Identification of different culture media

 

EXPERIMENT -2

IDENTIFICATION OF DIFFERENT CULTURE MEDIA

Objective: To identify and understand different culture media's composition, uses, and preparation methods (Nutrient Agar, MacConkey Agar, Nutrient Broth, Mueller Hinton Agar).

Theory: Culture media are substances that provide a favorable environment for the growth and multiplication of microorganisms in the laboratory. They are classified based on their composition, function, and purpose. Different culture media are used to cultivate specific types of microorganisms, depending on their nutritional requirements and growth characteristics.

Culture media may be classified into several categories depending on their composition, physical state, or use.

1.         Based on the physical state of the media, it can be classified as

a)      Solid media

b)      Liquid media

c)      Semi- solid media

a)         Solid media:- Solid media are those media which are solid in state. Agar is the major solidifying agent which is added on the concentration of 1.5% for plates, slant media. Eg: Nutrient agar, Macconkey agar.

b)        Liquid media (broth):- Those media which are in liquid state are called liquid media. This form of media don't contain agar or any solidifying agent. Eg:- Nutrient broth, Macconkey broth.

c)         Semi- solid media:- This form of medium is prepared by adding a small amount of agar (0.2-0.5%)to a fluid medium. The consistency of this media is jelly like, the consistency between solid and liquid. Eg:- Hugh Leifson's media, SIM media.

 

2)         Chemical composition

On the basis of chemical composition, media are of two types.

a)  chemically-defined (synthetic) medium    b) complex (undefined) medium

A chemically-defined (synthetic) medium:  It is one in which the exact chemical composition is known. It is generally made from various chemicals that are highly purified and precisely defined.

A complex (undefined) medium is one in which the exact chemical constitution of the medium is not known. complex media usually contain complex materials of biological origin such as blood or milk or yeast extract or beef extract, the exact chemical composition of which is obviously undetermined.

 3)         Utility

   Based on Utility purpose, media are of the following types

a)         Basal Media( General Purpose Media):- These are simple media which support the growth of microorganisms that do not have special nutritional requirements. These are designed to grow as many as possible of the organisms as possible commonly used in the general bacteriological laboratory.

Uses

·       It is often used in the preparation of enriched media ( Nutrient agar +blood = Blood agar)

·       To maintain stock cultures

·       For subculturing pathogens from differential or selective media before performing biochemical tests. E.g., Nutrient agar, Nutrient broth.

b)        Enriched Media:- Enriched media are used for the cultivation of certain microorganism that have elaborated requirements for specific nutrients. Such micro-organisms are referred to as fastidious micro-organism such as Haemophilus influenza, Streptoccocus,  Neisseria etc. Fastidious organism are generally human pathogens.

In such media, some addition as supplementary foods are supplied besides the common nutrients. Such as blood, egg, serum, vitamins ,peptones, aminoacids to a basal media. e.g. Blood agar, chocolate agar, egg yolk medium.

c)         Selective Media:- A selective media is one which has a components added to it which will inhibit or prevent the growth of certain types or species of bacteria and/or promote the growth of desired species. Thus such media contains some selective substances that supports the growth of desired organism while inhibiting the growth of unwanted organism in a mixture. e.g. Macconkey Agar is selective media for Gm -ve bacteria because it contains selective agents like bile salts and crystal violet which inhibit the Gm+ve bacteria. XLD agar selects for Salmonella Shigella species.

d)        Differential Media:- Differential media consists of indicator, dyes or other substances to differentiate microorganism. These types of media are also known as indicator media and these media incorporate some substances i.e. changed visibly as a result of the metabolic activities of particular organism. Eg: Macconkey Agar contains the indicator neutral red which differentiate lactose fermenting from Non- Lactose fermenting organism.

e)         Enrichment media:- These are fluid media that increases the number of pathogens by containing enrichment and a substances that discourage the multiplication of unwanted bacteria.For e.g:- Alkaline Peptone Water is used as enrichment media for vibrio species. The pH of the media is high (8.5-9.2) that supports the growth of vibrio species and is inhibitory to other organisms.

f)         Transport Media:- These are media that are devised to maintain the viability of a pathogen and to avoid over growth of other contaminants during transit from the patient to the laboratory. Their use is particularly important when transporting microbiological specimens from health centre to the district microbiology lab. E.g:- Cary and Blair medium for preserving enteric pathogens

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Materials required :

·        Bottles of dehydrated culture media: Nutrient Agar, MacConkey Agar, Nutrient Broth

·        Mueller Hinton Agar

 

 

Procedure :

1. Reading the Composition and Uses

·        Carefully read the label on each bottle of dehydrated media.

·        Note down the composition and specific uses of each medium in the notebook.

Observation Table

S.No.	Culture Medium	Composition	Uses	Appearance/Texture
1	Nutrient Agar	Peptone, beef extract, agar	General-purpose medium for culturing bacteria	Solid, translucent, pale yellow
2	MacConkey Agar	Lactose, bile salts, neutral red dye, agar	Selective and differential medium for Gram-negative bacteria	Solid, pinkish-red
3	Nutrient Broth	Peptone, beef extract	Liquid medium for growing bacteria	Liquid, clear, amber
4	Mueller Hinton Agar	Beef extract, starch, agar	Used for antibiotic susceptibility testing	Solid, opaque, light brown

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Result

·        Nutrient Agar: A general-purpose medium that supports the growth of most non-fastidious bacteria. Its transparency makes it suitable for observing colony morphology.

·        MacConkey Agar: A selective and differential medium used to isolate Gram-negative bacteria. Lactose fermenters produce pink colonies, while non-fermenters produce colorless colonies.

·        Nutrient Broth: A liquid medium used for growing bacteria in suspension. It is simple and cost-effective, but does not allow colony differentiation.

·        Mueller Hinton Agar: Specifically designed for antibiotic susceptibility testing. Its uniform surface and composition ensure consistent results during disk diffusion assays (Kirby-Bauer method).

Conclusion: This practical session provided nursing students with a comprehensive understanding of the composition, preparation, and applications of four commonly used culture media: Nutrient Agar, MacConkey Agar, Nutrient Broth, and Mueller Hinton Agar.

Precautions

·        Always wear gloves, lab coats, and safety goggles while handling culture media.

·        Follow the directions given in the bottle for preparation of culture media.

 

References

Cappuccino, J. G., & Sherman, N. (2017). Microbiology: A Laboratory Manual,11th ed. Pearson.

Brown, A. E. (2015). Benson's Microbiological Applications: Laboratory Manual in General Microbiology, 13th ed. McGraw-Hill Education.