Bacteriological method — Anaerobic bacteria.pdf

Microbiology

1. Bacteriological method
Anaerobic bacteria

2. Methods of the oxygen reduction for anaerobic
bacteria culture:
Kitt-Tarozzimedium
MPB with 1% glucose and pieces of cooked liver
or fat-free meat on bottom for absorption of gases.
The medium should be heated before inoculation
for 10-20 minutes in order to remove the air
oxygen and then cool and inoculated. After
inoculation the medium should be covered with a
layer of sterile vaseline
The material is added into two test tubes, one of
which is heated to 80 °C for 20 minutes in order
to destroy the non-spore forms. The growth of
bacteria is determined by the turbidity of the
medium and the formation of bubbles of carbon
dioxide on the surface of a layer of liquid vaseline.
Non-spore bacteria grow only in a test tube
without heating, spore bacteria -clostridia -in
both tubes.

3. Methods of the oxygen reduction for anaerobic
bacteria culture:
Gas Pak

4. Methods of the oxygen reduction for anaerobic
bacteria culture:
Biological method

5. Isolation of anaerobic bacteriain pure culture from
clinical specimens for bacteria identification
Day 1
1.Patients’ samples are collected and inoculated into 2-4 tubes with
Kitt-Tarozzimedium (enrichment media). One tube is set unheated,
and the other is heated at 80
0
C for 20 minutes.
Incubate at 37
0
C for 18-24h
Day 2
1. Study of media turbidity and gases formation
-Whithoutheating -After 80
0
C heating for 25 min
Differentiation of non-sporingand sporinganaerobes,
anaerobes and aerobes, vegetative cells and
heat resistant spores

6. Isolation of anaerobic bacteriain pure culture from
clinical specimens for bacteria identification
Day 2
2. Smear preparation and staining:(Gram stain,
Special staining for spores demonstration)spores



Ozheshko stain




vegetative cells

7. Isolation of anaerobic bacteriain pure culture from
clinical specimens for bacteria identification
Day 2
3. Subculture of bacteria from enrichment media for pure culture
isolation may be run by two methods:
•Zeissler’smethod (transfer method).
On the firs and second plate the confluent growth may not allow
obtaining well-separated colonies. They will be seen then in the third
plate, where much less bacteria grow.
•Veinberg’smethod (sequential dilutions).
The colonies of anaerobes will appear black in this medium and the
separated colonies can be examined and extracted for subculture.
Incubate at 37
0
C for 18-24h

8. Scheme of pathogenic Clostridia pure culture isolation
(Zeisler’s method and Veinberg’s method) and identification
Specimen: ______________________________________________________________
1 stage Kit-Tarozzi liquid medium
After inoculation medium is heated
under 80
о
С for 25 min
1. 2.



Gram stain Ozheshko stain

2 stage Zeisler’s method Veinberg’s method


Sugar blood agar

Wilson-Blair agar


3 stage

9. Isolation of anaerobic bacteriain pure culture from
clinical specimens for bacteria identification
Day 3
•Veinberg’smethod (sequential dilutions).
The colonies of anaerobes will appear black in this medium and the
separated colonies can be examined and extracted for subculture.

10. Isolation of anaerobic bacteriain pure culture from
clinical specimens for bacteria identificationDay 3 Typical colonies should be examined and smear prepared for
checking of the purity of the colonies.
If the colonies are pure they
may be subcultured
Subculture of the typical pure colonies in Kitt-Tarozzi medium Obtaining of the enough
growth of bacteria for culture
identification
Day 4 Study of Kitt-Tarozzi media turbidity and gases formation

If positive – bacterial growth
occurs.
Smear preparation and staining:
Gram stain; Special staining for spores demonstration
The appearance of Gram+
rods with spores indicates the
presence of Clostridia
Subculture of bacteria from enrichment media in differential
(Hiss` media etc.) for pure culture identification.

If the bacterial growth is pure
culture the bacteria may be
subcultured
Day 5 Demonstration of sugarlytic and proteolytic properties,
Study of antigenic structure.
Run of neutralization reaction in lab animals.
Culture identification.
Identification of the type of
toxin for proper
immunotherapy.

11. Isolation of anaerobic bacteriain pure culture from
clinical specimens for bacteria identification
Species Carbohydrates fermentationSpores
location
Oxygen
requirements
Glu Lac Mal
1 Cl. botulinum
AG - AG
subterminalObligate
anaerobes
2 Cl. tetani
- - -
terminalObligate
anaerobes
3 Cl. perfringens
AG AG AG
subterminalmicroaerofilic
Differential properties of Clostridia spp.

12. Cultivation and identification of fungi.
Smear preparation and staining:
•Gram stain

13. Cultural properties of Candidapure culture when growing on
Sabouraund’s media.
Incubate at 28-30
0
C for 48h

14. Species GluLacMal Suc Gal Tregalose
C.albicans + - + - + +
C.tropicalis + - + + + +
C.pseudotropicalis + - - - - -
C.crusei + - - - - -
C.guilliermodii + - + + + +
C.parapsilosis + - - - + -
Identify Candida spp. according to diagnostic table 2.
Table 2
Biochemical properties of Candida spp.

15. Cultivation and identification of fungi.