Microbiology: A Laboratory Manual

561 Pages·2016·57.751 MB·English

by  Cappuccino, James G;Welsh, Chad T

A flexible approach to the
modern microbiology lab
EXPERIMENT
Propagation of Isolated 39
Bacteriophage Cultures
NEW! “Propagation LEARNING OBJECTIVES CLINICAL APPLICATION
Once you have completed this experiment, With the increase in the rates of antibiotic resis-
of Isolated Bacteriophage you should be able to tance in clinically relevant bacteria, pharmaceutical
Genus CIudlteunrets”i fiexcpaertimioennt of Unk1. nIcmsuoalltnauitrpoeeu bfloaatrci otwleanrtseio.rp gheanngeetsic f rso tumd iae sp loarq ue ctnbhooaemwcrtEap elpoareiona3ukiXle itinisncg fa e tanrcPetdt ati orhtemnesE ewpenoaittsrhscsR ihianbe ivulriistrn1Iyu la isokrM.eef Cl tylruoe proarlkeatEiincnntegg rs fea.oN sTrree hnaseeriyscw Tthaa r net
has been added to the 2. Eisnoulamteedra ftreo mth ea np lianqduivei-dfouraml pinlagq uunei.ts iass e ax ammeianninsg o tfh tere caltininicga bl aucsteesr ioafl ibnafecctetiroionpsh iang tehse EXPERIMENT
Bacterial Cultures absence of antibiotics. 46
Eleventh Edition. This Microbial Fermentation
experiment (39) guides Principle
This exercise will demonstrate the procedure for AT THE BENCH
students to isolate isspoelcaiteins gf raonmd ap rsoinpgalgea ptilnagq uae s ppeicckifiecd bfraocmte rai olapwhang e
bacteriophages for pbgeleangtieent.i sBct eurefdocyroienm agb main niacetriwoob nbi oastcluotedgriisieots p oahrn av giinreod oliovr igbdiesutga imln s atyra in MCualtuterersials PART A Alcohol Fermentation gttihroaanpt sea asre rae rr eeins acpcroiednasss ieabdnle di n fmo trhi ntehe firean clishs hawrehadoc ptserero icdsotuinccct te aannsttdrea s-
LOEncAe RyoNu IhNagicmvuGeernp rceOoeotmnrBitctpa J lcnmeElttie aCntdnei TctcihapIhiVslun relEieaxqpstuieeoerain mri,nc eah nnt , auornbemdctcfbciktnhoileoaeouf elalcnleslns tontsbce;Wtee .wio t aa rsbbWehc noie eseduotadii fe mtfntico h uewsr wegberio awaletlehlelhd c sag epip tehlntcinhrleen eo e r eoerdsselsff vto .tpolea hueecTnohnrstendaymh atrasty hu.iguwmlei siTsnena liioiusghgilllnsoll fs e wy gsta vstbps l siisieubeehrmwsl pu l eaavg striss gylaiercepearaesmnhaiusrr eedoop e.snetbt n saao tiispecdnr o bwgtnr l f iavaan.e chesniccec laaoedttitTt iegti s d ctn mrh mhhfiefigfbraahesf uoooalu tte ws ma rssasteibpiri lni obl lnoael gne e gn-lb i lto cteeox h bpesAE2MP btbe r4uregxeyio-rfapephrf tdredoethrd orprieu itenivlesmrmuaade intgbe eanssunensaotast erlfrt ,ttii3 teene0 8isadinne. 9egot tfisr( h ambvtTtaruern,Btcla odd 2hpSte hfasema)lnr;v ro ct l et ttmurgpp uaiylrinet bpoEurdoerntu rxiot.evpupon in:dobed e1rfeu c i0aE; ma galmosr eaenpcnrlhdle aftodpe sqnrfl s uai3iTntLucOyte71Reheuos .hEnos i IutarWaa rcSrAanl c eye snecditt phnoRdiy vtla ose osoiiN utunBpi lee Ir.dhs oN a udovnGufe dcy cteeiOoroasmnsBtta pb JnclyeedE lttlehsCde. T tfheIirVsm eEexnpteartiimvee nt, actWgaommonrrnruuu ha ddappssis ltrTt behlteee oxh.toes sowwP.aedus c o e qricgentedutorixaetn eamatts gtporirsssam de ip eucops retxtm frohmir poodcce rdmuniie eafgus sfseolrtecs sot fp.reba wtertFdbehhon ti ieefdfrhstre s iuu jworrtoulc ,mmfic fiittn cotihae oeeetlcneho ,nsie te.wrsg e to Friidjahcfnauod i pwtwiacrdoce chiiree sntite dhdi hoedas e ft trb ic ow heswaja u e citaclhniihlt rrceileeeu toedsr,e s .nki t hahgi,ne e ds
you should abse aab lpeo tossible way to arenane iigstmhhbso feroinr gpb rcoepallusl.ss Sioiinnsc, es utohceh f vai sr tuas hfleasig hesallva e io ndro fi mmee-nch-tifiEcquaiptmioennt. HoweverP, ryinocuip le mgroalpdess, iann dth we ivlidn yeeyaasrdt .t hAa wt ainree -epnrdoodguecninogu ss ttroa in of
treat antibiotic-resistant shobthreiua seo, lftthd aeg p aanrr mtioceldetisu emmu tsott shrperlaye aodtn f droyifmfuo scieoulnl ttorh rc oebullg. hi ocBcheunnetsreimfnu gbeu irtuncberea,s w, 1la-t merrlb seattehsr,i ltueh peirlpmteotstme se,mtWt sehtriee,n r1jeuia. l5iiesc- m ygeals lap o srsfo gdruacpte osf a tnhde ontahtuerr aflr ufeitrsm, iennctlautdioinng o f yise uasste,d S taoc cinhoacruomlatyec eths ec emruevsit,s iwahei vcahr i. s e ltlhiepnso ideus,
1. Use previously studied staining, cultural noTtthhe i sbp ehxeaegr ec ipisader twiecilleln su sfrteo timhca at nao cisclou lratrteeondc p el tatoqh ureeo.mosvee shPdeaovsitcweeu,r t epnsipt et utitbeens ,r arcu kbT,b aeanrd b bgullabls,es mw ea3creh am1napyicre.ekaaa1ilscn pthg ;ic eppe seel,tnl ptsci.en iTlag.rh si,s pbliuomchs,e amnidca alp cpolnevs,e brsyi othne o af cjutiiocne of iant c2u1b°aCt etod 3fo2r° C3. tToh 5i sd iasy fso ullnodweerd a beryo abnic a cnoanedroitbioicn s
characterbisaticcst,e arniadl b iinocfheecmtiiocanls p.roce- they may vary because of variations in batdoce gwtriaendeer o tchiceau frrsul iw t hsuegna trhse, fyreuacstto csee lalsn edn gzlyumcoastiec, afillrys t i1nyceuabra ttoio 5n ypeearrios din. Tahgein wg itnaen kiss tohre wn oaogedde nfo bra rrels.
to acetaldehyde and then to alcohol, as illustrated During this time, the wine is clarified of any
dures for independent genus identification strains (subgroups of a species). Thereforein ,F iGgiurtarep e4s6 c.1o.ntaining 20% to 30% sugar concen- truersbpiodnitsyi,b tlhe efroerb cyh parroacdtuecriinstgi cv ofllaavtoilres e. sTtheers c tlhaaritfi aerde
of an unknown bacterial culture. becomes imperative to recall the specific bitorofa aticpopnhr owxeililm ymaieteldly- w 1i0n%e sto w 1i5th% a. nA lasloc ophreosl ecnotn itne nt p30romduincut tiess t, haennd fiblotetrtleedd,. pasteurized at 60°C for
ical tests that differentiate among the different
REVISED EXPERIMENTS include options for
genera of the test organisms. CH2OH
aalntedr ancactee smsiebdleia t,o m aallk sinizge st hoef leaxbpm peorrrioIemd geexernattneimfintscsasi vat.ei foE fpnoxr oorpfdc eeaadrnbiu murlneeke tnono tdw ifnfe cruelnttuiraete u bsianHOcHgtO eHa- OH OHH Genlyzcyomlyetisc 2CH3 CO COOH Decarboxylation Ac2eCtaHl3dCeHhOyde+ CCaOrb2on
Principle rial species is presented in Experiment 68. TheH OH 4H+ reduction dioxide
46 now includes both wine and lactic acid fermentation,
Identification of unknown bacterial cultures is one rationale for the performance of this exercise 2CH3 CH2 OH
looking at the production of winlaet earn ind t hyeo sgeumrets.ter is twofold. First, you will Glucose Pyruvic acid Ethyl alcohol
of themajor responsibilities of the microbiologist. Figure 46.1 Biochemical pathway for alcohol production
have acquired expanded knowledge of microbial
Samples of blood, tissue, food, water, and cosmet-
activities and will be more proficient in labora-
ics are examined daily in laboratories throughout
tory skills. Second, and more important, you will
the world for the presence of contaminants. In addi-
be more cognizant of and more critical in your
tion, industrial organizations are constantly screen- NEW! BioSafety Levels (BSLs) alert students to
approach to species identification using dichoto-
ing materials to isolate new antibiotic-producing
organisms or organisAmT TsH tEh aBtE NwCiHll increase the yield moTheurmosm etkereys supplemeantpedp rwoitph rBiaertgee ys’sa Mfeatnyu atel.chniques. The organisms within
of marketable produMcattse,r isaulsch as vitamins, solvents, this manual are mostly BSL-1 organisms, with any
and enzymes. Once Ciusltourleasted, these unknown organ- CBeakLer wIith NwaterICAL APPBLSILC-A2 ToIrOgaNn isms now marked within the text. The
48- to 72-hour nutrient broth cultures (50 ml
isms must be identipafieurre e2u5ds0 -m BaSl LEn -r2le dannmd ec Byealrca ifllalsuskss )c oeirffi eSutesa;p 7dh2y- .ltooc o9c6c-huos ur 1o0f -nmult rtieesntt tburboeth Eleventh Edition also reflects the most up to date
The science of cSEarlbleaonumsraesuydeir bfi flraocsthka )c outfl tiAuorsepsen r(g5 i0il lmussl pnceirga e2r5l 0al-nmedl d taxonomy AWirpe gapuzelication of Leasrnaefde Atys spayrso ttoo cIdoelnst iffyr om governing bodies such as the
and deals with the sSeacpchaarormaycteis ocernev isoiafe. living organisms anUnknown Bacterial Pathogen
into interrelated groMPuere pddeissaig.n aBtede strudgenet gyro’usp (pMairsa orn gruoupas olf has been TBuhnseen b urrnoerle of the clinicaEl PlaAbo,r aAtoSryM in, a a hnosdp itAalO isA toC , better preparing students for
the official, internatfaiogouarr)n p: lfiaavteel sn,l uaytnrdi e aonnt cea g1ca0r- mepllap ttuebts,e efi ovfde n Su atrbrioeeunrft abeurdor the.nce for quickly and efficientlyp irdoenfteifys sthieo cnaausla ltaivbe awgeontr k.
bacterial classificatiEoqnuip smienncte 1923. The current edi- of a patient’s infection. This will entail choosing the
tion, Bergey’s ManuM aicrlo inocifne rSatoyr osr tBeunmsen baurtnierc, 8 0B0-mal cterioFigluoreg 40y.2, W aterbath for mocisto herart eexpcertim aenstsays and performing them in the correct
arranges related bacwtuitbthee sh,r egaliata-srsew siiasnrtea ntmt oaprak di3,n tgh3 pere mngcoimrl, eaotnedur ,i nsptoecrsuillea ttciensgat lle6d. Slowly heat the water to 4o0°Cr;d cheecrk ttheo th elro-gically identify the genus and species
loop. mometer frequently to ensure that it does not
sections rather than into the classical taxonomecixucclteu erdes t hoef tdhees ierxepde treimmpenertoaalt uforregt.a hPnliaescme s at hinegt ofo etuhren t.
groupings of phylumP,r occleadsusr, eo rLdabe rO, naend family. Tbheakeer and maintain the temperature at 40°C
for 10 minutes. Remove the cultures and asep-
iisn tbearsreelda toionn cshhairpa ocft e1t.r hiLae2enx5sa°bdpCt e eSo lri( aitcmcbhoroeens ugnctrrto aaoavsull e)hd,nru e s4aa 0goict°a fsCt reeh ,pmam 6lca0p ht°ea eCorssa,fs, t 8tiuhn0i re°dmneC isnc, u taaoettonri nbideager n1 tut0pch s0aee°gh hdCa:r. osleoc7g.t yitaRroi,etcaep ai nseslleaey tc t ihtSnietooe ncwp uo a6lnta ef ttorehbr ee ata thtchweh toi eno mporglcpaautenelraisast tmliuaob rnTieen o l teifot dst 6 hI 4a0e0p° °tpPCCwr oa.onp driS - FOR SUCCESS NEW! Tips for Success
spthayinsiionlgo rgeya, ccteilolunlsa, rn 2cu. hS gOStlc.nar eoas triushmewtre et aihunreousei t usmrBnnieSadtnLre,rkt -r 2 iasnycig daga,n erup d oepa nftll ahcatnteilell u osipnd,t lhltaraoetb e rateb swB lw lo.o i cinstoecheer cesacthue iocshtn.a iOso.nner A a.m citce8.ar lipRrpe sllaaaptittseteeeeassi t t cllshSaaetbbtse eewp ll,e ea6 ddt ef 68orb00r °°atCCthh..e tienmocpuelraattiuorne Go tfo t r8h0ae° tCmw aon ds tain your unknown culture first and then aepxppeerairm thenrotsu gahnodu dtr tahwe
rkensouwAltstle tfdhogirse s ppooef icsnitfita cyin om3iu.n e nUabetghhgayitsegac i ma enrhpamr g aal tba ankeavtnsdisendtoee . eSg opt ahrtatlbi ge cishao ds ictnuneeircgoecsa olmheuennvd-d dliii nnq naSe suegi.te adcsll,,roe r ioropn eeiplopsvasp pciitsneueroicosleaa tocleitaulve.dbela date hst lieteeuosc dnint u iu2ocoo5tfnr° tfin Ceosfin f t. tcei91ec.0 .nh RrpIiannenlat-avpcditeu se erteabsht tae te lhtSda eSet b pe atewphobl eesao6id tut nef ir1uoorab0trnur 0a tifd°theohC near . t tgi2 enaa4mogr tacpporule al4prata8letat i hutoceornu euc ldtto trutuiefsol ref tt aeu1hysts0re t e30f itets7o°nw C°riaCn ro 4ga amntnnod y dino teuh rew b chaiictcrthae ttreeia st.et Fsso tw rw oeouxuladlmd b pbele eu ,os tfeh nfeuo lo uixnsi deida iesnne - amttiestnatkioens taon dco smtummobnli ng
niques, microbial n4u. tUtersri,i nttgra iano ssfteenrr i1l,e0 pmbipl eoitfto ee aaccnhdh cmueeltcumhraen tioic afcol uparip let -activ5i tdaiyes ast 2,5 °C in a moist chambeird. entifying a Gram positive cocci bacteria. blocks in the lab. Each
and characteristics osoftre gramilnei stmeisc ta ntrudb otehseo l atebrmelgpeedar watniuthrei t hs(4e0m n°Cam, s6e0 o°Ctf ,to he beP raobcleed ure Lab TwoSince many of the tests utilize agars that are tip explains why specific
80°C, and 100°C). 1. Observe all plates for the amount of growth of
to work independen5. tSleyt u pi t4nh0e.2 w,a ianttseertrbteainthmg aths epi ltluhtsetrirmantoemgd ein te tr oin ani den2.t itRfheeyc toe rsdt yoorguar nriessmuslt sa ti ne athche cohf athrte p streoimvmpideeridal tiaunr ers .in appearance, be sure to label all tubes techniques are necessary
the genus of an unknuoncwappned tucbeu ofl ntuutrirenet b.r oCth.haracteristhtei Lcabs R eport. and plates to ensure that results are collected
to yield accurate results
of the major organisms that have been used in for the correct test.
experiments thus far are given in Table 31.1. You and helps guide students
on how to perform crucial
procedural steps correctly.
Pearson Mastering Microbiology
prepares students for the
modern microbiology lab
The items mentioned here are available in the Study Area of
various Pearson Mastering Microbiology courses.
Pre-Lab Quizzes can be assigned for each of the 76 experiments in
Microbiology: A Laboratory Manual, Eleventh Edition. Each quiz
consists of 10 multiple-choice questions with personalized wrong
answer feedback.
MicroLab Tutors help instructors and students get the most out
of lab time and make the connection between microbiology
concepts, lab techniques, and real-world applications.
These tutorials combine live-action video and molecular animation
paired with assessment and answer-specific feedback to help
students to interpret and analyze lab results.
MicroLab Tutor
Coaching Activities
include the following
topics:
▶ Use and Application
of the Acid-Fast Stain
▶ Multitest Systems—
API 20E
▶ Aseptic Transfer of
Bacteria
▶ ELISA
▶ Gram Stain
▶ Use and Application
of Microscopy
▶ Polymerase Chain
Reaction (PCR)
▶ Safety in the
Microbiology
Laboratory
▶ Quantifying Bacteria
with Serial Dilutions
and Pour Plates
▶ Smear Preparation and
Fixation
▶ Streak Plate Technique
▶ Survey of Protozoa
▶ Identification of
Unknown Bacteria
Lab Technique Videos give students an
opportunity to see techniques performed
correctly and quiz themselves on lab
procedures both before and after lab time.
Lab Technique videos can be assigned as
pre-lab quizzes in MasteringMicrobiology
and include coaching and feedback.
Lab Technique Videos include:
▶ NEW! The Scientific Method
▶ NEW! How to Write a
Lab Report
▶ Acid-fast Staining
▶ Amylase Production
▶ Carbohydrate Catabolism
▶ Compound Microscope
▶ Differential and Selective Media
▶ Disk-diffusion Assay
▶ ELISA
▶ Gram Stain
▶ Hydrogen Sulfide Production
▶ Litmus Milk Reactions
▶ Negative Staining
▶ Respiration
▶ Serial Dilutions
▶ Simple Staining
▶ Smear Preparation
▶ Structural Stains
▶ Safety in the Microbiology
Laboratory
MicroLab Practical Activities assess
students’ observation skills and
give them extra practice to analyze
important lab tests, procedures, and
results.
Instructors: Tailor this lab manual
to perfectly fit your course!
EXPERIMENT
8
Name:
Date: Section: Lab Report
Observations and Results
NEW! Easy-to-adapt Lab Reports include blank
1. Draw representative fields of your microscopic observations.
spaces for individual course customization.
Instructors can select their preferred organisms.
M. luteus
2. Describe the microscopic appearance of the different bacteria using the
chart below.
Organism M. luteus
Shape EXP8ERIMENT
Negative StainAirnranggement
Magnification
NEW! Revised Experiments include
options for alternate media, reduced
volumes, and fewer bacteria, making the LEARNING OBJECTIVES CLINICAL APPLICATION
Once you have completed this experiment, Detecting Encapsulated Invaders
experiments affordable and accessible to you should be able to The principle application of negative staining is to
1. Perform a negative staining procedure. determine if an organism possesses a capsule (a
any-sized lab program. 2. Uvinsudaelrisztinangd u tnhseta bineende fimt iocbrotaoirngeadn firsomms . gitsoem lda emtimnoooreun ssv tiorrauutlteee nrs tlp)a,o yareeltr h ftoohruamgt ham tiiato kcnea. snT t haheles t ome cbichern ouiqosuergeda ins -
frequently used in the identification of fungi such as
Cryptococcus neoformans, an important infectious
Principle agent found in bird dropping that is linked to menin-
Negative staining requires the use of an acidic geal and lung infections in humans.
stain such as India ink or nigrosin. The acidic stain,
with its negatively charged chromogen, will not pen-
etrate the cells because of the negative charge on the
surface of bacteria. Therefore, the unstained cells are AT THE BENCH
easily discernible against the colored background.
The practical application of negative staining
iasn tdw tohfeo clde.l lFsi rasrte, nsiontc seu hbejeact tfiexda ttoio tnh eis d nisotto rretqinugir ed Materials
effects of chemicals and heat, their natural size and
shape can be seen. Second, it is possible to observe Cultures
REVISED! Instructor’s Guide for brsaitralaelcai ntn.e ioBrntiega kc ptiahlruloaestcde ea ahsrnseed, a dk tsi effilfiiexdcpaeu tislin tos tmnho oii snsu tdlnad oit nhbt, ae ds tohu tnachehne ddaolusre grdsiao nwnmgii stetmhh se sp i- TMawlitecerrnnotcayo-tfceoc buuars–c lthueortueiaur lsa ,cg uBalrat uscliralelnust.s c cuelrtueuress, aonf d other
care. Figure 8.1 shows a negative stain of bacilli. Reagent
Microbiology: A Laboratory Manual Nigrosin.
by James G. Cappuccino, Equipment
Microincinerator or Bunsen burner, inoculating
Chad T. Welsh lmoiocpr,o sstcaoinpien.g tray, glass slides, lens paper, and
© 2018 | 1-292-17581-8 • Procedure
Steps 1–4 are illustrated in Figure 8.2.
978-1-292-17581-2 1. Place a small drop of nigrosin close to one end
of a clean slide.
Updated to reflect changes in the 2. Using aseptic technique, place a loopful of
inoculum from the M. luteus culture in the
Figure 8.1 Negative staining: Bacilli (1000×) drop of nigrosin and mix.
lab manual, this guide is a valuable
teaching aid for instructors and
provides:
▶ NEW! Recommended readings for
each experiment
▶ Detailed lists of required materials
▶ Tables for calculating the amount
of media and equipment needed
for your class
▶ Procedural points to emphasize
▶ Suggestions for optional
procedural additions or
modifications
▶ Helpful tips for preparing or
implementing each experiment
▶ Answers to the Review Questions
in the lab manual
▶ Information on laboratory safety
protocol for instructional and
technical staff
M i c r o b i o l o g y
A L A b o r A T o r y M A n u A L
eleventh edition
Global edition
James G. Cappuccino
SUNY Rockland Community College
Chad Welsh
Lindenwood University
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Authorized adaptation from the United States edition, entitled Microbiology: A Laboratory Manual, 11th edition,
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ISBN 10: 1-292-17578-8
ISBN 13: 978-1-292-17578-2
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Contents
Preface 10 Part B: Capsule Stain (Anthony
Laboratory Safety 13 Method) 99
Laboratory Protocol 15
PART 4 Cultivation of Microorganisms:
PART 1 Basic Laboratory Techniques Nutritional and Physical
for Isolation, Cultivation, and Requirements, and
Cultural Characterization Enumeration of Microbial
of Microorganisms 17 Populations 107
Introduction 17 Introduction 107
Experiment 1: Culture Transfer Experiment 12: Nutritional
Techniques 23 Requirements: Media for the Routine
Experiment 2: Techniques for Cultivation of Bacteria 109
Isolation of Pure Cultures 31 Experiment 13: Use of Differential,
Part A: Isolation of Discrete Colonies Selective, and Enriched Media 115
from a Mixed Culture 31 Experiment 14: Physical Factors:
Part B: Isolation of Pure Cultures Temperature 125
from a Spread-Plate or Streak-Plate Experiment 15: Physical Factors: pH
Preparation 34 of the Extracellular Environment 131
Experiment 3: Cultural Experiment 16: Physical
Characteristics of Microorganisms 41 Factors: Atmospheric Oxygen
Requirements 135
Experiment 17: Techniques for
PART 2 Microscopy 47
the Cultivation of Anaerobic
Microorganisms 141
Introduction 47
Experiment 4: Microscopic Experiment 18: Serial Dilution–Agar
Plate Procedure to Quantitate Viable
Examination of Stained Cell
Cells 147
Preparations 49
Experiment 5: Microscopic Experiment 19: The Bacterial Growth
Curve 155
Examination of Living Microorganisms
Using a Hanging-Drop Preparation
or a Wet Mount 57
PART 5 Biochemical Activities
of Microorganisms 163
PART 3 Bacterial Staining 63
Introduction 163
Introduction 63 Experiment 20: Extracellular
Experiment 6: Preparation Enzymatic Activities of
of Bacterial Smears 67 Microorganisms 165
Experiment 7: Simple Staining 73 Experiment 21: Carbohydrate
Experiment 8: Negative Staining 79 Fermentation 171
Experiment 9: Gram Stain 83 Experiment 22: Triple Sugar–Iron
Agar Test 177
Experiment 10: Acid-Fast Stain 91
Experiment 23: IMViC Test 183
Experiment 11: Differential Staining
for Visualization of Bacterial Cell Part A: Indole Production Test 184
Structures 97 Part B: Methyl Red Test 185
Part A: Spore Stain Part C: Voges-Proskauer Test 186
(Schaeffer-Fulton Method) 97 Part D: Citrate Utilization Test 187
7
Experiment 24: Hydrogen Sulfide Experiment 41: Physical Agents
Test 195 of Control: Electromagnetic
Experiment 25: Urease Test 199 Radiations 301
Experiment 26: Litmus–Milk Experiment 42: Chemical Agents
Reactions 203 of Control: Chemotherapeutic
Agents 305
Experiment 27: Nitrate
Reduction Test 209 Part A: The Kirby-Bauer Antibiotic
Sensitivity Test Procedure 306
Experiment 28: Catalase Test 213
Part B: Synergistic Effect of 
Experiment 29: Oxidase Test 217
Drug Combinations 308
Experiment 30: Utilization of Amino
Experiment 43: Determination
Acids 221
of Penicillin Activity in the Presence
Part A: Decarboxylase Test 221
and Absence of Penicillinase 315
Part B: Phenylalanine Deaminase
Part A: MIC Determination Using
Test 223
a Spectrophotometer 316
Experiment 31: Genus Identification
Part B: MIC Determination Using
of Unknown Bacterial Cultures 227
a Plate Reader 317
Experiment 44: Chemical Agents
PART 6 The Protozoa 233
of Control: Disinfectants and
Antiseptics 321
Introduction 233
Part A: Disc Diffusion Testing
Experiment 32: Free-Living
of Disinfectants and
Protozoa 235
Antiseptics 324
Experiment 33: Parasitic
Part B: Modified Use Dilution
Protozoa 241
Testing of Disinfectants and
Antiseptics 325
PART 7 The Fungi 249
Introduction 249
PART 10 Microbiology of Food 331
Experiment 34: Cultivation
and Morphology of Molds 251 Introduction 331
Part A: Slide Culture Technique 251 Experiment 45: Microbiological
Part B: Mold Cultivation on Solid Analysis of Food Products: Bacterial
Surfaces 253 Count 333
Experiment 35: Yeast Experiment 46: Microbial
Morphology, Cultural Characteristics, Fermentation 337
and Reproduction 259 Part A: Alcohol
Experiment 36: Identification Fermentation 337
of Unknown Fungi 267 Part B: Lactic Acid
Fermentation 339
PART 8 The Viruses 273
PART 11 Microbiology of Water 343
Introduction 273
Experiment 37: Cultivation and Introduction 343
Enumeration of Bacteriophages 277 Experiment 47: Standard Qualitative
Experiment 38: Isolation Analysis of Water 345
of Coliphages from Raw Sewage 283 Experiment 48: Quantitative
Experiment 39: Propagation of Analysis of Water: Membrane
Isolated Bacteriophage Cultures 289 Filter Method 353
PART 9 Physical and Chemical Agents PART 12 Microbiology of Soil 359
for the Control of Microbial
Introduction 359
Growth 293
Experiment 49: Microbial Populations
Introduction 293 in Soil: Enumeration 361
Experiment 40: Physical Agents Experiment 50: Isolation of
of Control: Moist Heat 295 Antibiotic-Producing Microorganisms
8 Contents
and Determination of Antimicrobial Experiment 63: Identification
Spectrum of Isolates 367 of Streptococcus pneumoniae 467
Part A: Isolation of Experiment 64: Identification
Antibiotic-Producing of Enteric Microorganisms Using
Microorganisms 368 Computer-Assisted Multitest
Part B: Determination Microsystems 473
of Antimicrobial Spectrum Experiment 65: Isolation and
of Isolates 369 Presumptive Identification of
Experiment 51: Isolation Campylobacter 483
of Pseudomonas Species by Means of Experiment 66: Microbiological
the Enrichment Culture Technique 373 Analysis of Urine Specimens 487
Experiment 67: Microbiological
Analysis of Blood Specimens 493
PART 13 Bacterial Genetics 379
Experiment 68: Species Identification
Introduction 379 of Unknown Bacterial Cultures 499
Experiment 52: Enzyme
Induction 381
Experiment 53: Bacterial PART 16 Immunology 507
Conjugation 387
Introduction 507
Experiment 54: Isolation of a
Experiment 69: Precipitin Reaction:
Streptomycin-Resistant Mutant 393
The Ring Test 509
Experiment 55: The Ames Test:
Experiment 70: Agglutination
A Bacterial Test System for Chemical
Reaction: The Febrile Antibody
Carcinogenicity 397
Test 513
Experiment 71: Enzyme-Linked
PART 14 Biotechnology 403 Immunosorbent Assay 519
Experiment 72: Sexually
Introduction 403
Transmitted Diseases: Rapid
Experiment 56: Bacterial
Immunodiagnostic Procedures 523
Transformation 405
Part A: Rapid Plasma Reagin Test
Experiment 57: Isolation of Bacterial
for Syphilis 523
Plasmids 413
Part B: Genital Herpes: Isolation
Experiment 58: Restriction Analysis
and Identification of Herpes Simplex
and Electrophoretic Separation of
Virus 525
Bacteriophage Lambda DNA 423
Part C: Detection of Sexually
Transmitted Chlamydial
PART 15 Medical Microbiology 433 Diseases 526
Introduction 433
Appendices
Experiment 59: Microbial Flora
of the Mouth: Determination of
Appendix 1: Scientific Notation 531
Susceptibility to Dental Caries 435
Appendix 2: Methods for the
Experiment 60: Normal
Preparation of Dilutions 533
Microbial Flora of the Throat and
Appendix 3: Microbiological
Skin 439
Media 535
Part A: Isolation of Microbial
Appendix 4: Biochemical Test
flora 439
Reagents 541
Part B: Effectiveness of
Appendix 5: Staining Reagents 544
Handwashing 443
Appendix 6: Experimental
Experiment 61: Identification of
Microorganisms 545
Human Staphylococcal
Pathogens 451
Art & Photo Credits 547
Experiment 62: Identification of
Human Streptococcal Pathogens 459 Index 549
Contents 9