Culture Media

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 98^oC and usually solidifies at 42^oC 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

 

 Essential Ingredients of Culture Media

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.