Medical Microbiology

212 3 GeneralBacteriology

iodinecomplex.Inoldculturesinwhichautolyticenzymeshavebegunto

breakdownthecellwalls,Gram-positivecellsmaytestGram-negative

(“Gram-labile”bacteria).

AnotherdifferentialstainistheZiehl-Neelsentechnique.Itisusedtostain

mycobacteria,whichdonot“take”gramormethylenebluestainsduetothe

amountsoflipidsintheircellwalls.Sincemycobacteriacannotbedestained

withHCl-alcohol,theyarecalledacid-resistantrods.Themycobacteriaare

stainedredandeverythingelseblue.

&Fluorescencemicroscopyisanotherspecialtechnique.Afluorochrome

absorbsshortwavelightandemitslightwithalongerwavelength.Prepara-

tionsstainedwithfluorochromesareexposedtolightattherequiredwave-

length.Thestainedparticlesappearclearlyagainstadarkbackgroundinthe

coloroftheemittedlight.Thistechniquerequiresspecialequipment.Itsprac-

ticalapplicationisintheobservationofmycobacteria.Inimmunofluores-

cencedetection,afluorochrome(e.g.,fluoresceinisothiocyanate)iscoupled

toanantibodytorevealthepresenceofantigensonparticlesurfaces.

CulturingMethods........................................

Typesofnutrientmediums.Culturingisrequiredinmostcasestodetect

andidentifybacteria.Almostallhumanpathogenbacteriacanbecultivated

onnutrientmediums.Nutrientmediumsareeitherliquid(nutrientbroth)or

gelatinous(nutrientagar,containing 1 .5– 2 %ofthepolysaccharideagarose).

Enrichmentmediumsarecomplexmediumsthatencouragetheproliferation

ofmanydifferentbacterialspecies.Themostfrequentlyusedenrichmentme-

diumisthebloodagarplatecontaining 5 %wholeblood.Selectivemediums

allowonlycertainbacteriatogrowandsuppressthereproductionofothers.

Indicatormediumsareusedtoregistermetabolicprocesses.

Proliferationforms.Mostbacteriashowdiffuseproliferationinliquidme-

diums.Someproliferatein“crumbs,”otherformagrainybottomsediment,

yetothersabiofilmskinatthesurface(pseudomonads).Isolatedcoloniesare

observedtoformon,orin,nutrientagarifthecelldensityisnottoohigh.

Thesearepurecultures,sinceeachcolonyarisesfromasinglebacterium

orcolony-formingunit(CFU).Thepureculturetechniqueisthebasisofbac-

teriologicalculturingmethods.Theproceduremost frequentlyusedto

obtainisolatedcoloniesisfractionatedinoculationofanutrientagarplate

(Figs.3. 33 –3. 35 ).

Useofthistechniqueensuresthatisolatedcolonieswillbepresentinone

ofthethreesectors.Besidesobtainingpurecultures,theisolatedcolony

techniquehasthefurtheradvantageofshowingtheform,appearance,and

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Kayser, Medical Microbiology © 2005 Thieme