To study microorganisms properly, we have to be able to grow them. To accomplish this, it is necessary to transfer the specimens to an environment that will simulate the same conditions under which they occur in nature.
Nutritional requirements vary widely from one species of bacteria to another and in many cases are not clearly known. Much has been accomplished concerning the duplication of conditions necessary for the cultivation of microorganisms, and most microbes can now be cultivated on or in artificial media.
Ingredients in media are intended to supply the nutritional and growth requirements of microorganisms so that the cultures studied will present characteristics comparable to those that exist in nature.
1. Primary or Isolation Media: Media used for primary inoculation of specimen; usually prepared in Petri dishes so they can be streaked to obtain isolated colonies of any organisms present. Media used routinely in most laboratories are, Trypticase soy agar (TSA) and Nutrient agar
2. Enrichment Media: Media that has been enriched by the addition of extra ingredients to enhance the growth of fastidious microbes. Examples: blood agar, chocolate agar
3. Selective Media: Media used to grow one particular type of bacteria from a mixed culture by inhibiting the growth of the other bacterial species. Examples: Phenylethyl alcohol (PEA) agar-selects for gram-positive bacteria; Mannitol salt agar-selects for staphylococci; MacConkey agar-selects for gram-negative bacteria; Eosin methylene blue agar-selects for gram-negative bacteria
4. Differential Media: Media used to distinguish between species of bacteria which may look exactly alike or very similar by other methods, such as the Gram stain, or on TSA. Examples: MacConkey agar – distinguishes between lactose fermenters and non-lactose fermenters; Mannitol salt agar- distinquishes between Staphylococcus aureus and other Staphylococcus species; Eosin methylene blue – distinguishes between E.coli and other enteric bacilli
Most used media
Trypticase soy agar; nutrient primary isolation media; will grow many types of bacteria (both gram-positive and gram-negative bacteria)
Phenylethyl alcohol agar; selective media; grows only gram-positive bacteria. The phenylethylalcohol is inhibitory to gram-negative bacteria.
MacConkey agar; selective media; grows only gram-negative bacteria; gram-positive bacteria are inhibited by the crystal violet dye in the agar. MacConkey agar is also used as differential media to distinquish between lactose-fermenting and non-lactose fermenting bacteria. Incorporation of lactose, bile salts, and phenol red indicator causes lactose-fermenters to appear red, whereas non-lactose fermenters will appear colorless or transparent.
Mannitol salt agar; selective media; grows only Staphylococcus bacteria. 7.5% salt is inhibitory to most other bacteria. Mannitol salt is also differential media used to distinguish between Staphylococcus aureus and other Staphylococcus species. Mannitol fermentation with subsequent acid production by S. aureus is indicated by a change in the color of the phenol red indicator to yellow.
Eosin methylene blue; selective media; grows only gram-negative bacilli. Eosin methylene blue is also differential media used to distinguish E.coli from other gram-negative enteric bacilli. E.coli ferments the lactose in the agar, causing acid production, which precipitates the eosin and methylene blue dyes. This results in a metallic blue-black color with a greenish sheen. Other gram-negative enteric bacilli will appear pink or transparent.
Blood agar plate; enrichment media used to grow a variety of fastidious microorganisms such as Streptococcus. Blood agar is also used to demonstrate different types of hemolysis:
Beta hemolysis → complete lysis of the red blood cells by streptolysin 0 and streptolysin S enzymes.
Alpha hemolysis → incomplete lysis of red blood cells resulting in the breakdown of hemoglobin, which produces a greenish halo around the bacterial colonies.
Gamma hemolysis → no lysis of the red blood cells; no significant change in in the color of the agar surrounding the colonies.
Specimens submitted to the laboratory for microbiological examination often contain a mixture of microorganisms. In order to study the characteristics of a microorganism, it is first necessary to separate it from other microorganisms present in the mixture; we must isolate the suspected organism in pure culture.
A pure culture is one in which all of the cells present in the culture originated from a single cell type. The streak plate method is the method classically used for isolating a pure culture from a mixed culture.
With this method you will attempt to purify a mixed broth culture containing several different species of bacteria. Once isolated, the bacterial colonies can be differentiated from each other. An essential component for isolating a pure culture is aseptic technique, which involves the transfer of microorganisms from one environment to another in such a way that neither you nor the environment around you is contaminated with the specimen that you are transferring and that the pure culture you are making is not contaminated with other organisms from the environment. In the aseptic preparation of pure cultures, the transfers are usually made with sterile inoculating loops or needles or with sterile pipettes.