Intranet.tdmu.Edu.ua_data_kafedra_internal_micbio_classes_stud_en_pharm_prov_pharm_ptn_Microbiology With Basis Immunology_2_Lesson 3. the Main Methods, Principles and Steps of Isolation of Bacteria’s Pure Cultures

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Themain
THEME6.MAINMETHODS,PRINCIPLESANDSTEPSOFISOLATINGOF
BACTERIASPURECULTURES.

Forselectionthepurecultureof,itfollowstoseparatenumerousbacteriawhicharein
testedmaterial, one from other. It is possible to attain by methods which are based on two
principlesmechanicalandbiologicalseparationofbacteria.

Mechanicalprinciple Biologicalprinciple
METHODS METHODS
1.Factionaldilutions(L.Pasteurs Takeintoaccount:
technique) Respirationtype(Fortnersmethod)
2.Pourplatetechnique bacterialmotility(Shukevichsmethod
(Dilutioninsolidnutrientmediaby resistancetoacids(acidfastbacteria)
R.Kochstechnique) sporulation
3.Spreadplatetechnique temperatureoptimum
(Superficialdispersionsby selectivsensitivenessoflaboratoryanimalsto
Drigalskystechnique) thebacteriaandsoon.
4.Streakplatetechnique

Methodsbasedonmechanicalprinciple
Method of factional dilutions Pasteurs technique) is based on mechanical
disconnectionofmicroorganismsbyserialdilutioninliquidnutrientmedia.Themainlackof
thistechnique:wecannotmakecontroltheamountofmicrobalistestedtubes.
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Pourplatetechnique(DilutioninsolidnutrientmediabyR.Kochstechnique) is
basedondilutionofmicrobesandpouringthetestedmaterialwithgelatin.Aftercoolingthe
gelatinisolatedcoloniesofmicroorganismsareformedandtheyeasilycanbetransferredon
afreshnutrientmediumbyaplatinumloopforobtainingamicrobialpureculture.

Spread plate technique (Superficial dispersions by Drigalskys technique) is more
perfect method which is widely in everyday microbiological practice. There is quantitative
techniquethatallowsthedeterminationofthenumberofbacteriainasample.
Stages:
Pipettetherequiredamountofbacteria(fromyourdilution)onthesurfaceofthe
Petriplate.
Spreadtheinoculumoverthesurfaceoftheagarmediumusingahockeystick
(spatula).
Repeatthisactionon34Petriplateswithoutsterilizationofthehockeystick.
Incubatetheplateinvertedat37oC.
TheremustbedifferentnumberofmicrobialcolonyonthePetriplates.

Streakplatetechnique.
ADVANTAGES:
Spreadmillionsofcellsoverthesurface
Individualcellsdepositedatadistancefromallothers
Divideformingdistinctcolonies
Distinctcoloniesdonottouchanyothercolonies
Cloneofasinglebacteria pureculture

Youstreaktheplateon3differentportionofthePetriplate,soyoucandrawthesection
thatyouwillstreakonthebottomofyourplate.
Stages:
Usingasterilelooptakealoopfulofyourbacteriafromthebroth
Streakaverticalline
Thenstreakgentlyacrosssection1
Zigzagpatternuntila1/3oftheplateiscovered
Donotdigintotheagar
Sterilizetheloop letitcool
Rotatetheplateabout90degreesandspreadthebacteriafromthefirststreakintoa
secondarea
Doonlyonestreak(orveryfew)inthefirstareaandonceyouareinthesecond
areadonotgobacktothefirst
Doazigzagpatternuntilthe2ndareaiscovered
Sterilizeagain dothesamefor3rdarea
Makesurethatyourredhotloopiscoolenoughpriortotouchthebacteria
Afteryouwaitedafewseconds
Stabitintotheagarinapositionawayfrombacteria willcoolit
Ifyoustabwherebacteriaare productionofaerosol
Incubatetheplateinvertedat37oC.
Inadayitisnecessarytoexaminethecoloniesforfutureinvestigation.

Or:
Methodsbasedonbiologicalprinciple
Biological principle of disconnection of bacteria foresees the purposeful search of
methodswhichtakeintoaccountthenumerousfeaturesofmicrobalspecies.Amongthemost
widespreadmethodsitispossibletoselectthefollowings:
1. Respiration type. All of microorganisms according to the type of respiration are
divided into two basic groups: aerobic(Corynebacterium diphtheriae, Vibrio holerae and
others) and anaerobic (Clostridium tetani, Clostridium botulinum, Clostridium perfringens
andsoon).Iftestedmaterialfromwhichitfollowstoselectanaerobicbacteriatowarmup
preliminary,andthencultivateinanaerobicterms,thesebacteriawillgrowexactly.
2.Sporulation.Itisknownthatsomemicrobes(bacilliandclostridia)formendospores.
There are Clostridium tetani, Clostridium botulinum, Clostridium perfringens, Bacillus
subtilis, Bacillus cereus among them. Spores are resistant against different external
environment factors. Thats why, if tested material would be heated previously and then
inoculatedinnutrientmediumsporeformingbacteriawouldbegrown.
3. Resistance of microbes against acids and alkali. Some microbes (Mycobacterium
tuberculosis, Mycobacterium bovis) as a result of their chemical structure features are
resistantagainsacids.Thatswhytestedmaterialwiththisbacteriapreviouslyistreatedwith
10 % sulfuric acid and later inoculated on proper nutrient medium. An extraneous flora
perishes,andmycobacteriaasaresultoftheirresistancetoacidsgrow.
Vibrio holerae is a halophylic bacterium, and for its growth it is inoculated in 1 %
alkaline peptone water. Already in 46 hrs it growth like a tender bluish pellicle on the
surfaceofmedium.
4. Bacteria motility. Some microbes (Proteus vulgaris) have a tendency to creeping

growthandisabletospreadquicklyonthesurfaceofmoistnutrientmediumbecausethey
have flagella. So such bacteria are inoculated in the drop of condensation liquid which
appears after the cooling the slant agar. In 1618 hrs they spread on all surface of nutrient
medium.Ifmaterialfromtheupperpartofagarwouldbetakenwewillhaveapureculture
ofmicrobe.
5. A susceptibility of microbes different chemicals, antibiotics etc. As a result of
featuresofmetabolismsomebacteriahaveadifferentsusceptibilitytosomechemicalfactors.
Forexample,staphylococci,aerobicbacillicangrowinnutrientmediawhichhave7,510%
tothesodiumchloride.Thatiswhyfortheselectionofthesebacteriathissubstanceisadded
into yalksalt agar and mannitolsalt agar for their selection. Other bacteria under the
influenceofsuchconcentrationofsodiumchloridedonotgrowpractically.
Some antibiotics (nistatin) is used for inhibition for pathogenic fungi growth if it is
necessarytoobtainonlybacteria.AddingthePenicillininnutrientmediuminhibitthegrowth
only grampositive bacteria. Presence of Furazolidon makes favorite condition for
CorynebacteriaandMicrococci.
6.Abilityofmicroorganismstopenetratethroughunharmedskin.Somepathogenic
bacteria (Yersinia pestis) as a result of presence a lot of aggression enzymes are able to
penetratethroughanintactskin.Forthispurposebodywooloflaboratoryanimalisshaven
and tested material with different bacteria a rubbed in this skin area. Later some microbes
maybeobtainfromthebloodorinternalorgans.
7. A sensitiveness of laboratory animals is to the exciters of infectious diseases.
Somelaboratoryanimalsshowahighsusceptibilitytothedifferentmicroorganisms.
Forexample,afteranymethodofStreptococcuspneumoniae introduction into a mouse
generalized pneumococcal infection are developed. An analogical picture is observed after
injectionofMycobacteriumtuberculosis into Guinean pig or Mycobacterium bovis into the
rabbit.
8.Temperatureoptimum.Thecardinaltemperatures:
Minimum
Optimum
Maximum
Microorganismscanbegroupedbythetemperaturerangestheyrequire
Psychrophiles,lowtemperatureoptima(4C)Polaromonasvacuolata
Mesophilsmidrange(39C)Escherichiacoli
Thermophileshigh(60C)Bacillusstearothermophilus
Hyperthermophilesveryhigh(>80C)Thermococcusceler
In everyday practice bacteriologists use such concepts as a species, a strain and pure
cultureofmicroorganisms.
Speciesacollectionofbacterialcellswhichshareanoverallsimilarpatternoftraitsin
contrasttootherbacteriawhosepatterndifferssignificantly
Astrainisasubsetofabacterialspeciesdifferingfromotherbacteriaofthesame
speciesbysomeminorbutidentifiabledifference.Astrainis"apopulationoforganismsthat
descendsfromasingleorganismorpurecultureisolate.Strainswithinaspeciesmaydiffer
slightlyfromoneanotherinmanyways."
Culture:populationofmicroorganismsgrownunderwelldefinedconditions.
Purecultureonethatcontainsonetypeofmicroorganism.

IsolationandIdentificationofPureCultureofAerobicBacteria


Firstday.Preparesmearsofthetestedmaterialandstudythemunderthemicroscope.
Then,usingaspatulaorabacteriologicalloop,streakthematerialontoasolidmediumina
Petri dish. This ensures mechanical separation of microorganisms on the surface of the
nutrientmedium,whichallowsfortheirgrowthinisolatedcolonies.Inindividualcasesthe
materialtobestudiedisstreakedontotheliquidenrichmentmediumandthentransferredto
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Petridisheswithasolidnutrientmedium.Placethesedishesina37 0Cincubatorfor1824
hrs.
Incubator

Secondday.Followinga24hourincubation,theculturalpropertiesofbacteria(nature
oftheirgrowthonsolidandliquidnutrientmedia)arestudied.
Secondday.Followinga24hourincubation,theculturalpropertiesofbacteria(nature
oftheirgrowthonsolidandliquidnutrientmedia)arestudied.

Macroscopic examination of colonies in transmitted and reflected light. Turn the
dishwithitsbottomtotheeyesandexaminethecoloniesintransmittedlight.Inthepresence
ofvarioustypesofcoloniescountthemanddescribeeachofthem.Thefollowingproperties
arepaidattentionto(a)sizeofcolonies(largo,45mmindiameterormoremedium,24
mm small, 12 mm minute, less than 1 mm) (b) configuration of colonies {regularly or
irregularly rounded, rosetteshaped, rhizoid, etc.) (c) degree of transparency (non
transparent,semitransparent,transparent).
In a reflected light, examine the colonies from the top without opening the lid. The
following data are registered in the protocol: (a) colour of the colonies (colourless,
pigmented, the colour of the pigment) (b) nature of the surface (smooth, glassy, moist,
wrinkled,lustreless,dry,etc.)(c)positionofthecoloniesonthenutrientmedium(protruding
abovethemedium,submergedintothemediumflat,atthelevelofthemediumflattened,
slightlyabovethemedium).
Microscopicexaminationofcolonies.Mountthedish,bottomupward,onthestageof
the microscope, lower the condenser, and, using an 8 x objective, study the colonies,
registering in the protocol their structure (homogeneous or amorphous, granular, fibriliar,
etc.)andthenatureoftheiredges(smooth,wavy,jagged,fringy,etc.).
Use some portion of the colonies to prepare Gramstained smears for microscopic
examination. In the presence of uniform bacteria, transfer the remainder of colonies to an
agarslantforobtainingasufficientamountofpureculture.Placethetesttubeswiththein
oculatedmediumintoa37Cincubatorfor1824hrs.
Third day. Using the culture which has grown on the agar slant prepare smears and
stain them by the Gram method. Such characteristics as homogeneity of the growth, form,
size,andstainingofmicroorganismspermitdefinitejudgementastopurityoftheculture.To
identify the isolated pure culture, supplement the study of morphological, tinctorial, and
cultural features with determination of their enzymatic and antigenic attributes, phago and
bacteriocinosensitivity, toxigenicity, and other properties characterizing their species
specificity.
Todemonstratecarbohydratesplittingenzymes,Hiss'mediaareutilized.Whenbacteria
ferment carbohydrates with acid formation, the colour of the medium changes due to the
indicator present in it. Depending on the kind and species of bacteria studied, select media
withrespectivemonoanddisaccharides(glucose,lactose,maltose,sucrose),polysaccharides
(starch,glycogen,inulin),higheralcohols(glycerol,mannitol).Intheprocessoffermentation
oftheabovesubstancesaldehydes,acids,andgaseousproducts(CO2,H2,etc.)areformed.
Todemonstrateproteolyticenzymesinbacteria,transferthelattertoagelatincolumn.
Allow the inoculated culture to stand at room temperature (2022 C) for several days,
recordingnotonlythedevelopmentofliquefactionpersebutitscharacteraswell(laminar,in
theformofanailorafirtree,etc.)
Proteolyticactionofenzymesofmicroorganismscanalsobeobservedfollowingtheir
streakingontocoagulatedserum,withdepressionsformingaroundcolonies(liquefaction).A
caseinclotissplitinmilktoformpeptone,whichismanifestedbythefactthatmilkturns
yellowish(milkpeptonization).
More profound splitting of protein is evidenced by the formation of indol, ammonia,
hydrogen sulphide, and other compounds. To detect the gaseous substances, inoculate
microorganisms into a meatpeptone broth or in a 1 per cent peptone water. Leave the
inoculatedculturesinanincubatorfor2472hrs.
To demonstrate indol by Morel's method, soak narrow strips of filter paper with hot
saturatedsolutionofoxalicacid(indicatorpaper)andletthemdry.Placetheindicatorpaper
betweenthetesttubewallandstoppersothatitdoesnottouchthestreakedmedium.When
indolisreleasedbythe2nd3rdday,thelowerpartofthepaperstripturnspinkasaresultof
itsinteractionwithoxalicacid.
Thetelltalesignofthepresenceofammoniaisachangeinthecolourofapinklitmus
paper fastened between the tube wall and the stopper (it turns blue). Hydrogen sulphide is
detected by means of a filter paper strip saturated with lead acetate solution, which is fast
enedbetweenthetubewallandthestopper.Uponinteractionbetweenhydrogensulphideand
leadacetatethepaperdarkensasaresultofleadsulphideformation.
Todeterminecatalase,pour12mlofa1percenthydrogenperoxidesolutionoverthe
surfaceofa24hourcultureofanagarslant.Theappearanceofgasbubblesisconsideredas
apositivereaction.Useacultureknowntocontaincatalaseasacontrol.
The reduction ability of microorganisms is studied using methylene blue, thinning,
litmus,indigocarmine,neutralred,etc.Addoneoftheabovedyestonutrientbrothoragar.
Themediumdecolorizesifthemicroorganismhasareductionability.Themostwidelyem
ployed is Rothberger's medium (meatpeptone agar containing 1 per cent of glucose and
severaldropsofasaturatedsolutionofneutralred).Ifthereactionispositive,aredcolourof
the agar changes into yellow, yellowgreen, and fluorescent, while glucose fermentation is
characterizedbycracksinthemedium.
Antigenpropertiesoftheisolatedcultureareinvestigatedbytheagglutinationtest(see
p.37)andotherserologicaltests.
Species identification of aerobic bacteria is performed by comparing their
morphological,cultural,biochemical,antigenic,andotherproperties.

Isolationandidentificationofapureculture
Firstday
1.Microscopicexaminationofthetestedmaterial.
2.Streakingofthematerialtestedontonutrientmedia(solid,liquid).
Secondday
1.Investigationoftheculturalproperties.
2.Subinoculationofcoloniesontosolidmediatoenrichforapureculture.
Thirdday
1.Checkingofthepurityoftheisolatedculture.
2.Investigationofbiochemicalproperties:(a)sugarlytic,(b)proteolytic.
3.Determinationofantigenicproperties.
4. Study of phagosensitivity, phagotyping, colicinogensitivity, colicinogenotyping,
sensitivitytoantibiotics,andotherproperties.

IsolationandIdentificationofPureCultureofAnaerobicBacteria
First day. Inoculate the studied material into KittTarozzi medium (nutrient medium):
concentratedmeatpeptonebrothorHottinger'sbroth,glucose,0.15percentagar(pHl7.2
7.4).
Toadsorboxygen,placepiecesofboiledliverormincedmeattoforma11.5cmlayer
andpiecesofcottonwoolonthebottomofthetesttubeandpourin67miofthemedium.
Priortoinoculationplacethemediumintoboilingwaterfor1020mininordertoremoveair
oxygen contained in it and then let it cool. Upon isolation of spore forms of anaerobes the
inoculatedcultureisreheatedat80'"Cfor2030mintokillnonsporeformingbacteria.The
culturesareimmersedwithpetrolatumandplacedintoanincubator.ApartfromKittTarozzi
medium,liquidmediacontaining0.51percentglucoseandpiecesofanimalorgans,casein
acidandcaseinmycotichydrolysatescanalsobeemployed.
Caseinacidmedium',caseinacidhydrolysate,0.5110percentyeastextract,0.351:
20percentcornextract,0.151millet,240gcottonwool,25g.Themediumispouredinto
flaskswithmilletandcottonwoolandsterilizedfor30minat110 0C. Use caseinmycotic
hydrolysatetoobtaincaseinmycoticmedium.
Secondday.Takenoteofchangesintheenrichmentmedium,namely,theappearanceof
opacificationoropacificationincombinationwithgasformation.Takebrothculturewitha'
Pasteurpipetteandtransferitthroughalayerofpetrolatumontothebottomofthetesttube.
Prepare smears on a glass slide in the usual manner, then flame fix and Gramstain
them.DuringmicroscopicexaminationrecordthepresenceofGrampositiverodforms(with
orwithoutspores).Streaktheculturefromtheenrichmentmediumontosolidnutrientmedia.
Isolatedcoloniesarepreparedbytwomethods.
1.Preparethreeplateswithbloodsugaragar.Todoit,meltandcoolto45C100mlof
2percentagnronllottinger'sbroth,thenadd1015mlofdeftbrinatedsheeporrabbitblood
and 10 ml of 20 per cent sterile glucose. Take a drop of the medium witli microorganisms
intothefirstplateandspreaditalongthesurface,usingaglassspatula.Usethesamespatula
tostreaktliccultureontotliesecondandthenthirdplatesandplacethemintoananaerobic
jarorothersimilardevicesat37''Cfor2448hrs(Zoisslcr'smethod).
2, Anaerobic microorganisms are grown deep in a solid nutrient medium (Veinherg's
methodofsequentialdilutions).TheculturefromthemediumistakenwithaPasteurpipette
withasoldcdtipandtransferredconsecutivelyintothe1st,2nd,and3rdtesttubeswith10ml
of isotonic sodium chloride solution. Continue to dilute^ transferring the material into the
4th,5th.and6ththinwalledtesttubes(0.8cmindiameterand18cminheight)withmelted
andcooledto50CmeatpeptoneagarorWilsonBlairmedium(to100mlofmeltedmeat
peptoneagarwith1percentglucoseadd10mlof20percentsodiumsulphitesolutionand1
mlof8percentferricchloride).Alteragarhassolidified,placetheinoculatedcultureintoan
incubator.
Onthethirdday,studytheisolatedcoloniesformedintlieplatesandmakesmearsfrom
themosttypicalones.TheremainderisinoculatedintoKittTarozzimedium.Thecoloniesin
thetesttubesareremovedbymeansofasterilePasteurpipetteortheagarcolumnmaybe
pushedoutofthetubebysteamgenerateduponwarmingthebottomofthetesttube.Some
portionofthecolonyisusedtopreparesmears,whileitsremainderisinoculatedintoKitt
Tarozzi medium to enrich pure culture to be later identified by its morphological, cultural,
biochemical,toxicogenic,antigenic,andotherproperties.
The VinyaleVeyones method is used for mechanical protection from oxygen. The
seedingaremadeintotubewithmeltingandcooling(at420C)agarmedia.

BasesofbacterialIdentification

Identificationisthedeterminationofwhetheranorganism(orisolateinthecaseof
microorganisms ) should be placed within a group of organisms known to fit within
someclassificationscheme.

Morphologicalidentification(accordingtothebacterialmorphology)
Microscopicmorphology

Anumberofmorphologicalcharacteristicsareusefulinbacterialidentification.These
includethepresenceorabsenceof:
cellshape
CocciRods
cellsize
endospores
Spores
flagella
Flagella
glycocalyx
etc.
The techniques used at the earliest stages are relatively simple. An unknown sample
may contain different bacteria, so a culture is made to grow individual bacterial colonies.
Bacteriatakenfromeachtypeofcolonyisthenusedtomakeathinsmearonaglassslide
andthisisexaminedusingalightmicroscope.Viewingthebacteriashowsiftheyarecoccior
bacillioroneoftherarerforms,suchasthecorkscrewshapedspirochaetes.

GramStaining
Cocci and bacilli can be either gram positive bacteria or gram negative bacteria,
dependingonthestructureoftheircellwall.TheGramStainisnamedafterHansChristian
Gram,abacteriologistfromDenmarkwhodevelopedthetechniqueinthe1880s.Thetestis
performedonathinsmearofanindividualbacterialcolonythathasbeenspreadontoaglass
slide. Gram positive bacteria retain an initial stain, crystal violet, even when the bacterial
smear is rinsed with a mixture of acetone and ethanol. The solvent removes the dark blue
colour from gram negative bacteria, dissolving away some of the thin cell wall. When a
secondstain,apinkdyecalledfuchsinisthenadded,grampositivebacteriaareunaffectedby
this, as they are already stained dark blue, but the gram negative bacteria turn bright pink.
Thecolourdifferencecanbeseeneasilyusingalightmicroscope.
Gramstain
Placeadropofsterilewateronamicroscopeslide.
Makealightsuspensionoftestcultureinthewater.
AllowtodrythenfixthefilmbypassingtheslidethreeorfourtimesthroughaBunsen
flamewiththefilmuppermost.
Allowtocool.
Floodslidewithcrystalvioletandallowtoreactfor60seconds.
Washwithwater.
FloodwithLugol's(orGram's)iodinefor60seconds.
Washasbeforeanddrainoffexcesswater.
Decolourisewithacetoneandwashoffimmediately.
Counterstainwithdilutecarbolfuchsinfor30seconds.
Washandblotdry.
Grampositive(left)andgramnegative(right)microbes

AcidFastBacteria
SpirochaetessuchastheMycobacteriathatcausetuberculosisandleprosydonotstain
well using the Gram Stain. Other stains that do not wash away with dilute acid are used
instead. The bacteria are deeply stained, either bright red against a blue background or red
against a green background. Because the stain cannot be removed by washing with acid,
organismsstainedbythesemethodsaretermedacidfastbacteria.

ZiehlNeelsenStain
Floodafixedslidewithstrongcarbolfuchsin.
Heattheslideuntilitsteamsandkeepsteamingfor5minutes
WarningDoNOTBOIL.
DoNOTdothiswithacetonebeingusedinthesamesink.
DoNOTallowtheslidetodryout.
Washslidewithwater(preferablyfilteredwatertapwatermaycontainmycobacteria
leadingtofalsepositiveresults).
Treatwith3%acidalcoholfor10minutesoruntilonlyasuggestionofpinkremains
onthefilm.
Washfilmwithwater.
Counterstainfor1530secondswithmethyleneblue.
Washandblotdry.
Acidfastbacilliappearbrightredwhiletissuecellsandotherbacteriastainblue.

Thisstainsacidalcoholfastbacteria,e.g.mycobacteria.

SporeStain(ModifiedZiehlNeelsenmethod)
Stainthefixedfilmwithstrongcarbolfuchsinfor35minutes,heatinguntil
steamrises.
Washwithwater.
Treatwith0.25%sulphuricacidfor1560seconds.
Washwithwater.
Counterstainwith1%aqueousmethylenebluefor5minutes.
Washandblotdry.
Bacterialsporesareseenasredstructures:vegetativecellsstainblue.

Bacterialspores(SchaefferFultonstaintechnique)

Motility
Placeadropofliquidcultureonamicroscopecoverslip.
Invertoveraplasticineringonamicroscopeslide.
Examineunderx40objective(highpowerdrylens).
Accidentalspillsmayoccurduringthisprocedure.
Wetmounttechnique

Leftnegativetest,rightpositive

Capsulestaining(reliefstainingwitheosin)
Placeadropofbrothcultureononeendofamicroscopeslide.
Addonedropofeosinsolutionandleaveforoneminute.
Takeasecondslideanddrawitsedgebacktocontactthestainedsuspension.
Holdingthesecondslideat45degrees,spreadathinlayeroffluidalongthefirstslide
byacontinuousforwardmovement.
Allowthefilmtoairdrythenexamineunderoilimmersion.

Backgroundmaterialandcellsstainred.Capsularmaterialappearsasan
unstainedhaloaroundthecells

AerobicorAnaerobic?
Findingoutwhetherbacteriaareaerobicoranaerobichelpsseparatethemintodifferent
categories.Itisrelativelyeasytodiscoverwhetherabacterialculturegrowsinthepresence
or absence of oxygen. Bacteria that only grow if oxygen is available are called aerobic
bacteria. Anaerobic bacteria can grow without oxygen, and some species are killed by
oxygen,onlysurvivingincompletelyoxygenfreeenvironments.Thesespeciesaredescribed
asobligateanaerobes(seeabove)

Cultural(accordingtothebacterialgrowthsignsin/ondifferentnutrientmedia)
colonymorphology
Mixedcolonies

Biochemical(accordingtothebacterialabilitytoutilizedifferensubstrates)
EnzymeTests
Withinbroadtypesofbacteria,individualspecieshavedifferentmetabolicsystemsand
are able to grow using a range of nutrients. Testing bacteria to find out whether they are
positive or negative for specific enzymes helps narrow down their identity. For example,
Staphylococcus aureus tests positive for the enzyme coagulase, but Staphylococcus
epidermidisisnegativeforthisenzyme.
Fermentativepropertiesofmicrobesareusedinthelaboratorydiagnosisofinfectious
diseases,andinstudyingmicrobesofthesoil,water,andair.
Toidentifytheisolatedpureculture,supplementthestudyofmorphological,tinctorial,
and cultural features with determination of their enzymatic and antigenic attributes, phago
and bacteriocinosensitivity, toxigenicity, and other properties characterizing their species
specificity.
To demonstrate carbohydratesplitting enzymes, Hiss' media are utilized. When
bacteriafermentcarbohydrateswithacidformation,thecolourofthemediumchangesdueto
the indicator present in it. Depending on the kind and species of bacteria studied, select
media with respective mono and disaccharides (glucose, lactose, maltose, sucrose),
polysaccharides (starch, glycogen, inulin), higher alcohols (glycerol, mannitol). In the
process of fermentation of the above substances aldehydes, acids, and gaseous products
(CO2,H2,etc.)areformed.

TSI(TripleSugarIron)andKIA(Kligler'sIronAgar)
Triple Sugar Iron Agar (TSI) and Kligler's IronAgar (KIA) are used to determine if
bacteriacanfermentglucoseand/orlactoseandiftheycanproducehydrogensulfideorother
gases.(Ifanorganismcanfermentglucose,itis"glucosepositive".Ifitfermentslactose,itis
"lactose positive".) In addition, TSI detects the ability to ferment sucrose. These
characteristics help distinguish various Enterobacteriacae, including Salmonella and
Shigella,whichareintestinalpathogens.
TSIcontainsthreesugars:glucose,lactoseandsucrose.Lactoseandsucroseoccurin10
times the concentration of glucose (1.0% versus 0.1%). Ferrous sulfate, phenol red (a pH
indicator that is yellow below pH 6.8 and red above it), and nutrient agar are also present.
Thetubeisinoculatedbystabbingintotheagarbutt(bottomofthetube)withaninoculating
wire and then streaking the slant in a wavy pattern. Results are read at 18 to 24 hours of
incubation.
ReadingtheResults
AyellowslantonTSIindicatestheorganismfermentssucroseand/orlactose.OnKIAa
yellowslantindicatestheorganismfermentslactose.(BecauseKIAdoesnotcontainsucrose,
sucrosefermentationisnotdetectedwithKIAtests.)OtherresultsarethesameforTSIand
KIA.Ayellowbuttshowsthattheorganismfermentedglucose.Blackpreciptateinthebutt
indicates hydrogen sulfide production. Production of gases other than hydrogen sufide is
indicatedeitherbycracksorbubblesinthemediaorthemediabeingpushedawayfromthe
bottomofthetube.
UnderstandingtheResults
Ifanorganismfermentsglucoseonly,theentiretubeturnsyellowduetotheeffectofthe
acid produced on phenol red. Because there is a minimal amount of glucose present in the
tube, the organism quickly exhausts it and begins oxidizing amino acids for energy.
AmmoniaisthusproducedandthepHrises.Within24hoursthephenolredindicatorreverts
toitsoriginalredcolorontheslant.BecauseTSI/KIAmediaispouredasadeepslant,the
butthaslimitedoxygenandbacteriaareunabletooxidizeaminoacidsthere.Thebuttthus
remainsyellow.
Ifanorganismcanfermentlactoseand/orsucrose,thebuttandslantwillturnyellow(as
they do from glucose fermentation). However, they remain yellow for at least 48 hours
becauseofthehighlevelofacidproductsproducedfromtheabundantsugar(s).
KIA resembles TSI in all respects except that KIA contains two sugars (lactose and
glucose)whileTSIcontainthreesugars(lactose,glucoseandsucrose).LikeTSImedia,KIA
contains 10 times as much lactose as glucose. Thus KIA tests for an organism's ability to
fermentglucoseorlactosebutnotsucrose.
Ifthegasbeingproducedishydrogensulfide(H2S),itreactswiththeferroussulfateand
preciptatesoutasablackprecipitate(ferricsulfide)inthebutt.Organismsproducinglarge
amounts of hydrogen sulfide (e.g. Salmonella and Proteus) may produce so much black
precipitatethatitmaskstheyellow(acid)colorofthebutt.

1.Readingresults

2.InterpretingresultsonTSI

3.InterpretingresultsonKIA

To demonstrate proteolytic enzymes in bacteria, transfer the latter to a gelatin
column. Allow the inoculated culture to stand at room temperature (2022 C) for several
days, recording not only the development of liquefaction per se but its character as well
(laminar,intheformofanailorafirtree,etc.).
Serratiamarcescensontheleftispositiveforgelatinaseproduction,asevidencedbythe
liquidationofthemedia.
Salmonellatyphimuriumontherightisnegative,asevidencedbythesolidityofthe
media.

Proteolyticactionofenzymesofmicroorganismscanalsobeobservedfollowingtheir
streakingontocoagulatedserum,withdepressionsformingaroundcolonies(liquefaction).A
caseinclotissplitinmilktoformpeptone,whichismanifestedbythefactthatmilkturns
yellowish(milkpeptonization).
Moreprofoundsplittingofproteinisevidencedbytheformationofindol,ammonia,
hydrogen sulphide, and other compounds. To detect the gaseous substances, inoculate
microorganisms into a meatpeptone broth or in a 1 per cent peptone water. Leave the
inoculatedculturesinanincubatorfor2472hrs.
TodemonstrateindolbyMorel'smethod,soaknarrowstripsoffilterpaperwithhot
saturatedsolutionofoxalicacid(indicatorpaper)andletthemdry.Placetheindicatorpaper
betweenthetesttubewallandstoppersothatitdoesnottouchthestreakedmedium.When
indolisreleasedbythe2nd3rdday,thelowerpartofthepaperstripturnspinkasaresultof
itsinteractionwithoxalicacid.
The telltale sign of the presence of ammonia is a change in the colour of a pink
litmuspaperfastenedbetweenthetubewallandthestopper(itturnsblue).
Hydrogen sulphide is detected by means of a filter paper strip saturated with lead
acetatesolution,whichisfastenedbetweenthetubewallandthestopper.Uponinteraction
between hydrogen sulphide and lead acetate the paper darkens as a result of lead sulphide
formation.

Todeterminecatalase,pour12mlofa1percenthydrogenperoxidesolutionover
thesurfaceofa24hourcultureofanagarslant.Theappearanceofgasbubblesisconsidered
asapositivereaction.Useacultureknowntocontaincatalaseasacontrol.
Thereductionabilityofmicroorganismsisstudiedusingmethyleneblue,thionine,
litmus,indigocarmine,neutralred,etc.Addoneoftheabovedyestonutrientbrothoragar.
Themediumdecolourizesifthemicroorganismhasareductionability.Themostwidelyem
ployed is Rothberger's medium (meatpeptone agar containing 1 per cent of glucose and
severaldropsofasaturatedsolutionofneutralred).Ifthereactionispositive,aredcolourof
the agar changes into yellow, yellowgreen, and fluorescent, while glucose fermentation is
characterizedbycracksinthemedium.

Bilesolubilitytest(Pureculture)
Emulsifyafewcoloniesofthetestculturein1mlofsalinetoformasmooth
suspension.
Addonedropof10%sodiumdeoxycholatesolution.
Incubateat37
oC.
Examineforclearingat15minutes,30minutesand60minutes.
Clearingshouldoccurwithin30minutes
Inamixedcultureplaceonedropof10%sodiumdeoxycholatesolutionontothetest
colony..

Thisshouldlysewithin30minutes.Thismethodisnotentirelyreliable,anditis
bettertopurifyanysuspectedcolony

CatalaseTest
Usingaglasscapillarytube,pickasmallamountofculturefromtheplate.
Ifpossibledonotpickfromabloodcontainingmediumasthepresenceofcatalasein
themediumitselfmaygiveafalsepositiveresult.Thissometimescannotbeavoided.
Carefullyinvertthetubeandinsertitintothehydrogenperoxidesolution.
Tiltthetubesothefluidflowsontotheculturematerial.
Lookfortheimmediateformationofoxygenbubblesinthetubeindicatingthe
activityofcatalase.

Catalasetestpositive(left)andnegative(right)


Catalasetest

CoagulaseTest
A.Slidemethod:
Thistestdetectsthepresenceof"clumpingfactor"andisnotatruecoagulasetest.
Placethreeseparatedropsofsalineonacleanslide.
Suspend a loopful of test colony in two of these, and a loopful of control

Staphylococcusaureusinthethird.
With a sterile loop, add a drop of citrated rabbit plasma to one test and the control
suspension.
Clumpingoccurringwithin10secondsindicatesapositiveresult.
Thesalinecontrolshouldremainevenlysuspended.
B.Tubemethod:
Emulsify a few colonies of control Staphylococcus aureus and the test isolate into
appropriatelylabelledtubescontaininga1/10dilutionofplasmain0.85%saline.
Incubateat37
oC.
Examineforcoagulationat1,3and6hours.
Conversionoftheplasmaintoasoftorstiffgel,seenontiltingthetubetoa
horizontalpositionindicatesapositiveresult.

DNaseTest
InoculatesectionsoftryptoseagarmediumcontainingDNAwithmaterialfrom
testcolonies.
ControlsofknownStaphylococcusaureusandStaphylococcusepidermidisshould be
inoculatedaspositiveandnegativecontrols.
Incubatetheplateat37
oCfor1824hours.
FloodtheplatewithlMHClthatprecipitatesDNAandturnsthemediumcloudy.
ThepresenceofazoneofclearingroundtheareaofgrowthindicatesDNase
productionthathashydrolysedtheDNA.

Lecithinaseactivityresultsintheproductionofanopaquezoneofprecipitationaround
the area of growth. This precipitation should not be present on that side of the plate
previouslyinoculatedwithspecificantitoxin.
NaglerTest
Clostridium perfringens elaborates a variety of exotoxins, one of which is toxin

(lecithinaseorphospholipaseC).Thefollowingtestisusedtodemonstrateproductionofthis
specifictoxin.
Divideaneggyolkplateintotwoequalsections.
SpreadaloopfulofClostridiumperfringensantitoxinoverhalftheplateandallowto
dry.
Withasinglestreak,inoculatetheplatewithaloopfulofthetestculture,beginningon
theuntreatedsideoftheplate.
Incubateat37
oCunderanaerobicconditions.
Lecithinaseactivityresultsintheproductionofanopaquezoneofprecipitation
aroundtheareaofgrowth.Thisprecipitationshouldnotbepresentonthatsideofthe
platepreviouslyinoculatedwithspecificantitoxin
OptochinTest
Divideabloodagarplateintothreeequalsections.
Inoculate one with a known Streptococcus pneumoniae, another with a viridans

streptococcusandthethirdwiththetestisolate.
Caremustbetakentokeeptheculturesseparate.
Place a 5 microgram Optochin disc (ethylene hydrocupreine hydrochloride) in the
centreoftheplate.
Incubateat37
oCovernightandobservethezoneofinhibition.

OxidaseTest
Dipasterileswabinfreshlypreparedoxidasereagent(1%tetramethylpara
phenylene diamine dihydrochloride) then touch the target colony. A positive reaction is
indicated by the rapid appearance of a purple colour on the swab where the test bacteria
adhere.

Alternatively
Place a drop of freshly prepared oxidase reagent (1% tetra methylparaphenylene
diaminedihydrochloride)onapieceoffilterpaperinaPetridishoronaglassslide.
Leavefor1minute.
Usingawoodenstickoraglassslide(notawireloop)rubasmallamountofthetest
colonyontothemoistenedpaper.
Again,apositivetestisindicatedbytherapidappearanceofapurplecolouratthissite.

Indoleproductionmeasuretheabilitytohydrolyseanddeaminatetryptophan
Klesiellaenterobactersalmonellaserratiaaremostlynegative
positiveredcolour

Methylredmethylred,apHindicatorwitharangebetween4.4(red)and6.0(yellow)
onlyspeciesthatproducesuffiicientacidscanmaintianthepHatbelow4.4againstthe
buffersystemofthetestmedium
mostspeciesofEnterobacteriaceaeproducestrongacids.Enterobacterserratiadonot
produceenoughacids

Positivestableredcolourinthesurfacelayerofthemedium

Vogesproskauerreactiontest
this test is based on the conversion of acetoin to a red coloured complex through the
actionofKOH,atmospheric02andalphanapthol
Klesiellaenterobacterserratiaisabletoperformthispathway

Vogesproskauerreactiontest.Redcolouratthesurfaceofthemediumafter15mins
followingtheadditionofreagents

Citrate utilisation test some bacteria have the ability to utilize citrate as the sole
carbonsourcandturnthemediumallkalineduetoproductionofammonia
EscherichiaEdwardisellashigellasalmonella cannot utilise citrate as the sole source
ofcarbon

Positivefromcolourgreentoblue

Ureasetest some species posses the enzyme urease and able to hydrolyze urea with
thereleaseofammoniaandcarbondioxide
thisisusedmainlytodifferentiateureasepositiveProteusspeciesfromothermember
ofEnterobacteriaceae
positiveyellowishorangetopink

Positiveyellowishorangetopink
The API20E test kit for the identification of enteric bacteria (bioMerieux, Inc.,
Hazelwood,MO)providesaneasywaytoinoculateandreadtestsrelevanttomembersofthe
FamilyEnterobacteriaceaeandassociatedorganisms.Aplasticstripholdingtwentyminitest
tubes is inoculated with a saline suspension of a pure culture (as per manufacturer's
directions).Thisprocessalsorehydratesthedessicatedmediumineachtube.Afewtubesare
completely filled (CIT, VP and GEL as seen in the photos below), and some tubes are
overlaidwithmineraloilsuchthatanaerobicreactionscanbecarriedout(ADH,LDC,ODC,
H2S,URE).
Afterincubationinahumiditychamberfor1824hoursat37C,thecolorreactionsare
read(somewiththeaidofaddedreagents),andthereactions(plustheoxidasereactiondone
separately) are converted to a sevendigit code. The code is fed into the manufacturer's
database via touchtone telephone, and the computer voice gives back the identification,
usually as genus and species. The reliability of this system is very high, and one finds
systemsliketheseinheavyuseinmanyfoodandclinicallabs.
Note: Discussion and illustration of the API20E system here does not necessarily
constituteanycommercialendorsementofthisproduct.Itisshowninourlaboratorycourses
as a prime example of a convenient multipurpose testing method one may encounter out
thereinthe"realworld."
Inthefollowingphotos:
Noteespeciallythecolorreactionsforaminoaciddecarboxylations (ADH through
ODC)andcarbohydratefermentations(GLUthroughARA).
o The amino acids tested are (in order) arginine, lysine and ornithine.
Decarboxylationisshownbyanalkalinereaction(redcoloroftheparticularpHindicator
used).
oThecarbohydratestestedareglucose,mannitol,inositol,sorbitol,rhamnose,sucrose,
melibiose, amygdalin and arabinose. Fermentation is shown by an acid reaction (yellow

colorofindicator).

Hydrogen sulfide production (H2S) and gelatin hydrolysis (GEL) result in a black
colorthroughoutthetube.
Apositivereactionfortryptophandeaminase(TDA)givesadeepbrowncolorwiththe
additionofferricchloridepositiveresultsforthistestcorrelatewithpositivephenylalanine
and lysine deaminase reactions which are characteristic of Proteus, Morganella and
Providencia.
Inthefirstsetofreactions:
Culture "5B" (isolated from an early stage of fermentation) is identified as
Enterobacteragglomeranswhichhasbeenaconvenientdumpinggroundfororganismsnow
beingreassignedtobetterdefinedgeneraandspeciesincludingthenewgenusPantoea.This
particularisolateproducesreddish(lactose+),"pimply"coloniesonMacConkeyAgarwhich
exude an extremely viscous slime as may be seen this appearance is certainly atypical of
organismsidentifiedasE.agglomeransorPantoeaingeneral.
Culture "8P44" is identified as Edwardsiella hoshinae. The CDC had identified this
culture(in1988)astheultrarareBiogroup1ofEdwardsiellatardawhichmaynotbeinthe
API20Edatabase.Thissystemprobablywouldnotbeabletodifferentiatebetweenthesetwo
organisms.


Serologicalidentification(accordingtothebacterialantigens)
All immunological tests are based on specific antibodyantigen interaction. These
testsarecalledserologicalsincetomakethemoneshoulduseantibodycontainingsera.
Serological tests are employed in the following cases: (a) to determine an unknown
antigen (bacterium, virus, toxin) with the help of a known antibody (b) to identify an
unknownantibody(inbloodserum)withthehelpofaknownantigen.Hence,onecomponent
(ingredient)inserologicaltestsshouldalwaysbeaknownentity.
The main serological tests include tests of agglutination, precipitation, lysis,
neutralization,andtheirvariousmodifications.

AgglutinationTests
Every individual species of bacterium has a unique collection of 3D shapes on its
surface,calledantigens.Theseareformedbythemoleculesontheoutsideofthecellwall.
Whenabacteriuminfectsahumanorananimal,theimmunesystemreactstotheseantigens,
makingaspecificantibodytoeachone.Antiserumraisedagainstaknownbacterialspecies
canthereforebeusedtopositivelyidentifyifthatspeciesispresentinanunknownculture.A
smallamountofdifferentantiserum,specificfordifferentbacteria,isusedtotestasample.
When the result is positive, the bacteria clump together, or agglutinate when the result is
negative,noclumpingoccurs.

LancefieldGrouping(StreptexMethod)
Emulsifyaloopfulofthetestculturein0.4mlofextractionenzyme.
Incubateat37
oCfor1hour.
Addonedropoflatexreagenttotheappropriatecircleofablacktile.
Next,addonedropofextracttoeachcircleandmix,usingawoodenstick.
Rockgentlyforoneminute.
Clumpingindicatesapositivereaction.

Presumptiveagglutinationtest.ApresumptiveATisperformedonglassslides.Using
aPasteurpipette,transferseveraldropsofserumoflow(1:101:20)dilutionsandadropof
isotonicsalineforcontrolonagreasefreeglassslide.Intoeachdropoftheserumaswellas
inthecontroldrop,inoculatealoopfulof24hourlivingcultureofthemicroorganismpicked
from the surface of a solid nutrient medium or pipette one drop of the suspension of dead
microorganisms(diagnosticum).Theinoculatedcultureisthoroughlymixeduntilthedropof
liquidisuniformlyturbid.
Thereactiontakesplaceatroomtemperature.Inspectvisuallytheresultsin510min
occasionally one may use a 5 X magnifying lens for this purpose. If the glass slides are
placed into a humid closed chamber to prevent evaporation, the results of the test may be
readin3040minaswell.
Apositivetestisindicatedbytheappearanceinthedropwithserumoflargeorsmall
flakes, readily visible upon rocking of the coverslip. In a negative test, the fluid remains
uniformlyturbid.

Slideagglutination

In cases where the number of microorganisms is small and the results of the test are
difficult to interpret, dry the drop of the inoculated serum, fix the preparation, stain it with
Pfeifier'sfuchsine,andstudyunderthemicroscope.Inapositivetest,amicroscopicfieldis
largelyfreeofmicroorganismsbuttheyareaccumulatedinsomeplaces.Inanegativetest,
microorganisms are uniformly distributed throughout the microscopic field. This test is
knownasmicroagglutination.

Bilologicalidentification
Biologicalexamination.Biologicalstudyconsistsofinfectinganimalsforthepurpose
of isolating the culture of the causative agents and their subsequent examination for
pathogenicityandvirulence.
Choiceofexperimentalanimalsdependsontheaimofthestudy.Mostfrequentlyused
arerabbits,guineapigs,albinomice,andalbinorats.Thisisexplainedbythefactthatthey
aresusceptibletothecausativeagentsofvariousinfectionsdiseasesinman,easytohandle,
and propagate readily. Hamsters, polecats, cotton rats, monkeys, birds, etc. may also be
occasionallyinfected.
Specialized,particularlyvirological,laboratories,makeuseofgeneticallystandardized,
socalledinbredanimals(mice,rabbits,guineapigs,andothers).
Working with experimental animals, one should keep it in mind that they may have
spontaneous bacterial and viral diseases and latent infections activated as a result of
additional artificial inoculation. This hinders the isolation of pure culture of the causative
agentanddeterminationofitsaetiologicalrole.Gnotobiotes(withoutmicroflora)andanimals
freeofpathogenicmicroorganismshavenosuchdrawback.Currentlytheyincludechickens,
rats,mice,guineapigs,pigs,etc.
Laboratory animals are distinguished by their species, age, and individual sensitivity
toward microorganisms. Thus, in selecting animals for study it is necessary to take into
account their species and age. For instance, sensitivity in mongrel animals may show con
siderable individual variations. The use of inbred animals with a definite constant
susceptibilitytowardmicroorganismsexcludesindividualvariationsinsensitivityandallows
forreproducibleresults.
Animalsareinfectedfor.isolatingpurecultureofthecausativeagentincaseswhereitis
impossible to obtain it by any other method (for example, in contamination of the studied
objectsbyextraneousmicroflorawhichinhibitsgrowthofthecausativeagentandincaseof
insignificantamountsofmicroorganismsortheirtransformationintofilteringforms).Thus,
in studying decayed corpses of rodents for the presence of plague causative agents, one
inoculates(withsuspensionoftheorgansorblood)guineapigswhichdie37dayslaterwith
manifestationsofsepticaemia.Purecultureofthecausativeagentisreadilyisolatedfromthe
bloodofinternalorgans.
Contaminationofsusceptibleanimalsforreproducingtheinfectiousprocessisusedin
diseasescausedbyRickettsiaandviruses.
Injection to mice of material from a patient with tickborne encephalitis brings about
paralysis and death in these animals. To determine pathogenicity and virulence of the
causative agents of plague, tularaemia, botulism, anthrax, and some viral diseases, cultures
obtainedfrompatientsarcinoculatedintoalbinomice,guineapigs.rats,orsucklingmice.
Mousewithtetanussigns

Guineapigwithbotulismsigns

PhageTyping.Bacteriophage(orphage)arevirusesthatinfectbacteria.Phagecanbe
veryspecificinwhatbacteriatheyinfectandthepatternofinfectionbymanyphagemaybe
employedinphagetypingtodistinguishbacterialspeciesandstrains.The molecules on the
surfaceofthebacterialcellarealsotargetsforbacteriophages(phagesforshort).Theseare
virusesthatinfectbacteriaandthatassociatewithdifferentbacterialspeciesveryspecifically.
Itisthereforepossibletoidentifybacteriabyinvestigatingwhichbacteriophagescanbindto
theirsurface.

Proteinanalysis[gelelectrophoresis,SDSPAGE,establishmentofclonality]
I. The size and other differences between proteins among different organisms may be
determinedveryeasilyemployingmethodsofproteinseparationusingmethodscollectively
knownasgelelectrophoresis.
II.SDSPAGE:
OnepopulartechniquegoesbythenameSDSPAGEwhichstandsforsodiumdodecyl
sulfatepolyacrylamidegelelectrophoresis
NotethatanothernameforSDSissodiumlaurylsulfate,adetergentyouwillfindin
manyshampoos.
Such methods are very good at detecting small differences between isolates and are
especiallygoodatestablishingclonality.

ProteinandDNASequencing
Inthelast25years,molecularbiologyhasdevelopedrapidlyanditisnowpossibleto
sequencetheproteinsfromdifferentbacterialspecies,makelargedatabasesofthesequences,
andusethemasverypowerfulidentificationtools.Similardatabasehavebeendevelopedfor
bacterialDNAandbacterialRNA,particularlytheRNAthatformsthestructuralcomponents
ofbacterialribosomes.
Such techniques are also being used to follow the development of strains of bacterial
speciesthatarecurrentlyevolvingataveryrapidrate.StrainsofChlamydiatrachomatis,for
example, are known to be exchanging large numbers of genes, forming completely new
strains in a very short time. This is worrying this bacterium is responsible for taking the
sightof8millionpeoplelivingindevelopingcountriestoday.Identifyingthenewstrainsand
studying how they have arisen so quickly is crucial to controlling infection and preventing
newcasesofblindness.
There are a few basic things regarding 16S ribosomal RNA gene analysis. The actual
mechanicsofthevariouspartsofthistestcanbefoundelsewhereontheweborinanupto
date textbook, and they may be summarized here in the future. With this comparative test,
differences in the DNA base sequences between different organisms can be determined
quantitatively, such that a phylogenetic tree can be constructed to illustrate probable
evolutionaryrelatednessbetweentheorganisms.
The nucleotide base sequence of the gene which codes for 16S ribosomal RNA is
becoming an important standard for the definition of bacterial species. Comparisons of the
sequencebetweendifferentspeciessuggestthedegreetowhichtheyarerelatedtoeachother
a relatively greater or lesser difference between two species suggests a relatively earlier or
latertimeinwhichtheysharedacommonancestor.
A comparison between eleven species of gramnegative bacteria is illustrated on a
separate sequencecomparisonpage,wherethesequencesarealignedsuchthatsimilaritiesand
differencescanbereadilyseenwhenonescrollstotherightorleft.Gapsandinsertionsof
nucleic acid bases (the result of "frameshift" mutations occuring over eons of time as the
organismsdivergefromcommonancestors)whichaffectlongstretchesofDNAhavetobe
takenintoaccountforaproperalignment.
Inanearlierversionoftheabovementionedsequencecomparisonpage,whenonlyfour
specieswerecomparedwitheachother,arelativelyshortsegmentstoodoutasappearingto
be"frameshifted"whencomparingPseudomonasfluorescenswithagroupofthreeenterics.
This situation is shown as follows with the nucleotide bases of the segment in question
showninred.
Pseudomonasfluorescens ...gctaataccgcatacgtcctacgggagaaagcagggg...
Our new organism, shown below as
...gctaataccgcataacgtcgcaagaccaaagcggggg...
"AH"
Budviciaaquatica ...gctaataccgcgtaacgtcgaaagaccaaagcggggg...
Edwardsiellatarda ...gctaataccgcataacgtcgcaagaccaaagtggggg...
One can surmise that a frameshift mutation if the bases are not misplaced to the
extentthatthemutationbecomessilentorlethalcouldbea"cheap"waytoeffectamajor
change in the genotype and subsequent phenotype perhaps resulting in one of those
infamous "leaps" in evolution one hears conjectured about from time to time. Even though
the specific sequence within a shifted segment of DNA may not be changed, the shift will
result in the nucleotide bases being regrouped into different triplet codes and read
accordingly,andtheresultinggenemayproduceavastlydifferentproteinwhichcanchange
theappearanceorfunctionofacelltoasignificantextent.So,whensequencesbetweentwo
species are compared, the organisms may appear to be a bit more closely related if these
relativelyshortframeshiftedsegmentsweretakenintoconsideration.(Withlongstretchesof
DNA,onewouldnotexpectindependantgenesfartheralongthechromosometobeaffected.)
When a 1308base stretch of that part of the chromosome which codes for 16S
ribosomal RNA was lined up and analyzed ("manually" when I had a little time to kill) to
find the extent to which the above four organisms differed from each other, the percent
difference between any two organisms was determined, and the results are summarized as
follows:
PFPF
AH14.8*AH
BA14.53.2BA
ET14.94.35.0ET
*Anexample:Thesamebasesappearinthesamesequence,positionbyposition,for
eachofthetwoorganismsexceptfor14.8%ofthetime.
Withthepercentdifferencesusedtodenoteprobableevolutionarydistancesbetweenthe
organisms,aphylogenetictreewasroughedouttoillustratetherelationships.Thedistances
betweenanytwoorganisms,whenreadalongthehorizontallines,correspondscloselytothe
percentdifferences.(Thebaratthebottomsignifiesapproximately1%basedifference.)
Databases of various gene sequences are found on the web. Genbank's database was
usedasthesourceoftheabovesequences.Andratherthanhavingtolineupthesequences
anddeterminethedifferencesmanually,asetofprogramstoanalyzesequencedataandplot
treesareavailable.

References
1.ReviewofMedicalMicrobiology/E.Jawetz,J.Melnick,E.A.Adelberg/Lange
MedicalPublication,LosAltos,California,2002.P.4687.
2.MedicalMicrobiologyandImmunology:ExaminationandBoardRewiew/W.
Levinson,E.Jawetz.2003.P.1416
3.HandbookonMicrobiology.LaboratorydiagnosisofInfectiousDisease/Edby
Yu.S.Krivoshein,1989,P.2974.
4. EssentialsofMedicalMicrobiology/W.A.Volkatal.,LippincottRaven,
PhiladelphiaNewYork

Intranet.tdmu.Edu.ua_data_kafedra_internal_micbio_classes_stud_en_pharm_prov_pharm_ptn_Microbiology With Basis Immunology_2_Lesson 3. the Main Methods, Principles and Steps of Isolation of Bacteria’s Pure Cultures

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Intranet.tdmu.Edu.ua_data_kafedra_internal_micbio_classes_stud_en_pharm_prov_pharm_ptn_Microbiology With Basis Immunology_2_Lesson 3. the Main Methods, Principles and Steps of Isolation of Bacteria’s Pure Cultures

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3/8/2017 intranet.tdmu.edu.ua/data/kafedra/internal/micbio/classes_stud/en/pharm/prov_pharm/ptn/Microbiologywithbasisimmunology/2/Lesson3.

4. Bacteria motility. Some microbes (Proteus vulgaris) have a tendency to creeping

Suspend a loopful of test colony in two of these, and a loopful of control

Clostridium perfringens elaborates a variety of exotoxins, one of which is toxin

Inoculate one with a known Streptococcus pneumoniae, another with a viridans

Place a 5 microgram Optochin disc (ethylene hydrocupreine hydrochloride) in the

Place a drop of freshly prepared oxidase reagent (1% tetra methylparaphenylene

melibiose, amygdalin and arabinose. Fermentation is shown by an acid reaction (yellow

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