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MLT 2324 MedicalMicrobiology 3
1
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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$$ue$
4 Negati6e aerobic culture$ o $.ecimen$ , .u$ cell$
13
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The accepte# specimens for
anaerobic processing are as
follo%s&
Site$
CNS
Dental'(NT
Acceptable
specimen
CS%@ ab$ce$$@ 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
1
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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
34
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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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