Sunday, 11 October 2020

Culture Media - Definition, Principle, Preparation and Types and Microbiology

Culture media can be defined as the environment from which the organisms satisfy entire nutritional requirements for growth. They are used for the cultivation of microorganisms. 

Principles involved in construction of culture media -

Construction of a cultural medium requires consideration of following criteria.

1). The medium should consist of a balanced mixture of all the required nutrients at a concentration that permits good growth. This requires a thorough knowledge of nutritional requirements and habitat of the organism to be cultivated.

2). The medium should not possess nutrients in great excess. This is due to inhibitory effect of many nutrients at a higher concentration. e.g. fatty acids. certain metal lons etc.
  High concentration may also create unfavorable osmotic conditions for the organisms to be cultivated.

3). Due to the growth and metabolic activities of the organisms, unfavorable conditions gradually develop into the medium. This may be reflected as
a). increase in concentration of toxic metabolites,
b). depletion of availability of dissolved oxygen,
c). change in pH etc.
This results into the inhibition of growth. Hence, more supply of nutrients in excess is meaningless.

4). The microorganisms differ widely in their nutritional requirements. Hence, the medium should consist of those ingredients only. which can be attacked and utilized by the organisms to be cultivated.


Ingredients used for the preparation of bacteriological media -

A bacteriological medium should contain
1). Suitable sources of carbon, nitrogen, energy and electron donor. 
2). Mineral sources
3). Growth factors, if required
4). Water

Ingredients used as sources of carbon -

Various carbohydrate and non carbohydrate compounds are used as the sources of carbon in the medium. They include:  
a. Carbohydrates like
1. Monosaccharides such as glucose. fructose and others.
2. Disaccharides such as lactose, sucrose, maltose etc.
3. Polysaccharides such as starch, cellulose and others.

b. Noncarbohydrates like
1. Protein and protein hydrolysates
2. organic acids
3. hydrocarbons

Ingredients used as sources of nitrogen -

A wide variety of nitrogen compounds, both organic and Inorganic, can be used as sources of nitrogen in the medium construction.
Organic sources of nitrogen include proteins and protein hydrolysates such as peptone, peptides, amino acids, meat and meat extract, urea etc.
  Inorganic sources of nitrogen include nitrate, nitrite. ammonium salts, liquor ammonia etc.

Ingredients used as sources of energy and electron donor -

  Energy source is another important ingredient as nutrient, which is used for driving cellular metabolism. A wide variety of substances are used as sources of energy. Chemotrophs obtain energy from chemical which include both organic
and inorganic.
The substances incorporated into medium as sources of energy are as under.

a). Usually organic substances are used as sources for energy by chemoorganotrophs. Usually these organisms use same organic matter as source of both carbon and energy.

b). Usually inorganic substances are used as sources of energy by chemolithotrophs. These substances include reduced sulfur and iron compounds, ammonia or molecular hydrogen.

Organisms normally use the same chemical as electron donor, which is used as energy source. Upon oxidation of these compounds, they generate reducing power, required for biosynthesis.

Mineral sources -

Minerals are usually supplied as corresponding salts into the medium. Often, the Ingredients used as sources of carbon, nitrogen and energy as well as water used to prepare medium may contain some of the minerals needed by organisms.

Growth factors -

Growth factors may be added to the medium in pure form or materials containing them. Their addition supports growth of fastidious organisms. It also allows luxuriant growth of other organisms. Generally, substrates like meat and beef extract, yeast extract are used as source of growth factors.

Water -

All media ingredients are dissolved in water. The water used may be distilled water or tap water. Tap water is used, where media are used for commercial (fermentation) purposes.

Buffers -

  Apart from all these nutritional requirements, presence of suitable buffer is necessary in the medium. Buffer is the system that resists any change in pH of the medium. It is a mixture of weak acid and its conjugate base. Buffers have maximum buffering capacity at a pH. where concentration of the acid equals to Its conjugate base.

  When bacteria grow in the medium their metabolic activity affects the pH of the medium. This in turn affects the growth. Buffers, when incorporated into the medium, help in maintenance of optimum pH required for the growth. A variety of buffer systems can be incorporated in media. A few examples are listed below.
-COOH / -COO¯ e.g. acetate buffer
H2PO4¯/ HPO4²¯ e.g. phosphate buffer
NH3+ / -NH4 e.g. proteins

Of these, phosphate buffers are widely used In bacteriological media because they have Optimum buffering capacity at pH 6-8.
Proteins and peptones, used in the preparation of media as source of carbon, nitrogen and energy can also act as buffers.

Solidifying agents -

  Certain media are convenient to use if they are solid. The bacteriological media, therefore, can be solidified by dissolving a suitable solidifying agent into the medium. The solidifying agents used are agar, gelatin and silica gel.
- Agar is the most commonly used solidifying agent.
- Gelatin was the first solidifying agent employed in construction of solidified media. However, Its use became limited due to following reasons,
1). Property of gelatin to melt at temperature above 35°C.
2). Easy degradation by many microorganisms.
- Silica gel is inorganic substance and it is widely used for preparation of solidified media meant for cultivation of chemoautotrophs.


Some important ingredients used in construction of bacteriological media -

Following are the most widely used media ingredients, which are used for construction of media.

• Bacteriological peptone

  Peptone is the product obtained from hydrolysis of native proteins, such as gelatin, casein etc.
They are hydrolysed by acids or enzymes.
These digested proteins are easily attacked by most microorganisms.
They can serve as source of organic nitrogen. In addition, it can also satisfy the need of carbon, energy as well as growth factors for the organisms.

• Beef extract

It is the extract of beef and obtained as paste, when concentrated.
It contains water soluble substances of the animal tissues, organic nitrogenous compounds, some carbohydrates, vitamins and growth factors. It is supplied to the medium mainly as the source ofr growth factors.

• Yeast extract

  It is the extract obtained from yeast and available as powder. It is rich source of vitamins and used as source of growth factors in the medium.

• Agar

Agar is the most widely used solidifying agent in preparation of bacteriological media. It is obtained from cell walls of marine algae. It is hetero polysaccharide in nature. Following characteristics of agar make it most suitable as the solidifying agent.
1. Ability to melt at 92°C - 93°C and gets solidified at 42°C.
2. Resistance to degradation by microorganisms.
3. Non interfering with the growth of microorganisms.


Types of culture media :

The culture media can be classified into different types on the basis of various criteria:
A). Solid, solidified, semisolid and liquid media.
B). Natural, synthetic and complex media.
C). Routine and specialized media.

A. Solid, solidified semi solid and liquid media -

Based on physical status, the media can be classified as solid, solidified, semisolid or liquid media.

Solid media

  Solid media are prepared by mixing solid ingredients. Water is used to provide adequate moisture to the organisms to be cultivated.
  These media are commonly used in industry for the fermentative production of enzymes as well as preparation of inoculum.
e.g. use of moistened rice bran, dried bread powder, grass soaked in buffer etc.

Liquid or broth media

  A liquid medium, without any solidifying agent is called broth medium.
  They are prepared by dissolving nutrients into water. They are used mainly for
1. Cultivation of microorganisms.
2. Primary cultivation of microorganisms from natural sources.
3. Study of various biochemical and physiological characters of organisms etc.

Solidified media

  Solidified media are prepared by dissolving suitable solidifying agent Into the liquid media. Depending on the type of solidifying agent used, these media are commonly called as agar media, gelatin media or silica gel media. These media are commonly used for
1. Isolation and cultivation of microorganisms.
2. Preservation of microorganisms.
3. Enumeration of microorganisms by viable count. bloassay etc.

Semi solid media

  Semi solid midea are prepared by dissolving suitable solidifying agent into the liquid media. But, in this case, the concentration of solidifying agent is much less. AS a result, the gel strength of medium remains low. These media are widely used for study of motility of organisms.


B. Natural, synthetic and complex media

Based on the composition, the bacteriological media can be classifled as natural, synthetic and complex media,

Natural media

  These media are prepared from natural ingredients and extract, which can provide natural environment to the organisms aimed for isolation and cultivation.
  These media were first employed by Winogradsky to isolate organisms from soil. The type of natural extracts used depends on the type of organisms to be Isolated.
e.g. Soil extract for isolation of soil organisms. Potato extract for isolation of plant pathogens etc.
  These media are usually preferred for primary isolation of organisms from their natural habitats.

Synthetic media

  These media are commonly known as chemically defined media. These are the media where the exact chemical nature and composition of all the ingredients used in the preparation of media is known. These media are prepared by adding
- Known concentration of sugar or other source of carbon and energy, whose chemical composition is known.
- Nitrogen source, usually as ammonium or nitrate salt or Liquor ammonia.
- Salt solutions to provide mineral nutrients requirements.
- Amino acids and growth factors, If required, in known concentration.

These media are widely used to study
1. influence of various chemicals on the growth
2. bioassay etc.

Complex media

  The media, where, the exact chemical composition of all the Ingredients of the medium is not known, are called complex media.
  These media are widely used for growth and cultivation of microorganisms. These media are relatively easy to prepare, comparatively cheaper than synthetic media and allow better growth of organisms. Media containing peptone, beef extract etc. fall in this category.


C. Routine and specialized media 

  Based on the nature of use. the media can be classifled into two broad categories: routine and specialized.

Routine media

  The media, which are routinely used for cultivation of micro organisms are called routine media. They are designed in a manner that they permit growth of most of the types of organisms. Nutrient agar and nutrient broth media fall in this category because they are used widely in the laboratory.

Preparation of n-agar & n-broth
Composition of nutrient agar and nutrient broth

Specialized media

  The media which are used for specialized purposes fall in this category, Following are the diferent categories of such specialized types of media.

Enriched media -

  Certain mieroorganisms possess complex nutritional requirements. They grow poorly on the routine laboratory media. They need supply of specific growth factors. Therefore, special media are constructed which are supplemented with highly nutritious substances such as blood, serum, egg. yeast extract etc.
  Such media are nutritionally enriched. Hence, they are called enriched media. They favor luxuriant growth of microorganisma, Including fastidious organisms.
e.g. Blood agar medium, Chocolate agar medium, Glucose yeast extract agar medium, Egg media etc.

Enrichment media -

  These are the media which favor growth of destred type of organisms, without affecting normal growth of other organisms. These media are constructed by using those
growth of desired group of organisms.
e.g.
a) Incorporation of aromatie hydrocarbon in the medium as the sole source of carbon will favor growth of hydrocarbon degrading organisms.
b). Adjusting pH of the medium at a very low value, 2 to 3 will favor growth of acidophiles.

Usually, these media are used as liquid media and are used for improving probability of Isolation of desired type of organisms from natural populations.

Selective media -

  These are the media which allow growth of only selective group of microorganisms and Inhibit growth of other undesired types of organisms. These media are prepared by adding speciflc inhibitors into the medium, which inhibit particular class of organisms.
e.g.
a). Crystal violet or bile salt may be used to construct selective media for isolation of gram- negative bacteria. These agents are able to inhibit growth of gram-positive bacteria.
b). Streptomycin is used in preparation of rose bengal agar medium for inhibiting growth of bacteria and increase probability of isolation of fungi.

Selective media are normally used as agar media and are widely used for the isolation of desired types of microorganisms from a mixed population.

Differential media -

  The media, which allow differentiation of two groups of organtsms on the basis of their growth and cultural characteristics are called differential media.
  These media are prepared by, Incorporating suitable reagents or chemicals or Indicators Into the medium, which helps in differentiation of different groups of organisms.
e.g. Mac Conkey's agar medium. It is incorporated with pH Indicator dye Neutral red to allow differentiation of lactose fermentor and non fermentor colonies.

Assay media -

  The media which are used for the purpose of bioassay of antibiotics or vitamins and growth factors are called assay media. These media have a specifically prescribed composition.
  These media do not contain any substance that bind with substance to be assayed and interfere with it's estimation. Further these media do not contain even a trace of a substance to be assayed.

Enumeration media -

  Media, which are used for enumeration of bacteria from the specimen are called enumeration media. These media allow most types of bacteria to grow. Usually these media are used as agar media.

Maintenance media -

  Special media are used for the maintenance of culture. These media are designed in a manner that the properties of organisms are not affected and organisms grow slowly such that the intervals between two subculturing can be longer.

   Usually these media are solid. The use of solid /agar media allow diffusion of toxic metabolites away from the growing culture and protect them from their toxic effect.


Some differential and selective media :

A). Mac-Conckey agar -

   Mac-Conkey agar is a selective as well as a differential medium, used for selective Isolation of Enterobacteriaceae and related enteric gram-negative rods.
  The medium contains bile salt and/or crystal violet, which inhibit the growth of gram-positive bacteria and some fastidious gram- negative bacteria. Thus, the medium is a selective medium.
 
The medium also incorporates lactose as a sole fermentable carbohydrate and neutral red as pH indicator dye. The organisms, capable of fermenting lactose produce acid.
  Hence their colonies become acidic, which appear pink because of the conversion of neutral red (Neutral red is pink under acidic conditions).
  Colonies of lactose non-fermenting bacteria appear colorless. Thus, the medam ls also differential, allowing differentiation of bacteria as lactose fermentor and non-fermentor.

B). Eosin Methylene Blue (EMB) agar -

  EMB agar is also a selective and differential medium. useful for differentiation of Enterobacterlaceae and related coliforms. The medium incorporates aniline dyes; eosin and methylene blue, which inhibit gram-positive and fastidious gram-negative bacetria and hence it acts as selective medium.

  The medium also incorporates lactose as a sole fermentable carbohydrate and neutral red as pH Indicator dye. Organisms that ferment lactose and produce large amount of acid, allow precipitation of aniline dyes on their colonies.

Typical coliforms like E. coli produce colonies with greenish blue metallic sheen. Atypical coliforms like Enterobacter produce pink colonies but not metallic sheen.
  Enterobacter can convert acid in to a neutral compound, 2.3 butanediol and hence they do not produce sufficlent acidity to allow precipitation of anfline dyes. Pink colonies are due to presence of neutral red in medium.
  Lactose non fermentor organisms produce colorless colonies. Thus, the medilum also acts as a differential one.

C). Deozycholate citrate agar (DCA) -

  DCA agar is also selective and differential medium, used for isolation of members of Enterobacteriaceae.The medium contains
- About three times the concentration of bile salts than Mac-Conkeys agar medium. This permits selective isolation of Salmonella from mixed cultures contaminated with coliforms and other gram-positive bacteria.
- Sodium and ferrie citrate that retard growth of coliforms.
- Lactose and neutral red. Their presence in the medium helps in differentiation of lactose fermentors from lactose non-fermenting bacteria.

D). Endo agar -

  The medium is a differential one, used for differentiation of lactose fermentors and non-fermentors. Incorporation of sodium sulfite and basic fuchsin inhibits growth of gram- positive bacteria.
  Lactose fermentors produce pink colonies due to release of basic fuchsin from neutralized sodium sulfite-basic fuchsin complex. Acetaldehyde. produced as one of the intermediates during fermentative metabolism is trapped by sodium sulfite allowing basic fuchsin to be released from the complex, that impart pink color to the colonies.

E).Salmonella - Shigella (SS) agar -

  SS agar medium is highly selective for Isolation of Salmonella and Shigella from clinical specimens. Presence of high concentration of bile salts and sodium citrate Inhibits gram- positive and many gram-negative bacteria, Including coliforms.
   Presence of lactose and neutral red allows differentlation of organisms as lactose fermentor and non- fermentors.
  Similarly. presence of sodium thiosulfate and ferric citrate allows the detection of bacteria that produce H₂S. Organisms that use sodium thiosulfate and reduce it to produce H₂S develop black colonies due to formation of iron sulfide.


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