1. Culture Media (Types, Preparation & Sterilization)
Microbiology Lab (5)
Culture Media (Types, Preparation & Sterilization)
Abdelraheem BA

2. Introduction Microorganisms (MOs) are ubiquitous.
Culture: growth in media containing living MOs.
Pure culture Vs Mixed culture.
Culture media: solution which contains nutrients needed for bacterial growth.

3. Nutrients in media Water. Source of C. Source of N. Buffer system.
To dissolve materials to be transported across cytoplasmic membrane.
Source of C.
Required for the construction of all organic molecules.
Usually glucose.
Source of N.
Examples:
Proteins: broken down into amino acids.
Beef extract: broken down into nucleic acids.
Buffer system.
Most bacteria grow at pH 7.0.
This is achieved by using a buffer system;
Carbonate buffer.
Phosphate buffer.
Source of minerals.
Required in small amounts (Iron, Sulfur, phosphorus… etc)

4. Types of media
Media can be classified according to any of the following criteria:
Composition.
Content / percent of agar.
Purpose.
Agar:
The usual solidifying agent for media.
A hydrocolloid derived from red algae.
Properties:
Melting point: 100 degrees.
Remains liquid until cooled to 40 degrees.
Cannot be metabolized by most bacteria.

5. Types of media
Culture media can be classified into two categories according to their composition;
Chemically-defined (Synthetic).
The exact chemical composition is known.
Prepared from biochemicals off the shelf.
Example: Minimal Salt Medium.
Complex (Undefined).
The exact composition is not known.
Usually contain complex materials of biological origin, such as;
Blood.
Milk.
Yeast extract.
Beef extract.

6. Types of media According to percent of agar: Broth media (liquid).
No agar is added.
Example: Trypticase soy broth media.
Semisolid media.
<1% agar.
Example: Motility media.
Solid media.
% agar.
Example: Trypticase soy agar.

7. Solid media Solid media are divided into three forms:
Slanted agar position.
Deep agar position.
Agar plate media.

8. Agar plate Provide large surface area.
Used for isolation and subculture purposes.
Always incubated in inverted position to avoid water condensation that forms on the surface of petridishes.

9. Slant & Deep Agar Slant agar:
Used for maintenance and preservation of pure cultures for subculture purposes.
Deep agar:
Used for storage.
Also used for studying the gaseous requirements for MOs.

10. Types of Media According to purpose: All purpose medium (simple).
Enriched medium.
Differential medium.
Selective medium.
Selective-Differential medium.
Enrichment-Differential medium.

11. Simple media These media support growth of most bacteria.
They don’t have inhibitors or pH indicators.
Examples:
Trypticase Soy broth.
Nutrient broth.

12. Enriched media These media contain: Examples: The base growth medium.
Special supplements.
Examples:
Blood Agar (also differential).
Chocolate Agar.

13. Differential media More than one type of MOs grow on it.
Separation is based on color and other characteristic differences.
Example:
Blood Agar.
It separates bacteria according to the type of hemolysis. (Next Slide)

14. Hemolysis in blood agar
Some bacteria have hemolytic substances which breakdown red blood cells (Hemolysin).
Types of hemolysis:
Alpha (partial) hemolysis.
Beta (complete) hemolysis.
Gamma or no hemolysis.

15. Hemolysis in blood agar
Alpha hemolysis:
Greenish zone surrounding colonies.
These bacteria have hydrogen peroxide which oxidizes Hb into MetHb.
Examples:
Streptococcus pneumoniae.
Streptococcus viridans.

16. Hemolysis in blood agar
Beta hemolysis:
Yellow zone surrounding colonies.
These bacteria contain Streptolysin (an exotoxin).
Streptolysin O:
Oxygen sensitive, results in β-hemolysis under the surface.
Streptolysin S:
Oxygen stable, results in β-hemolysis on the surface.
Example:
Streptococcus pyogenes / GAS (Group A Streptococci).

17. β-hemolysis

18. Selective media They allow the growth of one or more species.
They also suppress the growth of other species, by one of the following:
Incorporating dyes.
Antibiotics.
Adjusting pH.
Bile salts.
Example:
Salmonella-Shigella agar (S-S)
This medium contains Bile salts and brilliant green dye as selective agents.

19. Selective-Differential media
Containing inhibitors (Selective) and pH indicators (Differential).
Examples:
MacConkey agar.
Mannitol Salt agar.

20. MacConkey agar Selective; Differential;
It contains Bile salts and Crystal violet.
Prevent the growth of Gram +ve bacteria.
Differential;
Lactose fermenter Vs Non-lactose fermenter.
It contains lactose & Neutral red (a dye).
If bacteria ferment lactose, the medium will acidify and the dye turns red.
If bacteria don’t ferment lactose, no color change occurs.

21. MacConkey agar Examples of Lactose fermenters:
Escherichia coli, Enterobacter and Klebsiella
Examples of Non-Lactose fermenters:
Salmonella , Proteus species , Yersinia , Pseudomonas aeruginosa and Shigella

22. Mannitol Salt agar
Is a selective differential medium, because it contains:
High concentration of salt;
Only Staphylococcal species will tolerate.
Mannitol;
S. aureus, unlike other staphylococcal species will ferment mannitol and change the pH.
A pH indicator dye;
A yellow halo will surround S. aureus colonies due to changes in pH.

23. Mannitol Salt agar