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UROGENITALIAFatimah Yunikartika Akbar
Fakultas Kedokteran
Universitas Hasanuddin
2011
Penjelasan Praktikum Mikrobiologi
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ASISTEN MIKROBIOLOGI 2011 -2012
ASEPMETRIKA : Kel. A1-B1
MUH.AYYUB PRIMADI : Kel. A2-B2
MOH. DIMASISMAIL : Kel. A3-B3
MUH. YOGIPRATAMA : Kel. A4-B4
ANA ZAHARINA HASYIM : Kel. A5-B5
FATIMAH YUNIKARTIKA AKBAR : Kel. A6-B6
NUR HAFIZAH BT. HATTA : Kel. A7-B7 WINDAAGUSTIN : Kel. A8-B8
SUHAILAH BINTI MOHD JAMIL : Kel. A9-B9
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TATA-TERTIB LABORATORIUMMIKROBIOLOGI
Laki laki : Memakai celana panjang kain, no polo shirt, rambut
tidak boleh kena kerah baju
Perempuan :Memakai rok panjang kain, no polo shirt, rambut
diikat ke atas. Bagi yg memakai jilbab, kasi masuk di dalam jas lab
Alat yang harus dibawa saat praktikum :
Jas lab
ID Card
Penuntun praktikum + Foto 4x6 berwarna
Masker Handscoen
Penggaris panjang 1 / kelompok
Pensil Warna
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Urogenital infectionscaused bypathogenic bacterial infection
Bacteriuriathe presence of asmany as 105bacteria in one ml mid
stream urinePyuriathe presence of white blood
cell in the urine
URINARY TRACT INFECTIOS(UTI)
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AGENTS THAT CAUSE URINARYTRACT INFECTIONS
Escherichia coli: 80%
Coagulase-negative staphylococci: 7%
Staphylococcus saprophyticus in youngwoman
Other Gram-negative bacteria: 4%
Other Gram-positive bacteria: 3%
Streptococcus Group B in pregnant woman
Proteus mirabilis: 6%
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Other organism
Anaerobic bacteriarare(2%)
Mycobacterium tuberculosis Mycobacterium spp
Campylobacter spp
Chlamydia trachomatis
Gardnerella vaginalis
Haemophilus influenzae
Legionella pneumophila
Mycoplasma hominis
Ureaplasma urealyticum Salmonella spp
Shigella spp
Adenoviruses
URINARY TRACTINFECTIONS
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ESCHERICHIA COLI
- Enterobactericeae
- Negative Gram
- Rod Shaped- Non Spora
- Fakultative Anaerobic
- Indole (+)
- Citrate (-)
- Urease (-)
- H2S (-)- MR (+)
- VP (-)
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KLEBSIELLA PNEUMONIAE
- Enterobactericeae
- Negative Gram
- Non capsule
- Non motile- Facultative anaerobic
- Indole (-)
- Antigen O and K
- H2S (-)
- Citrate (+)
- Urease (+)
- MR (+)
- VP(-)
- Lactose fermentation
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ENTEROBACTERAGGLOMERANS
- Enterobactericeae
- Negative Gram
- Rod Shaped
- H2S (-)
-
VP (+)- MR (-)
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PROVIDENCIA
Providencia Stuartii
- Enterobactericeae
- Negative Gram
- H2S (-)
- Indole (+)
- Citrat (+)
- MR (+)- VP(-)
- Motile
ProvidenciaAlkalifaciens
- Enterobactericeae
- Negative Gram- Rod Shaped
- H2S (-)
- Indole (-)
- MR (+)- VP (-)
- Motile
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PROTEUS
Proteus Vulgaris
- Enterobactericeae
- Negative Gram
- Rod Shaped- Motile
- H2S (+)
- Indole (+)
- MR (+)
- VP (-)
Proteus Mirabilis
- Enterobactericeae
- Negative Gram
- Rod Shaped- Fakultative Anaerobic
- Motile
- H2S (+)
- Indole (-)- MR (+)
- VP (-)
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ALKALIGENES FAECALIS
- Alcaligenaceae
- Negative Gram
- Rod Shaped
- Motile
- H2S (-)
- MR (-)
-
Citrate and Gas (+)- Oxidase (+)
- Catalase (+)
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PSEUDOMONASAERUGINOSA
- Pseudomonadaceae
- Negative Gram
- Rod Shaped
- Obligative Aerob
- Normal flora in human skin
- Have 3 kind pigmen :
- Pyocyanin (bluegreen)
- Pyoverdine (yellowgreenand fluorescence)
- Pyorubin (redbrown)
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URINE SAMPLE
Must be collected in a sterile manner, to beput in a sterile container.
Sterilization cannot be done with:
1. Wet heat sterilizationmay change thevolume of urine sample leading to dilution ofurine sample
2. Chemical sterilizationwill leave thechemical substance at the wall of thecontainer, thus will kill the bacteria
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URINE COLLECTION
Good urine for bacteriological examination ismorn ing ur ine, except for Mycobacteriumtuberculosis examination, 24 hoursurine isusually used.
Urine samples are collected in the followingways :
1. Mid Stream Urine
2. Urine Pocket3. Supra-Pubic Punction
4. Catheterization
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Most commonly used urine sample in
bacteriological examination of urine
If properly done, may minimizing the
contamination or urethral normal flora
MID STREAM URINE
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Area surrounding the external meatus is cleanwith diluted detergent and clean water (do notused disinfectants)
After removal the first excreted urine, nexturination is directly patched into a sterile bottle(without touching the container or other bodyparts of the patient)
Bottle is labeled and sent or kept in a cool place(refrigerator, or with ice blocks)
MID STREAM URINE
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Sterile bag plastic bag adhered around/in front
of external orificium to patch urine excreted
anytime by the patient
Sterile urine bag pocket available in market
Urine bag pocket is labeled and sent or kept in
a cool places (refrigerator, or in ice blocks)
URINE POCKET
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URINE COLLECTION:
BAGGED OR CLEAN CATCHContamination rate of 10%
Incooperative patien>105CFUinfection likely
104105CFUsuspicious
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The most sterile way of collecting urine. (Goldstandard99% specificty)
Requires to be done in the morning, as thebladder must be in full condition
Supra-pubic is cleaned & sterilized with 70%alcohol and bethadine or salvon.
By using 5ml sterile spoeit, puncture needle asdeep as finger on pubis until it penetrates the
bladder.Suck urine as many as 2-3ml, inject into a sterile
bottle, label and send to laboratory.
SUPRA-PUBIC PUNCTION
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Urine catheterization should be avoided,
except for patients with permanent catheter.
Catheterlead to irritation of at urogenital
tract mucosa & enhance the infection
For patients with permanent catheteruse
spoiet from the basal of disinfected catheter
CATHETERIZATION
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URINE COLLECTION:TRANSURETHERAL CATHERIZATION
>105Colony Forming Units - 95%
specificity
104105CFUinfection is likely
103104CFUSuspicious
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Gram staining of direct preparation
Bacterial count
i) Pour plate method
ii) Surface drop method
iii) Dip slide method
Isolation & identification
Antibiotic sensitivity testingSerotype / biotyping of bacterial agents
BACTERIOLOGICALEXAMINATION
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Direct preparation can be made from urine
sediments or urine without centrifugation.
From this preparation, we can see;
- The number of bacteria per field
- The number of leucocytes per field(used as a criteria for pyuria)
GRAM STAINING OFDIRECT PREPARATION
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a) Pour plate method
BACTERIAL COUNT
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b) Surface drop method
Urine is dipped with a sterile Pasteur pipette as much as one
to two droplets on the dry surface of McConkey & Nutrient
agar. Let the medium cool in room temperature and incubateovernight at 350C
Bacterial growth:
Grade 0no bacterial at the edge of droplet; 1 to 2
colonies only in the center of droplet
Grade 3 & 4growth 105cfu/ml; the edges of the droplet
will appear fully bacteria growth
BACTERIAL COUNT
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c) Dip slide method
Sterile slide layered with agar medium at
both sides is dipped into urine(sample).
Dry the specimen & incubate overnight.
Bacterial growth then compared with
standard pattern.
BACTERIAL COUNT
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Urine is dripped onto the surface of McConkey/Nutrient agar/Blood agar/Chocolate agar.
Strike with a loop or a swab on the media
surfaceCulture is incubated overnight & the appearance
of growing colony observed.
*If there are 2 types of bacterial growth;considered as contaminated (repeat)
ISOLATION &IDENTIFICATION
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Must be done forobserving thebacterialsensitivity toantibiotics,primarily to those
commonly usedagainst urogenitalinfections.
ANTIBIOTIC SENSITIVITYTEST
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For epidemiological purposes,
sometimes it is necessary to determine
the serotype and biotype of isolatedbacterial agents.
Bacterial biotype can be also determinedby observing the pattern of antibiotic
sensitivity called biogram.
SEROTYPE & BIOTYPE ofBACTERIAL AGENTS
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BIOCHEMICAL
TEST
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TSI (TRIPLE SUGAR IRON)TEST
Slunt & Butt
Alcali : Red
Acid : Yellow
Neutral : Orange
H2S : (+) : Black
(-) : Basic Colour
Gas : (+) : Medium terangkat(-) : Medium tidak
terangkt
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SIM (SULPHUR INDOLEMOTILITY) TEST
Sulphur (H2S) : (+) : Hitam
(-) : Tidak ada
Indole :
Reagen kovacsberekasi dengan
indole dan menghasilkan warnamerah di bagian atas dari tabung.
bacteria used trypthophanaseenzyme to degrade tryptophan toindole
(+) : Terdapat cincin merah
(-) : Tidak ada cincin merah
Motility :
(+) : Bergerak (Stab line)
(-) : Tidak bergerak
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METHYL RED /VOGES PROSKAUER (MR/VP)
Methyl Red
(+) : Red (-) : Yellow
Voges Proskauer
(+) : Red (-) : Yellow
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CITRATE & UREAHYDROLYSIS
Citrate
(+) : Blue (-) : Green
Urea Hydrolysis
(+) :Pink (-) : Yellowbacteria used urease enzymeto
degrade urea to NH4
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SUGAR FERMENTATIONTEST
Lactose Fermentation
SucroseFermentation
GlucoseFermentation
Mannitol
(+) : Yellow (-) : Red
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RESPON!!!
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WRITE DOWN
YOUR NAME, ID,GROUP,
ASSISTANTSNAME, and CODEA
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1
Write down 5 agents
that cause UrinaryTract Infections!
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2
Explain about
bacteriuria!
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3
What is the characteristic of :
a. Escherichia colib. Providencia stuartii
Minimal 5
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4
Write down 3 methods of
bacterial count!
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5
Good urine for bacteriological
examination is (..a..), except
for (..b..) examination, (..c..) is
usually used.
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6
Write down predisposition
factors of Urinary Tract
Infection (UTI) ! (min.5)
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7
Write down 3 kinds pigmen of
Pseudomonas aeruginosa!
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8
Biochemical test :Alcali acid,
H2S (-), and Indole (-).
Bacteria?
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9
Write down the biochemical test and
the interpretation of :
a. b.
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10
Biochemical test :Alcali
neutral, Mc Conkey Brown.
Bacteria?
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RESPON!!!
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WRITE DOWN
YOUR NAME, ID,GROUP,
ASSISTANTSNAME, and CODE B
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1
Write down 5 agents
that cause UrinaryTract Infections!
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4
Write down 3 methods of
bacterial examination!
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5
a. Write down 4 methods of
collecting urine specimen!
b. What is the best method to
collect the urine specimen?
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6
Write down predisposition
factors of Urinary Tract
Infection (UTI) ! (min.5)
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7
Write down 3 kinds pigmen of
Pseudomonas aeruginosa!
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8
Biochemical test :Alcali acid,
H2S (+), and Indole (+).
Bacteria?
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9
Write down the biochemical test and
the interpretation of :
a. b.
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