Culture Methods Culture Methods Indications for culture Isolate

Culture Methods

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.

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

Aseptic technique

Streak Culture • Routinely employed for isolation • Platinum / Nichrome loops

Dr Ekta, Microbiology

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

ANTIBIOTIC SUSCEPTIBILITY TESTING

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

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

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.

05. 10. 08 Dr Ekta, Microbiology

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

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

Displacement Method • Displacement of O 2 with gases like H 2 , N 2 , He or CO 2. • Rarely produces complete anaerobiosis. e. g. Candle jar

Chemical or Biological Methods • Alkaline pyrogallol ( pyrogallic acid in Na. OH) absorbs O 2 • Yellow phosphorous • Rosenthal method - Mixture of chromium & sulphuric acid • Gaspak

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

Ø Germinating seeds or chopped vegetables

DISPLACEMENT AND COMBUSTION OF OXYGEN • Most reliable & widely used anaerobic method • Complete anaerobiosis • Catalyst – palladinised asbestos

Mc. Intosh - Fildes’ Jar

Gaspak • Method of choice for preparing anaerobic jars. • Commercially available as disposable envelope, containing chemicals which generate H 2 , CO 2 with the addition of water.

Reduction of Oxygen Using reducing agents: • 1% glucose • 0. 1% thioglycollate • 0. 1% ascorbic acid • 0. 05% cysteine

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

ANAEROBIC CULTURE MEDIA THIOGLYCOLLATE BROTH ROBERTSON’S COOKED MEAT MEDIUM

Automated Methods

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