Suitable culture media are required to grow and maintain microorganisms in laboratory. A culture medium is a solid or liquid preparation used to grow, transport and store microorganisms. To be effective, the medium must contain all the nutrients the microorganism requires for growth. Specialized media are essential in the isolation and identification of microorganisms, the testing of antibiotic sensitivities, water and food analysis, industrial microbiology and other activities. Although all microorganisms need sources of energy and macro and micronutrients, the precise composition of a satisfactory medium depends on the nutritional requirements of the species being cultivated.
A medium is used to select and grow specific microorganisms or to help identify a particular species. Media can also be specifically designed to facilitate the growth of one type of microbe present in a sample from culture (enrichment). This is useful in isolating a species of interest from a mixed sample.
Microorganisms usually exist as mixed population in clinical material or as resident flora. They need to be cultivated in pure form for several reasons such as
- To
demonstrate their properties so as to identify them for their clinical
significance
- To
determine their sensitivity to antibiotics
- To
study their physiological virulence and genetic properties
- To
obtain sufficient growth for preparing antigens and vaccines
- To
type isolates for epidemiological purposes
- To archive them for research purposes
Culture media supports all these and are of different types.
Culture media can be classified based on several parameters : the chemical constituents from which they are made, their physical nature, and their function.
Classification
Bacterial culture media
are classified based on
- 1)
Their consistency
- 2)
Their constituents
- 3)
The functional requirement by bacteria
|
Sl
No. |
Type
of Culture Media |
Examples |
|
Based
on Consistency |
||
|
1.
|
Liquid |
Nutrient
broth, Peptone water, Brain heart infusion broth |
|
2.
|
Solid |
Nutrient
agar, Blood agar, Chocolate agar |
|
3.
|
Semisolid |
Sloppy
agar |
|
Based on Composition |
||
|
4.
|
Simple |
Nutrient
agar |
|
5.
|
Complex |
TCBS
agar – Thiosulphate citrate bile salt sucrose agar |
|
6.
|
Synthetic/Defined |
Hanck’s
balanced salt solution (for viral transport) Dubos’ medium with Tween 80 (for cultivation of Mycobacterium tuberculosis) |
|
Based
on their functional requirement |
||
|
7.
|
Enriched |
Todd-Hewit
Broth (for fastidious, nutritionally demanding organisms) |
|
8.
|
Enrichment |
Selenite
F Broth (for fecal samples to suppress gut commensals) |
|
9.
|
Selective |
Salmonella
Shigella agar (isolates Salmonella & Shigella in fecal samples) |
|
10.
|
Indicator |
MacConkey
agar (differentiates lactose fermenters from non-lactose fermenters) |
|
11.
|
Differential |
Mannitol
salt agar (differentiates Staphylococcus aureus from
Staphylococccus epidermidis) |
|
12.
|
Transport |
Stuart’s
transport medium (for transporting throat swabs and genital tract swabs from
which pathogens are to be isolated) |
|
13.
|
Anaerobic
media |
Robertson’s
cooked meat medium and thioglycolate medium (for cultivation of Bcateroides,
Clostridia etc) |
Classification based on
consistency
Liquid Media
Liquids are used when
large volumes of clinical material need to be inoculated to obtain a culture
(eg., blood). They are also used for preparing bulk cultures of antigens or
vaccines. Bacteria grow diffusely in liquids.
Solid media
On solid media, bacteria
have distinct colony morphology and exhibit several characteristic features
such as pigmentation (on nutrient agar) or hemolysis (on blood agar), making
identification easy.
Agar or agar-agar forms
the base of solid media. Agar is obtained from a type of sea-weed. It has
virtually no nutritive value and is not affected by the growth of bacteria. It
melts at 98oC and usually solidifies at 42oC depending on
the agar concentration. Approximately 2% agar is used for solid media.
Semisolid media (sloppy
agar)
This has low
concentration of agar (1.5%). It may be used to demonstrate motility of the
bacteria-motile bacteria exhibit diffuse growth.
Classification based on
the constituents
Simple media (basal
media)
It is a general – purpose
media that supports the growth of non-fastidious microbes. It is primarily used
for the isolation of microorganisms. Examples are nutrient broth, peptone water
and nutrient agar.
Nutrient Broth, an
example of simple media. It consists of peptone, meat extract, sodium chloride
and water. It is used to grow bacteria and to demonstrate motility.
Nutrient Agar
made by adding 2% agar to nutrient broth, is the simplest and most common solid
medium used as basal media in clinical, diagnostic laboratories. It is also
used for culturing and demonstrating the pigment producing ability of bacteria.
|
Nutrient broth |
Nutrient agar |
|
Peptone |
Peptone |
|
Yeast extract/Beef extract |
Yeast extract /Beef extract |
|
Sodium chloride |
Sodium chloride |
|
Distilled water |
Distilled water |
|
----- |
Agar |
Culture media contains a carbon source, nitrogen source, a variety of inorganic salts including phosphates, potassium and magnesium. Certain accessory growth factors such as riboflavin are also present. Blood, serum and yeast extract are other common ingredients of culture media.
Peptone is a common ingredient of culture media. It is a complex mixture of partially digested proteins and has proteoses, polypeptides and amino acids, a variety of inorganic salts including phosphates, potassium and magnesium and certain accessory growth factors such as riboflavin.
Beef extract is aqueous extract of lean beef tissue concentrated to paste like form. It has nutritive value and consists of water soluble substances of animal tissue such as carbohydrate, organic nitrogen compounds, water soluble vitamins and salts.
Yeast extract It is aqueous extract of yeast cells available commercially in powder form. It is nutritious with rich source of B Vitamins, also contains organic nitrogen and carbon compounds.
Sodium chloride The presence of sodium chloride in nutrient agar maintains a salt concentration in the medium that is similar to the cytoplasm of the microorganisms.
Agar Agar (or agar-agar) is used to prepare solid media. Approximately 2% agar is used for solid media. Agar is obtained from seaweed and has virtually no nutritive value. It melts at 980C and usually sets at 420C depending on the agar concentration. Agar is an excellent hardening agent since microorganisms cannot degrade it.
Distilled water Water is necessary for the growth of living organisms. Usually, distilled water is used for preparation of culture media
Complex media
These have added
ingredients for special purposes such as bringing out certain characteristics
or for providing special nutrients required for growth of the bacterium under
study. Most of the media other
than basal media, used in microbiology laboratory are complex media. e.g., chocolate
agar, Blood agar, MacConkey agar, Robertson’s cooked meat (RCM) medium,
Lowenstein–Jensen (LJ) medium (Cultivation and isolation of Mycobacterium),
etc.
Complex media is also called rich media and have a mixture of many
different organic compounds including glucose as carbon source, a source
of amino acids and nitrogen (e.g., beef and yeast extract), various salts,
growth factors, vitamins etc. and water needed for bacterial growth.
Synthetic or defined
media
These media are prepared
from pure chemical substances and the exact composition of the medium is fully
documented. It does not contain
any animal, yeast, or plant tissue or their extracts, hence called synthetic
media. The exact quantity of all ingredients used in the media is known,
hence also called defined media. They are used for various
special purposes such as studying the metabolic requirements of
the organisms. This media consists of a defined carbon and nitrogen source,
trace elements (Mn, Mo, Cu, Co, Zn etc.) and vitamins. Glucose or glycerol
is often used as carbon source and ammonium salts or nitrates used as inorganic
nitrogen sources. Dubos’ medium with Tween 80 used for cultivation of Mycobacterium
tuberculosis is an example of this medium.
Minimal media (sometimes
called Mineral media) is also an example of synthetic media. Minimal
media contain just enough ingredients, generally without the
presence of amino acids, to support growth of wild type microorganisms. Minimal
media typically consists of a carbon source (sugar like glucose or less
energy source like succinate), various salts with essential element like Mg, N,
P, S which help bacteria to synthesize protein and nucleic acids and water.
Classification based on functional requirements
General purpose/Supportive
media
Tryptic soy broth and
tryptic soy agar- sustain the growth of many microorganisms,
hence called supportive media
Enriched Medium
Blood and other nutrients
when added to supportive media / basal medium help in the growth of fastidious/exacting
bacteria. Nutritional requirements such as blood, serum or eggs can be added. These
fortified media are called enriched media. Examples are blood agar, chocolate
agar and brain-heart infusion broth.
Blood Agar
This is a solid culture
medium consisting of agar, peptone and blood. Hose sheep blood is commonly
used, horse, cow and pig blood may also be used.
Blood agar supports the
growth of most aerobic and anaerobic bacteria (vitamin K, cysteine and hemin
supplementation enhances the growth of anaerobic bacteria) and fungi. Blood
agar can indicate the degree of hemolysis caused by hemolysin. Based on this,
it is used to differentiate among gram-positive cocci. Hence it is also known
as a differential medium.
Beta hemolysis
refers to the complete lysis of red blood cells and hemoglobin; this results in
the complete clearing of the blood agar medium surrounding the colonies, e.g.,
group A Streptoccocci.
Alpha hemolysis
refers to the partial lysis of red blood cells and hemoglobin; this results in
a greenish discoloration of the blood agar around the colonies. e.g., Streptococcus
pneumoniae.
Gamma hemolysis is the absence of lysis.
Chocolate Agar This
is made by heating a mixture of sheep blood and nutrient agar. This results in
the release of hemoglobin, a related substance hemin (also called X factor) and
nicotinamide adenine dinucleotide 9NAD, also called V factor), during the
process of heating. Chocolate agar is used to grow fastidious organisms,
including H influenzae, N meningitidis, N. gonorrhoeae, Pneumococcus
Brain-heart infusion
broth (BHIB)
This is a highly
nutritious, buffered, fluid culture medium prepared by non-enzymic infusion
from calf brain and cow heart, often with added peptone and dextrose. It is
suitable for the cultivation of fastidious organisms.
Enrichment media
These media are used to
suppress commensal bacteria while allowing the pathogen to remain viable and to
grow. It is employed for specimens with mixed flora e.g., fecal sample from
which diarrheagenic bacteria need to be isolated. Substances that have an
inhibitory effect on the commensals are incorporated in the medium. Examples of
enrichment media are Tetrathionate broth and Selenite F broth. Tetrathionate inhibits
coliforms while allowing typhoid-paratyphoid bacilli to grow. Selenite F broth is used for fecal samples in
cases of dysentery.
