One of the most important systems for the identification of microorganisms is to observe their growth in artificial food substances prepared in the laboratory. The food material in which the microorganisms grow is the Culture Medium and the growth of the microorganisms is the Culture. More than 10,000 different culture media have been prepared.
For bacteria to grow properly in an artificial culture medium, it must meet a series of conditions such as: adequate temperature, humidity and oxygen pressure, as well as a correct degree of acidity or alkalinity. A culture medium must contain the necessary nutrients and growth factors and must be free of all contaminating microorganisms.
Most pathogenic bacteria require complex nutrients similar in composition to the organic fluids of the human body. For this reason, the base of many culture media is an infusion of meat extracts and Peptone to which other ingredients will be added.
Agar is a solidifying element widely used for the preparation of culture media. It liquefies completely at the temperature of boiling water and solidifies upon cooling to 40 degrees. With few exceptions it has no effect on the growth of bacteria and is not attacked by those that grow on it.
Gelatin is another solidifying agent but it is used much less since enough bacteria cause its liquefaction.
In the different culture media there are numerous enrichment materials such as carbohydrates, serum, whole blood, bile, etc. Carbohydrates are added for two main reasons: to increase the nutritional value of the medium and to detect fermentation reactions of the microorganisms that help to identify them. Serum and whole blood are added to promote the growth of the least resistant microorganisms.
Dyes that act as indicators to detect, for example, the formation of acid or as growth inhibitors of some bacteria and not others are also added (Phenol Red is used as an indicator since it is red at basic pH and yellow at acid pH Gentian Violet is used as an inhibitor since it prevents the growth of most Gram-positive bacteria).
General conditions for the cultivation of microorganisms
The proper development of microorganisms in a culture medium is affected by a series of factors of great importance and, in some cases, are completely unrelated to the medium itself.
What are the most important conditions?
- availability of adequate nutrients
- adequate consistency of the medium
- presence (or absence) of oxygen and other gases
- adequate humidity conditions
- Ambient light
- pH
- Temperature Sterility of the medium
1- availability of adequate nutrients
A culture medium suitable for microbiological research must contain, at a minimum, carbon, nitrogen, sulfur, phosphorous and inorganic salts. In many cases, certain vitamins and other growth-inducing substances will be necessary. The right substances to act as donors or electron scavengers for the chemical reactions that take place must always be present.
All these substances were originally supplied in the form of infusions of meat, extracts of meat or extracts of yeast. However, the preparation of these substances for application to the culture media caused the loss of labile nutritional factors.
Currently, the most widespread way of contributing these substances to the media is to use peptone, which also represents an easily available source of nitrogen and carbon since most microorganisms, which do not usually use natural proteins directly, have the ability to attack amino acids and other simpler nitrogen compounds present in peptone.
Certain bacteria have specific nutritional needs, so substances such as serum, blood, ascitic fluid, etc. are added to many media. Likewise, certain carbohydrates and mineral salts such as calcium, magnesium, manganese, sodium or potassium and growth promoting substances, generally of a vitamin nature, may be necessary.
Very often certain dyes are added to the culture medium, either as indicators of certain metabolic activities or because of their ability to act as selective inhibitors of certain microorganisms.
2- adequate consistency of the medium
Starting from a liquid medium we can modify its consistency by adding products such as albumin, gelatin or agar, with which we would obtain media in a semi-solid or solid state.
The gelatin solidified media have the great drawback that many microorganisms do not develop properly at temperatures below the melting point of this solidifier and that others have the ability to liquefy it.
Solid media are currently in universal use for their versatility and convenience, but there are also a large number of liquid media that are widely used in the laboratory.
3- presence (or absence) of oxygen and other gases
Large numbers of bacteria can grow in an atmosphere with normal oxygen tension. Some can obtain oxygen directly from various substrates. But strict anaerobic microorganisms will only thrive properly in an atmosphere without ambient oxygen. At an intermediate point, microaerophilic microorganisms grow best in partially anaerobic atmospheric conditions (greatly reduced oxygen tension), while facultative anaerobes have a metabolism capable of adapting to any of these conditions.
4- adequate humidity conditions
A minimum level of humidity, both in the medium and in the atmosphere, is essential for the proper development of microbial vegetative cells in crops. The maintenance of these minimum conditions must be foreseen in the culture stoves at 35-37ºC, providing an adequate source of water that maintains the humidity necessary for the growth of the crops and thus prevents the medium from drying out.
5- Ambient light
Most microorganisms grow much better in the dark than in the presence of sunlight. There are obvious exceptions such as the case of photosynthetic microorganisms.
6- pH
The concentration of hydrogen ions is very important for the growth of microorganisms. Most of them develop better in media with a neutral pH, although some require more or less acidic media. It should not be forgotten that the presence of acids or bases in amounts that do not impede bacterial growth can, however, inhibit it or even alter its normal metabolic processes.
7- Temperature
Mesophilic microorganisms grow optimally at temperatures between 15 and 43ºC. Others like psychrophiles grow at 0ºC and temophiles at 80ºC or even at higher temperatures (hypertemophiles). In general, human pathogens grow in much shorter temperature ranges, around 37ºC, and saprophytes have broader ranges.
8- Sterility of the medium
All culture media must be perfectly sterile to avoid the appearance of life forms that can alter, mask or even prevent the normal microbial growth of the specimen or inoculated in said media. The classic system for sterilizing culture media is the autoclave (which uses pressurized steam as a sterilizing agent)
Basic types of culture media
Depending on your physical condition:
- liquids
- semi-solids
- solids
Considering its practical utility:
- For general uses: non-selective, for cultivation of a wide variety of organisms that are difficult to grow. They are often enriched with materials such as: blood, serum, Hemoglobin, FX, FV, glutamine, or other accessory factors for the growth of bacteria (Blood Agar, Schaeadler, etc.)
- Selective: (they can be moderate or highly selective) substances are added that inhibit the growth of certain groups of bacteria, while allowing the growth of others. By varying the added substances, the type and degree of selectivity is varied (Mac Conkey, Kanamycin-Vancomycin)
- Enriched: they slow down / suppress the growth of normal competitive flora, enhancing the desired growth and culture (Selenite, medium with Vitamin K).
- For specialized isolates: special nutritional formulations that satisfy the requirements of specific groups of bacteria, helping their identification (Lowenstein).
Means of identification:
Differentials: special formulations in which the specific physiological peculiarities (nutrition and respiration especially) of bacteria are studied. Selecting the appropriate means, it is possible to identify almost any bacterium (Oxidation-Fermentation)