26 zn staining finh

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InstitutionLaboratory nameLocationHead/Responsible person

Standard Operating Procedure (SOP)

ZiehlNeelsen staining

Code!ersion no"#ate o$ release

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Content

1. Scope

2. Definitions and abbreviations

3. Personnel qualifications%"& 'edical $itness%" ducation and training

4. Procedure*"& Principle*" Sa+ples

*"% ,uip+ent and +aterials*"* Reagents and solutions*"- #etailed instructions*". Reading recording and reporting*"0 1ualit2 control and e3aluation o$ s+ear ,ualit2*"4 5aste +anage+ent

5. elated documents

!nne" 1. equest and reportin# form for sputum e"amination!nne" 2. $icroscope components

Co+piled b2 6a+ined b2 7ppro3ed b2 Replaced Ne8 3ersion

Na+e Code Code

#ate

Signature

Laborator2 area No o$ copies Reason $or change

1. Scope


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9his SOP describes the ZiehlNeelsen (ZN) staining techni,ue $or the detection o$acid:$ast bacilli (7;


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8earing and use o$ protecti3e e,uip+ent and clothing?

laborator2 design including air$lo8 conditions?

use o$ e,uip+ent (operation identi$ication o$ +al$unctions +aintenance)?

good laborator2 practice and good +icrobiological techni,ues? organi@ation o$ 8or> $lo8?

procedures?

i+portance o$ laborator2 results $or patient +anage+ent?

i+portance o$ laborator2 results $or the N9P"

9he training shall be

gi3en be$ore a sta$$ +e+ber ta>es up his/her post?

strictl2 super3ised?

adapted to ta>e account o$ ne8 or changed conditions? and

repeated periodicall2 pre$erabl2 e3er2 2ear"

4. Procedure

4.1 Principle

9he propert2 o$ acid:$astness is based on the presence o$ +2colic acids in the cell 8allo$ +2cobacteria" Pri+ar2 stain ($uchsin) bindsto cell:8all +2colic acids" Intensedecolouri@ation (strong acid or acid/alcohol) does not release the pri+ar2 stain $ro+ thecell 8all and the +2cobacteria retain the red colour o$ $uchsin hence acid:$astness"Counterstaining (8ith +eth2lene blue) pro3ides a contrasting bac>ground"

5hile +2cobacteria are 7;l2 (e"g" Nocardia)" 7;< $ound in respirator2 speci+ens o$

patients $ro+ countries 8ith high 9< pre3alence are al+ost al8a2s 9< bacilli" Non:9ing area" Label the slides properl2 using the laborator2 register serial nu+ber +ar>ed on the

sputu+ container"

Place each slide on its corresponding container"

Proceed to s+earing ta>ing the labelled slides and opening containers one b2 one?do the s+earing behind the $la+e o$ a ness o$ the s+ear should be such that a ne8spaper held under the slidecan barel2 be read through the dried s+ear"

#isin$ect the 8or>ing area a$ter s+ear preparation"

Let the s+ears air:dr2 at roo+ te+perature? do not use heat to speed the dr2ing"

5here hu+idit2 is high gentle 8ar+ing 8ill be needed on a slide 8ar+er (or locall2+ade bo6 8ith glass top under 8hich there is a A:5 light bulb)"

5hen dr2 hold the slides in $orceps and $i6 the+ b2 passing three ti+es slo8l2through the $la+e o$ a spirit la+p or ,uic>l2 through that o$ a


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Place the slides s+ear up8ards on the staining rac> o3er a sin> about & c+ apart"

Place a ne8 $ilter paper in a s+all $unnel >eep it o3er the $irst slide and $ill it up 8ithcarbol:$uchsin staining solution"

Let the solution $ilter through the paper co3ering each slide co+pletel2"

Prepare the torch b2 dipping its cotton 8ool end in burning spirit? light it"

Heat all the slides >eeping the torch a little belo8 the+ and +o3ing it continuousl2bac> and $orth along the line until stea+ rises" 9his is repeated t8ice at inter3als o$%- +inutes" #o not allo8 the staining solution to dr2 on the slides add $reshsolution as re,uired" Contact ti+e o$ the staining solution 8ith the s+ear shouldtotal at least &A +inutes"

Bsing $orceps tilt each slide to drain o$$ the stain solution" Rinse the slides 8ell 8ithdistilled 8ater or clean tap 8ater $ro+ a bea>er (not directl2 $ro+ the tap)"

Pour the acid solution o3er the s+ears co3ering the+ co+pletel2 and allo8 to act

$or % +inutes"

Bsing $orceps tilt each slide to drain o$$ the acid solution" entl2 rinse each slideagain 8ith distilled 8ater or clean tap 8ater $ro+ a bea>er (not directl2 $ro+ the tap)"

I$ necessar2 repeat the acid treat+ent and rinsing until all +acroscopicall2 3isiblestain has been 8ashed a8a2"

;lood s+ears 8ith +eth2lene blue solution $or & +inute"

Bsing $orceps tilt each slide to drain o$$ the +eth2lene blue solution" Rinse theslides 8ell 8ith distilled 8ater or clean tap 8ater $ro+ a bea>er (not directl2 $ro+ thetap)"

Bsing $orceps ta>e each slide $ro+ the rac> and let the 8ater drain o$$" Stand the

slide on edge on the dr2ing rac> and allo8 to air:dr2"

Note: The stained smear should show a light blue colour. ( dar# blue colour usuall!indicates that the smear is too thic# or that the meth!lene blue staining time wastoo long this will hide the red (30 in the bac#ground.

4.! Reading" recording and reporting

4..1 5eading 6see (nne7 28

Set the 3ariable 3oltage regulator to +ini+u+ and s8itch the po8er on"

Slo8l2 ad=ust the light until the desired intensit2 is reached"

nsure that the lenses +irrors and other light:conducting sur$aces are clean

9urn the coarse ad=ust+ent >nob to +o3e the stage a8a2 $ro+ the ob=ecti3e lens"

Place a stained slide on the stage s+ear up8ards"

Rotate the nosepiece to the &A6 ob=ecti3e and ad=ust the light intensit2 as re,uired"

7d=ust the inter:pupillar2 distance until the right and le$t i+ages +erge"

;ocus the i+age 8ith the right e2e b2 loo>ing into the right e2epiece and ad=usting 8iththe $ine $ocus >nob"


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;ocus the i+age 8ith the le$t e2e b2 loo>ing into the le$t e2epiece and turning thedioptre ring"

Open the condenser iris diaphrag+ so that the $ield is e3enl2 lit (about 4AE open)"

9urn the coarse $ocus >nob to bring the &A6 ob=ecti3e lens close to the slide? do notallow the obective lens to touch the smear"

5hile loo>ing into the e2epieces slo8l2 turn the coarse $ocus >nob to separate theob=ecti3e lens and the stage" 9he s+ear should co+e into $ocus 8ithin a $e8 turns"9hen turn the $ine $ocus >nob until the s+ear is seen +ost clearl2" (lwa!s use thefocusing adustment #nobs to lower the stage awa! from the lens.

Place a drop o$ i++ersion oil on the le$t edge o$ the s+ear? do not touch the slide 8iththe oil applicator but allo8 the drop o$ oil to $all $reel2 onto it" 9hen rotate the &AA6ob=ecti3e into place"t is a professional error to focus directl! with this obective.

5ith par$ocal lenses the i++ersion ob=ecti3e 8ill no8 be in the oil? i$ not lo8er it

slightl2 until it =ust touches the oil (loo>ing $ro+ the side)" Raise the condenser as high as possible" Increase the brightness o$ the light until the

$ield is 8ell:lit but still co+$ortable $or the e2e"

;ocus b2 ad=usting 8ith the $ine $ocus >nob" Bse a +a6i+u+ o$ one turn in onedirection? i$ this is not success$ul repeat in the other direction"

Scan the stained s+ear s2ste+aticall2 $ro+ le$t to right side co3ering one length (&AA&-A +icroscopic high:po8er $ields depending on the length o$ the s+ear or % c+)"9his is the +ini+u+ that +ust be scanned be$ore reporting a negati3e result? theprocess should ta>e about - +inutes" Count 7;< in positi3e s+ears $or ,uanti$ication"

7l8a2s search $or use$ul areas i"e" those containing +ucoid threads and pus cells? dothis b2 +o3ing up or do8n 8hen arri3ing at an al+ost e+pt2 area until another use$ul

@one has been $ound then continue +o3ing to the right"7cid:$ast bacilli appear bright red against the bac>ground +aterial counterstained inblue" Report as positi3e $or 7;< 8hen the bac>ground is bluish and at least one red

7;< is seen in a 8ell de:stained s+ear (e3en i$ the 7;< +a2 be +2cobacteria otherthan tubercle bacilli)" 9ubercle bacilli are ,uite 3ariable in shape $ro+ 3er2 short$rag+ents to elongated t2pes" 9he t2pical appearance is usuall2 o$ rather long andslender slightl2 cur3ed rods" 9he2 +a2 be uni$or+l2 stained or 8ith one or +an2gaps or e3en granular" 9he2 occur singl2 or in s+all groups and rarel2 in largeclu+ps"

Once the s+ear has been read rotate the &AA6 ob=ecti3e a8a2 8ithout changing$ocus and re+o3e the slide"

Place the slide s+ear:do8n on a piece o$ absorbent paper (e"g" $olded toilet paperne8spaper) to soa> up the oil? do not +o3e the slide once it is on the absorbent paper"

9a>e the ne6t slide put a drop o$ oil on one end o$ the s+ear $i6 the slide on thestage turn the &AA6 ob=ecti3e into the oil and continue reading" I$ slides are o$ thesa+e thic>ness $ocusing this (and subse,uent) s+ears should re,uire onl2 slightad=ust+ent o$ the $ine $ocusing >nob"

5hen all slides ha3e been e6a+ined reset the 3oltage regulator control to +ini+u+and turn the po8er o$$"


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Store the slides in a slide bo6 in order o$ the nu+bers o$ the laborator2 register? the28ill be needed $or e6ternal ,ualit2 assess+ent" #o not 8rite results on the slides"

Clean the ob=ecti3e lens at the end o$ each da2 using lens tissue or other suitable so$t

tissue"4..2 5ecording

;inding RecordingNo 7;< $ound in at least &AA $ields negati3e&F 7;< per &AA $ields e6act $igure/&AA&AFF 7;< per &AA $ields G&&A 7;< per $ield (count at least -A $ields) GG'ore than &A 7;< per $ield (count at least A $ields) GGG

Some national #uidelines require 3(( fields to be read before a smear isreported ne#ative) *+ic+ ta,es about 15 minutes. !d-ust t+e SP if

necessary.

4..+ 5eporting

Results +ust be reported in a special register o$ 9< laborator2 e6a+ination" Bse red in>$or positi3e results" Reports +ust be pro3ided as soon as possible"

;or a negati3e result report 7cid:$ast bacilli 8ere not seen"

;or a positi3e result report ,uanti$ication o$ 7;< seen"(It should not be assu+edthat 7;< are tubercle bacilli")

Never report 9No T0: 6or e;uivalent wording8.

4.# $ualit% control and e&aluation of smear qualit%

4.ness as possible gi3ing each series the sa+e 1C identi$ication nu+ber" Chec>% o$ each a$ter good staining and note the a3erage nu+ber o$ 7;< $or the &G inthe 1C logboo>

Chec> e3er2 ne8l2 prepared staining solution 8ith unstained control s+ears usingat least one positi3e 8ith >no8n appro6i+ate nu+ber o$ 7;under the batch nu+ber (and/or preparation date) o$ the ne8 solutions"


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Bnacceptable control results include the $ollo8ing

Positi3e control 7;< are not stained strongl2 red or are clearl2 too $e8 innu+ber"

Positi3e control bac>ground re+ains red or contains precipitates" Negati3e control sho8s 7;< (possibl2 $ro+ conta+inated 8ater)"

Stain deposit is present on the 1C slides"

I$ one or +ore o$ these are $ound chec> 8hether so+ething 8ent 8rong 8ith thesolution preparation" I$ this see+s unli>el2 repeat the controls 8ith t8o +ore slideso$ each control pa2ing attention to correct staining techni,ue" 7ccept i$ thesecontrols gi3e the e6pected results" I$ the repeat controls also gi3e unacceptableresults discard the staining solutions and prepare ne8 ones"

Internal 1C o$ staining solutions in use and o$ staining procedure

Include positi3e and negati3e controls 8ith each da2Js reading" Read control slides

be$ore patient s+ears" I$ results are unacceptable (as described abo3e) re:stain s+ears o$ that da2

together 8ith ne8 controls pa2ing attention to correct techni,ue? i$ these controlsare also unacceptable prepare ne8 staining solutions and repeat the staining"

Internal 1C indicators

'onitor laborator2 per$or+ance b2 +onthl2 counts plotted on a graph o$

nu+ber o$ s+ears

positi3it2 rate

positi3e cases detected"

9hese indicators pro3ide an earl2 8arning o$ proble+s and signal the need $orcorrecti3e actions" 9he2 contribute to sta$$ +oti3ation and sel$:reliance"

7+ong the possible reasons $or false/positive resultsare

re:use o$ containers or positi3e slides?

conta+inated stain prepared 8ith 8ater containing en3iron+ental +2cobacteria?

use o$ scratched slides?

7;< $loated o$$ one slide and beca+e attached to another during the stainingprocedure because there 8as no space bet8een ad=acent slides?

inade,uate decolouri@ation?

lac> o$ e6perience con$usion 8ith arte$acts (especiall2 i$ stains are not or poorl2$iltered)?

+icroscope (la+p) in poor condition or poorl2 ad=usted interpreting glitter as7;


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e6cessi3e decolouri@ation?

poorl2 prepared staining solution?

too little ti+e staining 8ith carbol $uchsin?

o3er:staining 8ith +eth2lene blue? o3erheating during $i6ing?

reading less than one length?

slide e6posed to da2light $or too long?

too long an inter3al bet8een staining and reading particularl2 i$ slides 8erepoorl2 stained or not >ept in the dar>"

4.7ternal ;ualit! control

17 is described in a separate SOP"

4.' (aste management

7t the end o$ each da2 seal conta+inated +aterial (used sputu+ containers stic>setc") in a bag and incinerate as soon as possible" Keep the bag in a sa$e closed bin orlarge buc>et until it can be incinerated"

In inter+ediate or central laboratories 8here there is an autocla3e in$ectious 8asteshould be collected in an autocla3able bag and should be autocla3ed be$oreincineration"

5. elated documents

7ngra P et al" Ziehl:Neelsen staining strong red on 8ea> blue or 8ea> red understrong blue nternational ?ournal of Tuberculosis and @ung Disease AA0 &&&&.A&&.&"

Health Protection 7genc2" nvestigation of specimens for ,!cobacterium species.LondonStandards Bnit 3aluations and Standards Laborator2 AA. (NationalStandard 'ethod


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!nne" 1. equest and reportin# form for sputum e"amination

Request and reporting form for sputum smear examination9reat+ent BnitMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMM #ateMMMMMMM

Patients na+eMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMM

7geMMMMMMMM Se6 ('/;)MMMMMMMM

7ddress (precise) MMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMM

MMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMM

MMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMM

Reason $or e6a+ination diagnosis O $ollo8:up OI$ $ollo8:up e6a+ination 'onth MM

Registration nu+ber MMMMMMMMMM

Signature o$ person re,uesting e6a+ination

esults 0to be completed in t+e laboratory1

Laborator2 serial No"MMMMMMMMM 9echni,ue used Ziehl:Neelsen / ;luorochro+e

Result (chec> one)

#ate Speci+en 7ppearance neg &:F G GG GGG

&

%

3isual appearance o$ sputu+ (blood:stained purulent +ucous +ucopurulent sali3ar2

#ateMMMMMMMMMMMMM 6a+ined b2 MMMMMMMMMMMMMMMMMMMMMMMMMMMMMM

SignatureMMMMMMMMMMMMMMMMMMMMMMMMMMMM

9he co+pleted $or+ (8ith results) should be sent pro+ptl2 to the treat+ent unit


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!nne" 2. $icroscope components

26 zn staining finh

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