CULTURE MEDIAMuhammad Salman Siddique

Tanveer-ul-Hassan

WHAT IS A CULTURE MEDIA?

A growth or culture medium is a gel or liquid substance designed to support the growth of micro organisms or cells.

There are different types of media used for different types of micro organisms or cells.

The most commonly used growth media for micro organisms are nutrient broths and agar plates.

Some fastidious organisms need specialized media for there growth.

WHAT IS AGAR?

Used for preparing solid medium. Obtained from seaweeds. No nutritive value. Not affected by the growth of Bacteria. Melt at 98⁰C and sets at 42⁰C. Approx. 2% agar is employed in solid

medium.

TYPES OF CULTURE MEDIA

Based on their consistencya) Solid medium b) Liquid mediumc) Semisolid medium

o Based on constituentsa) Simple mediumb) Complex mediumc) Synthetic and defined medium d) Special media

TYPES OF CULTURE MEDIA (CONT.)

Based on oxygen requirementsa) Aerobic mediab) Anaerobic media

Special mediaa) Enrichment mediab) Enriched mediac) Selective mediad) Indicator mediae) Differential mediaf) Sugar mediag) Transport mediah) Media for bio-chemical reaction

COMPLEX MEDIA

Media other than basal media. They have added ingredients. They provide special nutrients.

Synthetic or Defined Media Media prepared from pure chemical

substances and its exact composition is known. (E.g. peptone water - 1% peptone + 0.5% NaCl in water)

NUTRIENT AGAR

It contains all the elements that most bacteria need for growth.

It is a undefined and non-selective medium.

CONTENTS Peptone Lab-Lemco powder Yeast extract Sodium chloride Agar

PREPARATION

It is usually used at a concentration of 2.8 gm in every 100 ml distilled water.i. Prepare as instructed by the manufacturer.ii. Sterilize by autoclaving at 121⁰C for 15

minutes.iii. Pour aseptically to agar plate.iv. Date the medium and give it a batch

number.v. Store in a cool dark place.

Note:

Shelf life is up to 2 years.

pH of medium should be within the range of 7.2 to 7.6

USES

It is used for culturing almost every micro organism.

Non-fastidious organisms grows well on nutrient agar.

It is used for routine culturing in microbiology lab.

In the early 1900’s it is suggested for water testing.

BLOOD AGAR

It is an enriched and differential media. It is used to grow fastidious organisms. It contains defibrinated mammalian blood

(usually sheep or horse blood).

Note:

Sheep blood may contain inhibitors to Heamophilus Influenzae.

Expired, citerated, donor blood should not be used because this may contain substance inhibitory to the growth of some pathogens (e.g. β Hemolytic Streptococci).

CONTENTS

Blood agar base Enzymatic Digest of Casein ........... 15 g

Enzymatic Digest of Animal Tissue ........... 4 g

Yeast Extract ........... 2 g

Corn Starch ............ 1 g

Sodium Chloride ........... 5 g

Agar ............ 14 g Columbia agar Tryptone soya agar

PREPARATION

i. Prepare as instructed by the manufacturer.ii. Sterilize by autoclaving at 121⁰C for 15

minutes.iii. Transfer to a water bath at 50⁰C.iv. When the agar is cooled to 50⁰C, add the

blood aseptically and mix gently.v. Pour aseptically 15 ml in the sterile petri dish.vi. Date the medium and give it a batch number.vii. Store plates at 2⁰C - 8⁰C preferably in sealed

plastic bags to prevent loss of moisture.

Note:

Shelf life up to 4 weeks.

pH ranges from 7.2 to 7.6 at room temperature.

USES

It is used for the growth of fastidious organisms.

It is a differential medium based on the hemolytic reaction.

There are 2 types of hemolytic reactions shown by the organisms o blood agar plate.• α-Hemolysis• β-Hemolysis

CHOCOLATE AGAR

When blood agar is heated, the red cells are lysed and medium becomes brown in color. It is

referred to as chocolate agar and supplies the factors required for the growth Haemophilus influenzae. It is also used to culture nutritionally demanding pathogens such as Neisseria meningitides and Streptpcoccus pneumoniae

CONTENTS

Blood agar base Enzymatic Digest of Casein ........... 15 g Enzymatic Digest of Animal Tissue ........... 4 g Yeast Extract ........... 2 g Corn Starch ............ 1 g Sodium Chloride ........... 5 g Agar ............ 14 g Columbia agar Tryptone soya agar

PREPARATION

1. Prepare as describe for blood agar except after adding the blood, heat the medium in a 70⁰C in water bath until it becomes brown in color. This takes about 10-15 minutes during which time the medium should be mixed gently several time.

2. Allow the medium to cool to about 45⁰C, remix and pour in sterile petri dishes as describe for blood agar.

Note:

3. Care must be taken not to over heat or prolong the heating of the medium because this will cause it to become granular and unfit for use.

4. Date the medium and give it a batch number store the plates as describe for blood agar.

5. pH ranges from 7.1 to 7.5

PERFORMANCES

Test the medium by inoculating it with Haemophilus influenzae. After overnight incubation in a candle jar at 35–37 ⁰C and record the growth.

Uses To isolate Haemophilus influenzae & Neisseria gonorrhoeae.

TRIPLE SUGAR IRON AGAR (TSI)

o It contains 3 sugars – Glucose, Lactose and Sucrose.

o It is a composite media used to study different properties of a bacterium – sugar fermentation, gas production and H2S production.

o It is an orange red medium with a slant and a butt.

o It is recommended for the identification of member of Enterobactriaceae and Salmonella.

CONTENTS

It Contains three sugars:-I. GlucoseII. LactoseIII. Sucrose The Iron salt (Ferric citrate indicates H2S

production) Phenol red is the indicator. Agar pH of the medium – 7.4 ±0.2

TSI REACTIONS

Yellow – Acid Pink - Alkaline Yellow slant / Yellow butt – Lactose fermenters. Pink slant / Yellow butt – Non lactose fermenters. Pink slant / no colour change – Non fermenters Black colour – H2S production. Gas bubbles or crack in the medium – gas

production.

Note LF – E.coli, Klebsiella NLF – Salmonella, Shigella H2S - Proteus

TSI REACTIONS

Orgaism Slant Butt H2S Gas

E. Coli Yellow Yellow -ve +ve

Klebsiella Yellow Yellow -ve +ve

Citrobacter Red/Yellow Yellow d +ve

Enterobacter

Yellow Yellow -ve +ve

Salmonella Red Yellow Weakly +ve

-ve

Shegella Red Yellow -ve -ve

Proteus Red Yellow +ve d

SELENITE F BROTH

It is recommended as enrichment media for the growth of Salmonella from feces, urine and other pathological specimens

It is a selective medium.

COTENTS

Part A Casein enzymic hydrolysate Lactose Sodium phosphate Part B Sodium hydrogen selenite

PREPARATION

Suspend 4 grams of part B in 1000 ml of distilled water.

Add 19 grams of part A in suspension and mix well.

Sterilize in boiling water bath or free flowing steam at 100⁰C for 10 minutes (DO NOT AUTOCLAVE).

Note:

1. Store below 30⁰C in tightly closed container and prepared medium at 2⁰C – 8⁰C.

2. Use before expiry date on the label.

3. Date the medium and give it a batch number.

REACTIONS

Organism Recovery Color of colony

Escherichia coli No increase in number

Pink with bile precipitation

Salmonella typhii Good - luxuriant Colorless

Salmonella typhimurium

Good - luxuriant Colorless

TO