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ISOLASI, IDENTIFIKASI DAN

TES SENSITIVITAS
Salmonella thypi
Kelompok 4
How does the bacteria cause disease ?

Ingestion of contaminated food or water

Salmonella bacteria

Invade small intestine and enter the bloodstream

Carried by white blood cells in the liver, spleen, and bone marrow

Multiply and reenter the bloodstream

Bacteria invade the gallbladder, biliary system, and the lymphatic tissue of the bowel and
multiply in high numbers

Then pass into the intestinal tract and can be identified for diagnosis in cultures from the
stool tested in the laboratory
Diagnosis

Diagnosis of typhoid fever is made by

Blood, bone marrow, or stool cultures test


Widal test
Slide agglutination
Antimicrobial susceptibility testing
CULTURE TEST
Flow diagram for detection of
Salmonella
Tahap pre-enrichment dan selective enrichment. Tahap pre-
enrichment dilakukan dalam Buffered peptone water (BPW) dan
medium selective enrichment selenite cystine broth (SC)
Seleksi dalam medium agar selektif. Sesudah melalui tahap selective
enrichment, dilakukan subculture/plating ke dalam medium agar
selektif: Chromocult Coliform Agar (CCA) MacConkey Agar, dan
Salmonella Shigella Agar (SS Agar)
Untuk menguji kemurnian kultur tunggal yang tumbuh maka koloni
tersebut ditumbuhkan kembali dengan digoreskan pada media Brain
Heart Infusion Agar (BHIA). Koloni tunggal yang muncul diinokulasikan
ke dalam Trypticase Soy Agar (TSA)
Dalam medium CCA, koloni Salmonella,Shigella, dan Yersinia berwana
jernih transparan sampai biru terang tergantung pada ekspresi enzim
-glucoronidase Dalam medium MacCONKEY Agar, koloni Salmonella
dan Shigella terlihat jernih dan transparan karena tidak menghasilkan
enzim -galactosidase. Sedangkan dalam medium SS Agar, koloni
typical Salmonella juga akan terlihat jernih dan transparan
Uji konfirmasi dalam medium Triple Sugar Iron Agar (TSIA)

SELEKSI BAKTERI SALMONELLA TYPHI DARI KULTUR DARAH


PENDERITA DEMAM TIFOID : Charis Amarantini dkk
Photographs of Salmonella growth on
various media and positive reactions of
biochemical tests.

The picture shows an


uninoculated plate on
Brilliant green agar
(BGA)
Salmonella on BGA Agar. The colonies are
red because the bacterium does not
ferment lactose or sucrose.

This medium is used to isolate


Salmonella from pathological
material, foodstuffs, etc. by
separating lactose- and/or
sucrose-positive bacteria from
lactose- and sucrose-negative
bacteria. Brilliant-green in the
medium inhibits accompanying
micro-organisms.
Salmonella Typhi In Mcconk
ey Agar

MacConkey agar is both a selective and differential medium


frequently used in culture testing.
It contains crystal violet dye and bile salts, both of which inhibit the
growth of most gram-positive bacteria. It contains lactose (a sugar)
and neutral red indicator (a pH indicator which is yellow in a neutral
solution, but turns pink to red in an acidic environment), which
allow for differentiation
. On MacConkey agar, Escherichia coli and Enterobacter
aerogenes would ferment the lactose producing acid and would
form colonies pink to red in color.
On the same medium, Salmonella, Shigella, and Pseudomonas
species would not ferment the lactose and would form off-white
colonies.
The red colored colonies show that acid was produced from
lactose, meaning the bacteria could utilize lactose as a carbon
source.

Growth on the plate indicates the organism, Salmonella


typhimurium, is not inhibited by bile salts and crystal violet and is
a gram-negative bacterium. The absence of color in the bacterial
growth indicates S. typhimurium is unable to ferment lactose
Salmonella identification
Gram Negatif

Coccobacili coccus bacilli

Lactosa Lactosa
+ -

Oksidasi + Oksidasi -

Urease + Urease -
Salmonella
sp
Produce
Motile H22S
Gram negative small rods arranged
singly or pairs on Grams staining
(100X)
Fermentasi karbohidrat
Tujuan
Membedakan bakteri yang mampu mengubah gula menjadi
asam-asam melalui proses fermentasi.
Prinsip
Monosakarida berupa glukosa mengalami fermentasi menghasilkan asam-
asam organik dan/atau gas, sehingga mengubah warna indikator pH
dan/atau ada gelembung gas di tabung durham.
Medium
Glukosa broth, lactose broth dan sucrose broth
Interpretation:
If the medium changes from colorless to yellow and gas bubble is found in
Durhams tube then it indicates acid and gas production. In some cases gas may
not be evolved during the process. If no change observed in the colour of medium
then sugar is not degraded by the organism.
CARBOHYDRATE FERMENTATION
TEST (DURHAM TUBES)

Nutrient Broth + Respective Sugar


From left to right: uninoculated
positive, and negative.
Triple Sugar Iron Agar (TSIA)
Reaksi pada uji TSIA
1.Butt (kuning) dan Slant (merah) : Menunjukkan
fermentasi dari glukosa (Indikator Merah fenol berubah warna
menjadi kuning akibat penurunan pH dari hasil fermentasi
glukosa di bagian dasar/butt). Bagian Slant tetap berwarna
merah, karena jumlah glukosa yang terbatas (hanya dari
laktosa dan sukrosa) dalam medium, akibatnya jumlah asam
yang terbentuk juga terbatas.
2. Butt (kuning) dan Slanta (kuning) : Menunjukkan juga
ada fermentasi laktosa dan/atau sukrosa serta glukosa (Warna
kuning pada bagian Slant dan Butt menunjukkan banyak gula
yang difermentasi menghasilkan asam).
3. Butt (merah) dan Slant (merah) : Menunjukkan tidak ada
gula yang difermentasikan menghasilkan produk yang bersifat
asam.
4. Pembentukan gas : Pembentukan gas ditandai dengan
Triple Sugar Iron Agar (TSIA)
Hasil positif
Uji indol
Tujuan
Mengidentifikasi bakteri yang mmpu
menggunakan triptofan sebagai sumber
energi (memiliki enzim triptofanase).

Prinsip
para-dimetil-aminobenzaldehida +
indol rosindol (merah).
para-dimetil-aminobenzaldehida
terdapat pada semua jenis reagen
yang mungkin digunakan : kovacs,
Gore, Ehrilch, dan Ehrilch-Bohme.
Methylen Red
Tujuan
Mengetahui kemampuan bakteri
dalam memfermentasikan asam
campuran.

Prinsip
karbohidrat berupa glukosa
mengalami fermentasi menghasilkan
berbagai asam-asam organik (asam
campuran), sehingga menurunkan pH
medium secara drastis dan
menyebabkan indikator methylen red
tetap berwarna merah.

Medium
Medium MRVP
Voges Proskauer
Tujuan
Mengidentifikasi bakteri yang
memfermentasi 2,3-butanadiol.

Prinsip
karbohidrat berupa glukosa mengalami
fermentasi menghasilkan 2,3-
butanadiol.
Asetoin + 40% KOH + 5% alphanaftol
kompleks merah muda

Medium
Medium MRVP
Uji penggunaan sitrat
Tujuan
Uji ini digunakan untuk melihat kemampuan
m.o menggunakan sitrat sebagai satu-
satunya sumber karbon dan energi
Prinsip
Kemampuan bakteri disebabkan bakteri
memiliki enzim citrate permease yang
memfasilitasi transport sitrat ke dalam
bakteri. Ketika masuk ke dalam bakteri,
sitrat akan berubah menjadi asam piruvat
dan CO2.
Simmons citrate agar mengandung Na-
sitrat sebagai satu-satunya sumber karbon,
NH4+ sebagai sumber N, dan indikator BTB
sebagai indikator pH. Jika sitrat dioksidasi,
maka sitrat akan hilang dan dihasilkan CO2
yang akan berreaksi dengan natrium (dari
Uji H2S dan motility
Tujuan
Mengidentifikasi bakteri yang mampu
bergerak dan menghasilkan H2S pada
medium SIM .

Prinsip
Banyak protein kaya akan asam amino
sistein dan metionin. Asam amino ini
dihasilkan sewaktu dihidrolisiskan
untuk memenuhi kebutuhan zat hara
mikroorganisme. Mikroorganisme yang
memiliki enzim desulfurase, sewaktu
dibiakkan dalam medium yang kaya
akan asam amino yang kaya akan sulfur
(asam amino sistein dan metionin
misalnya) akan menghasilkan H2S.
Uji oksidase
Tujuan
Uji ini berfungsi untuk menentukan
adanya oksidase sitokrom yang
ditemukan pada mikroorganisme
tertentu

Prinsip
Bila mikroorganisme yang bersifat
oksidase positif diberi reagens
oksidase (dimetil-p-fenilendiamin
oksalat), maka warna koloni berubah
menjadi ungu kehitaman dalam
waktu 30 menit. Perubahan warna
ini desebabkan oksidase sitokrom
mengoksidasikan larutan reagens.
Urea Hydrolysis (Urease test)

Property it tests for: This Results:


test is done to determine a Urea broth is a yelloworange
bacterias ability to color. The enzyme urease will be
hydrolyze urea to make used to hydrolyze urea to make
ammonia using the ammonia. If ammonia is made,
enzyme urease. the broth turns a bright pink
color, and is positive. If test is
Media and Reagents Used:
negative, broth has no color
Stuarts Urea broth (pH change and no ammonia is
6.8). made.
Principle : To determine the
ability of the organism to
split urea forming 2
molecules of ammonia by
the action of the enzyme
Urease with resulting
alkalinity
Widal Test
A test involving agglutination of typhoid bacilli when they are
mixed with serum containing typhoid antibodies from an
individual having typhoid fever; used to detect the presence
of Salmonella typhi and S. paratyphi.
Standard test tube method

Take four sets of 8 test tubes and label them 1 to 8 for O,H,AH and BH antibody detection.

Pipette in to the tube No.1 of all sets 1.9 ml of isotonic saline.

To each of the remaining tubes (2 to 8) add 1.0 ml of isotonic saline.

To the tube No. 1 tube in each row add 0.1 ml of the serum sample to be tested and mix well.

Transfer 1ml of the diluted serum from tube no.1 to tube no.2 and mix well.
Tube no.8 in all sets,serves as a saline control. Now the dilution of the serum sample achieved in each set is as follows:

Tube no. 1 2 3 4 5 6 7 8 (control)

Dilutions 1:20 1:40 1:80 1:160 1:320 1:640 1:1280

To all tubes (1 to 8) of each set add one drop of the respective WIDAL TEST antigen suspension (O,H,AH,BH) from
reagent vials and mix well.

Cover the tubes and incubate at 37 C overnight (approx. 18 hrs).

Dislodge the sedimented button gently and observe.


How do you read Widal test results for typhoid fever?

The highest dilution of the patients serum in which agglutinations occurs is


noted, ex. if the dilution is 1 in 160 then the titer is 169.

Agglutination in dilution up to <1:60 is seen in normal individuals .


Agglutination in dilution 1:160 is suggestive of Salmonella infection.

Agglutination in dilution of and more than 1:320 is confirmatory of Enteric


fever .
Slide agglutination
Antimicrobial susceptibility testing
Antimicrobial susceptibility testing

Antimicrobial susceptibility testing of the Salmonella


isolates to various routinely used antibiotics was
determined by disc diffusion technique on Muller Hinton
agar
The panel of antimicrobials were included, ciprofloxacin
(5 0g), norfloxacin (100g), nalidixic acid (300g),
ampicillin (100g), chloramphenicol (300g), co-
trimoxazole (1.25/23.750g), tetracycline (300g),
ceftriaxone (300g), cefotaxime (300g) and gentamicin
(100g).
MIC against ampicillin, nalidixic acid, ciprofloxacin,
cefotaxime and ceftriaxone was determined by agar
dilution method following CLSI guidelines 2008.

STUDIES ON ANTIMICROBIAL SUSCEPTIBILITY PATTERN OF SALMONELLA ISOLATES


FROM CHENNAI, INDIA
Antimicrobial susceptibility was
determined by the Kirby-Bauer disc
diffusion method (Collee et al., 2006).
The antibiotic discs used were
Cotrimoxazole (25 mcg), Ciprofloxacin (5
mcg), Ceftriaxone (30 mcg),
Azithromycin (15 mcg), Nalidixic acid (30
mcg), Amoxycillin (30 mcg), Cefixime (5
mcg) and Ofloxacin (5 mcg).

Prevalence and Antibiotic Sensitivity test of Salmonella Serovars from Enteric Fever Suspected
Patients Visiting Alka Hospital by Kalpana Pandey, et al, 2015
Prevalence and Antibiotic Sensitivity test of Salmonella Serovars from Enteric Fever Suspected
Patients Visiting Alka Hospital by Kalpana Pandey, et al, 2015
Prevalence and Antibiotic Sensitivity test of Salmonella Serovars from Enteric Fever Suspected
Patients Visiting Alka Hospital by Kalpana Pandey, et al, 2015
ISOLATION, IDENTIFICATION AND CHARACTERIZATION
OF SALMONELLA SEROVARS FROM DIARRHOEIC STOOL
SAMPLES OF HUMAN
PCR RFLP and nucleotide
sequence analysis
A 630bp fragment containing the
QRDR of the gyrA gene of the 41
clinical isolates were amplified
by PCR and sequenced.
The 630bp PCR based fragment
of gyr A gene has three
HinfIrestriction sites, one of
which lies at Ser83.
The codon for Serine (TTC) is
mutated to phenylalanine (TCC)
there by leading to the loss of
restriction site for HinfI.
Restriction analysis of the PCR
product revealed that 100% (41
isolates) of S.paratyphi A were
showing Ser 83 mutation in the
gyrA gene.

MOLECULAR ANALYSIS OF gyrAMUTATIONS IN SALMONELLA PARATYPHI ABY PCR-RFLP AND SEQUENCING


METHOD
The enzymeHinfI cuts
the 630bp of gyrA
gene product at three
sites, therefore
thegyr A gene
product from NAR
strains showed a
HinfI restriction
pattern consisting of
three fragments of
sizes 343bp, 144bp
and 138bp.
MOLECULAR ANALYSIS OF gyrAMUTATIONS IN SALMONELLA PARATYPHI ABY PCR-RFLP AND SEQUENCING
METHOD
References
MuthuG,SureshA,SumathyG,SrivaniR,Studies on antimicrobial
susceptibility pattern ofSalmonellaisolates from Chennai, IndiaInt J
Pharm Bio Sci2011 2:435-42.
Kalpana Pandey . Prevalence and Antibiotic Sensitivity test of Salmonella
Serovars from Enteric Fever Suspected Patients Visiting Alka Hospital by,
et al, 2015
M. K Nesa, ISOLATION, IDENTIFICATION AND CHARACTERIZATION OF
SALMONELLA SEROVARS FROM DIARRHOEIC STOOL SAMPLES OF HUMAN
GOPAL MUTHU et al , MOLECULAR ANALYSIS OF gyrAMUTATIONS
IN SALMONELLA PARATYPHI ABY PCR-RFLP AND SEQUENCING
METHOD. 2014
Beishir, L., 1991,Microbiology in Practice: A Self-Instructional Laboratory
Course, 5th ed. HarperCollins Publishers Inc, New York, USA. 223 - 229,
356 - 361, 446 - 450.
Lay, Bibiana W. 1994. Analisis Mikrobiologi Di Laboratorium.Raja Grafindo
Persada.Jakarta.

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