Medical Microbiology

Medical Microbiology
Department of Microbiology, HMU
Laboratory Diagnosis
For Bacterial Infections


Zhao-Hua Zhong, Prof.
Department of Microbiology
Harbin Medical University
Outline
Laboratory diagnosis for bacterial infections
Morphological -- microscopic
Molecular
Serological
Animal
Biosafty
Laboratory procedures
for infectious disease in humans
Morphologic identification of the agent in stains of specimens or sections of tissues (microscopy)
Culture isolation and identification of the agent.
Detection of antigen from the agent by immunologic assay (latex agglutination, EIA, etc) or by fluorescein-labeled (or peroxidase-labeled) antibody stains.
DNA-DNA or DNA-RNA hybridization to detect pathogen-specific genes in patients’ specimens.
Demonstration of meaningful antibody or cell-mediated immune responses to an infectious agent.
Assays
Morphological assays
Light or electron microscopies
Isolation and differentiation
Serological assays
Antigen-antibody assays
Molecular assays
Microorganism’s gene (DNA & RNA)

Principles for Specimen Collection
Sterile manipulation, avoid contamination
Obtain a specimen
from the infected site
according to the disease phase
before administrating antibiotics
Double sera for antibody detections, acute and recovery phase each
Positive if the titer of the later specimen is above 4X to the first specimen
Transport, and store correctly
as soon as possible
Store at -4oC for most bacterial specimens, but keep warm for Neisseria meningitidis and Neisseria gonorrhoeae
Store at -4oC ~ -70oC for viral specimens
General Rules
apply to all specimens
The quantity of material must be adequate.
The sample should be representative of the infectious process (eg, sputum, not saliva; pus from the underlying lesion, not from its sinus tract; a swab from the depth of the wound, not from its surface).
Contamination of the specimen must be avoided by using only sterile equipment and aseptic precautions.
General Rules
apply to all specimens
The specimen must be taken to the laboratory and examined promptly. Special transport media may be helpful.
Meaningful specimens to diagnose bacterial and fungal infections must be secured before antimicrobial drugs are administered. If antimicrobial drugs are given before specimens are taken for microbiologic study, drug therapy may have to be stopped and repeat specimens obtained several days later.

Bacteriological Diagnosis
Identifying the organism

Morphological
Isolation & culture
Biochemical reaction
Serological assays

Pathogenesis & antibiotics susceptibility
Animal experiment
Virulence test
antibiotics susceptibility
Morphological
Non-stained microscopic observation
Dark-field microscopy
Observing the movement of live bacteria
Stained microscopic observations
Gram stain
Acid-fast stain
Fluorescence stain
Isolation & Culture: Colony
Size
Shape
Color
Surface features
Smooth - Rough
Transparency
Hemolysis
Biochemical Reactions
Sugar Fermentation H2S Test Citrate utilization
Serological Assays
Detection antibody in the patient’s serum
A current infection should be
IgM positive
A 4-fold or greater rise on antibody titer between the acute serum sample and the convalescent serum sample
Major drawbacks
A single IgG antibody titer is difficult to interpret because it is unclear whether it represents a current or a previous infection
the convalescent sample is usually taken 10-14 days after the acute sample. By this time, the patient has often recovered and the diagnosis becomes a retrospective one
Some exceptions
In certain diseases, a single titer of sufficient magnitude can be used as presumptive evidence of a current infection
Animal experiment
Animals
　Mouse　Guinea Pig　 Rabbit Dog Monkey
Inoculation routes
Intradermal Subcutaneous
Intraperitoneal　 Intravenous　
Intracranial / intracerebral Intraspinal
Intranasal Lavage
Virulence Test
Median lethal dose, LD50
Median infective dose, ID50

Elek Plate
Diphtherotoxin
Corynebacterium diphtheriae　　
Enterotoxin
enterotoxigenic E. coli
Antibiotic Susceptibility Test
Method
MIC & MBC
Minimum Inhibitory Concentration，MIC
Minimum Bactericidal Concentration，MBC

Bactericidal drugs usually have an MBC equal or very similar to the MIC
Bacteriostatic drugs usually have an MBC significantly higher than the MIC

Bacterial Proteins, DNA & RNA
Antigens (Proteins)
Known antibodies
Agglutination, coagulation, precipitation, ELISA, Immunofluorescence, radioimmunoassay
DNA & RNA
PCR
Nucleic acid hybridization
Detection of Bacterial Antigens
Precipitation test
Coagglutination test
Immunoflorecence, IF
McAb technique
Bacterial DNA & RNA
PCR
DNA probe & hybridization
DNA hybridization (southern blot)
Plasmid fingerprint analysis
Restrict multimorphologic analysis
PCR
Gene Chip / microarray
Diagnosis Strategy for Bacterial Infection
Control, Prevention, & Treatment for Bacterial Infections
Control
Sterilization
Disinfection
Prevention
Vaccines
Kill vaccine
Live vaccine, attenuated vaccine
Genetic engineering vaccine
Subunit vaccine
Polypeptide vaccine
DNA vaccine

Active immunization
Immunological products
Antiserum
Antitoxin
Immunoglobulin




Passive immunization
Therapy
Antibiotics