Culture Methods 631t6i

Culture methods The indications for culture are : 1) isolate bacteria in pure culture 2) demonstrate their properties 3) preparation of antigens & other tests 4) type isolates by bacteriophage & bacteriocin susceptibility 5) sensitivity to antibiotics 6) estimate viable counts 7) maintain stock cultures

Methods Streak, lawn, stroke, stab, pour plate & liquid cultures Special method for anaerobic bacteria

Sweep plate method for bacteria in the dust on clothing

Streak culture (surface plating)  Routinely employed  A platinum loop (high cost) or nichrome resistance wire charged with the specimen  A loopful transferred to the periphery of a well dried plate

Streak culture

contd…

 Distributed thinly by streaking parallel lines  Then to different segments of the plate  The loop should be flamed & cooled between the different sets of streaks  Incubation  Growth confluent at the original inoculation  Well separated colonies over the final streak

The lawn or carpet culture  Provides a uniform surface growth  Useful for phage typing & antibiotic sensitivity testing (disc method)  To prepare bacterial antigens & vaccines  Prepared by flooding the surface with a liquid culture  Pipetted off the excess inoculum  Incubate  Inoculation by a swab soaked in bacterial suspension-alternate method

Lawn or carpet culture

Stroke culture Made in tubes containing agar slope Employed for providing pure growth for slide agglutination & other diagnostic tests

Stab culture  Medium punctured with a long ,straight charged wire  Employed for demonstration of gelatin liquefaction & oxygen requirement  To maintain stock cultures

Pour plate culture  Tubes containing 15 ml of the agar  Melted & left to cool in water bath at 45 – 50 c  Appropriate dilutions of the inoculum are added in 1 ml volume to the molten agar  Mixed well & poured into a sterile petridish  Allowed to set & incubate  Colonies enumerated using colony counters  Gives an estimation of viable bacterial count in a suspension  Recommended method for quantitative urine cultures

Pour plate method

Sweep plate method  Edges of the petridishes containing the medium are rubbed over the fabric  Colonies develop on incubation  Counted & estimates made

Liquid cultures  Made by touching with a charged loop in a broth  Large inocula can be employed  Method adopted for blood culture & for sterility tests – concentration of bacteria are small  Used for inocula containing antibiotics & other antibacterial substances – rendered ineffective by dilution in the medium  Preferred when large yields are desired  Disadvantage, does not provide a pure culture from mixed inocula.

Anaerobic culture methods Principles : 1.exclusion of oxygen or production of a vacuum 2.displacement of oxygen with other gases 3.absorption of oxygen by chemical or biological means 4.reduction of oxygen

Cultivation in vacuum  Inoculating cultures in a vacuum desiccator  Unsatisfactory as some oxygen always remains behind  Fluid cultures may boil over  The media may get detached from the plates  Not in use now

Displacement of oxygen with gases  H2, N2, helium or CO2  Popular but ineffective method is candle jar  Inoculated plates placed in a airtight container  Lighted candle kept & the lid sealed  Lighted candle expected to use oxygen  But some always left behind  Candle jar provides CO2 which stimulates the growth of most bacteria

Candle jar

Chemical methods  Alkaline pyragallol absorbs oxygen  Pyrogallic acid added to NAOH in a test tube  Placed inside an airtight jar  Disadvantage – small amount of CO formed inhibitory to some bacteria  Mixture of chromium & sulphuric acid (Rosenthal method)  Mixture of chromium & yellow phosphorous

Mc Intosh & Fildes jar  stout glass or metal jar with a metal lid

 Clamped airtight with screw  Lid has 2 tubes with taps  One act as the gas inlet,other as the outlet  2 terminals to connect electrical supply  Underside of the terminals of the lid a small grooved porcelain spool  A palledinised asbestos wraps around it  Inoculated plates placed inside the jar  Outlet tube connected to vacuum pump & air inside evacuated

Mc Intosh & Fildes jar

Mc Intosh contd……  The outlet tap closed  The inlet tube connected to a H2 supply & fill with it  Terminals are connected to a current supply  Heated asbestos acts as a catalyst,combination of H2 with the residual O2  Risk of explosion but rare  eliminated by alumina pellets coated with palladium,contained in a gauze sachet act as catalyst at room temperature

Gas pack  Method of choice  Disposable envelop has chemicals,generates H2 & CO2 on the addition of water  Inoculated plates are kept in the jar with the envelop  Water added  The lid screwed tight  Simple & effective  Indicator - reduced methylene blue,colorless anaerobically

Biological methods Incubation along with aerobic bacteria, germinating seeds or chopped vegetables Anaerobiosis is slow & ineffective with biological methods

Reduction of oxygen  Various reducing agents – 1% glucose, 1% thyoglycolate, 0.1% ascorbic acid & 0.05% cysteine  Pieces of red hot iron in broth – easy method  Broth containing many animal tissue s the growth of anaerobes  Robertson’s cooked meat medium is most widely used, consists of fat free minced cooked meat in broth  Even strict anaerobes grow  Indicates saccharolytic activity – red  Proteolytic activity - black

Glove box  Air-tight, glassfronted cabinet filled with inert gas  An entry lock for the introduction & removal of materials  Gloves for the insertion of hands for working

Methods of isolating pure cultures 1. Surface plating 2. Enrichment, selective & indicator media for the isolation of of pathogens from specimens like feces 3. Pretreatment of specimens with bactericidal substances which destroy unwanted bacteria E.g :isolation of T.B bacilli from sputum by treatment with alkali 4. Obligate aerobes & anaerobes,cultivation under aerobic & anaerobic conditions 5. Separation of bacteria with different temperature optima E.g :only thermophiles grow at 60c

Pure culture contd…. 6. By heating at 80 c vegetative forms can be

eliminated E.g : isolation of tetanus bacilli from dust 7. Separation of motile & nonmotile bacteria by Craigie’s tube.Motile traverse & appear at the top of medium 8. Inoculation into animals, pathogens produce fatal septicemia.Cultured pure from the heart blood 9. By using selective filters 10.Use of micromanipulators by which a single bacterium can be separated