Diagnosis of Bacterial Infection Bacterial Cultivation

1. Diagnosis of Bacterial Infection Bacterial Cultivation

2. Outline • General procedureof bacteriological Diagnosis • Culture medium • concept • categories • bacterial growth patterns • Inoculation and transfer techniques

3. Specimenscollection General Procedureof Bacteriological Diagnosis General rules The specimens should be representative of the infectious process; Sufficient material; Avoid the contamination of specimens; Be sent to the lab immediately in an appropriate method and examined ASAP. Be collected before using antimicrobial agents, e.g. antibiotics.

4. specimens Morphologic identification microscopy & staining Isolation, identification Biochemical tests Antigen detection EIA, ELISA, IF test, agglutination test Serological diagnosis (Ab titer) convalescent phase / acute phase≥4 Molecular Biological Diagnosis(hybridization, PCR, RT-PCR,etc) General Procedureof Bacteriological Diagnosis

5. Experiment 1 Bacterial Cultivation

6. Requirements for bacterial growth • Nutrients • Temperature H2O, C-source, N-source, Inorganic salts, Growth factors • pH • Gas incubator Temperature, gas culturemedium  Nutrients, pH

7. Culture medium • is the mixture of various nutrients that is suitable for the growth of microorganisms. • Types of Culture Media • based on the function and chemical components • based on the physical state

8. Based on the function and the chemical components: • Basic Medium --contains the basic nutrients for the most bacterial growth; --the base of other kind of media. --e.g. broth. • Nutrient Medium/Enriched Medium • Additional or special nutrients(e.g., serum, growth • factors, trace elements) are added to support some • fastidious bacterial growth. • e.g. blood agar.

9. Selective Medium the medium that can prevent the certain bacterial growth while permitting others. e.g. SS agar • Differential Medium Some special substrates and indicators are added into the media in order to produce a visual differentiation when several bacteria grow on the same kind of medium. e.g. EMB agar (Eosin-methylene blue agar).

10. S.dysenteriaeon EMB E.coli on EMB

11. Citrate slant Double sugar iron slant

12. Anaerobic Medium a medium for the cultivation of certain anaerobes. The medium contains reducing agent, such as non-saturation fatty acid.

13. Based on the physical state • Liquid medium: • Without agar. • for the proliferation of bacteria. • Solid medium: • 1.5-2.5% agar. • for the isolation and identification of bacteria • e.g., slant, Petri dishes/plates. • Semisolid medium: • 0.3-0.5% agar. • for the observation of bacterial motility and preservation of bacteria.

14. Bacterial growth patterns • In liquid medium: Superficial growth; Turbidity/diffuse; Precipitate growing; (sediment) • In solid medium: Confluent growth / Smear; Colony: a cluster of microorganisms growing on a solid medium. It is directly visible and arises from a single cell.

16. In semi-solid medium: • Only grow along the line of inoculation • Grow diffusely

18. General procedureof bacteriological Diagnosis • Culture medium • concept • categories • bacterial growth patterns • Inoculation and transfer techniques

19. Inoculation and Transfer Techniques • Streak-plate technique • Slant inoculation • Liquid medium inoculation • Semisolid medium inoculation

20. 1/10 II I I 1/5 Rotate 90 III 1/4 IV Streak-plate technique four-area streak plate technique Rotate plate 90 Flame loop Flame loop Rotate 90

21. Slant inoculation

22. Liquid medium inoculation

23. 2 students/group Inoculation method Culture mediaBacterial strains Streak plate 2 plates (1plate/student) A mixed liquid of S.aureus & E.coli Slant inoculation 2 slants (1slant/student) 1 E.coli slant Liquid medium 1 liquid medium 1 E.coli slant inoculation 1 liquid medium 1 B.subtitis slant Semisolid medium 1 semisolid medium 1 Proteus slant inoculation (Stabbing) 1 semisolid medium 1 S.dysenteriae slant Mark your freshly inoculated plate/tube; Shake up themixedliquid of bacterial strains before using it. Prepare Gram Stain & Acid-fast Stain. NOTE!!!