bacterial culture methods
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8/7/2019 Bacterial Culture Methods
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Dr.T.V.Rao MD
BACTERIAL CULTURE
METHODSBASICS
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• Based on bacterial
metabolism*• Culture pH
• Culture oxidation- reduction
potential.• Gaseous requirements
• Oxygen, Carbon dioxide
and other gases
BACTERIAL NUTRITION AND THE DESIGN
OF CULTURE MEDIA
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• Used to grow bacteria
• Can be used to:
• Enrich the numbers of
bacteria
• Select for certain bacteriaand suppress others
• Differentiate among
different kinds of bacteria
CULTURE MEDIA
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• Aerobs
• Anaerobs (do not require
oxygen)
• Obligate anaerobs (die inthe presence of Ooxygen
)
• Facultative anaerobs(E.coli)
• Microaerophilic bacteria
OXYGEN CONCENTRATION
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• Isolation
• Properties of bacteria• To create antigens for
laboratory use
• Typing with Bacteriophagesand Bacteriocins susceptibility
• To test for Antibiotic sensitivity
• Estimate viable counts• Maintain stock cultures
PURPOSE OF CULTURING
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• 1. Surface plating
• 2 Enrichmentmedium
• 3 Selective medium• 4 Indicator medium
METHODS OF ISOLATION OF PURE
CULTURE WITH ..
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• Psychrophiles: with optimumgrowth T around 20 C
• Mesopihles: between 15 and45 with optimum around 37 C
• Thermophiles: between 30and 75 with optimum around
55 C• Hyperthermophiles: T grater
than 100C
TEMPERATURE ( CHARACTERISTIC RANGES)
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TEMPERATURE AND BACTERIAL
GROWTH
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• pH
• Most bacteria grow
between pH 6.5 and
7.5
• Molds and yeasts grow
between pH 5 and 6
• Acidophilic grow inacidic environments
THE REQUIREMENTS FOR GROWTH:
PHYSICAL REQUIREMENTS
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CULTURING • Used to grow bacteria
• Can be used to:• Enrich the numbers of
bacteria
• Select for certain bacteriaand suppress others
• Differentiate amongdifferent kinds of bacteria
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• Streak culture
• Stroke
• Stab
• Pour plate• Liquid culture
• Special methods for
anaerobic cultures
METHODS TO ISOLATE THE
BACTERIA
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HOW TO INOCULATE A CULTURE PLATE
• Plate: provide largesurface for isolation andobservation of colonies
• Using a sterile loop or asterile swab streak your sample on the Petri plate
• Important let your sterilizedloop cool before you pick up
your sample
How to inoculate a plate
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DIFFERENT METHODS OF CULTURING
BACTERIA
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MACCONKEY AGAR• Example:: MacConkey agar
has color indicator thatdistinguishes presence of
acid. Bacteria that ferment a
particular sugar (e.g.,glucose in culture media) will
produce acid wastes on
plates, turn pH indicator red.
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• Shape
• Size
• Elevation
• Edge• Surface
• Opacity
• Consistency
COLONIES - MAKE A OBSERVATION
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LIQUID MEDIALiquid media: easiest to
prepare and use. Good for growing quantities of
microbes needed for
analysis or experiments.Unless inoculated with
pure culture, cannot
separate differentorganisms.
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SLANT OBSERVATION
http://www.rlc.dcccd.edu/MATHSCI/reynolds/MICRO/lab_manual/slant_patterns.jpgDR.T.V.RAO MD 20
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STREAK CULTURE• Lawn or carpet culture
to create uniformsurface of organisms
• Bacteriophages typing• To obtain large
amount of antigens
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CULTURING THE MICROBES NEEDS SKILLS
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STREAK PLATE
Figure 6.10a–bDR.T.V.RAO MD 23
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METHODS OF ISOLATION OF PURE
CULTURE
• 1. Surface plating
• 2 Enrichment
medium
• 3 Selective medium
• 4 Indicator medium
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LIQUID CULTURING• Liquid cultures are done
in
• Tubes
• Bottles
• Flasks
• Blood culture• Water analysis
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STAB CULTURE• Puncturing suitable
medium such asnutrient agar, gelatin,
• Observe gelatinliquefaction
• Preserving the stockculture.
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MULLER HINTON AGAR FOR ANTIBIOTIC TESTING
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MEASURING THE ZONE OFINHIBITION
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MINIMUM INHIBITORY CONCENTRATION
DETECTS ANTIBIOTIC SENSITIVITY PATTERNS
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•Anaerobic Bacterial Isolationand Identification Needs
specified conditions
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DESICCATOR• In Desiccator some oxygen
is left
Not suitable for fluid culture
Displacement of oxygen isdone with
HydrogenNitrogen
Helium
Co2
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CANDLE JAR• Inoculated plates are kept
• Burning candle use up alloxygen
• But a little o2 is left
• But presence of Co2
stimulates the most
bacterium
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MAC IN TOSH FILDES ANAEROBIC JAR
• Contain inlet and outlet
• Electrical supply
• Inoculated culture plates
• When electrifiedpalladinised asbestos
heating acts as catalyst for
combination of hydrogen
with residual oxygen causes
complete anaerobiasis
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OTHER REDUCING AGENTS
• Reducing agents
O.1% Thiglyclolate
0.1% Ascorbicacid
0.05 % cysteine
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LOWENSTEIN JENSEN MEDIUM - CULTIVATION OF
MYCOBACTERIUM TUBERCULOSIS
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WORKING WITH MYCOBACTERIUM NEEDS
BIOSAFTEY CONCERNS
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