cucunawangsih

COMMUNICATION BETWEEN PHYSICIAN AND LABORATORY

The laboratory needs to select appropriate test on
each specimen according to the clinical information given
(available on the request form)
Information routine:
Age
Main clinical condition
The date of onset of the illness
Information about antibiotic therapy
Other information such as antibiotic allergy, immunization,
suspected contact

Prior discussion with the microbiologist

SPECIMEN COLLECTION-GOOD QUALITY SPECIMEN

POOR-QUALITY SPECIMEN

USELESS RESULT
Optimal time of specimen collection
Correct site /types of specimen
Minimum contamination from the normal flora of
the patient or person collecting the specimen
Adequate quantities of specimen
Clearly labeled and safe specimen

SPECIMEN COLLECTION-GOOD QUALITY SPECIMEN

Sampling Sites, The Normal Flora and Interpretation of Result
Body sites that are normally sterile
Blood
Bone marrow
LCS
Tissue
Lower respiratory tract
Bladder

Body sites that have a normal commensal flora

Mouth, nose and upper respiratory tract
Skin
Gastrointestinal tract
Female genital tract

The responsibility of the clinician does not end with

collection of the specimen and requesting test.
Good communication with the microbiologist is essential.

TRANSPORT OF SPECIMENS TO THE LABORATORY

Specimen should be as fresh as nossible for the optimal
isolation of microbes
Transport as soon as
Organisms contaminating specimen from
normal flora may rapidly grow in specimen
kept at temperature room
Many pathogenic organisms do not survive
in temperature room
(Haemophilus influenzae, gonococci,
most viruses)
Urine or sputum should reach within 2 hours
of collection

Refrigerator
Medium transport
Stuart
Amies

Route from patient to

microbiologic diagnosis.
This scheme shows an
overview of the key
step in specimen
processing.
Culture min 18 h
incubation
Antibiotic susceptibility
test need more incubation
period
Alternative Ag/Ab
detection

LABORATORY PROCEDURES
Morphologic Identification

Light and electron microscopic

Staining (Gram, ZN)
Immunofluorescent microscopy for viral

Culture isolation and identification

Serology

Detection of Ag/Ab by immunologic assay:

Latex agglutination
EIA, ELISA

Molecular technique
Antibiotic resistance

DNA-DNA, DNA-RNA hybridization

PCR, RT-PCR

NON-CULTURAL TECHNIQUES
Microscopy is an important first step in the examination of all specimens
LIGHT MICROSCOPY
Wet preparation are used to demonstrate:
Blood cells/microbes in fluid specimen (urine, feces, CSF)

Differential staining technique:

a. Gram stain Gram positive (stain purple);
Gram negative (stain pink)
b. Acid fast staining used to detect mycobacteria
Special staining used to demonstrate particular
features of bacterial cells. Ex, granule in
Corynebacterium spp; lipid in Bacillus spp.
DARK FIELD MICROSCOPY
observing motility and thin cells such as
spirochaetes

NON-CULTURAL TECHNIQUES STAINING

a

g
c

a.
b.
c.
d.
e.
f.
g.

Gram (+) cocci in chain (streptococci)

Gram (+) cocci in cluster (staphylococci)
Gram (-) rods (ex. Escherichia sp.)
Gram (+) rods (bacillus)
Spirochaeta appearance
Large Gram (+) bacillus with subterminal
endospores
Acid fast bacill

NON-CULTURAL TECHNIQUES
NON-CULTURAL TECHNIQUE FOR DETECTION OF MICRIOBIAL PRODUCT
Non-specific techniques

Toxin detection

Fatty acid end-products of metabolism of

anaerobes can be detected in fluid
specimen by gas liquid chromatography

Detection of exotoxin
Clostridium botulinum toxin by injection of patients
serum into mice
Clostridium difficile cytotoxin in feces by addition of
suspension to cell culture
Clostridium perfringens and S.aureus enterotoxin in
feces by agglutination of antitoxin-coated latex particles
E.coli enterotoxin detected by tissue culture/animal
model

Antigen detection

Detection of soluble carbohydrate Ag by

agglutination of antibody-coated latex
particles or RBC, e.g.:
S.pneumoniae capsule in CSF/urine
Hib capsule in CSF/urine
Strep. pyogenes group Ag in throat swab Detection of endotoxin
Endotoxin from cell wall of Gram-negative bacteria
Detection of particular antigens by binding detected by Limulus lysate assay (clotting of extracts of
amebocytes of the horseshoe (Limulus) crab)
to antibodies labeled with:
ELISA for hepatitis B, rotavirus
Fluorescent molecule
Detection of genes encoding microbial
products with DNA probes

CULTURAL TECHNIQUES
Bacteria can be cultured on solid nutrient or liquid media
Different species of bacteria have different growth requirements
Viruses, Chlamydia, and rickettsia must be grown in cell or
tissue culture
Bacteria are indentified by simple characteristics and
biochemical properties
Ability to produce enzymes that can be detected by simple test
Ability to metabolized sugars oxidatively or fermentatively (aerobically or
anaerobically)
Ability to use a range of substrates for growth (e.g. glucose, lactose, sucrose,
maltose)

Antibiotic susceptibility can only be determined after the bacteria

have been isolated in a pure culture

TERIMAKASIH