1. CULTURE MEDIA
Muhammad Salman Siddique
Tanveer-ul-Hassan

3. 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.


4. 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.


5. TYPES OF CULTURE MEDIA
Based on their consistency
a) Solid medium
b) Liquid medium
c) Semisolid medium

Based on constituents
a) Simple medium
b) Complex medium
c) Synthetic and defined medium
d) Special media
o

6. TYPES OF CULTURE MEDIA (CONT.)

a)
b)

a)
b)
c)
d)
e)
f)
g)
h)
Based on oxygen requirements
Aerobic media
Anaerobic media
Special media
Enrichment media
Enriched media
Selective media
Indicator media
Differential media
Sugar media
Transport media
Media for bio-chemical reaction

8. 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)

9. NUTRIENT AGAR
It contains all the elements that most bacteria need
for growth.
 It is a undefined and non-selective medium.


10. CONTENTS
Peptone
 Lab-Lemco powder
 Yeast extract
 Sodium chloride
 Agar


11. 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

12. 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.


13. 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).

15. 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


16. PREPARATION
i.
ii.
iii.
iv.
v.
vi.
vii.
Prepare as instructed by the manufacturer.
Sterilize by autoclaving at 121⁰C for 15 minutes.
Transfer to a water bath at 50⁰C.
When the agar is cooled to 50⁰C, add the blood
aseptically and mix gently.
Pour aseptically 15 ml in the sterile petri dish.
Date the medium and give it a batch number.
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.

17. 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

19. 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


21. 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


22. PREPARATION
1.
2.
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.
Allow the medium to cool to about 45⁰C, remix and
pour in sterile petri dishes as describe for blood agar.
Note:
1.
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.
2.
Date the medium and give it a batch number store the plates as
describe for blood agar.
3.
pH ranges from 7.1 to 7.5

23. 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.

24. TRIPLE SUGAR IRON AGAR (TSI)
o
o
o
o
It contains 3 sugars – Glucose, Lactose and
Sucrose.
It is a composite media used to study different
properties of a bacterium – sugar fermentation,
gas production and H2S production.
It is an orange red medium with a slant and a butt.
It is recommended for the identification of member
of Enterobactriaceae and Salmonella.

25. CONTENTS
It Contains three sugars:I.
Glucose
II.
Lactose
III. Sucrose
 The Iron salt (Ferric citrate indicates H2S
production)
 Phenol red is the indicator.
 Agar
 pH of the medium – 7.4 0.2


26. 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

27. 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

29. 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.


30. COTENTS
Part A
Casein enzymic hydrolysate
Lactose
Sodium phosphate
 Part B
Sodium hydrogen selenite


31. 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.

32. 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

34. TO