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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
Grams/litre
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
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.
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
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
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
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
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
Grams/litre
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
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
COMPLEX MEDIA
Ingredients added for special purposes to bring out certain characteristics and to provide
certain nutrients.
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
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
2. Enrichment 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
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
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
4. Indicator media :
Grams/litre
51.53
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