1. Diagnosis of Bacterial Infection Bacterial Cultivation

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

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

5. Bacterial Cultivation
Experiment 1
Bacterial Cultivation

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

7. Culture medium Types of Culture Media
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. E.coli on EMB
S.dysenteriae 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:
% agar.
for the isolation and identification of bacteria
e.g., slant, Petri dishes/plates.
Semisolid medium:
% 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 procedure of 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. Streak-plate technique
four-area streak plate technique I
1/10 II I
1/5
Rotate plate 90
Flame loop
Flame loop
Rotate 90
III
1/4 IV
Rotate 90

21. Slant inoculation

22. Liquid medium inoculation

23. Inoculation method Culture media Bacterial strains
2 students/group
Inoculation method Culture media Bacterial 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 E. coli slant
inoculation 1 liquid medium 1 B. subtitis slant
Semisolid medium 1 semisolid medium 1 Proteus slant
inoculation (Stabbing) 1 semisolid medium S.dysenteriae slant
Mark your freshly inoculated plate/tube;
Shake up the mixed liquid of bacterial strains before using it.
Prepare Gram Stain & Acid-fast Stain.
NOTE!!!