Enriched, Enrichment, Selective, Differential, Indicator & Transport media

III. 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. Horse 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 -NAD, 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

Haemophilus influenzae on chocolate agar

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/non-target 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 or a stimulating effect on the target bacteria to be grown 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.

 Selective medium: Media which favor the growth of particular microorganisms are selective media. They enhance the growth of a particular kind of microorganism and do not promote or even inhibit the growth of other microorganims that may be present. For example, antimicrobials, alcohol, bile salts or dyes like basic fuchsin and crystal violet can favor the growth of gram-negative bacteria by inhibiting the growth of gram-positive bacteria; the dyes have no effect on gram negative organisms.

Endo agar, Eosin methylene blue agar, Mannitol Salt agar and MacConkey agar are three media widely used for the detection of E. coli and related bacteria in water supplies etc. These media contain dyes that suppress gram positive bacterial growth. MacConkey agar also contains bile salts which inhibits gram-positive bacteria

Bacteria can also may be selected by providing nutrients that they specifically can use. A medium containing only cellulose as a carbon and energy source is useful in the isolation of cellulose-digesting bacteria. Starch agar can be used to isolate amylase producing microorganisms. Thus, the possibilities for selection are many, and there are numerous special selective media in use.

Some media may possess both selective and differential properties and can be powerful diagnostic tools in both medical and environmental settings.  

 

Differential medium are those that distinguish microorganisms from one another based on growth characteristics when grown. Different organisms show visible differences in growth when placed on differential media. Differential media has substances incorporated in it, enabling it to bring out differing characteristics of bacteria and thus helping to distinguish between them. Different bacteria can be recognized on the basis of their colony colour. Because of the presence of certain dyes, metabolic substrates or chemicals in the media, those bacteria that utilize them appear as differently coloured colonies. These characteristic changes or growth patterns are used for identification or differentiation of microorganisms. Examples are MacConkey agar, EMB agar, Blood agar and Mannitol salt agar

 

MacConkey agar: It is both differential and selective media. This medium is selective for Gram-negative species (taurocholate/bile slat inhibits Gram positive bacteria) and differential with respect to lactose fermentation.  MacConkey medium consists of peptone, lactose, agar, neutral red and taurocholate which is used to differentiate lactose fermenters and non-lactose fermenters. Lactose-fermenting bacteria appear red to pink while non-lactose fermenting bacteria appear as colorless or transparent colonies. MacConkey agar is used for the detection of coliforms and enteric pathogens based on their ability to ferment lactose.  

MacConkey agar

Blood agar : It is both an enriched media and differential media. This is a nutritive medium with differential properties in respect to hemolysis.. It is used to differentiate haemolytic and non –haemolytic bacteria. Blood agar can indicate the degree of hemolysis caused by hemolysin- Complete breakdown of the RBCs is termed beta (β) hemolysis and is recognized by clearing around the colonies. Partial destruction of the RBCs leads to a greenish brown color on the agar and is termed alpha(α)  hemolysis. Gamma (γ) hemolysis is the term applied to growth on blood agar that causes no damage to the RBCs and no change in the medium. Blood agar permits demonstration of the hemolytic properties of certain microorganisms, such as Streptococci.

Mannitol salt agar (MSA) : Mannitol salt agar is commonly used selective and differential medium. MSA is differential because it distinguishes bacteria based on their ability to ferment mannitol. MSA has 7.5%Nacl which allows the growth of halophiles while inhibiting the growth of non-halophiles. Mannitol is the fermentable carbohydrate source, fermentation of which leads to acid production. Staphylococcus aureus grows on this medium and ferments mannitol to produce yellow coloines. Most coagulase – negative species of Staphylococci and Micrococci do not ferment mannitol and grow as small red colonies. The colour of the colonies and the medium is due to phenol red (indicator) which is red at alkaline pH and yellow at acidic p H.

Eosin Methylene Blue agar (EMB agar): It is both differential and selective media. It contains the dyes eosin and methylene blue and inhibit Gram-positive organisms.  This medium is selective for Gram-negative species.  Lactose-fermenting organisms such as E. coli produce small, nucleated/dark-centred colonies with a greenish/dark metallic sheen on EMB.   Non-lactose fermenters such as Proteus sp., Salmonella sp., or Shigella sp. appear pink or uncolored. 

E. coli on EMB agar

 Indicator media: These media contain an indicator that changes colour when a bacterium grows in them; for example, due to incorporation of sulphite in Wilson-Blair medium. colonies of S. typhi grow with a black metallic sheen. This is because S. typhi reduces sulphite to sulphide in the presence of glucose. Potassium tellurite in McLeod’s medium is reduced to metallic tellurium by the Diphtheria bacillus to produce black colonies. MacConkey agar consists of neutral red, a pH indicator that turns pink under acidic conditions. Lactose fermenting bacteria produce acidic byproducts that lower the pH of the agar causing the colonies to turn pink. Non-lactose fermenting bacteria don’t cause a pH change and their  colonies remain colourless.

  

                                                                                       S. typhi  on Wilson-Blair medium 

Transport media : Used in case of delicate organisms (like gonococci) which may not survive the time taken for transporting the specimen to the laboratory or may be overgrown by non-pathogens (such as dysentery or cholera organisms in faeces). Special media are formulated for transporting such specimens. These are transport media, for example, Stuarts medium-a non-nutrient soft agar gel containing a reducing agent to prevent oxidation, and charcoal to neutralize certain bacterial inhibitors –for gonococci, and buffered glycerol saline for enteric bacilli. Venkatraman Ramakrishnan medium is used for Vibrio cholera.