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Culture Media

MSc. Sarah Ahmed

Types of culture media

Based on their consistency a) Solid medium b) Liquid medium c) Semi solid medium Based on the constituents/ ingredients a) Simple medium b) Complex medium c) Synthetic or defined medium d) Special media


Special media Enriched media Enrichment media Selective media Indicator media Differential media Sugar media Transport media Media for biochemical reactions Based on Oxygen requirement - Aerobic media - Anaerobic media


Solid media – contains 2% agarColony morphology, pigmentation, hemolysis can be appreciated.Eg: Nutrient agar, Blood agarLiquid media – no agar. For inoculum preparation, Blood culture, continuous culture.Eg: Nutrient brothSemi solid medium – 0.5% agar. Eg: Motility medium

Solid medium

Semi-solid medium
Liquid medium


Simple media / basal media - Eg: NB, NA (nutrient broth,nutrient agar )- NB consists of peptone, yeast extract, NaCl, NB + 2% agar = Nutrient agarComplex mediaMedia other than basal media.They have added ingredients.Provide special nutrients Synthetic or defined mediaMedia prepared from pure chemical substances and its exact composition is knownEg: peptone water – 1% peptone + 0.5% NaCl in water


Enriched media Substances like blood, serum, egg are added to the basal medium. Used to grow bacteria that are exacting in their nutritional needs. Eg: Blood agar, Chocolate agar
Chocolate agar
Blood agar


Enrichment media Liquid media used to isolate pathogens from a mixed culture.Stimulate growth of desired bacteriumInhibit growth of unwanted bacteriumMedia is incorporated with inhibitory substances(rarly) to suppress the unwanted organism → increase in numbers of desired bacteriaEg: Selenite F Broth – for the isolation of Salmonella, Shigella Tetrathionate Broth – inhibit coliformsAlkaline Peptone Water – for Vibrio cholerae

Selenite F Broth

Alkaline Peptone water
Tetrathionate Broth


Selective mediaThe inhibitory substance is added to a solid mediaStimulate growth of desired bacteriumInhibit growth of unwanted bacteriumIncrease in number of colonies of desired bacteriumEg:Desoxycholate citrate medium for dysentery bacilliMac Conkey’s medium for gram negative bacteriaTCBS – for V. choleraeLJ medium – M. tuberculosis

TCBS Thiosulphate citrate bile salt sucrose

Mac Conkey’s medium

LJ media (Lowenstein-Jensen)

Indicator mediacontain an indicator which changes its colour when a bacterium grows in themEg:Wilson-Blair medium – S. typhi forms black coloniesMcLeod’s medium (Potassium tellurite)– Diphtheria bacilli Wilson-Blair Medium
McLeod’s medium


Urease medium
Urease producing bacteriaUreaseUrea → CO2 + NH3NH3 → Medium turns pink

Differential mediaSubstances incorporated in it enabling it to distinguish between bacteria.Eg: Mac Conkey’s mediumDistinguish between lactose fermenters & non lactose fermenters.

MacConkey agar:Lactose fermenters – Pink coloniesNon lactose fermenters – colourless colonies

Transport mediaMedia used for transporting the samples.Delicate organisms may not survive the time taken for transporting the specimen without a transport media.Eg: Stuart’s medium – non nutrient soft agar gel containing a reducing agent & charcoalused for GonnococciBuffered glycerol saline – enteric bacilli


Anaerobic mediaThese media are used to grow anaerobic organisms.Have specific substances that absorb oxygen Eg: Robertson’s cooked meat medium, Thioglycolate medium.

PURPOSE OF CULTURE

To isolate bacteria in pure culture. Demonstrate their properties. Obtain sufficient growth for preparation of antigens and for other tests. Type isolates by methods such as bacteriophage and bacteriocin susceptibility. Determine sensitivity to antibiotics. Estimate viable counts and Maintain stock cultures.

METHODS

Streak culture. Lawn culture. Stroke culture. Stab culture. Pour plate culture. Liquid culture.



STREAK CULTURE
Routinely used method to isolate bacteria. One loopful of culture is made as a primary inoculum and is then distributed thinly over the plate by streaking it with the loop in a series of parallel lines in different segments of the plate. Loop flamed and cooled between the different sets of streaks. On incubation growth may be confluent at the site of the original inoculation but becomes progressively thinner and well separated colonies are obtained over the final series of streaks

LAWN CULTURE

Also called as carpet culture. Provides a uniform, growth of the bacterium. Useful for bacteriophage typing and antibiotic sensitivity testing. Also used in the preparation of bacterial antigens and vaccines. Prepared by flooding the surface of the plate with a liquid culture or suspension of the bacterium, pipetting off the excess inoculum and incubating the plate. Alternatively the surface of the plate may be inoculated by applying a swab soaked in the bacterial culture or suspension.

COTTON SWAB IS DIPPED IN CULTURE NEAR FLAME

SWAB CHARGED WITH CULTURE IS SWABBED ON PLATE

ANTIBIOTIC SENSITIVITY TESTING

STROKE CULTURE Stroke culture is made in tubes containing agar slope / slant. Uses Provide a pure growth of bacterium for slide agglutination and other diagnostic tests.


STAB CULTUREPrepared by puncturing a suitable medium – gelatin or glucose agar with a long, straight, charged wire.UsesDemonstration of gelatin liquefaction.Oxygen requirements of the bacterium under study.Maintenance of stock cultures.

POUR PLATE CULTURE Agar medium is melted (15 ml) and cooled to 45oC. 1 ml of the inoculum is added to the molten agar. Mix well and pour to a sterile petri dish. Allow it to set. Incubate at 37oC, colonies will be distributed throughout the depth of the medium. Uses Gives an estimate of the viable bacterial count in a suspension. For the quantitative urine cultures.

COLONY NUMBERS DECREASES WITH SERIAL DILUTION

COLONY COUNTER

LIQUID CULTURES Liquid cultures are inoculated by touching with a charged loop or by adding the inoculum with pipettes or syringes. Uses Blood culture Continuous culture methods

Blood culture bottles

ANAEROBIC CULTIVATION METHODS
To isolate anaerobic bacteria – which grow only in the absence of oxygen but grow only in the presence of carbon-di-oxide (20%)Creating Anaerobic condition called anaerobiosis established by various methods.In one way, Anaerobic condition can be created in a air-tight closed container – anaerobic jar (McIntosh & Filde’s jar)- made upof either stainless steel or polycarbonate/polypropyleneIn another way, anaerobic condition can be created by using anaerobic media in test tubes (contain reducing substance) provide anaerobic condition.


COLONY MORPHOLOGYForm- shape of coloncolor, surface, texture and sizeElevation-side view Edge- margin

Colony shape on agar plates

Rhizoid
Circular
Pinpoint

Margin of bacterial colony

Undulate
filamentous

Elevation of bacterial colony

Raised margin
Umbonate

Size of the bacterial colony Small Medium large

Texture of bacterial colony Dry Moist Viscid (stick to loop) Mucoid (mucus-like)


Colour of the colonies (pigmentation) Some bacteria produce pigment when they grow in the medium.


Opacity of the bacterial colony Opaque (not clear) Translucent (clear) Iridescent(shine)

THE END