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

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

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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.

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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 Clostridium tetani Clostridium histolyticum

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

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

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.

Germinating seeds or chopped vegetables

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

McIntosh - Fildes’ Jar

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

Using reducing agents: 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 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

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.

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