rotation. The radius used is the distance from the center of the axis of rotation to the middle of the centrifuge tube. The forces created at low speeds are small (e.g. 600 X g) and only very large or dense particles will fall out of solution (nuclei, whole cells and large cellular debris). At high speeds, the force created can be quite great (e.g. as much as 300,000 X g). At these speeds, most particles will fall out of solution and only very small, highly soluble molecules will remain in solution.


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A piece of tissue is homogenized by physically grinding it.

The cell homogenate contains large and small organelles.

A centrifuge is used to separate the organelles based on size and density.

Golgi Mitochondria Nuclei

The heaviest organelles can be removed and the remaining suspension recentrifuged until the next heaviest organelles reach the bottom of the tube.

Figure 1 Cell Fractionation. The organelles can be separated from one another after cells are broken open and centrifuged. Diagram: Life, the science of biology (6 th Ed.). William K. Purves, David Sadava, Gordan H. Orians, and H. Craig Heller (2001)


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Differential centrifugation schemes (Figure 1) involve stepwise increases in the speed of centrifugation. At each step, more dense particles are separated from less dense particles, and the successive speed of centrifugation is increased until the target particle is pelleted out. The final supernatant is removed, the pellet is re-suspended, and further study or purification can be done on it. The fractionation of rat liver is an example of how this process works. An important thing to note is that there is cross contamination between the second and third pellets. Mitochondria show up in Pellet 3 and lysosomes show up in Pellet 2. This shows that the separations made by this technique aren't absolute purifications, but relative enrichments of organelles. In order to develop a differential centrifugation scheme to isolate a particular organelle, a marker must be used to follow its isolation. The marker can be the activity of an enzyme that is confined to that organelle. For example, enzymes of the electron transport chain are membrane bound and confined to the inner membrane of the mitochondria. Therefore, after a centrifugation to isolate mitochondria, both the pellet and supernatant can be analyzed to see which part has more of the activity associated with these enzymes. The fraction with more of the activity has been "enriched" with mitochondria. Purification of the organelle is accomplished by following the enrichment through successive steps. Another way to follow enrichment is by binding a radioactive label to protein on the organelle. For example, cell surface membranes can be isolated by first binding a radioactive drug that has its protein receptor in the cell membrane. The drug binds tightly to the receptor and remains there, so the fractions that contain the most radioactivity are enriched for cell surface membranes.


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Figure 2 Centrifugation Scheme. Homogenized liver tissue is subjected to low centrifugation force to separate larger cell structures and then higher centrifugation force to separate smaller organelles. Procedures: Create a flowchart before you enter the lab in order to understand the steps in this experiment. Show this to your tutor before starting the experiment. 1. Obtain an uncut piece of liver (~ 3 cm x 3 cm). Be sure to remove any associated fat or surrounding foreign (non-liver) tissue. 2. Using a mortar and pestle, grind the liver piece into a pulp. This is called homogenization and the product the homogenate. 3. Add 10 ml or more isotonic solution as you grind, to increase the volume and dilute the pulp. [Use distilled or tap water if no isotonic solution if provided.] (Why is isotonic solution needed?) 4. Remove and dispose any ungrounded tissue or fat. 5. Using labelling paper (or masking tape), label the 15 ml graduated plastic tube provided with your group’s name. Lab manual version 4_201405 Biology I & Fundamentals of Cell Biology

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6. If the pulp is very thick, pour about 2-4 ml into the 15 ml graduated plastic tube and top up to 14 ml of the tube with isotonic solution. The graduated tube has markings to guide you. 7. If the pulp is not too thick, pour 5-8 ml of the pulp (homogenate) into the 15 ml graduated plastic tube and top it up till 14 ml with tap water. The graduated tube has markings to guide you. Note: It is extremely IMPORTANT that each tube is topped up to this volume as precisely as possible, so that the centrifuge will not be damaged during spinning. 8. Wipe off any spillage on the tube exterior so as not to contaminate the centrifuge. 9. With the support of lab technical staff, spin the tube for 5 minutes. While waiting, prepare for the next experiment. 10. After 5 minutes, collect your centrifuged sample. Note: DO NOT shake the tube. 11. Use one hand to hold the 50 ml graduated plastic tube and another to hold a glass pipette. 12. Press the air out of the rubber bulb of the pipette. Slowly insert the pipette tip into the supernatant, being careful not to mix up the contents. 13. By slowly releasing the pressure on the bulb, aspirate out the supernatant into one 15 ml graduated plastic tubes in roughly equal volumes. 14. Unless there is enough supernatant, top up just one of 15 ml graduated plastic tubes to 10 ml with distilled H 20. Note: It is extremely IMPORTANT that each tube is topped up to this volume as precisely as possible, so that the centrifuge will not be damaged during spinning. 15. Label the tube and wipe off any spillage on the tube exterior so as not to contaminate the centrifuge. 16. Centrifuge the graduated plastic tube for about 20 minutes. While waiting carry on with the next experiment. 17. After 20 minutes, collect your centrifuged sample. Note: DO NOT shake the tube. 18. Use one hand to hold the 15 ml graduated plastic tube and another to hold a glass pipette. 19. Press the air out of the rubber bulb of the pipette. Slowly insert the pipette tip into the supernatant, being careful not to mix up the contents.


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20. By slowly releasing the pressure on the bulb, aspirate out the supernatant into one 15 ml graduated plastic tube. 21. Unless there is enough supernatant, top up just one of the 15 ml graduated plastic tubes to 10 ml with distilled H 20. Note: It is extremely IMPORTANT that each tube is topped up to this volume as precisely as possible, so that the centrifuge will not be damaged during spinning. 22. Label the tube and wipe off any spillage on the tube exterior so as not to contaminate the centrifuge. 23. Centrifuge the labelled 15 ml graduated plastic tube for about 1 hour. While waiting carry on with the next experiment. 24. After 1 hour, retrieve the tube and take a look at it. Due to shortage of time and limitations of the centrifuge model used, this experiment will stop here. 25. Dispose of the excess liver pulp or pieces into the given plastic bag ( not the dustbin).


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Practical 7 (FHSC 1214 Cell Biology); Practical 5 (FHSB 1214 Biology I) Determination of the Solute potential of the Potato Cell Sap ______________________________________________________________________ Objectives: To find out the solute potential of potato cell sap as a function of changes in the concentration of sucrose solution and in the length of potato strips. To prepare sucrose solutions of various concentrations from a stock solution of 1.0 M by dilution technique for the determination of the solute potential. Introduction You are advised to read the following carefully before writing your lab report. Water potential, The tendency of water molecules to diffuse across a membrane is affected by:  the concentration of the solution on either side of the membrane  the resultant pressure in the solution on each side of the membrane Water potential (denoted by the Greek letter  , psi) is used to describe combined

effects of concentration and pressure in a solution on the tendency of water molecules to diffuse across a membrane. Water potential i s d e f i n e d a s t h e n e t t en d e n c y o f w a t e r t o d i f f u s e o u t o f a s o l u t i o n by osmosis

It is measured in pressure units such as kPa (kilopascals) and MPa (megapascals). Water potential of a solution = effect of the solute concentration of that solution + effect of pressure on that solution

where  =

= s + p water potential of the cell (It’s a -ve pressure, i.e., inward force like that created on solutions or water when the plunger of a syringe is pulled to draw liquid up 1; see picture arrows)

s = solute potential of the cell

(It’s a -ve pressure also, i.e., inward force; see picture arrows) p = pressure potential, due to wall pressure

(It’s a +ve pressure; outward force like that created on solutions or water when the plunger of a syringe is pressed to expel liquid; see picture arrows)

1

Campbell (2002). Chp 36. (6th Ed.).


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Solute potential is the tendency of a solution to gain water The potential of a solution to gain water is always negative The more concentrated a solution, the m o r e n e g at i v e its solute potential

For the purposes of comparison, pure water at atmospheric pressure has a water potential of 0. If a solute, such as sugar, is added to this water, its water potential is effectively decreased or lowered. This is because  water concentration in a given space is decreased  water molecules are attracted to the solute molecules and so move less freely  Water potential of a solution is always negative owing to the presence of solutes . T h e m o r e c o n c e n t r a t e d t h e s o l u t i o n , t h e l o w e r i t s w a t e r p o t e n t i a l i . e. , t h e m o r e n e g a t i v e i s i t s w a t er p o t e n t i a l.

(Pic. Hoh, 2003. A Level biology) In pure water, water molecules are free to move about at random. In a solution, solute particles attract water molecules and restrict their movement. Thus, water molecules cannot leave a solution so easily, the presence of solute particles lowers the water potential. The movement of water molecules across a selectively-permeable membrane 2 from a solution with a higher water potential to a solution with a lower water potential is known as osmosis. 2

Never use the term ‘semi-permeable’ membrane as it means that only water gets through and no other solutes. Lab manual version 4_201405 Biology I & Fundamentals of Cell Biology

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For example, if a plant cell is immersed in a solution with a higher water potential than the cell, then osmotic uptake of water will cause the cell to swell. By moving, water can perform work. Therefore the potential in water potential refers to the potential energy that can be released to do work when water moves from a region with higher psi to lower psi. Plant biologists measure psi in MPa, where one MPa is equal to about 10 atmospheres of pressure. 3

3

Campbell (2002). Chp 36. (6th Ed.).


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F o r y o u r u n d e r s t a n d i n g o n l y : t h e c o n c e p t o f tu r g o r p r e s s u r e •

•

•

Any solution at atmospheric pressure has a negative water potential. For instance, a 0.1-molar (M ) solution of any solute has a water potential of -0.23 MPa. 4 In contrast to the inverse relationship of psi to solute concentration, water potential is directly proportional to pressure. •

•

•

•

•

•

Physical pressure - pressing the plunger of a syringe filled with water, for example - causes water to escape via any available exit. If a solution is separated from pure water by a selectively permeable membrane, external pressure on the solution can counter its tendency to take up the water due to the presence of solutes or even forcing the water from the solution to diffuse into the compartment with pure water. It is also possible to create negative pressure, or tension when the plunger of a syringe is pulled up.

If a 0.1 M solution is separated from pure water by a selectively permeable membrane, water will move by osmosis into the solution. •

•

p

Water will move from the region of higher psi (0 MPa) to the region of lower psi (-0.23 MPa). (Fig. 36.3a)

If a 0.1 M solution (psi = -0.23 MPa) is separated from pure water (psi = 0 MPa) by a selectively permeable membrane, then water will move from the pure water to the solution. (Fig. 36.3d) Application of physical pressure can balance or even reverse the water potential (Fig. 36.3). A negative pressure can make water potential more negative (Fig. 36.3d).

Fig. 36.3. Values for & s in the left and right arms of the U-tube respectively are given for initial conditions, before any net movement of water. (a) The addition of solutes makes water potential more –ve. (b,c) Application of physical pressure increases water potential. (d) A –ve 4 Pic. and text from Campbell (2002). Chp 36. (6th Ed.). pressure (tension) decreases water potential Lab manual version 4_201405 Biology I & Fundamentals of Cell Biology

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E s s e n t i a l k n o w l e d g e f o r p r a c t i c a l : Osmosis in living plant cells


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The figure below shows the changes in water potential, pressure potential and solute potential of a plant cell as it takes up or loses water and so changes in volume.

0 kPa

- ve kPa

Ψs

Ψp = Ψs Ψ = 0

At full turgor  The plant cell is fully turgid  The cell wall is stretched fully  The pressure of the cell contents resists the uptake of water  Turgor pressure (Ψp) is at a maximum  There is no net tendency of water to move in or out of the cell, i.e., water potential (Ψ) = 0, because the rate of water movement into the cell equals that of water out of the cell. Incipient plasmolysis  Is when the shrinkage of the protoplasm reaches the point where the cell surface membrane is just about to pull away from the cell wall  No pressure is exerted by the protoplast (= cytoplasmic contents + membrane) against the cell wall, i.e. (Ψp) = 0, i.e. (Ψ)= (Ψs).  The cell is flaccid. Full plasmolysis  Occurs when the cell surface membrane is pulled away from the cell wall with the cytoplasm contracted. The curves for water potential (Ψ) and solute potential (Ψs ) are the same when the water content of this cell falls below 50%.


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As the cell contents shrink, there is no more force pushing on the cell contents. i.e., the pressure potential (Ψp) = 0. Hence, water potential (Ψ) = solute potential (Ψs) [Note: This is a very important equation.] In your lab report, be sure to use the proper terms (e.g., plasmolysis, the various ‘potentials’ etc.) when explaining the concepts herein. Apparatus: Boiling tubes (5 per pair) Ruler Knife Forceps Glass rod

Boiling tube rack Petri dish White tile Graduated glass pipette, 10cm 3

Materials: Sucrose solution, 1.0 M Distilled water Potato Tissue paper Graph paper Flowchart Students will be allowed to proceed with the experiment only if they have come into the laboratory with a flowchart of the day’s experiment. Procedures: Create a flowchart before you enter the lab in order to understand the steps in this experiment. Show this to your tutor before starting the experiment. 1. Each group should have 35cm 3 of sucrose. In labelled boiling tubes, prepare a series of 20 cm3 of sucrose solutions at the concentrations of 0.1 M, 0.2 M, 0.3 M, 0.4 M, and 0.5 M from the stock solution using dilution technique. Record the volumes of solution and of distilled water used in the table below. You may find this formula helpful: M = molarity V = volume M1V1=M2V2 Existing solution: 1 M (M 1) of sucrose Desired solution: 0.1M (M 2) of 20cm 3 sucrose What is the volume of 1 M sucrose in cm 3? (1M) (V1) = (0.1M) (20 cm 3) V1 = ?


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Molarity

0.1 M

0.2 M

0.3 M

0.4 M

0.5 M

Volume of 1.0 M sucrose solution (cm 3) Volume of distilled water (cm3)

2. Prepare 15 strips of potato tissue of about equal length (choose any single length between 4 cm to 6 cm) with a cross-section of about 0.5 cm x 0.5 cm. Using a table, record the average length of the potato strips. (Note: Don’t spend too much time cutting – dimensions can be approximated instead of being identical. Ideally, it is important for each strip to be of equal dimensions. Why?) 3. Using a ruler and simple eye estimation, measure the initial level of 0.1 M sucrose solution in the boiling tube before adding the potato strips. Record this in your manual. Take 3 potato strips, record the length of each strip, and place the strips into the boiling tube. Repeat these steps using 0.2 M, 0.3M, 0.4 M and 0.5 M sucrose solutions. 4. After 30 minutes, remove the strips with the forceps provided. Wipe them gently with tissue paper and record the final length and change in length of each potato strip in your table. 5. In your manual, record the final level of the sucrose solution in each boiling tube and record any changes to the physical condition of the potato strips. 6. Calculate the average change in the length of the potato strips and record it in your table. 7. On a piece of graph paper, plot a standard graph of solute potential against the concentration of the sucrose solution to determine the solute potential of the potato cell sap. Concentration (M) Solute potential (atm)

0.0 5

0.1 0

0.1 5

0.2 0

0.2 5

0.3 0

0.3 5

1.3

2.6

4.0

5.3

6.7

8.1

9.6

0.4 0 11. 1

0.4 5 12. 6

0.5 0 14. 3

0.5 5 16. 0

8. Plot a second graph of the change in average length of the potato strip against the concentration of the sucrose solution. Use a best fit curve/ line.


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Assignments Please check with your tutor which option is required of you. Option 1: (please refer to WBLE/ Turnitin for instructions which may incorporate other options below) Option 2: Skills-Based Assessment: Tabulation of quantitative data Your table should have the following: 1. initial, final and change in lengths of each strip and their averages 2. initial, final and change in levels of sucrose solution in the boiling tubes before the addition of potato strips 3. physical condition of the potato strips 4. Adherence to the specifications mentioned in the introduction of this manual. Length of potato Sucrose solution (M) strips (mm) 0.1M 0.2M 0.3M 0.4M 0.5M Initial length Average initial length Final length Average final length Change in average length The above table is just a suggested format. You may present in another way if suitable. Option 3: Skills-Based Assessment: Graphing of quantitative data Present your graph (pasted from Excel) of the change in average length of the potato strip against the concentration of the sucrose solution. Use a best fit curve. To get full marks, please observe the guidelines given on pp6-7 as well. No need to write procedure, draw table, write a discussion or conclusion. Option 4: Skills-Based Assessment: Discussion Write your discussion in prose form and without numbering. Excluding your cover page, your discussion and conclusion should NOT exceed ONE A4 page of Word document (standard/ default size). Anything in excess will NOT be graded. • Font Arial, size 11. • Margins: 1 inch from top, bottom, left and right (no need to change if you’re using the standard/ default size when MS Word opens). • Theory to apply: Refer to relevant information from lecture topics which may or may not have been covered yet.


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General

Note: Students must relate results with theory (no separate paragraphs on theory without reference to expected results) Definition of relevant terms: a. Osmosis b. Water potential c. Solute potential d. Turgor pressure

Positive change in length

Zero change in length

Terms above used correctly below. Comment on: a. Change in length b. Tonicity (e.g., is solution hypertonic? etc.) c. Condition of cells (e.g., turgid, etc.)? d. (Net) movement of water? Comment on: a. Change in length b. Tonicity (e.g., is solution hypertonic? etc.) c. Condition of cells (e.g., turgid, etc.)? d. (Net) movement of water? e. Relationships among w plant cell , s plant cell , w sucrose, s plant cell , s sucrose

Negative change in length (shortening of strip)

Comment on: a. Change in length b. Tonicity (e.g., is solution hypertonic? etc.) c. Condition of cells (e.g., turgid, etc.)? d. (Net) movement of water?

Conclusion

State the: a. The concentration of sucrose solution at which the water potential of the potato tissue is equal to the water potential of sucrose solution (determine from graph). b. The solute potential in atm at which the water potential of the potato tissue is equal to the water potential of sucrose solution (determine from graph). c. Relationship between change in length and sucrose concentration


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Practical 8 (FHSC 1214 Cell Biology); Practical 6 (FHSB 1214 Biology I) Effects of various treatments on pieces of stained potato tissues ______________________________________________________________________ Objective: To investigate the effects of various treatments on pieces of stained potato tissues Apparatus and Materials: 1 pen-knife or scalpel (if provided) 1 white tile A pair of forceps A thermometer (if no incubator used) A plastic ruler 10 test tubes 3 syringe (5ml) 2 beakers 1 stop watch

1 Potato rod (if provided) Methylene blue solution 50% ethanol

Flowchart Students will be allowed to proceed with the experiment only if they have come into the laboratory with a flowchart of the day’s experiment. Procedures: Create a flowchart before you enter the lab in order to understand the steps in this experiment. Show this to your tutor before starting the experiment. Unless otherwise instructed by your tutor, you may conduct this experiment in pairs. 1. Use a sharp scalpel, cut four cubes from the potato rod provided, each approximately 1 cm (breadth) x 1 cm (length) x 2 to 5 mm thick (select one size of thickness. Ensure all cubes are the same size and thickness). Trim off any peel which is still attached. Why should the surface area be kept constant for each piece of tissue?

2. Place them in a 50mL size small beaker (or petri dish or specimen tube), immerse them in methylene blue solution for 10 minutes. Use only enough methylene blue (maximum 10mL) to cover them (Swirl the contents to ensure that all surfaces of the cubes are exposed to the stain). Always read through your lab notes once before starting and look out for waiting time. What can you do while waiting?

3. After 10 minutes, pour off the methylene blue solution and wash the cubes with tap water until the water contains little or no stain. Then cover the cube with tap water. Why is a coloured stain chosen?


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4. Label four test tubes A, B, C and D. To each of the tubes A, B and C, add 5cm 3 of distilled water, to tube D add 5 cm 3 of 50% ethanol. State the reason for using equal volumes of liquids in all tubes. 5. Place tube A in boiling water or water bath (95 oC), tube B in a water bath of 38 oC to 42oC and tube C and D at room temperature. What purpose does having tube C placed in water at room temperature? Why isn’t tube D placed at high temperature?

6. After 2 minutes, add one stained potato cube to each of the four test tubes. Start the stop watch immediately. Explain the significance of the 2 minutes of incubation before adding the tissue in.

7. After 2 minutes, remove tubes A and B from the water baths and place them in the rack with tubes C and D. Shake the tubes. Explain the significance of the 2 minutes of incubation.

8. Immediately separate the tissue from solutions. For each tube, quickly pour away the liquid into another the corresponding test tubes labeled A’ to D’. Immediately record your observations of each test tube. [Some guidelines (non- exhaustive): What’s the texture (i.e., soft/ flaccid, hard/ turgid?) and colour o f the tissue? What’s the colour of the liquid and how much light can pass through? How will you record the difference in intensity of colouration of the liquid?] Explain the reason for separating the tissue from solutions after adding the tissue in.

9. Repeat the experiment using fresh potato cubes. Why repeat the experiment again?


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Results and Discussions: Table of Observations Data to tabulate:  Tube A, B, C  Observations (please number observations according to meaning assigned to values in table below):  Compile at least 2 sets of data. Unless otherwise instructed by tutor, you may calculate average where necessary. *

Texture of tissue

Colouration of tissue Colouration of solution (potato cubes) 1 Very soft/ very flaccid Colourless Colourless 2 Soft/ flaccid Light blue Light blue 3 Hard/ turgid Blue Blue 4 Very hard/ very turgid Dark blue Dark blue * You may use just 3 numbers if the differences between tubes are not that clear.


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Assignments Please check with your tutor which option is required for you. Option 1: (please refer to WBLE/ Turnitin for instructions which may incorporate other options below) Option 2: Skills-Based Assessment: Tabulation of qualitative data Option 3: Skills-Based Assessment: Discussion Write your discussion in prose form and without numbering. Excluding your cover page, your discussion and conclusion should NOT exceed ONE A4 page of Word document (standard/ default size). Anything in excess will NOT be graded. • Font Arial, size 11. • Margins: 1 inch from top, bottom, left and right (no need to change if you’re using the standard/ default size when MS Word opens). • Theory to apply: Refer to relevant information from lecture topics which may or may not have been covered yet. From the data you have collected in the practical, account fully for the observations you have made and draw clear conclusions, using your knowledge and understanding. You will need to use the following questions as guidelines. 1. Why are the potato cubes stained with methylene blue? 2. What happens to the stained potato cubes when they are placed in water at room temperature? 3. What are the effects of temperature on potato cubes ’ cell components in tubes A, B and C? 4. What are the effects of the ethanol on the potato cubes ’ cell components? [Hint: is ethanol hydrophobic or hydrophilic? Which part of it?] 5. State the reason for using equal volumes of liquids in all tubes. 6. Explain why the potato cubes added after the tubes are left at the respective conditions for 2 minutes. 7. What is the purpose of repeating the experiments by doing 2 sets?


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Practical 9 (FHSC 1214 Cell Biology); Practical 8 (FHSB 1214 Biology I) Respiration of Germinating Beans ______________________________________________________________________ Objective: To investigate the aspects of respiration in germinating mung bean seeds. To understand the concept of respiratory quotient. Apparatus and Materials: Syringe Gauze rubber tube

capillary tube colour dye beans

Introduction: Respiratory quotient Metabolic energy comes primarily from oxidative reactions. As a result, the more highly reduced a respiratory substrate, the higher potential it has for generating biological energy. When a respiratory substrate (eg. glucose) is oxidized for energy, carbon dioxide is produced. The volume (or moles) of carbon dioxide produced with reference to the volume (or moles) of oxygen consumed during oxidation of a respiratory substrate for a fixed period of time is termed as the respiratory quotient (RQ).

RQ =

volume of CO 2 produced volume of O 2 consumed

RQ gives indication of the  type of respiration,  nature of respiratory substrate, and hence amount of metabolic energy that can be produced.  For example, the complete oxidation of glucose is represented by the following equation: C6H12O6 + 6O2

6CO2 + 6H2O + energy

RQ for glucose = 6/6 = 1.0


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In general, the lower the respiratory quotient, the more oxygen is required for complete oxidation of a respiratory substrate, and hence the greater the potential for generating ATP from that respiratory substrate. The table given illustrates some common values and their significance. When RQ is >1.0

Carbohydrates are used as a respiratory substrate with some anaerobic respiration occurring simultaneously.

1.0

Carbohydrates are used as the respiratory substrate.

0.7

Mainly fats are being used as the respiratory substrate.

0.8 – 0.9

A mixture of carbohydrates, lipids and proteins are used as respiratory substrate

0.85

A mixture of carbohydrates and lipids are used as respiratory substrates.

0.9

Proteins are the respiratory substrates. Note that the composition of proteins is too varied for them to give the same RQ. However, most of them have a value around 0.9

You are required to investigate some aspects of respiration in germinating green bean seeds, using the apparatus shown in Figure 1.

Figure 1 Warning: Do not remove the soda lime from the syringe as it will burn your skin. Flowchart Students will be allowed to proceed with the experiment only if they have come into the laboratory with a flowchart of the day’s experiment. Procedures: Create a flowchart before you enter the lab in order to understand the steps in this experiment. Show this to your tutor before starting the experiment. 1. You are provided with some germinating green bean seeds and a coloured liquid. 2. Place four or five of these green bean seeds into the barrel of the syringe and carefully replace the plunger. 3. Attach the length of glass capillary tube to the syringe, using the rubber tubing provided.


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4. Dip the end of the glass capillary tube into the coloured liquid so that a drop of liquid enters the capillary tube. Remove any excess liquid with paper towelling. 5. Place the apparatus on a sheet of white paper alongside a milimeter ruler. Your assembled apparatus should now look like that shown in Fig. 1. 6. Wait until the drop of coloured liquid starts to move. 7. Mark the position of the coloured liquid on the capillary tube with a marker pen. 8. Measure how far the liquid moves in one minute. Repeat the measurement every minute for the next nine minutes. 9. If you do not get any liquid movement after 3 minutes, adjust the apparatus (e.g., add one more germinating seed, readjust the rubber connecting tube and syringe tip etc.). Do NOT touch the point of connection between the tube and glass capillary. 10. If you’ve obtained reasonable readings, you may dismantle the apparatus and dispose the used soda lime (without touching it) in the syringes into the beakers provided. 11. Record your results also on the whiteboard.

Results and Discussions: If you are required to prepare an individual report, complete procedures, results and discussion. The results and discussion answers to questions ‘a’ to ‘h’. Questions ‘c’ to ‘h’ not exceed 2 conclusion is not required. 1. Construct a table and record your results of how far the liquid over ten minutes.

with a write up on section will be your pgs of your report. A moves in one minute

2. Plot your results on a piece of graph paper. Use a best fit line. 3. From your table, calculate the mean distance travelled in mm min -1. Show your working. 4. Assume the diameter of the capillary tube hollow is 0.2 mm. The area of the end of the capillary tube can be calculated by using the formula πr 2, where π = 22/7. Calculate the volume of gas that is absorbed by the seeds in one hour. Show your working. 5. With reference to respiration of the green bean seeds, explain why the drop of liquid moves along the capillary tube. 6. The formula used for calculating the RQ (respiratory quotient)is given as follows: RQ = Vol. of carbon dioxide evolved during respiration Vol. of oxygen absorbed during respiration Lab manual version 4_201405 Biology I & Fundamentals of Cell Biology

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Explain how you would use or modify the apparatus in our experiment to calculate the RQ of the seeds. 7. Experiments of this kind are very susceptible to changes in temperature. Explain how you could compensate for any temperature changes during the experiment. 8. Discuss the sources of errors and ways to improve the experiment. You may provide answers other than the ones below as long as you follow this tabulated format.

Sources of error

Explanation

Improvement

Explanation This will ensure that the change in volume of air in the syringe is due to oxygen absorbed by the green bean during the experiment. This will ensure constant rate of respiration as oxygen is not a limiting factor. This will ensure a more accurate measurement of the rate of respiration of the green beans in the specified environment. Movement in liquid will be more sensitively detected.


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Assignments Please check with your tutor which option is required for you. Option 1: (please refer to WBLE/ Turnitin for instructions which may incorporate other options below) Option 2: Skills-Based Assessment: Tabulation of quantitative data Tabulate a table on how far the liquid moves in one minute over ten minutes Option 3: Skills-Based Assessment: Graphing of quantitative data Present your graph (pasted from Excel). Use a best fit curve. To get full marks, please observe the guidelines given on pp6-7 as well. No need to write procedure, draw table and write a discussion or conclusion. Option 4: Skills-Based Assessment: Discussion Write your discussion in prose form and without numbering. Excluding your cover page, your discussion and conclusion should NOT exceed ONE A4 page of Word document (standard/ default size). Anything in excess will NOT be graded. • Font Arial, size 11. • Margins: 1 inch from top, bottom, left and right (no need to change if you’re using the standard/ default size when MS Word opens). • Theory to apply: Refer to relevant information from lecture topics which may or may not have been covered yet.


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Practical 9 Exercise 1 (FHSB 1214 Biology I ONLY) Microscopic Examination of Cells at Various Stages of Plant Mitosis

Objective: To examine the cell at various stages of the mitotic cell cycle microscopically. Equipment: Binocular microscope Slides provided: Onion mitosis Root tip, Allium l.s. Onion mitosis Root tip, Allium c.s. Note to instructor: this practical may be divided into two sessions, one for mitosis and another for meiosis.


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Introduction The primary root system i. The apical meristem of the root. The most obvious differences in appearance between longitudinal section of stem and root apices is the absence of bulges comparable to leaf and bud primordial on the root apex. The root apex is also covered by root cap (Fig 24.6). There is, however, a marked similarly in appearance and behaviour of the apical cells which constantly divide by mitosis, in most roots it is possible to distinguish a number of zones of cells at the apex. The outermost zone is called the protoderm. It produces cells which differentiate into the root epidermis and root cap. Inside the protoderm is the ground meristem, the derivatives of which differentiate into the root cortex. Just behind the root apex a single procambial strand can be seen at the centre of the root. Some roots have an additional meristematic layer, the calyptrogen, which gives rise to the cells of the root cap. The meristematic zones radiate from a clump of cells called the quiescent centre situated immediately behind the root cap. The significance of the quiescent centre is not as yet fully understood. Its cells divide slowly and it is probably the site from which the other meristematic layers arise. ii. Tissue differentiation in the root. Differentiation of vascular tissue begins near the root apex. Several strands of sieve-tube elements and companion cells appear near the outside of the procambial strand. Shortly afterwards a similar number of strands of protoxylem cells alternating with the primary phloem strands differentiate, Metaxylem cells differentiate last of all at the centre of the procambial strand. The outermost procambial cells undergo litter change and retain their ability to divide. They become the pericycle which may later produce lateral roots.


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Acknowledgment: Student name: Hon Lair Teng Foundation in Science, CFS-PJ Intake: Jan 2010 Title: Photo of onion mitosis root tip, allium longitudinal section Magnification power: 10x.100x


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Acknowledgment: Student name: Joanne Liew Hui Qi Foundation in Science, CFS-PJ Intake: Jan 2010

Title: Detailed Photos of Onion Mitosis Root Tip, Allium (Longitudinal Section) Magnification power: 100x.10x

Photo 1

Photo 2


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Photo 3 Assignment: Option 1: (please refer to WBLE/ Turnitin for instructions which may incorporate other options below) Consult your lecturer on which assignment is required. Students are allowed to make either drawings or photographs. For either presentation, please ensure the general conventions are followed (please refer to Notes on Biological Drawings) with at least the following appearing:  Title  Magnification power  Labels, where visible, plus annotations (descriptive): chromosomes, nucleolus, nuclear envelope, cell wall etc. Option 2: Photography 1. Take one or more photographs showing any one of the following:  At least three cells each showing any 3 mitotic phases  At least two cells each showing any 2 mitotic phases, for each l.s. and c.s. slide viewed 2. Label in a Word or Powerpoint file. 3. Upload to the given WBLE link. 4. As part of an on-site assessment, identify for or show your lecturer 2 stages of mitosis when called on. Option 3: Drawing 1. Make one detailed drawing showing at least one cell undergoing 1 mitotic phase. Include also several surrounding cells (they can be of the same or different phase). Please refer to Sem 2 lab manual, “ In making high-pow er detailed , repeated features need not all be drawn but only a small drawings portion showing a few large accurate cells (3 or 4 adjacent cells) representative of each type must be indicated.” 2. As part of an on-site assessment, identify for or show your lecturer 2 stages of mitosis when called on.


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Option 4: Answer the following questions 1. From the slides viewed, which is the most: a. Frequently observed phase? Why?

(2 marks)

b. The least? Why?

(2 marks)

2. Figure 1 shows drawings of cell at various stages in the mitotic cell cycle.

Figure 1 a) List the letter shown in Figure 1 in the order in which these stages occur during a mitotic cell cycle. The first stage has been entered for you. (1 mark) A  _____



_____  _____



_____

Explain what is happening in stage D in Figure 1.

(2 marks)

b) Describe in outline what happens to the DNA in the nucleus during stage A in Figure 1. (2 marks) c) State the importance of mitosis in the growth of a multicellular organism, such as a flowering plant or a mammal. (2 marks) 3. Figure 2 shows four animal cells in different stages of mitotic division.

Figure 2 a) Name the structures labelled A, B, C and D.

(4 marks)

b) Name the stages of division shown by cells 1 and 3.

(2 marks)

c) Using the number given to each cell above, arrange the stages as they occur in the mitotic sequence. (1 mark)


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Practical 9 Exercise 2 (FHSB 1214 Biology I ONLY) Reproductive Tissues in Plants (Histology of Plant - Lily Reproductive Structures) [Meiosis]

Slides provided: Lily Anther early Prophase c.s. Lily Anther late Prophase c.s. Lily Anther First Meiotic division c.s. Lily Anther second Meiotic division c.s. Lily Anther Pollen Tetrad

Introduction: This final histology schedule will deal with structures directly associated with reproduction. Unlike asexual reproduction, where only single parent is involved and where offspring are identical in hereditary characters to the parent, sexual reproduction involves production of male and female gametes in specialized organs. This schedule is concerned with these organs in mammals and angiosperms. Fusion of the nuclei of these gametes results in the formation of the zygote that ultimately develops into the offspring showing a combination of characteristics from both parents.

Brooker 1e


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Brooker 1e Lily Flower Bud TS: This slide should be examined with the naked eye and then under low power. Prepare a tissue map only. You may draw more than one ovule. However, you need only label one ovule. Note the following: 1. There are 6 stamens (anthers specifically), each a 4-lobed structure. Note the pollen grains within each lobe. 2. The 3-loculate (i.e., chamber of three parts) ovary in the centre with the ovules.

Transverse Section of Flower Bud Lab manual version 4_201405 Biology I & Fundamentals of Cell Biology

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Lilium Anther 2-cell Stage TS:

This is a dehisced stage of the anther. Comparison with the 4-celled stage seen in the preceding slide should be made. Dehiscence is usually preceded by breakdown of the partition between the locules of one half. Note the following: 1. The bilayered wall of each locule – made up of the outer epidermis and an inner fibrous layer (endothecium). 2. The stomium or opening from the break is slit-like. 3. The single vascular bundle (connective) found in between the two lobules. 4. The thick exine of each pollen grain. In many of the pollen grains two nuclei will be seen – the vegetative and generative nuclei. 5. Use an appropriate objective lens with which you can make a detailed drawing with labels such as the anther filament, wall-tissue, pollen sac and grains.

http://www.cartage.org.lb/en/themes/Sciences/BotanicalSciences/ClassificationPlants /Spermatophyta/Angiosperms/FlowerStructure/anther.gif

Microspore tetrads in Lilium anther. http://sols.unlv.edu/Schulte/Anatomy/Repro/LiliumAntherDiv1.jpg


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Second division in microsporocytes of a Lilium microsporangium http://sols.unlv.edu/Schulte/Anatomy/Repro/LiliumAntherDiv2a.jpg


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Lilium Embryo Sac, 4-nucleate Stage TS:

This slide is a TS of the entire ovary which is 3 loculate and has 2 ovules per locule. Compare this with the younger stage seen in the slide of Lily flower bud TS where the ovules are only small groups of undifferentiated cells or ovular buds. Ovules in this slide are already differentiated into the t ypical shape of an anatropous ovule. 1. Focus the embryo sac under high power and note, in some of them, the 4 nuclei present. This will be the stage just before the formation of the typical 8-nucleate embryo sac seen in most angiosperms. The 4-nuclei seen will probably be in interphase. 2. Draw a tissue plan map of the embryo sac as viewed under high power (your choice) to the best of your ability. Be sure to draw the nucleated portions of the embryo sac distinctly. Include relevant labels for portions related to the ovule, to the best of your ability.

Lilium embryo sac development - four nucleate stage.

http://sols.unlv.edu/Schulte/Anatomy/Repro/LiliumFourNucleate.jpg

Lilium embryo sac development - two nucleate stage

http://sols.unlv.edu/Schulte/Anatomy/Repro/LiliumTwoNucleate.jpg


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Lilium Early Embryo TS:

After fertilization, in general, the ‘triple fusion nucleus’ divides to form endosperm tissue first and the zygote divides shortly afterwards to form the embryo. 1. Observe, under low power, the young embryo (proembryo) embedded in the endosperm tissue. Cells of both embryo and endosperm are densely stained. Note the very large nuclei relative to cell size in the young embryo. No distinction into basal cell, suspensor, etc. can be made here. 2. Using an appropriate objective lens, draw a tissue map clearly indicating the position of embryo and approximate the region of the endosperm as well. If visible, include relevant labels for portions related to the fertilized ovule, to the best of your ability.

http://www.botany.hawaii.edu/faculty/webb/Bot201/Angiosperm/MagnoliophytaLab99/ EmbLilyEndoOverview240Lab.jpg

Figure Transverse Section of Lily Ovary For your assignment, one to two slides may be drawn [please consult your lecturer]. If two drawings are to be done, each student is allowed only 30 mins per slide for drawings to be done in the 1 st half. You are required to label according to the instructions given. Lab manual version 4_201405 Biology I & Fundamentals of Cell Biology

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Title: Meiotic phases of lily anther cross section Magnification: 10x.60x

(Picture & labels by Amy Tan En Ling, 2010)


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Title: Meiotic phases of lily anther cross section. Magnification: 10x.60x



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Title: Meiotic phases of lily anther cross section. Magnification: 10x.60x



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Title: Meiotic phases of lily anther cross section Magnification: 10x•60x



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Title: Photo of Lily Anther Second Meiotic Division C.S. Magnification Power: 10x.40x

(Picture & labels by Gan Hui XIn, 2010)


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Practical 10 Exercise 1 (FHSB 1214 Biology I ONLY) Mitosis and Meiosis Modelling

Objective: To understand and compare the different events occurring in Mitosis and Meiosis. Apparatus and Material: 6 pairs of chromosomes 16 triangular genes 16 circular genes Introduction: Comparison between Mitosis and Meiosis Mitosis Meiosis I Prophase Chromatin condense Synapsis and to form chromosome Crossing over occur Metaphase Individual Homolog align at chromosome align metaphase plate at metaphase plate Anaphase Separation of sister Separation of chromatids homolog Telophase Form 2 daughter Form 2 daughter cells cells

Meiosis II Chromatin condense to form chromosome Individual chromosome align at metaphase plate Separation of sister chromatids Form 4 daughter cells

Procedure: Students are divided into 3 groups to carry out the following activities. Group 1: Mitosis 1. You are provided with a pair of duplicated homologous chromosome in Prophase of a cell. The homologs contain different combination of genes. 2. Based on the given chromosomes, model the cell when it is in Metaphase, Anaphase and Telophase. You are provided with the empty cells (without chromosomes) in each phase to assist you in the modelling. Group 2: Meiosis I 1. You are provided with a pair of duplicated homologous chromosome in Prophase I of a cell. The homologs contain different combination of genes. 2. Based on the given chromosomes, model the cell when it is in Metaphase I, Anaphase I and Telophase I. You are provided with the empty cells (without chromosomes) in each phase to assist you in the modelling. Group 3: Meiosis II 1. You are provided with a duplicated chromosome in Prophase II in each daughter cell arisen from Meiosis I. 2. Based on the given chromosomes, model the cell when it is in Metaphase II, Anaphase II and Telophase II. You are provided with the empty cells (without chromosomes) in each phase to assist you in the modelling.


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Discussion: 1. From the model cell that you have assembled, identify the following structures to your instructor: sister chromatids, chromatins, chromosomes, centromere, centrosome, kinetochore microtubules, non-kinetochore microtubule, aster and metaphase plate. 2. Predict the possible chromosome for the daughter cells of Meiosis I and Meiosis II if crossing over does not occur at Prophase I.


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Practical 10 Exercise 2 (FHSB 1214 Biology I ONLY) DNA replication modelling ___________________________________________________________________ Objective: To study and lear n to construct a replication “bubble” using DNA Simulation Student kit Apparatus and Material: 44 Red beads – phosphate 44 White beads – Deoxyribose sugar 13 Orange beads – Adenine 8 Pink beads – Ribose sugar

13 Yellow beads – Thymine/ Uracil 13 Green beads – Guanine 13 Blue beads – Cytosine 24 Clear connectors – Hydrogen bonds

Introduction: The structure of DNA A nucleotide consists of the pentose sugar deoxyribose, a phosphate, and one of four nitrogenous bases. The nucleotides are linked by covalent bonds to form an alternating sugar-phosphate backbone. No matter how long the chain may be, the 5’ end has a 5’ carbon attached to a phosphate and the 3’ end has a 3’carbon attached to a hydroxyl group.

DNA replication Replication starts at origins of replication, where the two DNA strands are separated, forming a replication “bubble”. DNA polymerases add nucleotides only to the free 3’ end of a growing strand or RNA primer. Thus, a new DNA strand can elongate only in the 5’ to 3’ direction. Procedure: 1. Group yourselves into 2 to 3 students per group. 2. Construct a single stranded DNA with the base sequence as follows: 5’ – AGCACGTAACGTTCGA – 3’ 3. Construct the complementary DNA strand based on the base sequence shown in Step 2. 4. Join the two strands together by using clear connectors (hydrogen bonds). 5. Slightly twist the DNA molecules to observe the double helix structure of DNA. 6. Lay the DNA molecule flat on your bench. 7. Break open 12 base pairs from the two strands in the middle (origin of replication) to form a replication bubble.


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8. Attach a 2-nucleotide RNA primer (use pink beads to represent ribose sugar) to each of the leading strand and lagging strand. 9. Add the respective DNA nucleotides one by one based on the template strand. 10. Before dismantling your completed DNA replication bubble, show and identify the template strand, leading strand, lagging strand, Okazaki f ragments and replication fork to your instructor.

Discussion: 1. Based on the procedure, name the enzymes that participate in: a) Step 7 b) Step 8 c) Step 9 2. Predict how long it will take to produce a 100-nucleotides-long DNA if the elongation process is done by a) you b) the enzyme involve in Step 9.


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Practical 10 (FHSC 1214 Cell Biology ONLY) Respiration of Yeast ___________________________________________________________________ Objective: To investigates the effect of different nutrients on anaerobic respiration of yeast. Apparatus and Equipment: Incubator Test-tubes (5 units) Test tube rack Fermentation tubes (Durham tube) Wash bottle Materials: 20% sucrose solution 20% lactose solution 10% yeast suspension

20% glucose solution 10% starch suspension Distilled water

Procedures: Create a flowchart before you enter the lab in order to understand the steps in this experiment. Show this to your tutor before starting the experiment. 1. Label five test tubes used for fermentation from A to E . 2. Using teat pipettes, place the following solutions into each fermentation glass tube. Fermentation Tube A B C D E

Solution 5 drops distilled water 5 drops glucose solution 5 drops sucrose solution 5 drops lactose solution 5 drops starch solution

3. Add 5 drops of yeast suspension into each fermentation tube. Add distilled water to fill up the fermentation tubes. 4. Using pencil, support the fermentation tube as shown in Diagram I. Without removing the pencil, inverse the fermentation tube. Take care not to spill out the fluid in the fermentation tube.

5. Record the height of fluid in fermentation tubes A to E, as shown in Diagram II.


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6. Place all the tubes in the incubator at 37 oC – 40 oC for 30 minutes (in the absence of an incubator, place your tubes outside the lab or in a non-airconditioned room, being careful not to bump into anything that will displace your Durham tubes). 7. Remove the tubes from the incubator and measure the final height of the fluid in each fermentation tube. 8. Record the difference in height between the initial and final readings for each of the tubes. Results: (title) Tube

Nutrient

A

Distilled water

B C D E

Initial height of fluid, x /mm

Final height of fluid, y /mm

Difference (x -y )/mm

Glucose Sucrose Lactose Starch solution

Washing up Please rinse the small fermentation glass tubes within the beaker provided so that none will slip down the sink hole.


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Biology 1 Lab Manual 201405

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