These slides explain how media preparation in microbiology lab for bacterial culture, history of culture media, types of culture media based on concentration and ingredients and practical section.

1. Preparation of Culture
Media
By: Reem Riyadh , BSC

2. Topics
 History of bacterial culture media
 Basic requirements and components of culture media.
 Types of culture media.
 Tools used in the preparation of media
 Tips for success media culture preparation

3. culture media :
Culture media gives artificial environment stimulating natural
conditions necessary for growth of bacteria.
Can be used to :
1) Enrich the numbers of bacteria.
2) Select for certain bacteria and suppress other.
3) Differentiate among different kinds of bacteria.

4. Major contribution to culture media

5. Basic requirements for culture media
 Nutrients:
• Energy source
• Carbon source
• Nitrogen source.
 Mineral Salt- Sulphate, phosphates, chloride & carbonate of K, Mg and Ca
 Optimal PH (7.2-7.4)
 Optimal Temperature (35-37C)
 Adequate oxidation-reduction potential
 Growth factor :
• Tryptophan for Salmonella Typhi
• X &V factor for H. influenza-

6. Components of culture media:
1. Agar-agar (gelling agent) :
• Isolated from seaweed, is not digested by bacteria.
• prepared by treating algae with boiling water
the extract is filtered, concentrated and then dried.
• Agar contain long chain polysaccharide.
• Melt at 95°C and Solidifies at 43°C.

7. Gelatin
Agar agar
Animal
plant
Source
YES
NO
Digested by microbes
28° C
95°C
Melt degree

8. 2. Water source for hydrogen and oxygen.
3.Protein source.
• Peptone
(complex mixture of partially digested enzyme protein)
• Meat extract .
4. Mineral salt – sodium chloride

10. Types of culture media
based on agar concentration (consistency)
Solid media (2% agar) Liquid media (absence of agar)
Semi solid media (0.2 -0.5% agar)

11. Types of culture media
Based on the ingredients
Remember: all media must be sterile before use

12. 1. Synthetic or defined medium
These type of media is prepared from ‘CHEMICALS’. Therefore,
their exact chemical composition is known. Defined synthetic
media are commonly used for research purposes. Further, they
are of two types: simple and complex.
Eg : peptone water-1% peptone+ 0.5%Nacl in water .

13. 1.1 Simple Synthetic Media
Simple synthetic media contain a carbon and energy source, an
source of nitrogen and various inorganic salts in buffered
1.2 Complex Synthetic Media
This media contains all the ingredients of simple synthetic
addition, it also contains amino acids, purines, pyrimidines and
growth factors depending upon the nature and growth
organism.
the chemical composition of complex media is unknown

14. 2. Routine laboratory media

15. 2.1 Simple or Basal medium
 Examples: Nutrient agar ,Nutrient broth and peptone water
 Consist of peptone, meat extract, sodium chloride and water
 Used to isolate separate colonies for studying.
1.Colony morphology
2.Pigmentation
3.Biochemical identification test

17. 2.2 Enriched media:
• Enriched media refers to the media which contain the nutrients
required to support the growth of a wide variety of organisms,
including some fastidious ones.
• This media are enriched by adding blood , serum or egg.
• Enriched media are solid media as they are agar-based.
• The examples of enriched media are blood agar, chocolate
agar, Loeffler’s serum, etc.

19. 2.3Enrichment media:
• Enrichment media refers to the liquid media that inhibits the
growth of unwanted bacteria.
• The examples of enrichment media:
 Selenite F broth: isolation salmonella and shigella
Tetrathionate broth: isolation salmonella, proteus and inhibit
coliform
alkaline peptone water (APW) :for vibrio cholera

21. 2.2Enriched Media 2.3Enrichment Media
Enriched media refers to the media which contain the nutrients required to
support the growth of a wide variety of organisms, including some fastidious
ones.
Enrichment media refers to the liquid media that inhibits the growth of
unwanted bacteria.
Enriched media allow the growth of a wide variety of microorganisms Enrichment media allow the growth of a particular type of microorganism in
the medium.
The enriched media facilitate the growth of fastidious microorganisms The enrichment media inhibits the growth of unwanted commensal or
contaminating bacteria
Enriched media are solid media as they are agar-based Enrichment media are liquid media
The examples of enriched media are blood agar, chocolate agar, Loeffler’s
serum, etc.
The examples of enrichment media are Selenite F broth, tetrathionate broth,
alkaline peptone water (APW), etc.

22. 2.4Selective media:
Principle: Differential growth suppression.
Selective medium is designed to suppress the growth of some
allowing the growth of others.
Selective medium is an agar-based (solid) medium so that individual
isolated.

23. Thayer Martin Agar
For Neisseria sp
Lowenstein Jensen Medium
For M. Tuberculosis

24. Thiosulfate citrate bile salts
sucrose(TCBS), Alkaline PH
Inhibit of most throat organism except
Diphtheria bacilli

25. Sabouraud Dextrose Agar (SDA)
PH5.4 isolate fungi

26. 2.5 Differentiate (Indicator) media :
These media contain an indicator which changes its colour when
bacterium grows in them.
E.g.:
Blood agar
Mac Coney's medium
Christensen’s urease medium

28. Mannitol salt agar
PH indicator: phenol
red
7.5% NaCl
Yellow :coagulase (+)
S.aureus
Pink: coagulase(-)
(epidermidis,saprophticus)
inhibit Gram (-)
MacConkey agar
PH indicator: Neutral Red
Crystal violet ,Bile salt
Pink: lactose fermenter
Yellow :non lactose fermenter
Inhibit gram+

29. 2.6Transport media :
These media are used when the sample will not be cultured soon
after collection.
Examples:
• Cary-Blair medium for faeces
• Amies medium for gonococci
• Stuart medium.
non nutritional medium which maintains the
viability of organisms without significant
multiplication.

30. 2.7 Storage media:
• These media are useful for storing the bacteria for a long period
of time. For Example, Dorset egg medium.

31. 2.8 Sugar media:
• Sugar media contains 1% “sugar” such as glucose, sucrose, lactose,
dextrose, mannitol, etc.
• in peptone water beside an appropriate indicator

32. •The indicator used in sugar media is Andrade’s indicator,
•A small tube (Durham’s tube) is kept inverted in the sugar
tube to detect gas production.

33. Based on oxygen requirement:
Anaerobic media:
These media are used to grow anaerobic organism.
Eg: Robertson’s cooked meat medium, Thioglycolate medium .

34. Practical culture media

37. Biological Safety Cabinet Pipette

38. Tips for success for media preparation
• Storage
• Selective materials are high quality
• Check the expired date

39. Problems caused by faulty culture media
preparation
+ Reduced growth
+ Atypical colonial morphology
+ Inhibition of target organism
+ Failure to inhibit competing flora
+ Reduced shelf life of prepared medium

42. Thank you
Any questions ??