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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

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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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