Post on 17-Jan-2016
description
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 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