The artiﬁcial culture and maintenance of microorganisms is the basic necessity of the modern day research. Culturing of microorganism requires a supply of the necessary nutrients in the form of a culture media, together with the provision of appropriate conditions such as temperature, pH and oxygenconcentration.A culture medium is a solid or liquid preparation that contains nutrients that are essential for the growth, transport and storage of microorganisms.
Culture media may be categorised into
1. Chemically defined media: This is the medium whose exact chemical composition is known. Such media are used in the research laboratories for growth of specific bacteria. For ex. Glucose is used in media used for the growth of chemo-heterotrophic E.coli
2. Complex Media: This is the medium where the exact chemical composition is not known as it is made up of nutrients including extracts from yeasts, meat, or plants, or digests of proteins from these and other sources. Ex. Nutrient agar- it is one of the most common complex media used for the growth of heterotrophic bacteria.
3. Selective and Differential Media: In clinical and public health microbiology, it is very necessary to detect the presence of specific microorganisms associated with disease or poor sanitation. For this task, selective and differential media are used.
Selective media favour the growth of particular microorganisms. Selective media are designed to suppress the growth of unwanted bacteria and encourage the growth of the desired microbes.For example, bismuth sulphite agar (BSA) is one medium used to isolate the typhoid bacterium, the gram-negative Salmonellatyphi, from faeces. Bismuth sulphite inhibits growth of grampositive bacteria and other gram-negative intestinal bacteria.
Differential media make it easier to distinguish colonies of the desired organism from other colonies growing on the same plate. For ex. Blood agar (which contains red blood cells) is used to identify bacterial species as Streptococcuspyogenesthat destroy red blood cells.
4. Enrichment Culture media: This media is used to enhance the growth of a specific microorganism present in a sample. It is particularly employed in pathological microbiology. Enrichment medium is usually combined with incubation conditions (temperature, aerobic/anaerobic, etc) to select for growth of the desired organism. Foe ex. Use of bile salts in media designed for enrichment of Enterobacteriaceae such as E. coli.
5. Media for growth of anaerobic bacteria: Anaerobic bacteria require absence of oxygen to grow, hence they must be placed in a special medium called a reducing medium.These media contain ingredients, such as sodium thioglycolate, that chemicallycombine with dissolved oxygen and deplete the oxygen in the culture medium.
Microorganisms occur as mixed populations in natural habitats. However, pure cultures are needed to characterize and study any organism thoroughly. Some of the ways to prepare pure cultures are
1. Spread plate method:
In this method, a small volume of dilute microbial mixture containing around 30 to 300 cells is transferred to the center of an agar plate and spread evenly over the surface with a sterile bent-glass rod.
2. Streak plate method : In this method a microbial inoculum is transferred to the edge of an agar plate with an inoculating loop or swab and then streaked out over the surface in one of several patterns.
In both spread-plate and streak-plate techniques, successful isolation depends on spatial separation of single cells.
3. Pour plate method : In the pour plate the original sample is diluted several times to reduce the microbial population sufficiently to obtain separate colonies after plating.
There are four basic phases of microbial growth: the lag phase, the log phase, the stationary phase, and the death phase.
- In the lag phase represents the initial state when there is very little or no multiplication of cells. This phase can last from zero to one hour to several days.
- Log phase starts with the logarithmic multiplication of the cells, when the cell number starts increasing exponentially. At this stage the cells are the most active metabolically.
- The stationary phase is the state of equilibrium where the growth rate slows down and the number of dead microorganisms equals the number of new microorganisms, and the population stabilizes.
- In this phase the number of dead cells exceeds the number of new cells. This phase continues until the population is diminished or dies out entirely.
The figure below represents a typical growth curve.
Points to remember
- Different types of media are used for culturing of different microorganism.
- Culture media can be prepared from chemically defined components (defined media or synthetic media) or may contain constituents like peptones and yeast extract whose precise composition is unknown (complex media).
- Culture media are classified based on function and composition as general purpose media, enriched media, selective media, and differential media.
- Pure cultures usually are obtained by isolating individual cells with any of three plating techniques: the spread-plate, streak-plate, and pour-plate methods
- Microbial growth can be represented by a typical curve known as the growth curve.
- The growth curve usually has four phases: the lag, exponential or log, stationary, and death phases