Medical Microbiology

LaboratoryDiagnosis 211

Microscopy................................................

Bacteriaaresosmallthatamagnificationof 1000 !isrequiredtoviewthem

properly,whichisatthelimitoflightmicroscopecapability.Atthismagni-

fication,bacteriacanonlybediscernedinapreparationinwhichtheirden-

sityisatleast 104 –1 05 bacteriaperml.

Microscopicexaminationofsuchmaterialrequiresaslidepreparation:

&Nativepreparations,withorwithoutvitalstaining,areusedtoobserve

livingbacteria.Thepoorcontrastofsuchpreparationsmakesitnecessaryto

amplifythisaspect(darkfieldandphasecontrastmicroscopy).Nativepre-

parationsincludethecoverslipandsuspendeddroptypes.

&Stainedpreparationsarericherincontrastsothatbacteriaarereadily

recognizedinanilluminatedfieldat 1000 !.Thestainingprocedurekills

thebacteria.Thematerialisfirstappliedtoaslideinathinlayer,driedin

theair,andfixedwithheatormethylalcohol.Simpleanddifferentialstain-

ingtechniquesareused.Thebest-knownsimplestainingtechniqueemploys

methyleneblue.Gramstainingisthemostimportantdifferentialtechnique

(Table3. 7 ):Gram-positivebacteriastainblue-violet,Gram-negativebacteria

stainred.TheGram-positivecellwallpreventsalcoholelutionofthestain-

Table3. 7 ProcedurefortheThreeMostImportantTypesofStaining

Methyleneblue Gramstaining Ziehl–Neelsenstaining

Methyleneblue

1 – 5 minutes

Gentianvioletorcrystal

violet, 1 minute

Concentratedcarbolfuchsin;

heatthreetimesuntilvaporis

observed

Rinseoffwithwater Pouroffstain,rinseoffwith

Lugol’ssolution,thencover

withLugol’ssolutionfor

2 – 3 minutes

Rinseoffwithwater

DestainwithHCl(3%)/alcohol

mixture

PouroffLugol’ssolution Counterstainwithmethylene

blue, 1 – 5 minutes

Destainwithacetone/ethyl

alcohol(1:4)

Rinseoffwithwater

Rinseoffwithwater

Counterstainwithdilute

carbolfuchsin, 1 minute

Rinseoffwithwater

3

Kayser, Medical Microbiology © 2005 Thieme