LAB 2: ASEPTIC TECHNIQUE AND TRANSFER OF MICROORGANISMS DISCUSSION In natural environments, microorganisms usually exist as mixed populations. However, if we are to study, characterize, and identify microorganisms, we must have the organisms in the form of a pure culture. A pure culture is one in which all organisms are descendants of the same organism. Techniques for obtaining pure cultures from a mixed population will be described in ab !. In wor"ing with microorganisms we must also have a sterile nutrient# containing#medium containing#medium in which to grow the organisms. Anything Anything in or on which we grow a microorganism is termed a medum. medum. A !terle medium is one which is free of all life forms. It is usually sterilized by heating it to a temperature at which all contaminating contaminating microorganisms are destroyed. $inally, $inally, in wor"ing with microorganisms, we must have a method of transferring growing organisms %called the "#culum& "#culum& from a pure culture to a sterile medium without introducing any unwanted outside contaminants. This method of preventing unwanted microorganisms from gaining access is termed $!eptc tec%"&ue. tec%"&ue. 'eep " m"d t%$t (#u mu!t )e$r t%e c#rrect Per!#"$l Pr#tect#" E&upme"t *PPE+ " *PPE+ " $ll l$,! )%ere (#u $re u!"- mcr#,$l culture!. !t$"!. c%emc$l!. $"d -l$!!)$re #r mcr#!c#pe !lde!/ 'oncept map for ab () Terminology
A0 ASEPTIC TECHNIQUE The procedure for aseptically transferring microorganisms is as follows) *. U!"*. U!"- $ mcr#"c"er$t#r t# !terl1e t%e "#cul$t"- l##p Mcr#"c"er$t#r! enable Mcr#"c"er$t#r! enable the sterilization of inoculating loops without having to use an open flame of a +unsen burner. The microincinerator uses infrared heat at a temperature of *-' to sterilize the wire portion of the inoculating loop. Be c$reul "#t t# t#uc% t# uc% t%e t#p p#rt#" # t%e mcr#"c"er$t#r0 mcr#"c"er$t#r0 It ,ec#me! 3er( %#t/
a. At t%e !t$rt # cl$!!0 tur" #" t%e mcr#"c"er$t#r $"d )$t 45 m"ute! #r t t# %e$t up0 b. Pl$ce t%e e"tre )re p#rt#" # t%e "#cul$t"- l##p "t# t%e #pe""# t%e mcr#"c"er$t#r $"d %#ld t t%ere #r 45 !ec#"d! %see $ig. *A&. In this way all contaminants on the wire are incinerated. T# $3#d ,ur""- (#ur %$"d #" t%e %$"dle # $" #3er%e$ted "#cul$t"- l##p. "e3er l$( t%e l##p d#)" " t%e mcr#"c"er$t#r $"d t%e" $ttempt t# pc6 t up. /ever lay the loop down once it is sterilized or it may again become contaminated. c. All#) t%e l##p t# c##l 45725 !ec#"d! before removing the inoculum. (. Rem#3e t%e "#culum0 $0 Rem#3"- "#culum r#m $ ,r#t% culture %organisms growing in a liquid medium&) *. Hold the culture tube in one hand and in your other hand, hold the sterilized inoculating loop as if it were a pencil %see $ig. *&. (. Rem#3e t%e c$p # t%e pure culture tu,e )t% t%e lttle "-er # (#ur l##p %$"d. %see $ig. *+ and $ig. *+(&. Ne3er l$( t%e c$p d#)" or it may become contaminated. !. Pl$ce t%e lp # t%e culture tu,e $t t%e #pe""- # t%e mcr#"c"er$t#r #r 278 !ec#"d! %see $ig. *'&. This heats the glass and creates a convection current which forces air out of the tube and prevents airborne contaminants from entering the tube. 0. I"!ert t%e "#cul$t"- l##p $"d rem#3e $ l##pul # "#culum %see $ig. *1&. 2. Again pl$ce t%e lp # t%e culture tu,e $t t%e #pe""- # t%e mcr#"c"er$t#r #r 278 !ec#"d! %see $ig. *3&. -. Immed$tel( repl$ce t%e c$p %see $ig. *$&. Rem#3"- t%e "#culum r#m $ ,r#t% tu,e ! !umm$r1ed " F-0 40
b. Rem#3"- "#culum r#m $ pl$te culture %organisms growing on an agar surface in a petri plate&) *. Sterl1e t%e "#cul$t"- l##p ,( pl$c"- t " t%e mcr#"c"er$t#r #r 45 !ec#"d!. %see $ig. !A&. (. ift the lid of the culture plate slightly and stab the loop into the agar away from any growth to cool the loop. !. 4crape off a !m$ll $m#u"t of the organisms and immediately close the lid %see $ig. !+&. !. Tr$"!er t%e I"#culum t# t%e Sterle Medum0 a. Tr$"!err"- t%e "#culum "t# $ ,r#t% tu,e: *. 5ic" up the sterile broth tube and rem#3e t%e c$p )t% t%e lttle "-er # (#ur l##p %$"d %see $ig. (A&. D# "#t !et t%e c$p d#)". (. Pl$ce t%e lp # t%e culture tu,e $t t%e #pe""- # t%e mcr#"c"er$t#r #r 278 !ec#"d! %see $ig. (+&. !. Pl$ce t%e l##pul # "#culum "t# t%e ,r#t%, and withdraw the loop %see $ig. ('&. D# "#t l$( t%e l##p d#)"/ 0. Again pl$ce t%e lp # t%e culture tu,e $t t%e #pe""- # t%e mcr#"c"er$t#r #r 278 !ec#"d! %see $ig. (1&. 2. Repl$ce t%e c$p %see $ig. (3&. -. Re!terl1e t%e "#cul$t"- l##p ,( pl$c"- t " t%e mcr#"c"er$t#r #r 45 !ec#"d! %see $ig. ($&. /ow you may lay the loop down until it is needed again. Tr$"!er"- t%e "#culum "t# $ ,r#t% tu,e ! !umm$r1ed " F-0 20 b. Tr$"!err"- t%e "#culum "t# $ petr pl$te: *. If the agar surface of the plate is visibly wet, use a sterile swab to gently remove the water. (. 6n the bottom of the petri plate, d3de t%e pl$te "t# t%rd! )t% (#ur )$9 m$r6er $"d l$,el $! !%#)" ,el#) . This will guide your strea"ing.
!. Lt t%e ed-e # t%e ld u!t e"#u-% t# "!ert t%e l##p. 0. Stre$6 t%e l##p $cr#!! t%e !ur$ce # t%e $-$r medum u!"- t%e et%er t%e p$tter" !%#)" " $ig. 0 #r t%e p$tter" !%#)" " $ig. 2. These strea"ing patterns allow you to #,t$" !"-le !#l$ted ,$cter$l c#l#"e! originating from a single bacterium or arrangement of bacteria%see $ig. -&. In order to avoid digging into the agar as you strea" the loop over the top of the agar you must 6eep t%e l##p p$r$llel t# t%e t%e $-$r !ur$ce. Always start strea"ing at the 7*()88 position7 %see $ig. !'& of the plate and strea" side#to#side as you pull the loop toward you. As you follow either $ig. 0 or $ig. 2, each time you flame and cool the loop between sectors, rotate the plate countercloc"wise so you are $l)$(! )#r6"- " t%e ;42:55 p#!t#"; # t%e pl$te. This "eeps the inoculating loop parallel with the agar surface and helps prevent the loop from digging into the agar. 2. 9emove the loop and immediately close the lid. -. Re!terl1e t%e "#cul$t"- l##p ,( pl$c"- t " t%e mcr#"c"er$t#r #r 45 !ec#"d! %see $ig. !1&. $lash animation showing how to strea" an agar plate for isolation) ! sector method. html2 version of animation for i5ad showing how to strea" an agar plate for isolation) ! sector method. :I$ animation showing how to strea" an agar plate for isolation)
2 sector method. ;ouTube movie showing how to strea" an agar plate for isolation) 0 sector method. +lue 9idge 'ommunity 'ollege,
See t%e l"6! ,el#) t# 3e) !#l$ted c#l#"e! #" t%ree !ect#r !tre$6 pl$te!. •
4trea" plate =*) Serratia marcescens on trypticase soy agar.
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4trea" plate =() Escherichia coli on trypticase soy agar.
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4trea" plate =!) $ecal specimen on blood agar.
I" t%e uture. e3er( pr#cedure " t%e l$, )ll ,e d#"e u!"- !ml$r $!eptc tec%"&ue0
B0 FORMS OF CULTURE MEDIA *. +roth tubes are tubes containing a liquid medium. A typical nutrient containing broth medium such as Trypticase 4oy broth contains substrates for microbial growth such as pancreatic digest of casein, papaic digest of soybean meal, sodium chloride, and water. After incubation, -r#)t% %development of many cells from a few cells& may be observed as one or a combination of three forms) a. Pellcle) A mass of organisms is floating on top of the broth %see $ig. >A&. b. Tur,dt() The organisms appear as a general cloudiness throughout the broth %see $ig. >+&. c. Sedme"t) A mass of organisms appears as a deposit at the bottom of the tube %see $ig. >'&. (. 4lant tubes %see $ig. A&are tubes containing a nutrient medium plus a solidifying agent, agar#agar. The medium has been allowed to solidify at an angle in order to get a flat inoculating surface %see $ig. +&.
!. 4tab tubes %deeps& are tubes of hardened agar medium which are inoculated by 7stabbing7 the inoculum into the agar %see $ig. ?&. 0. Agar plates are sterile petri plates that are aseptically filled with a melted sterile agar medium and allowed to solidify. 5lates are much less confining than slants and stabs and are commonly used in the culturing, separating, and counting of microorganisms. 4ingle colonies of microorganisms on agar plates can be described using the terms found in Appendix A. 'oncept map for ab () Terminology
C0 O<=GEN REQUIREMENTS FOR MICROBIAL GRO>TH @icroorganisms show a great deal of variation in their requirements for gaseous oxygen. @ost can be placed in one of the following groups) *. O,l-$te $er#,e! are organisms that grow #"l( in the presence of oxygen. They obtain energy from aerobic respiration. (. Mcr#$er#p%le! are organisms that require a low concentration of oxygen for growth. They obtain energy from aerobic respiration. !. O,l-$te $"$er#,e! are organisms that grow #"l( without oxygen and, in fact, oxygen inhibits or "ills them. They obtain energy from anaerobic respiration or fermentation. 0. Aer#t#ler$"t $"$er#,e!, li"e obligate anaerobes, cannot use oxygen for growth but they tolerate it fairly well. They obtain energy from fermentation. 2. F$cult$t3e $"$er#,e! are organisms that grow with or without oxygen, but generally better with oxygen. They obtain energy from aerobic respiration, anaerobic respiration, and fermentation. @ost bacteria are facultative anaerobes. 'oncept map for ab () Terminology
D0 TEMPERATURE REQUIREMENTS
@icroorganisms have a minimum and maximum temperature at which they can grow, as well as an #ptmum temper$ture )%ere t%e( -r#) ,e!t. @icroorganisms can be divided into groups on the basis of their preferred range of temperature) *. P!(c%r#p%le! are cold#loving bacteria. Their optimum growth temperature is between #2' and *2'. They are usually found in the Arctic and Antarctic regions and in streams fed by glaciers. (. Me!#p%le! are bacteria that grow best at moderate temperatures. Their optimum growth temperature is between (2' and 02'. @ost bacteria are mesophilic and include common soil bacteria and bacteria that live in and on the body. !. T%erm#p%le! are heat#loving bacteria. Their optimum growth temperature is between 02' and >8' and are comonly found in hot springs and in compost heaps. 0. H(pert%erm#p%le! are bacteria that grow at very high temperatures. Their optimum growth temperature is between >8' and **8'. They are usually members of the Archae and are found growing near hydrothermal vents at great depths in the ocean. 'oncept map for ab () Terminology
E0 COLON= MORPHOLOG= AND PIGMENTATION A colony is a visible mass of microorganisms growing on an agar surface and usually originating from a single organism or arrangement of organisms. 1ifferent microorganisms will frequently produce colonies which differ in their morphological appearance %form, elevation, margin, surface, optical characteristics, and pigmentation&. 4ingle colonies can be described using standard terms, as listed in Appendix A. •
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4canning electron micrograph of a microcolony of Escherichia coli . Animation of +acterial 'olony @orphology. Hussein 4hoeb, author. icensed for use, A4@ @icrobeibrary.
5robably the most visual characteristic is p-me"t$t#" %color&. 4ome microorganisms produce pigment during growth and are said to be c%r#m#-e"c. 6ften, however, formation of pigment depends on environmental factors such as temperature, nutrients, pH and moisture. $or example, Serratia marcescens produces a deep red pigment at (2', but does not produce pigment at !>'. 5igments can be divided into two basic types) water#insoluble and water# soluble. If the pigment is )$ter7"!#lu,le %see $ig. *8A&, as is the case with most chromogenic bacteria, it does not diffuse out of the organism. As a result, the colonies are pigmented but the agar remains the normal color. If the pigment is )$ter7!#lu,le as in the case of Pseudomonas aeruginosa %see $ig. *8+ and $ig. *8'&, it will diffuse out of the organism into the surrounding medium. +oth the colonies and the agar will appear pigmented. +elow is a list of several common chromogenic bacteria) •
Staphylococcus aureus # goldB water#insoluble
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Micrococcus luteus # yellowB water#insoluble
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Micrococcus roseus # pin"B water#insoluble
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Mycobacterium phlei # orangeB water#insoluble
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Serratia marcescens # orangeCredB water#insoluble
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Pseudomonas aeruginosa $ig. *8+ and Pseudomonas aeruginosa $ig. *8+ # greenCblueB water#soluble 'oncept map for ab () Terminology
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MEDIA Trypticase 4oy +roth tubes %0&, Trypticase 4oy Agar slant tubes %0&, Trypticase 4oy Agar stab tubes %0&, and Trypticase 4oy Agar plates %>&.
ORGANISMS Trypticase 4oy +roth cultures of Bacillus subtilis, Escherichia coli and Micrococcus luteus, and Trypticase 4oy Agar plate cultures of Mycobacterium phlei . PROCEDURE *t# ,e d#"e " p$r!+ *. Aseptically inoculate one Trypticase 4oy +roth tube, one Trypticase 4oy Agar slant tube, one Trypticase 4oy Agar stab tube, and one Trypticase 4oy Agar plate withB. subtilis. %4ee $ig. **& 9emember to label all tubes with a wax mar"er. Dhen strea"ing the agar plates, use either of the patterns shown in $ig. 0 or $ig. 2. This procedure is termed!tre$6"- #r !#l$t#" and has a diluting effect. The friction of the loop against the agar causes organisms to fall off the loop. /ear the end of the strea"ing pattern, individual organisms become separated far enough apart on the agar surface to give rise to !#l$ted !"-le c#l#"e! after incubation. $lash animation showing how to strea" an agar plate for isolation) ! sector method. :I$ animation showing how to strea" an agar plate for isolation) 2 sector method. ;ouTube movie showing how to strea" an agar plate for isolation) 0 sector method. +lue 9idge 'ommunity 'ollege,
(. Aseptically inoculate one Trypticase 4oy +roth tube, one Trypticase 4oy Agar slant tube, one Trypticase 4oy Agar stab tube, and one Trypticase 4oy Agar plate withE. coli. %4ee $ig. **& !. Aseptically inoculate one Trypticase 4oy +roth tube, one Trypticase 4oy Agar slant tube, one Trypticase 4oy Agar stab tube, and one Trypticase 4oy Agar plate withM. luteus. %4ee $ig. **& 0. Aseptically inoculate one Trypticase 4oy +roth tube, one Trypticase 4oy Agar slant tube, one Trypticase 4oy Agar stab tube, and one Trypticase 4oy Agar plate withM. phlei . %4ee $ig. **&
2. Incubate all the tubes and plates inoculated with B. subtilis, E. coli , M. luteus, and M. phlei $t 8?C. 5lace the tu,e! " (#ur dedd$ted te!t tu,e r$c6. I"cu,$te t%e petr pl$te! up!de d#)" %lid on the bottom& $"d !t$c6ed " t%e petr pl$te %#lder #" t%e !%el # t%e 8?C "cu,$t#r c#rre!p#"d"- t# (#ur l$, !ect#". Incubating the plates upside down prevents condensing water from falling down on the growing colonies and causing them to run together. %4tore your test tube rac" on your incubator shelf when not in use.& -. In order to illustrate that microorganisms are all around us and to demonstrate the necessity for proper aseptic technique, c#"t$m"$te three Trypticase 4oy Agar plates as follows) a. 9emove the lid from the first agar plate and place the exposed agar portion in or out of the building for the duration of todayEs lab. 9eplace the lid, label the plate 7air7, and incubate it up!de7d#)" $t r##m temper$ture0 D# t%! pl$te r!t0 b. Fsing a wax mar"er, divide a second petri plate in half. ;ou and your partner both moisten a sterile cotton swab in sterile water. 9ub your swab over some surface in the building or on yourself. Fse this swab to inoculate your half of the second agar plate. abel the plate and incubate up!de7 d#)" $t r##m temper$ture. c. Dith a wax mar"er, divide a third petri plate into quartersand label as shown in $ig. *(. 6n your half of the plate, first rub the fingers of one of your gloved hands over your 7glove7 quadrant. 9emove that glove and rub your fingers over your 7fingers7 quadrant. ;our partner will do the same on his or her half of the plate. abel the plate and incubate up!de7d#)" " (#ur petr pl$te %#lder $t 8?C. D# t%! pl$te l$!t0 Retur" t# Me"u #r L$, 2
RESULTS *. 1raw and describe the growth seen in each of the four broth cultures.
Bacillus subtilis
Escherichia coli
Micrococcus luteus
:rowth G
:rowth G
:rowth G
Mycobacterium phlei
:rowth G
(. 6bserve the growth in the slant cultures and stab cultures for pigmentation and purity. !. Fsing the terms in the Appendix A, compare a single colony of B. subtilis with a single colony of M. luteus. Fse a hand lens or a dissecting microscope to magnify the colony. •
single colonies of Bacillus subtilis
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single colonies of Micrococcus luteus
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single colonies of Escherichia coli
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single colonies of Mycobacterium phlei
c%$r$cter!tc!
B. subtilis
M. luteus
$orm of colony 3levation @argin %edge& 4urface 6ptical characteristics 5igmentation
0. 6bserve the results of the three 7contamination7 plates and note the differences in colony appearances. 2. 6bserve the demonstration plates of chromogenic bacteria and state the color and water#solubility of each pigment. #r-$"!m
C#l#r
S#lu,lt(
Micrococcus luteus Micrococcus roseus Mycobacterium phlei Serratia marcescens Staphylococcus aureus Pseudomonas aeruginosa $ig. *8+ andPseudomonas aeruginosa $ig. *8'
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PERFORMANCE OB@ECTIES FOR LAB 2 After completing this lab, the student will be able to perform the following obectives) DISCUSSION
*. 1efine the following terms) pure culture, sterile medium, inoculum, aseptic technique, and colony. (. 4tate and define the three types of growth that may be seen in a broth culture. !. 1efine the following terms) obligate aerobe, microaerophile, obligate anaerobe, aerotolerant anaerobe, and facultative anaerobe. 0. 1efine the following terms) psychrophile, mesophile, thermophile, and hyperthermophile. 2. 1efine the following terms) chromogenic, water#soluble pigment, and water# insoluble pigment. PROCEDURE *. Fsing an inoculating loop, demonstrate how to aseptically remove some inoculum from either a broth tube, slant tube, stab tube, or petri plate, and inoculate a sterile broth tube, slant tube, stab tube, or petri plate without introducing outside contamination. (. abel all tubes and plates and place them on the shelf in the incubator corresponding to your lab section. !. 9eturn all class pure cultures to the instructors lab bench. 0. 1ispose of all materials when the experiment is completed, being sure to remove all mar"ings from the glassware. 5lace all culture tubes in the plastic bas"ets in the biohazard hood and all petri plates in the buc"ets in the biohazard hood. RESULTS *. 9ecognize and identify the following types of growth in a broth culture) pellicle, turbidity, sediment, and any combination of these. (. 4tate the color and water#solubility of pigment seen on a plate culture of a chromogenic bacterium.
