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Microbiology Practical

Explanation
Gastroenterohepatology system
2014
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, KH 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)
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 production
FERMENTATION
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
acid Methyl Red: left – and right +
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). 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
Tube 1: Negative acid /Negative gas fermentation
Tube 2A: Must incubate longer
(ambiguous result) Mannitol
Tube 2B: Positive acid /Negative
gas
Tube 3A: Positive acid/ Positive gas
Lactose Fermentation
 This tests for the bacteria’s ability to
ferment lactose.
 (+) = yellow (indicating lactose
fermentation).
 (-) = red
Sucrose Fermentation
 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.
 (+) = yellow (indicating
sucrose fermentation).
 (-) = red
Glucose Fermentation & Gas
Production
 This tests for the bacteria’s ability to
ferment glucose and produce gas and/or an
acid end-product.
 (+) = yellow (indicating
glucose fermentation)
 for gas (+) = a bubble in the
Durham tube.
 Complete (-) = red and no
bubble.
To observe,,,,,,,,,
 Escherichia coli
 Shigella flexneri
 Salmonella typhii
 Vibrio cholera
Special selective and differential agar
medium:

 Mc Conkey agar plate


 SS agar plate
 TCBS
Mc 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


Principle:
•SS agar is often used to help
isolate salmonellae and
shigellae 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)
 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 : (+)
Escherecia coli
Pathogenic group epidemiology Laboratory diagnosis
Enteropatogenic E coli (EPEC) EPEC strains belong to particular O Isolate organism from feces
serotypes cause sporadic cases and Determine serotype of several colonies
outbreaks of infection in babies and withpolyvalent antisera for known EPEC
young children importance in adults less types
clear Adhesion to tissue culture cells can be
demonstrated
By a fluorescence actin staining test
DNA-based assays fro detection of
attacment
(virulence) factors
Enterotoxigenic E coli Most important bacteria cause of Isolate organisms from feces
(ETEC) diarrhea in children in developing Tests commercially available for
countries most common cause of immunologic detection of toxins from
traveler’s diarrhea water contaminated culture supernatants
by human or animal sewage may be Genes probes specific for LT and ST genes
important in spread available for detection of ETEC in feces
and in food and water samples
Enterohemorrhagic (verotoxin Serotype O157 most important EHEC in Isolate organisms from feces
producing) E coli (EHEC) human infections Proportion of EHEC in fecal sample may be
Outbreaks and sporadic cases occur very low (often <1% of E coli colonies)
worldwide Usually sorbitol non-fermenters
Food and unpasteurized milk impotant in Shiga toxin production and associated genes
spread detected by biological, immunological and
May cause haemolytic uremic syndrome nucleid acid based assays
(HUS)
Enteroinvasive E coli (EIEC) Important cause of diarrhea in areas of Isolate organisms from feces
poor hygiene Test for enteroinvasive potential in tissue
Ionfection usually foodborne, no culture cells or nucleid acid based assays
evidence of animal or environmental for invasion associated genes
reservoir
Enteroaggregative E coli (EAEC) Characterisitic attachment to tissue Issue culture assays for aggregative of
Diffuse aggregative E coli culture cells diffuse adherence
(DAEC) Cause diarrhea in children in developing
counties
Role of toxins uncertain
Escherecia coli Enterotoksigenik (ETEC)
 Menyebabkan Traveller diarrhea
 ETEC melekat pada usus halus melalui pili
dan mengeluarkan 2 toksin:
Toksin LT(heat labile) : mengkatalisis
ribsilasi-ADP, meningkatkan aktivitas siklasa
adenilat
Toksin ST(heat stable): 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
Vibrio cholera
 Best enrichment
medium :
TCBS agar
 Yellow colonies for
vibrios due to
oxidase reaction
 Comma shaped in
electron micrograph
MEDIUM TRANSPORT

◦ Stuart medium: dipakai untuk transportasi


specimen yang mengandung bakteri positif
Gram dan negatif Gram.
◦ Carry and Blair medium: dipakai untuk
transportasi tinja atau apusan rectum yang
dicurigai mengandung Vibrio atau
Enterobacteriaceae yang patogen.
 Medium enrichment untuk bakteri
enterik:
◦ Air penton 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)

Watery diarrhea (nausea, vomiting, fever, abdominal pain and


intestinal fluid loss—caused by VC and ETEC in proximal small
intestine)—the “run”
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”
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
INSTRUKSI!!!
 AMBIL KERTAS SELEMBAR & PULPEN!!!
 TULISKAN NAMA DAN STAMBUK!!!
1.Sebutkan 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 !!!
6
 What is the function of transport
medium?
 Give 2 examples of transport medium.
1.Berikan 4 zat atau kandungan di dalam mc
conkey beserta fungsi
Soal no. 2
 Namakan medium
 Berikan nama proses
yg menukarkan dr
hijau ke kuning
3.Berikan 5 kelompok strain E.coli
 4.kenapa TCBS dikatakan medium
selectives?
 5.berikan specimen yg boleh digunakan
dalam proses diagnosis microbiology.
 Berikan tahapan dalam pemeriksaan
microbiology