dr.

Irham
 PUKUL KEGIATAN
07.30 – 08.20 Penjelasan Praktikum
08.20 – 09.10 Praktikum Mikrobiologi
09.10 – 10.00 Praktikum Mikrobiologi
10.00 – 10.50 ISTIRAHAT
10.50 – 11.40 Kuliah oleh Prof. dr. Muh. Nasrum Massi, Ph.D
11.40 – 13.00 Kuliah oleh Prof. dr. Muh. Nasrum Massi, Ph.D
13.00 – 13.50 Kuliah oleh Prof. dr. Muh. Nasrum Massi, Ph.D
13.50 – 14.40 Kuliah oleh Prof. dr. Muh. Nasrum Massi, Ph.D
14.40 – 15.30 Praktikum Mikrobiologi (Lanjutan)
 Diagnosing in Microbiology

* Specimen : Depend on symptoms and timing (blood, pus, spinal

fluid, or stool)

* Gram staining

* Biochemical Tests (TSI, SIM, VP, Methyl Red, Citrat, Urea, CH

test)

* Cultures (Blood Agar, EMB, MacConkey, SS Agar, TCBS)

* Nucleic Acid Methods (PCR, hybridization, blots)

* Antibody Mediated (ELISA, slide/latex agglutination, Widal

reaction, tagged fluorescence, titer)
 To observe……….

* Escherichia coli
* Shigella flexneri
* Salmonella typhii
* Vibrio cholera
 Biochemical Test

*TSI (Triple sugar iron)

*SIM (sulphur Indole motility)
*Methyl Red (MR)
*Voges Proskauer (VP)
*Citrate
*Urea hydrolysis
*Lactose fermentation
*Sucrose fermentation SUGAR FERMENTATION
*Glucose fermentation
& gas production
 Triple Sugar Iron (TSI) Test

* In TSI test we observe the :

- Slant
- Butt
- Sulfur (H2S)
- Gas

Acid = yellow
Alkali = red
Netral = orange
* In SIM we observe :
- Sulfur (H2S)
- Indole
- Motility

SIM media contains the sulfur containing amino acid,

cysteine, sodium thiosulfate, & peptonized iron or ferrous
sulfate.
 SIM (Sulphur Indole Motility) cont’

* H2S will react with the

iron or ferrous sulfate and
produce a black
precipitate. A positive
result has a black
precipitate present and a
negative result has no
black precipitate.

*Black = (+) *
 SIM (Sulphur Indole Motility) cont’

* Kovac’s reagent reacts

with indole and creates a
red color at the top part
of the test tube.

*Red ring = (+)

*
 SIM (Sulphur Indole Motility) cont’

* If bacteria is motile, there

will be growth going out
away from the stab line, and
test is positive. If bacteria
is not motile, there will only
be growth along the stab
line.

*
*MR : tests for acid end
products from glucose
fermentation.
• (+) = red (indicating
pH below 6) and
• (–) = yellow (indicating
no acid production)

Methyl Red: left – and right +

*VP : tests for acetoin
production from glucose
fermentation.
*(+) = red
*(–) = no color change
(yellow)

VP: left + and right –

 Citrate Test

* It is selective for bacteria

that has the ability to
consume citrate as its sole
source of carbon and
ammonium as sole
nitrogen source.
* (+) = blue (meaning the
bacteria metabolised
citrate and produced an
acid end product). *
* (–) = green.

Left positive and right negative.

* This test is done to
determine a bacteria’s ability
to hydrolyze urea to make
ammonia using the enzyme
urease.
* (+) = a bright pink color.
* (-) =yellow

Left positive and right negative.

 • Glucose fermentation
• Lactose fermentation
• Sucrose fermentation
• Mannitol fermentation

(+) = Yellow (indicating carbohydrate fermentation)

(-) = Red
Special Selective and Differential Agar Medium

1. Mac Conkey agar plate

2. SS agar plate
3. TCBS
 * Mac Conkey Agar
* MacConkey agar is a culture medium designed to grow Gram-
negative bacteria and stain them for lactose fermentation.

Ingredients:
* Bile Salt (to inhibit most Gram-positive bacteria, except
Enterococcus and some species of Staphylococcus i.e.
Staphylococcus aureus),
* Crystal Violet dye (which also inhibits certain Gram-positive
bacteria),
* Neutral Red dye (which stains microbes fermenting lactose),
* Lactosa & Peptone.
Macconkey agar with lactose(left) and non-lactose(right) fermenters
PRINCIPLE
Lactose (+)
* By utilizing the lactose available
in the medium, Lac+ bacteria
such as Escherichia coli,
Enterobacter and Klebsiella will
produce acid, which lowers the
pH of the agar below 6.8 and
results in the appearance of
red/pink colonies. The bile salts
precipitate in the immediate
neighborhood of the colony,
causing the medium surrounding
the colony to become hazy.
Lactose (-)
* Non-Lactose fermenting bacteria
such as Salmonella, Proteus species,
Pseudomonas aeruginosa and
Shigella cannot utilize lactose, and
will use peptone instead. This forms
ammonia, which raises the pH of the
agar, and leads to the formation of
white/colorless colonies formed in
the plate. But they can also look
golden to brown with dark centers.
They are circular colonies and
arranged randomly.
 * SS agar plate:
Ingredients:
Nutrient agar 100ml
Lactose 1.00g
Bile salts 0.85g inhibitor

Sodium citrate 0.85g inhibitor

Sodium thiosulfate 0.85g

10% ferric citrate 1ml
1% Neutral red 0.25ml indicator

1% brilliant green 0.033ml inhibitor

 *

*Used for culture Vibrio

cholerae
*Yellow colonies for
vibrios due to oxidase
reaction
* TCBS Agar:
* Yeast Extract ........ 5.0 g
* Pancreatic Digest of Casein ........ 5.0
* Peptic Digest of Animal Tissue .... 5.0
* Sodium Citrate .. 10.
* Sodium Thiosulfate......................................... 10.0
* Ox Bile Extract ................................................ 5.0
* Sodium Cholate ............................................... 3.0
* Sucrose .......................................................... 20.0
* Sodium Chloride ............................................. 10.0
* Ferric Citrate ................................................... 1.0
* Agar ................................................................ 14.0
* Bromthymol Blue ............................................ 40.0 mg
* Thymol Blue ................................................... 4.0
* Final pH 8.6 ± 0.2 at 25°C

(2) Alkaline Peptone Water:

* Pancreatic Digest of Casein .......................... 10.0 g
* Sodium Chloride .............................................. 5.0
* Final pH 8.5 ± 0.2 at 25°C
* The bile salts in the media inhibit the growth of gram-
positive microbes; the presence of sucrose allows for
the differentiation of those vibrios, which can utilize
sucrose with the aid of bromthymol blue, and thymol
blue indicators. The high 8.6 pH of TCBS Agar
suppresses other intestinal flora while allowing
uninhibited growth of vibrios.
 *Escherichia coli
*Cocco bacil, gram (-)
*Katalase (+)
*H2S (-)
*Aerob or could be facultative anaerobic
*The major flora in human intestine
*Could be motile or nonmotile
*Oksidase production (-)
*Glucose fermentation releasing gas
*Not using citrate as its energy source
 Biochemical test of Escherichia coli

 TSI
◦ Slant : asam
◦ Butt : asam
◦ Gas : (+)
◦ H2S : (-)
 SIM :
◦ Indol : (+)
◦ H2S : (-)
◦ Motility : (+)
 VP : (-)
 Citrat : (-)
 Urea : (-)
 MR : (+)
 Menyebabkan Traveller diarrhea
 ETEC melekat pada usus halus melalui pili dan
mengeluarkan 2 toksin:
1. Toksin LT : mengkatalisis ribsilasi-ADP, meningkatkan
aktivitas siklasa adenilat
2. Toksin ST: mengaktifkan siklasa guanilat,
meningkatkan kadar guanosin monofosfat siklik
(cGMP) dan mengakibatkan terjadinya hipersekresi
cairan dan elektrolit)
 Enteropathogenic E. Coli/EPEC

* Like
ETEC, EPEC also causes diarrhea, but the molecular
mechanisms of colonization and aetiology are different.
* EPEC lack fimbriae, ST and LT toxins, but they use an adhesin
known as intimin to bind host intestinal cells. This virotype
has an array of virulence factors that are similar to those
found in Shigella, and may possess a shiga toxin. Adherence
to the intestinal mucosa causes a rearrangement of actin in
the host cell, causing significant deformation.
* EPEC cells are moderately invasive (i.e. they enter host cells)
and elicit an inflammatory response. Changes in intestinal
cell ultrastructure due to "attachment and effacement" is
likely the prime cause of diarrhea in those afflicted with
EPEC.
 E. Coli Enterohemorragik/EHEC

 EHEC ditemukan pada tinja sapi dan ditularkan melalui

daging sapi giling setengah matang atau air minum
yang tercemar tinja sapi
 Mensekresi verotoksin: toksin ini mencungkil rbosom
60s sel usus halus, menghentikan sintesis protein,
merusak selaput lendir kolon dan menyebabkan
perdarahan ke dalam lumen usus
 Tidak mampu meragikan sorbitol
 Serotipe yang dominan adalah 0157:H7
 Enteroinvasive E. coli (EIEC)

* EIEC infection causes a syndrome that is identical to

shigellosis, with profuse diarrhea and high fever.
 *Shigella
* Basil Gram –
* Fakultatif Anaerob
* Tidak bergerak aktif
* Sangat tahan terhadap asam
* Bersifat infasiv menyebabkan tukak dangkal
* S. dysentriae tipe 1 Toksin shiga
 * Shigella dysentriae
* TSI
* Slant : alkali
* Butt : asam
* Gas : (-)
* H2S : (-)
* SIM :
* Indol : (-)
* H2S : (-)
* Motility : (-)
* VP : (-)
* Citrat : (-)
* Urea : (-)
* MR : (-)
 * Salmonella typhii
*TSI
* Slant : alkali
* Butt : asam
* Gas : (+/-)
* H2S : (+)
*SIM :
* Indol : (-)
* H2S : (+)
* Motility : (+)
*VP : (-)
*Citrat : (-/+)
*Urea : (-)
*MR : (+)
 * Vibrio Cholera
* Organisme air yang berbentuk berbentuk batang
bengkok gram-negatif seperti koma,oksidase positif
* Ditemukan di dalam air dan ditularkan melalui air atau
di bawah cangkang kerang yang dibiakkan di dalam air
yang tercemar
 Toksin
* Toksin kolera (koleragen) memiliki fragmen A (bagian
toksik) dan fragmen B (mengikat sel); toksin ini
khususnya mlekat pada sel-sel epitel mikrovili lapisan
sikat pada usus halus
* Komponen A : menggagnggu keseimbangan cairan dan
elektrolit, menyebabkan terjadinya hipersekresi klorida
dan bikarbonat
*Best enrichment
medium :
TCBS agar
*Yellow colonies for
vibrios due to oxidase
reaction
*Comma shaped in
electron micrograph
 Medium Transpor

* Stuart medium: dipakai untuk transportasi specimen yang

mengandung bakteri Gram positif dan Gram negatif.
Spesimen: Feses/rectal swab

* Carry and Blair medium: dipakai untuk transportasi tinja

atau apusan rectum yang dicurigai mengandung Vibrio atau
Enterobacteriaceae yang patogen.
Spesimen: Feses/rectal swab
* Medium enrichment untuk bakteri enterik:
* Air pepton alkalis: untuk Vibrio species dalam tinja
* Selenite: Salmonella dan Shigella species dalam tinja.
* Medium bile pepton: untuk Salmonella typhi dalam darah
 * Acute infection of GI tract
Clinical Features
(fever, vomiting, abdominal pain and
diarrhea)
1.Watery diarrhea (nausea, vomiting, fever, abdominal pain and
intestinal fluid loss—caused by VC and ETEC in proximal small
intestine)—the “run”
2.Dysentery (fever, abdominal pain, cramps, tenesmus, feces
with blood and pus—produce inflammatory or destructive
changes in colonic mucosa by direct invasion or produce
cytotoxins– the “squirt”
3.Enteric fever (fever and abdominal pain—mild diarrhea—
bacteremia common and cause metastatic infection in other
organs---typhoid fever in distal small bowel
 *Food poisoning
The most common causes:
1. Intoxication
Bacillus cereus (vomiting toxin), Clostridium
botulinum, Staphylococcus aureus, Chemical
2. Infections
Clostridium perfringens, Salmonella, Shigella,
Vibrio parahaemolyticus, Trichinella spiralis,
Hepatitis A
* AMBIL KERTAS SELEMBAR & PULPEN
* TULISKAN NAMA DAN NIM

*INSTRUKSI
1. Tuliskan semua mikroorganisme yang akan
dipraktikumkan
 Soal No. 2
A. Medium Apa Ini?
B.Mengapa medium ini
dikatakan selektif
diferensial?
A.Medium apakah ini?
B. Sebutkan zat yang
digunakan sebagai
inhibitor bakteri gram
positif?
 Soal no. 4
Didalam medium ini
terdapat salah satu jenis
karbohidrat yang bisa
berguna untuk
membedakan species
vibrio yang satu dengan
yang lain. Karbohidrat apa
yang dimaksud?
5. Sebutkan 2 toksin yang dihasilkan oleh
Escherecia coli enterotoksigenik
*