Objectives

At the end of the session the students should be able

to:
 Describe the use of bacterial cultivation in identifying bacteria.

Cultivation of Bacteria  List the basic ingredients of culture media.

 Describe the use of basic, enriched, selective, differential and

transport media.

 Describe the use of the three forms of (solid, semi solid, and
fluid) culture media.
Aishath Majidha Hassan
 Describe the method of preparing commonly used culture media.
• Agar plates
• Slopes
• Butts/stabs/deeps
• Broth cultures

Use of cultivation of bacteria

Bacteria may be cultivated on artificial media
to:

 Identify the cause of an infection

Bacteria may be cultivated on
artificial media to
 Isolate bacteria in pure culture from a mixed
culture

 Perform antibiotic susceptibility testing

 Obtain a broth culture

Based on the composition, culture media can

Culture Media be classified as:

The nutrient material prepared for the growth of  Chemically defined media
microorganisms outside their natural habitat.

Culture media should provide:  Complex media

 Source of energy
Chemically defined Complex

 Source of carbon, nitrogen, sulphur,

sulphur, phosphorus and minerals Component Amount Component Amount
• Usual source of: NH4Cl 0.52 g Beef extract 1.5 g
 Carbon is citrate
 Nitrogen is peptone KH2PO4 0.28 g Yeast extract 3.0 g
MgSO4 7H2O 0.25 g Peptone 6.0 g
 Growth factors CaCl2 2H2O 0.07 g Glucose 1.0 g
Elemental Sulfur 1.56 g Agar 15.0 g
 Requirement of water
CO2 5%* water 1000 ml
 Requirement of proper pH, ionic strength & osmotic pressure water 1000 ml

Basic ingredients of culture media:
Culture Media
 Water

Types of media:
 Agar
 Basic
 Peptone
 Enriched
 Yeast or meat extracts
 Selective

 Mineral salts  Differential

 Carbohydrates  Transport

Types of Media Types of Media

Enriched:

Basic:  Contains specific nutrients required for the growth of
bacteria (fastidious) that has additional nutrient
 Simple media that supports the growth of requirements & facilitates the growth of these bacteria.
organisms that do not have special nutritional
requirements.  Basic media can be enriched with:
• Whole or lysed blood
• Examples: Nutrient agar & broth • Serum
• Peptone
• Yeast extracts
 Use: • Vitamins and other growth factors
• Used in the preparation of enriched media
 Used for the rapid multiplication of fastidious organisms
• Used to maintain stock cultures for control strains and organisms which may be present in small numbers.
• Used for sub-
sub-culturing
 Examples: blood agar, chocolate blood agar

Types of Media Types of Media

Selective:
 Contains substances which inhibits the growth
Differential:
of bacteria other than the intended bacteria.  Media containing certain substances such as
dyes to differentiate bacteria.
 Substances used include:
• Bile salts, chemicals, dyes, antibiotics
 Differentiation between bacteria is achieved by
adding indicators to the media which changes
 Used when culturing specimens with normal
color when acid is produced by fermentation of
flora, to prevent overgrowing of contaminants.
carbohydrates.

 Examples:
• Example: MacConkey agar
• Deoxycholate citrate agar, SS agar
 Types of Media

Transport:
 Media designed to prevent overgrowth of organisms
and ensures the survival of the organisms during
transport.

 Mostly semi-
semi-solid media

 Useful when there is delay in the transport of

specimens to lab

 Examples:
• Cary
Cary--Blair,
• Amies transport media

Culture media may be classified based on the

Solid media is usually contained in:
consistency:
 Solid:
• Contains agar  Petri dishes
• Cells are immobilized

 Semi--solid:
Semi
• Contains small amount of agar  Tubes:
• Mainly used for motility testing and as transport media. • Slants

 Fluid: • Butts
• Most commonly used as enrichment
• May be used for:
 Biochemical testing
 Looking at typical surface growth
 Protein / DNA isolation

Media preparation Activity: Complete the following table

Use of dehydrated media: Name of media Type of media Use
 Ensures good performance and reproducibility Blood agar Enriched Used for the rapid multiplication of
 Less expensive fastidious bacteria such as
Streptococcus sp.
 Easy to use
Mannitol salt agar

Preparation of culture media: (general method)

Selenite broth
 Weigh the dehydrated culture media
 Measure the required amount of water
 Dissolve the dehydrated media in water, heat if required. Cary--Blair media
Cary

 Sterilize the dissolved media.

 Cool, if heat sensitive ingredients need to be added. Buffered charcoal-
charcoal-yeast exatract
 Dispense into sterile containers, using aseptic precautions. agar

 Keep the containers undisturbed, until set. Xylose lysine desoxycholate

 Store the media in refrigerator until use. agar