Urine, faecal, wound, genital specimens,

Blood, Lower respiratory

Urine

Saluran kemih Normalnya steril.

Infeksi saluran kemih adalah serangan pada jaringan

saluran kemih oleh satu atau lebih mikroorganisme,
menghasilkan respons peradangan (dengan leukosituria)
dan gejala yang bervariasi.
BACTERIA MOSTLY INVOLVED
IN URINARY TRACT INFECTIONS
BACTERIA MOSTLY INVOLVED
IN URINARY TRACT INFECTIONS
 Koleksi pagi hari
Koleksi midstream
Collection Pengiriman dalam 2 jam
Suprapubic aspiration (SPA)
Uriline

Pemeriksaan makroskopik
Macroscopic
warna
examination
Kekeruhan

Test strip examination

Test strip Solved compounds
examination pH
cells
 Gram staining

Microscopy
examination
staining

Specific research

Ziehl Kinyoun-Gabett
staining
 Culture
isolation

Uriline
 Culture
Systematic research
media

E. coli / Proteeae Non-fermentative Staphylococcus Group B Yeasts

Enterobacteria Gram negative rods Streptococcus

CLED
agar

or

MacConkey agar MacConkey agar BAP

Culture Specific research
media

Streptococcus Corynebacterium Mycobacterium

Gardnerella

Columbia CNA agar +

5% sheep blood
 Culture Culture
Direct
Manual streaking
Manual streaking exam

1. Streak the specimen

35˚C* 18-24h*
2. Without
using a 1 or 10 µL calibrated
loop straight through the adding more 3. Still without adding
half of the plate. urine, spread more urine, inoculate
Begin with chromID the specimen the Columbia CNA agar
CPS ELITE on all the + 5% sheep blood
surface.
Incubation 18-24h at 35oC
Enumeration
Reading and interpretation for 10µL inoculum

Probable
contamination Doubtful test Probable infection
Faecal Samples
POLYMICROBIAL

Commensal flora vs
infectious diarrhea
Pathogen flora
 Shigella Salmonella

Yersinia

Campylobacter enterocolitica
(in certain
countries)
Adults
 Delivery within 24 hours
Collection Keep at 2/8°C

Liquid/solid
Macroscopic Aspect
examination Color
 Systematic research

Salmonella Shigella Yersinia Campylobacter

SS agar

Yersinia CIN agar Campylosel agar

Mac Conkey agar

 Specific research

E.coli E.coli O157 Clostridium difficile Vibrio

chromID® Vibrio
or chromID® Or
Mac Conkey agar SMAC CT agar TCBS
C.difficile agar
Anaerobic atmosphere

Eosin-Methylene Blue agar

Wound / pus swabs

POLYMICROBIAL

Primary Bites
& Wounds
Secondary
infection Abscess
…
 Skin infections Traumatic Suppurations from Subcutaneous
wound subcutaneous areas suppurations
which have a superficial
opening

Staphylococcus
Pasteurella Anaerobic bacteria E.coli
aureus
Streptococcus
Eikenella Various aerobic bacteria Salmonella
pyogenes
Staphylococcus Enterococcus

Clostridium Anaerobic bacteria

Various aerobic
Aeromonas
bacteria

Pseudomonas
Staphylococcus Streptococcus Enterobacteriaceae Anaerobes
Non-Enterobacteriaceae

Schaedler agar
+5% sheep blood
BAP Mac Conkey agar
LOWER RESPIRATORY
TRACT INFECTIONS
 PATHOGENS INVOLVED ACCORDING TO
CLINICAL DIAGNOSIS

• Streptococcus pneumoniae
Most • Haemophilus influenzae
frequently • Staphylococcus aureus
involved • Gram negative rods

Comunity • Neisseria meningitidis

acquired • Branhamella catarrhalis

Pneumonia

Hospital-
• Neisseria spp
acquired • Branhamella catarrhalis,
respiratory • Legionella pneumophila
infection • Legionella spp

Intensive care
unit,
immuno-
compromised • Pseudomonas aeruginosa
patients • Burkholderia cepacia
23
 Macroscopic Microscopic Culture Culture
Collection
examination examination isolation media

 
Delivery within 2 hours

 
Immediately if possible
Collection 
Storage at 2 to 8ºC

 
 Sample collection site






 
Origin
 
Macroscopic Aspect
 
Volume
examination 






 
 Gram staining for microbiology

Microscopic  
examination  Wet mount for cytology
/ staining  
 Giemsa for cytology
 
24
Ziehl (Kinyoun Gabett)
 Macroscopic Microscopic Culture Culture
Collection
examination examination isolation media

Liquefaction
dilution
Culture
isolation

sputolysin

25
 Macroscopic Microscopic Culture Culture
Collection
examination examination isolation media

Systematic research

Haemophilus influenzae Streptococcus Klebsiella pneumoniae Staphylococcus

Neisseria meningitidis pneumoniae Other Enterobacteriaceae
Branhamella catarrhalis

Haemophilus Columbia agar + Mac Conkey chromID®

chocolate 2 agar 5% sheep blood agar S. aureus

or

Chocolate agar + Chocolate agar + Columbia agar +

Poly ViteX™ Poly ViteX™ 5% sheep blood

or

Chocolate agar +
Poly ViteX™ VCAT3 agar
26
Blood
Orientation tests
oxidase
catalase
• To identify the family of bacteria, it is important to perform:

• Gram staining: differentiation between two families: and

• Respiratory enzymes research: Catalase and Oxidase tests

Classification of microorganisms

Gram Neg

Catalase Oxidase

? ?
catalase

Orientation test for Gram Positive Cocci

Intended Use: Differentiate Gram Positive Cocci
Staphylococcus spp.
Streptococcus spp.
 Uji Katalase

Reagent
3%

Streptococcus (-) from

Micrococcus–Staphylococcus (+)

(Madigan, et al., 2013)

Slide or tube testing Direct testing

H2O2

slide

O Immediate reaction
2
(within 5 sec) O2
Catalase + Catalase +
 Gram Positive cocci

Catalase Positive Catalase Negative

Bubbles= O2 emission No bubbles observed

Presence of catalase Absence of catalase

Staphylococcus spp Streptococcus spp

Oxidase

Orientation test for Gram Negative Bacilli

Intended Use: Differentiate Gram Negative Bacilli
Enterobacteriaceae
Non Enterobacteriaceae
Uji Oksidase
 Gram Negative bacilli

Oxidase Positive Oxidase Negative

Violet to purple color No color

Presence of oxidase Absence of oxidase
Non Enterobacteriaceae Enterobacteriaceae
 Main families of microorganisms

Unknown bacterium

Gram Pos Gram Neg

Cocci Bacilli

Catalase Pos Catalase Neg Oxidase Pos Oxidase Neg

Staphylococci Streptococci Non Enterobacteriaceae

Enterobacteriaceae
 CLASSIFICATION OF MICROORGANISMS*

Gram Pos Gram Neg

Cocci
Bacilli Cocci Bacilli
Catalase Pos

Diplococci (cofee beans) Oxidase Neg Oxidase Pos

Chains
Catalase Neg Clusters Spore
forming
Catalase Pos Non spore
forming
Streptococci Bacillus Enterobacteriaceae
Non
Neisseria Enterobacte
Staphylococci Corynebacterium
riaceae
* Aerobes or facultative aerobes
IDENTIFICATION API is an identification system combining:
Biochemical and enzymatic tests
®
API Specific data base (evolution new species, new classification)

1. ONPG 11. Gelatinase

2. Arginin dihydrolase (ADH 12. Glukosa
3. Lysine Decarboxylase Test (LDC) 13. Mannitol
4. ornithine decarboxylase test 14. Inositol
5. Citrate test 15. Sorbitol
6. H2S 16. Rhamnosa
7. Urea Test 17. Sucrosa
8. Tryptophane DeAminase 18. Melibiosa
9. Indole test 19. Amigdalin
10. VP 20. arabinose
 • Perform Gram staining and orientation tests to choose the
appropriated product for the identification

Unknown bacterium

Gram Pos Gram Neg

Cocci Bacilli

Catalase Pos Catalase Neg Oxidase Pos Oxidase Neg

Staphylococci Streptococci Non fermenters Enterobacteriaceae

API staph API 20 strep API 20 NE API 20 E

Name of the strip Reference Identification of Results in

API 20 E 20100 / 20160 Gram negative bacilli 24 hours

API 10 S 10100 Enterobacteria 24 hours

RapiD 20 E 20701 Enterobacteria 4 hours

API 20 NE 20050 Non Enterobacteria 24-48 hours

API staph 20500 Staphylococci 24 hours

API 20 Strep 20600 Streptococci 4-24 hours

API 20 C AUX 20210 Yeasts 48-72 hours

API Candida 10500 Yeasts 48-72 hours

API 20 A 20300 Anaerobes 24-48 hours

API Campy 20800 Campylobacter 24 hours

API Coryne 20900 Corynebacteria 24 hours

API NH 10400 Neisseria/Haemophilus 2 hours

API Listeria 10300 Listeria 24 hours

API 50 CHB/E 50300 / 50430 Bacillus 24-48 hours

API 50 CHL 50300 / 50410 Lactobacillus 24-48 hours

 STEP 1: Choice of the strip API workflow
According to microscopic examination (morphology and Gram staining) and
orientation tests catalase/oxidase

STEP 2: Inoculation

STEP 3: Incubation
4 hours – 24 hours at 30 - 37ºC,
according to the strip used.

STEP 4: Reading
Add revealing reagents requested in IFU
 Inoculation step
There is two parts in API wells: the tube and the cupule.
 
Two different atmosphere

Cupule
Aerobic zone

Tube
«Anaerobic » zone
 Inoculation step
anaerobic like strict anaerobic aerobic

Oil

Oil

GLU ADH CIT

 Reading step
Reactions are detected after incubation
• by spontaneous color changes

• or addition of revealing reagent

 Reading of the strips

• Read each test according to the reading table (in the IFU of the strip
used)

++ -+- -
1 2 4 1 2 4

• ++ -+- -
Convert the results to a digit profile
1 2 4 1 2 4

3 1
contoh
 Identification status

STATUS % of identification typicity

CORRECT

Excellent ≥ 99,9 ≥ 0,75

Very good 99≤ %ID ≤99,9 0,5 ≤ T ≤ 0,75
Good 90 ≤ %ID ≤ 99 0,25 ≤ T ≤ 0,5
Acceptable 80 ≤ %ID ≤ 90 0 ≤ T ≤ 0,25

STATUS WHY
Low discrimination the system cannot separate 2 taxa
 Complementary tests
INCORRECT

Doubtful profile Against test 0% or 100%

Unreliable identification %Id < 80
Unacceptable profile No taxon profile selected
APIWEB RESULT SHEET