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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 Refrigerator

Medium transport Stuart Amies

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 hoursof collection

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

Culture isolation and identification

Serology

Molecular technique

Antibiotic resistance

Light and electron microscopic Staining (Gram, ZN) Immunofluorescent microscopy for viral

Detection of Ag/Ab by immunologic assay: Latex agglutination EIA, ELISA

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. Gram (+) cocci in chain (streptococci)b. Gram (+) cocci in cluster (staphylococci)c. Gram (-) rods (ex. Escherichia sp.)d. Gram (+) rods (bacillus)e. Spirochaeta appearancef. Large Gram (+) bacillus with subterminal

endosporesg. Acid fast bacill

a

c

e

b

d

f

g

NON-CULTURAL TECHNIQUES

NON-CULTURAL TECHNIQUE FOR DETECTION OF MICRIOBIAL PRODUCT

Non-specific techniques Fatty acid end-products of metabolism of anaerobes can be detected in fluid specimen by gas liquid chromatography

Antigen detectionDetection 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/urineStrep. pyogenes group Ag in throat swab

Detection of particular antigens by binding to antibodies labeled with: ELISA for hepatitis B, rotavirus Fluorescent molecule Detection of genes encoding microbial products with DNA probes

Toxin detectionDetection of exotoxin Clostridium botulinum toxin by injection of patient’s 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

Detection of endotoxin Endotoxin from cell wall of Gram-negative bacteria detected by Limulus lysate assay (clotting of extracts of amebocytes of the horseshoe (Limulus) crab)

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