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Isolation and Culture

 

Cultures
Types of Cultures
Classifications

Some Microbial Media

 

Cultures

Since the microorganisms are too small to be seen with the aid of a microscope, it is not generally practical to work with a single microorganism. For this reason we study cultures that contain thousands, millions, or even billions of microorganisms. A culture that consists of a single kind of microorganisms (one living species), regardless of the number of individuals, in an environment free of other living organisms is called auxenic culture. Microbiologists customarily refer to such a culture as a pure culture, although in a strict technical sense a pure culture is one grown from, a single cell. If two or more kinds (species) of microorganisms grow together, as they commonly do in nature, this mixed population is referred to as mixed culture.

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Types of Cultures (especially pure cultures): 

Streak culture (fig.8)

Streak culture of a microorganism is that which has developed by drawing an inoculated needle in a st­raight line over the surface of a medium.

Stab or Stick culture (fig. 9)

These cultures are prepared in solidified (by agar or gelatin) or liquid media by inserting straight an inoculation

Fig.8. Streak    Cultures   showing   various   types   of    microbial growths

 

Fig.8.Gelatin   stab   cultures: a.crateriform; b.napiform; c.infundi-buliform; d. ssccateje, snatiform.

 needle for some distance. The main points to note in these cultures are the growth of microorganisms along the line of puncture (insertion) and the changes occurring in the medium.

Stock culture:

Stock   cultures   are    those   cultures   of   known   species of microorganisms, which are maintained in laboratory for a longer duration of time. Actually, these cultures are the stocks of particular microorganisms or their strains and one can take samples from them for study.

Starter culture:

The starter culture consists of some species or com­binations of species best studied for the manufacture of the desired product. For instance, several commercial laboratories specialize in preparing starter culture for the dairy industry; the principal microorganisms involved are the species of Lactobacillus, Streptococcus and Leuconostoc, which are responsible for the desirable changes in manufacturing fermented rnilk products.

Enrichment culture:

Enrichment   culture   is   that  in   which   the growth   of a particular microorganism is favoured as against a mixed population by adjusting the nutritional environment. For example, if we have to isolate the microorganism W from a mixture of microorganisms WXYZ, the nutritional environment is modified by enrichment technique whereby the growth of W is favoured. Now the microorganism W can be transferred to other medium and a pure culture of it can be obtained.

Batch culture:

Growth of microorganisms in a limited volume of liquid medium is generally termed as a batch culture. This culture is helpful in studying the growth character­istics mainly of bacteria. When bacteria are transferred to a known volume of batch culture, their population undergoes a characteristic sequence in its rate of increase in cell number. Four recognizable 'phases' are seen when the increase in cell number is determined in relation to time (Fig.10). These are:

i. Lag phase:

In this phase there is no increase in the number of viable cells but the cells increase in- size due to ext­ensive synthesis of macromolecules. Lag phase represents a period of active growth without cell division; the cells being ready for division.

Fig.10.Growth curve of bacteriain batch culture.

 ii. Log phase:

            In the log phase the cell population increases logarithmically and the cells divide at the maximum rate; growth rates are measured during this period since growth occurs at a maximum rate.

iii. Stationary   phase:

In   this   phase the total   number   of viable cells   remains   constant as   a result of no increase in viable cell number due  to stopage  of  their division.

iv. Death or Decline phase:

This phase is characterized by exponential decrease in the number of their viable cells.

When a population of cells from a stationary phase or death phase in batch culture is transferred to fresh growth medium, the cells reenter the lag phase and initiate new growth.

Continuous culture:

A culture which permits growth of microorganisms at a constant rate (steady state growth) is called continuous culture the latter are extreme useful for genetical and biochemical studies of microorganisms because in these studies it is necessary to have microbial cells with a constant composition. Novick and Szillard (1959) developed a method (the chemostat), which permits growth of microorganisms at a constant rate.

Synchronous culture:

Synchronous culture represents population of cells of particular microorganisms that are at the same stage of their life cycle. All the cells in the culture divide at the same time, grow for a generation time and all divide again at the same time. Thus in a synchronous culture, the entire population is kept uniform respect to growth and division. The most widely used method for obtaining synchronous culture is the Helmstetter-Cummings technique, though a number of other techniques are available now. It is very difficult to analyze a single microbial cell to obtain the information about growth behaviour the synchronous cultures are used because growth behavior of such cultures are equivalent to indivi­dual cells.

2.2.2.Culture media:

Any nutrient or combination of nutrients so prepared as may be directly used for the growth or multiplication of microorganisms is referred to as ‘culture medium’(pl.culture media).

 Microbiological culture media consist of various nutrient substances supporting the growth of particular types of microorganisms. Some media contain solutions of inorganic salts and may be supplemented with one or more organic compounds. Other media are prepared from complex in-gradients such as extracts or digests of plant and animal tissues. The propagation of rickettsias and viruses requires living host cells (a tissue culture) within which they multiply, Bacteriophages propagate within certain bacterial cells in which the bacterium provides nutrients and other factors required for the synthesis of more bacteriophages. In this instance the bacterial cell serves as the "medium" (host) for the bacteriophage.

Culture media would, hereinafter, be called 'media' (sing, medium).

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Classification:

1. Media may be grouped principally as follows:

-Those media that require living cells or tissues, the latter get parasitized by the microorganisms to be cultured.

-Those media that do not require living cells or tissues. These media may again be divided into two:

                          a.  Non-synthetic media

                          b.  Synthetic media

a.  Non-synthetic media:

              A medium in which the exact chemical composition of each of the constituents is not certainly known is ref­erred to as non-synthetic medium. Potato-Dextrose-Agar (GM-25), Soil-Extract-Agar (SM-1), Oatmeal-Agar (GM-24), Malt-Extract-Agar (GM-19b), Waksmans medium (GM-40) are   some   of   the   most   widely   used   non-synthetic   media.

b.  Synthetic media:

             A medium in which only pure chemicals in definite concentrations are used is called synthetic medium. On account of their known chemical compositions these media are useful for nutritional and metabolic studies. Czakek's-Dox Medium (GM-9) and Richard's solution (GM-27) etc. are the most widely used synthetic media.

2. Media  may  also be divided into four categories on  the basis of consistency i.e.,  their physical slate, they are :

                                a.  Liquid media

                                b.  Semisolid media

                                c.  Liquefiable solid media

                               d.  Solid media

a.  Liquid media:

            These media are used in liquid form. Examples-Nutrient Broth, Skimmed Milk, Peptone Solution etc.

b.  Semisolid media:

These media contain a smaller amount (0.5% or less) of agar which imparts a "custard consistency". Examples-Cystine Trypticase Agar Medium.

c.  Liquefiable solid media:

This medium is also called solid reversible to liquid medium. These media are prepared by adding suitable amount of gelatin or agar to the liquid medium and remain solid when cool but become liquid when warm or vice versa. Example-Nutrient-Agar Medium, Nutrient-Gelatin Medium, Potato-Dextrose-Agar Medium.

d.  Solid media:

These media always remain solid. Example-Potato slices (used for special cultivation of bacteria), Coagulated Blood Serum, Coagulated Egg, Trypticase-Soy-Agar Medium.

 

3.   Media used  for  bacteria (bacteriological  media)  may be classified, on  the basis of   their application or function, as follows :

                a.  Cultivation media

                b.  Storage media

                c.  Enrichment media

                d.  Differential media

                e.  Assay media

                f.  Maintenance media

a.  Cultivation media:

              Medium which is used for the general cultivation of bacteria. Example-Nutrient Broth, Nutrient Agar etc.

b.  Storage media:

Medium in which bacteria are stored in "stock culture" condition for longer periods to provide a source of viable cultures are called storage medium. Example-Yeast Extract Mannitol Agar Medium.

c.  Enrichment media:

Enrichment medium (enriched medium) is that in which nutritional environment is adjusted in such a manner as to enhance selectively the growth of a certain bacterial type within a given mixed inoculum. This approach cons­titutes a powerful tool for the bacteriologist in the isolation and identification of pure cultures from an initially mixed population of bacteria. Example-Addition of extracts of plant or animal tissues to Nutrient Broth or Nutrient Agar media provides additional nutrients and the media start favouring the growth of fastidious heterotrophic bacteria.

d.  Differential media:

Medium employed to determine differential reactions which may permit presumptive identification of bacterial species is called differential medium. Blood Agar Medium is a good differential medium. If a mixture, of bacteria is inoculated on a Blood Agar Medium, some of the bacteria may hemolyze (destroy) the red blood cells while others do not show hemolytic reactions. The observer may see a clear zone of hemolyzed red blood cells around certain colonies of bacteria while the non-hemolyzed bacterial colonies do not develop such a clear zone around them.

e.  Assay media:

Certain media of prescribed composition have pro­found influence on the bacterial cell with respect to for­mation of enzymes, toxins, antibiotics and other products. Such media are called assay media or media for special purposes. Example- Pyridoxine-deficient Growth Medium for Streptococcus faecalis which yields ceils containing, large amounts of tyrosine decarboxylase apoenzyme.

f.  Maintenance media:

These media are different from growth media and are required to maintain the viability and physiological characteristics of bacteria.

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Some Microbiological Media

1. General purpose media for bacteria:

a. Nutrient Broth (or Beef-extract Peptone Broth):

This   liquid   medium    is   widely   used   for   the   general cultivation   of   aerobic   bacteria   and   as   a   basal   medium for a variety of physiological tests.

Composition

 

Beef-extract       3 g
Peptone 5 g
Distilled water   1000 ml
Final pH 6.8 - 7.2 ±

The water containing beef-extract and peptone is heated to 60 C to promote solubilization of the ingredients. It is now cooled and the said pH is adjusted; is dispensed in tubes or other containers and is autoclaved at 15 Ib pressure (or 120 C temperature) for 15 minutes.

b.  Nutrient Agar (or Beef-extract Agar):

This medium supports the growth of a variety of common heterotrophic bacteria. It is commonly used for enumeration of bacteria from various substrates

Composition:

Beef-extract 3 g
Peptone 5 g
Agar 15 g
Distilled water 1000 ml
Final pH 6.8 - 7.2±

The ingredients are added to water which is heated to 60 C to promote solubilization of the ingredients. It is cooled and the pH is adjusted to 6.8 - 7.2. The medium is dispensed in tubes or other containers and is autoclaved at 15 Ib pressure (or 120°C temperature) for 15 minutes.

c.  Trypticase Soy Agar:

This is a general purpose solid medium which favors better growth of more bacteria than does Nutrient Agar Medium.

Composition:

 

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