Culture Media

- DR ARSHEYA
 INTRODUCTION
• Bacteria have to be grown (cultured) for them to be identified, as only
rarely can they be recognized by their morphology alone.
• The study of bacteria involves the study of bacterial populations rather
than of single bacterial cells.
• Bacteria occur as mixed populations. By appropriate procedures they
have to be grown separately (isolated) on culture media and obtained
as pure cultures for study.
• Culture media are required to grow the organisms from
infected material to identify the causative agent. The basic
constituents of culture media are:
1. Water
2. Electrolyte
3. Peptone
4. Meat extract
5. Blood or serum
6. Agar
 TYPES OF MEDIA
• Media are classified in many ways:
1. Based on physical state:
i) Liquid media
ii) Semisolid media
iii) Solid media
• 2. On the basis of presence of molecular oxygen and reducing
substances in the media:
i) Aerobic media
ii) Anaerobic media
3. Based on nutritional factors :
i) Simple media
ii) Synthetic media
iii) Special media
(a) Enriched media
(b) Enrichment media
(c) Selective media
(d) Differential media
(e) Indicator media
(f) Transport media
 LIQUID MEDIA
• The original media used by Louis Pasteur were liquids such
as urine or meat broth.
• They have limitations in identifying bacteria and isolating
different types from mixed populations.
• However, they are useful for testing large samples, like blood
or water, and preparing bulk cultures for antigens or vaccines.
• In contrast, solid media allow bacteria to grow as visible
colonies, making it easier to study and identify them.
 SOLID MEDIA
• Solid media, first developed by Robert Koch using potatoes
and gelatin, became more effective with the introduction of
agar, suggested by Frau Hesse.
• Agar, derived from seaweed, is ideal for solidifying media due
to its melting and setting properties, lack of nutritive value, and
stability during bacterial growth.
• Peptones, partially digested proteins, and other additives like
meat extract, blood, or yeast extract are also commonly used in
culture media for bacterial growth and identification.
 Simple Media
• Nutrient broth is an example of simple medium.
• It contains peptone water and meat extract 1%.
• When glucose is added to nutrient broth, it becomes glucose broth
• When 2-3% agar is added to nutrient broth, it becomes nutrient agar.
This is the simplest and routinely employed medium in laboratory for
diagnostic purposes.
• If concentration of nutrient agar is reduced (0.2 to 0.4%), semisolid
medium is obtained which enables motile bacteria to spread.
• Brain-Heart Infusion (BHI) broth is another liquid medium used for the
culture of non-fastidious (which have simple nutrient requirements) and
moderately fastidious (which need additional nutrients) organisms.
 COMPLEX MEDIA
• These have added ingredients for special purposes or for
bringing out certain characteristics or for providing special
nutrients required for the growth of the bacterium under
study
 Synthetic Media
• These are prepared from pure chemicals and the exact
composition of the medium is known. This allows precise
control over the nutrients provided.
• These are used for special studies
• Dubo's medium with tween 80 is one example of a synthetic
medium.
 Special Media
a) Enriched Media
When basal medium is added with some nutrients such as blood, serum or
egg, it is called enriched medium. Some examples are blood agar ,
chocolate agar and egg media
1. Blood agar : This is a solid culture medium consisting of agar peptones
and blood. It supports the growth of most aerobic and anaerobic bacteria ,
and fungi.
The degree od hemolysis caused helps to differentiate among gram
positive cocci.
A) Beta hemolysis B) Alpha hemolysis C) Gamma hemolysis
Beta hemolysis and alpha hemolysis
2. Chocolate agar : This is a solid culture medium made by heating a
mixture of sheep blood and nutrient agar, during which the RBCs are
disrupted to release their contents.
Beta hemolysis Alpha hemolysis

Gamma hemolysis
b) Enrichment Media
These media are used to suppress commensal bacteria while
allowing the pathogen to remain viable and grow.
Some substances are incorporated in the liquid medium which have
a stimulating effect on the bacteria to be grown or inhibits its
competitors. This results in an absolute increase in the number of
wanted bacteria in relation to other bacteria.
It is employed for specimens with mixed flora, e.g.,fecal sample to
isolate diarrheagenic bacteria.
Such media are called enrichment media.
Examples are 'tetrathionate broth’ (tetrathionate inhibits coliforms
while allowing typhoid – paratyphoid bacilli to grow) and 'selenite
F broth’(for the bacteria that cause desentry).
c) Selective Media
Selective media are solid and contain substances that inhibit all but a
few types of bacteria and facilitate the isolation of a particular species.
Examples are deoxycholate citrate agar (DCA) for fecal samples and
Thiosulphate Citrate Bile Sucrose agar (TCBS) for vibrio species in
cholera.
Organisms that ferment sucrose appear yellow, while those that do
not, appear green.
d) Differential Media
When a medium contains substances which help to distinguish
differing characteristics of bacteria, it is called "differential medium".
Example is MacConkey's agar. On MacConkey's agar, the lactose
fermenters (LF) form pink-coloured colonies whereas non-lactose
fermenters (NLF) produce colorless or pale colonies.
e) Indicator Media
These media contain an indicator which changes colour when a
bacterium grows in them.
Examples are sulphite in Wilson and Blair medium. Salmonella
typhi reduces sulphite to sulphide to give black metallic colonies
on Wilson and Blair medium.
f) Transport Media
These are used in the case of delicate organisms (e.g. gonococci)
which may not survive the time taken for transit or may be
overgrown by non-pathogenic bacteria (e.g. cholera organisms).
Example is Stuart's transport medium (a non-nutrient, soft agar
gel).
 Anaerobic Media
• These are used for cultivation of anaerobic bacteria, e.g.
(a) Cooked meat broth (CMB): It is also known as
Robertson's cooked meat broth (RCM).
(b) Thioglycollate broth
This medium supports the growth of all organisms with
varied oxygen requirements: anaerobes, aerobes, and
facultative anaerobes.