CULTURE MEDIA

A nutrient material prepared for the growth of microbes in the lab is called a culture medium. The microbes that grow are called a culture. Bacteria vary in their requirements: Some grow on almost any medium Some have special, unusual requirements Some do not grow on any nonliving medium Requirements for a culture medium: 1. Must have the right nutrients for the microbe. 2. Must supply sufficient moisture. 3. Must have the proper pH, and often a buffer to maintain it. 4. Must supply suitable level of oxygen. 5. Medium must be sterile before intended microbes are added. 6. Must be incubated at the proper temperature. Modern media are purchased in a dried form. For use, the proper amount of water is added and the mixture is sterilized. Media can be used in a liquid formthis is called a broth. This type is generally used in a test tube. Media can also be solid. Usually a polysaccharide called agar, derived from seaweed, is added to solidify a medium. Agar is chosen because: 1. Microbes do not break it down, so the medium remains solid as microbes grow. 2. Once solidified, agar does not melt until it reaches 100o C (boiling). This means it remains solid in all normal incubation temperatures. 3. Once melted, agar remains liquid until the temperature reaches about 40o C. Bacteria can be added to liquid agar which has been held at a temperature slightly above 40o C. After adding the bacteria, the agar can be quickly cooled and most bacteria are not harmed.

Agar media are used in test tubes or Petri dishes. If the agar in a test tube is solidified while the tube is tilted, the tube is called a slant. This gives a larger surface area for growth. If the tube is left vertical while the agar sets, this is called an agar deep.

A Petri dish containing agar is called a plate.

CHEMICALLY DEFINED MEDIA

Chemically defined mediumone whose exact chemical composition is known. This means that every substance present, no matter how small the amount, is known, and also that exactly how much of every substance present is known. Media of this type are not used for general lab work. Since these media must be exceptionally pure, they are difficult and expensive to prepare, so they are only used when special work requires them. If the organism is a chemoheterotroph, the medium must contain organic growth factors that serve as a source of carbon and energy. Escherichia coli, is a good example of a chemoheterotroph with very simple requirements. Glucose is the only organic growth factor needed. With it plus some electrolytes and water, E. coli can synthesize everything else it needs. TABLE 6.2 P. 164 The other extreme is microbes that are described as fastidious. These require many organic growth factors and sometimes have unusual needs. An example of one of these, Neisseria gonorrhoeae is shown in TABLE 6.3 P. 165 Fastidious microbes known to require a certain organic growth factor such as a particular vitamin can be used to perform a microbiological assay to determine the amount of that factor present in a medium. This method could be used to check the actual amount of a vitamin present in a vitamin pill, for example.

COMPLEX MEDIA
Used for most routine work, these media are prepared without such exacting standards as the chemically defined media. Ingredients such as yeasts, meat, plant products, and sometimes partly digested proteins from these sources are included. Other ingredients sometimes include blood and milk. All of this provides an energy source as well as carbon, nitrogen, sulfur, vitamins, and other growth factors, and will support growth of the great majority of microbes. Relatively large amounts of protein and fragments of proteins (peptones) are included . This general growth medium is called nutrient broth if left liquid and nutrient agar if agar is added to solidify it.

ANAEROBIC GROWTH MEDIA AND METHODS

Remember, strict or obligate anaerobes do not tolerate the presence of oxygen at normal atmospheric levels, although aerotolerant anaerobes can. Special media and techniques are required for strict anaerobes. 1. Special reducing media such as fluid thioglycolate medium can be used. Although this medium is slightly thickened with a small amount of agar, it basically is a liquid medium in tubes. Individual colonies cannot be seen. 2. If solid media are required, the plates are inoculated and quickly put into a special jar. Modern technique involves then activating a packet of chemicals and sealing the jar. The chemical reactions occurring in the sealed jar use up all the oxygen and leave anaerobic conditions. 3. In large labs, an anaerobic glove box chamber is used. This is a sealed transparent chamber which has all room air pumped out and replaced with inert gases after all microbes and necessary supplies have been placed inside. From this point on, work is done by using rubber gloves built in to the walls of the chamber with air-tight seals. Worker insert their hands into the gloves and work without opening the chamber to room air. This means that the microbes do not get any exposure to oxygen at all, which is ideal.

SPECIAL CULTURE TECHNIQUES

1. Some bacteria have never been successfully grown on artificial media. These must be grown in cultures of living cells called tissue cultures, or in living lab animals. Mycobacterium leprae Pathogenic strains of Treponema pallidum Rickettsias Chlamydias All viruses 2. Some microbes require higher than normal levels of carbon dioxide. They are called capnophiles. For these, special carbon dioxide incubators, candle jars, or jars or bags with special carbon dioxide-producing chemical packets can be used. These produce conditions similar to those of the intestinal tract, for example. Campylobacter Neisseria

SELECTIVE AND DIFFERENTIAL MEDIA

1. Selective mediasuppress the growth of unwanted bacteria and encourage the growth of a desired microbe or group of microbes. Bismuth sulfite agar---Salmonella typhi Sabourauds dextrose agarfungus organisms Brilliant green agar---Salmonella and other gram-negative bacteria 2. Differential media---these media cause colonies of certain microbes to grow with a characteristic appearance. Just by observing growth, the presence of a certain type of microbe can be determined. Streptococcus pyogenes---blood agar (a clear ring forms around colonies)

3. Some media combine the two types and are both selective and differential. Mannitol salt agar---Staphylococcus aureus Levine's EMB agar McConkey's agar Desoxycholate agar

More about selective media for fungi. These media in general contain a higher level of sugar (4%) and a lower pH (3.8 - 5.6) than bacterial media. These conditions discourage growth of bacteria, which we need to do because fungi often grow very slowly and bacteria will overun them if we don't intervene. To make the medium even more selective for fungi, an antibacterial antibiotic an be added.

ENRICHMENT CULTURING
Most samples of bacteria taken from natural environments contain many different species. Use of selective and differential media can aid in isolation and identification of certain species or groups. Unfortunately, good selective and differential media are simply not available for all possible species. Sometimes, it is possible that among large numbers of various bacterial species in a sample, just a very small number of a particular species, which we will call Species X, might be present. On a general growth medium, this small number of Species X would very likely be overwhelmed by large numbers of other species. If there were a special selective medium on which only Species X would grow, this would be an easy solution, but unfortunately this time there is no such selective medium. (That is frequently the case.) The next best thing for locating Species X would be an enrichment medium, which would contain nutrients that suit Species X particularly well and maybe dont suit the other microbes quite so well, although they will probably still grow fairly well. Maybe this medium could also be incubated in conditions which particularly suit Species X. The idea is that while many types of microbes could grow, maybe the total percentage of the microbes that belong to Species X would increase, making them easier to isolate.

OBTAINING PURE CULTURES

most samples from natural conditions contain many different species of bacteria, and often other types of microbes as well. Each microbe placed on the surface of a solid medium grows and forms a visible colony. Colonies formed by different microbes may have different appearances. In obtaining, a pure culture, one that contains only one type of microbe, the goal is to thin out the original microbes so that the resulting colonies are separate from each other. This can be done in several ways: 1. Streak plate 2. Pour plate 3. Spread plate 4. Micromanipulator

METHODS OF PRESERVING BACTERIAL CULTURES

1. Refrigerationshort-term 2. Deep-freezing in liquid suspending medium-- temperature quickly lowered to -50o C to -95o C. Years. 3. Lyophilization (freeze-drying)microbes are quickly frozen while the water content is removed at the same time (formation of ice crystals is damaging to cells). The result is a dry powder sealed in a vacuum which can be stored for decades.

GROWTH OF BACTERIAL CULTURES

Growth of bacteria is an increase in the number of cells, not in the size of cells. Bacteria normally reproduce by binary fission: 1. Cell elongates and chromosome is replicated. One copy of the chromosome moves to each end of the cell. 2. Cell wall and cell membrane begin to grow inward between the 2 chromosomes.

3. Cell walls meet, forming 2 separate cells, each identical to parent. A few bacterial species reproduce by other means: 1. Buddinga small outgrowth forms and splits off the parent cell 2. Spores--some filamentous bacteria produce chains of reproductive spores at the tips of the filaments. These are reproductive spores, different from endospores. 3. Fragmentationa few filamentous bacteria fragmentbreak into pieces and each piece forms a new cell