Table Of ContentC O N C I S E V E R S I O N
Benson’s
Microbiological
Applications
Laboratory Manual in General Microbiology
Fourteenth Edition
Alfred E. Brown
Emeritus Professor, Auburn University
Heidi R. Smith
Front Range Community College
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BENSON’S MICROBIOLOGICAL APPLICATIONS: LABORATORY MANUAL IN GENERAL MICROBIOLOGY, CONCISE VERSION,
FOURTEENTH EDITION
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About the Authors
Alfred Brown Heidi Smith
Emeritus Professor of Front Range Community College
Microbiology B.A. Biology/Pre-Medicine,
Auburn University Taylor University, IN
B.S. Microbiology, California M.S. Biology, Bowling Green
State College, Long Beach State University, OH
Ph.D. Microbiology, UCLA
Heidi Smith is the lead fac-
Teaching Dr. Brown has been ulty for microbiology at Front
a member of the American Range Community College
Society for Microbiology for 50 years, and he has taught in Fort Collins, CO, and teaches a variety of biology
various courses in microbiology over a teaching career that courses each semester including microbiology, anatomy/
spans more than 30 years. Courses have included general physiology, and biotechnology. Heidi also serves as the
microbiology, medical microbiology, microbial physiol- principal investigator on a federal grant program designed
ogy, applied and environmental microbiology, photosyn- to increase student success in transfer and completion
thesis, microbiological methods, and graduate courses of STEM degrees at the local university. In that role,
such as biomembranes. In 2008, Dr. Brown retired from Heidi works directly with students to train them for and
the Auburn University faculty as an emeritus professor of support them through summer undergraduate research
microbiology. experiences.
Administration During his tenure at Auburn Uni- Student success is a strategic priority at FRCC and
versity, Dr. Brown served as the director of the University a personal passion of Heidi’s. She continually works to
Electron Microscope Facility. He also served as the chair develop professionally in ways that help her do a better job
of the Department of Botany and Microbiology and the of reaching this important goal. During the past 7 years,
chair of the Department of Biological Sciences. Heidi has had the opportunity to collaborate with faculty
Research My research has focused on the physiology throughout the United States in developing digital tools,
of the purple nonsulfur bacteria. This has involved how such as SmartBook, LearnSmart Labs, and Connect, that
bacteriochlorophyll and photosynthetic membrane syn- measure and improve student learning outcomes. This col-
thesis are coordinated. Herbicides, such as atrazine, have laborative experience with these tools has revolutionized
been used to determine the binding site for ubiquinone in her approach to teaching in both face-to-face and hybrid
photosynthetic electron transport. Binding occurs on the courses. The use of digital technology has given Heidi the
L-subunit, a protein in the photosynthetic reaction center. ability to teach courses driven by real-time student data
Resistance to atrazine involves a single amino acid change and with a focus on active learning and critical thinking
in the L-subunit that prevents the herbicide from binding to activities.
the protein and inhibiting electron transport. This is com- Heidi is an active member of the American Society for
parable to how atrazine inhibits electron transport in plants Microbiology. She has presented instructional technology
and how resistance to these herbicides develops. My labo- and best online and face-to-face teaching practices on nu-
ratory also investigated how the sulfonylurea herbicides in- merous occasions at the annual conference for undergradu-
hibit acetolactate synthase, a crucial enzyme in the pathway ate educators. She also served as a member of the ASM
for branched-chain amino acids. Recently, I consulted for Task Force on Curriculum Guidelines for Undergraduate
a company that manufactures roofing shingles. Because of Microbiology Education, assisting in the identification of
the presence of calcium carbonate in shingles, cyanobac- core microbiology concepts as a guide to undergraduate
teria can easily grow on their surface, causing problems of instruction.
contamination. My laboratory isolated various species of Off campus, Heidi spends as much time as she can en-
cyanobacteria involved in the problem and taxonomically joying the beautiful Colorado outdoors with her husband
characterized them. We also tested growth inhibitors that and three young children.
might be used in their control.
Dr. Brown and his wife have traveled extensively in
Europe since his retirement. His three children have fol-
lowed him in science, having earned doctorates in phys-
ics, chemistry and anatomy.
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Contents
Preface vi PART 7 Environmental Influences
Digital Tools for Your Success x and Control of Microbial Growth 175
Basic Microbiology Laboratory Safety xi
24 Effects of Oxygen on Growth 177
Biosafety Levels for Selected Infectious Agents xii
25 Temperature: Effects on Growth 185
Microorganisms Used or Isolated in the
Lab Exercises in This Manual xiv 26 pH and Microbial Growth 193
27 Water Activity and Osmotic Pressure 197
PART 1 Microscopy 1
28 Ultraviolet Light: Lethal Effects 201
1 Brightfield Microscopy 3 29 The Effects of Lysozyme on
2 Darkfield Microscopy 13 Bacterial Cells 205
3 Phase-Contrast Microscopy 17 30 Evaluation of Alcohol: Its Effectiveness
4 Microscopic Measurements 25 as an Antiseptic 211
31 Antimicrobic Sensitivity Testing:
PART 2 Survey of Microorganisms 31 The Kirby-Bauer Method 215
5 Microbiology of Pond Water—Protists, Algae, 32 Evaluation of Antiseptics and Disinfectants:
and Cyanobacteria 33 The Filter Paper Disk Method 227
6 Ubiquity of Bacteria 47 33 Effectiveness of Hand Scrubbing 233
7 The Fungi: Molds and Yeasts 51
PART 8 Identification of Unknown
PART 3 Manipulation of Bacteria 241
Microorganisms 61 34 Morphological Study of an Unknown
Bacterium 243
8 Aseptic Technique 63
35 Cultural Characteristics 249
9 Pure Culture Techniques 73
36 Physiological Characteristics: Oxidation
PART 4 Staining and Observation and Fermentation Tests 253
of Microorganisms 85 37 Physiological Characteristics: Hydrolytic
and Degradative Reactions 265
10 Smear Preparation 87
38 Physiological Characteristics:
11 Simple Staining 93
Multiple Test Media 271
12 Negative Staining 97
39 Use of Bergey’s Manual 279
13 Capsular Staining 101
14 Gram Staining 105
PART 9 Miniaturized Multitest
15 Spore Staining: Two Methods 111
Systems 287
16 Acid-Fast Staining: Kinyoun Method 117
40 Enterobacteriaceae Identification:
17 Motility Determination 121
The API® 20E System 289
41 Enterobacteriaceae Identification:
PART 5 Culture Methods 127
The EnteroPluri-Test System 295
18 Culture Media Preparation 129
42 Staphylococcus Identification: The API®
19 Enumeration of Bacteria: Staph System 305
The Standard Plate Count 139
20 Slide Culture: Fungi 151 PART 10 Applied Microbiology 311
43 Bacterial Counts of Foods 313
PART 6 Bacterial Viruses 155
44 Bacteriological Examination of Water: Most
21 Determination of a Bacteriophage Titer 159
Probable Number Determination 317
22 Isolation of Phages from Flies 165
45 Bacteriological Examination of Water: The
23 Phage Typing 171 Membrane Filter Method 327
iv
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COntEntS
46 Reductase Test 331 PART 13 Immunology and
47 Temperature: Lethal Effects 335 Serology 411
48 Microbial Spoilage of Canned Food 341 56 Slide Agglutination Test: Serological
49 Microbiology of Alcohol Fermentation 347 Typing 415
57 Slide Agglutination Test for S. aureus 417
PART 11 Bacterial Genetics
58 Slide Agglutination Test for
and Biotechnology 351 Streptococcus 423
50 Polymerase Chain Reaction for Amplifying 59 Enzyme-Linked Immunosorbent Assay
DNA 353 (ELISA) 429
51 Bacterial Transformation 361 60 Blood Grouping 435
Appendix A Tables A-1
PART 12 Medical Microbiology 367
Appendix B Indicators, Stains, Reagents A-5
52 The Staphylococci: Isolation and
Appendix C Media A-8
Identification 369
Appendix D Identification Charts A-12
53 The Streptococci and Enterococci: Isolation
and Identification 381 Appendix E Metric and English Measurement
Equivalents A-18
54 Gram-Negative Intestinal Pathogens 395
55 A Synthetic Epidemic 403
Index I-1
Indicates a LearnSmart Lab® activity is available for all or part of this
exercise. For more information, visit http://www.mhhe.com
/LearnSmartLabsBiology/
MICROBIOLOGY
v
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Effects of Oxygen on Growth EXERCISE 27
Preface
EXERCISE 27 Effects of Oxygen on Growth
Benson’s Microbiological Applications has been the relevancy message within these introductions, explain-
the threaded hole. Unless this is done, it will
gold standard of microbiology laboratory manuals for ing to studbeen itms pwoshsyib tleh etoy e snhgoaguel dth ce aluregs a obfo thuet jtahre w liathb .
over 35 years. This manual has a number of attractive the outer lid.
features that resulted in its adoption in universities, col- First andf. SPelaccoen tdhe P oeurtieor dlisd on the jar directly over the
leges, and community colleges for a wide variety of mi- In manIny a dcidnaitnsioeenrs ,tlo idi dn oasinntdrgu raco stttaiuotdeny tsoh fea t hrlieed o psxlyrieggeshnet lrnye tqteuodi rae l-floowr tiwbt root ho cru ltures for even-numbered students:
crobiology courses. These features include user-friendly momree ntcsl oafs stsiox opdrregoarpni oisidnmtsso i nsp otlha icsye eo.x puNe orciwma enrno t,tp ayrtoeou cmtheaeey dlhi advt ehf irromulyg h● Baanc illus subtilis, Escherichia coli,
an opporttuon ietyn gdaugrien gt hthee lsuegcso.n dT hpeer iloidd tmo adyo ab em ircortoa-ted in and Clostridium beijerinckii (ATCC 14950)
diagrams that students can easily follow, clear instruc- exesrccEoiXpsiEce R sitCnueId iSytahE noe 7 fr a tdhp ierp Ueercbnotdiiqpooursnpii.taoyr teoesf ffBoaarsmchteeidor iabny. three spore- 1. Set up a 45°C water bath at your station in which
tions, and an excellent array of reliable exercises suitable formergs. uTseidg hinte tnhe tihneo ctuhlautmionbss.c Prreowce ebdy a st ufornllionwgs :it clock-
you can keep your tubes of TGYA shakes from
for beginning or advanced microbiology courses. environwmiseen. tI f itnh ev oauritoeur sli dw raayisse. sA unpy, thbea clutegrsi aalr ec enlolts thsaotl idifying3. O. nMe woiasteter nba ath s ftoerr yiloeu sawnda ybo buyr l aimbom- ersing it into a tube
In revising the lab manual for the fourteenth edi- occ uFri rpisnrt o tPpheeerr leiyon devnirgoangmede.nt and are deposited on the agraarto ry partner owfi lnl usutrfifeicnet. (bNrootteh i an nthde e mxaptreerisaslisn g most of the broth
8. Place the jar (containing the plates of anaerobic list that the agar shakes have been liquefied for
tion, we have tried to maintain the proven strengths of (Inmoceudlaiutimon so arn idn I ntchueb abtrioonth) will subsequently undergo cell out of it by pressing the swab against the inside
agar) and the tubes in a 37°C incubator. After 2 or you prior to lab time.)
the manual and further enhance it. We have updated Sidnicvei 3ssi iohxno umtoris cp,r royooodrugu aocneri s cymooslu oran niindes st trohurrce cetao urk sisnehd toshu eol dfb rcmohteeh-c tko tbheec 2o.m Leab el the threwe taulbl eos fo tfh FeT tMub aen.d three tubes of
the introductory material in many exercises to reflect ditau arbrejia dirn. vtTooh lvdeee ditde irenma t ihinsi set oeixf a ptpehpreir meincedniati,ct eait t towhrai ltsl tbrbiaep c ntheeacrsiea sl -oasret iutsb iquTiG-YA with4 .t hRe uobrg tahneis mmso aisssteignneedd stow yaobu o(voneer a part of your body
changes in scientific information and increase rel- sstautcro9dyuue .r fnsroIbe tnri ln nwucet uecost hbo rcknaeion ot wlee mo nittrtyhvh.h eie oro fotna untliiybrmm. e tesheI rtnaae ntnei dsa do n mrpidgmlaa actnpteaeirosnsimra tflbaosse .nr t Tots2 ph4hteroa e tv moaea da 4let s8beaor cyh ih- ouc uonrndsve.erscttaiono ndrg aNniostme: pHera ntsduduobleceo )htr ha keann sdtou yabboe fuso irgn rie gnnitetteliryal elotsop.r h aeovaonride, otamrk iosnougm thep ioebceje, cat nsdu crhe tausr na
evancy for students. Critical thinking questions have als list for this period indicates how the organisms will on any unwanted oxygen into the media.
that the morphology of individual bacterial colonies that the swab to the tube of broth. It may be necessary
also been added to increase the Bloom’s level of the be distributed. 3. Heavily inoculate each of the TGYA shake tubes
laboratory reports. Finally, the names and biosafety deDveu lr oiSnpge otchnios a npned rai oPgdae, rer amicohed dstiuudmen tu wsuilal linlyo cduilfaftee rt hfrreoem one spwe-ith several lotoop fburles aokf toheff a pthpero sptriicatke eorngda noisfm th e swab so that you
ofc ieeasc ht ot yapneo othf etru.b Te hmise dmiuamy iannvdo olvnely d oifnfee rpelnatte a ospf ects of thfoer that tube. Tcoa gne tr egopoladc deis tpheers icoanp o fo tnh et hoerg taunb-e.
levels of microorganisms used in the manual are con- B(rcCeowulleotrun’sry ea nsEauvecarholub aaicst i aotghnaser. aArnelgdl ou Sfl pathroiert eyp l Saottefa sii nwtsini legl db)eg ep,l apceigd mentatioinsm, s in the 5m.e Ldiaubme, lr othll ee atcuhb teu bwe igtehn ytlyo uber tiwneietina ls and the source of
sistent with those used by the American Type Culture int hae G caosPnafkig juarr.a tBioonth atnhed GteaxsPtuakre jaorf ciotnst asiunirnfga cteh,e and its ethl-e palms as tshheow bna cinte rfiigau.re 27.3. To prevent
Collection. This4 .i sIn iomcuplaotret aeancth f oofr tthheo FsTeM u steubrse sw whitoh rtehely a ppro- pwlRheialotvlee lbamsde tais ooni nvdntche .tu h ebEet ah iFxtneeTadn mM elairt pd a 3l nl7eifdd°sr CT ofoG.m iSfrY tmutAthdhl eyteeuns betiGsen sw ca tphhisllaPala trsac ayhkecoa, rut Eeej bia rnrriXreoes.s ctauEiuIkclfltsas R.tt vehaCdaer ce uIv gSuaicmvE-e n oAinxlyl ogwen t huepste6a kt.u eEb, edxsop t ono osstoe ol itvdheifreyl y ba atl goroiotoadmte a ttgehmea rmp eperldaaituutemre ..b y coughing onto it.
MICoROnBI OtLhOGeY ATCC for apr siaoteu rocrgea onifs mcus.ltures. uEuxmer cbiys es l3id8i,n fgig tuhree l3id8 .t4o. the edge. W1hen tr2ansport- Label the bottom of the plate with the initials of
Si5m. Usipng laesept icS tecthnaiquien, strienak ygour three organ- iMnga tteYhreoi aupl lwsatiells b aen pdr otuvbideesd t ow yitohu trh rdeees kki, ntdask eo fc satreer inleo bt acte- the individuals who cough onto it. Be sure to date
Guided Tour Tihsmrso oun gthhe pala tLe aobf a nEaxereobricc iasgear in the manner troio aloggitiacatel mtheed FiaT tMha tt uyboeus w. Tilhl eex ppoossiet itoon t hoef egnrvoiwrothn ment the plate.
shown in figure 27.4. Note that only three straight- ●iin n GvtahaserPi aomku aesn dawieuraomybsi c. c jTaar,no 3 eGbnaess Puearakes gitelhynae trc ayhtooaru nergn veeedxlo ppiofe ss,hu arensd cleodv er as 7. Incubate the plates and tube at 37°C for 48 hours.
Learning Outcomes 1 GasPak anaerobic generator strip, scissors, one
line streaks, well separated, are made. Be sure to cwair1d0ee lme lsa ps ilspype.tetectrum as possible, specific assignments will
Each exercise opecnlesa rwlyi tlhab Lel eyaorunri nplga tOe ountc tohme beost,t owmh wicihth your ●b ew mateard bea thfos ra t estaucdhen st tsutadtieonnst .( eTlehctirsic hmota pyl aiten,v olve the use of
list what a studentit niositnhisao.l suP alladncd eb t hethe ea o bargglaeanr tipsoml adtse’o n( iaanmfvteeersrt ea cdt )ot hwme ictphol retrhete-c tp lloacteas- Mapteearr Msisbatwuealdasaekt beenNr tr :toeoifa e wrldeastmeerd,o avned tbhaercmteormieat efrr)om an object or surface; in Evaluation
ing thLee eaxerrnciisne6g.. oOiOtf ni oncutteho te atcrlh lseo tt uhjmadere .snettussd ienn tths’e pGlaatsePsa kar jea ri nra tchke. rack, place Trehqius●●●owni r●●●tusieh 331lme dpeltTiii cunnnlrbGa bb ccctttieYeieeouuunAor sbbbso e ncaaatosfwx ahtttfBo eeenpafirlmldddlkeucolie wi estsBds pudethse trutbser laehdab’ees wkis oe spetiattegsg eon heo lrt(yna ’lutfseictrt qeab Fohhi arute TolepenelelasbM faa l tiiaaoeeyceebtd ifroxma eroo)Tg ue ebcGriardrniaorYc ic ut naAadiomstsgiasrfeaiiyfrng.e pinrnelmmagnte eata nt enetn,ur vitariasriloe sdnn etm ttmhaeiaenledtt sdi.ua miArne Aewnfittt heborr u4ot8t h wh aoentutdrins ’tg wi ntohc eup blcaaattpeios. .nI s,S ehixta akcmleo iutnhdeey t htouerb tecu lbeveai gro?of r Cnouuotsmrliy--
After comple7t.i naTgso t fhoailsclo teiwvxaset:rec isaen,d y osue aslh othueld Gbea saPbalke tojar, proceed br●to h●the S tccasupphltohuayrrreeltos- csb fotoeacrlic noouiwdnsd ga.- unkruietmsu sba,en E6rdne tdEse lrisXdotuecEodsRcec6nCutssI: fSaeEcalis pisa rteh eit swiginthif iacna nucnec oonft acmloiundatiende stsu?b eD oof yboruo ths.e eW ahnayt
1. PrepaMrIeCR OaBI OtLahOG.iY nP semeleB aarp aoafr cbt attchetee rrfiaoili acl ealllts Toannrde asetnanidn soff ao GramsPaak tion1Ma. nOda bCtsleoesrrtviriadei lutsmhe s pgorroowgetnhe spatterns in the six FTM and colonies growing on the blood agar plate? Are the
them with a siimnpdliec asttoairn s.trip and pull it halfway down. The Coccobactihlleu ssix TGYAB ashciallkie tubes thaFt uysiofour mand your colonies all the same size and color? If not, what
2. Understand whiny dsitcaaintionrg wisi nlle tcuersns abrlyu teo oonb seexrpvoes ure to the air. per lsatubdoerantto:ryR poadr t(nBearc iilnluosc)ulated and incubated. Use does this indicate? Group toge1th9e7r a set of TSA plates
bacteria with aP blraigchet ftiehled imnidcircoastcoorp set.rip in the jar so that the ● f1i gtuubree 2o7f .n1u tarniedn tt hbero pthhotographs in figures 27.5 representing all nine types of exposure. Record your
Introdu3c. tOiobnserve thLebe .d awCgPiffrelueianncrtcee kioenrn faittgsf thm o tveOhromi esruep i cbinhtnovlcoe retolon.hlmogeerpi e jeeoasssrf o iefwn ab icaathhnc t oeuafrp iatrphil garceiheretl l spoG.ofa sssiPtciiaoskns o.gclraaosbms.o praettoernyt ,mO 2●●●bab. u njtaqCaei 11Spngu ctohpsuhaditmtlraieer aevey rt2tppermpri io7ilaslci aenepe. ra6t. en nl ecma Ot otttasbhaeotb netore jo dokef hmnig fcntet hrttahlsgiropdeveyw we pe pp asy ettliclbiohaxnecou tn am(eo sso predoergesef ta ls etyoaenonofcrityt ksmue mva rnoii g anlsfba a.e ebgr c ort(oThormaSewt Aeotoh )rxsy)oy oipglnaeedr nyt nboreyuer r-. raensdu Ylptsloa uotern s it Bnharsiregte rhL uttfcaioetb lodbo rMer aw icutorisolrelsy cdio n Rpfdyoei rpc aomEtreXta. EkwRiChnIegSt Ehs 1elird tehse isne Etuxbeers-
Because bacterAicaf.t el rP cciopemelptltlsee t in1ag0r te hm isc le oxoemfr ctipsaeop, s yooeurd sd hioosuftli dl labeepd ap bwrleoa xttoeir- into the Dippml3eo.arc toRtewcreciocai olosr rdf mr oyomorue sr l sirdteuesdsu elotnrst sfiin:n gLearbs.o Ara tpoireyc eR oefp loernts 2 t7is.sue cise 12 (Simple Staining). If the plates and tubes are to
Them iantetrloy d8u0c%ti wona ted1re., soUtchpndreeeinrrbes tec ainossd r vtnimheeperro y rots alfnui tt bertelajgeceio hccn otse ononf tvtfrhe aetl spohtGp ebLe Oe.e tpxAwlaesvemroeciidndi s efo rcinRge coamndb mciT4●ene.o pa tiItrns1afat btdep tlsnDeeiemt Nsrdioe Awpl v lTpiaetenetheScrt sg mtho rrmneefi ptoeebstnnlol,oo tcsigomoooydcnaac peakid gae pna rdrae vwciaootumSesrtl,bay poi,n hrw yeoildnlol ec us ooslciufcd atiehll eyw arecit--h be saved, containers will be provided for their storage
otehTrrceh teitwed h hsixreneeheettc.rf ia o ccioeimTdhlemrsbehl mi mlenaei alnaas oynfn tts oti adpdthonul iacant frshetafm t ileenqwicld esuedus2 3 pniee.u..oll GtotdltPmDoratIcTcc nhhLh eeenrutfectlibsosrOdlloeao allosutd sset p ek c gud f hpnutoorl dvehui aesn.sneriotnrnics u eiip oot nmdTeeathsgtdtjues ihe aiuv itme guhdihpton cpenrxhpt.aeeesipee en g rde sttelsseprr ihrniie aiiusr nGttizlhansngnesmisfaaLnses ngqiotcraOiei fcoooecc oenxeuasr eonkcrpr nea mm ntt e lirtoie sahnie ttcshoaofasmle uieetbd timilesfinuh o sasscte oi .eiennpcsdhc Oi t crnt.g npi oIeorte o eeovl-tarooagrni r nnnmaesc tv iillmtottlh v pnnaarceohmeoiieeadicg bcrpepdrfil rbsell bls oleoe iig tndy urstrtt.i ne r.hhai eg mtdnaagoimsersattonek tn h albpwostaeles aotkeiittnncnfrt sdoeur tgisaytetp pnee s drlo osri i lalaseiooendnn l tl oxvfn ttif-tthhdh eeee s rjj Thbgpriaaaiemeiuharronnnr e.,tpe cte PTiahosecnb a shrithietnsaonl siktetoet oceryxl( o fupe Epumwtstigiogr12u.pcrdmy ei ou cmom..ttsn uro vhet(soRsiaaecaSEEmpehlB eo t nrkr itnaeae s erld6ghceaxxwanfip peaied o6pdsaatrsneecpfsent ohr.ou tunacuoD1e iir o lalrittciln)isfbacnmv frh Ntlti.ssotweiig lc euiee eecmiepnA2Isdpvetn Sr sa nsoEia ie7 e,oa l t pystmt rt tmlaa. rro.ehyrhath wwh9oecn adaicra ia nveeondnes,u didytgncto tltlre te d eoi A relhhcolt roitC r su htyc enh ehswouoioTttmms areslm ooie dantecroeooebl S h udol ra snertsppeeco(tieA rrt kb hirCtnnaiacct tz clr jlrebhnioihireihid boeeinccn pts ssmdeadscnceaoegilf rhllclotneatitsopT saidaithouiord.dseilEvcekf) emetsfwmn Sst te reem e etxptc,f o ohgnhri t .i pyebhressiehay umtpeos adoLr, lprnmact uotdc ciav uieohnpatcicwcn tniuahoti eqa ssv.beaiocnddlf-rl),rmeiuer lei ,reetee p andedhedulea ss u)l1sie srt sas.an i sht6ia onpiini nag s.egnrdc o gs nL gocnisturb Ds lotteeameeiotohis-ob d.rntueyt faen osAto df oowoolns erysrtapuiafmt cwm, la btoeratndona t aedrosohabyuree-rn.rysfe sm- tta s a yi(tbigoosleenuerredy-r, idneL staihgben oaretrefadrti goceorrnyat taRoirne. epPrsol.arcte the plates and tubes in the
c(jefoilnglmssytuaocfriiieos nc hstp5r ha0oaene. 1,sdgy ) ceia. nnoT ctersph aomoiednsfu . eg icTnbeaetndoeere r i eneoewv4st.ntfo.a i sshkesUaOpmc un urfiabbeoensofarsdantoewelsefcillulcicacratinnsvmeztterc e(e ri Geaee e idivtatswFhns.d h elPerf tipr ) chep eotsglhweha xmreeec pseendGaim roe e tapwlFt hsni whdaiPmstw thih u orura aegaenhsmarne r iidoea snnncbbbfngt ee etir sno h l olisocepng fs argre reherbe.aewt seag fsnr mcioeo efwtldfuelin odc rt rdohrei naoosem calws e ocicnncutoe rtlpoeolrens- laid c ulenatinl acCtoimvfwCimaLtoiicaatenehbas d ol.noeefd nnrths sat eehtt iroces os rdunyDead uRteSiessnpet .si drpoeeirflosl;tiu ertrmnhat bualsce, c.wuimpiunlgat Sietsp o iorfofn c ohtchaceeat estosiopn saullry- Freigtauirnee 1r. R6le evTcehroe ir ssd lmi dyoeov muedru strote bstheue lp trrsiog pihnetr. lLy apboosirtiaotnoerdy a Rs tehpeo rt 7.
recombinant DNA techniques. The GFP gene codes Spiral or Curved
for sal girdeeen a flnudor euscseunat lplryot ehitneh taeht a-etf xiispx voeissdieb ldteo we naheddn h iesex r-ceo mthpel ecteellyls wtoit hthdrea wn into Complete Laboratory Report 27.
presssleidd bey abnacdte,r iiaml cpolooHrntioearsn itzhtolaytn p,t oatsols e Gpsser tenhsee ep Trlarvsamen itsdh.f eeri rM setrcuhcatnuirsaml si n- Procedures
teIng trhiitsy e.x eSrcmisee,a thrse i pnar reBesea ntchcteee onrfi atshtea GinFePd g eunse ionng various dyes to Figure 12.E1 XBPaOcStUerRiEa lM mEoTrHpOhDo lFoOgRy. TSA PLATE coarse adjustment knob. If SyToUu DaEreN uT sNinUgM aB bEiRnoc-
the pGLO plasmid w19ill8 be verified by two molecular If your microscope has three objectives, you have ular microscope, it will also be necessary to adjust
enhance cell fAeacqtuuirseitsio an nodf gsetnreuticc tmuraetesr.ia l, in addition to muta- th1.r eTeo mthaeg naiifri icna tliaobno orapttoioryn sf:o (r1 3) 0l omwi-npuotweser, or 100× the interocular distance and dio1p, t1e0r, a1d9j,u 2s8tment to
The use ofti oan soifn ggelnee ss atnadi nlo tsos ocf oulnonre cae sbsaarcyt geernieasl, icse alnl iims - or taperte2od.t a Telo mn tdahsge.n aiTfiirhc ieant ifrouonso,i m(f2o )or hmthig ehbr- atdhcrtayen mr ilaaagb anorirfeai ctroaotriydo snfo ,w rw 3iht0ihc mh inutes match your eyes. 2, 11, 20, 29
referred to as psoirmtanpt lpea rstt oafi bnaicntegri.a lC aodamptmatioonn layn du esveodlu tdioyne. sIn tapered ies n4d0s0 ×a ntodta al rwei tph rae 4v0a×le onbt jeinct itvhee, ahnudm (3a) n1 0m00o×u ttoht.a l 7. For optimal viewing, it is necessary to focus the
many cases, acquisition of new genes (i.e., antibiotic 3. To the air outside of building for 30 minutes 3, 12, 21, 30
for performing simple staining are methylene blue, Cocci mmayag oncifcicuart isoinn gwliyth, ian 1 c0h0a×i nosil, iimn mteetrrsaiodns o(bpjaeccktievtes. condenser and adjust it for maximum illumination.
resistance) may mean the difference between survival
basic fuchsin, aannd dd ecarthy sotfa aln v oirogalenits.m T ihne tshee eanrvei rroenfmeernrte. dT htuos , of four), 4o. rB iWlno hwire rtdheuegsru ty loaonru t coul sueesx tptehoress e.l odMw m-opesotdw siuetrmre opbtjoeccoticvce io or ct-he This procedure should be perfo4rm, 1e3d, e2a2c,h 3 t1ime the
as basic dyesb abcetecraiau hsaev et dheevyel ohpaevd ev acriooulos rm-ebcehaanriisnmgs , iioncnluicd - cur in cho5ai.li nMismo, miwsteh rleispirose nap sro etbhsjseeec sdtti vaaepg hawyinillsol tc dmoecepcedini uodm cocnu rh ionw c lmusu-ch objective lens is changed. Raise5 ,t h1e4 ,i r2is3 d, i3a2phragm
groups (chrominog pthraonsrfeosrm) attihoant, tararnes dpucotisoint,i vaenldy cocnhjaugragteiodn , ters thatm raegsneimficbalteio gnr iasp neesc.e Tssharey .s Gpiernael-raslhlya pspeeda kbiancgt,e hroiaw - to its highest position. Close the iris diaphragm
vi to acquire external genetic information. Bacterial e6v.e Fr,i nitg eisr tbipess tp troe ssstaerdt lwigihthtl yth oen l omwe-dpiouwmer objective until the edges of the diaphr6ag, m15 , i2m4a,g 3e3 appear
(cationic). Thteryan swfoormrka twione lrle fewrsi ttho tbhea cuptetarkiea lo fc nealklesd tDhNaAt can exist as spirochaetes, as spirilli, or as comma-
have chemicalm golreocuulpess boy nb atchteeriira l scuerllfsa. cTersan tshduactt iocno ninfveorl vaes shaped, asc7tnu.ud dSr yvep evrpodergor arrgeolr secdssos si.ten osTs . r tpCehrpeoe noshssniiegdedhem ret aret h mmpe paafoolgllrniladoirfwuiilcymian ot,gi no ts nhmuseg e gacdesais uutymisoo-nusr fkunzozby .u nLtoilw tehre tehdeg ecso nodf ethnese dri aup7sh,i nr1ga6g ,i mt2s5 aa,r de3j 4ubsrotmugehntt
transfer of genetic material carried by bacteriophages,
net negative cohr abragctee ritaol vtihrues ecs.e Cllo. nTjuhgaetiroenf oinrveo,l vtehs etrraen sifser ao f ative agf8eon.r t Hs eoatftiir n scgyo mpuhpb ielyidos u,o rvi sem r iaec xrsoppsocisroeopdce h maaneeddte ium mtahk a(i1tn0 g is stm rtoiockoreo s-) into sharp focus. You should n8o, w17 ,c 2le6a,r l3y5 see the
pronounced egleencettrico mstaatteircia l afrtotrma contieo linv inbge btawcteeerinum t htoe a ncoethlel r thin to bs9ec. o oOpbipcst eoiorbvnseaedlr:v Abatnyioy b nmrsi.egthhtofdie nldo tm lisicterdo sacboopvye. Vibrio sides of the diaphragm expand 9b,e 1y8o,n 2d7 t,h 3e6 field of
and the cationusiicn gc ahnr eoxmteronaplh aoppreen doafg et khneo wsnt aaisn a, pialunsd. the cholerae, the bacterium responsible for cholera, is a view. Refocus the specimen using the fine adjust-
bro1979X_fm_i-xvi.rinedsdu l6t is staineind tchBeea lclptsea.rt ihIaonl g etcrnaoincns tfboraarcmsttea rttiiouomn c awStatirsoe pnftioircsct o ccdhciurssco ompvneoreeu-d- comma-iLsnohgwa wp-Peiotdhw btehare cE tlxoeawrmi-upimnoaw.t eior no bjTechteiv me aiisn t ore aesnoanb lfeo ry ostua rtto- mredenu8tc/.1e 0N /1toh6t ee 7 tl:5hig1a htP tMa sin ytoenus citlyo,s eth teh ec iornist rdaisatp ihmrapgrmov etos
ptriehvpoeelrlyel sec,dh dabyryge ebsd atmpcc)chaea toaplatelnhrsstrioeu aiglh baeeceea.-lan cf Wvocilaclermeh—em leedalinS sn n .av ng aipio coprnnnuianedd-ltcuehi iancmsocpt tgo sacdenubnihiyleniaercce-e oa tfcs uohmwe.sr leeTmelos r b,eihpt nt haeheghecxes—oy pken roago aessnncretsdpqoad aut i(uhttinnhornueso esdkd g gani eloarotnlheen-fidec - claisceti Cs1,u 1l5Bt)enlou x6ainorpocegklfios il nrlt oSueg( tsoftaso hrtpre mu, hs dysemyylogi.ldu eoaOe acct eanrtoonsrcc i eplcua orumyloorsocke, ue afea aoddhunry a trdtvo eh e phue Cri sfgoeoo,h bpurejSanyerrdtc nmre tee dwaybp gohatinuoanci tcf t aieoyrEcercoax itucpuie uolmraans-nrs e-. adhneindfdi nt heaed f odacse upthsthee d or afin mfgiaeegl doe f i wndciistrhteaianns cewes .hi nDic fehrp ootnhtth oeofrf aofnibedjle dbc etiss-
the slide instegaedn.e s for capsule formation from the dead virulent xerosis Uwsiell tbhee fuoslleodw iinng t shtiesp ss iwmhpelne esxtpalionriinngg ae xsleirdcei swei.t h will appear clear and sharply defined.
Simple staceilnlss. cTahins abbeil iutys eodf at ob adcteetreiurmm tion eta kteh eup m naokre-d C. xerostihse i lso rwe-lpaotwede rt oob Cjeocrtiyvne:ebacterium diphtheriae, 8. Once an image is visible, move the slide about to
phology of bacDtetNreiaAr,i iamnlco cllueedcluilnlseg.s S Fi.s i ptgenuremurmeed o1 n“2ciao.1em aipnleldtu eBnsactcreai.”llt ueSsos sm mueb otbiaslicts -, the bacte1r.i uPmos irteiosnp othnes isblildee foonr tdhiep shttahgeer wiai tihn t hheu mmaatnesri.a l search out what you are looking for. The slide is
of the commoarne nsahtuarpalelys coomf pbetaecntt earnida cl anc etalklse .u pT nhaekyed cDaNnA The basic dytoe bme estthuydlieedn eo nb ltuhee (umppeethr ysluernfaec+e cohfl othreid sel−id)e . moved by turning the knobs that move the me-
be grouped inmtool ectuhlrees ef rommo trhpe heonvloirgonicmaeln t.t yMpaensy: obthaerc iblalci - will be useFdig utore s1t.a6i nil lutshterastee s cheolwls ,t hael lsoliwdei nmgu syt obue hteol d chanical stage.
(rods), cocci te(rsiap, hinecrliucdainl)g, Eascnhder icshpiai rcaollis, a(rec onrokt sncartuerwal-ly visualize thine iprl adcief fbeyr ethnet mmecohrapnhicoallo sgtaiegse raentadin ecre llelv earr.- 9. Check the cleanliness of the ocular, using the
2. Turn on the light source, using a minimum amount procedure outlined earlier.
shaped rods). Rods or bacilli can have rounded, flat, rangements. It is informative to do the simple stain
of voltage. If necessary, reposition the slide so 10. Once you have identified the structures to be stud-
435
that the stained material on the slide is in the e xact ied and wish to increase the magnification, you may
center of the light source. proceed to either high-dry or oil immersion magni-
3. Check the condenser to see that it has been raised fication. However, before changing objectives, be
101 sure to center the object you wish to observe.
to its highest point.
4. If the low-power objective is not directly over the
center of the stage, rotate it into position. Be sure High-Dry Examination To proceed from low-power
that as you rotate the objective into position it to high-dry magnification, all that is necessary is to
clicks into its locked position. rotate the high-dry objective into position and open
5. Turn the coarse adjustment knob to lower the ob- up the diaphragm somewhat. It may be necessary
jective until it stops. A built-in stop will prevent to make a minor adjustment with the fine adjust-
the objective from touching the slide. ment knob to sharpen up the image, but the coarse
6. While looking down through the ocular (or ocu- adjustment knob should not be touched.
lars), bring the object into focus by turning the Good quality modern microscopes are usually
fine adjustment focusing knob. Don’t readjust the both parfocal and parcentral. This means that the
7
PREfACE
Illustrations
Illustrations provide visual instructions for perform-
Laboratory Report 22 Student:
ing steps in procedures or are used to identify parts of Date: Section:
instruments or specimens.
22 Isolation of Phages from Flies
A. Results
1. Plaque Size Increase
With a Sharpie, circle and label three plaques on one of the plates and record their sizes in millimeters
at 1-hour intervals.
TIME
Oculars (eyepieces)
3 hours
5 hours
12 hours
Diopter
adjustment 24 hours
Rotatable ring
head
a. Were any plaques seen on the negative control plate?
b. Do the plates show a progressive increase in number of plaques with increased amount of
Nosepiece fly-broth filtrate?
Arm c. Did the phage completely “wipe out” all bacterial growth on any of the plates?
If so, which plates?
Stage Objective 2. Observations
adjustment Count all the plaques on each plate and record the counts in the following table. If the plaques are very
Mechanical nu merous, use a colony counter and hand counting device. If this exercise was performed as a class
Coarse stage pcoroujnetcst fwroimth yinoduirv cidlausaslm stautedse ntots c doominpgle oten ltyh eo ntaeb oler. two plates from a common fly-broth filtrate, collect
adjustment
knob
Diaphragm control
Fine Condenser
adjustment
knob Illuminator
Stage
adjustment
knob Base
On/off switch
169
Figure 1.2 The compound microscope.
© Charles D. Winters/Science Source
(1) Two loopfuls of the organism are (2) The smear is allowed to only air-dry;
used to prepare a thin smear. do not heat-fix.
Safety
In the fourteenth edition, we have followed the re-
cent recommendations of the American Society for
Microbiology concerning the biosafety levels of organ-
(3) Stain the smear with 1% crystal violet (4) The crystal violet is washed off the (5) Blot dry with bibulous paper or allow
for 2 minutes. splriodpee wr witha s2te0 %co cnotapipneerr .s uDlofa nteo ti nwtoa sah it to fully air-dry. isms used in the exercises in this laboratory manual.
copper sulfate into the sink.
The Basic Microbiology Safety section in the intro-
Figure 13.1 Steps of the Anthony capsule staining method.
ductory pages of the lab manual has been completely
revised to align with ASM’s Guidelines for Biosafety
in Teaching Laboratories released in 2012. The BSL
classifications of all organisms used in each exercise
Laboratory Reports have been updated according to ATCC. Where pos-
sible, BSL-1 organisms replace BSL-2 organisms
A Laboratory Report to be completed by students im-
in many exercises. However, in some exercises, it is
mediately follows most of the exercises. These Labo-
necessary to use BSL-2 organisms such as specific
ratory Reports are designed to guide and reinforce
staphylococci, streptococci, and some of the Entero-
student learning and provide a convenient place for
bacteriaceae due to specific tests, stains, and concepts
recording data. These reports include various types
that involve these organisms. For these exercises, we
of review activities, tables for recording observations
recommend that safety procedures be followed as sug-
and experimental results, and questions dealing with
gested by ASM, such as the use of biosafety cabinets.
the analysis of such data.
As a result of these activities, students will in-
crease their skills in gathering information by obser- Changes to This Edition
vation and experimentation. By completing all of the
Exercises
assessments in the Laboratory Reports, students will
be able to determine if they accomplished all of the ∙ Many photographs and some diagrams have been
learning outcomes. replaced, upgraded, or revised for clarity.
vii
bro1979X_fm_i-xvi.indd 7 8/10/16 7:51 PM
PREfACE
∙ Even if the procedure itself did not change, the ∙ Added new figure of possible endospore positions
text was revised to clarify areas where students in bacterial cell.
traditionally struggle through an exercise.
Exercise 36 Physiological Characteristics:
∙ New critical thinking questions have been added
Oxidation and Fermentation Tests
to the end of many of the exercises.
∙ Rearranged procedural sections and the process
Part 2 Survey of Microorganisms figures to make it easier for students to complete
all of the required inoculations for both controls
∙ Updated diagram of Domains Bacteria, Archaea,
and unknown test bacteria.
and Eukarya.
∙ Added a flowchart to explain the steps of the ni-
Exercise 5 Microbiology of Pond Water trate reduction test and the correct analysis after
each step.
∙ Revision of introductory material.
Exercise 37 Physiological Characteristics:
Part 3 Manipulation of Microorganisms
Hydrolytic and Degradative Reactions
Exercise 9 Pure Culture Techniques
∙ Revised figures, photos, and their associated cap-
∙ A photo of a pour plate was added with a tions to facilitate better student analysis of results
description of surface versus subsurface colony for each of these tests.
growth. Exercise 38 Physiological Characteristics:
Multiple Test Media
Part 4 Staining and Observation of Microorganisms
∙ Removed litmus milk test.
Exercise 13 Capsular Staining
Part 9 Miniaturized Multitest Systems
∙ A new photo of a capsule stain replaces the old
photos. Exercise 40 Enterobacteriaceae Identification:
∙ The introductory material was revised to explain The API® 20E System
how each step of the staining process works to
∙ Simplified the tabulation process and enlarged
allow for visualization of the capsule.
the photo for easier analysis and identification by
Exercise 15 Spore Staining: Two Methods students.
∙ Added a new photo of a spore stain. Exercise 41 Enterobacteriaceae Identification:
∙ The procedures for the various staining methods The EnteroPluri-Test System
were rearranged and revised for greater clarity.
∙ Completely rewrote this exercise to align with
Part 7 Environmental Influences and Control newly available test system from Becton-
Dickinson due to discontinuance of the Entero-
of Microbial Growth
tube II system.
Exercise 24 Effects of Oxygen on Growth
Part 10 Applied Microbiology
∙ Revised line art and photos throughout the exer-
cise to clarify oxygen classifications. Exercise 44 Bacteriological Examination of
Water: Most Probable Number Determination
Exercise 25 Temperature: Effects on Growth
∙ Updated introduction to the exercise.
∙ Added a figure depicting microbial temperature
∙ Revised the MPN table to make it easier for stu-
classification groups.
dents to analyze and understand their results.
∙ Updated temperature ranges to most currently
accepted upper and lower limits. Exercise 46 Reductase Test
Exercise 28 Ultraviolet Light: Lethal Effects ∙ The introductory section to the exercise has been
revised and updated.
∙ Replaced line art figure of UV lamp with photo of
plate half covered by white card. Exercise 49 Microbiology of Alcohol
Fermentation
Part 8 Identification of Unknown Bacteria
∙ The introduction to the exercise has been revised,
Exercise 34 Morphological Study of an
emphasizing the history of fermentation and how
Unknown Bacterium
fermentation is a means to preserve foods.
∙ New, easy-to-use descriptive chart for unknown ∙ Added an explanation of how off-flavors pro-
identification. duced by sulfides occur in the fermentation of
viii
bro1979X_fm_i-xvi.indd 8 8/10/16 7:51 PM
PREfACE
wines and how they can be monitored by a lead Exercise 60 Blood Grouping
acetate strip placed in the fermentation flask.
∙ Added table figure to clearly explain the ABO
groups.
Part 11 Bacterial Genetics and Biotechnology
∙ Revised introductory material, adding informa-
∙ This section was added to provide two exercises tion on the Rh factor and the use of RhoGAM in
that are appropriate for general microbiology stu- Rh-negative mothers to prevent hemolytic dis-
dents. These exercises can be done individually or ease in newborns.
the techniques of PCR, gel electrophoresis, and ∙ Revised the procedure for the use of artificial blood
bacterial transformation can be done as a con- available in kits that can be purchased from biologi-
nected sequence. cal supply companies. The use of finger sticks by
students to collect blood and the use of human blood
Part 12 Medical Microbiology in laboratories has become a serious safety issue.
Exercise 52 The Staphylococci: Isolation and
We would like to thank all the people at
Identification
McGraw-Hill for their tireless efforts and support
∙ Upgraded photos of mannitol salt agar. with this project. They are professional and competent
and always a pleasure to work with on this manual.
Exercise 53 The Streptococci and Enterococci:
A special and deep thanks to Darlene Schueller, our
Isolation and Identification
product developer. Once again, she kept the project fo-
∙ Upgraded photos of blood agar plate hemolysis. cused, made sure we met deadlines, and made sugges-
tions that improved the manual in many ways. Thanks
Part 13 Immunology and Serology as well to Marija Magner, brand manager; Mary Jane
Lampe, content project manager; Kristine Rellihan,
Exercise 59 Enzyme-Linked Immunosorbent
marketing manager; Matt Backhaus, designer; Keri
Assay (ELISA)
Johnson, assessment content project manager; and Me-
∙ Added this exercise which simulates the com- lissa Homer and Lorraine Buczek, content licensing
monly used screening test for HIV. specialists; and many who worked “behind the scenes.”
ix
bro1979X_fm_i-xvi.indd 9 8/10/16 7:51 PM
