AFB Microscopy

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Module

AFB MICROSCOPY


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Content Overview

Collection of sputum specimens Quality of sputum specimens Processing of sputum specimens

Registration of sputum specimens

Sputum smearing and staining Smear examination Recording and reporting


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Collection of Sputum Specimens

Collect 1-3 sputum specimens for diagnosticpurposes according to NTP policy

ollo! NTP policy for declaring or excluding asmear-positi"e case

ollo! NTP policy for follo!-up smears


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Instructions to Patients

$i"e clear instructions to patients on %o! tocollect t%e sputum&

'mportance of sputum examination for T(

diagnosis

Need for real sputum) not sali"a

*o! to produce good sputum

*o! to open and close t%e container

*o! to a"oid contamination of outside of t%econtainer


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Characteristics

of Good Sputum Specimen

3-+ ml ,sually t%ic and mucous) .ut may .e fluid !it%

pieces of purulent material

Color "aries from opa/ue !%ite to green) reddis% to.ro!n !%en .lood is present

Clear sali"a is not suita.le0 .ut examine sali"a if a.etter specimen can not .e produced) especially forfollo!-up examinations

specimen mainly containing .lood s%ould not .eexamined0 patient s%ould see a doctor for immediatemanagement


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Processin Sputum Specimens

Process specimens as soon as possi.le fort%e .enefit of t%e patients

*o!e"er) for microscopy time .et!een

collection and staining matters little a refrigerator is not needed to eep samples

fter preparation of a fixed smear) furt%er

processing can !ait


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Collection and !ransportation

of Sputum

4ptions if t%e %ealt% facility attended .y t%epatient does not perform smear microscopy

Refer patients to nearest microscopy facility

*a"e t%e specimens taen .y assigned person orsent .y pu.lic transport to microscopy facility

*a"e t%e specimens collected) smears prepared

and fixed and taen to microscopy facility .y

assigned person


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Reistration of Sputum Specimens

Register all specimens for ( microscopy in t%e T(6a.oratory Register

4ne line for t%e diagnostic smears of eac% patient

4ne line for eac% follo!-up

4ne or more results may .e registrered on one line

6a.el t%e sputum containers on t%e side) not on t%elid) !it% la.oratory serial num.er and t%e column

num.er or code for diagnostic smears

6a.el t%e slides !it% t%e la.oratory serial num.erand t%e column code


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Sputum Smearin "#$

Select ne!) clean) grease-free) unscratc%edslides) free of fingerprints

,se a pencil in case of a frosted slide and a

diamond pencil in case of unfrosted slides to!rite t%e serial num.er

8nsure t%at t%e num.er on t%e slide is t%e

same as on t%e sputum container


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Sputum Smearin "%$

Select and pic up yello!is% :or !%itis%;particles !it% an applicator stic) e


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Sputum Smearin "&$

Place t%e used stic in a discard container0use a ne! stic for eac% specimen

>ip t%e !ire loop in a sand-alco%ol .ottle0

mo"e t%e loop up and do!n to remo"e excesssputum0 %eat in flame until red-%ot

ir-dry t%e smear at room temperature 'f t%e smear is completely dry) pass t%e slide

2-3 times) 2-3 seconds o"er a flame) %oldingt%e slide up!ards


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Smear Stainin

with 'Iehl()eelsen Stain "#$

!ell stained ?N smear s%o!s strong red ()against a !ea .lue .acground

(acground s%ould not s%o! muc% remaining redand no red artifacts

Proposed tec%ni/ue is to use 1@ car.ol fuc%sin:strong red; and 9estaining !it% 3@ %ydroc%loric acid in alco%ol or 29-2+@ sulp%uric acid in !ater

must .e complete can .e repeated to ensure complete a.sence of red color

from t%e .acground


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') Stained Smear


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ST8PS&

rrange slides in a serial order on a staining .ridge) smearside up) a"oid slides touc%ing eac% ot%er

Co"er slides completely !it% car.ol fuc%sin solution :t%roug%filter paper;

$ently %eat to steam 6ea"e for 19-1+ minutes Rinse !it% !ater and drain Co"er !it% decoloriAing solution) 3-+ min) rinse !it% !ater)

drain< Repeat if needed) rinse and drain pply met%ylene .lue solution for 1) at most 2 minutes Rinse !it% !ater and drain ir dry on a slide rac

Smear Stainin

with 'iehl()eelsen Stain "%$


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Smear Stainin with Auramine "#$

ST8PS

rrange slides in a serial order on a staining .ridge) slide sideup) a"oid slides touc% eac% ot%er

pply filtered auramine solution) lea"e for at least 1+) max< 39minutes

Rinse !it% !ater and drain pply decoloriAing solution) 1-3 minutes) rinse !it% !ater and

drain) repeat if needed) rinse !it% !ater and drain

pply counter stain for max< 1 minute

Rinse !it% !ater and drain ir dry on a slide rac


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( s%o! yello! or green fluorescenceagainst a more or less dar .acground)depending on t%e lamp and filters used

>estaining solution must %a"e alco%ol0 acidusually 9


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Auramine Stained Smear "#$


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Auramine Stained Smear "%$


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*+amination of ') Smears

8xamine 1 lengt% of t%e smear :2 cm; or 199 fields !it%t%e .rig%t field microscope) 1999x magnification

>eclare t%e smear as negati"e if no ( are found

'f 19 ( are found) count t%e num.er of ( 'f DE 19 ( are found) grade t%e smear 1F) 2F) or 3F or %ig% positi"es examination of 29-39 fields may

suffice


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*+amination of

Auramine Stained Smears

8xamine 1 lengt% of t%e smear :2 cm; !it% G .est use a 29x o.Hecti"e& 1 lengt% is 39 fields !it% t%is

o.Hecti"e) .ut 3 times t%e area seen t%roug% a 199I

o.Hecti"e

if a #9x o.Hecti"e is used) one lengt% counts #9-+9 fields or 2

times t%e area seen t%roug% a 199I o.Hecti"e

'f necessary) confirm () typical cur"ed rod s%ape)

turning to a %ig%er po!er o.Hecti"e :#9I if screening

!it% 29I;


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Gradin of

Auramine Stained Smears

8xamine one lengt% if no or fe! ( are seen declare negati"e if none at all are seen

if less t%an 39 ( are found using 29x o.Hecti"e) or less t%an29 ( using #9x o.Hecti"e) t%e result is scanty< Report t%eexact num.er of ( per lengt%

if ( per lengt% count 39 to 277 using 29I o.Hecti"e) or 29 to177 using #9I o.Hecti"e) grade t%e smear as 1F

8xamine a.out 19 fields if many ( are seen if on a"erage t%ere are more t%an 199 ( per field !it% 29I

o.Hecti"e) or more t%an +9 !it% #9I) grade t%e smear as 3F

if per field .et!een 19 and 199 ( are seen !it% 29Io.Hecti"e) or .et!een + and +9 !it% #9I o.Hecti"e) grade as 2F


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Recordin and ReportinRecordin and Reportin

Jrite t%e smear results in t%e T( 6a.oratoryRegister

Jrite t%e smear results on t%e sputumexamination re/uest forms

or!ard t%e filled sputum re/uest form to t%eperson re/uesting t%e sputum examinations


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Gradin of ') Smears

I,A!-./01O scale ') #222+3

# lenth4#22 1PF

)eative 2 AFB / # lenth

Scant5 #(6 AFB /

# lenth#7 #2(66 AFB /

# lenth

%7 #(#2 AFB /

# 1PF on averae

&7 8#2 AFB /

# 1PF on averae

Gradin of Smears in FM


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,sin %29 O:;ectiveI,A!-./01O scale FM %2+3

# lenth 4 &2 fields# lenth 4 &22 1PF

)eative 2 AFB / # lenth

Scant5 #(%6 AFB /

# lenth

#7 &2(%66 AFB /

# lenth

%7 #2(#22 AFB /

# field on averae

&7 8#22 AFB /

# field on averae



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,sin


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-a:orator5 Reister for Sputum Smear Microscop5


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Re>uest and

Reportin

Form for

SputumSmear

*+amination

?e5 Messaes?e5 Messaes


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?e5 Messaes?e5 Messaes

$ood /uality specimens) smearing) staining and readingare all essential for correct diagnosis of tu.erculosis

?ie%l-Neelsen and fluoroc%rome met%ods are .ot%suita.le) .ut fluorescence is .etter in case of %ig%

!orload and=or fe! (

$rading scales for /uantification of ( in smears "ary!it% t%e magnification system and t%us o.Hecti"e used

T%e specific re/uest form and t%e la.oratory register

must al!ays .e used to record and report smearexamination results

AFB Microscopy

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