Culture Media: Types, Preparation and Requirements

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

Types of Culture Media:

The first medium prepared was meat-infusion broth. As most pathogenic microbes require complex food similar in composition to the fluids of the animal body, it was Robert Koch and his colleagues who used meat infusion and meat extracts as basic ingredients in their culture media for the isolation of pathogenic microbes, while one of his assistant named Petri designed and developed glass dishes, known today as Petri dishes, are used in microbiological work.

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

(i) Synthetic or chemically defined medium:

These media are prepared by mixing all the pure chemicals of known composition for e.g. Czapek Dox medium.

(ii) Semi-synthetic or undefined medium:

Such are those media, where exact chemical composition is unknown e.g. potato dextrose agar or MacConkey agar medium.

On the basis of consistency, the culture media are of three types:

(a) Solid medium or synthetic medium:

When 5-7% agar agar or 10-20% gelatin is added the liquid broth becomes solidified. Such media are used for making agar slants or slopes and agar stab.

(b) Liquid medium or broth:

In 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 or floppy agar medium:

Such media are prepared by 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 organism and retards the growth of the other organism. On the other hand, there are differential media which serve to differentiate organisms growing together.

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 (Table 3.3).

Many microorganisms resemble animals and humans, using organic compounds. These are called chemoorganotrophs but when they use inorganic chemicals as energy source, called chemolithotrophs.