culture tb

8/17/2019 Culture Tb

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CULTURE

PURPOSE :

To grow AFB(Acid Fast Bacilli) in a culture medium.

PRINCIPLE :

AFB present in the sputum will grow very well when provided with selective

media. They can be identified based on their colony morphology, presence/absence

of pigment and certain biochemical tests.

PRIMARY SAMPLE :

As in case of pulmonary TB sputum

!ulmonary specimens sputum, transtracheal aspiration, laryngeal swab,bronchoscopic specimens.

EQUIPMENT :

"#$ incubator

Biological safety cabinet class %%%

PROCEDURE :

The specimen (sputum) is decontaminated by !etroff&s method.

Petroff’s method :

'. Transfer about ml of sputum using a pipette to centrifuge tube$. Add eual volume of *+ sterile a#-. orte0 well and place in 12c water bath for $3 to $ minutes.*. 4ha5e well at '3 minutes interval to facilitate digestion of the mucopurulent

material.. Then, add a drop of sterile 3.33*+ !henol red indicator.6. Then, add sterile 7+ -"l slowly till yellow colour is formed.1. Bac5 titrate with *+ sterile a#- until pin5 colour appears.7. "entrifuge at 33 rpm for ' min.8. 9se sediment for smear preparation : culture inoculation.

'3. %noculate ;owenstein


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Typical growth of Mycobacterium tuberculosis is seen as warty, dry buff

coloured colonies.

%f no growth after 8 wee5s, culture to be reported negative.

=hen and where needed, species to be confirmed by appropriate biochemical

tests

Culture report :

"ulture positive when AFB morphologically typical of Mycobacterium

tuberculosis is grown

=hen there is growth not typical of Mycobacterium tuberculosis confirmation

of species to be done with biochemical tests.

Atypical mycobacteria can be confirmed by repeated isolation of the same

organism only.%n case of growth, growth can be graded as

$3 colonies 4canty

$3C'33 colonies Doderate

"onfluent growth -eavy

Qul!t" #o$trol pro#edure :

4terility of ;< medium is chec5ed before use.

Sfet" pre#ut!o$s :

4afety cabinet class %%%, with -?! A filters should be used while inoculating

specimen.

=ear face mas5, glove : apron while handling specimens.

PROCESSIN% O& SPECIMENS OT'ER T'AN SPUTUM :

CS& :

To 8 ml of "4F add ' ml of *+ sodium citrate

"entrifuge at 33 rpm for 3 min

9se sediment for inoculation and smears

%str!# (sh!$) :

4pecimen should be processed within an hour


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"entrifuge the entire specimen

The specimen to be digested using petroff&s method and then inoculated into

appropriate media

Other flu!ds * As#!t!#+ ,o!$t $d pleurl :

4pecimen to be collected with $.+ sodium citrate to prevent clotting

?ntire specimen is centrifuged at 33 rpm for 3 min

4pecimen to be used for smear

;oopful of sediment is inoculated onto Blood agar and supernatant in

Thioglycollate broth

%f no growth, inoculate on to ;< slopes

Pus :!reliminary culturing on Blood agar is done

Then, concentrate the specimen as mentioned previously and inoculate into

;< medium

T!ssue -.!ops!es/ :

@rind the tissue in a sterile grinder by adding ' to drops of sterile saline

"entrifuge and do preliminary culture inoculation

%f no growth, inoculate onto ;< medium

S(0 :

4wabs are not satisfactory

But laryngeal swab from only children can be accepted

>ip swab in a centrifuge tube control. * ml of *+ a#- solution. 4wirl the

swab

!lace the tube at 1 c in a water bath for '3 min

eutralise it with 7+ -"l : centrifuge at 33 rpm for 3 min

%noculate onto the ;< medium

Ur!$e :

"entrifuge early morning clean catch urine

4ediment is ta5en and digestion is carried as mentioned

Then smears are prepared and inoculated onto culture media

.lood :

ml of blood is aseptically collected in 3 ml liuid medium


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%ncubate at 1 c

4mear is made once a wee5 and is subcultured onto ;< slope at $ wee5s

interval

.IOC'EMICAL TESTS :

iacin accumulation C !ositive

itrate reduction C !ositive

!yraEinamidase activity C !ositive

9rease activity C !ositive

"atalase test C egative (heatClabile)

%ron upta5e C egative

a"l tolerance C egative

Tellurite reduction C egative

N!#!$ produ#t!o$ :

The niacin test, which detects niacin (nicotinic acid) in aueous e0tracts of the

culture, is one of the elements in the identification of M. tuberculosis. Almost every strain of M. tuberculosis e0cretes a large amount of niacin

(nicotinic acid) into culture media. iacinCnegative M. tuberculosis strains are very

rare, and very few other mycobacterial species yield positive niacin tests.

The conventional method reuires the use of cyanogen bromide, an e0tremely

haEardous chemical that is banned in most countries, and should be discontinued.

-owever, the niacin test can still be performed, using commercially available testC

strips.

The test must be carried out on pure cultures otherwise it will yield false results.

• Add ' ml of sterile distilled water to the culture.

• Tighten the caps.

• !lace the tubes horiEontally so the fluid covers the entire surface of the medium.

• ;eave for at least 3 minutes.

• Turn the slants upright for minutes to let fluid to drain to the bottom.

• !repare a clean screwCcap tube for each culture tube and mar5 it with the laboratoryregister number.

• emove 3. ml of fluid and transfer it to the clean screwCcap tube.

•

To it add 3. ml of *+ aniline followed by 3. ml of '3+ cyanogen bromide.• "lose the tube immediately.


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• ;eave at room temperature for '$3 minutes, agitating the tube occasionally.

• #bserve the colour of the liuid in the bottom of the tube against a white bac5ground.

The test is negative if no colour develops and positive if a yellow colour appears.

(A) !ositive C Dycobacterium tuberculosis strains

(B) egative D#TT

N!trte Redu#t!o$ :

Ta5e few drops of sterile distilled water in a tube and emulsify a loopful

of colours in the water. Add $ ml of ao and place at 13c in a water bath for $ hours.

Add one drop of '$ dilution of concentrated -"l and sha5e.

Add $ drops of '+ aueous nCnaphtha ethylene diamine

dihydrochloride solution. >evelopment of red colour indicates positive reaction.

=hen reaction is negative, test can be confirmed by addition of Ginc

dust into the tube. >evelopment of red colour confirms negative test. M.kansasii , M.szulgai , M.fortuitum are clinically significant species

which reduce nitrate.

!ositive D.tb H egative C DA"

&lo(#hrt for spe#!me$ pro#ess!$) for Isolt!o$ of M"#o0#ter!


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Commo$l" used L!1u!d med! s"stems to #ulture $d dete#t the )ro(th of

M"#o0#ter!


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Co$trols $d med! used for the 0!o#hem!#l !de$t!f!#t!o$ of M"#o0#ter!

O2er2!e( of #o$2e$t!o$l methods to determ!$e sus#ept!0!l!t" ofM.tuberculosis !soltes to $t!m"#o0#ter!l )e$ts


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ANTITU.ERCULOSIS A%ENTS COMMONLY TESTED A%AINST M.tuberculosis

PRIMARY DRU%S

4treptomycin

%soniaEid

ifampin

?thambutol

!yraEinamide

SECONDARY DRU%S

?thionamide

"apreomycin

"iproflo0acin

#flo0acin

Ianamycin

"ycloserine

ifabutin

Me#h$!sm of #t!o$ of A$t!3T. dru)s


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10/16

Re2!sed RNTCP DOTS Strte)" for T. tretme$t


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MULTI3DRU% RESISTANT TU.ERCULOSIS -MDR3T./ :

D>CTB C atleast resistant to isoniaEid and rifampicin

D>CTB can be caused by

• %ncomplete treatment• %nterrupted treatment• ;ac5 of adeuate antiCTB treatment

The D>CTB strains are resistant to conventional antiCTB treatment

D>CTB is commonly seen in

• -% patients• -omeless populations• %ndividuals living in crowded environments

T?ATD?T BA4?> # >#T4 !;94

• INTENSI4E P'ASE 536 MONT'S

IAADJ"%, #F;#KA"%, "J";#4?%?, ?T-%AD%>?, ?T-ADB9T#;,

!JAG%AD%>?

• CONTINUATION P'ASE 78 MONT'S

#F;#KA"%, "J";#4?%?, ?T-%#AD%>?, ?T-ADB9T#;

E9TENSI4ELY DRU% RESISTANT T. -9DR T./ :

esistance to %soniaEid and ifampicin L resistance to any of the Fluorouinolones L

resistance to any one of the inMectible second line drugs

Treatment

• INTENSE P'ASE -537 mo$ths/

"aperomycin, !A4, Do0iflo0acin, "lofaEimine, ;ineEolid, Amo0icillin / "lavulanate

• CONTINUATION P'ASE -78 mo$ths/

!A4, Do0iflo0acin, %soniaEid, "lofaEimine, ;ineEolid, Amo0icillin / "lavulanate


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ATYPICAL MYCO.ACTERIA

N Inown by several termsO

ontuberculous mycobacteria (TD)

Atypical mycobacteria

Dycobacteria other than tuberculosis ?nvironmental mycobacteriaOall refer to mycobacteria other than

Dycobacterium tuberculosis, its close relatives (M. bovis, M. caprae, M.

africanum, M. pinnipedii, M. canetti), and M. leprae.

N The number of 5nown species currently e0ceeds '3. TD are highly adaptable and can

inhabit hostile environments, including industrial solvents.

Ep!dem!olo)"

N The true international epidemiology of infections due to TD is hard to determine.

N TD are ubiuitous in soil and water.

N Dost TD cause disease in humans only rarely unless some aspect of host defense is

impaired, as in bronchiectasis, or breached, as by inoculation (e.g., liposuction, trauma).

N -umanCtoChuman transmission of TD is not 5nown.

N >isseminated disease denotes significant immune dysfunction (e.g., advanced -%

infection), whereas pulmonary disease, which is much more common, is highly associated

with pulmonary epithelial defects but not with systemic immunodeficiency.

Ru$"o$ Clsss!f!#t!o$

N 4low growing

!hotochromogens, which develop pigments in or after being e0posed to light. ?0amples

include M. kansasii, M. simiae and M. marinum.

4cotochromogens, which become pigmented in dar5ness. ?0amples include M.

scrofulaceum and M. szulgai.

onCchromogens, which includes a group of prevalent opportunistic pathogens called D.

avium comple0 (DA"). #ther e0amples are M. ulcerans, M. xenopi, M. malmoense, M.terrae,M. haemophilum and M. genavense.

N apid growers

%nclude four well recogniEed pathogenic rapidly growing nonCchromogenic species D.

chelonae, M. abscessus, M. fortuitum and M. peregrinum. #ther e0amples cause disease

rarely, such as M. smegmatis and M. flavescens.


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.!o#hem!#l Tests :

N Aryl sulphatase test !ositive in Atypical Dycobacterium

N "atalase pero0idase test >ifferentiates Atypical from Typical. Dost Atypical are strongly

"atalase positive. Tubercle bacilli are wea5ly positive.

N Tubercle bacilli are pero0idase positive not atypical %- resistant strains are negative for

test

;e" .!o#hem!#l re#t!o$s to help d!st!$)u!sh M"#o0#ter! 0elo$)!$) to the

sme M"#o0#ter!l )roup


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Culture :

%f cultures are negative and a positive acidC fast stain,

slowly growing atypical mycobacteria are suspected then a set of inoculated media

should be incubated at a lower temperature (eg, $* P") and both sets incubated for '$

wee5s.

D.tuberculosis D.5ansasii D.gordonae

(!hotochromogen) (4cotochromogen)


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CLSI re#omme$dt!o$s for sus#ept!0!l!t" test!$) of NTM

Tretme$t :


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N DA" infection often reuires multidrug therapy, one is a macrolide (clarithromycin or

aEithromycin), ethambutol, and a rifamycin (rifampin or rifabutin).

N For disseminated nontuberculous mycobacterial disease in -%Cinfected patients, the use

of rifamycins poses special problemsOi.e., rifamycin interactions with protease inhibitors.

N For pulmonary DA" disease, thriceCwee5ly administration of a macrolide, a rifamycin, andethambutol has been successful.

N Therapy is prolonged, generally continuing for '$ months after culture conversionH

typically, a course lasts for at least '7 months. #ther drugs with activity against DA"

organisms include % and aerosoliEed aminoglycosides, fluorouinolones, and clofaEimine.

N D. 5ansasii lung disease is similar to tuberculosis and is also effectively treated with

isoniaEid (33 mg/d), rifampin (633 mg/d), and ethambutol (' mg/5g per day).

N #ther drugs with very highClevel activity against D. 5ansasii include clarithromycin,

fluorouinolones, and aminoglycosides.

N Treatment should continue until cultures have been negative for at least ' year.

N D. 5ansasii infection is easily cured.

N Treatment of the other TD is less well defined, but macrolides and aminoglycosides are

usually effective.

culture tb

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