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Culture Media

Introduction:
Difficult to identify bacteria by morphology alone.
They occur as mixed populations.
Separated on culture media & grown as pure cultures for study.

Types of media

Classification
1)

Solid media
Liquid media
Semisolid media

2)

Simple media
Complex media
Synthetic media
Special media

Special media
1. Enriched media
2. Enrichment media
3. Selective media
4. Indicator media
5. Differential media
6. Sugar media
7. Transport media
8. Aerobic media

9. Anaerobic media
SIMPLE MEDIA (BASAL MEDIA)
a.Nutrient broth

SIMPLE MEDIA (BASAL MEDIA)


a.Nutrient broth
Composition:

Grams/litre

Peptic Digest of animal tissue

10.00

Beef extract

10.00

Sodium chloride

5.00

Procedure
To prepare 1 litre of Nutrient Broth
Suspend 28 grams of M 001 powder in 1000 ml of Distilled Water
Boil to dissolve the medium completely
Dispense into the required number of test tubes (12x 75mm)
Sterilise by autoclaving at 15 lbs pressure (121 0C) for 15 minutes
2% agar added to this makes nutrient agar.
When reduced to 0 .2-0.5%-semi solid agar.
Enable motile organisms to spread.
When increased to 6%,prevents spreading or swarming by organisms such as
proteus.
Storage & Shelf life
Store below 300C
Store prepared media at 2-80C

b. Nutrient agar

Composition:

Grams/litre

Peptic Digest of animal tissue

10.00

Beef extract

10.00

Sodium chloride

5.00

Agar

15.00

Procedure
To prepare 1 litre of Nutrient Agar
Suspend 28 grams of M 001 powder in 1000 ml of Distilled Water
Boil to dissolve the medium completely
Sterilise by autoclaving at 15 lbs pressure (121 0C) for 15 minutes
Dispense into sterile petri plates in a laminar air flow cabinet
Storage & Shelf life
Store below 300C
Store prepared media at 2-80C

COMPLEX MEDIA
Ingredients added for special purposes to bring out certain characteristics and to provide
certain nutrients.

SYNTHETIC OR DEFINED MEDIA


Prepared from pure chemical substances
The exact composition is known
Used for various studies like metabolic requirements
E.g : Simple peptone water medium - 1% peptone with 0.5% sodium chloride
in water

COMPLEX MEDIA
1. Enriched media :
Blood, serum, egg are added to a basal medium-for bacteria more exacting in
their nutritional needs.
E.g : Blood agar, chocolate agar & egg media.
Blood and chocolate agar

a.Blood agar
Composition:

Grams/litre

Peptic Digest of animal tissue

10.00

Beef extract

10.00

Sodium chloride

5.00

Agar

15.00

Procedure I
To prepare 1 litre of Nutrient Agar
Suspend 28 grams of M 001 powder in 1000 ml of Distilled Water
Boil to dissolve the medium completely
Sterilise by autoclaving at 15 lbs pressure (1210C) for 15 minutes
Procedure II
Melt the required amount of Nutrient agar
Cool in a water bath at 500C
Add 5-10% sterile blood (human/sheep/horse)
Mix the blood and agar by gentle agitation from time to time
Pour as plates in a laminar air flow cabinet

Blood for use in the media must be collected with aseptic precautions to avoid
bacterial contamination.
It must be defibrinated to render it non-coagulable (sterile glass beads are used)
The sterile blood is immediately dispensed in 5 10 ml screw capped bottles &
stored in the refrigerator
Shelf life is 2 months
It must not be frozen
Culture response:
Organisms:
Staphylococcus aureus - Good luxuriant growth, showing beta hemolytic colonies
Streptococcus pyogenes Good growth, showing beta hemolysis

Store below 300C,Store prepared media at 2-80C


b.Chocolate agar:
Composition: Grams/litre
Peptic Digest of animal tissue 10.00
Beef extract 10.00
Sodium chloride 5.00
Agar 15.00
Procedure I
To prepare 1 litre of Nutrient Agar
Suspend 28 grams of M001 powder in 1000 ml of Distilled Water
Boil to dissolve the medium completely
Sterilise by autoclaving at 15 lbs pressure (1210C) for 15 minutes
Procedure II
Melt the required amount of Nutrient agar
Cool in a water bath at 750C
Add 10% sterile blood (human/sheep/horse)

Allow to remain at 750C.


Mix the blood and agar by gentle agitation from time to time till the blood becomes
chocolate brown in colour, within 10 minutes
Pour as plates in a laminar air flow cabinet
Culture response
Organisms
Escherichia coli Good luxuriant growth
Staphylococcus aureus - Good luxuriant growth
Pseudomonas aeruginosa Good luxuriant growth
Storage & Shelf life
Store below 300C
Store prepared media at 2-80C
2. Enrichment media :

In mixed cultures, unwanted bacteria overgrow the bacterium to be isolated.


Commensal bacteria overgrow pathogens.
E.g : S.typhi by E.coli in feces culture.
Substances incorporated to suppress unwanted bacteria.
But stimulating effect on bacteria to be grown.
Is a liquid medium
E.g : tetrathionate broth - inhibits coliforms but stimulate typhoid bacilli.
Selinite f broth.
3. Selective media :
Inhibiting substances added to a solid medium
Greater number of colonies formed by the required bacterium

E.g :Deoxycholate citrate medium for dysentery bacilli


a. TCBS media

Composition:

Grams/litre

Proteose Peptone

10.00

Yeast extract

5.00

Sodium thiosulphate

10.00

Sodium citrate

10.00

Ox gall

8.00

Sucrose

20.00

Sodium chloride

10.00

Ferric citrate

1.00

Bromothymol blue

0.04

Thymol blue

0.04

Agar

15.00

Procedure
To prepare 1 litre of TCBS Agar
Suspend 89 grams of M 189 powder in 1000 ml of Distilled Water
Boil to dissolve the medium completely

Immediately cool to 500C


DO NOT AUTOCLAVE
After cooling pour into sterile petriplates
Culture response
Culture characteristics after 18 to 24 hours at 37 0C - Organisms
Vibrio cholerae good to luxuriant, yellow

Vibrio parahemolyticus good to luxuriant, blue


Escherichia coli inhibited
Shigellae inhibited
Streptococcus faecalis inhibited
Storage & Shelf life
Store below 300C
Store prepared media at 2-80C
b.Xylose Lysine Deoxycholate Agar

It is a selective medium recommended for the isolation of Salmonella species


It is also a differential medium

Composition:

Grams/litre

Yeast extract

3.00

L Lysine

5.00

Lactose

7.5

Sucrose

7.5

Xylose

3.5

Sodium chloride

5.00

Sodium deoxycholate

2.5

Sodium thiosulphate

6.8

Ferric ammonium citrate

0.8

Phenol red

0.08

Agar

15.00

Procedure
To prepare 1 litre of XLD Agar
Suspend 56.68 grams of M 031powder in 1000 ml of Distilled Water
Heat with frequent agitation until the medium boils
Immediately transfer to a water bath at 500C

DO NOT AUTOCLAVE OR OVERHEAT


After cooling pour into sterile petri plates
Culture response
Culture characteristics after 18 to 24 hours at 37 0C - Organisms (ATCC)
Proteus mirabilis good to luxuriant, Yellow
Escherichia coli fair, yellow
Salmonella typhi good to luxuriant, red with black centres
Shigellae good to luxuriant, red
Staphylococcus aureus inhibited
Storage & Shelf life
SIMPLE MEDIA (BASAL MEDIA)
a.Nutrient broth
Composition:

Grams/litre

Peptic Digest of animal tissue

10.00

Beef extract

10.00

Sodium chloride

5.00

Procedure
To prepare 1 litre of Nutrient Broth
Suspend 28 grams of M 001 powder in 1000 ml of Distilled Water
Boil to dissolve the medium completely
Dispense into the required number of test tubes (12x 75mm)
Sterilise by autoclaving at 15 lbs pressure (121 0C) for 15 minutes
2% agar added to this makes nutrient agar.
When reduced to 0 .2-0.5%-semi solid agar.
Enable motile organisms to spread.
When increased to 6%,prevents spreading or swarming by organisms such as
proteus.
Storage & Shelf life
Store below 300C
Store prepared media at 2-80C

b. Nutrient agar
Composition:

Grams/litre

Peptic Digest of animal tissue

10.00

Beef extract

10.00

Sodium chloride

5.00

Agar

15.00

Procedure
To prepare 1 litre of Nutrient Agar
Suspend 28 grams of M 001 powder in 1000 ml of Distilled Water
Boil to dissolve the medium completely
Sterilise by autoclaving at 15 lbs pressure (121 0C) for 15 minutes

Dispense into sterile petri plates in a laminar air flow cabinet


Storage & Shelf life
Store below 300C
Store prepared media at 2-80C

COMPLEX MEDIA
Ingredients added for special purposes to bring out certain characteristics and to provide
certain nutrients.

SYNTHETIC OR DEFINED MEDIA


Prepared from pure chemical substances
The exact composition is known
Used for various studies like metabolic requirements
E.g : Simple peptone water medium - 1% peptone with 0.5% sodium chloride
in water

COMPLEX MEDIA
1. Enriched media :
Blood, serum, egg are added to a basal medium-for bacteria more exacting in
their nutritional needs.
E.g : Blood agar, chocolate agar & egg media.
Blood and chocolate agar

a.Blood agar
Composition:

Grams/litre

Peptic Digest of animal tissue

10.00

Beef extract

10.00

Sodium chloride

5.00

Agar

15.00

Procedure I
To prepare 1 litre of Nutrient Agar
Suspend 28 grams of M 001 powder in 1000 ml of Distilled Water
Boil to dissolve the medium completely
Sterilise by autoclaving at 15 lbs pressure (1210C) for 15 minutes
Procedure II
Melt the required amount of Nutrient agar
Cool in a water bath at 500C
Add 5-10% sterile blood (human/sheep/horse)
Mix the blood and agar by gentle agitation from time to time
Pour as plates in a laminar air flow cabinet
Blood for use in the media must be collected with aseptic precautions to avoid
bacterial contamination.
It must be defibrinated to render it non-coagulable (sterile glass beads are used)
The sterile blood is immediately dispensed in 5 10 ml screw capped bottles &
stored in the refrigerator
Shelf life is 2 months
It must not be frozen
Culture response:
Organisms:
Staphylococcus aureus - Good luxuriant growth, showing beta hemolytic colonies
Streptococcus pyogenes Good growth, showing beta hemolysis

Store below 300C,Store prepared media at 2-80C


b.Chocolate agar:
Composition: Grams/litre
Peptic Digest of animal tissue 10.00

Beef extract 10.00


Sodium chloride 5.00
Agar 15.00
Procedure I
To prepare 1 litre of Nutrient Agar
Suspend 28 grams of M001 powder in 1000 ml of Distilled Water
Boil to dissolve the medium completely
Sterilise by autoclaving at 15 lbs pressure (1210C) for 15 minutes
Procedure II
Melt the required amount of Nutrient agar
Cool in a water bath at 750C
Add 10% sterile blood (human/sheep/horse)
Allow to remain at 750C.
Mix the blood and agar by gentle agitation from time to time till the blood becomes
chocolate brown in colour, within 10 minutes
Pour as plates in a laminar air flow cabinet
Culture response
Organisms
Escherichia coli Good luxuriant growth
Staphylococcus aureus - Good luxuriant growth
Pseudomonas aeruginosa Good luxuriant growth
Storage & Shelf life
Store below 300C
Store prepared media at 2-80C

2. Enrichment media :

In mixed cultures, unwanted bacteria overgrow the bacterium to be isolated.


Commensal bacteria overgrow pathogens.
E.g : S.typhi by E.coli in feces culture.
Substances incorporated to suppress unwanted bacteria.
But stimulating effect on bacteria to be grown.
Is a liquid medium
E.g : tetrathionate broth - inhibits coliforms but stimulate typhoid bacilli.
Selinite f broth.
3. Selective media :
Inhibiting substances added to a solid medium
Greater number of colonies formed by the required bacterium
E.g :Deoxycholate citrate medium for dysentery bacilli
b. TCBS media

Composition:

Grams/litre

Proteose Peptone

10.00

Yeast extract

5.00

Sodium thiosulphate

10.00

Sodium citrate

10.00

Ox gall

8.00

Sucrose

20.00

Sodium chloride

10.00

Ferric citrate

1.00

Bromothymol blue

0.04

Thymol blue

0.04

Agar

15.00

Procedure
To prepare 1 litre of TCBS Agar
Suspend 89 grams of M 189 powder in 1000 ml of Distilled Water
Boil to dissolve the medium completely

Immediately cool to 500C


DO NOT AUTOCLAVE
After cooling pour into sterile petriplates
Culture response
Culture characteristics after 18 to 24 hours at 37 0C - Organisms
Vibrio cholerae good to luxuriant, yellow
Vibrio parahemolyticus good to luxuriant, blue
Escherichia coli inhibited
Shigellae inhibited
Streptococcus faecalis inhibited
Storage & Shelf life
Store below 300C
Store prepared media at 2-80C
b.Xylose Lysine Deoxycholate Agar

It is a selective medium recommended for the isolation of Salmonella species


It is also a differential medium

Composition:

Grams/litre

Yeast extract

3.00

L Lysine

5.00

Lactose

7.5

Sucrose

7.5

Xylose

3.5

Sodium chloride

5.00

Sodium deoxycholate

2.5

Sodium thiosulphate

6.8

Ferric ammonium citrate

0.8

Phenol red

0.08

Agar

15.00

Procedure
To prepare 1 litre of XLD Agar
Suspend 56.68 grams of M 031powder in 1000 ml of Distilled Water
Heat with frequent agitation until the medium boils
Immediately transfer to a water bath at 500C

DO NOT AUTOCLAVE OR OVERHEAT


After cooling pour into sterile petri plates
Culture response
Culture characteristics after 18 to 24 hours at 37 0C - Organisms (ATCC)
Proteus mirabilis good to luxuriant, Yellow
Escherichia coli fair, yellow

Salmonella typhi good to luxuriant, red with black centres


Shigellae good to luxuriant, red
Staphylococcus aureus inhibited
Storage & Shelf life
Store below 300C

4. Indicator media :

Has an indicator which changes color when a bacterium grows.


E.g : sulphite in Wilson & Blair medium
S.typhi reduces sulphite to sulphide,a black metallic sheen
Mc Leods medium black colonies due to tellurite reduction by Diphtheria
bacillus
Blood agar both indicator & differential
Bacteria lysing red cells show a clearing around their colonies
5. Differential media :
Substances incorporated bring out differing characteristics of bacteria & thus
distinguish them
a. Mac Conkeys medium
shows lactose fermenters as pink colonies,non lactose fermenters as colorless or pale
Composition :

Grams/litre
51.53

Peptic Digest of animal tissue

17.00

Proteose peptone

3.00

Lactose

10.00

Bile salts

1.5

Sodium chloride

5.00

Neutral red

0.03

Agar

15.00

Procedure
To prepare 1 litre of Mac Conkey Agar
Suspend 51.53 grams of M008 powder in 1000 ml of Distilled Water
Boil to dissolve the medium completely with gentle swirling
Sterilise by autoclaving at 1210C , 15 lbs pressure for 15 minutes
Cool to 45 50 0 C
Dispense into sterile petri plates in a laminar air flow cabinet

Culture response
Organisms
Escherichia coli luxuriant growth, pink colonies
Staphylococcus aureus growth inhibited
Proteus vulgarisluxuriant growth, colourless
Salmonella typhi - luxuriant growth, colourless

Storage & Shelf life


Store below 300C
Store prepared media at 2-80C

b. Mueller Hinton Agar


Mueller Hinton Agar is used for testing susceptibility of common and rapidly growing
bacteria using antimicrobial discs by Kirby Bauer method.
Kirby- Bauer recommended the use of this medium for performing antibiotic
susceptibility tests
Composition: Grams/litre
Casein acid hydrolysate 17.50

Beef heart infusion 2.00


Starch soluble 1.5
Agar 17.00
Procedure
To prepare 1 litre of Mueller Hinton Agar
Suspend 38grams of M1084 powder in 1000 ml of Distilled Water
Mix well & Boil to dissolve the medium completely
Sterilise by autoclaving at 1210C , 15 lbs pressure for 15 minutes
Dispense into sterile petri plates in a laminar air flow cabinet
Culture response (after 18 -24 hours at 37 0C)
Organisms
Escherichia coli luxuriant growth
Staphylococcus aureus luxuriant growth
Salmonella typhi - luxuriant growth
Storage & Shelf life
Store below 300C
Store prepared media at 2-80C