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Friday, December 27, 2019

Culture Media: Definition ,Types, classification and Preparation




CONTENTS:
·        Introduction to culture media
·        Types and classification of culture media
·        Preparation of culture media

SELECTIVE MEDIUM

The culture media (nutrients) consist of chemicals which support the growth of culture or microorganisms. Microborganisms can use the nutrients of culture media as their food is necessary for cultivating them in vitro.

CHARACTERISTICS OF MEDIUM
The medium should neither be acidic nor alkaline. It should contain all kinds of nutrients in suitable amounts. It must be sterilized (free from microbes) before' use.

TYPES OF MEDIA
The first medium prepared was meat-infusion broth. Almost all pathogenic microorganisms required complex food similar in composition to the fluids of the animal body,
 Robert Koch and his colleagues who has used the meat infusion and meat extracts as the basic ingredients in their culture media for the isolation of pathogenic microorganisms, while one of his assistant name Petri who designed and developed glass dishes, known today as Petri dishes, are used in microbiological work.

On the basis of chemical composition, the media are classified into two types:

(i) Synthetic medium or chemically define medium: These media are prepare  by the mixing all the pure chemicals of known composition for e.g. Czapek Dox medium.
(ii) Semi-synthetic medium  or undefined medium : Such are those media, where exact chemical composition are  unknown for example potato dextrose agar or MacConkey agar medium.

On the basis of consistency, the media are of three types:
(a) Solid or synthetic medium : When 5-7% agar agar or 10-20% gelatin is added in the liquid broth become solidified. Such media are used for making agar slants or slopes and agar stab which is used in MicrobialMicrobial work.
(b) Liquid or broth medium: Such cases no agar is added or used while preparing the medium. After inoculation and later incubation, the growth of cells becomes visible in the form of small mass on the top of the broth.
(c) Semi-solid medium or floppy agar medium : Such media are prepare by the adding half quantity of agar (1/2 than required for solid medium) i.e. about 0.5% in the medium. This type of medium may be selective which promote the growth of one organisms and retards the growth of the other organisms. On the other hand, there are differential media which serve to differentiate organisms growing together.
Classification of Bacterial Culture media 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.
o   General purpose media/ Basic media
Basal media are basically simple media that supports most non-fastidious bacteria. Peptone water, nutrient broth and nutrient agar (NA) are considered as basal medium. These media are generally used for the primary isolation of microorganisms.
o   Enriched medium (Added growth factors): Addition of extra nutrients in the form of blood, serum, egg yolk etc, to basal medium makes  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.
o   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.
Ø SELECTIVE MEDIUM

Principle: Differential growth suppression Selective medium is designed to suppress the growth of some microorganisms while allowing the growth of others. Selectivemedium are agar based (solid) medium so that individual colonies may be isolated.
Examples of selective media include:
1.     Thayer Martin Agar used to recover Neisseria gonorrhoeae contains antibiotics; vancomycin, colistin and nystatin.
2.     Mannitol Salt Agar and Salt Milk Agar used to recover S.aureus contains 10% NaCl.
3.     Potassium tellurite medium used to recover C.diphtheriae contains 0.04% potassium tellurite                                                    .
4.     MacConkey’s Agar used for Enterobacteriaceae members contains bile salt that inhibits most gram positive bacteria.
5.     Pseudosel Agar (Cetrimide Agar) used to recover P. aeruginosa contains cetrimide (antiseptic agent).
6.     Crystal Violet Blood Agar used to recover S. pyogenes contains 0.0002% crystal violet.
7.     Lowenstein Jensen Medium used to recover M.tuberculosis is made selective by incorporating malachite green.
8.     Wilson and  Blair’s Agar for recovering S. typhi is rendered selective by the addition of dye brilliant green.
9.     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.

Ø 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. 

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

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.
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.
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.

PREPARATION OF MEDIUM:

The liquid medium or broth is prepared by dissolving the known amounts of chemicals in distilled water; the pH is adjusted by adding N/10 HCl or 1N NaOH. The liquid medium is dissolved into either Erlenmeyer flasks or rimless clean test tubes.
In 15 ml capacity of test tube, 5 ml medium should be poured while in flask of 250 ml capacity, the amount of the medium should be 100 ml. These are then plugged with non-adsorbent cotton plugs. The plugged tubes or flasks should be wrapped by brown paper and placed for sterilization by autoclaving at a pressure of 15 lbs/inch2 (at temperature 121°C), for 15 min.
The heat sensitive substances (protein or enzymes etc.) should be sterilized by using membrane filters (millipore). The agar agar is to be dissolved separately and dispensed after dissolving all ingredients of the medium. It is first to be noted that all the glassware in use should be sterilized in oven at 170°C for 3 h before using them. Such sterilized glassware is needed for pouring the medium used for culturing the microor­ganisms.
Each and every biological process requires energy for their vital activities. The basic cell building requirements are supplied by the nutrition, which is ma­nipulated according to its requirement. Nutrition not only provides energy but also acts as precursors for growth of microorganisms.
The nutritional require­ment of an organism depends upon the biochemical capacity. If an organism is capable of synthesizing its own food using various inorganic components, requires a simple nutritional diet whereas organism unable to meet such synthesis requires complex organic substances.

Minimal Requirements:

Every microbe has its own specific minimal nutritional requirement. If it is not provided, they do not grow. This minimal requirement consists of a carbon source, nitrogen source, sulphur source, phosphorus source besides energy source.
They grown better in the presence of particular amino acids or vitamins or other compounds, so that the species could grow or develop better. Microbes can utilize a wide range of substrates from complex form of compounds (lignin etc.) that are generally not used by other forms of life.
Carbon source (glucose etc.) is essential for the basic cell structure because each and every biomolecule is made up of carbon along with other compounds. Nitrogen source is required for the biosynthesis of amino acids, nucleic acids, enzymes etc. Sulphur and phosphorous required for synthesizing nucleic acids, vitamins, and certain amino acids.
A photosynthetic microorganism eg. Cyanobacteria do not require a energy source. They use sunlight and trap the form of chemical energy, used frequently. With the help of CO2 and water, they synthesize food in the form of carbohydrate. But many microorganisms need some energy sources. This is met out by organic compounds. Some microbes have special capacity. They can harvest energy from redox potential for their vital activities.

Nutritional Types of Microorganisms:


Based on the way of harvesting energy, they are classified into two major groups. Those organisms that can make use of external energy sources and assimilate inorganic carbon are called as autotrophs.
Blue green algae and some chemosynthetic bacteria belong to this group.
They can make use of sunlight/ redox potential as their energy source. CO2 is the main and sole carbon source. Nitrogen is assimilated in the form of NH4+, sulphur as SO4– – and phosphorus in PO4– – from their surroundings.
Further, autotrophs may be of two types:
Photoautotrophs are bacteriochlorophyll containing microorganisms, while chemoautotrophs, utilize various oxidation-reduction reactions as their energy source. During oxidation, energy is released hence; the microbes oxidize the reduced traditional compounds and make use of the released electrons i.e. energy in case of sulphur bacteria (Thiobacillus spp.) and nitrifying bacteria (Nitrosomonas spp.). The phototrophs utilize solar energy to oxidizes from O (singlet) stage to O2 stage and thus utilizes the electrons released 
Many microorganisms resemble animals and humans, using organic compounds. These are called chemoorganotrophs but when they use inorganic chemicals as energy source, called chemolithotrophs.

Reference:        
            Ø  Dr.RC.dubey eds(2013) text of microbiology revised edition , S.Chand publication, ISBN:81-219-2620-3
Ø  Madigan M, Martinko J, eds. (2005). Brock Biology of Microorganisms (11th ed.). Prentice Hall. ISBN 0-13-144329-1.
Ø   Birgit Hadeler, Sirkka Scholz, Ralf Reski (1995) Gelrite and agar differently influence cytokinin-sensitivity of a moss. Journal of Plant Physiology 146, 369–371
Ø  Ryan KJ, Ray CG, eds. (2004). Sherris Medical Microbiology (4th ed.). McGraw Hill. ISBN 0-8385-8529-9.
Ø  Hans Günter Schlegel (1993). General Microbiology. Cambridge University. p. 459. ISBN 978-0-521-43980-0. Retrieved 6 August 2013.


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