Culture Media - Nutrients for Microbial Growth

Chromogenic Culture Media: Principle, Composition and Results

September 4, 2018 Nisha Rijal Bacteriology, Culture Media used in Microbiology, laboratory diagnosis of Bacterial Disease, Microbiology, Microbiology for Beginners 0

Chromogenic Agar Media are culture medium used to isolate, identify, and differentiate specific microorganism from a heterogeneous population. The medium contains chromogenic substrate which is utilized by the microorganism to give colored colonies that is specific for each microorganism. Depending on the color of the result, the presence or absence of target organism is determined and also accurately differentiated from others.

Classical culture media are based on the principle of change in color of the pH indicator whereas Chromogenic media are based on enzymatic utilization of chromogenic substrates.

Principle of Chromogenic Media

Chromogenic media contains soluble colorless molecules called chromogens. Chromogens are composed of two parts: a substrate ( which is the target of a specific enzymatic activity of the microorganism) and a chromophore.

Principle of Chromogenic Agar Medium (source: chromagar.com)

When the bond between the substrate and chromophore is split by a specific enzyme produced by the target microorganism, chromophore is released. In its unconjugated form, the chromophore shows distinctive colour. Due to reduced solubility, chromophore forms a precipitate that imparts unique color to the colony.

Composition of Chromogenic Agar Media

Like traditional culture media, chromogenic media also contains nutrients such as peptones, amino acids, yeast extract, minerals, vitamins and solidifers (agar). Depending on the purpose chromogenic media may also contain inhibitors. Unlike traditional media, they contain chromogenic substrates or chromogens. These chromogenic substrates such as ONPG, X-Gal, or X-Glu, together with a specified selectivity of the medium, is the simple principle behind chromogenic media.

Chromogenic Agar Media (Source:biomerieux)

Advantages of Use of Chromogenic Agar Media:

Less Labor intensive and more economic:
Though people perceive chromogenic media as expensive alternative, use of a single chromogenic medium rather two-three selective ones, reduces the cost of sample processing. Chromogenic media may eliminate the need of subculture and further biochemical test for the identification of the isolates.
Less time consuming: As chromogenic media eliminate various steps of sample processing (e.g. subculturing, biochemical testing) results are available within 24 hours as compared to 48 hours or more by conventional methods. Timely diagnosis not only ensures better outcome for the patients but also helps in prevention and spread of infections.
Easy identification: On chromogenic agar medium, target colonies of specific microorganisms can be recognized by their colour at a glance. No specialized equipment needed.

Examples of Chromogenic Agar Medium

Most of the commercially available chromogenic media utilize substrates like indoxyl and its halogenated derivatives, metal chelators like Esculin, 8‐Hydroxyquinoline, substrates for β‐glucosidase enzyme etc. Based on these a number of companies have formulated several variations depending on need and usage. Following are some of the examples

S.N	Target organism	Media Available
1.	Candida	CHROMagar Candida, Albicans ID, CandiSelect , Candida ID, Pourmedia Vi Candida, Chromogenic Candida agar , Brilliance Candida Agar and HiCrome Candida differential agar.
2.	Pseudomonas aeruginosa	Chrom ID Pseudomonas
3.	Urinary Tract pathogens	CHROMagar Orientation, UriSelect medium, Rainbow Agar UTI medium , Chromogenic UTI medium , USA agar , Harlequin CLED and Urichrom agar
4.	Bacillus cereus	Bacillus cereus ChromoSelect Agar
5.	Escherichia coliserotype O157:H7	CHROMagar O157
6.	Methicillin resistant Staphylococcus aureus (MRSA)	CHROMagar MRSA II
7.	Carbapenem-resistant Enterobacteriaceae	Brilliance CRE Agar

Culture media contains nutrients and physical growth parameters necessary for microbial growth. All microorganisms cannot grow in a single culture medium and in fact many can’t grow in any known culture medium.

Organisms that cannot grow in artificial culture medium are known as obligate parasites. Mycobacterium leprae, rickettsias, Chlamydias, and Treponema pallidum are obligate parasites. Bacterial culture media can be distinguished on the basis of composition, consistency and purpose.

Classification of culture media used in Microbiology laboratory on the basis of consistency

Solid medium
solid medium contains agar at a concentration of 1.5-2.0% or some other, mostly inert solidifying agent. Solid medium has physical structure and allows bacteria to grow in physically informative or useful ways (e.g. as colonies or in streaks). Solid medium is useful for isolating bacteria or for determining the colony characteristics of the isolate.
Semisolid media
They are prepared with agar at concentrations of 0.5% or less. They have soft custard like consistency and are useful for the cultivation of microaerophilic bacteria or for determination of bacterial motility.
Liquid (Broth) medium
These media contains specific amounts of nutrients but don’t have trace of gelling agents such as gelatin or agar. Broth medium serves various purposes such as propagation of large number of organisms, fermentation studies, and various other tests. e.g. sugar fermentation tests, MR-VR broth.

Classification of culture media based on the basis of composition

Synthetic or chemically defined medium
A chemically defined medium is one prepared from purified ingredients and therefore whose exact composition is known.
Non synthetic or chemically undefined medium

Non-synthetic medium contains at least one component that is neither purified nor completely characterized nor even completely consistent from batch to batch. Often these are partially digested proteins from various organism sources. Nutrient broth, for example, is derived from cultures of yeasts.

Synthetic medium may be simple or complex depending up on the supplement incorporated in it. A simple non-synthetic medium is capable of meeting the nutrient requirements of organisms requiring relatively few growth factors where as complex non-synthetic medium support the growth of more fastidious microorganisms.

Classification of Bacterial Culture Media based on the basis of purpose/ functional use/ application

Many special purpose media are needed to facilitate recognition, enumeration, and isolation of certain types of bacteria. To meet these needs, numerous media are available.

1. General purpose media/ Basic media
Basal media are basically simple media that supports most non-fastidious bacteria. Peptone water, nutrient broth and nutrient agar are considered as basal medium. These media are generally used for the primary isolation of microorganisms.

2. Enriched medium (Added growth factors):

Addition of extra nutrients in the form of blood, serum, egg yolk etc, to basal medium makes them enriched media. Enriched media are used to grow nutritionally exacting (fastidious) bacteria. Blood agar, chocolate agar, Loeffler’s serum slope etc are few of the enriched media. Blood agar is prepared by adding 5-10% (by volume) blood to a blood agar base. Chocolate agar is also known as heated blood agar or lysed blood agar.

3. Selective and enrichment media are designed to inhibit unwanted commensal or contaminating bacteria and help to recover pathogen from a mixture of bacteria. While selective media are agar based, enrichment media are liquid in consistency. Both these media serve the same purpose. Any agar media can be made selective by addition of certain inhibitory agents that don’t affect the pathogen of interest. Various approaches to make a medium selective include addition of antibiotics, dyes, chemicals, alteration of pH or a combination of these.

a. Selective medium

Principle: Differential growth suppression
Selective medium is designed to suppress the growth of some microorganisms while allowing the growth of others. Selective medium are agar based (solid) medium so that individual colonies may be isolated.

Examples of selective media include:

Thayer Martin Agar used to recover N.gonorrhoeae contains antibiotics; vancomycin, colistin and nystatin.
Mannitol Salt Agar and Salt Milk Agar used to recover S.aureus contains 10% NaCl.
Potassium tellurite medium used to recover C.diphtheriae contains 0.04% potassium tellurite.
MacConkey’s Agar used for Enterobacteriaceae members contains bile salt that inhibits most gram positive bacteria.
Pseudosel Agar (Cetrimide Agar) used to recover P. aeruginosa contains cetrimide (antiseptic agent).
Crystal Violet Blood Agar used to recover S. pyogenes contains 0.0002% crystal violet.
Lowenstein Jensen Medium used to recover M.tuberculosis is made selective by incorporating malachite green.
Wilson and Blair’s Agar for recovering S. typhi is rendered selective by the addition of dye brilliant green.
Selective media such as TCBS Agar used for isolating V. cholerae from fecal specimens have elevated pH (8.5-8.6), which inhibits most other bacteria.

b. Enrichment culture medium
Enrichment medium is used to increase the relative concentration of certain microorganisms in the culture prior to plating on solid selective medium. Unlike selective media, enrichment culture is typically used as broth medium. Enrichment media are liquid media that also serves to inhibit commensals in the clinical specimen. Selenite F broth, tetrathionate broth and alkaline peptone water (APW) are used to recover pathogens from fecal specimens.

4. Differential/ indicator medium: differential appearance:
Certain media are designed in such a way that different bacteria can be recognized on the basis of their colony colour. Various approaches include incorporation of dyes, metabolic substrates etc, so that those bacteria that utilize them appear as differently coloured colonies. Such media are called differential media or indicator media. Differential media allow the growth of more than one microorganism of interest but with morphologically distinguishable colonies.
Examples of differential media include:

Mannitol salts agar (mannitol fermentation = yellow)
Blood agar (various kinds of hemolysis i.e. α, β and γ hemolysis)
Mac Conkey agar (lactose fermenters, pink colonies whereas non- lactose fermenter produces pale or colorless colonies.
TCBS (Vibrio cholerae produces yellow colonies due to fermentation of sucrose)

5. Transport media:
Clinical specimens must be transported to the laboratory immediately after collection to prevent overgrowth of contaminating organisms or commensals. This can be achieved by using transport media. Such media prevent drying (desiccation) of specimen, maintain the pathogen to commensal ratio and inhibit overgrowth of unwanted bacteria. Some of these media (Stuart’s & Amie’s) are semi-solid in consistency. Addition of charcoal serves to neutralize inhibitory factors.

Cary Blair transport medium and Venkatraman Ramakrishnan (VR) medium are used to transport feces from suspected cholera patients.
Sach’s buffered glycerol saline is used to transport feces from patients suspected to be suffering from bacillary dysentery.
Pike’s medium is used to transport streptococci from throat specimens.

6. Anaerobic media:
Anaerobic bacteria need special media for growth because they need low oxygen content, reduced oxidation –reduction potential and extra nutrients.

Media for anaerobes may have to be supplemented with nutrients like hemin and vitamin K. Such media may also have to be reduced by physical or chemical means. Boiling the medium serves to expel any dissolved oxygen. Addition of 1% glucose, 0.1% thioglycollate, 0.1% ascorbic acid, 0.05% cysteine or red hot iron filings can render a medium reduced. Before use the medium must be boiled in water bath to expel any dissolved oxygen and then sealed with sterile liquid paraffin.

Robertson Cooked Meat (RCM) medium that is commonly used to grow Clostridium spps contains a 2.5 cm column of bullock heart meat and 15 ml of nutrient broth. Thioglycollate broth contains sodium thioglycollate, glucose, cystine, yeast extract and casein hydrolysate.

Methylene blue or resazurin is an oxidation-reduction potential indicator that is incorporated in the medium. Under reduced condition, methylene blue is colorless.
7. Assay media
These media are used for the assay of vitamins, amino acids and antibiotics. E.g. antibiotic assay media are used for determining antibiotic potency by the microbiological assay technique.
Other types of medium includes

Media for enumeration of Bacteria,
Media for characterization of Bacteria,
Maintenance media etc.

This index page contains information about various culture media used in diagnostic Microbiology laboratory. All the relevant links related to culture media, especially bacteriological culture media are posted in an alphabetical order.

To get information about a particular culture media, click its name, then a new detail/description page about that media will open.

In the description page, you will find relevant information about the culture media e.g. guidelines on the preparation, uses, and colony characteristics of the organisms that grow on these media.