Media that by virtue of their ingredients distinguish organisms growing together are differential media. Examples include eosin-methylene blue (EMB) agar and MacConkey agar, used in the differentiation of the gram-negative bacteria of the intestinal tract. The incorporation of lactose into such differential media makes possible a sharp separation in colonial characteristics between the organisms that ferment this sugar and those that do not. The colonies of the lactose fermenters are deeply colored red or possess a metallic sheen; colonies of the nonfermenters are colorless. This point is important because generally pathogens in the intestinal tract such as the Salmonella and Shigella species do not ferment lactose, whereas certain of the normal inhabitants, the coliforms, do.
Media that by virtue of their complexity of com¬position promote the growth of one organism and retard the growth of others are selective media. Their inhibitory component is highly selective in many instances. Selective media may also differentiate among certain genera of microorganisms. Examples of selective media are Deoxycholate-citrate agar and Lowernstein-Jensen medium. Deoxycholate-citrate agar inhibits the growth of most coliforms, along with many strains of Proteus, while favoring the selective isolation of intestinal pathogens, especially those of the Salmonella and Shigella species. What coliform colonies do appear on the medium is readily separated from the nonfermentors of lactose (the pathogens) by their color and opacity. Lowenstein-Jensen medium promotes the growth of tubercle bacilli but retards the growth of any other organism present.
Although EMB agar and MacConkey agar are primarily thought of as differential media, they display a selective function in that they allow the growth of gram-negative bacteria but prevent the growth of gram-positive ones.
Selective and differential media are of great value in the diagnosis of infections in such areas of the body as the respiratory and intestinal tracts, where normally a variety of organisms reside and where the presence of pathogenic bacteria may not disrupt the normal bacterial pattern for the area (the normal flora). In test specimens taken from such an area, the pathogens tend to be mixed with many other microorganisms, and only with the use of selective and differential media is it possible to isolate and identify the disease producers.
Synthetic media
The culture media just discussed are made up of components of variable composition, such as meat infusions, serum, or other body fluids. Such media are nonsynthetic (complex) media. Synthetic (chemically defined) media, on the other hand, contain ingredients of definite chemical composition. Used primarily in research work, such media have the advantage that, wherever prepared, their composition is the same. Therefore the results of microbial action on these media in one laboratory are strictly comparable with those in other laboratories thousands of miles away.
CULTURE METHODS
Inoculation
A culture is made or inoculated when the specimen to be cultured, such as sputum, urine, or pus, is placed into a fluid medium or is rubbed gently over the surface of a solid medium with either a sterile swab or a flame-sterilized inoculating wire loop The inoculated medium is then incubated for a period of time, routinely 24 to 48 hours and then examined under a microscope such as a compound binocular microscope.
Bacteriologic incubator
A bacteriologic incubator consists of an insulated cabinet fitted with an electrical heating element and a thermoregulator to maintain the temperature at a set point. A good thermoregulator placed in a properly constructed incubator maintains the temperature constant from day to day to within 0.5° C. The incubator must be properly ventilated. It may be constructed so that circulation of air is brought about by the combined effects of gravity and the difference in weight of warm and cold air. Incubators are also ventilated with blowers or fans. All are fitted with perforated shelves and most with sets of double doors. While keeping out cold ail, the inner ones of glass allow the contents of the incubator to be viewed. Incubators may be fitted with mechanisms to maintain a controlled environment within the incubator, such as a desired carbon dioxide tension at constant temperature and humidity, thereby facilitating the growth of carbon dioxide-dependent and other fastidious bacteria with special requirements for growth. To support the growth of anaerobes, the anaerobic incubator is constructed so that after a vacuum has been pulled for the interior, the environment of the evacuated chamber may be charged with whatever gas is desired-nitrogen, hydrogen, or carbon dioxide, alone or in various mixtures.
Inspection of cultures
Each microbe in the medium inoculated and incubated multiplies rapidly, and within a few hours there are many more microorganisms in the culture than there were in the original sample when examined under a compound binocular microscope. Consequently, bacteria or fungi are readily identified in cultures using a compound binocular microscope. When in smears of the same test sample, they may he found with difficulty or not at all. Diphtheria bacilli can be found in throat cultures twice as often as in throat smears. When a culture is made on a solid medium, all the bacteria that grow from each individual bacterium which are de¬posited on the medium cling together to form a mass that is visible to the naked eye, called a colony. The colony has characteristics that can be seen using a compound binocular microscope such as texture, size, shape, color, and elevation. These features are fairly constant for each species and are valuable in differentiating one species from another. Theoretically each organism should give rise to one colony, but two or more may cling together and, when planted on a medium, give rise to but a single colony. If organisms that cling together are different, the colony will contain the different kinds. Petri dish cultures permit good observation of colonies using a microscope, since the large surface area favors separation of individual microorganisms. Petri dish cultures are usually spoken of as plates and the process of making such cultures is referred to as plating or streaking.
Pure cultures
A pure (axenic) culture contains only one kind of bacteria. A mixed culture on the other hand contains two or more kinds. As a rule, infectious material contains more than one kind of bacteria. So that the pathogen can be studied, it must be separated from all other kinds and grown alone as a pure culture. Pure cultures are usually obtained by the pour plate or streak plate method. The pour plate method is as fol¬lows:
1. Melt at least 3 (or more if needed) tubes of agar in boiling water and then allow cooling to around 42° C. If an enriching material that is injured heat, such as serum or whole blood, is needed, add it to the medium at this time.
2. Transfer to 1 tube a loopful of the test material from which you expect to obtain a pure culture.
Replace the cotton plug and roll the tube between the palms of the hands to distribute the bacteria throughout the medium.
3. Flame-sterilize the loop thoroughly. Transfer 3 loopfuls of the contents of the inoculated tube to the second tube.
4. Mix the contents of the second tube with the inoculum. Sterilize the inoculating loop. Transfer 5 loopfuls of the mixture to the third tube and mix.
5. Pour the contents of each tube of medium into separate Petri dishes and allow solidifying.
6. Incubate the Petri dishes for 24 to 48 hours. Observe the colonies using a microscope.
Successive dilution of the specimen in the three tubes reduces the number of bacteria and disperses them in the medium so that the colonies in the Petri dish cultures are more likely to be distinctly separated from each other. Those that are to be studied further are removed from the Petri dish with an inoculating needle and rubbed over the surface of one or more slants of suitable media. This maneuver is known as fishing. If the colonies are not separated from each other, it is impossible to fish one colony without touching other colonies and thus mix in other types of bacteria in the transfer.
The inoculated slants (subcultures) are allowed to incubate for 24 to 48 hours and studied further using a microscope. In the majority of cases all bacteria growing on a slant will be alike because they all grew from the members of a single colony on the plate, and these in turn grew from a single bacterium in the original material. If, as may sometimes happen, the original colony on the plate contains two or more kinds of bacteria, the same two or more kinds will grow on the slant. Subsequent separation is made by suspension of some of the growth from the slant in sterile salt solution and replating.
