Microbiology Practical

Review
SISTEM GASTROENTEROHEPATOLOGI
Diagnosing in Microbiology
Specimen : Depend on symptoms and
timing
• Urine, blood, pus, spinal fluid, sputum, or
stool.

Gram staining

Biochemical Tests (TSI, SIM,VP,

Methyl Red, Citrat, Urea, KH test)

Cultures (Blood Agar, EMB,

MacConkey, SS Agar, TCBS)
Diagnosing in Microbiology

Nucleic Acid Methods (PCR,

hybridization, blots)

Antibody Mediated (ELISA,

slide/latex agglutination,
Widal reaction, tagged
fluorescence, titer)
 Biochemical Test
◦ TSI (Triple sugar iron)
◦ SIM (sulphur Indole motility)
◦ Methyl Red (MR)
◦ Voges Proskauer (VP)
◦ Citrate
◦ Urea hydrolysis
◦ Lactose fermentation
◦ Sucrose fermentation SUGAR
◦ Glucose fermentation & gas FERMENTATION
production
 Triple Sugar Iron (TSI) Test

 In TSI test we observe the :

- Slant
- Butt
- Sulfur (H2S)
- Gas
 Acid = yellow
 Alkali = red
 Netral =orange
SIM
(Sulfur Indole Motility)
 In SIM we observe :
- Sulfur (H2S)
- Indole
- Motility

SIM media contains the sulfur containing

amino acid, cysteine, sodium thiosulfate, &
peptonized iron or ferrous sulfate.
 Sulfur
 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 = (+)
Indole
 Kovac’s reagent
reacts with indole
and creates a red
color at the top part
of the test tube.

 Red ring = (+)

 Motility
 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.
 Stab line red = (+)
 Methyl Red (MR)
 MR—tests for acid
end products from
glucose
fermentation.
• (+) = red
(indicating pH
below 6) and
• (–) = yellow
(indicating no Methyl Red: left – and right +

acid production)
Voges Proskauer (VP)
 VP—tests for
acetoin
production from
glucose
fermentation.
 (+) = red
 (–) = no color
change (yellow)
VP: left + and right –
Citrate
 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
Urea Hydrolysis

 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.

 Sugar Fermentation Tests

• Lactose
fermentation
• Sucrose
fermentation
• Glucose
fermentation
Tube 1: Negative acid /Negative gas
Tube 2A: Must incubate longer
(ambiguous result)
• Mannitol
Tube 2B: Positive acid /Negative gas
Tube 3A: Positive acid/ Positive gas
Lactose Fermentation
 How to Perform Test: Inoculate lactose broth with
inoculating loop.

 Property it tests for: This tests for the bacteria’s ability

to ferment lactose.

 Media and Reagents Used: Lactose broth contains

beef extract, gelatin peptone, and lactose. A phenol red indicator is added
to indicate acid production from fermentation.
 Results
◦ A positive result is yellow after indicator is added
(indicating lactose fermentation)
◦ A negative result will have no color change or will
be redish.
Sucrose Fermentation
 How to Perform Test: Inoculate sucrose broth with
inoculating loop.

 Property it tests for: This test is done to help

differentiate species of the family Enterobacteriaceae. This tests for the
bacteria’s ability to ferment sucrose and production of acid end-product

 Media and Reagents Used: Sucrose broth contains

beef extract, gelatin peptone, and sucrose. Phenol red indicator is added
to indicate an acid end-product.
 Results
◦ A positive result is yellow after indicator is added
(indicating sucrose fermentation)
◦ A negative result has no color change or is reddish.
Glucose Fermentation & Gas
Production
 How to Perform Test: Inoculate broth with inoculating loop.
 Property it tests for: This test is done to help differnetiate
species of the family Enterobacteriaceae. This tests for the bacteria’s ability to
ferment glucose and produce gas and/or an acid end-product..
 Media and Reagents Used: Glucose broth contains beef
extract, gelatine peptone, and glucose. A phenol red indicator is added to
indicate an acid enproduct. A Durham tube is added to indicate gas
production.
 Results
◦ A positive result for acid is yellow after indicator is
added (indicating glucose fermentation)
◦ A positive result for gas is a bubble in the Durham
tube.
◦ A completely negative result has no color change or
reddish color and no bubble.
Special selective & differential agar
medium

Mc Conkey agar
plate

SS agar plate

TCBS
 Principle:
Lac+
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.[2][3]
Lac-
Non-Lactose fermenting bacteria such as
Salmonella, Proteus sp., Pseudomonas
MacConkey agar with aeruginosa and Shigella cannot utilize
lactose(left) and non- lactose, and will use peptone instead.This
lactose(right) fermenters 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

Principle: Kultur Salmonella typhii
• SS agar is often used to help
isolate Salmonella and
Shigella from other enteric
gram-negative bacteria.
• Neutral red is the indicator.
• In acid condition it turns red,
and it turns yellow or brown
in base condition.
• E.coli that ferment lactose
can produce acid so the
colonies turns red . while
other enteropathogen cannot
ferment lactose, but can use
peptone to produce base, so
the colonies turns yellow.
TCBS
(Thiosulfate-Citrate-Bile-Sucrose)
Escherichia Shigella
coli dysentriae

Salmonella Vibrio
typhii cholera
 Escherichia coli
 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 : (+)
 Escherichia coli
Pathogenic group
Enteropatogenic E coli (EPEC)
Enterotoxigenic E coli
(ETEC)
Enterohemorrhagic (verotoxin
producing) E coli (EHEC)
Enteroinvasive E coli (EIEC)
Enteroaggregative E coli (EAEC)
Diffuse aggregative E coli
(DAEC)
epidemiology
EPEC strains belong to particular O
serotypes cause sporadic cases and
outbreaks of infection in babies and
young children importance in adults less
clear
Most important bacteria cause of
diarrhea in children in developing
countries most common cause of
traveler’s diarrhea water contaminated
by human or animal sewage may be
important in spread
Serotype O157 most important EHEC in
human infections
Outbreaks and sporadic cases occur
worldwide
Food and unpasteurized milk impotant in
spread
May cause haemolytic uremic syndrome
(HUS)
Important cause of diarrhea in areas of
poor hygiene
Ionfection usually foodborne, no
evidence of animal or environmental
reservoir
Characterisitic attachment to tissue
culture cells
Cause diarrhea in children in developing
counties
Role of toxins uncertain
Laboratory diagnosis
Isolate organism from feces
Determine serotype of several colonies
withpolyvalent antisera for known EPEC
types
Adhesion to tissue culture cells can be
demonstrated
By a fluorescence actin staining test
DNA-based assays fro detection of
attacment
(virulence) factors
Isolate organisms from feces
Tests commercially available for
immunologic detection of toxins from
culture supernatants
Genes probes specific for LT and ST genes
available for detection of ETEC in feces
and in food and water samples
Isolate organisms from feces
Proportion of EHEC in fecal sample may be
very low (often <1% of E coli colonies)
Usually sorbitol non-fermenters
Shiga toxin production and associated genes
detected by biological, immunological and
nucleid acid based assays
Isolate organisms from feces
Test for enteroinvasive potential in tissue
culture cells or nucleid acid based assays
for invasion associated genes
Issue culture assays for aggregative of
diffuse adherence
 Shigella dysenteriae
 Gram negative
 Rod shape
 Facultative anaerob
 Non motile, non spore
 Contamination via fecal-oral
 Shigella dysenteriae
 TSI  MR : (+)
◦ Slant : alkali  VP : (-)
◦ Butt : asam  Citrat : (-)
◦ Gas : (-)
 Urea : (-)
◦ H2S : (-)
 SIM :
◦ Indol : (-)
◦ H2S : (-)
◦ Motility : (-)
 Salmonella typhi
 Gram negative
 Rod shape
 Facultative anaerob
 Motile ( flagel & fimbria )
 Characterized by O, H, and Vi antigens
 Salmonella typhi
 TSI
◦ Slant : alkali
◦ Butt : asam
◦ Gas : (+/-)
◦ H2S : (+)
 SIM :
◦ Indol : (-)
◦ H2S : (+)
◦ Motility : (+)
 MR : (+)
 VP : (-)
 Citrat : (-/+)
 Urea : (-)
 Vibrio cholerae
 Comma-shaped/curve rods
 Gram negative, oksidase +
 Highly motile with a single polar flagellum
 Tolerate alkaline but sensitive to acid
 Vibrio cholerae serogroup O139 & O1 is
toxogenix produce a heat-labile enterotoxin.
 Vibrio cholerae O grup 1
◦ Serotype : inaba (AB), Ogawa (AC), Hikojima (A,B,C)
◦ Biotype : Classical and El Tor
 Direct dark- field examination of the stool or phase
microscopy  “shooting stars”
 TSI
◦ Slant : asam
◦ Butt : asam
◦ Gas : (-)
◦ H2S : (-)
 SIM :
◦ Indol : (+)
◦ H2S : (-)
◦ Motility : (+)
 MR : (+)
 VP : (+)
 Citrat : (+)
 Urea : (-)
 TSI

Asam/Asam Alkali/Asam

Gas/Laktosa H2S/Motility

+ - + -
Salmonella Shigella
Escherichia coli Vibrio cholerae
typhi dysenteriae
 MEDIUM TRANSPORT
 Medium transport untuk tinja
◦ Stuart medium: digunakan
untuk transportasi
specimen yang
mengandung bakteri Gram
positif dan Gram negatif.
◦ Carry and Blair medium:
dipakai untuk transportasi
tinja atau apusan rectum
yang dicurigai mengandung
Vibrio sp. atau
Enterobacteriaceae yang
patogen.
 Medium enrichment untuk
bakteri enterik
◦ Air penton alkalis: untuk
Vibrio species dalam tinja
◦ Selenite: Salmonella sp.
dan Shigella sp. dalam
tinja.
◦ Medium bile pepton: untuk
Salmonella typhi dalam
darah
 Acute infection of GI tract
Clinical Features
(fever, nausea, 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, 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
RESPON
1. a. Jelaskan mengapa MacConkey disebut
sebagai medium selective differential
b. tuliskan dan jelaskan fungsi dari
komposisi MacConkey
2. a. tuliskan karakteristik dari Escherichia
coli
b. tuliskan 5 pathotype Escherichia coli
3. a. tuliskan karakteristik dari Vibrio
cholerae
b. tuliskan medium yang digunakan
untuk kultur Vibrio cholerae, dan
medium transport yang digunakan
pada spesimen yang dicurigai
mengandung Vibrio cholerae
4. Tuliskan bakteri yang menyebabkan
intoksikasi pada kasus keracunan
makanan (min. 2)
5. a. Tuliskan medium enrichment yang
digunakan pada bakteri enterik dan fungsinya
masing-masing

b. Tuliskan bakteri yang bersifat invasive

6. a. Tuliskan semua nama asisten dan
kesanmu pada asisten Mikrobiologi
b. Tuliskan kesan dan saran untuk Bagian
Mikrobiologi
RESPON
1. a. Tuliskan dan jelaskan fungsi komposisi
SS agar
b. jelaskan interpretasi pada SS agar
2. a. tuliskan 5 pathotype Escherichia coli
b. Sebutkan toxin yang dihasilkan oleh
Enterotoxigenic Escherichia coli !
3. Jelaskan secara singkat patomekanisme
diare yang disebabkan oleh Vibrio
cholerae
4. Tuliskan bakteri yang menyebabkan
infeksi pada kasus keracunan makanan
(min. 3)
5. a. Tuliskan medium transport yang
digunakan untuk spesimen feses dan
fungsinya masing-masing

b. Tuliskan nama bakteri yang bersifat

invasif !
6. a. Tuliskan nama asisten dan kesanmu
pada asisten Mikrobiologi
b. Tuliskan kesan dan saran untuk Bagian
Mikrobiologi