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Culture MethodsCulture Methods
II MBBS
Dr Ekta ChourasiaLecturer, Microbiology
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05.10.08 Dr Ekta, Microbiology
Culture MethodsCulture MethodsIndications 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.
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05.10.08 Dr Ekta, Microbiology
Types of culture methodsTypes of culture methodsStreak culture or surface platingLawn or carpet cultureStroke cultureStab culturePour plate methodAnaerobic methods of culturing
bacteria
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05.10.08 Dr Ekta, Microbiology
Aseptic techniqueAseptic technique
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05.10.08 Dr Ekta, Microbiology
Streak CultureStreak Culture
Routinely employed for isolation
Platinum / Nichrome loops
http://vudat.msu.edu/fileadmin/user_upload/vudat/flash/streakplate.swf
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05.10.08 Dr Ekta, Microbiology
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05.10.08 Dr Ekta, Microbiology
Lawn or Carpet CultureLawn or Carpet Culture Uniform surface growth
Bacteriophage typing
Antibiotic sensitivity testing
Preparation of bacterial antigens & vaccines
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05.10.08 Dr Ekta, Microbiology
Stroke CultureStroke CultureTubes containing agar slopesFor slide agglutination & other
diagnostic tests.
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05.10.08 Dr Ekta, Microbiology
Stab CultureStab Culture By puncturing a
suitable medium with a long, straight charged wire.
For gelatin liquefaction, stock cultures & motility
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05.10.08 Dr Ekta, Microbiology
Pour Plate MethodPour 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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05.10.08 Dr Ekta, Microbiology
Broth CultureBroth CultureInoculated by a
charged loop, pipette or syringes.
For blood cultures & sterility testing.
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05.10.08 Dr Ekta, Microbiology
Anaerobic Culture Anaerobic Culture MethodsMethods
Anaerobic condition can be achieved by
1. displacement of O2 with other gases.2. absorption of O2 by chemical or
biological means3. reduction of O2
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05.10.08 Dr Ekta, Microbiology
1. Displacement Method1. Displacement Method Displacement of O2
with gases like H2 , N2 , He or CO2 .
Rarely produces complete anaerobiosis.
e.g. Candle jar
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05.10.08 Dr Ekta, Microbiology
2. Chemical or Biological 2. Chemical or Biological MethodMethod
Alkaline pyrogallol ( pyrogallic acid in NaOH) absorbs O2.
Yellow phosphorous.Rosenthal method - Mixture of
chromium & sulphuric acid.McIntosh - Fildes’ Jar.Gaspak
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05.10.08 Dr Ekta, Microbiology
McIntosh - Fildes’ JarMcIntosh - Fildes’ JarMost reliable & widely
used anaerobic method
Complete anaerobiosis
Catalyst – palladinised asbestos
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05.10.08 Dr Ekta, Microbiology
GaspakGaspak Method of choice for preparing
anaerobic jars.
Commercially available as disposable envelope, containing chemicals which generate H2 , CO2 with the addition of water.
Reduced methylene blue is used as indicator. Remains colorless anaerobically but turns blue on exposure to O2
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05.10.08 Dr Ekta, Microbiology
3. Reduction of Oxygen3. Reduction of Oxygen Using reducing agents like 1% glucose,
0.1% thioglycollate, 0.1% ascorbic acid.
Robertson’s Cooked Meat Medium most widely used fluid medium Fat free minced cooked meat in broth,
with a layer of sterile vaseline over it.
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05.10.08 Dr Ekta, Microbiology
Automated MethodsAutomated 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.