Institution

Laboratory name
Location
Head/Responsible person
Standard Operating Procedure (SOP)
Identification of Mycobacterium
tuberculosis
Code:
Version: no.
ate: of release
Page: 1 of 8
Content
1. Scope
2. Definitions and abbreviations
3. Personnel qualifications
!." #edical fitness
!.$ %ducation and training
4. Procedure
&." Principle
&.$ Sa'ples
&.! %(uip'ent and 'aterials
&.& Reagents and solutions
&.) Process of identification of M. tuberculosis
&.* Interpretation and identification
. !elated documents
Co'piled b+ %,a'ined b+ -ppro.ed b+ Replaced /e0 .ersion
/a'e Code: Code:
ate
Signature
Laborator+ area: /o of copies: Reason for c1ange:
Institution
Laboratory name
Location
Head/Responsible person
Standard Operating Procedure (SOP)
Identification of Mycobacterium
tuberculosis
Code:
Version: no.
ate: of release
Page: 2 of 8
1. Scope
21is SOP describes t1e differentiation of tubercle bacilli fro' ot1er '+cobacteria and
t1e p1enot+pic identification of Mycobacterium tuberculosis based on bioc1e'ical
tests.
21e .ast 'a3orit+ of strains p1enot+picall+ identified as M. tuberculosis are genuine M.
tuberculosis strains4 alt1oug1 p1enot+pic traits 'a+ not al0a+s differentiate bet0een
M. africanum strains and M. tuberculosis. Ho0e.er4 t1is 1as no 'a3or i'pact on
patient 'anage'ent and follo05up.
6or precise epide'iological studies in a geograp1ical region 01ere M. africanum or M.
bovis is fre(uent or for a stud+ of patients fro' suc1 regions4 a lin7 s1ould be
establis1ed for reference purposes 0it1 a laborator+ t1at carries out genot+pic
identification of tubercle bacilli.
Identification based on bioc1e'ical tests 1as se.ere li'itations: tests are ti'e5
consu'ing4 can be perfor'ed onl+ on solid cultures4 and re(uire large (uantities of 28
bacilli if t1e+ are to gi.e reliable results.
8+ contrast4 i''unoc1ro'atograp1ic assa+s enable rapid identification of tubercle
bacilli fro' eit1er solid or li(uid cultures and 'ini'i9e t1e 1andling of large (uantities
of tubercle bacilli t1at is re(uired in bioc1e'ical tests. :HO reco''ends t1e use of
i''unoc1ro'atograp1ic tests for t1e rapid identification of cultures of 28 bacilli.
21ese tests are co''erciall+ a.ailable; if used4 t1e 'anufacturer<s instructions 'ust
be follo0ed.
2. Definitions and abbreviations "
P/8: p5nitroben9oate.
/-: not applicable
3. Personnel qualifications
3.1 Medical fitness
In accordance 0it1 national la0s and practices4 arrange'ents s1ould be 'ade for
appropriate 1ealt1 sur.eillance of 28 laborator+ 0or7ers:
before enrol'ent in t1e 28 laborator+;
at regular inter.als t1ereafter4 annuall+ or bi5annuall+;
after an+ bio1a9ard incident;
at t1e onset of 28 s+'pto's.
Ideall+4 indi.idual 'edical records s1all be 7ept for up to "= +ears follo0ing t1e end
of occupational e,posure.
Laborator+ 0or7ers s1ould be educated about t1e s+'pto's of 28 and pro.ided
0it1 read+ access to free 'edical care if s+'pto's arise.
Institution
Laboratory name
Location
Head/Responsible person
Standard Operating Procedure (SOP)
Identification of Mycobacterium
tuberculosis
Code:
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ate: of release
Page: 3 of 8
Confidential HIV counselling and testing s1ould be offered to laborator+ 0or7ers.
Options for reassign'ent of HIV5positi.e or i''uno5suppressed indi.iduals a0a+
fro' t1e 1ig15ris7 areas of t1e 28 laborator+ s1ould be considered.
-ll cases of disease or deat1 identified in accordance 0it1 national la0s and/or
practice as resulting fro' occupational e,posure to biological agents s1all be
notified to t1e co'petent aut1orit+.
3.2 Education and training
8asic education and training 'ust be gi.en on t1e follo0ing topics:
potential ris7s to 1ealt1 (s+'pto's of 28 disease and trans'ission);
precautions to be ta7en to 'ini'i9e aerosol for'ation and pre.ent
e,posure;
1+giene re(uire'ents;
0earing and use of protecti.e e(uip'ent and clot1ing;
1andling of potentiall+ infectious 'aterials;
laborator+ design4 including airflo0 conditions;
pre.ention of incidents and steps to be ta7en b+ 0or7ers in t1e case of
incidents (bio1a9ard incidents4 c1e'ical4 electrical and fire 1a9ards);
good laborator+ practice;
organi9ation of 0or7 flo0;
procedures;
i'portance of laborator+ results for patient 'anage'ent;
i'portance of laborator+ results for t1e national 28 progra''e.
21e training s1all be:
gi.en before a staff 'e'ber ta7es up 1is/1er post;
strictl+ super.ised;
adapted to ta7e account of ne0 or c1anged conditions; and
repeated periodicall+4 preferabl+ e.er+ +ear.
4. Procedure
4.1 Principle
21e process in.ol.es t1e p1enot+pic identification of cultures of acid5fast bacilli
gro0n on solid 'edia. Identification c1arts are based on t1e co'bination of
obser.ed colon+ 'orp1olog+4 inabilit+ to gro0 on a culture 'ediu' containing
P/84 and results of bioc1e'ical tests specific for detection of niacin4 1eat5labile
catalase and nitrate reductase.
4.2 Samples
Institution
Laboratory name
Location
Head/Responsible person
Standard Operating Procedure (SOP)
Identification of Mycobacterium
tuberculosis
Code:
Version: no.
ate: of release
Page: 4 of 8
Cultures gro0n on solid 'edia. 21ese could be pri'ar+ cultures if gro0t1 is
abundant.
Subcultures4 e.g. control tubes used for drug susceptibilit+ testing.
Identification 'ust be carried out on pure cultures onl+.
4.3 Equipment and materials
/-
4.4 Reagents and solutions
/-
4.5 Tests of identification of M. tuberculosis
4.5.1 Observation of colonies: morphology and growth rate
21e follo0ing c1aracteristics of isolated colonies 'a+ +ield a tentati.e identification of
M. tuberculosis:
for'ation of .isible colonies "=>$? da+s after incubation at !@ AC;
gre+is1 01ite or buff4 dr+4 cauliflo0er5li7e colonies 0it1 no late production of
pig'ent (up to $? da+s).
4.5.2 Interpretation of the growth test on !"
Bro0t1 is in1ibited on LC0enstein>Densen 'ediu' containing P/8 ()== Eg/'l).
4.5.# Interpretation of the catalase test
21e catalase acti.it+ is not 1eat5stable.
4.5.4 Interpretation of the nitrate reductase test
21e nitrate reductase test is positi.e.
4.5.5 Interpretation of the niacin test
21e niacin test is positi.e.
4. !nterpretation and identification
4.$.1 Identification of M. tuberculosis
20o c1arts (see belo0) can be follo0ed:
/iacin test not used
Criteria &.)." to &.).& defined abo.e 'ust be 'et for t1e specific identification of
M. tuberculosis.
/iacin test used
Criteria &.)." and &.).) defined abo.e allo0 t1e specific identification of 'ore t1an
F)G of M. tuberculosis strains.
Institution
Laboratory name
Location
Head/Responsible person
Standard Operating Procedure (SOP)
Identification of Mycobacterium
tuberculosis
Code:
Version: no.
ate: of release
Page: of 8
"ote# %he niacin test should be performed using commercially available strips. %he
conventional method re&uiring the use of cyanogen bromide' an e(tremely
ha)ardous chemical' is banned in most countries and should be discontinued.
21e .ast 'a3orit+ of tubercle bacilli are M. tuberculosis. Ho0e.er4 if 'ore precise
identification is re(uired4 strains s1ould be referred to laboratories t1at perfor'
genot+pic tests.
Identification c1art 0it1out niacin test

Institution
Laboratory name
Location
Head/Responsible person
Standard Operating Procedure (SOP)
Identification of Mycobacterium
tuberculosis
Code:
Version: no.
ate: of release
Page: # of 8
Institution
Laboratory name
Location
Head/Responsible person
Standard Operating Procedure (SOP)
Identification of Mycobacterium
tuberculosis
Code:
Version: no.
ate: of release
Page: $ of 8
Identification c1art 0it1 niacin test
-cid5fast bacilli
Colonies .isible
H @ da+s
Pig'entation %&S
'(
"= da+s > ? 0ee7s
P/8
niacin
)
I tubercle bacilli
J ot1er '+cobacterial species
I tubercle bacilli
J ot1er '+cobacterial species
I tubercle bacilli
J ot1er '+cobacterial species
/O tubercle bacilli
I ot1er '+cobacterial species
*
Catalase
M. bo$is
M. africanum
J ot1er '+cobacterial species
M. tuberculosis
M. africanum
)
*
Heat5labile
M. bo$is
M. africanum
Heat5stable
/O tubercle bacilli
I ot1er '+cobacterial species
Institution
Laboratory name
Location
Head/Responsible person
Standard Operating Procedure (SOP)
Identification of Mycobacterium
tuberculosis
Code:
Version: no.
ate: of release
Page: 8 of 8
. !elated documents
Collins C4 Brange D4 Kates #. Organi)ation and practice in tuberculosis
bacteriology. London4 8utter0ort1s4 "F?).
de Lantor I/4 Las9lo -. ("FF@). 2uberculosis laborator+ procedures for de.eloping
countries. In: Bangadar1a' P4 Den7ins P-4 eds. Mycobacteria: basic aspects. *ol.
1. /e0 Kor74 C1ap'an and Hall4 "FF@.
Hasega0a / et al. ($==$). /e0 si'ple and rapid test for culture confir'ation of
#+cobacteriu' tuberculosis co'ple,: a 'ulticenter stud+. +ournal of ,linical
Microbiology4 $==$4 &=(!):F=?>F"$.
Lent P24 Lubica BP. ublic health mycobacteriology: a guide for the level III
laboratory. -tlanta4 B-4 Mnited States epart'ent of Healt1 and Hu'an Ser.ices4
Centers for isease Control4 "F?).
Leao S et al. ractical handboo- for the phenotypic and genotypic identification of
mycobacteria. 8rugge4 8elgiu'4 Van den 8roele4 $==).
Vincent V4 ButiNrre9 #C. Mycobacterium: laborator+ c1aracteristics of slo0l+
gro0ing '+cobacteria. In: Manual of clinical microbiology. :as1ington4 C4
-'erican Societ+ for #icrobiolog+4 $==@:)@!>)??.
.aboratory services in tuberculosis control. art III: ,ulture. Bene.a4 :orld Healt1
Organi9ation4 "FF?.