Institution

Laboratory name
Location
Head/Responsible person
Standard Operating Procedure (SOP)
Catalase test
Code:
Version: no.
Date: o release
Page: 1 o 5
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
&.) Detailed procedure
&.* Result and interpretation
&.+ ,ualit- control
&.. /aste 'anage'ent
5. elated documents
Co'piled b- %0a'ined b- 1ppro2ed b- Replaced 3e4 2ersion
3a'e Code: Code:
Date
Signature
Laborator- area: 3o o copies: Reason or c5ange:
1. Scope
Institution
Laboratory name
Location
Head/Responsible person
Standard Operating Procedure (SOP)
Catalase test
Code:
Version: no.
Date: o release
Page: 2 o 5
65is SOP describes t5e procedure or t5e bioc5e'ical test t5at detects t5e acti2it- o
catalase ro' bacterial colonies 45ic5 is used or t5e identiication o Mycobacterium
tuberculosis.
7ecause t5e test in2ol2es 5andling suspensions containing 5ig5 concentrations o 2iable8
inectious bacteria8 strict co'pliance 4it5 saet- and protection 'easures is 'andator-. 65e
test 'ust be peror'ed in a laborator- 'eeting t5e /HO standards or biosaet- le2el ! 4it5
access restricted to aut5ori9ed personnel onl-.
2. Definitions and abbreviations
#/: 'olecular 4eig5t
7SC: biological saet- cabinet
3. Personnel qualifications
3.1 Medical fitness
In accordance 4it5 national la4s and practices8 arrange'ents s5ould be 'ade or
appropriate 5ealt5 sur2eillance o 67 laborator- 4or:ers:
beore enrol'ent in t5e 67 laborator-;
at regular inter2als t5ereater8 annuall- or bi<annuall-;
ater an- bio5a9ard incident;
at t5e onset o 67 s-'pto's.
Ideall-8 indi2idual 'edical records s5all be :ept or up to "= -ears ollo4ing t5e end o
occupational e0posure.
Laborator- 4or:ers s5ould be educated about t5e s-'pto's o 67 and pro2ided 4it5 read-
access to ree 'edical care i s-'pto's arise.
Conidential HIV counselling and testing s5ould be oered to laborator- 4or:ers. Options or
reassign'ent o HIV<positi2e or i''uno<suppressed indi2iduals a4a- ro' t5e 5ig5<ris:
areas o t5e 67 laborator- s5ould be considered.
1ll cases o disease or deat5 identiied in accordance 4it5 national la4s and/or practice as
resulting ro' occupational e0posure to biological agents s5all be notiied to t5e co'petent
aut5orit-.
3.2 Education and training
7asic education and training 'ust be gi2en on t5e ollo4ing topics:
potential ris:s to 5ealt5 (s-'pto's o 67 disease and trans'ission);
precautions to be ta:en to 'ini'i9e aerosol or'ation and pre2ent e0posure;
5-giene re(uire'ents;
4earing and use o protecti2e e(uip'ent and clot5ing;
5andling o potentiall- inectious 'aterials;
laborator- design8 including airlo4 conditions;
Institution
Laboratory name
Location
Head/Responsible person
Standard Operating Procedure (SOP)
Catalase test
Code:
Version: no.
Date: o release
Page: 3 o 5
use o biological saet- cabinets (operation8 identiication o 'alunctions8
'aintenance);
use o autocla2es8 4ater<bat5s8 pH 'eters (operation8 identiication o 'alunctions8
'aintenance);
pre2ention o incidents and steps to be ta:en b- 4or:ers in t5e case o incidents
(bio5a9ard incidents8 c5e'ical8 electrical and ire 5a9ards);
good laborator- practice and good 'icrobiological tec5ni(ues;
organi9ation o 4or: lo4;
procedures;
4aste 5andling;
i'portance o laborator- results or patient 'anage'ent;
i'portance o laborator- results or t5e national 67 progra''e.
65e training s5all be:
gi2en beore a sta 'e'ber ta:es up 5is/5er post;
strictl- super2ised;
adapted to ta:e account o ne4 or c5anged conditions; and
repeated periodicall-8 preerabl- e2er- -ear.
4. Procedure
4.1 Principle
65e *. >C catalase test8 45ic5 'easures t5e 5eat stabilit- o t5e en9-'e acti2it-8 is one o
t5e ele'ents in t5e identiication o M. tuberculosis and ot5er tubercle bacilli.
Catalase con2erts 5-drogen pero0ide to 4ater and o0-gen8 generating o0-gen bubbles in a
li(uid solution. Virtuall- all '-cobacteria e0cept certain isonia9id<resistant tubercle bacilli are
catalase<positi2e. #oreo2er8 t5e catalase en9-'e present in 'ost o t5e '-cobacterial
species is 5eat<stable at pH +.= e0cept in tubercle bacilli and a e4 ot5er '-cobacterial
species suc5 as M. fallax, M. chelonae, M. gastri, M. marinum, M. malmoense, M.
haemophilum.
65e test 'ust be carried out on pure cultures ot5er4ise it 4ill -ield alse results.
4.2 Samples
Pure cultures o acid<ast bacilli8 gro4n on solid 'edia8 'ini'u' age "& da-s
4.3 Equipment and materials
7SC8 class I or II8 annuall- certiied
/ater<bat5 4it5 lid8 5eater and t5er'ostat (or te'perature bloc: 5eater) at *. >C
pH 'eter
1utocla2e
Scre4<cap reaction tubes8 "! 0 .) ''
Reerence strains o M. tuberculosis (or a pre2iousl- identiied strain) and a reerence strain
o M. terrae (saprop5-tic strain)
Institution
Laboratory name
Location
Head/Responsible person
Standard Operating Procedure (SOP)
Catalase test
Code:
Version: no.
Date: o release
Page: 4 o 5
4.4 Reagents and solutions
4.4.1 Phosphate buffer, 0.067 mol/litre, pH 7.0
Reagent ". Dissol2e ?.=+ g @H
$
PO
&
('onopotassiu' di5-drogen p5osp5ate8 #/ "!*.")
in "=== 'l distilled 4ater.
Reagent $. Dissol2e $!.*. g 3a
$
HPO
&
."$H
$
O (disodiu' 5-drogen p5osp5ate
dodeca5-drate8 #/ !).."&) in "=== 'l distilled 4ater.
#i0 !..? 'l o reagent " 4it5 *"." 'l o reagent $. C5ec: pH using a pH 'eter.
4.4. Hy!rogen peroxi!e "H

$, %0&
'ot t5e !A H
$
O
$
obtainable ro' p5ar'acies.
/ear plastic glo2es and a protecti2e e-e s5ield 45en 5andling H
$
O
$
.
4.4.% ()een *0 solution, 10&
#i0 "= 'l 64een .= 4it5 ?= 'l distilled 4ater; autocla2e at "$">C or "= 'inutes. Store in
t5e rerigerator up to ! 'ont5s.
4.4.4 +ubstrate
#i0 e(ual 2olu'e o !=A 5-drogen pero0ide and "=A 64een .=. 1l4a-s prepare t5e
substrate res5 Bust beore t5e test.
4.5 Detailed procedure
(he entire proce!ure must be carrie! out in a biological safety cabinet.
Dispense =.) 'l o p5osp5ate buer8 =.=*+ 'l/litre8 pH +.=8 into t4o "* 0 "$) '' scre4<
cap test<tubes.
%'ulsi- se2eral loopuls o bacterial colonies scraped ro' solid 'edia into t5e buer.
Place one tube in t5e 4ater<bat5 at *. >C or $= 'inutes. Lea2e t5e ot5er tube at roo'
te'perature.
1llo4 t5e 5eated tube to cool to roo' te'perature.
1dd =.) 'l res5l- prepared 64een .=Cpero0ide substrate to eac5 tube and cap loosel-.
Obser2e or t5e or'ation o bubbles. Retain negati2e tubes or $= 'inutes beore
discarding. ,o not sha-e the tubes. ()een *0 alone may form bubbles on sha-ing,
resulting in false/positi0e results.
4.6 Result and interpretation
I o0-gen bubbles or' in t5e un5eated tube but not in t5e 5eated tube8 t5e test strain
produces 5eat<labile catalase and probabl- belongs to t5e M. tuberculosis co'ple0.
I o0-gen bubbles or' in bot5 t5e 5eated and un5eated tubes8 t5e test strain produces
5eat<stable catalase and is unli:el- to be a 'e'ber o t5e M. tuberculosis co'ple0.
I no o0-gen bubbles or' in eit5er t5e 5eated or t5e un5eated tube8 eit5er t5e test 5as
ailed or t5e strain 'a- be catalase<negati2e (suc5 as rare isonia9id<resistant strains o
M. tuberculosis). Repeat t5e test 4it5 a larger (uantit- o bacilli to eli'inate t5e
possibilit- o a alse<negati2e result.
4. !ualit" control
Institution
Laboratory name
Location
Head/Responsible person
Standard Operating Procedure (SOP)
Catalase test
Code:
Version: no.
Date: o release
Page: 5 o 5
4.7.1 Morphology of colonies
Obser2e t5e 'orp5olog- o colonies on solid 'ediu' to c5ec: culture purit-.
4.7. Positi0e control. no bubble formation after heating
Carr- out t5e test 4it5 a pre2iousl- identiied M. tuberculosis strain or a reerence strain ro'
a culture collection.
4.7.% 'egati0e control. bubble formation after heating.
Carr- out t5e test 4it5 a reerence strain suc5 as M. terrae (saprop5-tic strain) ro' an
international culture collection.
4.# $aste management
1utocla2e all conta'inated 'aterials.
5. elated documents
@ent P68 @ubica DP. Public health mycobacteriology. a gui!e for the le0el 111 laboratory.
1tlanta8 D18 Enited States Depart'ent o Healt5 and Hu'an Ser2ices8 Centers or Disease
Control8 "?.).
2aboratory ser0ices in tuberculosis control. Part 111. 3ulture. Dene2a8 /orld Healt5
Organi9ation8 "??..
Rieder HL et al. Priorities for tuberculosis bacteriology ser0ices in lo)/income countries.
Paris8 International Enion 1gainst 6uberculosis and Lung Disease8 $==+.
Vincent V8 DutiFrre9 #C. Mycobacterium: laborator- c5aracteristics o slo4l- gro4ing
'-cobacteria. In: Manual of clinical microbiology. /as5ington8 DC8 1'erican Societ- or
#icrobiolog-8 $==+:)+!C)...