1. Department of Microbiology
Culture Methods
Dr. Mohit Bhatia
Assistant Professor
Department of Microbiology
AIIMS, Rishikesh

2. Culture Methods Indications for culture -
Isolate bacteria in pure cultures.
Demonstrate their properties.
Obtain sufficient growth for preparation of antigens & for other tests.
Typing bacterial isolates.
Antibiotic sensitivity.
Estimate viable counts.
Maintain stock cultures.

3. Types of culture methods
Streak culture or surface plating
Lawn or carpet culture
Stroke culture
Stab culture
Pour plate method
Anaerobic methods of culturing bacteria

4. Aseptic technique

5. Streak Culture Routinely employed for isolation
Platinum / Nichrome loops

6. Dr Ekta, Microbiology

7. Lawn or Carpet Culture Uniform surface growth Bacteriophage typing
Antibiotic sensitivity testing
Preparation of bacterial antigens & vaccines

8. ANTIBIOTIC SUSCEPTIBILITY TESTING

9. Stroke Culture Tubes containing agar slopes
For slide agglutination & other diagnostic tests.

10. Stab Culture
By puncturing a suitable medium with a long, straight charged wire.
For gelatin liquefaction, stock cultures & motility

11. Pour Plate Method
1 ml of appropriately diluted inoculum is added to 15 ml of molten agar and poured on petridish.
Colonies appear through out the depth of medium.
Used to estimate viable count, recommended method for quantitative urine cultures.

12.
Dr Ekta, Microbiology

13. Broth/Liquid Culture
Inoculated by a charged loop, pipette or syringes.
For blood cultures & sterility testing.

14. Anaerobic Culture Methods
Anaerobic condition can be achieved by:
Cultivation in vacuum
Displacement of oxygen with other gases
Chemical or biological methods
By displacement and combustion of oxygen
By reducing agents
Anaerobic chamber
Clostridium tetani
Clostridium histolyticum

15. Displacement Method
Displacement of O2 with gases like H2 , N2 , He or CO2 .
Rarely produces complete anaerobiosis.
e.g. Candle jar
Cultivation in vacuum was attempted by incubating cultures in a vacuum desiccators but it proved to be unsatisfactory. This method is not in use now .
Displacement of oxygen
Displacement of oxygen by inert gases like hydrogen, nitrogen,
carbon dioxide or helium is sometimes employed. Oxygen can never be removed completely by this method. A popular but ineffective method is use of candle

16. Chemical or Biological Methods
Alkaline pyrogallol ( pyrogallic acid in NaOH) absorbs O2
Yellow phosphorous
Rosenthal method - Mixture of chromium & sulphuric acid
Gaspak
Chemical Methods
Pyrogallol
First introduced by Buchner (1888)
Principle:-Alkaline pyragollol absorbs oxygen
Procedure:- a large tube containing solution of NaOH and pyragollol acid placed inside air tight jar produce an anaerobic conditions
Disadvantages:- small amount of CO is formed during the reaction, may be inhibitory to some bacteria
Chromium and sulphuric acid (Rosenthal Method)
Mixture of chromium and sulphuric acid is used for producing anaerobiosis
Principle:- two chemicals react in presence of oxygen
[O2]
Chromium + Sulphuric acid Chromous + anaerobic
sulphate condition

17. BIOLOGICAL METHODS
Absorption of oxygen from small closed systems has been attempted by incubation along with
Aerobic bacteria EXAMPLE:- Pseudomonas aeruginosa
Anaerobiosis produced by this method is slow and ineffective.

18. Germinating seeds or chopped vegetables

19. DISPLACEMENT AND COMBUSTION OF OXYGEN
Most reliable & widely used anaerobic method
Complete anaerobiosis
Catalyst – palladinised asbestos
Alumina pellets coated with palladium- catalyst at room temperature
Reduced methylene blue is used as indicator. Remains colorless anaerobically but turns blue on exposure to O2

20. McIntosh - Fildes’ Jar

21. Gaspak Method of choice for preparing anaerobic jars.
Commercially available as disposable envelope, containing chemicals which generate H2 , CO2 with the addition of water.
Gas pack
Commerically available disposable packet containing pellets of sodium borohydride, cobalt chloride, citric acid and sodium bicarbonate
Principle :- These chemicals generate hydrogen and carbon dioxide when water is added then hydrogen combines with oxygen in the presence of a catalyst
Now this technique is widely used for preparing anaerobic jars
Procedure:- after the inoculated plates are placed inside an air tight jar, the packet of “gas pack “ with water added is kept inside and the lid is tightly closed

22. Using reducing agents:
Reduction of Oxygen
Using reducing agents:
1% glucose
0.1% thioglycollate
0.1% ascorbic acid
0.05% cysteine

23. ANAEROBIC CHAMBER It is an anaerobic incubation system
It provides oxygen free atmosphere for inoculating culture media and for incubation
It is fitted with airtight rubber gloves to insert hands for working with specimens
The anaerobic chamber contains catalyst, desiccant, hydrogen gas, carbon dioxide gas, nitrogen gas and an indicator

24. ANAEROBIC CULTURE MEDIA
THIOGLYCOLLATE BROTH
ROBERTSON’S COOKED MEAT MEDIUM
Anaerobic broth is an easily prepared anaerobic medium into which pieces of red hot metallic iron are introduced. It is then layered over with sterile vaseline.
Anaerobic broth containing fresh animal tissue, such as rabbit kidney, spleen, testes or heart called Smith-Noguchi medium, supports the growth of many anaerobes.
The most widely employed anaerobic liquid culture are:-
-Thioglycollate broth
-Robertson’s cooked meat medium

25. Automated Methods Bactec - blood culture method
The sample to be tested is inoculated into one or more vials which are inserted into the BACTEC fluorescent series instrument for incubation and periodic reading.
Each vial contains a chemical sensor which can detect increases in CO2 produced by the growth of microorganisms.
The sensor is monitored by the instrument every ten minutes for an increase in its fluorescence, which is proportional to the amount of CO2 present.
A positive reading indicates the presumptive presence of viable microorganisms in the vial.

26. METHODS OF ISOLATING PURE CULTURES
Surface plating
Enrichment, selective & indicator media
Incubation at different temperatures
Heating
Craigie’s tube/U-tube
Animal inoculation
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