Types of Media:
Selective Media: Are designed to suppress the growth of unwanted bacteria and encourage the growth of the desired microbes.
Examples: MacConkey agar inhibits growth of gram positive bacteria and thus is selective for gram-negative bacteria. Phenylethyl alcohol (PEA) agar and Colistinalidixic acid (CAN) agar are selective for gram positive bacteria.
Manitol salt agar contains a concentration of 7.5% NaCl that is quite inhibitory to most human pathogens. Bile salts, a component of fecus, inhibit most gram positive bacteria while permitting many gram negative rods to grow. This media is used for selecting intestinal pathogens which contain bile salts. Dyes such as methylene blue and crystal violet also inhibit certain gram positive bacteria (Staphylococcus can produce acid from mannitol and turn the phenol red dye to bright yellow.
A medium containing acetate as a carbon source would be selective for organisms that grow on acetate. Sabaraud's dextrose agar is used to isolate fungi and cellulose for cellulose digesting bacteria.
Differential media : Makes it easier to distinguish colonies of the desired organism from other colonies growing on the same plate.
MacConkey agar is also a differential and selective medium to distinguish between lactose fermenting organisms (e.g, E.coli) and other non-fermenters (e.g, Shigella sp.). Lactose in the medium is fermented by E.coli producing acid which causes an indicator dye to change color to red and colonies that do not ferment lactose are white. Dyes can be used as differential agents because many of them are pH indicators that change color in response to the production of an acid or base. MacConkey agar contains neutral red, a dye that is yellow when neutral and pink or red when acidic.
Mannitol salt agar is used to screen for Staphylococcus aureus, and it turns the originally pink medium to yellow due to its ability to ferment mannitol. In a sense, blood agar is also differential because it is used to determine the hemolytic and non-hemolytic bacteria.
Blood agar is both differential and an enriched one. It distinguishes between hemolytic and non-hemolytic bacteria. Hemolytic bacteria (eg., many Streptococci and Staphylococci) produce clear zones around their colonies because of red blood cell destruction.
Isolation of pure cultures:
In natural habitats, microorganisms usually grow in complex, mixed populations containing several species. One needs a pure culture, a population of cells arising from a single cell, to characterize an individual species. Robert Koch introduced pure culture techniques.
Spread plate and streak plate:
Spread plate - Mixture of cells is spread out on an agar surface so that every cell grows into a completely separate colony, each colony representing a pure culture. A small volume of around 30 to 300 cells (mixed) is transferred to the center of the plate and spread evenly over the surface with a sterile bent-glass rod.
Streak plate – the microbial mixture 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 these techniques, successful isolation depends on spatial separation of single cells.
The pour plate : Extensively used with bacteria and fungi. The original sample is diluted several times to reduce the microbial population sufficiently to obtain separate colonies when plating. The microbes are mixed with molten agar which has been cooled t 45° Cand then poured into pertidishes. All these techniques require petridishes (special culture dishes) after their inventor Julius Richard Petri (1887). They consist of two round halves, the top half overlapping the bottom. Each bacterium replicates to form a colony that is visible to the naked eye. A colony contains up to 109 copies of the original bacterium.
Colony morphology and growth:
Colony development on agar surfaces aids the microbiologist in identifying bacteria because individual species often form colonies of characteristic size and appearance. Structure of bacterial colonies also has been examined with the scanning electron microscope. Generally the most rapid cell growth occurs at the colony edges. Growth is much slower in the center and cell autolysis takes place in the older central portion of some colonies. These differences in growth appear due to gradients of oxygen, nutrients and toxic products within the colony. At the colony edge, oxygen and nutrients are plentiful. Bacteria growing on solid surfaces such as agar can form complex and intricate colony shapes. Nutrient diffusion and availability, bacterial chemotaxis and the presence of liquid on the surface all appear to play a role in pattern formation. These patterns vary with nutrient availability and the hardness of the agar surface.
REFERENCES:
Text Books:
1. Jeffery C. Pommerville. Alcamo's Fundamentals of Microbiology (Tenth Edition). Jones and Bartlett Student edition.
2. Gerard J. Tortora, Berdell R. Funke, Christine L. Case. Pearson - Microbiology: An Introduction. Benjamin Cummings.
Reference Books:
1. Lansing M. Prescott, John P. Harley and Donald A. Klein. Microbiology. Mc Graw Hill companies.