MLT 2324 Medical

Microbiology 3
1
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 : , )

Learning Objective
3

/no0 laboratory tec!ni1ue$

concerning bacteria .at!ogen$

-er2orm $erological te$t

3&.lain no$ocomial in2ection

3&.lain t!e 1uality control and

a$$urance in microbiology lab
Topics
No Topic
1 #naerobic tec!ni1ue$
2 #naerobic bacteria
3 4e$.iratory in2ection
4 5a$trointe$tinal in2ection
, 3&cretory $y$tem in2ection
' Central ner6ou$ in2ection
7 8rinary $y$tem in2ection
9 Se&ually tran$mitted di$ea$e
: No$ocomial in2ection
M;<
Term
Topics
No Topic
1( Cut and .u$
11 Mycological diagno$tic Laboratory
12 Sa2ety in 6irology laboratory
13 Collection and tran$.ort o2 6iral $am.le
14 =irology diagno$tic laboratory
1, Serological te$t #S>/#N#/;--
1' 3merging and re?emerging .at!ogen$ in2ection$
17 *uality control .rogram$
%inal
Let$ t!e
learning
Start
Anaerobic bacteria
Basic culture methods
7
Nora+li 5!adin
Learning Objectives
9

To de$cribe 0!at i$ anaerobic bacteria

To di$cu$$ 0!at i$ re1uirement 2or anaerobic

bacteria culture

To li$t a..ro.riate $am.le$ 2or anaerobic bacteria

e&amination

To no0 t!e correct tec!ni1ue 2or anaerobic

bacteria !andling

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

e&amination
What Are Anaerobic Microorganisms

#naerobic
microorgani$m$ are
0ide$.read and
6ery im.ortant

<o not re1uire

o&ygen 2or gro0t! ?
o2ten e&tremely to&ic
:
Defining Anaerobes

Facultative anaerobes ? can gro0 in

t!e .re$ence or ab$ence o2 o&ygen

>btain energy by bot! re$.iration and

2ermentation

>&ygen not to&ic@ $ome u$e nitrate

AN>
3
?
B or $ul.!ate AS>
4
2?
B a$ a terminal
electron acce.tor under anaerobic
condition$
1(
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.torC

#rc!aea $uc! a$
met!anogen$ and
Dacteria@ eCg Clo$tridia@
Dacteriode$ etcC etcC
11
Culturing of anaerobes nee# special
s$ills

Culture o2 anaerobe$ i$ e&tremely di22icult

becau$e

need to e&clude o&ygen@

$lo0 gro0t! and

com.le& gro0t! re1uirement$

12
Clinical signs suggesting possible
infection %ith anaerobes

1C %oul $melling di$c!arge

2C ;n2ection in .ro&imity to a muco$al $ur2ace

3C 5a$ in ti$$ue$

4C Negati6e aerobic culture$ o2 $.ecimen$

,C .u$ cell$C
13
The accepte# specimens for
anaerobic processing are as
follo%s&
Site$

CNS

Dental'(NT

Acceptable
specimen

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

#b$ce$$@ a$.irate$@
ti$$ue$
14
The accepte# specimens for anaerobic
processing are as follo%s&

Local ab$ce$$

)ulmonar*

Nee#le aspirates

Tran$ trac!eal
a$.irate$@ lung
a$.irate$@ .leural
2luid@ ti$$ue@

-rotected bronc!ial
0a$!ing
1,
The accepte# specimens for anaerobic
processing are as follo%s

#bdominal

8rinary tract

5enital tract

8lcer$/0ound$

>t!er$

#bdominal #b$ce$$
a$.irate@ 2luid and ti$$ue$

Su.ra.ubic bladder
a$.irate

Culdocente$i$ $.ecimen@
endometrial $0ab$

#$.irate/$0ab .u$ 2rom dee. .ocet$

or 2rom under $in 2la.$ t!at !a6e been
decontaminated

<ee. ti$$ue or bone le$ion$@ blood@

bone marro0@ $yno6ial 2luid@

ti$$ue$
1'
Sample collection
Collection by needle
aspiration is
preferable than swab
culture because of
A. Better survival of
pathogen
B. Greater quantity of
specimen
C. Less contamination with
extraneous organism
are often achieved
17
Sample collection
+f a s%ab must be use#, a - tube s*stem
must be use#
1
st
tube contains swab in ! free C!
!
nd
tube contains "#A$ %pre&reduced
anaerobically sterili'ed culture media(
Specimen shoul# be place# in anaerobic
transport #evice %ith gas mi.ture
19
/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 2lora

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

immediate .roce$$ing i$ doneC

1:
Transporting

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

al0ay$ be a6ailable at t!e >4 and 34C

S.ecimen$ $!ould be .laced

in lea?.roo2 container 0it! tig!t 2itting ca.$C

.ro.er label 2or identi2ication 0it! date and time o2

collection $!ould accom.any all $.ecimen$ $ubmitted
2or cultureC

-ut $am.le$ in room tem.erature 0!ile 0aiting 2or

deli6ery to t!e laboratoryC Some anaerobes are $ille#
b* refrigeration2
2(
Anaerobic Culture Metho#s
1C -roduction o2 a
6acuum
2C <i$.lacement o2
>&ygen 0it! ot!er
ga$e$
3C #b$or.tion o2 >&ygen
by c!emical or
biological met!od$
4C Dy u$ing reducing
agent$
21
P. aeruginosa Strict aerobe
Enterococcus Facultative
Grows aerobic or anaerobic.
Bacteriodes fragilis
22
Obligate Anaerobes nee#s Optimal
Metho#s

Anaerobic
chambers

Obligate
anaerobes can be
culture in special
anaerobe
chambers an#
han#le# in
anaerobe hoo#s

Can create vacuum

env2

2
23
Displacement of O.*gen

Dy inert ga$e$ lie

Hydrogen@ Nitrogen@
Carbon dio&ide or
Helium
24
Absorption of O- b* Chemical metho#

-yrogallol

EC!romium and
$ul.!uric acid

E5a$?.a

?a6ailable
commercially
2,
Absorption of O- b* Chemical metho#
A. A)A*#B+C ,A#
1C Candle Far
? reduce$ >2
en6ironment
? only G C>2
ten$ion


2C 5a$ -a Far
aC -alladium aluminum
coated .ellet$
? a$ cataly$t to
c!emically reduce$
>2
? react$ 0it! re$idual
>2 in t!e .re$ence
o2 H2 to 2orm H
2
>
2'
5a$ -#/ F#4
Candle F#4
* re#ucing agents

T!iglyclolate brot!
>t!er met!od$

4obert$onH$ Cooed
Meat A4CMB brot!

contain$ nutrient brot!

0it! .iece$ o2 2at?2ree
minced cooed meat o2
o& !eartC
A4CMB brot!
Anaerobic Glove Chamber
bC 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#al gas ! CO- 4 /- "
%oul# come out
3 place test tube #irectl* to the outlet
3(
Culture of strict anaerobes

%or culture o2 $trict anaerobe$ all trace$ o2 o&ygen

mu$t be remo6ed 2rom medium and $am.le mu$t
be e.t entirely anaerobic during mani.ulation$

3&am.le: Met!anogenic arc!aea 2rom rumen and

$e0age treatment .lant$ illed by e6en a brie2
e&.o$ure to >
2

Medium u$ually boiled during .re.aration and

reducing agent added@ $tored under >
2
?2ree
atmo$.!ere
31
Choosing the Optimal Me#ia

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

3&am.le
1C anaerobic blood agar .late$ enric!ed 0it!
$ub$tance$ $uc! a$ brain?!eart in2u$ion@ yea$t
e&tract@ amino acid$@ and 6itamin /C
2C a $electi6e medium $uc! a$ anamycin?
6ancomycin A/=B blood agar or laed blood agarC
3C brot! $uc! a$ brain !eart in2u$ion brot! 0it!
T!iglyclolate or ot!er reducing agentC
32
Me#ia chosen accor#ing to our
nee#s

T!e c!oice o2 media de.end$ u.on t!e ty.e o2

$.ecimenC 3&am.le
aC -rereduced .e.tone?yea$t e&tract?gluco$e brot! ?
2or analy$i$ o2 6olatile .roduct$ by ga$C
bC egg yol agar I2or detection o2 lecit!ina$e acti6ity
o2Clostridiumspp.
cC cyclo$erine?ce2o&itin?2ructo$e agar ACC%#B 2or
i$olation o2 Clo$tridium difficile 2rom $tool
dC Dacteroide$ bile e$culin agar 2or i$olation o2 t!e
Bacteroidesfragilisgroup.
33
IDENTIFICATION o
ANAEROBE!
-late$ are c!eced at
J 19?24 !our$ 2or 2a$ter gro0ing $.ecie$ lie
ClC -er2ringen$ " DC2ragili$ &dailythereafterupto

J ,?7 day$ 2or $lo0ly gro0ing $.ecie$ lie
#ctinomyce$@ 3ubacterium " -ro.ionibacterium
Genus i$ determined by
? gram $tain@ cellular mor.!ology@ 5a$?li1uid
c!romatogra.!y
$pecies determination i$ ba$ed on 2ermentation o2
$ugar$ " ot!er bioc!emical determination
34
+#entification of Anaerobes is Comple.

;denti2ication o2 anaerobe$ i$ !ig!ly com.le&@ and

may u$e di22erent identi2ication $y$tem$C

-artial identi2ication i$ o2ten t!e goalC %or e&am.le@

t!ere are $i& $.ecie$ o2 t!e Bactericidesgenu$
t!at may be identi2ied a$ t!e Bactericidesfragilis
grou. rat!er t!an identi2ied indi6iduallyC

>rgani$m$ are identi2ied by t!eir colonial and

micro$co.ic mor.!ology@ gro0t! on $electi6e
media@ o&ygen tolerance@ and bioc!emical
c!aracteri$tic$ and #STC

#-; 2(# it
3,
A#vance# +#entification Techni6ue

Matri.3Assiste# Laser Desorption +oni7ation8

Time of Flight Mass Spectrometr* MALD+3TOF

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

se6uences anal*sis