Introduction to

Microbiology Laboratory Practice

Retno Kadarsih S

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Introduction

Respiratory tract diseases and pathogens

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Specimens
 Upper Respiratory Tract Infections
◦ Pharyngitis :
 Group A β hemolytic streptococci
 Acute cases
 Specimen : Posterion pharynx
 Culture and direct antigen detection tests
 Bordetella pertussis :
 fragile m.o  immediate culture
 Specimen : posterior nasopharynx mucus
 Corynebacterium diptheriae :
 swab of posterior nares and posterior pharynx
 Viral :
 Nasal Swab/washings  viral transport medium

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◦ Laryngitis
 Primary caused by viruses
 Specimen : swab/nasal washing with viral transport
medium
◦ Epiglottitis
 Culture not indicated
 Touching the inflamed epiglottis may precipitate
complete obstruction of the airway
 Specimen of choice : blood culture
◦ Sinusitis
 Needle aspirate of sinuses after decontamination of the
nasal cavity
 No specimen other than an aspirate is recommended

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 Lower Respiratory Tract Infections
◦ Pneumonia
 Specimen :
 Sputum
 Aspirate/lung biopsy
 Bronchial lavage
 Transtracheal aspirate
 Blood
 In bacterial pneumonia  sputum may not be
the specimen of choice for determining
etiologic agent

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 Specimen handling
 Type : swab, sputum or blood ?
 How much : adult VS pediatric ?
 How many : 1 or more ?
 When : morning VS spot ?
 Colleting : proper specimen ?
 Labeling : critical item to be added ?
 Transportation : time needed, packaging?
 Storage before handling in laboratory :
RT vs. COOL vs. immediate handling ?

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 Throat swab
• Using a tongue blade
to hold the tongue
down, look at the back
of the throat and the
tonsillar area for
localized areas of
inflammation and
exudates (theses
areas are the most
productive for
producing cultures of
the etiologic agents of
acute pharyngitis)

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 Label the swab container with patient
identification data, including the time of
collection
 Transport the swab to the laboratory as
soon as possible (if transport is to be
delayed beyond 1 hour, refrigerate the
swab)

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SPUTUM – Collecting
 Good Container
 Sterile
(except for tuberculosis)
 Clean
 Widemouth
 Clear
 Tight screwcap
 Leak proof

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SPUTUM – how to collect it correctly
Explain the patient how to
collect sputum properly
Patient should be given time
to produce bronchial
sputum from deep in the
chest
Never collect sputum
specimens inside the clinic
or laboratory or in a closed
circulation room
Collect the sputum outdoor
and far from other people
Put the container to the
lower lip, collect the
sputum and close the
container immediately

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 Sputum quality

Mucoid Purulent Bloody Sputum Saliva

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 SPUTUM – Labeling
 Container labeling  Patient’s data : name, age, sex
 Time and date of specimen
collection, Specimen No,
Physician’s name
 A request form must
accompany each specimen.
 The information on the form
must exactly match the
information on the container.
 Always label the container on
the side, never on the lid.
 Using an indelible marking
pen, write the name of the TB
suspect, the date of collection
and health institution

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 SPUTUM – Labeling
Request Form
Patient’s Data :
name, age, sex, address
Physician :
name, address, hospital, phone no,
Specimen:
Special procedure
Date and time of specimen collection
Antibiotic administrations
Working Diagnosis
Type of laboratory examination
Other relevan data
post operation,
imunodefisiency status
Special case : HIV, urgent case, etc

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SPUTUM – Keterlambatan pengiriman

 Sebaiknya dikirim secepat mungkin ke

laboratoriumsecepat mungkin
 Bila terjadi keterlambatan pengiriman
(>1–2 jam setelah pengambilan sputum)
simpan pada 4°C

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SPUTUM - catatan
 Untuk pemeriksaan infeksi saluran nafas bawah
bakterialis  pengambilan spesimen cukup 1 x A
single
 Untuk pemeriksaan Mycobacteria atau Jamur 
pengambilan sputum pagi hari 3 X berturut-turut
(untuk TB dapat dilakukan pengambilan sputum SPS
untuk mempermudah)
 Pemeriksaan anaerob pada sputum TIDAK
DILAKUKAN
 Deteksi langsung atau uji molekuler dari sputum
dapat dilakukan pada beberapa pemeriksaan tertentu
dan pada laboratorium yang kompeten

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Making a smear slide for staining …
Always use new slides
especially when preparing
AFB smears. Never reuse
sputum smear slides for TB
work!!!

They should be cleaned with

alcohol and wiped dry…or
passed briskly through a
flame. (This will remove any
residue of oil that could interfere
with staining.)

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  Properly identify
each slide with
patient’s name, date
and specimen
number
 Use a lead pencil to
write these
numbers on the
frosted end of the
slide
 Make a 3x2cm2 oval
zone in the middle
of the slide

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For AFB staining :
Wet slides can create aerosols if disturbed.
Place them in a protected area where they can air dry for 15-30 minutes.
Avoid direct sun drying
DO NOT flame them to induce drying. This can also produce aerosols.

For other staining : Flaming slides direclty is allowed

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If it is too thin, the If it is too thick, the
specimen may yield smear may wash off the
false negative slide during the staining
results. procedure and also may
yield false negative
results

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 Fixing the smear by heating
 When dried, fix the slides
using the blue flame of a
Bunsen burner.

 With forceps, pass the slide

briefly through the flame 3
times, smear side up

 Heat fixation ensures that the

sputum will stick to the glass
slide

 Excessive heating could

damage bacterial structure

 Under heated fixing will make

insufficiently fixed and parts
of the material will be washed
off during staining

 Over and under heated fixing

will make false negative
results
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Gram staining
 To examine sputum microorganism
microscopically
 To evaluate a good specimen ( SPUTUM
and NOT SALIVA)

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 Sputum Gram Stain Good Quality
1. PMN > 25 (40X10
magnitude field)

2. No epithelial cells

3. Single microorganism
found

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 Sputum contamination with mouth flora

1. Sel epitel > 25

2. Polymorphonuclear (PMN) < 10

pada pembesaran 40x

3. Ditemukan beragam jenis

mikroorganisme(Bartlett, 1987)

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Sputum Gram Stain Unacceptable

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Ziehl Neelsen Staining
 Staining reagents,
 Clock/Timer
 Forceps
 Cotton/alcohol
holder or bunsen
burner
 Tray containing fixed
slides to be stained.
 A slide rack should
be placed in the
sink.

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All reagents must have expiration dates. Discard all expired dates

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.

 Using forceps, place slides

on a staining rack with the
smear side up.

 Place all slides in uniform

orientation.

 Slides must not touch each

other. This avoids the
possibility of stain running
over and causing cross-
contamination.

 Include a + and - control slide

daily for quality control
purposes.

 Never stain more than 12

slides at a time

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 Cover the entire surface of each
slide with carbol fuchsin.

 Using a Bunsen burner or cotton

and alcohol flame, gently heat the
slides until vapor rises.

 Do not allow them to boil or dry.

Boiling will alter the shape of the
TB bacilli and could result in a
false negative reading.

 Allow the stain to remain on the

slides for 5 minutes.
Maintain heat throughout this
period.

Adequate time is required for the

carbol fuchsin to penetrate and
stain the cell wall.

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 Rinse slides again
carefully with water and
tilt each slide to remove
excess water.

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 Cover each slide with
decolorizing solution such
as acid alcohol.

 Leave this on the slides

until no remaining carbol
fuchsin solution out of the
slides for 2 seconds (or 3
minutes maximum)

 If under-decolorized,
sputum contents other
than the TB bacilli may
remain stained. This could
lead to a false positive
result.

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 Rinse slides again
carefully with water and
tilt each slide to remove
excess water.

 If the slide is still pink,

an additional amount of
decolorizing solution
can be reapplied for 1 to
3 minutes.

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  counter-stain with
methylene blue
for 1-2 minutes

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 Rinse slides again carefully
with water and tilt each slide
to remove excess water

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 Finally, tilt each slide and
place in a slide block to air
dry
 DO NOT blot
 After smears are stained,
clean off the back of each
slide if necessary with some
alcohol on a paper towel
 Do not examine slides until
they are thoroughly dried.
 Cover the stained slides to
protect them from sunlight
which can fade acid-fast
bacilli.

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Good stained smear

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Bad Smears Too small

Too thick

Small/
Slough-off

Large/uneven

Slough-off

Large/uneven
poor destained

Too thin

Bad smearing
Uneven/
Poor
decolorized
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 Before examining the
next slide, wipe the
immersion lens with
lens paper. This will
protect from carry
over of organisms
that could create a
false positive
reading.

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 Use the 40X objective to
focus and determine a
suitable reading area of the
slide.

 Place a drop of immersion

oil on the stained smear.
Let the drop fall freely onto
the slide.

 Never touch the slide

with the oil applicator.
This could lead to carry
over of AFB in the
immersion oil to the next
slide.

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 Turn the nosepiece to bring the 100X objective
into place.
 Now, gently lower the 100X objective.
 It should barely touch the oil. Never allow the
lens to touch the slide. This can damage the
lens and possibly break the slide.
 While looking through the eye-piece, adjust the
immersion
lens slowly and focus until the image on the
smear appears.
 To fine focus, turn the fine adjustment knob
carefully.

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 Polimorphonuclear cells
and other bacterias
appear as counterstain
color (blue)
Acid-fast bacilli (AFB)
typically appear as slender,
rod-shaped bacilli but may
appear curved or bent and
retain the first stain color
(red)

 Ensure to have a good specimen : Sputum not saliva

 PMN >25 and Epithelial cell < 10 per 100 fields
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 At least 100 microscopic
fields should be examined
before reporting as a
negative smear. Examining
fewer than 100 fields may
cause a false negative
report. Fewer than 100
fields can be read if the
slide is positive for AFB.
 Read systematically to
avoid overlap, moving the
slide lengthwise so that the
next field to the right can
then be examined.

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Reporting
The WHO and IUATLD recommended method for reporting
results is as follows:

Negative:
Report: “Negative for acid-fast bacilli” where no organisms
have
been observed in 100 fields.

Positive:
Report: “Positive for acid-fast bacilli.”
Provide AFB quantization.

The number of AFB found is an indication of the degree of

infectivity as well as severity of disease. Results must be
quantitated.
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Recording & Reporting by
WHO/IUATLD recommended method
Acid Fast Bacilli (AFB) appear red or pink colored rod and the
background is stained in blue colour

More than 10 AFB per field in 20 fields ( +++ )

1 – 10 AFB per field in 50 fields ( ++ )
10 – 99 AFB per 100 fields (+)
1 – 9 AFB per 100 fields Exact number
Request repeat specimen
No AFB found in at least 100 fields (-)

(Never report as M. tuberculosis by smear result only)

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 After completing the
reading, remove the oil from
the slide with organic
solvent.

 When dried, place the slides

carefully in a slide storage
container for transfer to a
referral laboratory for quality
control. This must be done
on a regular basis as
determined by the National
Tuberculosis Programme.

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 False Positive
 Errors in specimen handling or recording information
 Re-use of containers or positive slides
 Unfiltered fuchsin
 Contaminated immersion oil
 Inadequate decolorization

 If a False positive result is reported, a patient will be placed on

treatment unnecessarily.

 If it is a Follow-up, treatment is lengthened.

Valuable medication is wasted and, sadly
It can cause emotional trauma to patients and their families.
Patients may lose confidence in the program itself.

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 False Positive
 Errors in specimen handling or recording information
 Re-use of containers or positive slides
 Unfiltered fuchsin
 Contaminated immersion oil
 Inadequate decolorization

 If a False positive result is reported, a patient will be placed on

treatment unnecessarily.

 If it is a Follow-up, treatment is lengthened.

Valuable medication is wasted and, sadly
It can cause emotional trauma to patients and their families.
Patients may lose confidence in the program itself.

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 False Negative
 Errors in specimen handling or recording
information
 Poor quality sputum
 Excessive decolorization
 Reading less than 100 microscopic fields

A false negative can result a catastrophic set of

events
A patient with TB is not treated
If the sample was a follow-up specimen,
the intensive phase of therapy would not be extended
causing inadequate treatment.
This can lead to additional suffering, spread of TB to family
and community, and even end in patient death.

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Other respiratory tract disease’s
pathogens

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 based on haemolytic activity

Classification aerobic Streptococcus species

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Alpha-haemolytic Streptococci
Colonies are surrounded by an area of partial haemolysis and
a green-brown color (reduced haemoglobin)
– Very common in the respiratory tract as normal flora
– Also called S. viridans
– No inhibition with Optochin disk

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Streptococcus pneumoniae
(Pneumococcus)
The optochin (ethyl
hydrocupreine) test is a
presumptive
S. pneumoniae is an
optochin sensitive
Streptococcus,
a zone of inhibition will
develop around the disk
where the
bacteria have been lysed.

This zone is typically >14mm

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Beta-haemolytic Streptococci
Colonies are surrounded by a zone of complete
haemolysis with decolorization of haemoglobin
- BACITRACIN TEST
- Group A Beta-haemolytic Streptococci known as
S. pyogenes (left) is sensitive to bacitrasin disk
a zone of inhibition will develop around the disk

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Non-haemolytic (Gamma) Streptococci

- Colonies show neither typical

alpha- nor beta-haemolysis.

- There maybe, however,

slight discoloration in the
medium

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Classification aerobic Streptococcus
species based on antigenic activity :

Lancefield grouping of beta hemolytic streptococci

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Lancefield grouping of
beta hemolytic streptococci
 Slide agglutination
test
 Grouping according
to their surface
carbohydrate
antigens

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Identification and classification of
aerobic Staphylococcus species

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Staphylococcus aureus
Is carried in the nose of 40 % or more of healthy people
- Produce yellow to cream or occasionally white on blood agar
- Gram positive cocci of uniform size, that occur characteristically
in groups but also singly an in pairs

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Staphylococcus epidermidis

Colonies are usually non-haemolytic

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Mannitol Salt Agar (MSA)
MSA is both a selective and an indicator medium, contains 1%
mannitol, 7.5% NaCl and 0.0025% phenol red in nutrient agar.

Most strain of S. aureus ferment mannitol and so form colonies

surrounded by yellow zones due to acid production, whilst most
other staphylococci fail to ferment mannitol and form colonies
with red or purple zones

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 Coagulase Test for Staphylococci

Coagulase producing strains of S.aureus

- Form a clot when grown in plasma
- The free coagulase turns citrated plasma into firm gel
and bound coagulase (clumping factor) which
agglutinates the cocci.
- the best single test to identify a Staphylococcus
as belonging to the pathogenic species, S.aureus.
- The only non-aureus Staphylococci to produce
coagulase are the animal parasites, S. intermedius
and S. hyicus, rarely found in man, whilst most other
S. aureus failing to produce coagulase are very rare.

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Identification and classification of
Haemophillus species

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 HaemophilusMicroscopy :
Satellite growth of 

Haemophilus species small, non-motile, Gram

around a streak of negative rods or
coccobacili, long thread-
S. aureus like forms from CSF

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  H.influenzae growth
in the presence of
both X and V factors
 Haemophilus sp can
growth in the
presence of only X
or V factor in the
nutrient agar
-

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 X factor or hemin is provided by the lysed sheep
erythrocytes surrounding the staphylococcus streak,
while the staphylococci provide the V factor of NAD
(nicotinamide adenine dinucleotide).

 These factor enable the tiny dew-drop colonies of

haemophilus to grow adjacent to staphylococcus
streak.

 This test provide a presumptive identification of

Haemophilus species.

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 Haemophilus influenzae is formally
identified by demonstrating the
dependence of its growth on a supply of
both X and V factors.

 This is usually done in a disk test wherein

the presence of absence of growth is
observed around paper disk impregnated
with X factor alone,V factor alone, or X
and V factor placed on a nutrient agar
deficient in both X and V factors

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Identification of
Klebsiella pneumoniae

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 Klebsiella pneumoniae

Klebsiella produce large and usually

mucoid colonies when cultured on
blood agar

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Identification of Corynebacterium
diphteriae

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 Corynebacterium diphteriae
Neisser Stains
 to detect Babes-Ernest granules in diphtheriae

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The colonies are grey-
black and are
surrounded by a dark
brown halo. The halo is
due to the formation
of hydrogen sulphide
from cystine (cystinase
activity) on tellurite
agar

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Identification and classification of
Mycobacteria

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 Microscopy :
 Is non-sporing, non-
capsulated,straight or
slightly curved
 rod measuring.
 - The organism stains
red and is acid fast
(AFB)

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Uji Niasin

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 Uji Niasin ………

Dasar reaksi :

Semua Mycobacterium memproduksi asam nikotinat.

M. tuberculosis; M. Chelonae dan M. simiae tidak

memetabolisme asam nikotinat lebih lanjut sehingga
asam tersebut terakumulasi di media

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 Uji Niasin ………
Hasil Positif :
Berwarna kuning apabila kedalam tabung yang ditumbuhi
bakteri M. tuberculosis dituangi dengan cairan Niasin (KCN
1% dan Chloramida T 5 % dalam jumlah yang sama)
dalam waktu 3 – 5 menit

Arti :
bakteri yang diteliti adalah M. tuberkulosis patogen
jenis human

Hasil Negatif :
Warna tidak berubah setelah 3 – 5 menit.

Arti : bakteri yang diteliti bukan M. tuberculosis atau

M. tuberculosis yang telah berkurang patogenesisnya

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 Uji Peroksidasa

Dasar reaksi :

Sama dengan Katalasa.

Ensim peroksidasa selain dapat memisahkan H2O2 pun dapat

memisahkan peroksidasa lain sehingga umumnya percobaan
Ini lebih peka dibandingkan dengan percobaan katalasa

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 Uji peroksidasa ….
Hasil Positif :
Jika setelah 10 menit bakteri M. tuberculosis berubah warna
menjadi tengguli sampai hitam setelah dimasukkan campuran
larutan penyangga asetat, katekol dan H2O2 3 % sama
banyak

Arti :
bakteri yang diteliti mempunyai ensim peroksidasa / katalasa
adalah M. tuberkulosis yang sensitif terhadap INH

Hasil Negatif :
Jika setelah 10 menit tidak terjadi perubahan warna.

Arti : bakteri M. tuberculosis resisten terhadap INH dan

kemungkinan akan berkurang virulensinya
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Identification of yeats and molds

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Jamur ragi (kamir)
 Bersel tunggal berbentuk bulat lonjong

 Mempunyai dinding sel yang tebal

 Berkembang biak dengan tunas

 Dapat diisolasi dari kulit, mulut,

mukosa vagina, sputum

 Pertumbuhan  3 hari

 Misal :
A. berkapsul: Cryptococcus
B. tidak berkapsul : Candida

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 Tersusun dari benang sel yang memanjang (Hifa)
 Ada 2 macam Hifa :
A. Bersepta
 Hifa menjadi multiseluler dengan masing-masing inti

 Misal : Penicillium

B. Tidak bersepta
 Hifa berinti banyak tanpa sekat

 Misal : Mucor, Aspergillus

Pertumbuhan jamur membentuk suatu massa

yang tersusun dari anyaman-anyaman hifa : (mycellium)

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Aspergillus

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Drug Susceptibility Testing for
M. tuberculosis

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Proportional Method

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 INH INH Amikacin 1/100 Control Control
0.2 μg/ml 0.1 μg/ml 5 μg/ml
Drug
Susceptibility
Culture Plate Cut off conc No Antibiotic No Antibiotic

(DSCP) RIF RIF INH INH INH

0.2 μg/ml 0.1 μg/ml 2.0 μg/ml 1.0 μg/ml 0.5 μg/ml

(Cut off conc)

PAS RIF RIF RIF RIF

1 μg/ml 5.0 μg/ml 2.0 μg/ml 1.0 μg/ml 0.5 μg/ml

Cut off conc Cut off conc

ETB ETB ETB ETB ETB

20 μg/ml 10 μg/ml 5.0 μg/ml 2.0 μg/ml 1.0 μg/ml

Cut off con

STR STR STR

STR STR
5.0 μg/ml 2.O μg/ml 1.P μg/ml
20 μg/ml 10 μg/ml

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Liquid-based methods :
MGIT 960

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Molecular-based diagnostics of
respiratory tract infections

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Polymerase Chain Reaction
PRINCIPLE
1. dNTP
2. PCR buffer
DNA rDNA
3. Taq Pol.
4. Primers
extraction

Physical
rupture tech.
Bacterial cell DNA

Denaturation step
1 cycle Annealing step DNA Thermal-cycler
Extension step
2n
(n = number of cycle)

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Spoligotyping
DR locus

BCG

H37Rv

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PCR- RFLP

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PCR-RFLP of Mycobacterias

DNA size marker M., tuberculosisr Mycobacteria sp

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 Spoligotyping :
Direct Repeat Unique Spacer 1 Unique Spacer 2 Unique Spacer 3 Unique Spacer 4

PCR primers

Example of possible PCR

products

PCR products hybridized to a

prefabricated membrane with the
sequence of each unique spacer
covalently bonded to it in columns.
Unique Spacer: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 etc

Isolate #1
Isolate #2
etc

Excess PCR product removed,

autoradiography film exposed
and developed.

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Reverse Line probe Hybridization

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PCR - Hybridization

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Myc
TB

w1
w2
w3
w4
w5
M1
M2
M3
M4
M5

DNA Size Marker Strip type Blotter type

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What is Genotype® MTBDRplus
assay
 PCR-hybridization based rapid detection method
 Short turn around time ( O’Brien – Cape Town 2008)
◦ TAT from specimen collection to receipt at lab was 2 days (range 0-15
days)
◦ TAT of MGIT culture and DST was median of 41 days (range 21 to 69
days)
 Easy to perform
 Allow to detect mutation genes involved in both Rifampicin
and INH resistance M. tuberculosis strains which major
antitubercular drugs in MDR-TB
 Another similar kit can detect only mutation genes in
Rifampicin Resistance M. tuberculosis strains

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 Conjugated control (CC)
 Amplification control (AC)
 M. tuberculosis complex (TUB)
 rpoB Locus Control
 rpoB wild type probe 1 (rpoB WT1)
 rpoB wild type probe 2 (rpoB WT2)
 rpoB wild type probe 3 (rpoB WT3)
 rpoB wild type probe 4 (rpoB WT4)
 rpoB wild type probe 5 (rpoB WT5)
 rpoB wild type probe 6 (rpoB WT6)

Specific Probes
 rpoB wild type probe 7 (rpoB WT7)
 rpoB wild type probe 8 (rpoB WT8)
 rpoB mutation probe 1 (rpoB MUT1)


rpoB mutation probe 2A (rpoB MUT2A)
rpoB mutation probe 2B (rpoB MUT2B)
in MTBDRplus
 rpoB mutation probe 3 (rpoB MUT3)
 katG Locus Control
 katG wild type probe (katG WT)
 katG mutation probe 1 (katG MUT1)
 katG mutation probe 2 (katG MUT 2)
 inhA Locus Control
 Inh wild type probe 1 (inhA WT1)
 Inh wild type probe 2 (inhA WT2)
 Inh mutation probe 1 (inhA MUT1)
 Inh mutation probe 2 (inhA MUT2)
 Inh mutation probe 3A (inhA MUT3A)
 Inh mutation probe 3B (inhA MUT3B)
 Colored marker UNISBA APRIL 2011
rpoB

Wild Type (WT1 – WT8) Mutan (MUT)

 Codon 505 – 533  MUT 1 : codon D516V
 MUT2A : codon D526Y
 MUT2B : codon H526Y
 MUT 3 : codon S531L

Specific probes
UNISBA APRIL 2011
katG

inhA

Wild Type wild-type probes

 Codon 315  WT1 position 15
 WT2 position 16
Mutan (MUT) Mutan ( MUT)
 katGMUT1 AGC-ACC (S315T1)  MUT1 : C T at position 15
 katGMUT 2 AGC-ACA  MUT2 : A  G at position 16
(S315T2)  MUT3B : T C at position 8
 MUT3A : T A at position 8

Specific probes
UNISBA APRIL 2011
Detection of H5N1

UNISBA APRIL 2011

Prosedur pemeriksaan
Bahan pemeriksaan
 Throat swab
 Nasal swab/wash
 Nasopharingeal swab/aspirate
 Bronchoalveolar lavage

UNISBA APRIL 2011

 Conventional PCR

Primer Principle of Conventional PCR

Template
Primer and Real-time PCR

PCR

Electrophoresis analysis

Positive Sample
PC NC #1 #2 #3 PC : Positive control
band NC : Negative control

None-specific
bands
g UNISBA APRIL 2011
Judge Neg Pos Neg
 1 kb Ladder 1 kb Ladder

480 NS 054 TS
Mutiara

481 TS 068 BS
Gel: 07-12-05

Maya
482 TS 483 TS

RNA Std 1 484 NS

Firdaus

RNA Std 2 485 TS

UNISBA APRIL 2011

RNA Std 3 486 TS

RNA Std 4 052 TS

Run: 07-12-05, BioRad PCT100

RNA Std 5 053 NS

Herdi Setiawan

NTC 476 TS

477 NS
In Juju

478 P

479 TS
Vervanya

306 bp
452 bp
306 bp
452 bp
 Probe

1) Denature
Primer F Q
Fluorescence Quencher

F Q
2) Annealing Polymerase
Hybridize

F
Q
3) Extension
Cleavage

F
Q

UNISBA APRIL 2011

 Real-time PCR

Primer Probe

Template ×
Primer Mutation

PCR

Probe is cleaved Probe is not cleaved

Increased fluorescence None fluorescence
fluorescence intensity

Real-time monitoring Real-time monitoring

Positive False-negative

Negative
UNISBA APRIL 2011
PCR cycle number
Hasil rRT-PCR . A12-A15 pengenceran RNA 10 3, 10 4, 10 5 dan 10 6
UNISBA APRIL 2011
 Test Target Specimen Senstvy Specty Time

RT-PCR RNA Swabs, Tissue +++++ +++++ <5 hr

Viral Isolation Virus Swabs, Tissue +++++ +++++ 3-5 dy

Rapid Test Antigen Swabs ++ ++ <1 hr

HI Antibody Sera +++ ++++ 48 hr

Micro Neutralisasi Antibody Sera ++++ +++++ 72 h

UNISBA APRIL 2011

UNISBA APRIL 2011