Describes plainly the steps of the routine Ziehl-Neelson staining steps

1. Title:Sputum smearpreparationandAFBstainingprocedure
Author:Negash
Occupation:Clinicallabscientist
E-mail:drnegash@gmail.com
Facebook:NegashRealis
Date:Dec15,2018

2. Title:SputumsmearpreparationandAFBstainingprocedure
Principle:Sputumsamplefromthedensepartofthesampleistakenoutofthe
vialinasafetycabinetunderhighprecautionandanovalsmearispreparedona
glassslidewithanapplicatorstickanditisallowedtoairdryandthenstainedby
theroutineAFBmethod.
Materials
1-Asputumsample.
2-PPEincludingglovesgownsandhypochloritetoemulsifyandkilltheAFB
sampleincontainerpriortosmearing.
3-Frostedslide
4-Pencil
5-Swabtomakeasmear
6-ZiehlNeelsenreagents[1]
*Fuchin:
Basicfuchin...3.0gm
95%Ethanol...100ml
Thisissolution...one
*Phenol
Phenolcrystals...5gm
Distilledwater...100ml
Thisissolution...two
Workingsolution:10mlofsolution1madeupwith90mlofsolution2.
*decolorizingagent:3%acidalcohol
ConcentratedHCL...3ml
95%ethanol97...ml
or

3. Concentratedsulphuricacid...25ml
Steriledistilledwater...100ml
Note:Alwaysaddacidtowatertoavoidexplosivesplash!
Counterstain
Methleneblue
Methylenebluechloride...0.3gm
Distilledwater...100ml
Procedure
1-Carefulopenthevialcontainingthesampleinsideasafetycabinet!Donotopen
AFBsamplesintheopen!AFBistransmittedviaaerosols.
2-Transferanappropriateportiontothelabeledslidebyanapplicatorstick;
choosethesolid,bloodyandgrayishpartofthesputum.Makea2.0by1.0cm
ovaltranslucentsmear.
3-Allowtoairdryfor15minutes.
4-Placeonaslideandfixbyflaming.Donotoverheat!
5-Coolbeforestaining
6-Floodwithfilteredcarbolfuschin
7-Heattosteamuntilvaporcomesoutbutnotboil;thenwaitfor5minutes
8-Nowrinsewithwateranddecolorizewithacidalcoholandwaitfor3minutes.
9-Rinsetheslidethoroughlywithwaterandfloodwithcounterstainwaitfor1
minute.
10.Airdryandexamine100xoilemersion.
Result:AFBbacteriaareredthreadlikepatternsinabluebackground.

4. Countingandreportingtechnique:
NoAFBper100fields.....NoAFBobservedorNoAFBper100fields
1-9AFBper100fields...Reportexactnumber
10-99AFBper100fields...1+(10to99AFBper100fields)
1to10AFBperfield...2+(1-10AFBperfieldin50fields)
Morethan10AFBperfield...3+(morethan10AFBperfieldin20fields)
Sourcesoferror:
-Inadequatesampleorsalivasample
-Labelingproblems
-Scratchedslides
-OtheracidfastbacillilikeRhodococcusandNocardiaspp
-Stainpreparationproblems(unfilteredstains)
-Foodartifacts
-Contaminatedcontainersandloopsfrompositivesamplestransferredto
negativeones.
References:
LaboratoryServicesintuberculosiscontrol;IsabelNarvaizdeKantoretal:forthe
GlobalTuberculosisprogramme,WHO,Geneva,Switzerland:2002[1].