Introductory Microbiology

Laboratory Manual

MB 230 Winter 2018

You MUST attend lab during the first week of the term or you will automatically be dropped from MB 230.

Instructors: Jesse J. Coutu

coutuj@oregonstate.edu 106A Dryden Hall

Copyright © 2013 Oregon State University

Lab Rooms: Nash 304/316 Labs start week of 1/08/18

agarside¦

lid¦

Petriplatewithagar

Label plates on the agar side, not the lid. Place plates agar-side up for most exercises.

lid¦

glass¦

Label culture tubes on the glass, not the lid.

culturetube

MB 230 Laboratory Schedule || indicates completion of the exercise

Reading/Methods ResultsWeek 1 Introduction to MB 230 lab.......................................................................... iv

Lab Safety and Equipment Care.................................................................. v-vi Exercise 1: Use of the Microscope||.................................................... 1-8.................. 50 Exercise 2: Microorganisms in the Air............................................... 9-10................ 51

Week 2 Exercise 3: Morphology of Bacteria||................................................. 11-14............. 52 Exercise 4: Pure Culture Technique................................................... 15-17............. 53 Exercise 5: Environmental Sampling................................................. 18-20............. 54 Exercise 2: Examine PCA plate||

Week 3 Exercise 6: Gram Stain||...................................................................... 21-22............. 55 Exercise 7: Rhizobium & Nitrogen Requirements............................. 23-24............. 56 Exercise 4: Examine streak plate (Gram stain)|| Exercise 5: Return and incubate RODAC and swab PCA plates

Week 4 Exercise 8: Isolation of Bacteriophage............................................... 25-26............. 57 Exercise 9: Koch’s Postulates & Crown Gall Disease (Gram stain) 27-28............. 58 Exercise 10: Relation of Oxygen to Microbial Growth....................... 29-30............. 59 Exercise 5: Examine RODAC and PCA plates||

Week 5 Exercise 11: Microbes of the Mouth (Gram stain) ............................. 31-32............. 60 Exercise 12: Normal Throat Culture.................................................... 33-34............. 61 Exercise 8: Examine bacteriophage pour plates|| Exercise 9: Examine MGY plate & Gram stain Exercise 10: Examine thioglycollate tubes||

Week 6 Exercise 13: Water and Coliforms....................................................... 35-36............. 62 Exercise 14: Conjugation & Antibiotic Resistance............................. 37-39............. 63 Exercise 11: Examine sucrose plate and Gram stain|| Exercise 12: Examine blood agar plate and Gram stain||

Week 7 Exercise 15: Staphylococcus aureus in Potato Salad.......................... 40-42............. 64 Exercise 16: Metabolism and Fermentation........................................ 43-44............. 65 Exercise 13: Examine water bottles, set-up EMB plates Exercise 14: Examine antibiotic plates||

Week 8 Exercise 17: Cultured Dairy Products (Gram stains) ......................... 45-46............. 66 Exercise 18: The Fungi||........................................................................ 47-48............. 67 Exercise 13: Examine EMB plates|| Exercise 15: Set up potato salad dilutions; plate on MSA agar Exercise 16: Examine BCP sugar tubes||

Week 9 Exercise 7: Examine clover plants for root nodules & Gram stain|| stain||Exercise 9: Examine carrots for crown gall & Gram stain||

Exercise 15: Examine MSA plates & Gram stain|| Exercise 17: Examine tube of milk & Gram stain||

Week 10 Turn in results pages; Take lab final.

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Point Breakdown Per Week

These are the points you would miss if you don’t attend lab in the given week. There is no way to earn Results Points unless you attend the given lab. If you turn in your post-lab BEFORE the deadline by 1) sending it with a friend, 2) dropping it off to the instructor or 3) some other means so that your TA has it in hand BEFORE the deadline, you can still earn the post-lab points.

The Results packet is due Week 10, so if you fail to turn in your results packet you will not earn ANY of your results points.

Week Quiz Points Results Points Participation Points

Lab Final Lab Safety

Quiz Pre-Lab Points

Post-Lab (PL) Points

1 1 3 1.0 1 2 3 3 (WK1 PL) 1.5 1 3 4 3 (WK2 PL) 1.5 1 4 4 4 (WK3 PL) 1.5 1 5 4 4 (WK4 PL) 2.5 1 6 4 4 (WK5 PL) 2.5 1 7 4 4 (WK6 PL) 1.0 1 8 4 4 (WK7 PL) 4.5 1 9 4 (WK8 PL) 4.0 1

10 10 Total Points 1 30 30 20 9 10

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MB 230 Laboratory Syllabus Winter 2018

LAB SECTIONS: Wednesday 10:00 – 11:50 am, Friday 12:00 – 1:50 pm Lab Rooms: Nash 304/316

LAB INSTRUCTORS: Jesse J. Coutu coutuj@oregonstate.edu 106A Dryden Hall

GRADING POINTS: Lab-Safety Quiz ................................................ 1 Pre-Lab Quizzes ................................................ 30 Post-Lab Questions ............................................ 30 In-Lab Results ................................................... 20 Lab Participation ................................................ 9 Lab Final ........................................................... 10 TOTAL ............................................................. 100

TOTAL LAB POINTS = 25% of MB 230 grade There is no curve for lab points

ATTENDENCE in lab is crucial! If more than one lab session is missed, you will receive a failing grade (F) for the entire MB 230 course. Except for extremely exceptional circumstances (as determined by the instructor), there is no way to make up a missed lab! All weeks count towards attendance.Lab Participation Points: The participation points will be equally distributed for all the lab classes except the week 10 lab. These points will be given based on the signed attendance of the students on the attendance sheet. REMINDER: Students will not get the ‘Participation points’ for any make-up lab class.

Lab-safety Quiz: Lab-safety quiz will be available on Canvas before the first session of the scheduled lab and need to be submitted before the second lab session. All questions will be asked about the general safety of the lab that are discussed in this lab manual in the ‘Laboratory Safety’ section.

Pre-Lab Quizzes: Lab exercise quizzes will be available on Canvas before the scheduled lab sessions in which the exercises will be performed. The quizzes must be completed BEFORE coming to lab. Quizzes are available at the end of the previous lab session until the start of the next lab session.

In-Lab Results: Results will be recorded throughout the lab exercises and briefly checked by TAs in lab. The Results pages will then be turned in as a single packet on the final day of lab. Graded results will be available for pick-up from the instructor’s office during normal office hours.

Post-Lab Questions: A document containing all post-lab questions will be available on Blackboard and can be accessed throughout the term. Data for the post-labs will be collected during lab by your teaching assistant and posted to Blackboard. Post-lab questions should then be completed using the data during the week and turned in to your lab TA during the next lab session.

Lab Final: The lab final will be given on the last lab date of the session. The lab final will cover material from the all of the lab exercises.

Grade Appeals: Students have one week from the time that scores are given to contest a score.

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Introduction to MB 230 Lab

The experiments in this manual have been selected to teach some of the basic techniques and organisms of microbiology. This course is appropriate for any student who wishes an introduction to the science of microbiology.

ATTENDANCE

ATTENDENCE in lab is crucial! If more than one lab session is missed, you will receive a failing grade (F) for the entire MB 230 course. Regular attendance is vital because all subsequent labs build upon prior weeks' work. There are no make-up labs.

FORMAT

Each laboratory period begins with a lead-in lecture with explanations and demonstrations for that period's exercises. However, these lead-in lectures are not meant to replace your own preparations. Read the exercises before coming to lab!

On some exercises you will work independently and on others you will work in pairs or in small groups of students. See specific experiments for detailed descriptions of experimental formats.

Every time you use your microscope, you should write your name on the microscope sign-in sheet (inside microscope cabinet) and have your TA check your microscope before leaving lab.

Please ask questions at any time! The instructor and the teaching assistants will circulate and be available for help during the entire lab period.

LAB CLOTHING

Lab coats and safety goggles are not required. However, many reagents can produce permanent stains on your clothes and caution should be exercised to avoid this. To protect your belongings from such stains or microbial contamination, store your coat and book bag under your workbench. Only your MB 230 lab manual and lab supplies should be left on the workbench's surface during the laboratory period.

You will be working with open flame. Do not wear loose-fitting clothing, especially cloth bracelets or sleeves with dangling material. Long hair should be tied back.

Closed-toe shoes are REQUIRED; sandals and other such shoes are NOT allowed. Pants are recommended and shorts are advised against.

Double-check your belongings at the end of the lab period to make certain you have collected everything. The Microbiology Department will not be responsible for any items brought to lab.

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Tardiness: Students are expected to be on time and be fully participatory for the scheduled lab time. Arriving at lab more than 10 minutes late two times will count as 1 absence. Arriving greater than 30 minutes late to lab will be considered an absence

Attending a different MB 230 lab section: Attending other MB 230 lab sections is possible only with prior permission of your instructor and is subject to availability of open seats. Email request to your instructor as soon as you are aware of a time conflict with your scheduled lab time.

LABORATORY SAFETY

1) DO NOT eat, drink or chew gum/tobacco. Open beverage and food containers must be lefton the hallway shelf outside lab. Keep your hands out of your mouth, nose and eyes.

2) KEEP YOUR WORKSPACE CLEAR – Keep only the lab manual and necessary labsupplies on your bench top; everything else should be placed under the bench, keeping theaisles clear.

3) WEAR APPROPRIATE CLOTHING – a shirt and closed-toe shoes are REQUIRED.Flip-flops and halter tops are not recommended. If your attire is deemed inappropriate forlab work, you will be offered a lab coat for use or asked to leave the lab.

4) NO UNAUTHORIZED VISITORS in the lab. NO ANIMALS in lab.

5) KNOW THE LOCATION of the fire extinguisher in the hallway and the fire blanket &eye wash in the lab.

6) CLEAN desktop with DISINFECTANT at the beginning and end of class. Carefully washhands with soap before leaving the lab.

7) BUNSEN BURNERS in the lab have almost invisible flames - turn them off completelywhen finished. Long hair must be tied back during lab to avoid contact with flame. Loosefitting clothing and clothing or jewelry with dangling material should not be worn.

8) BROKEN GLASSWARE - Call the instructor or TA to clean up the glass. Do notdispose of any glassware in the regular garbage cans.

9) INJURY - If you cut yourself in the lab, inform the instructor or TA so that the wound canbe properly disinfected. If the injury needs professional assistance, you will be escorted tothe Health Center or proper facility.

10) SPILLAGE – If you spill anything in lab, inform the instructor or TA so that they canclean up the spill. If culture is spilled on your clothing or belongings, they may requiredecontamination to assure your safety.

11) INCUBATING - Carefully label all materials to be incubated with your name/initials, seatnumber, and organism identification. Place materials to be incubated in the incubation tubat the front of your bench, unless otherwise directed.

a. Label culture plates on agar side with your name/initials, seat #, and organismidentification. Place plates in incubation tubs agar side up, unless otherwise stated.

b. Label culture tubes on the glass (not plastic caps) with your name/initials, seatnumber, and organism identification. Place tubes in racks in incubation tub.

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12) DISCARDING CLASSROOM MATERIAL - All materials used in this class that arecontaminated with culture (tubes, plates, pipettes, etc.) must be autoclaved before cleaningor disposal; DO NOT throw these materials away in the metal cans on your bench tops norin the normal garbage cans.a. Contaminated ‘Soft Materials’: Plastic Petri plates, transfer pipettes, swabs and other

‘soft’ materials go into an autoclave bag in a metal container (‘coffin’) on the discardtable. Do not discard glass items in the autoclave bag!

b. Contaminated Non-Broken Glass: Glass culture tubes go into wire baskets in metalcontainers (‘coffins’) at the discard table. Glass bottles go into metal containers(‘coffins’) at the discard table. Loosen screw caps before autoclaving.

c. Contaminated Broken Glass/Sharp Materials: Used razor blades/pins go in themetal can for contaminated sharps.

d. Uncontaminated ‘Soft Materials’: Paper towels used to clean the bench top withdisinfectant or lens paper used to clean microscopes can be placed in the metalcontainer on the lab bench. The metal container should be emptied into the maingarbage cans at the end of each lab session.

e. Uncontaminated Non-Broken Glass: Used slides should be cleaned with slide cleanerand returned to slide container.

f. Uncontaminated Broken Glass/Sharp Materials: Used cover slips go in thecardboard box for glass waste.

13) LEAVING THE LABa. Have TA check your microscope.b. Clear lab bench of all cultures, plates and other supplies.c. Empty the metal can containing uncontaminated ‘soft’ waste into a large garbage can.d. Clean desktop with disinfectant and wash hands with soap.e. Make sure you have all of your belongings before leaving lab.

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Exercise1:UseoftheMicroscope

Objectives • Identifythecomponentpartsofacompoundmicroscope. • Correctlyoperatethemicroscopeusingvariouspowersofmagnification.• Prepareandobservewetmountsandstainedpermanentslides.

Reading In MB 230, you will use a compound microscope almost every lab period. A compoundmicroscopeusestwotypesoflenses-theocularlensesandtheobjectivelenses.Theselensesworktogethertoprovidemagnificationorenlargementofobjects.Thetotalmagnification istheproductofthemagnificationoftheobjectivelensandthemagnificationoftheocularlens.Magnificationbyeachlensisindicatedbymarkingsonthelenses:l0X,40X/45X,orl00Xontheobjectives;l0Xontheoculars.Note:somemicroscopeshavea4Xobjectivelens.

In addition tomagnifying objects,microscopes enable the observer to distinguish structuresthat are separated by short distances. This function is called resolution. Resolution is moreimportant thanmagnification: it is not alwaysdesirable toobtain the largest imagepossible,but it is necessary to obtain sharp detail. The ordinary light microscope with oil immersion(100X) objective can distinguish or resolve two points approximately 0.2micrometers apart.Thisisdefinedasthelimitofresolution.Anotherconceptthatiscrucialtoproperutilizationofthe microscope is its’ capacity to be parfocal. Parfocal refers to the microscope’s ability toremaininapproximatefocuswhenswitchingfromoneobjectivetoanother.

Ofparamount importance to themicrobiologist is theoil immersion (100X) objective of themicroscope.Allobservationsofbacteria in labwillrequireuseoftheoil immersionobjective.Toproperlyusethisobjective,onemustactuallyplaceoilontheslidesurfaceandimmersetheobjectiveinitbyswingingitintoplace.Themaineffectofimmersionoilistocollectaberrantlight rays and allow them to enter the objective. Aberrant light rays are thosewhichwouldotherwisebelostduetodiffraction.

Specimenstobeviewedwiththecompoundmicroscopearemountedonglassslides.Astainedpermanent slide ispreparedby fixingwholemicroorganismsor sectionsoforganisms to theslide,stainingthepreparation,andcoveringitwithaglasscoverslip.Thecoverslipisattachedto theslidebya transparentadhesivematerial (glue).Semi-permanent slidesofbacteriaareprepared by fixing and staining the preparationwithout gluing on a coverslip.Most of yourmicroscope study in this course will involve semi-permanent slides that you will prepareyourself.Undernormalcircumstancesthemicrobesinthesepreparationsaredead.

Anunstainedwetmount ismadebysuspendingmicroorganisms inadropofwaterorotherliquid and covering thedropwitha coverslip.Awetmountdries after severalminutes, so itcannotbeusedoveralongperiodoftime.Inaddition,becauseoftheliquidbetweentheslideandthecoverslip,awetmountcanonlybeobservedusingthe10Xand40/45Xobjectivesonthemicroscope.However,awetmountenablestheviewertostudylivingmicroorganisms.

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Ourmicroscopes are Leica and Leitz compoundmicroscopes (see Figures 1.1 and 1.2 on thefollowingpages). Eachof the labeledparts in the figures shouldbe identifiableon yourownmicroscope.Thebasicpartsandtheirfunctionsare:

PartFunction

Oculars(Eyepieces) aseriesoflensesthatusuallymagnifyl0X

WidthAdjustment adjustforyoureyespan

RevolvingNosepiece rotatestochangefromoneobjectivetoanother

Objectives usuallythreemagnifications:l0X(lowpower), 40-45X(highdrypower),andl00X(oilimmersion); immersionoilusedwiththisobjectivehasarefractive indexapproximatelythesameasglass-lightpasses throughwithoutbeingbentorlost

MechanicalStageControls movestheslideontwohorizontalplanes–backward& forwardandsidetoside

MovableStage raisesandlowersduringfocusing

DiaphragmLever opensandclosesthediaphragmtocontroltheamount oflightstrikingtheobject

Condenser condenseslightwavesintoapencilshapedcone, therebypreventingtheescapeoflightwaves.Also controlsthelightintensitywhenraisedorlowered

CondenserKnob raisesandlowerscondenser

LightControl turnslightsourceonandoff

Arm supportsupperhalfofthemicroscope

CoarseAdjustment movesstage(orbodytube)upanddownrapidlyfor purposesofapproximatefocusing

FineAdjustment movesstage(orbodytube)upanddownveryslowly forpurposesofdefinitivefocusing

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FIGURE1.1:TheLeicaMicroscope

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FIGURE1.2:TheLeitzMicroscope

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GENERALRULESFORMICROSCOPEUSE

Themicroscopesarevaluableandshouldalwaysbeusedwithcare!

PREPARINGTHEMICROSCOPEFORUSE

1. When you are ready to use yourmicroscope, remove themicroscope from cabinet by

rollingshelfallthewayout,thencarefullyliftingmicroscopeontothetable.Bumpingtheocularsonthetopofthecabinetcandamagethem.

2. Unwindthecordfromaroundtheocularsandplugitintoareceptacleinthemiddleofthelabbench.Turnonthelightsourceatthebaseofthemicroscope.

ADJUSTINGLIGHTINTENSITY

Properlyadjustingtheamountoflightfordifferentmagnificationswillgreatlyenhanceyourabilitytoseedetailsandmakeusingthemicroscopemuchmorecomfortable. 1. Before examining a specimen make sure that your lamp is on and adjusted to full

intensity.Thisensuresthatalllightwavesreachyoursample.2. Findthecontrolforyourcondenserlensheightandadjustthecondensersothatitisup

closetothespecimen.3. Forexaminingspecimenswiththelow(10X)powerobjective,lowerthelightintensityby

closing the iris diaphragm. This is the best way to control light level.When viewing aspecimenusingthehighdry(40/45X)oroil immersion(100X)objectivelenses,opentheirisdiaphragmcompletely tocapturemaximumlight.Afteryouhavefoundanobject toexaminetryreducingthelightlevelusingtheirisdiaphragmlevertoseewhethercontrastisenhanced.

FOCUSINGTHEMICROSCOPE

Followthedetailedinstructionsonthefollowingpage.

PUTTINGTHEMICROSCOPEAWAY

1. Afteruse,cleanoiloffthe100Xobjectivebywipingwithflatlenspaper,followedbyflat

lenspapersaturatedwithlenscleaner.Crumpledlenspaperwillscratchthelens.Ifoilisleftontheobjective,itwilldestroythesealandthelenswillneedtobereplaced(>$300).

2. Ifoilisonthe40/45Xlens,lettheinstructororTAknowsoitcanhavespecialcleaning.3. Removedirtoroilfromthestage,condensersorocularsifneeded.4. Storemicroscopewithlowpowerorblankobjectiveinplaceandclosetostage.5. Wrapthecordaroundocularsneatly; replace incabinetwhenshelf iscompletelyrolled

out.6. Signanddatethesign-outsheetinthecabinet.7. HaveyourTAcheckyourmicroscopeandinitialsign-outsheetbeforeleavinglab.

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FOCUSINGAMICROSCOPE WetMount l. Putslideonstagewithinmovableslideholders. 2. Usingmechanicalstagecontrols,movetheslidesothatyourspecimenisdirectlyoverthe

lightsource.3. Movetherevolvingnosepiecesothatthelowpowerobjective(l0X)isinplace.4. Usingthecoarseadjustment,movethe10Xobjectiveasclosetotheslideaspossible.5. Slowly turn coarse adjustment, raising the objective until specimen is in focus.Use the

fineadjustmenttoobtainmaximumclarity.6. Withoutadjustingthefocus,carefullychangetothehighdryobjective(40/45X).Donot

usethisobjectiveifitwilltouchtheliquidonyourslide.7. Usingfineadjustment,focusuntilthespecimenisatmaximumclarity.8. Usethemechanicalstagecontrolstomovetooptimalviewingareas.Thefineadjustment

mayhavetobechangedasthefieldofviewchanges.9. DONOTUSETHEOILIMMERSIONLENS(100X)WITHAWETMOUNT. StainedPermanentSlide l. Putslideonstagewithinmovableslideholders. 2. Usingmechanicalstagecontrols,movetheslidesothatyourspecimenisdirectlyoverthe

lightsource.3. Movetherevolvingnosepiecesothatthelowpowerobjective(l0X)isinplace.4. Usingthecoarseadjustment,movethe10Xobjectiveasclosetotheslideaspossible.5. Slowly turn coarse adjustment, raising the objective until specimen is in focus.Use the

fineadjustmenttoobtainmaximumclarity.6. Withoutadjustingthefocus,carefullychangetothehighdryobjective(40/45X).7. Usingfineadjustment,focusuntilthespecimenisatmaximumclarity.8. Withoutadjustinganythingelse,carefullyrotatethenosepieceuntilthehighdryobjective

isoutoftheway.Donotputtheoilimmersionlens(100X)inplaceyet.9. Putasmalldropofoildirectlyoverthepathoflightonthespecimen.10. Carefullyswitchtotheoilimmersionlens(l00X).Theoilimmersionlensshouldjustbarely

touchtheoilonthespecimen.ONCETHEREISIMMERSIONOILONYOURSLIDE,YOUCANNOTGOBACKTOTHE40/45XOBJECTIVE!NOTIFYYOURTAIFYOUR40/45XOBJECTIVEGETSOILONIT.

11. Usingfineadjustmentonly,focusuntilthespecimenisagainatmaximumclarity.12. Usemechanicalstagecontrolstomovetooptimalviewingareas.Thefineadjustmentmay

havetobechangedasthefieldofviewchanges.13. Alwaysremembertocleantheoil immersionlenswithlenscleanerandlenspaperwhen

oneisfinished.AMICROSCOPESHOULDNEVERBEPUTAWAYWITHOILONIT.

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TABLE1.1:MICROORGANISMSFREQUENTLYOBSERVEDINHAYINFUSIONORPONDWATER(Pleasenote:drawingsareNOTtoscale!) Wet mounts of pond water or hay infusions contain large numbers of unstainedmicroorganisms. Youmay observe protozoa (amoebas, ciliates, or flagellates), various algae,somemicroscopicinvertebrates,andthelargerbacteria.Someofthesemicroorganismsmoveveryfast.Rememberthatonlythe10Xand40/45Xobjectivesshouldbeusedtoobservewetmounts!

COLONIAL

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EX.1:UsingaMicroscope–Materials

PartA:UnstainedWetMountsofFluidSpecimens

pondwater(containingalgaeandprotozoa) hayinfusionbroth(containingprotozoaandbacteria)microscopeslidesandcoverslips

PartB:StainedPermanentSlidesofOrganisms

eukaryote: Candidaalbicans (yeast) prokaryotes: Bacillussubtilis (largebacillusbacterium)

Staphylococcusaureus (smallcoccusbacterium) EX.1:UsingaMicroscope-Methods PartA:UnstainedWetMountsofFluidSpecimens l. Prepareawetmountofpondwaterorhayinfusionbrothbyplacingasmalldropoffluidon

acleanmicroscopeslide.Gentlypositionthecoverslipovertheliquid. 2. Usedirectionsonthepreviouspagesforexaminingyourunstainedwetmounts.Remember

toonlyusethe10Xand/or40/45Xobjectivesonthemicroscopewhileobservingyourwetmount.

3. Draw observations in the RESULTS section. Protozoans are generally motile and clear in

color.Algae,whichmayormaynotbemotile,tendtobegreenishorbrownishincolorduetotheirpigments.Thecyanobacteriaalsohavepigmentsandwillbegreenorblue-greenincolor.Mostbacteriawillbetoosmalltoseeclearlyatthesemagnifications.

4. Whenfinished,rinsetheslidewithwaterandreturntonearbyslideholder.Thecoverslip

maybedisposedofinthecardboardboxforbrokenglass. PartB:StainedPermanentSlidesofOrganisms 1. Thestainedpermanentslidesoforganismshavealreadybeenmadeforyou.Thecoverslips

have been glued on, so these slides can be used over and over again. Select a stainedpermanentslideofoneofthethreedifferentmicrobes.

2. Use directions on the previous pages for examining your stained permanent slide.

Remembertogoall thewayuptothe100Xobjectiveonthemicroscopewhileobservingyourstainedslide.DrawobservationsintheRESULTSsection.

3. Afteruse,theseslidesshouldbecleanedandreturnedtotheiroriginalbox.

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Exercise2:MicroorganismsintheAir Objectives • Acquireanawarenessofthediversityofmicroorganismsintheair. • Recognizethedifferenttypesofmicrobialgrowththatoccursonacultureplateexposedto

air. Reading Nomicroorganismsgrowintheatmosphere.Becauseairhasnonutrientsandlittlewater,itisaninhospitableenvironment.Whenwespeakofthemicrobialfloraofair,wearereferringtothose organisms which are found temporarily suspended in air or carried about on dustparticlesordroplets. Avarietyof techniqueshavebeendevised - some simple, someelaborate - for isolatinganddetecting microorganisms in the air. One of the simplest of these is the use of gravity (orsettling)plates.GravityplatesarePetriplatesfilledwithsterilemediawhichareleftopenwiththe agar surface exposed.Microorganisms carried ondust or droplets simply "settle" on theagarsurfaceand,followingincubation,reproduceoverandovertoeventuallyformindividualcolonies. ThenutrientmediumwewillbeusinginourPetridishesforthisexerciseiscalledPlateCountAgar (orPCA).Themediacontainstryptone(digestedmeatprotein),glucose(simple,easytometabolize carbohydrate), and yeast extract (containing B vitamins and protein). Theseingredients provide the basic nutrients required bymany heterotrophicmicroorganisms. Butnot all microbes will grow on a PCA plate - microbes with complex nutritional needs andautotrophic microorganisms would not find the things they need to grow. The media issolidifiedusingagar,acomplexcarbohydrateextractedfromseaweed.Becauseofitscomplexstructureandunusualoriginmostterrestrialmicroorganismsdonotdegradeit,unlikegelatin,ananimalproteinwhichisoftendegradedbycommonmicrobes.Agarreactstoheatingmuchlikegelatin.Todissolveagarinwateritmustbethoroughlyheated.Itwillremainliquidwhilewarm,solidifyingattemperaturesnear42oC(108oF).Thenutrientmediumhasbeensterilizedby autoclaving at 121oC and 15 pounds/in2 steam pressure. The combination of heat andpressureproducedbyanautoclaveeffectively kills nearly allmicroorganisms. Themedium isthenpouredaseptically(withoutintroducingunwantedorganisms)intotheplateandallowedtocool. Agarplatesarelabeledontheagarsideincaselidsareaccidentallyswitchedatsomepoint.Platesaretypicallyincubatedagarsideupsothatcondensationthatformsduringincubationwillnotdripbackdownontothecoloniesontheplate. You will find that microorganisms are common in the air and on body surfaces. Preventingcontaminationof culturemediaby theseorganisms isdifficult.Themicrobiologistmustworkcarefullyusingaspecialsetofprocedurescalledaseptictechniquetopreventorganismsfrom

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inadvertentlyenteringsterilemedia.Youwilllearnaseptictechniqueinalaterlaboratoryexercise.

EX2:MicroorganismsintheAir-MaterialsperStudent

1sterileplatecountagar(PCA)plate

EX2:MICROORGANISMSINTHEAIR:Methods

1. Labeltheplateontheagarsidewithyournameandseatnumber.

2. RemovethePetriplatelidandexposetheagarsurfacetotheairuntiltheendoflab.You

may gently touch the agar surfacewith one finger if youwould like. Note the delicatetexture of the agar. In later lab exercises you will be depositing organisms across thisratherfragilesurface.

3. Attheendoflab,replacethelid,turntheplateoversothattheagarsideisup,andplace

theplateinthecorrectboxforincubation.

4. Nextlab,examineyourplateandnotethetypesofmicrobialcoloniesthathavegrownontheagarsurface.Contrastthecolonialgrowthonyourplatewiththosearoundyou.Ifyoutouchedtheplate,noteanydifferencesinthedensityofthecoloniesonthatpartoftheplatewithotherareasoftheplate.RecordobservationsintheRESULTSsection.

Table2.1:CharacteristicsUsedforColonyEvaluationonAgarPlate

Size: Pinpoint Small Moderate Large Pigmentation: Colorofcolony Form: Circular:round,evencolonies shapeofthecolony Irregular:irregularlyshapedcolonies

Rhizoid:root-like,spreadinggrowth Margin: Entire:sharplydefined,even appearanceofcolonyouter Lobate:markedindentations edge Undulate:wavyindentations

Serrate:tooth-likeappearance Filamentous:threadlike,spreadingedge Elevation: Flat:elevationnotdiscernible degreetowhichcolony Raised:slightlyelevated growthisraisedonagar Convex:domeshapedelevation surface Umbonate:raised,withcenterofcolonymarkedlyelevated

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Exercise3:MorphologyofBacteria–TheSimpleStain Objectives • Preparebacterialsmearsandapplythesimplestaintechnique. • Distinguishamongbasicbacterialshapes(morphologies)andcellarrangements. Reading Whenbacteriaarestainedusingasingledye,itiscalledasimplestain.Thedyeenhancesthecontrastbetween the cell and its surroundings, showingus themorphologyor shapeof thecell.Dyesaregenerallysaltsinwhichoneofthechargedparticlesorionsiscolored.Asaltisacompoundcomposedofapositivelychargedionandanegativelychargedion.Thesimpledyemethylene blue is actually the salt methylene blue chloride, which dissociates in water toproduce:

MbCl methyleneblue++chloride

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The color of the stain is in the positively chargedmethylene blue ion. Bacterial cells have aslightnegativechargewhenthepHoftheirsurroundingsisnearneutrality(whichitgenerallyis).Thenegativelychargedbacterialcellcombineswiththepositivelychargedmethyleneblueion, with the result that the cell is stained. It is the difference in charge that produces anaffinitybetweenthedyeandthebacterialcell. Thethreegeneralmorphologies(shapes)ofbacteriaare:

coccus

bacillus

spiral Apart from these differences in cellular shape, definite patterns in cellular aggregations orarrangementsareknowntoexistamongdifferentbacteria. In thecaseofsphericalorcoccusforms, two important patterns include chains of four or more organisms (streptococci) andirregulargroupsofmicroorganismsresemblinggrapeclusters(staphylococci). Bacilli(sing.,bacillus)arealsoreferredtoasrod-shapedbacteria. Threegroupsofspiral-shapedbacteriaareknown.Thespirochetesconsistofflexible,wavingforms,withseveralcoils.Thesecondgroup,thespirilla,isagroupofrigidbacteriapossessingoneorseveralcurves.Thethirdgroup,thevibrios,consistsofshort,curvedbacteria. Variationsinthegeneralshapesandsizesofbacteriaarefrequentlyseen,andcanbeexplainedintermsofenvironmentalfactors.Pleomorphismisthetermusedtodenotethesevariations.

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FIGURE3.1:StepstoPrepareaBacterialSmear Thelaststepkillsthebacteriaandfixesthecellstotheslide.Donotoverheat!

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EX3:MorphologyofBacteria–Materials

PCAplatefromExercise2(perstudent)brothcultures:Escherichiacoli StaphylococcusepidermidispreparedslideofSpirillum bottleofmethylenebluestain

EX3:MorphologyofBacteria–Methods MakingaBacterialSmear Itisabsolutelyimperativethattheslidehasbeencleanedthoroughlyorbacteriawillnotadheretotheslideandwillbewashedoffduringthestainingprocess. CleaningaSlide:

a. Thoroughlywashwithslidecleaner.b. Rinsewithwaterandblotdry.c. LightlyflamewithBunsenburner.

Avoidtouchingthecleanedslidesurfacewithyourfingers,oroilswillbere-depositedontheslide.Instead,holdthecleanslidebytheedges. MakingaSmear:

1. Asmallamountofbacterialgrowthisplacedintheslide.2. Spreadtheorganismsoverthesurfaceofthecleanglassslide.3. Thethinfilm,orbacterialsmear,isair-driedontheslide.4. PasstheslidebrieflythroughtheflameofaBunsenburneronceortwice.

Thelaststepkillsthebacteriaandfixesthebacterialcellstotheslide.Donotheatthesmearbeforeitiscompletelyairdriedanddonotoverheatthesmearorthebacteriawillexplodeandlosetheirshape. Whenmakingasmearfromasolidmedium,youshouldplaceasmalldropofdistilledwaterontheslidefirst.Thelesswateryoucanuse,thefasterthesmearwilldry. MethyleneBlueSimpleStain 1. Applyenoughmethylenebluestaintocoverthefixedsmear.Letitsitforone(1)minute.2. Pouroffthestain.Rinseslidewithslowlyrunningwater.3. Blottheslidedrybyplacing itbetweentwopapertowelsandpressinggently.Donotrub

slide.

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EX3:MorphologyofBacteria–MicroscopicExamination 1. Make a smear from one colony of the PCA plate from exercise 2. Use a small drop of

distilledwatertomakethissmear.Stainthesmearwithmethyleneblue.Examinetheslidewithyourmicroscope.

2. Make a smear from the Staphylococcus epidermidis broth culture. Stain the smear with

methyleneblue.Examinetheslidewithyourmicroscope.3. MakeasmearfromtheEscherichiacolibrothculture.Stainthesmearwithmethyleneblue.

Examinetheslidewithyourmicroscope.4. Examinetheslideswithyourmicroscope.

a. Startwith the lowpower (10X)objective. Find themicroscope focal planewith color,bringthecoloredobject(s)intofocusandcenterinmicroscopefieldofview.

b. Turn the high dry objective (40/45X) objective into place over the specimen. Refocusandre-centerthecoloredobject(s).

c. Movethehighdryobjectiveoutoftheway,addadropofimmersionoilontopoftheslide and move the immersion oil (100X) objective into place above the specimen.Refocustheobjectsandrecordresults.

d. Cleanthe immersionoiloffof themicroscopebeforebeginningtheprocessoverwithanotherpreparedslide.

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Exercise4:PureCultureTechnique–PreparingaStreakPlate Objectives • Employthestreakplatetechniquetoproduceindividualcoloniesonanagarplate. • Practiceaseptictechnique. Reading In order to study microorganisms and observe their characteristics it is first necessary toobtaintheminpureculture(apurecultureisdefinedasaculturecontainingonlyonespeciesoforganism).This isvery importantbecause it is impossibletostudythecharacteristicsofamicroorganismwhencontaminants(unwantedorganisms)arepresent. Oneofthemostcommonmethodsemployedinthelaboratorytoobtainapurecultureisthepreparationofastreakplate.Toproperlystreakaplateforisolationyoumustspreadouttheorganism(s) bymeansof the inoculating loopuntil single colonies result. Each single colonyconsistsofaclusterofcellsthatoriginatebycelldivisionfromasinglebacterialcell.Thuseachisolatedcolonyrepresentsapurecultureofbacteria.Todothis,youmustfirstlearnaseptictechnique.Thisisaspecialsetofproceduresdesignedtopreventcontamination.Thismeansthatyoutransferonlythemicroorganismofinterestanddonotcontaminatethemwithothermicroorganismsfromthesurroundingenvironment. RulesforAsepticTechnique • Bacteriaarefoundeverywhere,usecommonsensetoavoidcontamination. • Youmustnotallowanypartofyourselforanyothernon-sterileobjecttotouchthegrowth

media. • DonotremovethelidfromyourPetridishcompletely,insteadliftitandholditabovethe

dishtoprotectthemediafromdust. • DonotputtesttubecapsorPetridishlidsdownonthecounter. • Sterilizeyourlooporinoculatingneedlebyheatingituntilitglows.Dothisbeforeandafter

eachtransfer.

15

Figure 4.2

16

EX4:StreakPlating–Materials

brothculture:mixedculturecontainingbothMicrococcusluteusandEscherichiacoliinoculatingloop 1trypticasesoyagar(TSA)plate(perstudent)

EX4:StreakPlating-Methods

1. Follow theprocedure in Figure 4.2 to aseptically pick up a loopful of bacterial brothculture. Follow theprocedure inFigure4.1 to spread the loopfulof cultureonto theagarsurface,covering≈⅓ofthesurfaceinaback-and-forthfashion.Keepyourloopontheagarsurfaceanddon’tgougetheagar.Inthisareayoushouldgetconfluentgrowthafterincubation.

2. Reflameyour loop,killingallofthebacteriaon itssurface.Touchthehot looptotheagar surface inorder to cool it downbeforegoingacross your first streakonce, andthenproceeding tostreak thesecondsectionof theagar. Insection two,youshouldgetmoderategrowthafterincubation.

3. Reflameyourloop,againkillingallofthebacteriaonitssurface.Touchthehotlooptotheagarsurfaceinordertocoolitdownbeforegoingacrosstheareaofyoursecondstreak once and then proceeding to streak the third section of the agar. In sectionthree,youshouldgetdiscretecoloniesafterincubation,becausethebacteriawillhavebeendilutedoutsufficientlyfromtheoriginalculture.

4. Label the agar-side of the plate with your initials/seat # and ‘Mix’ to indicate thebacterial cultureused. Incubateplateagar-sideup in theappropriate incubationboxuntilnextlabperiod.

5. Nextlab,observetheplatesandanswerquestionsinRESULTSsection.

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Exercise5:EnvironmentalSampling–MicrobialExaminationofSurfaces

Objectives • ApplyPublicHealthsanitarylevelstomicrobialsamplesgatheredfromtheenvironment. • Recognize the correlation between the number of microbial colonies obtained from an

objectwiththedegreeofsanitation.

Reading Thecontaminationoffoodserviceutensils,equipment,andfoodpreparationcountertopsisofconsiderable interest to individuals in a number of health-related occupations. Suchoccupations include people who are health inspectors, food processors, and hospitalepidemiologists.Thesepeopleneedtobeabletojudgeaccuratelythedegreeofcleanlinessofmanyobjects.Inordertoaccomplishthis,severaltechniqueshavebeendevised.

The most simple and rapid procedure is the use of the RODAC plate to obtain a directenvironmentalsample.TheRODACplate isaspecialplate inwhichtheagarhasbeenslightlyoverfilled.Whenthecoverisremoveditcanbeseenthattheagarprotrudesslightlyabovethelipoftheplate,givingaconvexsurface.Inuse,thisagarsurfaceispressedlightlyonthesurfacebeing sampled, the lid is replaced, and the plate is incubated. Organisms on the surfacesampledadheretotheagarandgrowasdiscretecolonieswhichmaythenbecounted.Thus,anapproximateideaastothedegreeofsanitizationcanbeobtained.RODACstandsforReplicateOrganismDetectionandCounting.ThelimitationoftheRODACplateisthatitcanonlybeusedsuccessfully on flat, impervious surfaces which are free of crevices. Additionally, not allmicrobescanutilizethenutrientagarintheRODACplateasfood.

Asecondmethodwhichhasthedistinctadvantageofbeingusefulonirregularsurfacesisthemoist swab (or utensil swab) technique. Here a sterile swab is moistened with a specialphosphatebufferedsolutionandisthenusedtowipeadefinedareaofeatingutensilssuchasglasses, forks,spoons,etc.Theswab is thenreturnedtothevialcontainingthediluting fluid,tightlycapped,andtransportedbacktothelaboratory.Thediluentkeepstheorganismsinanosmotically correct environment so that they remain alive during transport back to thelaboratory.Onceatthelab,0.25mlofthefluidisplatedoutonappropriatemedium.Followingincubation,theplatesareobservedandthecoloniescounted.

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EX.5:EnvironmentalSampling–MaterialsperStudent

1paperbagtobetakenhomecontaining: 1RODACplate 1tubeofbuffercontainingsamplingswab

1.0mlsteriletransferpipette(usedinlab) 1PCAplate(usedinlab) hockeystickandflamingalcohol(usedinlab)

EX.5:EnvironmentalSampling–Methods

TOBEDONEATHOME:Inoculatethenightbeforeormorningofyourlab.

RODACPlate:Useonanyflatsurface(e.g.cuttingboardorkitchencountertop).

1. Remove the lid,pressexposedagar firmlybutgentlyonto surfacebeing sampledusingaslightrockingmotion.Replace lidandre-tapetopreventthe lidfromfallingoff.Labeltheplatewithsurfacesampled.BringtheRODACplatebacktoclassthefollowinglabperiod.

SampleSwab:Useonfourspotsofanyirregularsurfaces(e.g.fourutensilsorfourspotsonakeyboard).

1. Bend the redsnapvalveuntil youhear thevalvebreak.Squeeze thebulbof theswab totransferalloftheliquidtothetubeendwiththeswab.Twistandpullapartthebulbendoftheswabfromthetubeendoftheswab.

2. Swab any irregular surface, by rolling the moist swab back and forth 3 times over thesurface.Afterthisisdone,theswabisimmersedbackintothebuffer,swirled,andpressedoutagainstthesideasbefore.

3. Usingthesameswabprocedureasbefore,repeatonanothercleanutensil/cupuntilaseriesof4 similar utensils/cups (or surfaces) havebeen swabbed.When the fourth surfacehasbeenswabbed,replaceswabinsamplingtubefortransporttolab.Besuretotightlycapthesamplingtube.

4. Label thesamplingtubewiththetypeofsurfacesswabbedsoyoudon’t forget.Bringthesamplingtubebacktoclassthefollowinglabperiod.Theinstructionsforplatingthesamplearelistedonthenextpage.

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BACKINTHELAB

RODACPlate:

1. LabeltheRODACplatewithyour initials/seat#andthesitesampledbeforeplacing inthecorrectincubationtub.Plateswillbeincubatedfor48hrsat37°C.

SampleSwab:

1. Before beginning, label your PCA (plate count agar) plate on the agar sidewith yourinitials/seat#andsurfacesampled.

2. Removea0.25mlaliquotofthebufferusinga1.0mlsteriletransferpipette.Placethealiquotontotheagarsurface.

3. Sterilizeaglass"hockey-stick"bydippingitintoflamingethanolandlightingtheethanolon fire. DO NOT HOLD THE HOCKEY STICK IN THE FLAME. Allow the ethanol tocompletelyburnoffandthenusethehockeysticktospreadtheliquidevenlyacrosstheagarsurface.Rotatetheplatetoallowforevendistribution.Placetheplateinthecorrectincubationtube.Iftheagarsurfaceisstillquitewet,doNOTplaceagarsideup.Plateswillbeincubatedat37oCfor48hrs.

InterpretationofResults:

AftercolonieshavegrowninterprettheRODACplateandPCAplateasfollows:

1. Countthecoloniesonyourplates.Ifcolonycountexceeds100,sanitationisdefinitelynotacceptable.

2. Describethemostcommoncolonytypeoneachplate(color,shape,size).

3. SelectasinglecolonyfromyourRODACplate;makeasmearandGramstain,followingtheinstructionsfromExercise6.Besuretoplaceasmalldropofwateronyourslidetomixinwiththeselectedcolony.

4. RecordyourresultsinRESULTSsection.

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Exercise6:TheGramStain

Objectives • PracticeGramstainingbacterialsmears. • Acquire a general understanding of the theoretical explanation for differing Gram stain

reactions.

Reading Simplestainsdonottellusmuchaboutthecellexceptformorphology(shape). Ifwewishtolearnmore about the cell, such aswhether it contains a spore or not, it’s cell wall type, orwhether it possesses a capsule,wemustuse adifferential stain.Differential stainsnormallyemployseveraldyes.TheGramstainisadifferentialstain;itallowsustodifferentiatebacteriaby theircellwall structure.Grampositive cells havea thick layerofpeptidoglycan, a sugar-aminoacidcomplex.Gramnegativebacteriahavea thin layerofpeptidoglycanandanouterlipidmembrane.AGramstainisoftenthefirststepinthedifferentiationandidentificationofbacterialspecies.

TheGramstainrequirestheuseof fourreagents.Thefirstdyeorprimarystainaddedtothesmeariscrystalviolet,whichisfollowedbyaniodinesolution.Theiodineiscalledthemordant(aspecializedtermusedindyeing),whichcombineswiththecrystalviolettoformaninsolublecoloredcompoundinthemicrobesbeingstained.Thisinsolubleprecipitateiscalledthecrystalviolet-iodinecomplex.Afterdecolorizing,usuallywithacetone-alcohol,safranindyeisappliedtothesmearasacounterstain.

Organismswhichresistdecolorizingandretainthecrystalviolet-iodinecomplexappearpurpleor blue and are calledGram positive. Conversely, those cells that decolorize or give up thecrystal violet-iodine complex will accept the safranin counterstain and appear red or pink.ThesearetheGramnegativeorganisms.

YoushouldrememberthatthedifferentiationoftheGramreaction isnotanabsolute,all-or-nonephenomenon. It isbasedon the rateatwhich thecells release thecrystal violet-iodinecomplextothedecolorizer.EvenGrampositiveorganismscanshowaGramnegativereactionifdecolorizedtoomuch.AnumberofotherfactorscanresultinvariableGramreactions(wheresomecellsappearGrampositiveandothersappearGramnegative),suchasthefollowing:

1. Improperheatfixingofthesmear.Ifasmearisheatedtoomuch,thecellwallscanrupture,causingG+cellstoreleasethecrystalviolet-iodinecomplexandacceptthecounterstain.

2. Cell density of smear. An extremely thick smearmay not decolorize as rapidly as one ofordinarydensity.

3. Lengthandthoroughnessofwashingaftercrystalviolet.4. Theamountofdecolorizerapplied.EventuallyGram+cellswilldecolorize.5. Ageofbacterialculture.Gramreactionsareonlyreliableforcultures24hoursoldor less.

Culturesolder than24hours have increased cellwall permeability and thusmay convertfromanoriginalGrampositivereactiontoaGramnegativereaction.

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EX.6:GramStaining-Materials

Gramstainreagents:crystalviolet,iodine,acetone-alcoholandsafraninbrothcultures:MicrococcusluteusandEscherichiacoli trypticasesoyagar(TSA)platefromexercise4

EX.6:GramStaining-Methods

GramStain:ADifferentialStain 1. Apply enough crystal violet (primary stain) to cover the fixed smear. Let it sit for one

minute.2. Pouroffthestain.Rinseslidewithslowlyrunningwater.3. Applyenoughiodine(mordant)tocoverthefixedsmear.Letitsitforoneminute.4. Pourofftheiodine.Rinseslidewithslowlyrunningwater.5. Tiltslideoverpapertowel.Dripacetone-alcohol(decolorizer)downtopofslide,sothat it

runs down the slide over the smear and onto the paper towel. Continue drippingdecolorizeruntilitrunsclearofftheslide(approximately4-5seconds).

6. Immediatelyrinseslidewithslowlyrunningwater.7. Applyenoughsafranin(secondarycounterstain)tocoverthefixedsmear.Letitsitforone

minute.8. Pouroffthesafranin.Rinseslidewithslowlyrunningwater.9. Blottheslidedrybyplacing itbetweentwopapertowelsandpressinggently.Donotrub

slide.

MicroscopicExamination

1. PrepareasmearfromeachoftheabovebacterialculturesandfromacolonyfromyourTSAplate. (Ex. 3 has specific instructions on how to make smears.) Air-dry and heat-fix thesmearsbeforebeginningthestainingprocedure.

2. StainthesmearusingtheGramstain(detailedabove)3. Examine your slidewith themicroscope, startingoutwith the lowpower (10X) objective

andworkingyourwayuptotheoilimmersionlens(100Xobjective)ofthemicroscope.Besuretogeteverythinginfocusbeforemovingontothenextobjective.

4. Underthe100xobjective,notewhetherthebacteriaareGrampositiveornegative,basedon their color. Also note themorphology (shape) of the bacteria under themicroscope.RecordobservationsinRESULTSsection.

SummarizedGramStainMethod Step Floodw/ Time Gram+ Gram-

1. primarystain crystalviolet 1minute Rinsew/water Purple Purple 2. mordant iodine 1minute Rinsew/water Purple Purple 3. decolorize acetone-alcohol ≈5-8seconds Rinsew/water Purple Clear 4. counterstain safranin 1minute Rinse.Blotdry. Purple Pink/red

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Exercise7:RhizobiumandNitrogenRequirementsforLegumes

Objectives • AcquireanunderstandingoftheimportanceofRhizobiumnitrogenfixationforthenitrogen

requirementsofleguminousplants• LocateRhizobiumbacteroidsinrootnodules

Results Plants,likeallorganisms,neednitrogentoproduceproteins,nucleicacidsandotherpolymerswhicharecrucialfortheircellstructuresandmetabolism.Nitrogengas(N2)constitutes80%ofthe earth's atmosphere, but plants cannot use nitrogen in this form. Nitrogen-fixingprokaryotes however can turn N2 into organic nitrogenwhich can then be used by plants.Someplantssuchaslegumes(peas,beans,cloverandalfalfa)nurtureamutualisticsymbiosiswithnitrogen-fixingbacillifromthegenusRhizobium.ThelegumeproducesenlargedsectionsofroottissuecalledrootnoduleswhereRhizobiumbacteriadevelopintoapleomorphicformknownasbacteroidsandfixnitrogenintoorganicnitrogenforusebythelegume.Rootnodulesare variable in size, 1-10 mm. Rhizobium bacteria actively induce root nodule formation byinvading and infecting root cells. The bacterium receives energy, usually in the form ofcarbohydrates from the host plant. Because of this symbiotic relationship, peas, beans, andcloverdonotneednitrogen-containingfertilizerstogrowwellandcanactuallyenrichthesoilwith fixed formsofnitrogenwhichcanbeusedbyotherplants.Rotating legumeswithothercrops is apopularagricultural strategywhichallows farmers tominimize theuseofnitrogenfertilizer.Without theirmicrosymbionts, legumesrequirenitrogen ina fixed formanddonotgrowwellinnitrogenpoorsoil.Theuseofnitrogenrichfertilizersmaydiscouragerootnoduleformation.

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EX.7:NitrogenUsage–MaterialsperPair

Lab1 Rhizobiuminoculum: Rhizobiumleguminosarumbiovarviceaeand

Bradyrhizobiumsp.biovarphaseoli 1 containerof6-8cloverseeds1 sterilepotwithpottingsoil1 tubeof5mlsterilewater2 plantmarkers

Lab2 butcherpaperforuprootedplant

EX.7:NitrogenUsage–Methods

Lab1:YouwillreceiveONEofthefollowingseedcontainers...

ControlSeeds:Add1tubeofsterilewatertocontainerholdingcloverseeds.Swirlgently.Plantseeds inpot containing sterile soil, approximately1cmbelowsoil surface.Use several seedsperpot.LabelmarkerstickwithCONTROL.

Inoculated Seeds: Add 1 tube of sterile water to container holding clover seeds. Pour ininoculum (fine black powder) and swirl gently to coat. Plant themixture in a pot containingsterilesoilapproximately1cmbelowsoilsurface.LabelmarkerstickwithINOCULATED.

Potswillbewateredandmaintainedatroomtemperaturewithouttheapplicationoffertilizer.

Lab2

1. Observe thehealth/vigor, and color of the control versus inoculatedplants.Measure theheightofeachplantfromwheretheplantcomesoutofthesoiltothetopmostleaves.

2. Carefullyuprootplants.Shakeoffexcesssoil, gently rinse roots with water andlookforthepresenceofsmall(1-10mm) grayish-whitenodulesattachedtotheroots.

3. Ifrootnodulesarepresent:removeonenodulewithfingers,brushoffdirtandplaceitonacleanslide.Addasmalldropofdistilledwater.Useanothercleanslidetocrushthenoduleandspreaditaroundonthefirstslide.Gramstainandobserveusing100XobjectiveforthebacteroidformofRhizobium.

4. RecordobservationsintheRESULTSsection.

5. Whenfinisheddiscardplantsandsoilinthetrashcan.Donotthrowawaypots!Stackpotsatthediscardcenterbytheautoclavebag.

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Exercise8:IsolationofBacteriophages

Objectives • Memorizestepsinthereplicationofbacteriophageinbacteria. • Acquireanunderstandingofhowplaquesareproducedbytheplaquemethod. • PracticecalculatingtheconcentrationofPFU/ml(plaque-formingunitspermilliliter)using

plaquecountsanddilutioninformation.

Reading Virusescapableofinfectingbacteriaarecalledbacteriophages,orphages.Tostartaninfection,thephageattachesontothesurfaceofabacterialcellbymeansofitstailfibersandbaseplate.Thephage injects itsDNAorRNAintothebacterialcellduringentry.Duringpenetration,thetailsheathcontracts,drivingitscorethroughthebacterialcellwallandthuspushingviralDNAintothebacterium.Inthesynthesisstage,thebacterialcellisforcedtomakeviralcomponents,suchasviralnucleicacidandproteins.Theseviralpartsareputtogetherduringassembly,andthecompletedvirusesarefinallyfreedfromthebacterialcellduringthereleasestage.

Bacteriophages can infect bacteria grown in liquid (broth) or solid cultures. The use of solidmediamakesvisualizationofthephagepossiblebytheplaquemethod.Intheplaquemethod,host bacteria cover the surface of an agar plate to produce a confluent "lawn" of growth.Bacteriophages are then added in a discrete spot on this bacterial lawn. At this point, eachbacteriophagethat infectsabacterialcellwillmultiplyuntil thebacterialcell isburstopenorlysed. Thenewviruses that are thus releasedwill infect adjacentbacterial cells and similarlyproducemoreviruses.Eventually,manybacteriainoneareawillbedestroyed,leavingaclearareaorplaquewithintheconfluentlawnofuninfectedbacterialcells.Thenumberofplaquesoneachplate iscountedandthis information,alongwithdilution informationandvolume, isused to determine the original concentration of virus as Plaque Forming Units (PFU) permilliliter(PFU/ml).Themostaccurateestimateisachievedusingplate(s)withaplaquecountinthe25-250range.Lessthan25plaquesonaplatecouldbeduetobubblesorbitsofsolidifiedagar,whilemorethan250plaquesonasingleplatearedifficulttodifferentiate.

In this exercise, Lactococcus lactis, a bacterium used in the acidification and clotting ofmilkduringcheeseproduction,willbeusedasahostforbacteriophagec2.Youwillallowthevirustoattachtothehostcellandthenaddthemtomoltenagar.TheagarwillbelayeredontopofanNA(nutrientagar)platewherevirusreplicationandhostcelllysiscanoccur.Thislayeringisreferredtoasapourplate.

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EX.8:Bacteriophages–MaterialsperPair

0.1 mlbacteriophagec20.2 mlLactococcuslactisbrothculture

1sterilelongplastictransferpipette 1tubeof5mlsoftagarat48°C 1trypticasesoyagar(TSA)plate

EX.8:Bacteriophages–Methods

Lab1

1. Writeyournames/seatnumbersandthedilutionfactor(104to109,recordedonyourvirustube)ontheagarsideofyourTSAplate.

2. Usingtheplastictransferpipette,removetheL.lactisfromtheirtubeandputthemintothetubecontainingthebacteriophagec2.Gentlyswirlmixture.

3. Allowmixturetositfor5minutesonyourlabbench.

4. Afterthe5minutesareup,removeonetubeofsoftagarfromthe48°Cwaterbath.a. Dry the bottom of the tube off so that the water from the water bath does not

contaminateyourcultureandworkquicklyfortheremainingsteps.b. Addthemixtureofvirusandbacteriatothetubeofsoftagarc. SwirlthetubeandpouritoutontotheTSAplate.Theagarwillsetupquickly,sodon't

wastetime!d. Gentlyswirltheplatesothattheagarwillspreadevenlyacrossthesurface.

5. Waitatleast10minutesfortheagartobecomecompletelysolidandplacetheplateintheboxtobeincubated.Plateswillbeincubatedat30°Cfor4-12hours.

Lab2

1. Examineanentiresetofdilutionplates (104 to109), looking forplaques formed in theL.lactislawn.Completelycloudyplateshavenoplaques(solidlawnofbacterianotdestroyedby bacteriophage); clear plates indicate that the bacterial lawn has been completelydestroyedbybacteriophage.Count thenumberofplaquesoneachplate, recording clearplatesormostlyclearplatesasTNTC(toonumeroustocount).

2. Record your plaque counts in the RESULTS section. Use the results to determine theconcentrationofvirusintheoriginalsuspensionasPlaqueFormingUnitsperml(PFU/ml).Themost accurate count is achieved using any plate with a plaque count in the 25-250range.

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Exercise9:Koch’sPostulates–CrownGallDiseaseinPlants

Objectives • IsolateAgrobacteriumtumefaciensandobserveitsmorphology. • ApplyKoch’sPostulatestoprovethatA.tumefacienscausescrowngalldiseaseinplants.

Reading Crowngalldiseaseiscausedbythesoilbacterium,Agrobacteriumtumefaciens.Crowngallmayoccur in trees, roses, tomato plants, sunflowers, and other broad-leaf plants. The diseaseprocessstartswhenA.tumefaciensbacteriaenterwoundedplantsurfaces,oftentheroots.A.tumefacienstransferssomeofitsDNAintotheplantcellDNA,usingaplasmidknownastheTi(tumor-inducing) plasmid, which contains genes for proteins that stimulate bacterialreproduction.ThebacterialDNAalsotransformshostplantcells intotumors cells,eventuallycausing the formation of galls, a tumor-like growth. Galls are most common on roots andshoots. Iftheyareatthecrown,wherethestemcomesoutofthesoil,oronthemainroots,plantsgrowpoorlyordie.Oncethediseaseisestablished,thetumorcontinuestogrow,evenifviableA.tumefaciensbacteriaareeliminated.

Wewill use this plant disease to illustrate Koch's postulates. Koch's postulates are used toestablish that a particular disease is caused by a particularmicroorganism. It workswell formany bacterial diseases and was instrumental in demonstrating that microbes can causedisease.Thisconceptissowidelyacceptedtodaythatitisdifficulttoappreciatetheconfusioninduced by contaminatingmicroorganisms, poorly designed experiments, and plain bad luck,whichhadbesetKoch’sfellowscientistsinthe1870’s.TheGermancountrydoctorpublishedapapercontainingthefollowingproof.Theparticulardiseasehewasinvestigatingwasanthrax.

Koch’sPostulates: 1. Observethemicroorganisminahostwithdiseasesymptoms.2. Themicroorganismmustbeisolatedfromthediseasedhostandculturedinpureform.3. Thepurecultureof themicroorganism,when inoculated intoahealthyorganism,must

causethesamediseasesymptoms.4. Themicroorganismmustbere-isolatedfromtheartificially-infectedhostandshowntobe

thesameastheoriginalisolate.

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EX.9:Koch’sPostulates–MaterialsperPair Firstlab: diseasedcarrots(carrotslicedemoandpieceoftumortissue)

buffer solution containing diced tumor MGYplate

Secondlab: healthycarrotslice Ziploc®bagwithfilterpaper

Third lab: 3mlsterilewater buffersolution with diced tumor

EX.9:Koch’sPostulates–Methods SPECIALPRECAUTIONS:Agrobacteriumtumefaciensisaplantpathogenwhichshouldbehandledcarefullyfollowinglaboratorysafetyrules.

Firstlab 1. Observediseasedplantsetupasdemo.RecordobservationsinRESULTSsection.2. Youwillbegivenatube containing a diced pieceofgalltissuefromadiseasedcarrot

3. Makeasmearonacleanslide,usingaloopfulofliquidfromthemincedgallandGramstain.RecordobservationsinRESULTSsection.

4. Putanother loopfulof liquidfromthemincedgallontoaproperly labeledMGYplateandstreakforisolation.Theplatewillbeincubatedat25oCfor2-3days.

Secondlab 1. ExaminetheMGYplatefortypicalAgrobacteriumcolonies:grayish-whiteandmucoid.2. Chooseonelikelycolony.Makeasmearbymixingpartofthecolonywithasmalldropof

waterandthenGramstain.RecordyourobservationsintheRESULTSsection.ProceedonlyiftheGramstainmorphologymatchesresultsobtainedlastweek.

3. Label the Ziploc® bag with initials & seat #s. Pour 3 ml of sterile water into the bagcontainingapieceoffilterpapertowetthefilterpaperunderthecarrotslices.

4. Usingasterileinoculationloop,carefullyspreadtheremainderofthecolonyonthesurfaceofbothofthecarrotslices.Carrotsliceswillbeexaminedafterfourweeks.

Thirdlab 1. Observe infected carrot slice. Record observations in RESULTS section.

3. Discard the carrot and filter paper in the autoclave bag. Stack Petri dishes in themetalcoffinsonthediscardtable.Placetherazorbladeinthemetalsharpscan.

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Make asmearofthegallmaterialandGramstain.RecordobservationsinRESULTSsection.2.

Exercise10:RelationofOxygentoMicrobialGrowth

Objectives • Recognizewhatthefollowingtermsmean:aerobe,anaerobe,andfacultativeanaerobe. • Employtheuseofsodiumthioglycollateandresazurintoshowtheoxygenrequirementsof

bacteria.• Classifybacteriaaccordingtotheiroxygenrequirements.

Reading Bacteria show considerable variation in their requirement for oxygen. An organism thatrequiresoxygeniscalledaerobic;onewhosegrowthisinhibitedbyoxygeniscalledanaerobic;onethatcangrowundereitheraerobicoranaerobicconditionsisafacultativeanaerobe.

Inour laboratoryexercisewewillusea reducingmediumcalled sodium thioglycollatebroth.Sodiumthioglycollateremovesoxygenfromthebrothmediabycombiningwithitchemically.As a result, oxygen is tied-up and is unavailable to the bacteria. However, as might beanticipated inatubeofcapped liquidbroth,atmosphericoxygen isconstantlycirculatinganddissolvingintothebrothsurface.Asthedepthinthebrothincreases,theamountofdissolvedatmosphericoxygendiminishes toapointwhere itcanbecompletelyboundbythereducingagent,sodiumthioglycollate.Belowthispoint,trueanaerobicconditionsexist.Toshowwherethisoccurs,anoxygensensitivedye,resazurin,isalsoaddedtothebroth.Thisdyeiscolorlesswhenoxygenisnotpresentandpinkwhenoxygenispresent.

Youshouldnoteapinkishtingeintheupperlevelofyourthioglycollatebrothtubes.Thisshowsyouhowfaroxygenhasdiffusedintothemediumwithoutbeingtotallyboundbythesodiumthioglycollate.Whenperformingthefollowinginoculations,besuretheorganismisplacedwellbelowthebottomofthepinkzone.

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EX.10:OxygenUsage–MaterialsperPair

3sodiumthioglycollatebrothtubeswithresazurindye 3PCAsoftagartubeswithresazurindyebrothcultures:Bacillussubtilis

Proteusvulgaris Clostridumsp.

EX.10:OxygenUsage-Methods

1. Labeltheglassportion(NOTthecap!)ofthethioglycollatetubeswithonenameofeachofthethreebacterialculturesandyourseatnumber.

2. Carefully inoculate each of the tubes with an inoculation needle (not a syringe), goingdown into the medium about ¾ of the way. Remember to use aseptic technique. Onebacteriumpertube!

3. One at a time and working quickly, carefully inoculate one liquefied PCA+resazurin softagartubewithaloopfuloftheappropriatebacterium,thendumptheliquefied,inoculatedsoftagarintotheappropriatethioglycollatetube.

4. Placethetubesintheracksatthefronttable.Theywillbeincubatedfor48hoursat37oC.

5. Nextlab,observethelocationandappearanceofgrowthineachtubefollowingincubation.

6. RecordobservationsinRESULTSsection.Firstsketchtheregionofgrowthforeachbacterialtypeinthediagramsprovided;thenidentifywhattypeofoxygenrequirementisshownbysuchgrowth.

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Exercise11:MicrobesoftheMouth–TheirRelationshiptoCavities

Objectives • Recognizethehighnumbersanddiversityofnormalmouthflora. • Describe the association between Streptococcusmutans, S. salivarius and S. sanguis and

cavityproduction.

Reading Themouth is an exceptionally hospitable environment formicrobial growth. Not only is themouth warm and moist, but a consistent supply of nutrients (food we eat) is present. Themouthalsohasmanydifferentmicro-nicheswhereadiversevarietyofmicrobescanflourish.These micro-niches include biofilms made of bacterial cells and extracellular slime whichdeveloponthetongue,teeth,andgums.Someofthesebacteriaaredifficulttogrowastheyhavestrictnutritionaloratmosphericrequirements.

An important member of normal mouth flora is the Streptococcus group of bacteria.Specifically, on the tooth surface, Streptococcusmutans is the predominant species, with S.sanguisbeingmuchlessprevalent.Onthetongue,S.salivariusisadominantspecies.Allthreeof these Streptococcus species are capable ofmetabolizing sucrose (table sugar). Sucrose, adisaccharide,isfirstbrokendownbythesebacteriaintoitstwomonosaccharides,glucoseandfructose. The glucose is then combined into a long chain of glucoses, producing a stickycompoundcalledglucan.Glucancoatsteethandiseventuallyconvertedintodentalplaqueasbacteria and other salivary debris combine with the glucan. This dental plaque cannot bewashed away by saliva. The fructose and other carbohydrates are fermented to a variety ofacidsbyS.mutansandotherbacteriainthedentalplaque.Theseacidsarecapableoferodingtoothenamel,thusleadingtocavities.Sucroseisactuallytheonlycarbohydratewhichcanbeutilizedbybacteria tomakeglucan.However,allothercarbohydratescanbemetabolizedtoproduce acids, thus promoting cavity formation. Interestingly, even sugarless candies, thosewhichcontainmannitolorsorbitol,canbemetabolizedtomakeacids.

Wewillswabtheteethandcultureitonasucroseagarplate.Onsucrosemedia,S.mutans,S.salivariusandS.sanguisconvertthesucrose intoglucan,producingcolonieswhicharevisiblygummyandsticky.Productionofglucanonteethsurfacesisanimportantinitialstepincavityproduction.ToprovideamorerealisticappreciationforthediversityofthisbiofilmontheteethwewillplacescrapingsfromtheteethdirectlyonaslideandperformaGramstain.

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EX.11:MouthMicroorganisms–MaterialsperStudent

1sucroseagarplate 2sterilecottonswabs

EX.11:MouthMicroorganisms-Methods

1. Takeasterilecottonswabandswabthesurfaceofyourteethfora fewminutes.Makea

smearwithoutaddingwater,airdry,andGramstain.RecordobservationsintheRESULTSsection.Note:cheekcellswillstainaslargepinkovalscontainingdarkerpinknuclei(fried-eggtypeappearance).Theywillbemuch largerthanthebacteriayouare lookingfor,buttheremightbebacteriaclingingtotheoutsideofthem.

2. Takeasecondsterilecottonswabandswabthesurfaceofyourteethagain.Swabthefirst

thirdofasucrose agarplateusing the swab.Streakout the secondand thirdpartof theplatewitha sterile loop (a loop thathasbeen flamedandcooled),using the streakplatemethodtaughtinExercisefour.Labelagarsideofsucroseplatewithyourname/seatnumberandplace inappropriate incubation tub,agar-sideup.Plateswillbe incubatedat37oCfor48h.

3. Next lab, examine your sucrose agar plate. S. mutans will form large gummy looking

colonies that stickupabove the surfaceof theagar (like tinygumdrops).Gramstainonecolonyfromyoursucroseplate(remembertouseasmalldropofwaterwhenmakingthesmear).RecordobservationsintheRESULTSsection.

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Exercise12:NormalThroatFlora Objectives • Toobserveourownnormalthroatflora. • Toobservethemostcommontypeofmicrobeanditsassociatedhemolysis. • Torealizethediversityofnormalthroatflora. Reading In this exercise,wewill study thenormal bacterial flora of the throat.Normal flora are theorganisms found in the throatsof healthypeople. The throat is amoist,warmenvironment,allowing many bacteria to flourish. Many different types of organisms are found there.Infectionbypathogenicbacteria isactuallyminimizedbythenormalflora.That is,bacteriaofthe normal throat flora suppress the growth of pathogenic bacteria through competition fornutrientsandproductionofinhibitorysubstances. Streptococcus bacteria are the predominant bacteria found in normal throat cultures. Onestreptococcal species,Streptococcus pyogenes, isapathogen. It causes strep throatandskininfections as well as several serious diseases including: rheumatic fever, scarlet fever, acuteglomerulonephritis,andnecrotizingfasciitis. Streptococci are classifiedaccording to thehemolytic reactionsblood-enriched agar. Hemolysis is the lysis of red blood cells.behindthecolonywillchangecolororbecomeclear. Threepatternsofhemolysiscanoccuronbloodagar:

theycausewhilegrowingonIfhemolysisoccurs,theagar

l. Alpha-hemolysis:Incompletehemolysis;agreen,cloudyzonearoundthecolony.2. Beta-hemolysis:Completehemolysis;aclearzonearoundthecolony.3. Gamma-hemolysis:Nohemolysis;nochangeinthebloodagararoundthecolony. Alpha-andgamma-hemolyticstreptococciareusuallynormalflora,whereasbeta-hemolyticstreptococciareoftenpathogens.Streptococcuspyogenesproducesbetahemolysis.

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EX.12:ThroatMicroorganisms–MaterialsperStudent

1bloodagarplate 1sterilecottonswab 1steriletonguedepressor

EX.12:ThroatMicroorganisms-Methods

l. Swabthebackofyourthroatwithasterilecottonswab.Theareatobeswabbedisbetween

the golden arches (glossopalatine arches). Do not hit the tongue, which should be helddownwithasteriletonguedepressor.

2. Usetheswabtoinoculateone-thirdofabloodagarplate.Streakoutthesecondandthird

part of the platewith a sterile loop, using the streak platemethod taught in Exercise 4.Label agar side of blood agar plate with your name/seat number and place in theappropriateincubationtub.Plateswillbeincubatedat37°Cfor48hours.

3. Next lab,observecolonymorphologiesandtype(s)ofhemolysisonyourbloodagarplate.

Observehemolysisbyholdingtheplateuptothelight,lid-sideup,andobservingtheareaimmediatelysurroundingcolonies.

4. SelectacolonyfromyourplateandperformaGramstain.5. It is notpossible to identifymicroorganisms reliablywithoutexactbiochemical tests. You

may, however,make a presumptive identification of one of your bacteria based on yourGramstainresultsandcolonymorphology.UseTable13.1asareference.

6. RecordobservationsintheRESULTSsection.

TABLE13.1:COMMONNORMALFLORAOFTHETHROAT Organism Gramreaction/morphology Colonydescription Moraxellacatarrhalis Gram-diplococci/diplobacilli Large,smooth,gray

Candida(yeast) Gram+spheres(large) Large,smooth,pasty,cream/off-white

Corynebacteriumsp. Gram+bacilli/coccobacilli Large,rough,white

Lactobacillussp. Gram+bacilli(slender) Medium,smooth,cream

Streptococcussp. Gram+streptococci Small,smooth,translucentwhite/grey

Haemophilussp. Gram-coccobacilli(tiny) Small,smooth,transparent

Neisseriasp. Gram-diplococci Small,smooth,greyoryellowish

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Exercise13:TestingWaterforPathogenicMicroorganisms

Objectives • Practicethestepsinvolvedintestingwaterforpurity. • Identifycoliformbacteria.

Reading Watercanbeaseriouscarrierofdisease.Municipalsystems in thiscountry treat theirwaterwithchlorineorozonetokillmicroorganisms;however,watermuststillberoutinelycheckedforthepresenceofpotentialpathogens.Pathogenscanbedifficulttogrowandforthisreasonanindicatororganismisusedinstead.Theindicatororganismmustgrowrapidly,bepresentifpathogensarepresentanddieoffatthesamerateaspathogens.Inthiscountry,theindicatororganisms used are coliform bacteria. Coliforms are Gram negative rods which growfacultativelyandfermentlactosewiththeproductionofgasat35°Cin48hours.

LTBrothwithDurhamTube:Thepreliminarytestforcoliformsuseslactosetryptone(LT)brothand a Durham tube to trap any gas produced. The coliform group contains several types ofbacteria.OneimportantmemberisEscherichiacoli,abacteriumfoundinthelargeintestineofmostwarmbloodedanimals.IfE.coliispresent,thereisahighchancethatfecalcontaminationofthewaterhasoccurredanditisunfittodrink.E.coliandorganismswhichtestasverysimilararecalledfecalcoliforms.

EMBPlates:WewilltestforthepresenceoffecalcoliformsusingEMB(eosinmethyleneblue)agarplates.Thismedia inhibitsGrampositiveorganismsbecause itcontainstoxicdyeswhichcan be absorbed through their porous cell wall. It also contains lactose. Organisms whichferment lactose, producing acid, will cause the dye in the surrounding media to crystallize,formingreadilyvisiblegreenmetallic-lookingcolonies.

AnalternativetestinvolvesgrowthoforganismsinEC+MUGbroth.ECisanabbreviationforE.coli.TheoriginalECbrothwasdevelopedtoimprovethedetectionofthecoliformgroupandE.coliinwaterandothersourcessuchaswastewater,shellfishandfood.Furtherresearchfoundthat adding 4-methylumbelliferyl-β-D-glucuronide orMUG to the EC broth can be used todistinguishE.colifromotherbacteriainthecoliformgroup.E.coliproducesanenzymecalledglucuronidase that convertsMUG into a fluorescent product that glows in the dark when along-wave (366nm)UV light is used. All coliforms grown in the brothwill have turbidity andproducegasbutonlyE.coliwillglowundertheUVlight.

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EX.13:WaterandColiforms–MaterialsperPair

Lab1 100mlof2XlactosetryptonebrothwithDurhamtube100mlofwatersample

Lab2 1EMBagarplate 1shortsteriletransferpipette

EX.13:WaterandColiforms-Methods

Lab1 1. Add100mlofwatersampleto100mlof2X(i.e.,doublestrength)lactosetryptonebroth

containingaDurhamtube.Donotshakethebottle–youdon’twanttointroduceabubbleintotheDurhamtube.

2. Label the bottle with your seat numbers and water sample number. Place in theappropriateincubationtub.Bottleswillbeincubatedat37°Cfor48hours.

Lab2 1. Examinebroth for turbidityorsedimentat thebottom,which indicatesmicrobialgrowth.

ExamineDurhamtubeforpresenceofgas,indicatedbyabubbletrappedinthetube.Onlycoliformswillproducegasfromlactose.RecordobservationsinRESULTSsection.

2. Gentlyswirl thebottletoredistributethebacteriathatmayhavesettled tothebottomofthebottle.Usingasterileinoculation loop,Collect a loopful of water sample and perform a T-streak on EMB plate.

3. Labeltheplatewithname/seatnumberandwatersamplenumber.Tubeswillbeincubatedat37°Cfor48hours.

Lab3 1. Examine the EMB plates for growth. Non-fecal-coliforms will produce dark pink-purple

colonies,whilefecalcoliformsproducecolonieswithagreenmetallicsheen.

2. RecordobservationsinRESULTSsection.

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Exercise14:ConjugationandtheTransferofAntibioticResistance

Objectives

• Identifythefollowingterms:conjugation,plasmid,pili/pilus,F+(donor),F-(recipient). • Observe how genes carried on either a chromosome or plasmid can affect the antibiotic

resistanceofabacterium.

Reading Bacteria have several natural mechanisms for exchanging genetic material. The naturalmechanism we will study is called conjugation. In conjugation, a self-replicatingextrachromosomal,circularpieceofDNAcalledaplasmidispassedfromonestrainofbacteria,thedonor(F+),toanotherstrainofbacteria,therecipient,(F-),throughahollowtubecalledthesexpilus.TheplasmidDNAcontainsgenesthatcanthenbeexpressedintherecipientbacterialcell. In our exercise, the donor strain of Escherichia coli carries genes for resistance to oneantibioticonitsplasmid.TherecipientstrainofE.colinaturallyhasageneforresistancetoasecondantibioticonitsDNAchromosome.Afterconjugationbetweentherecipientanddonorstrains of E. coli, a new strain of E. coli is produced, which should have resistance to bothantibiotics.Notethatbygainingtheplasmid,thisnewE.colihasbeenconvertedintoanF+typeofbacterium.

Thediagramonthenextpageshowstheprocessofconjugationinvolvingplasmidtransfer.

37

(blue) (red)

(blue) (red)

(blue) (red)

(blue) (red)

38

EX.14:Conjugation–MaterialsperPair

1mlofEscherichiacoliHT99(Donor) 1mlofEscherichiacoliJ-53R(Recipient)

1chloramphenicol(C)plate 1rifampicin(R)plate 2chloramphenicol+rifampicin(C+R)plates

1sterilelongtransferpipette 1sterileshorttransferpipette 1hockeystickandethanol(EtOH)

EX.14:Conjugation-Methods

1. Divideonechloramphenicol(C)plate,onerifampicinplate(R),andonechloramphenicol+rifampicin (C+R) in half by drawing a linewith your pen on the bottom surface of theplate. Label one half ‘HT-99’ and one half ‘J-53R’ for each plate. Also label with yournames/seatnumbers.SetyourremainingC+Rplateasideuntillater.

2. Aseptically inoculateoneloopfulofE.coliHT-99cultureontheappropriatehalfofeachplate (C plate, R plate, and C+R plate). Don’t worry about streaking for isolation, juststreakazigzaglineofculturedownonehalfoftheplate.

3. Aseptically inoculateone loopfulofE.coli J-53Rcultureontheotherhalfofeachplate,withastreakasbefore.

4. Pipettetheremainingculturefromthelargetubeintotheculturecontainedinthesmalltube;swirlthesmalltubeandsettubeasideforatleast20minuteswithoutdisturbing.Attheendofthe20minutes,swirlgently.

5. Pipette 0.5 ml of the mixed culture (HT-99 plus J-53R mixture) on your remainingchloramphenicol+rifampicinplate.Useasterilehockeysticktospreadtheliquid.Donotinvert the plate! Place in the appropriate incubation tub, lid-side up. All plates will beincubatedat37oCfor48hours.

6. Nextlab,examineallplatesforgrowthandrecordobservationsintheRESULTSsection.

Growthonanantibiotic-containingmediummeansthatthebacterialstrainisresistantto the antibiotic(s) in that medium. Non-growth on an antibiotic-containing mediummeansthatthebacterialstrainissusceptible(non-resistant)totheantibiotic(s)inthatmedium.

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Exercise15:StaphylococcusaureusinPotatoSalad–OneCauseofFoodPoisoning Objectives • Prepare a food, potato salad, and mishandle it to provide conditions for Staphylococcal

contaminationandgrowth. • AnalyzethefoodforthepresenceofS.aureusanddeterminethelevelofcontamination. Reading Staphylococcus aureus is one of the most common causes of food-borne poisoning in theUnited States, producing an illness called Staph food poisoning. Not all strains of S. aureuscausefoodpoisoning.Onlythosestrainsthatcanmakepoisonsknownasenterotoxinsleadtofood-borneillness.SuchS.aureusstrainsusuallygetintothefoodsduetofoodhandlers.Manyfood handlers have S. aureus on their hands and in their mouths or noses as part of theirnormalflora.Thus,whentouchingfoodduringpreparation,orbycoughingand/orsneezingonfood,foodhandlersinoculateS.aureusintothefood.FoodpoisoningresultsiftheS.aureusinsuch foods remainsata temperature thatallows it togrowandproduceenterotoxins. Staphfoodpoisoning results fromconsumptionof theenterotoxin, rather thanconsumptionof thebacteria themselves. Subsequent heating of the food can destroy the bacteria but will notinactivatethetoxin.ThefoodsmostofteninvolvedinStaphfoodpoisoningarethosewhicharehandledduring theirpreparation,have rich sourcesofprotein and fat, and have a high saltcontent.Specific foods includecreamysalads,suchaspotatosalad,cream-filledpastries,andsaltymeats,suchasham.FoodsthataresuspectedofcausingStaphfoodpoisoningoftenhavelarge numbers of S. aureus. To detect and enumerate these bacteria, one must perform adilutionbeforeplatingasampletoobtainanaccuratecount. In this exercise, studentswill prepare potato salad in a deliberately unsanitarymanner. Thepotato salad will also be allowed to remain at a warm temperature (37oC) for a period ofseveral hours. Similar poor temperature control often happens with potato salad taken onpicnics(wheremanycasesofStaphfoodpoisoningoccur).Thepotatosaladwillthenbedilutedandculturedasdescribedintheproceduresection.StudentswillthenbeabletodeterminetheactualnumberofS.aureuswhichgrewintheirpotatosalad. Anexcellentmediathatselectsforstaphylococciismannitolsaltagar (MSA).Thehigh(7.5%)salt concentration in thismedia inhibits all skin bacteria except staphylococci. An additionalingredient of this media, the sugar mannitol, helps differentiate between the two mainstaphylococcispecies,S.aureusandS.epidermidis.S.aureusfermentsmannitolproducingacid.MSA media also contains phenol red,which is pink under neutral or alkaline pH but turnsyellowinthepresenceofacid.IfS.aureusispresentitwillgrowontheMSAplateandfermentmannitolproducingacidthatwillturntheagaryellow.Ifmannitolisnotfermentedthemediawillremainpink.

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EX.15:PotatoSalad–MaterialsperGroupof4 Lab1:

2smallboiledpotatoes 1boiledegg 1foodcontainerbutcherpaperSalt,mayonnaise

Lab2:

1x99mldiluentbottle 3x9mldiluenttubes 2sterileshorttransferpipettes 4mannitolsaltagar(MSA)plates 1hockeystick,flamingEtOH 1tonguedepressor

EX.15:PotatoSalad-Methods Lab1-PreparationofPotatoSalad-workingroupsof4 1. DONOTWASHYOURHANDSBEFOREBEGINNING.2. Usingbarehands,mashthecookedpotatoesintosmallpiecesintothepapertub.Peelthe

eggandaddittothepotatoesinasimilarmanner.3. Addapproximately1teaspoonofsaltand2-3tablespoonsofmayonnaise.4. Mixthesaladwithbarehands,withallgroupmemberstakingaturnatmixing.Trynotto

betooclean.5. Labelthefoodcontainerwithagroupname,thelabdayandtime(e.gM10:30)andwith

the word TOXIC. Containers will be incubated at 37oC for 4 hours. The product will berefrigerated after that point, mimicking a potato salad at a park that is subsequentlybroughthomeandrefrigerated.

Lab2–SerialDilutionofPotatoSaladBacteria–DONOTTASTEPRODUCT 1. Prepareserialdilutionsofthepotatosaladbyremovingapproximately1g(¼tsp.)fromthe

centerofthecontainerandaddingittoa99mlbottleofdiluent.Avoidchunksofpotatoandtrytogetsomeofthesofterormoreliquidpartsofthepotatosalad.

2. Tightly cap the bottle and shake vigorously for 30 sec. This represents a 1:100 or 102

dilutionofthepotatosalad.Labelthisbottle102.Usingasterilepipette,transfer1mlofthe102dilutiontoa9mldiluenttubeandmixwell.Thisnewdilutionrepresentsa1:10dilutionofthe1:100dilution,forafinaldilutionof103.Labelthistube103.

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3. Usingthesamepipettefromstep2,transfer1mlofthe103dilutiontoanew9mldiluent

tubeandmixwell.Thisnewdilutionrepresentsa1:10dilutionofthe103dilutionforafinaldilutionof104.Labelthistube104.

4. Usingthesamepipettefromstep3,transfer1mlofthe104dilutiontoanew9mldiluent

tubeandmixwell.Thisnewdilutionrepresentsa1:10dilutionofthe104dilutionforafinaldilutionof105.Labelthistube105.

5. LabeltheMSAplateswithseat#sandfinaldilutions.UseaNEWsterilepipettetoplatethe

followingdilutionsofpotato saladonmannitol salt agar (MSA)plates in the order listedbelow: 0.5mlofthe10

5dilutiononthe1

stplate

0.5mlofthe104dilutiononthe2

ndplate

0.5mlofthe103dilutiononthe3

rdplate

0.5mlofthe102dilutiononthe4

thplate

(1:100,000dilutionofpotatosaladbacteria)(1:10,000dilutionofpotatosaladbacteria) (1:1000dilutionofpotatosaladbacteria)(1:100dilutionofpotatosaladbacteria)

6. Spreadthedilutedpotatosaladovertheplateswithaflame-sterilizedhockeystick.Donot

invertMSAplates.Incubatetheplatesat37oCfor48hours. Lab3-MicrobiologicalExamination

1. Countthecoloniesontheplates.Optimumplatesforfurthercalculationswillhaveatotalof

25-250colonies.Colonies that formonMSAplates typically representa species from theStaphylococcusgenus.Staphylococcusaureuscolonieswillformayellowhaloaroundthem.IfenoughcoloniesofS.aureusareonasingleplatetheycanchangetheentireplateyellow.

2. Using the colony counts, dilution values and volume plated calculate the number of

Staphylococcus(notjustS.aureus)pergram(Staph/g)ofpotatosalad(1ml≈1g).

Calculation:(numberofcoloniescountedonplatemultipliedbythefinaldilutionfactoroftheplate) divided by volume plated equals the number of Staphylococcus colony-formingunits(CFU)pergramofpotatosalad.

Forexample,ifthecolonycountsofStaphare78CFUonthe103dilutionplatethen: (78CFUx10

3)/0.5ml=78000CFU/0.5ml=156000CFU/ml

or1.56x105StaphCFU/gramofpotatosalad

3. SelectonecolonyonanMSAplatetoGramstain,toverifythepresenceofStaphylococcus.

RecordyourobservationsintheRESULTSsection.

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Exercise16:MicrobialMetabolism–FermentationofCarbohydrates Objectives • Describethebreakdownproductsofglucose,lactose,andsucrosecarbohydrates. • Interpretcarbohydratetubesandthefermentationproductsproduced. Reading Metabolismcanbedefinedas the sum totalof all chemical reactionswhichoccur inside thecell. Themetabolismof carbohydratesentails the catabolism (orbreakdown)of certain largemolecules, such as disaccharides (double sugars), into smaller ones calledmonosaccharides(single sugars). For such breakdown to occur, enzymes are required. In this exercise the 2disaccharides, sucrose and lactose, are broken down by the enzymes sucrase and lactaserespectively.Thesereactionsproceedasfollows:

Lactose(disaccharide) Glucose+Galactose (twomonosaccharides)

Sucrose(disaccharide) Glucose+Fructose (twomonosaccharides) Themonosaccharidesarethenfurtherdegradedbyacombinationofglycolysisandoneoftwoprocesses,fermentationorrespiration.Theyarefermentedbysomebacteriatoyieldorganicacids, alcohols and/or gases. Fermentation reactions also release a small number of energymolecules (suchasATP)whichareneededto"fuel"otherbacterialchemicalreactions.Otherorganisms process monosaccharides more completely. In a process known as aerobicrespiration, theyproduceabundantATP,water,andCO2gas.Aerobic respirationtakesplaceonlywhenO2isavailable. Thisexerciseillustratesasimplemethodtodetectacidandgasformationfromcarbohydratebreakdown.FormationofacidsisdetectedbyincludingapHindicatorinmicrobialgrowthmedia.Inthisexperiment,thesugarbrothscontainthepHindicatorbromcresolpurple(BCP).ThispHindicatorispurpleatpH6.8(nearneutralpH)andyellowatpH5.2(acidpH).Inthesebroths,gasformationisdetectedbytheuseofasmallinvertedtubereferredtoasaDurhamtube.

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EX.16:MetabolismandFermentation–MaterialsperPair

3tubesofglucose-BCPbrothwithDurhamtubes(redcaps)3tubesofsucrose-BCPbrothwithDurhamtubes(bluecaps)3tubesoflactose-BCPbrothwithDurhamtubes(greencaps)

brothcultures:

Bacillussubtilis Micrococcusluteus Klebsiellapneumoniae

EX.16:MetabolismandFermentation-Methods

1. Inoculateadifferent tubeofeachmediumwitha loopfulofBacillus subtilis,Micrococcus

luteus, and Klebsiella pneumoniae. Use aseptic technique throughout! If even a smallamountofbacteriaorsugargetstransferredtothenexttube,itwillscrewuptheresults!

Bacillussubtilis:inoculateonetubeeachofglucose,sucroseandlactose.Micrococcusluteus:inoculateonetubeeachofglucose,sucroseandlactose.Klebsiellapneumonia:inoculateonetubeeachofglucose,sucroseandlactose.

Note:Besuretolabeleachtubeontheglasswithnameofbacterium,yournames/seatnumbers,andmakecertainyoureplacethecaponthesametube.Placetubesintorackinappropriateincubationtube.Alltubeswillbeincubatedat37oCfor48h.

2. Nextlabperiod,observetheculturesforbothacidandgasproduction.3. RecordobservationsintheRESULTSsection.

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Exercise17:CulturedDairyProducts Objectives • Observemicroorganismswhichareusedtoproducecultureddairyproducts. • Comparethepropertiesofdifferentripenedcheeses. • Acquire knowledgeabout the typesof changesproducedby themicrobialmetabolismof

milkwhichresultintraditionalcultureddairyproducts. Reading Productionofcultureddairyproductsreliesonavarietyofmicroorganismsdependingontheproduct being made. Lactic acid bacteria are the most frequently utilized. They fermentcarbohydratestoproducelacticacid.

Milk/Yogurt - This traditional European dairy product ismade of pasteurized or boiledmilk.Two lactic acid bacteria, Streptococcus thermophilus and Lactobacillus bulgaricus are usedtogether during incubation at high temperature (45°C). Boiling and the temperature ofincubationhelppreventthegrowthofunwantedcontaminants.S.thermophilusproduceslacticacid fromthemilksugar lactose.ThisencouragesthegrowthofL.bulgaricuswhichproducesadditionalacidandproteolyticenzymes.Milkproteinsdenaturecausingthemilktosolidify.Themilkdevelopsadistinctivetangyflavorduetothefermentationproductslikelacticacid.ThepHofthemilkdropssignificantlybecauseoftheproductionoflacticacid.

Cheese - Many types and sources of milk are used all over the world to produce cheese.Additional variety is providedby theuseof different culturing agents.Lactococcus lactis andspeciesfromthegenusLeuconostocareoftenusedfortheinitialculture.Lacticacidproducedby the bacteria, along with the enzyme rennin which is added separately, curdles the milkseparating the curds (milk solids) from the whey (liquid). Additional microorganisms calledripening agents can be added to the curd. The final taste and texture of the cheese can bemodifiedbythelengthandconditionsoftheripeningperiod.

Type Cheese ExampleRipeningagent(s)* RipeningPeriod Soft: Brie Penicilliumcamemberti 4-6months

Brevibacteriumlinens Camembert Penicilliumcamemberti 4-6months Semi-soft: Blue Penicilliumroqueforti 3-6months

Limburger Brevibacteriumlinens 1-2months Micrococcussp. Mixedyeast Hard: Cheddar Lactococcuslactis 4-12months

Leuconostocsp. Swiss Propionibacteriumsp. 3-12months Parmesan Streptococcusthermophilus 24-48months Lactobacillusbulgaricus

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EX.17:DairyProducts–MaterialsperPair

traditionalBlue,Cheddar,andLimburgercheeses boiledmilkfortifiedwith3-5%powderedskimmilk,heldat45°Cinwaterbathcommerciallivecultureyogurt pHpaper

EX.17:DairyProducts-Methods Lab1 Yogurt:

In pairs, test the initial pHof a tubeof fortifiedboiledmilk.UsepHpaper and the colorguidetodeterminethepH.Usingasterileloopinoculatethemilkwithstarterculture(liveculturecommercialyogurt).Useseveralloopfuls.Rolltheinoculatedtubebackandforthinyourhandtomixandlabelitontheglasswithyournames/seat#s.Returnthetubetothe45°Cwaterbath.Theyogurtwillbeincubatedforabout4hoursandthenrefrigerated.

Cheese:

ExaminetheBluecheese.Youmaynotelongstraightholesdrilledintothecurdtoprovidethe secondary culture agent Penicillium roqueforti with the oxygen it requires. The blueveinsofthecheesearemadeofsporeswhichdevelopfromthemold’shyphalfilaments.Awetmountdemohasbeenpreparedbyscrapingsomebluematerialfromthecheeseblockandaddingadropofdistilledwater.Examinethiswetmount,whichhasbeenplacedunderthe 40/45X objective. Look for hyphal filaments or spores. Remember, the 100X oilimmersionobjectiveisnotusedforawetmount!

Examine theCheddar and Limburger cheeses. Limburger has a particularly characteristicodor.Usealittlewatertopreparesmearsofbothtypesofcheese.Trytogetsomeofthesurfacematerial (therind)fromtheLimburgercheese–mostofthemicrobesare locatedhere.Gramstainyoursmearsandexaminethem.Youmayfindthatde-stainingthesmearstakeslongerthanusualduetothefatcontentofthecheese.RecordallobservationsintheRESULTSsection.

Lab2 Yogurt:

Examine the tube of milk for consistency and odor. Test the final pH using pH paper.PrepareasmearoftheyogurtandGramstain.(LacticAcidBacteriaresponsibleformakingyogurtaretypicallyGrampositive).RecordobservationsintheRESULTSsection.

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Exercise18:TheFungi–MoldsandYeast Objectives • Recognizethedifferencebetweenfilamentousfungiandnon-filamentousfungi. • Identifythemacroscopicandmicroscopicfeaturesofcommonmoldsandyeast. Reading The study of fungi is referred to as mycology (Gr. "Mykes" = fungus). Fungi are a group ofeukaryoticmicroorganismswhichlivebydigestingorganicmaterial.Fungicanbedistinguishedfromalgaebecausethefungidonothavechlorophyllandthuscannotcarryoutphotosynthesis,evenifsomeappeargreenincolor.Fungicanbedifferentiatedfromprotozoainthatmostarenon-motileandhaveadistinctcellwall. Fungi canbedifferentiated frombacteria,whichareprokaryotes,bythefactthatfungalcellsaremuchlargerandhavenucleiandotherorganellestypicalofeukaryoticcells.Althoughthefungiarealargeandratherdiversegroupofeukaryoticorganisms,twoexamplesoffungiareexaminedinthisexercise:moldsandyeast. Moldsarefilamentousfungiwhicheveryonehasobservedgrowingonstalebread,cheeseorfruit.Thefilaments,calledvegetativehyphae(Gr."Hyphe"=web)usuallygrowtogetheracrossasurfacetoformacompactmat,collectivelycalledamycelium.Thecontinualbranchingandintertwiningoffungalfilamentsresultsintheformationofavisiblestructureonthesurfaceofthe substrate. A portion of the mycelium becomes aerial (aerial hyphae) and gives rise tospecializedcellsatitstips.This‘fuzzy’growthiswhatweseeandcallmold.Thesespecializedcellsare referred toasspores.Theyallowthemoldcolony to reproduceasexually, searchingoutnewlocationsandnutrients.Sporesareextremelylightweightandveryeasilydispersedbyeven the slightest air current (suchasopening the lidquickly fromaPetridish). Thismotionscatters them about, disseminating themold throughout the area. The color of a particularmoldisduelargelytothespores,whichmaybepigmented.Dependingonthedivisiontowhicha mold belongs the asexual spore carrying structure may be called a conidiophore or asporangiophore.Aconidiophoregivesrisetoconidiaontheoutsideoftheaerialhyphae,whileasporangiophoregivesrisetosporangiacontainedwithintheaerialhyphae.Theseareusedascharacteristicsforidentificationofindividualmoldspecies.Moldsareadditionallyidentifiedbywhether their hyphae may be described as septate or aseptate (or coenocytic), based onwhetherornotthehyphaecontaincrosswallsorpartitionsbetweencells.Moldsareusedformanyindustrialpurposesaswellascausingsomehumandiseases. Yeastareunicellular(singlecell)fungigenerallydistinguishedfrommoldsbytheirlackoftruefilaments.Theyeasthaveauniquetypeofasexualreproductioncalledbudding.Inthisprocessasmall,newcell(calledthebudordaughtercell)ispinchedofffromthemothercell.Yeastareextremelyimportanttous.Anexampleofausefulyeastwhichisresponsibleforproductionofbread,beerandwineisSaccharomycescerevisiae.Otheryeastcanbepathogeniccausingsuchdiseases as vaginal infections or thrush, a fungal infection of themucousmembranes of themouth.

47

EX.18:Fungi-Materials

potatodextroseagar(PDA)platesof: Aspergillussp. Penicilliumsp. Rhizopussp.

stainedslidesshowingmicroscopicstructureofabovemolds suspensioncultureoftheyeast: Saccharomycescerevisiae

EX.18:Fungi-Methods Note:thematerialsneededforthislabaresetupasdemonstrationsaroundtheroom.

1. Observe the molds on the plates as they appear under the dissecting microscopes.Notethecolor(pigment)andgeneralappearanceofthespores.

2. Examinethepreparedslidesofthemoldsunderthelightmicroscopes.Lookcloselyat

individualhyphalfilamentstodeterminewhethereachmoldisseptateornon-septate.Lookcloselyataerialhyphaetodeterminewhethereachmoldformsconidia(outsideaerialhyphae)orsporangia(insideaerialhyphae).

3. Observe the wet mount of the Saccharomyces cerevisiae suspension and note the

morphologyoftheyeastcells.Lookforindividualcellsthatarebudding.

4. DrawyourobservationsintheRESULTSsection.

48

MB230IntroductiontoMicrobiology

Results

Name:________________________________Section:_______________________________RoomNumber:_________________________SeatNumber:__________________________

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EX.1:UseoftheMicroscope–Results

PartA.Unstainedwetmounts.Drawwhatyouseeusingthe10Xor40/45Xobjective.

HayInfusionBroth PondWater (protozoaandbacteria) (algaeandbacteria)

PartB.Stainedpermanentslides.Drawwhatyouseeusingthe100Xobjective.

Staphylococcusaureus Candidaalbicans Bacillussubtilis (bacterium) (yeast) (bacterium)

TA Initials:

50

EX.2:MicroorganismsintheAir–Results

InformationfromPCAplateexposedtoair:

Numberofdifferentbacteriaandyeastcolonies_______________________ (identifiedbyvaryingcolor,shape,texture,size,etc)

Numberofdifferentmoldcolonies_______________________ (identifiedbyvaryingcolor,shape,texture,size,etc)

Chooseonecolonyonyouragarplateanddescribeittothebestofyourabilityusingtable2.1.DonotremovethelidofthePetridish,exceptforbriefperiodsoftime. size: pigmentation: form: TA Initials: margin: elevation:

Table2.1:CharacteristicsUsedforColonyEvaluationonAgarPlate Size Pinpoint

Small Moderate Large

Pigmentation Colorofcolony

Form Circular:round,evencolonies (shapeofthecolony) Irregular:irregularlyshapedcolonies

Rhizoid:rootlike,spreadinggrowth

Margin Entire:sharplydefined,even (appearanceofcolonyouter Lobate:markedindentations edge) Undulate:wavyindentations

Serrate:toothlikeappearance Filamentous:threadlike,spreadingedge

Elevation Flat:elevationnotdiscernible (degreetowhichcolony Raised:slightlyelevated growthisraisedonagar Convex:domeshapedelevation surface) Umbonate:raised,withcenterofcolonymarkedlyelevated

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EX.3:Morphology–Results

EXAMINATIONOFSTAINEDCELLS–Drawafewrepresentativecellsthatyouobserve.Makeyourdrawingssufficientlylarge.

TA Initials: TA Initials:

Staphylococcusepidermidis Escherichiacoli

colonyfromPCAplate Spirillum(Ex.2) (permanentslide)

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EX.4:StreakPlating–Results

Drawthebacterialgrowththatyouobserveonyourstreakplate:

TA Initials:

HowmanydifferentCOLONYtypesdoyouobserve? Howmanyisolatedcoloniesdoyouobserve?

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EX.5:EnvironmentalSampling–Results RODACPlate:

SurfaceSampled NumberofColonies

ColonydescriptionofthemostcommoncolonytypeonRODACplate:

Doyouhaveacceptablesanitation?(Plateswithlessthan100colonies.)

Gramreaction:____________________

Cellmorphology:___________________

Gramstainofasinglecolony TA Initials: SwabTechnique(PCAplate):

SurfaceSampled NumberofColonies

ColonydescriptionofthemostcommoncolonyonPCAplate: Doyouhaveacceptablesanitation?(Plateswithlessthan100colonies.)

TA Initials:

54

EX.6:GramStain-Results

Micrococcusluteus

Escherichiacoli

TSAplatecolony □ yellowcolonyor□ whitecolony

TSA plate colony bacterial species:

TA Initials:

Morphology:_________________

Gramreaction:_______________

TA Initials:

Morphology:_________________

Gramreaction:_______________

TA Initials:

Morphology:_________________

Gramreaction:_______________ 55

EX.7:NitrogenUsage–Results Observationsofplants:

Group Color Vigor/Health Height(cm) Nodules(y/n?)

Control

Rhizobium

Gramreaction:____________________

Cellmorphology:___________________

Gramstainofrootnodule

TA Initials:

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EX.8:Bacteriophage–Results

Determinehowmanybacteriophagewerepresentpermloftheoriginalculturebymultiplyingthenumberofplaquesontheplatebythedilutionfactor.

(plaquecount xdilutionfactor)/volumeofvirusplated=PFU(plaque-formingunit)/ml

Workwiththerestofyourtabletofillinthechartbelowandestimatetheoriginalphageconcentration.Plateswithbetween25and250plaquesgivethebeststatisticalestimateoftheoriginalpopulation.Recordcompletelycloudyplatesaszeroornoplaques.

1. Howmanyplaquesareontheplatemadebyyouandyourpartner?2. Whatisyourdilutionfactor?3. Filloutthefollowingchart,usingplatecountsfromtheentiredilutionseries:

PlaqueCount DilutionFactor VolumeofVirusPlated PFU/mLResult

4ontube=104 0.1mL 5ontube=105 0.1mL 6ontube=106 0.1mL 7ontube=107 0.1mL 8ontube=108 0.1mL 9ontube=109 0.1mL

4.Estimateoforiginalphageconcentration(PFU/ml),usingmostaccurateplaquecount(s):

TA Initials:

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EX.9:Koch’sPostulates-Results Lab1 1. Describeappearanceofcontrolcarrot:2. Describeappearanceofdiseasedcarrot:3. GramstainofA.tumefaciensfrommincedplanttissue(gall):

Gramreaction:_____________

Cellmorphology:____________ Lab2 1. DescribecolonymorphologyonMGYagarplate:2. GramstainofA.tumefacienscolonyfromMGYagarplate:

Gramreaction:_____________

Cellmorphology:____________ Lab3 1. Describeappearanceofcontrolcarrot:2. Describeappearanceofinoculatedcarrot:3. GramstainofA.tumefaciensfrommincedplanttissue(gall):

Gramreaction:_____________

Cellmorphology:____________

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TA Initials: TA Initials:

TA Initials:

EX.10:OxygenUsage–Results 1.Drawwhereeachbacteriumgrewinthethioglycollatetubes: Bacillussubtilis Proteusvulgaris Clostridiumsp.

TA Initials:

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EX.11:MouthMicroorganisms-Results Lab1:

Gramstainofdirectsmearofteeth:

TA Initials:

Gramreaction:________________

Cellmorphology:_______________ Lab2:

1. Describe up to three different colony types found on your sucrose plate (size, color,

texture,etc.):

Colony# ColonyDescription Approx.%oftotal

coloniesonplate

1

2

3

2.Gramstainofonecolonyfromsucroseagarplate:

Gramreaction:________________ TA Initials:

Cellmorphology:_______________

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EX.12:ThroatMicroorganisms-Results 1.Describethedifferentcolonytypesonyourbloodagarplate:

Colony# DescriptionofColony Hemolysistype Approx.%oftotal

coloniesonplate

1

2

3

2. Whattypeofhemolysisismostprevalentonyourbloodagarplate?3. Gramstainofonecolonyfrombloodagarplate:

Colony#:________ TA Initials:

Gramreaction:______________

Cellmorphology:______________

3.Presumptiveidentificationoftheorganism,usingTable11.1:

TABLE13.1:COMMONNORMALFLORAOFTHETHROAT Organism Gramreaction/morphology Colonydescription Moraxellacatarrhalis Gram–diplococci/diplobacilli Large,smooth,gray

Candida(yeast) Gram+spheres(large) Large,smooth,pasty,cream/off-white

Corynebacteriumsp. Gram+bacilli/coccobacilli Large,rough,white

Lactobacillussp. Gram+bacilli(slender) Medium,smooth,cream

Streptococcussp. Gram+streptococci Small,smooth,translucentwhite/grey

Haemophilussp. Gram–coccobacilli(tiny) Small,smooth,transparent

Neisseriasp. Gram-diplococci Small,smooth,greyoryellowish

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EX.13:WaterandColiforms–Results

Lab2: 1. Isgrowthpresentinthelactosetryptonebroth?2. Isgaspresentinthelactosetryptonebroth?

TA Initials:

3.Isyourorganismacoliformornon-coliform?

Lab3: 4. IsgrowthpresentontheEMBplate?5. Doesthegrowthhaveagreenmetallicsheen?6. Isyourorganismafecalcoliformandnon-fecalcoliformoranon-coliform?

TA Initials:

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EX.14:Conjugation–Results Fillinthechartbelow(growthornon-growth):

Platewith: chloramphenicol rifampicin chloram.+rifamp.

Donor:

Recipient:

“New”: NA NA

TA Initials:

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EX.15:PotatoSalad–Results

ColonyCountofMSAplates

ColonyCount Dilution Calculation Results

102 (___x102)/0.5ml

103 (___x103)/0.5ml

104 (___x104)/0.5ml

105 (___x105)/0.5ml

1.WhatisyourfinalestimateofStaphylococcuspergram(Staph/g)ofyourpotatosalad?

Showyourwork. 2. Based on the MSA colony

growth,approximatelywhatpercentageofthisisStaphylococcusaureus?3. GramstainofonecolonyfromanMSAplate:

Gram reaction: ______________ TA Initials:

Cell morphology:______________

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TA Initials:

T TA Initials: Initials:

EX.16:Fermentation–Results Recordtheresultsofthecarbohydratesugarfermentationasnegative(-)fermentation;Aforacidfermentation(brothchangestoyellow);orAGforacidandgasfermentation(brothchangestoyellowandbubblepresentinDurhamtube).

BACTERIUM GLUCOSE SUCROSE LACTOSE

(red) (blue) (green)

Bacillussubtilis

Micrococcusluteus

Klebsiellapneumoniae

TA Initials:

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EX.17:DairyProducts–Results

CheeseType Odor Consistency MicroscopicExamination Totalmag.(wetmount)=

BlueCheese Description: Totalmag.(Gramstain)=

Cheddar Description: Totalmag.(Gramstain)=

Limburger Description:

Microscopicobservationsofcheeses(Gramstainorwetmount)

Morphology:_________________

Gramreaction:_______________ TA Initials:

cheddarcheese

Bluecheese

(wetmount)

Morphology:_________________

Gramreaction:_______________

Limburgercheese TA Initials:

Milk/Yogurt Lab1 initialpH:____________

Lab2 finalpH:_____________

Odorofyogurt: Consistencyofyogurt:

Gramstainresults:

Morphology:_________________

Gramreaction:_______________

TA Initials:

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EX.18:Fungi–Results MakedrawingsoftheSaccharomycescerevisiaecells.Magnification:40/45Xobjective

Saccharomycescerevisiae Foreachofthemolds,describethefollowing:

Aspergillus Penicillium Rhizopus DissectingScope(agarplate)

Drawordescribemold’s appearance:

__________ __________ __________ TotalMagnification:

LightMicroscope(preparedslide)

Drawsporestructure:

TotalMagnification: __________ __________ __________ ConidiaorSporangia? Twoofthefungihaveconidia. septateornon-septatehyphae?

Colorofcolonyonagarplate?

GeneralizedStructuresforMolds:

non-septate/aseptate/coenocytic

septate

conidia sporangium TA Initials:

67