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  LAB NOTES FOR EXAM 1 SECTION EX. 2-1: DIVERSITY AND UBIQUITY OF MICROOGANISMSPurpo!: Microorganisms are found every where in the environment around us. To demonstrate this and to get a taste of the different types of organisms in our environment, we can culture these microorganisms by collecting them on a swab and transferring them to an agar plate. M! #$ % M$&!r#$': 1 Tryptic Soy Agar (TSA) plate, 1 sterile swab and 1 tube of sterile saline per person Pro(! ur!: 1.e will use a simplified version of the lab manual protocol. The lab manual describes several methods for sample collection. !onfer with your lab partners at your table so that each person uses a different method. our table should have a total of # plates ($ if you have an e%tra  person at your table).&.'abel the plate with your name, todays date, and the source of inoculum to be collected (for e%ample, destop, doornob, faucet, hair, sin, etc.). ou may also use any public body surfacee%cept for your mouth (this means any areas of sin that you might normally e%pose in polite company). Transfer any microbes you may have piced up to the agar plate by gently swabbing the surface of the plate with the swab. The instructor will demonstrate the method for spreading the cells on the plate. *.The plates will be incubated at *+! until ne%t lab period. #.-bserve your plates during our ne%t lab meeting. -n your data sheet, mae note of the various colors, te%tures, and shapes that are produced when microscopic organisms are allowed to reproduce in large numbers. The individual areas of growth you see are colonies and consist of millions of identical cells that all arose from a single parent cell.$.e sure to dispose of your cultures in the ioha/ard waste containers when your observations are completed. EX. 1-): ASEPTIC TRANSFER AND INOCULATION TEC*NIQUES Since this is the first time you will be woring with bacterial cultures, the procedure for handling and transferring microorganisms will be described in detail. Aseptic techni0ue, the procedure used to prevent contamination, is carried out so that you, your neighbors, and your belongings are not contaminated by the microbial culture and so that microbes from the environment do not contaminate your bacterial culture. Purpo!: n this e%periment you will be transferring living cells grown on two different types of media2  broth and slant. ou will use these cells to inoculate a fresh slant and a fresh broth culture. Cu'&ur!: a broth culture of  Serratia marcescens  and an $+$r '$,&  culture of  Serratia marcescens   M! #$: 1 tryptic soy agar (TSA) slant and 1 tryptic soy broth (TS) per student Pro(! ur!: 1.efore beginning, label all tubes of sterile media with the name of the organism, the date, and your names or initials. A'A S label before inoculating.&.3ollow the protocol described in the manual. e will be using only one organism today, Serratia marcescens . This organism produces a red pigment when incubated under the appropriate conditions. This red or pin color will help you to determine if your aseptic transfer has been successful when you loo for growth ne%t lab period. ou should not see any pin or red color in your freshly inoculated tubes today.1  *.4se the broth culture of Serratia marcescens  to inoculate the slant. 4se the slant culture to inoculate the broth.#. our instructor may demonstrate some techni0ues that are slightly different from the ones describedin the lab manual. These variations are acceptable as long as they are safe and produce the desired results. f you feel confused by any differences, as for clarification.$.After you replace the screw top on a culture tube, bac off the top about 15& turn before placing the tube in the incubator in order to allow air to circulate. 6o not incubate cultures with the top screwed down tightly. Those bugs need to breath, 7ust lie you89.ncubate all cultures at *+! until ne%t lab period.+.6uring the ne%t lab meeting, mae your observations on the data sheet located near the bac of your lab manual. 6ispose of your cultures in the appropriate ioha/ard container as directed by your instructor. EX. 1-: STREA PLATE ISOLATION OF PURE CULTURE /PART 10Purpo!: A pure culture is one that contains a single type of organism. ou must first isolate single colonies in order to cultivate a pure culture. Single colonies contain identical cells that have arisen from a single cell and are genetic clones of each other. Single colonies can only be obtained by spreading single cells far apart from each other on the surface of an agar plate, then allowing them to grow. There are several ways to separate single cells, but we will only be using the strea plate method using an inoculating loop. Cu'&ur!: a mi%ed broth culture   containing both  Serratia marcescens  and  Staphylococcus aureus   and a mi%ed broth culture containing both  Escherichia coli and  Micrococcus luteus . M! #$: & TSA plates per pair of students Pro(! ur!: T! S&r!$ P'$&! M!&o 1.:ach student will inoculate a mi%ed broth culture onto a separate agar plates according to the strea plate procedure described by the lab manual and demonstrated by the instructor. or with your lab partner so that each of you streas a different mi%ed culture.&.hen you mae the streas across the plate, do them gently so that you do not dig into the agar with the loop. This will cause growth to occur in streas, rather than in single colonies.*.ncubate your plates upside down (lid on the bottom, agar on top) in a wire baset at *+! for untilne%t lab period. asets can be shared between lab partners. These plates are considered mi%ed cultures because they will contain two different types of colonies. #.;e%t lab meeting2 mae your observations on the data sheet pages. 4se blan paper if you need additional space. nclude a drawing and use the information in :%. &<& to write a description of a colony from each different type of organism. -bserve the plates for visible differences in colony morphology (si/e, color, shape, elevation, margin, te%ture, optical characteristics). 6id you get niceisolated single colonies= They should be spaced far enough apart so that you can pic up an without touching more than one colony using a loop. Serratia marcescens  colonies should be pin or reddish. Staphylococcus aureus  form smaller, white colonies.  Escherichia coli  produces beige colonies & to * mm in diameter, while  Micrococcus luteus  will produce smaller yellow colonies. EX. 1- /$, EX. 2-20: STREA PLATE ISOLATION OF PURE CULTURE /PART 20Purpo!: solate a pure culture from mi%ed culture plates by restreaing a fresh strea plate from a single isolated colony Or+$,#3: 'ast period>s mi%ed culture plates with isolated colonies from :%. 1<#.&  M! #$: # TSA plates per pair of students Pro(! ur!: 1.'abel fresh plates with the names of the four organisms found on your mi%ed culture plates, your initials, and the date. 'oo at the two strea plates that you and your lab partner prepared from mi%ed cultures the last lab period. !hoose single colonies that are well separated from the others sothat they are easy to pic up.&.3rom a mi%ed culture plate, aseptically touch the edge of the sterile loop to the colony. t is not necessary to scoop up the entire colony. ou should not transfer a visible 0uantity of inoculum to the fresh plate. 4sing the strea plate dilution method that you used last wee, strea a fresh, labeled plate with the appropriate colony. ?emember to flame the loop in between streaing each 0uadrant of the plate. f the colonies are very small and close together, you can use a needle to pic up cells.  Note: Make sure that you pick up your inoculum from only one colony .*.4se the same method to strea plates of all four organisms.#.@lace the plates inverted in a baset. ncubate the four plates at *+! until ne%t lab period. $.;e%t lab period2 ou will use these plates as part of :%. &<&, !olony Morphology. ?ecord your observations on your data sheet. 4se additional paper for drawings or other information. :ach  plate should have only one type of colony. EX. 2-2: COLONY MORP*OLOGYPurpo!: -bserve possible difference in colony morphology between different bacterial species grown on solid culture media. Or+$,#3:  broth cultures of  Bacillus subtilis   and  Mycobacterium smegmatis M! #$: Two TSA plates per pair  Pro(! ur!: 1.ith a sterile loop, inoculate  Bacillus subtilis  and  Mycobacterium smegmatis  onto separate TSA  plates using the strea plate method. NOTE: B! ($r!4u' &o $5o# (ro-(o,&$3#,$&#o, 6!&7!!, p'$&!. B! !p!(#$''8 (o,(#!,&#ou #, 4'$3#,+ 8our 'oop $4&!r &r$,4!rr#,+ $,8  Bacillus  p!(#!97#( pro u(! por!. M$! ur! &$& &! 'oop $r! 4'$3! 'o,+ !,ou+ &o +'o7 or$,+!. T$!&! &#3! &o 4'$3! &! 'oop $, +o &rou+ &! pro(! ur! 'o7'8 $, ($r!4u''8. R!por& $,8 p#'' &o &! #,&ru(&or o &$& prop!r ('!$,up ($, o((ur.   &. ou have already streaed plates with the other organisms from :%. 1<#. ncubate all the plates *+! until ne%t lab period. The  Mycobacterium  and  Micrococcus  cultures may re0uire additional incubation time. Therefore, if the growth is scant and the colonies are very small, incubate these cultures for 1 to & more periods. *.;e%t lab period2 mae observations on the 9 cultures on your data sheets. 4se the terms we covered in class and on the study guide in your descriptions. There may be demonstration plates of other organisms to view as well. e sure to include observations on these cultures in your results.:; -4 A?: 3;S:6 MAB;C A'' -4? -S:?DAT-;S, !4'T4?:S S-4'6 : 6S@-S:6 -3 ; -4? ?:6 A4T-!'AD: ACS A;6 T-S: ACS @'A!:6 ; T: 'A?C: C?A -AEA?6 AST: !A;. EX. 2-): GROT* PATTERNS ON SLANTSPurpo!: -bserve possible differences in morphology between different bacterial species grown on slant media Or+$,#3: *  ;  Bacillus subtilis   and    Mycobacterium smegmatis ; 'ast period>s mi%ed culture plates with isolated colonies from :%. 1<# M! #$: 9 TSA slants Pro(! ur!: 1.'abel each slant tube with the date, organism name, and your initials.&.4sing the aseptic techni0ue for inoculating slants that we learned last wee, inoculate each tube.3or  Serratia marcescens ,  Micrococcus luteus ,  Escherichia coli  ,  Staphylococcus aureus,  carefully pic up cells of a single colony from your mi%ed plates. ;ormally, pure cultures would beused for transferring organisms to slants or broths, but we are using the mi%ed plates due to time considerations. f you do not have sufficient single colonies of each organism, there will be some  pure culture plates available as well.*.;e%t lab period2 mae observations on the 9 cultures on your data sheets. 4se the terms we covered in class and on the study guide in your descriptions. 3or your uninoculated control, use a fresh TSA slant, but return it to the cart when finished. EX. 2-: GROT* PATTERNS IN BROT*Purpo!: -bserve possible differences in morphology between different bacterial species grown in broth media Or+$,#3: ;  Bacillus subtilis   and    Mycobacterium smegmatis ; 'ast period>s mi%ed culture plates with isolated colonies from :%. 1<# M! #$: 9 TS broths Pro(! ur!: 1.'abel each broth tube with the date, organism name, and your initials.&.noculate these tubes with the same cultures used in :%. &<*. Again, be especially careful when transferring cells from your mi%ed culture plates. *.;e%t lab period2 mae observations on the 9 cultures on your data sheets. 4se the terms we covered in class and on the study guide in your descriptions. 3or your uninoculated control, use a fresh TS broth tube, but return it to the cart when finished. EX. 2-<: FLUID T*IOGLYCOLLATE: ATMOSP*ERIC OXYGEN REQUIREMENTSPurpo!: -bserve the growth patterns of different organisms according to their o%ygen re0uirements Or+$,#3 2 roth cultures of  Staphylococcus aureus 9  Escherichia coli,    Pseudomonas aeruginosa,  Neisseria sicca, and Clostridium butyricum M! #$: 3our tubes of thioglycollate broths and one tube of supplemented thioglycollate per pair  Pro(! ur! 2 1.Thioglycollate is a reducing agent that removes o%ygen from the broth. 'abel the tube of S4@@':M:;T:6 thioglycollate for the organism Clostridium butyricum  (dont forget your initials and the date). 'abel the other tubes for each of the remaining organisms. &.After observing the instructors demonstration, inoculate each broth using a sterile disposable bulb  pipette2 Transfer F.&$ ml (the first mar on the sterile pipette above the 7oint) of the appropriate culture by gently s0uee/ing the bulb to e%pel the inoculum into the broth from the bottom of the tube to the top. 6- ;-T A''- A; A? T- 4': ;T- T: ?-T. #


Jul 23, 2017
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