PENJELASAN PRAKTIKUM

SISTEM UROGENITALIA
Jorvin Kurniawan Tandiarta
BAGIAN MIKROBIOLOGI
FAKULTAS KEDOKTERAN
UNIVERSITAS HASANUDDIN
2014

MICROBIOLOGY LAB ASSISTANTS

ACADEMIC YEAR 2013/2014
HAFIZ HAZWAN
JORVIN KURNIAWAN TANDIARTA
MARYAM BURHANUDDIN
MUHAMMAD FADLILLAH FIRSTIOGUSRAN
MUHAMMAD FUDAIL
MUHAMMAD RAHMAT RIDHA
MUTIAH MUFTIHATURRAHMAH
SITTI AISHAH
SRI WAHYUNI RAMADHAN
WAN ASHEKAL NAZUAN
ZULYUDISIAWAN MUIN

TATA TERTIB
LABORATORIUM MIKROBIOLOGI

Laki-laki
: Memakai KEMEJA, CELANA PANJANG KAIN,
dan
SEPATU TERTUTUP. Rambut TIDAK BOLEH
menyentuh
kerah kemeja.
Perempuan : Memakai KEMEJA, ROK PANJANG KAIN, dan
SEPATU
TERTUTUP. Rambut diikat ke atas dan bagi yang
berjilbab, jilbabnya dimasukkan ke dalam jas lab.
Perlengkapan yang WAJIB dibawa saat praktikum :
1. Jas lab
2. Name tag
3. Penuntun praktikum + pas foto berwarna 4x6
4. Masker
5. Handscoen
6. Pensil warna
7. Penggaris 30 cm (per kelompok)

PERSYARATAN
ASISTENSI & RESPONSI

Telah mengumpulkan Tugas Pendahuluan

PRAKTIKUM

Telah mengikuti Asistensi & Responsi

Memenuhi Tata Tertib Laboratorium Mikrobiologi
Membawa Perlengkapan Praktikum
UJIAN PRAKTIKUM

Telah mengikuti Praktikum

Telah mengumpulkan Laporan Praktikum
Memenuhi Tata Tertib Laboratorium Mikrobiologi
REMEDIAL UJIAN PRAKTIKUM

Telah mengikuti Ujian Praktikum namun belum dinyatakan

lulus Ujian Praktikum

INTRODUCTION
Urinary Tract Infection
Bacterial Agents
Causing UTI
Urine Sample &
Collection
Bacteriological
Examination
Biochemical Test

INTRODUCTION
Escherichia coli
Klebsiella
pneumoniae
Enterobacter
agglomerans

Providencia
stuartii
Providencia
alcalifaciens
Alcaligenes
faecalis

Proteus vulgaris
Pseudomonas
aeruginosa

Proteus mirabilis

URINARY TRACT INFECTION

Terjadinya infeksi pada daerah urogenitalia yang disebabkan oleh

infeksi bakteri pathogen.

Bakteriuria adalah suatu kondisi dimana di dalam urin didapatkan

adanya bakteri

dalam jumlah 105 cfu dalam 1 ml urin porsi

tengah atau 103 cfu dalam 1 ml urin melalui aspirasi suprapubik.

Piuria adalah suatu kondisi dimana di dalam urin didapatkan

adanya pus, atau white blood cells 5 cells/HPF atau > 10
cells/mm3.

BACTERIAL AGENTS CAUSING

URINARY TRACT INFECTION

Escherichia coli (80%)

Coagulase-Negative Staphylococci (7%)

Staphylococcus saprophyticus in young woman

Proteus mirabilis (6%)

Other Gram-Negative Bacteria (4%)

Other Gram-Positive Bacteria (3%)

-Hemolytic Group B Streptococcus in pregnant

woman

BACTERIAL AGENTS CAUSING

URINARY TRACT INFECTION

Other organism

Anaerobic bacteria (2% rare)

Legionella pneumophila

Mycobacterium tuberculosis Mycoplasma hominis

Mycobacterium spp.

Campylobacter spp.

Chlamydia trachomatis

Salmonella spp.

Gardnerella vaginalis

Shigella spp.

Haemophilus influenzae

Adenovirus

Ureaplasma
urealyticum

Escherichia coli

Esc
heri
chi
a

Family enterobacteriaceae
Gram-negative bacteria
Facultative anaerobic
Rod shaped
Commonly
found
in
lowerintestine
Have O, H, and K antigens

Slant
& Butt

Acid/Ac

H2S

Gas

Indole

Motili
ty

MR

the

VP

Citrat
e

Sugar Fermentation
Urea

Gluco
se

Lacto
se

Sucro
se

Manni
tol

Klebsiella pneumoniae

Kle
bsi
ella
pne

Family enterobacteriaceae
Gram-negative bacteria
Facultative anaerobic
Rod shaped
Commonly found in the mouth,
skin, and intestine
Only have O and K antigens

Slant
& Butt

H2S

Gas

Indole

Motili
ty

MR

VP

Citrat
e

Sugar Fermentation
Urea

Gluco
se

Lacto
se

Sucro
se

Manni
tol

Proteus vulgaris

Pro
teu
s
vul

Family enterobacteriaceae
Gram-negative bacteria
Facultative anaerobic
Rod shaped
Inhabits the intestinal tracts of
humans, animals, and raw meat, can
also be found in soil, water, and fecal
matter
(Mendiami
saluran
usus
manusia, hewan, dan daging mentah,
juga dapat ditemukan di tanah, air,
dan kotoran)

Slant
& Butt

H2S

Gas

Indole

Motili
ty

MR

VP

Citrat
e

Sugar Fermentation
Urea

Gluco
se

Lacto
se

Sucro
se

Manni
tol

Proteus mirabilis

Pro
teu
s
mir

Family enterobacteriaceae
Gram-negative bacteria
Facultative anaerobic
Rod shaped
Can
lead
to
the
formation
ofcrystalsofstruvite
and
calcium
carbonate
(Dapat
menyebabkan
pembentukan kristal struvite dan
kalsium karbonat)
Part of the normal flora of human
gastro-intestinal tract, can also be
found free living in water and soil

Slant
& Butt

H2S

Gas

Indole

Motili
ty

MR

VP

Citrat
e

Sugar Fermentation
Urea

Gluco
se

Lacto
se

Sucro
se

Manni
tol

Enterobacter agglomerans

Ent
ero
bac
ter

Family enterobacteriaceae
Gram-negative bacteria
Also known as Pantoea agglomerans
Aerobic bacteria
Rod shaped
Inhabits plants (grows symbiotically
with various plants), soil, water, and
such
species
includes
bacteria
reported as both commensal and
pathogen of animals and humans

Slant
& Butt

H2S

Gas

Indole

Motili
ty

MR

VP

Citrat
e

Sugar Fermentation
Urea

Gluco
se

Lacto
se

Sucro
se

Manni
tol

Providencia alcalifaciens

Pro
vid
enc
ia

Family enterobacteriaceae
Gram-negative bacteria
Facultative anaerobic
Rod shaped
Could cause Travelers Diarrhea

Slant
& Butt

H2S

Gas

Indole

Motili
ty

MR

VP

Citrat
e

Sugar Fermentation
Urea

Gluco
se

Lacto
se

Sucro
se

Manni
tol

Providencia stuartii

Pro
vid
enc
ia

Family enterobacteriaceae
Gram-negative bacteria
Facultative anaerobic
Rod shaped
Commonly found in the intestine

Slant
& Butt

H2S

Gas

Indole

Motili
ty

MR

VP

Citrat
e

Sugar Fermentation
Urea

Gluco
se

Lacto
se

Sucro
se

Manni
tol

Alcaligenes faecalis

Alk
alig
ene
s

Family alcaligenaceae
Gram-negative bacteria
Obligat aerob
Rod shaped

Slant
& Butt

H2S

Gas

Indole

Motili
ty

MR

VP

Citrat
e

Sugar Fermentation
Urea

Gluco
se

Lacto
se

Sucro
se

Manni
tol

Pseudomonas aeruginosa

Family pseudomonadaceae
Gram-negative bacteria
Obligat aerob
Coccobacillus bacteria
Have O, H, and K antigens
Have 3 pigments :
Pyocyanin (blue - green)
Pyoverdine
fluorescent)

(yellow

green

and

Pyorubin (red - brown)

Pse
udo
mo
nas

Slant
& Butt

H2S

Gas

Indole

Motili
ty

MR

VP

Citrat
e

Sugar Fermentation
Urea

Gluco
se

Lacto
se

Sucro
se

Manni
tol

Pengambilan Sampel Urine

Sampel urine harus diambil secara steril

Sterilisasi tidak boleh dilakukan dengan menggunakan
metode :
1. Wet heat sterilization dapat mengurangi volume
sampel urin karena proses dilusi
2. Chemical sterilization dapat meninggalkan
substansi kimia di dinding penampung, yang akan
membunuh bakteri

URINE COLLECTION

Good urine for bacteriological examination is morning

urine,
except
for
Mycobacterium
tuberculosis
examination, 24 hours urine is usually used.
Urine samples are collected in the following ways :
1. Mid Stream Urine
2. Urine Pocket
3. Supra-Pubic Punction
4. Catheterization

MID STREAM URINE

Most

commonly used urine

sample in bacteriological
examination of urine

If

properly done, may minimizing

the contamination or urethral
normal flora

MID STREAM URINE

Area

surrounding the external meatus is

clean with diluted detergent and clean
water (do not used disinfectants)
After removal the first excreted urine, next
urination is directly patched into a sterile
bottle (without touching the container or
other body parts of the patient)
Bottle is labeled and sent or kept in a cool
place (refrigerator, or with ice blocks)

URINE POCKET
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)

Usually

used for uncooperative patient

URINE COLLECTION: BAGGED

OR CLEAN CATCH
Contamination

rate of 10%
Incooperative patien
>105 CFU infection likely
104 105 CFU suspicious
<104 infection unlikely

SUPRA-PUBIC PUNCTION
The

most sterile way of collecting urine.

(Gold standard 99% specificty)
Requires to be done in the morning, as the
bladder must be in full condition
Supra-pubic is cleaned & sterilized with
70% alcohol and bethadine or salvon.
By using 5ml sterile spoeit, puncture
needle as deep 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.

CATHETERIZATION
Urine

catheterization should be avoided,

except for patients with permanent catheter.

Catheter

lead to irritation of at urogenital

tract mucosa & enhance the infection

For

patients with permanent catheter use

spoiet from the basal of disinfected catheter

URINE COLLECTION:
TRANSURETHERAL CATHERIZATION
>105

Colony Forming Units 95% specificity

104 105 CFU infection is likely
103 104 CFU Suspicious
<103 CFU infection unlikely

Pengambilan, Penyimpanan, Transportasi Urine

URINE
PORSI TENGAH

URINE
PUNKSI SUPRA PUBIK

KANTONG URINE

Pengambilan

SECARA STERIL

PUNKSI DGN SPOEIT

KANTONG URINE

Penyimpanan

BOTOL STERIL
4oC

BOTOL STERIL
4oC

KANTONG URINE
4 oC

Transportasi
DIBAWA SEGERA
DLM TERMOS BERISI ES

Laboratorium
SENTIRIFUS

PREPARAT GRAM

ISOLASI &
IDENTIFIKASI

HITUNG BAKTERI

BACTERIOLOGICAL
EXAMINATION
Gram

staining of direct preparation

Bacterial count
i) Pour plate method
ii) Surface drop method
iii) Dip slide method
Isolation & identification
Antibiotic sensitivity testing
Serotype / biotyping of bacterial agents

GRAM STAINING OF
DIRECT PREPARATION

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)

BACTERIAL COUNT
a)

Pour plate method

BACTERIAL COUNT
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 incubate overnight at 350C

Bacterial

growth:

Grade

0 no bacterial at the edge of droplet; 1 to 2

colonies only in the center of droplet

Grade

3 & 4 growth 105 cfu/ml; the edges of the

droplet will appear fully bacteria growth

BACTERIAL COUNT
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.

ISOLATION &
IDENTIFICATION
Urine

is dripped onto the surface of McConkey

/Nutrient agar/Blood agar/Chocolate agar.

Strike

with a loop or a swab on the media

surface

Culture

is incubated overnight & the appearance

of growing colony observed.

*If there are 2 types of bacterial growth;

considered as contaminated (repeat)

ANTIBIOTIC SENSITIVITY
TEST
Must be done
for observing
the bacterial
sensitivity to
antibiotics,
primarily to
those
commonly used
against
urogenital
infections.

SEROTYPE & BIOTYPE of

BACTERIAL AGENTS
For

epidemiological purposes,
sometimes it is necessary to determine
the serotype and biotype of isolated
bacterial agents.

Bacterial

biotype can be also

determined by observing the pattern of
antibiotic sensitivity called biogram.

BIOCHEMICAL
TEST

TSI (TRIPLE SUGAR IRON) TEST

Slant &
Alkali :
Asam :
Netral
H2S

Butt
Merah
Kuning
: Orange

(+) : Hitam
(-) : Warna dasar
Gas
(+)
: Medium
terangkat/ pecah
(-) : Medium tidak
terangkat

SIM (SULFIDE INDOLE MOTILITY) TEST

H2S
(+) : Hitam
(-) : Warna dasar
Indol
(+)
: Terbentuk cincin
merah
(-)
: Tidak terbentuk
cincin merah
Motilitas
(+) : Stab line diffuse
(-)
: Stab line tidak
berubah

METHYL RED / VOGES PROSKAUER

(MR/VP)
Methyl Red

(+) :

Merah

(-) :

Kuning / Orange

Voges Proskauer

(+) :

Merah

(-) :

Kuning / Colorless

CITRATE UTILIZATION & UREA

HYDROLYSIS TEST
Citrate Utilization

Urea Hydrolysis

(+) :

Biru

(+) :

Pink

(-) :

Hijau

(-) :

Kuning

SUGAR FERMENTATION

Lactose Fermentation

Sucrose Fermentation

Glucose Fermentation

Mannitol
(+) :
(-) :

Kuning
Merah

TSI
Acid

Alcal
i
Acid

Acid

E. Coli K. Pneumoniae

Proteu
s

Providen
cia

Citrate
&
Urease

Neutr
al

H2 S

Indol
e
+

Alcali

Mc
Conkey
Brown

Alkaligene
s

Indol
e
-

K. Pneumoniae E. Coli

+
Prot.
Vulgaris
Prov.
Stuartii

Mc Conkey
Brown +
Green

Prot. Mirabilis
Prov.
Alkalifaciens

Pseudomon
as

PREDISPOSITION FACTORS UTI

Obstruction

Pregnancy

Cateterization

Diabetes Mellitus

Tumor

Iritation

Than
k you