36 growth on pnb fin

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InstitutionLaboratory nameLocationHead/Responsible person

Standard Operating Procedure (SOP)

Growth on PNB ediu

!ode"#ersion" no$%ate" o& release

Page" 1 o& 5

Content

1. Scope

2. Definitions and abbreviations

3. Personnel qualifications'$ edical &itness'$* +ducation and training

4. Procedure,$ Principle,$* Saples,$' +-uipent and aterials,$, Reagents and solutions,$. %etailed procedure,$ Result and interpretation,$0 1ualit2 control,$3 4aste anageent

5. elated documents

!opiled b2 +5ained b2 6ppro7ed b2 Replaced New 7ersion

Nae !ode" !ode"

%ate

Signature

Laborator2 area" No o& copies" Reason &or change"


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Growth on PNB ediu


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1. Scope

8his SOP describes the procedure &or the culturing test that detects the capacit2 &or bacterial

growth on solid culture edia containingp9nitroben:oate; which is used &or the identi&icationo& Mycobacterium tuberculosis$

Because suspensions with high concentrations o& 7iable; in&ectious bacteria are handled;strict copliance with sa&et2 and protection easures is andator2$ 8he procedure ust becarried out in a laborator2 eeting the 4HO standards &or biosa&et2 le7el ' with accessrestricted to authori:ed personnel onl2$

2. Definitions and abbreviations

BS!" biological sa&et2 cabinet%S8" drug susceptibilit2 testing

L


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wearing and use o& protecti7e e-uipent and clothing@

handling o& potentiall2 in&ectious aterials@

laborator2 design; including air&low conditions@

use o& biological sa&et2 cabinets (operation; identi&ication o& al&unctions;aintenance)@

use o& autocla7es; incubators (operation; identi&ication o& al&unctions; aintenance)@

pre7ention o& incidents and steps to be ta?en b2 wor?ers in the case o& incidents(bioha:ard incidents; cheical; electrical and &ire ha:ards)@

good laborator2 practice and good icrobiological techni-ues@

organi:ation o& wor? &low@

procedures@

waste anageent@

iportance o& laborator2 results &or patient anageent@ iportance o& laborator2 results &or the national 8B prograe$

8he training shall be"

gi7en be&ore a sta&& eber ta?es up his/her post@

strictl2 super7ised@

adapted to ta?e account o& new or changed conditions@ and

repeated periodicall2; pre&erabl2 e7er2 2ear$

4. Procedure

4.1 Principle

Inabilit2 to grow in the presence o& PNB is one o& the ?e2 eleents in the phenot2picdi&&erentiation o& tubercle bacilli &ro other 2cobacterial species and is part o& theidenti&ication process &or M. tuberculosis.

M. tuberculosisand other tubercle bacilli will not grow on culture ediu containing PNB;.AA g/l@ other 2cobacterial species; with the e5ception o& M. gastriand soe strains o&M. kansasiiand M. marinum; will grow in the presence o& PNB$

8he test ust be carried out on pure cultures otherwise it will 2ield &alse results$

4.2 Samples

Pure cultures o& acid!&ast bacilli grown on solid edia; age *>*3 da2s; with ore than .Acolonies$

4.3 Equipment and materials

BS!; class I or II; annuall2 certi&ied

Incubator

6utocla7e

Re&erence strains o& M. tuberculosis(or a pre7iousl2 identi&ied strain) and a re&erence straino& M. terrae(saproph2tic strain)$

4.4 Reagents and solutions


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4.4.1 PNB solution, 500 g/ml

Weigh *.A g PNB$ %issol7e in A l prop2lene gl2col while heating in a water9bath at'0C!$

4.4. !"#enstein$%ensen me&ium #it' PNB

6ddA l PNB solution (.AA Dg/l) to ,EA l o& L< ediu and i5 well be&ore dispensing$

!oagulate the ediu b2 inspissation as &or plain L< ediu (see rele7ant SOP)$

4.5 Detailed procedure

('e entire proce&ure must be carrie& out in a biological sa)ety cabinet.

Inoculate a slope o& L< ediu containing PNB and a slope o& L< ediu with no PNB(control) with bacterial suspension adFusted to carland turbidit2 standard No$ $

or con7enience; the PNB tube is added to drug9containing tubes and inoculated while

%S8 is carried out$ 8he results o& the PNB test and o& %S8 are there&ore a7ailable at thesae tie$

Incubate at '0 C! and copare growth on both slopes a&ter *3 da2s o& incubation$

4.6 Result and interpretation

6bundant growth on both slopes > 2cobacterial strain other than tubercle bacilli$

6bundant growth on the control tube and little or no growth on PNB ediu > M.tuberculosiscople5 strain$

No growth on either slope > non9interpretable result; test to be repeated$

4. !ualit" control

4.*.1 Morp'ology o) colonies

Obser7e the orpholog2 o& colonies on solid ediu to chec? culture purit2$

4.*. Positi+e control

No growth on PNB ediu > carr2 out the test with a pre7iousl2 identi&ied M. tuberculosisstrain or a re&erence strain &ro a culture collection$

4.*. Negati+e control

Growth on PNB ediu > carr2 out the test with a re&erence strain such asM. terrae(saproph2tic strain) &ro a culture collection$

4.# $aste management

6utocla7e all containated aterials$

5. elated documents

ent P8; ubica GP$ Public 'ealt' mycobacteriology- a gui&e )or t'e le+el laboratory$6tlanta; G6; nited States %epartent o& Health and Huan Ser7ices; !enters &or %isease!ontrol; E3.$

!aboratory ser+ices in tuberculosis control. Part - ulture$ Gene7a; 4orld Health

Organi:ation; EE3$


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#incent #; GutiJrre: !$Mycobacteriu" laborator2 characteristics o& slowl2 growing2cobacteria$ In"Manual o) clinical microbiolog2$ 4ashington; %!; 6erican Societ2 &oricrobiolog2; *AA0".0'>.33.

36 growth on pnb fin

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