1. Anaerobic Technique

8/11/2019 1. Anaerobic Technique

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MLT 2324 MedicalMicrobiology 3

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Course Outline

Subject Name : Medical Microbiology 3

Subject Code : MLT2324

Contact Hour/Wee : T!eory " tutorial 3 !our

Lab 2 !our

#$$e$$ment : %inal e&amination : '( )

Mid term : 2()

*ui+ : ,)

#ttendance " -artici.itation : , )


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Learning Objective

3

no0 laboratory tec!niue$

concerning bacteria .at!ogen$

-erorm $erological te$t

&.lain no$ocomial inection

&.lain t!e uality control anda$$urance in microbiology lab


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Topics

No Topic

1 #naerobic tec!niue$

2 #naerobic bacteria

3 e$.iratory inection

4 5a$trointe$tinal inection

, &cretory $y$tem inection

' Central ner6ou$ inection

7 8rinary $y$tem inection

9 Se&ually tran$mitted di$ea$e

No$ocomial inection

M;/#N#/;--

1' merging and re?emerging .at!ogen$ inection$

17 *uality control .rogram$

%inal


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Let$ t!e

learning

Start


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Anaerobic bacteria

Basic culture methods

7

Nora+li 5!adin


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Learning Objectives

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To de$cribe 0!at i$ anaerobic bacteria

To di$cu$$ 0!at i$ reuirement or anaerobic

bacteria culture To li$t a..ro.riate $am.le$ or anaerobic bacteria

e&amination

To no0 t!e correct tec!niue or anaerobic

bacteria !andling To no0 !o0 to inter.ret t!e anaerobic bacteria

e&amination


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What Are Anaerobic Microorganisms

#naerobic

microorgani$m$ are

0ide$.read and6ery im.ortant


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Defining Anaerobes

Facultative anaerobes ? can gro0 int!e .re$ence or ab$ence o o&ygen

>btain energy by bot! re$.iration andermentation

>&ygen not to&ic@ $ome u$e nitrateAN>3

?B or $ul.!ate AS>42?B a$ a terminal

electron acce.tor under anaerobiccondition$

1(


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Strict Anaerobic acteria

Obligate !strict"

anaerobes ? o&ygen i$to&ic to t!e$e

organi$m$@ do not u$e

o&ygen a$ terminal

electron acce.tor

#rc!aea $uc! a$

met!anogen$ andDacteria@ eg Clo$tridia@

Dacteriode$ etc etc

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Culturing of anaerobes nee# special

s$ills

Culture o anaerobe$ i$ e&tremely diicult

becau$e

need to e&clude o&ygen@ $lo0 gro0t! and

com.le& gro0t! reuirement$

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Clinical signs suggesting possible

infection %ith anaerobes

1 %oul $melling di$c!arge

2 ;nection in .ro&imity to a muco$al $urace

3 5a$ in ti$$ue$

4 Negati6e aerobic culture$ o $.ecimen$ , .u$ cell$

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The accepte# specimens for

anaerobic processing are as

follo%s&

Site$

CNS

Dental'(NT

Acceptable

specimen

CS%@ ab$ce$$@ ti$$ue

#b$ce$$@ a$.irate$@

ti$$ue$

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The accepte# specimens for anaerobic

processing are as follo%s&

Local ab$ce$$

)ulmonar*

Nee#le aspirates

Tran$ trac!eala$.irate$@ lung

a$.irate$@ .leural

luid@ ti$$ue@

-rotected bronc!ial

0a$!ing

1,


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The accepte# specimens for anaerobic

processing are as follo%s

#bdominal

8rinary tract

5enital tract

8lcer$/0ound$

>t!er$

#bdominal #b$ce$$

a$.irate@ luid and ti$$ue$

Su.ra.ubic bladder

a$.irate

Culdocente$i$ $.ecimen@

endometrial $0ab$ #$.irate/$0ab .u$ rom dee. .ocet$

or rom under $in la.$ t!at !a6e been

decontaminated


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Sample collection

Collection by needleaspiration ispreferable than swabculture because of

A. Better survival ofpathogen

B. Greater quantity ofspecimen

C. Less contamination withextraneous organismare often achieved

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Sample collection

+f a s%ab must be use#, a - tube s*stem

must be use#

1

st

tube contains swab in ! free C! !ndtube contains "#A$ %pre&reduced

anaerobically sterili'ed culture media(

Specimen shoul# be place# in anaerobic

transport #evice %ith gas mi.ture

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/AND+N0 AND T1ANS)O1T OF

CL+N+CAL S)(C+M(NS

T!e ba$ic .rinci.le$ to remember are

a6oid contamination 0it! t!e normal

microbial lora .rom.t tran$.ort to t!e laboratory

immediate .roce$$ing i$ done

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Transporting

#naerobic tran$.ort tube$ and/or de6ice$ $!ould

al0ay$ be a6ailable at t!e > and

S.ecimen$ $!ould be .laced in lea?.roo container 0it! tig!t itting ca.$

.ro.er label or identiication 0it! date and time o

collection $!ould accom.any all $.ecimen$ $ubmitted

or culture

-ut $am.le$ in room tem.erature 0!ile 0aiting ordeli6ery to t!e laboratory Some anaerobes are $ille#

b* refrigeration2

2(


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Anaerobic Culture Metho#s

1 -roduction o a

6acuum

2 &ygen 0it! ot!er

ga$e$

3 #b$or.tion o >&ygen

by c!emical orbiological met!od$

4 Dy u$ing reducing

agent$

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Obligate Anaerobes nee#s Optimal

Metho#s

Anaerobic

chambers

Obligate

anaerobes can beculture in special

anaerobe

chambers an#

han#le# in

anaerobe hoo#s

Can create vacuum

env2

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Displacement of O.*gen

Dy inert ga$e$ lie

Hydrogen@ Nitrogen@Carbon dio&ide or

Helium

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Absorption of O- b* Chemical metho#

-yrogallol

EC!romium and$ul.!uric acid

E5a$?.a

?a6ailable

commercially

2,


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Absorption of O- b* Chemical metho#

A. A)A*#B+C ,A#

1 Candle Far

? reduce$ >2en6ironment

? only G C>2

ten$ion

2 5a$ -a Far

a -alladium aluminum

coated .ellet$ ? a$ cataly$t to

c!emically reduce$

>2

? react$ 0it! re$idual>2 in t!e .re$ence

o H2 to orm H2>

2'


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5a$ -# F# Candle F#


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* re#ucing agents

T!iglyclolate brot!

>t!er met!od$

obert$on$ Cooed

Meat ACMB brot!

contain$ nutrient brot!

0it! .iece$ o at?reeminced cooed meat o

o& !eart

ACMB brot!


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Anaerobic Glove Chamber

b 0as )a$ envelope

3 generates CO- 4 /- gases

c2 Meth*lene blue strip

3 in#icator

blue !5" O-

%hite !3" O-

II. Anaerobic Glove Chamber

3 close s*stem

3 use# for premature babies

3 e2g2 incubator

III. Roll Tube

3 has a pe#algas ! CO- 4 /- "

%oul# come out

3 place test tube #irectl* to the outlet

3(


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Culture of strict anaerobes

%or culture o $trict anaerobe$ all trace$ o o&ygenmu$t be remo6ed rom medium and $am.le mu$tbe e.t entirely anaerobic during mani.ulation$

&am.le: Met!anogenic arc!aea rom rumen and$e0age treatment .lant$ illed by e6en a briee&.o$ure to >2

Medium u$ually boiled during .re.aration andreducing agent added@ $tored under >2?ree

atmo$.!ere31


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Choosing the Optimal Me#ia

Drot! and $olid media $!ould bot! be inoculated

&am.le

1 anaerobic blood agar .late$ enric!ed 0it!$ub$tance$ $uc! a$ brain?!eart inu$ion@ yea$t

e&tract@ amino acid$@ and 6itamin

2 a $electi6e medium $uc! a$ anamycin?

6ancomycin A=B blood agar or laed blood agar3 brot! $uc! a$ brain !eart inu$ion brot! 0it!

T!iglyclolate or ot!er reducing agent

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Me#ia chosen accor#ing to our

nee#s

T!e c!oice o media de.end$ u.on t!e ty.e o

$.ecimen &am.le

a -rereduced .e.tone?yea$t e&tract?gluco$e brot! ?

or analy$i$ o 6olatile .roduct$ by ga$

b egg yol agar Ior detection o lecit!ina$e acti6ity

oClostridium spp.

c cyclo$erine?ceo&itin?ructo$e agar ACC%#B or

i$olation o Clo$tridium difficilerom $tool

d Dacteroide$ bile e$culin agar or i$olation o t!e

Bacteroides fragilis group. 33


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IDENTIFICATION o

ANAEROBE!-late$ are c!eced at

J 19?24 !our$ or a$ter gro0ing $.ecie$ lie

Cl -erringen$ " Dragili$ & daily thereafter up to

J ,?7 day$ or $lo0ly gro0ing $.ecie$ lie #ctinomyce$@ ubacterium " -ro.ionibacterium

Genus i$ determined by

? gram $tain@ cellular mor.!ology@ 5a$?liuid

c!romatogra.!y $pecies determination i$ ba$ed on ermentation o

$ugar$ " ot!er bioc!emical determination

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+#entification of Anaerobes is Comple.

;dentiication o anaerobe$ i$ !ig!ly com.le&@ and

may u$e dierent identiication $y$tem$

-artial identiication i$ oten t!e goal %or e&am.le@

t!ere are $i& $.ecie$ o t!e Bactericides genu$

t!at may be identiied a$ t!e Bactericides fragilis

grou. rat!er t!an identiied indi6idually

>rgani$m$ are identiied by t!eir colonial and

micro$co.ic mor.!ology@ gro0t! on $electi6e

media@ o&ygen tolerance@ and bioc!emical

c!aracteri$tic$ and #ST

#-; 2(# it 3,


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A#vance# +#entification Techni6ue

Matri.3Assiste# Laser Desorption +oni7ation8Time of Flight Mass Spectrometr* MALD+3TOF

9:S3-;S r1NA intergenic spacer !+TS"

se6uences anal*sis

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1. Anaerobic Technique

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