This document provides information on microbial culture and growth conditions. It discusses that media provides the necessary nutrients to support microbial growth. Different types of media are used for different purposes, such as general purpose media, enriched media to support fastidious microbes, selective media that inhibits some bacteria, and differential media that distinguishes between organisms. Examples of common media like nutrient agar and specific media like MacConkey agar are described. The document also outlines culture techniques for anaerobic and microaerophilic bacteria that require specialized environments to grow.

1. Dr. Hashem Ahmed

2. Remember
NO FOOD OR DRINK ALLOWED IN THE LABORATORY
NO MOUTH PIPETTING
INDIVIDUALS NOT ALLOWED TO WORK IN THE LABORATORY
ALONE
WEAR REQUIRED PERSONAL PROTECTIVE EQUIPMENT
NO SMOKING IN LABORATORY
MAINTAIN GOOD HOUSEKEEPING HABITS

3. Microbial culture
Culture media
Culture conditions
Measurement of growth

4. Media
In order to recover or isolate bacteria for study, you
need to provide all of the growth demands and growth
conditions.
Combination of all of the ingredients that supports
the growth of microorganisms is called media

5. Types of media
Different approaches for classifications of types of
media used in microbiology are available.

6. Culture Conditions
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7. 1. Obligate Aerobes

8. 2. Obligate Anaerobes

9. 3. Facultative Aerobes , Facultative
Anaerobes

10. 4. Microaerophilic

11. Culture Media
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12. Culture Media
A. compositions
1. Chemically defined
The exact chemical composition is known
Used to grow fastidious organisms
2. Complex media
Exact chemical composition is not known
Most bacteria and fungi are grown with this

13. Culture Media
B. consistency
1. Broth
Media provided in liquid phase
used to measure motility, aeration and counting of
bacteria
2. solid Media
Media with solidifying agents like agar
Used for recovery of bacterial colonies

14. Agar :
Solid medium is made by adding Agar
 Agar is obtained from Sea weeds
Agar contains long chain polysaccharides.
Inorganic salts and protein like substances
It melts at 980c and sets at 420c
It is not degraded or metabolized by bacteria

15. Liquid media can be converted to solid media by
adding 1-2% of agar
If you add 0.6% agar, the media is called semisolid

17. Petri Dish With Media :
Petri dish with Media Plate: provide large surface for
isolation and observation of colonies
 Using a sterile loop or a sterile swab streak your
sample on the petri plate
Important let your sterilized loop cool before you pick
up your sample

19. Culture media
C. Purpose and use
1. General purpose media
Media provided in liquid phase or agar to provide the
minimal and basic growth requirements
Not all microbes can grow on such media
It is a complex, general purpose medium
Examples:
• Nutrient agar,
• Trypticase soy broth ((TSB, or tryptone soya agar (TSA)):
is frequently the base media of other agar plate type, i.e. blood agar plates (BAP) are made
by enriching TSA plates with blood
• Mueller Hinton agar: commonly used for antibiotic susceptibility testing, it
contains starch (Starch is known to absorb toxins released from bacteria, to prevent
interfere with the antibiotics. Second, it is a loose agar. This allows for better diffusion of
the antibiotics. BETTER DIFFUSION = TRUER ZONE OF INHIBITION.

20. 2. Enriched media
Is a medium with specific and known qualities that favors the
growth of a particular microorganism. Media provided with
additional growth requirements to enable the growth of
fastidious microorganisms(by adding blood, serum or egg).
Best obtained by additions of 5% sheep or horse blood
 Examples:
• Blood agar
•Selenite broth is used to selectively isolate Salmonella species.
• Alkaline peptone water is used for the cultivation of vibrio.

21. 3. Selective Media
Inhibits the growth of some bacteria while selecting
for the growth of others
Example:
 Brilliant Green Agar, MacConky or CLED (Cystine Lactose
Electrolyte Deficient Media))
 Dyes Inhibit The Growth Of Gram (+) Bacteria
 Selects For Gram (-) Bacteria
Most G.I. Tract infections are caused by Gram (-) bacteria

22. Selective Media
EMB (Eosin Methylene Blue)
Dyes inhibit Gram (+) bacteria
Selects for Gram (-) bacteria
G.I. Tract infections caused by Gram (-) bacteria

23. Selective Media
MacConky agar
Dyes inhibit Gram (+) bacteria
Delects for Gram (-) bacteria by bile salts
G.I. Tract infections caused by Gram (-) bacteria

24. 4. differential media
Differentiates between different organisms
growing on the same plate
Example:
Blood Agar Plates (TSA with 5% sheep blood)
 Used to differentiate different types of Streptococci
MacConky agar
 Used to differentiate between lactose and non lactose
fermenting bacteria

25. Alpha Hemolytic Streptococci
Incomplete lysis of RBC’s

26. Beta Hemolytic Streptococci
Complete lysis of RBC’s

27. Selective and Differential
Media
Mannitol Salt Agar
Used to identify Staphylococcus aureus
Mannitol Salt Agar
High salt conc. (7.5%) inhibits most bacteria
Sugar Mannitol
pH Indicator (Turns Yellow when acid)

28. Selective and Differential
Media
MacConkey’s Agar
Used to identify G-ve
MacConkey’s Agar
Bile salts and crystal violet (inhibits Gram (+)
bacteria)
lactose
pH Indicator
Many Gram (-) enteric non-pathogenic bacteria can ferment
lactose, Salmonella can not

29. Special Culture Techniques
1. Anaerobic Bacteria
a. Reducing Media
b. Anaerobic Container
c. Agar Stab: stab culture one in which the medium is
inoculated by thrusting a needle deep into its
substance.
d. Agar Shake: shake culture a culture made by
inoculating warm liquid agar culture medium in a tube
and shaking to distribute contents evenly. Incubation of
the resolidified culture allows the development of
separated colonies; especially adaptable to obligate
anaerobes.

30. Special Culture Techniques
2. Microaerophilic Bacteria
Grow best under reduced O2 levels and increased CO2
levels
Normal Atmosphere 21 % O2
 3 to .03 % CO2

31. Microaerophilic Bacteria
A. Candle Jar
16 % O2
4 % CO2

32. Microaerophilic Bacteria
B. CO2 Generating Packet

33. 33
The End