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- 1. 1 Laboratory Diagnosis ofBacterial Infection Laboratory Diagnosis of Bacterial Infection
- 2.
- 3. Basic Principles forSpecimen Collection Basic Principles for Specimen Collection Collecting the correct specimen Collecting the correct specimen Endocervical swabs for GC Pernasal swabs for pertussis whole EMU for TB Sputum, not saliva Blood culture bottles, not clotted blood
- 4. Getting the specimento the lab Problems in delay or inappropriate storage delay in dignosis and treatment ------pathogens die ------contaminants overgrow Blood culture directly into incubator ------not refrigerator CSF straight to lab Don't put an entire surgical specimen into formalin ------send a portion to microbiology in a sterile container
- 5. Collecting the specimencorrectly Take an mid-stream urine ------avoids contamination with perineal flora CSF ------Avoid contamination ------Avoid bloody tap Blood cultures ------Avoid contamination with skin organisms
- 6. Infection Control Please beconsiderate to lab staff ------Label hazardous specimens Don't send specimens to the lab without proper packing ------Leaking or blood-stained specimens are not acceptable
- 7. Factors limiting usefulnessof bacteriological investigations wrong sample ------saliva instead of sputum delay in transport/ inappropriate storage ------CSF overgrowth by contaminants ------blood cultures insufficient sample/sampling error ------in mycobacterial disease patient has received antibiotics
- 8. Specimen Etiologic diagnosis (blood,urine,stool, cerebrospinal fluid,pus,secreta) Examination ofimmune responese of the body to an infectious agent Direct examination of the specimen (blood,urine,stool) Isolation, culture and identification of the agent. susceptibility to antimicrobial drugs Tissue cells serum Histochemical stain Antibody detection (ELISA,WB,RIA) Microscopic examination Light microscopy, EM,IEM Component of microbes Antigens(immunofluores cenece,solid-phase radioimmunoassay and ELISA Poison and toxicity test Experimental animal Nucleic acid (nucleic acid electrophoresis, nucleic acid hybridization,PCR ,gene sequencing and gene chips Examination of metabolite (biochemical characteristics) Procedures for detection of pathogens
- 9. 9 The main examinationmethods in diagnosis of bacterial infection The main examination methods in diagnosis of bacterial infection ※ Morphological examination ※ Isolation, culture and identification ※ Biochemical Reactions ※Antibiotic Susceptibility Test ※ Antibody detection ※ Antigens or Nucleic acids assay
- 10. 10 Morphological examination Morphological examination ※ ※Non-stained microscopic observation Non-stained microscopic observation ▲ ▲ Dark-field microscopy Dark-field microscopy ▲ ▲ Observe the movement of live bacteria Observe the movement of live bacteria
- 11. 11 ※ ※ Stained microscopicobservations Stained microscopic observations ▲ ▲ Gram stain Gram stain ▲ ▲ Acid-fast stain Acid-fast stain ▲ ▲ Fluorescence stain(such Fluorescence stain(such as Auramine stain as Auramine stain) ) ▲ ▲ methylene blue methylene blue stain metachromatic granule
- 12. 12 Isolation & Culture Isolation& Culture 1.Size 2. Shape 3. Color 4.Surface features 1.Size 2. Shape 3. Color 4.Surface features 5.Transparency 6. Hemolysis 5.Transparency 6. Hemolysis How to describe the feature of bacterial colonies on an agar plate?
- 13. 13 Biochemical Reactions Biochemical Reactions EVERYTHINGthat a living organism does is the result of the activity of an ENZYME, the SUMMATION of the activities of all an organism's enzymes equals its BIOCHEMICAL FINGERPRINT. That is, an organism is the totality of its enzymes, so by determining which enzymes are present in an unknown organism one can DESCRIBE & IDENTIFY that organism The theoretic basis of biochemical reaction
- 14. 14 Common Tests Toidentify Bacterial isolates △ Indole assay △ Methyl Red/Voges Proskauer test △ Citrate utilization △ H2S production （ hydrogen sulfide ） △ Urea hydrolysis △ Motility △ Lactose fermentation △ Sucrose fermentation △ Glucose fermentation & gas production △ Oxidase test △ ……
- 15. 15 Citrate utilization Citrate utilization SugarFermentation Sugar Fermentation H H2 2S Test S Test Examples of Common Biochemical Reactions Examples of Common Biochemical Reactions
- 16. 16 Antibiotic Susceptibility Test AntibioticSusceptibility Test The wide variation in susceptibility and high frequencies of drug resistance among strains in many bacterial species necessitates the determination of levels of resistance or susceptibility as a basis for the selection of the proper antibiotic for chemotherapy Antimicrobial Susceptibility testing can be down by : Minimum Inhibitory Concentration (MIC) Disk Diffusion Method Minimum Bactericidal Concentration (MBC)
- 17. Principle: The tube dilutiontest is the standard method for determining levels of resistance to an antibiotic. Serial dilutions of the antibiotic are made in a liquid medium which is inoculated with a standardized number of organisms and incubated for a prescribed time. The lowest concentration of antibiotic preventing appearance of turbidity is considered to be the minimal inhibitory concentration (MIC).
- 18. Different concentrationsof Tetracycline in Nutrient broth: Conc. in mcg/ml （ microgramme ） 0.1 0.2 0.4 0.8 1.6 3.1 6.3 12.5 Tetracycline, generally considered a bacteriostatic antibiotic, for this bacterium, has an MIC of 1.6 mcg/ml 1.Minimum Inhibitory Concentration (MIC) :
- 19. 2. Disk-diffusion Method(Kirby-Bauer Method): The disk-diffusion method (Kirby-Bauer) is more suitable for routine testing in a clinical laboratory where a large number of isolates are tested for susceptibility to numerous antibiotics. An agar plate is uniformly inoculated with the test organism A paper disk impregnated with a fixed concentration of an antibiotic is placed on the agar surface. Growth of the organism and diffusion of the antibiotic commence simultaneously resulting in a circular zone of inhibition in which the amount of antibiotic exceeds inhibitory concentrations. The diameter of the inhibition zone is a function of the amount of drug in the disk and susceptibility of the microorganism.
- 20. This testmust be rigorously standardized since zone size is also dependent on: inoculum size, medium composition, temperature of incubation, excess moisture and thickness of the agar. Zone diameter can be correlated with susceptibility as measured by the dilution method. Further correlations using zone diameter allow the designation of an organism as "susceptible", "intermediate", or "resistant" to concentrations of an antibiotic which can be attained in the blood or other body fluids of patients requiring chemotherapy.
- 21. Staphylococcus aureus (MRSA) Note the yellowish pigmentation of the bacterial lawn, and the lack of inhibition by the Oxacillin disk
- 22. Streptococcus pneumoniae (Pneumococcus): The brownish tint of the blood agar plate outside the zones of bacterial inhibition is caused by alpha-haemolysis.
- 23. Pseudomonas aeruginosa: Thegreenish tint of the lawn and plate in general is caused by the diffusible pigment made by the Pseudomonas aeruginosa itself.
- 24. 24 Serological Assays Serological Assays ※ ※Detection antibody in the patient’s serum Detection antibody in the patient’s serum ※ ※ A current infection should be A current infection should be △ △ IgM positive IgM positive △ △ A 4-fold or greater rise on antibody titer A 4-fold or greater rise on antibody titer ■ ■ the convalescent sample is usually taken 10-14 the convalescent sample is usually taken 10-14 days after the acute sample. days after the acute sample. ※ ※ Major drawbacks Major drawbacks ■ ■ A single IgG antibody titer is difficult to interpret, A single IgG antibody titer is difficult to interpret, of course, In certain diseases, a single titer of of course, In certain diseases, a single titer of sufficient magnitude can be used as presumptive sufficient magnitude can be used as presumptive evidence evidence ■ ■ Some exceptions Some exceptions
- 25. Molecular Biology Techniques A-Genetic probes (DNA or RNA probes): Detection of a segment of DNA sequence (gene) in unknown organism using a labeled probe Probe: consists of specific short sequence of labeled single- stranded DNA or RNA that form strong covalently bonded hybrid with specific complementary strand of nucleic acid of organism in question B- Polymerase chain reaction (PCR): Amplification of a short sequence of target DNA or RNA Then It is detected by a labeled probe C- Plasmid profile analysis: Isolation of plasmids from bacteria and determination of their size and number compared with standard strains by agarose gel electrophoresis
- 26. Microbes and humans Veryfew microbes are always pathogenic Many microbes are potentially pathogenic Most microbes are never pathogenic
- 27.
- 28. How do weknow that a given pathogen causes a specific disease?
- 29. Diagnosis and effectivetreatment of infection depends not just on isolating an organism, but in establishing a plausible link between the laboratory findings, recognised syndromes and the patient's clinical condition potential pathogen isolated from or detected in clinical samples recognised syndromes e.g. meningitis pneumonia patient's clinic condition
- 30. Exercises: 1. What arethe basic principles for specimen collection basic principles for specimen collection? 2. What’s antibiotic susceptibility test and its use antibiotic susceptibility test and its use? 3.What are the main examination methods in diagnosis of main examination methods in diagnosis of bacterial infection bacterial infection?