Microorganisms in the nature around us are everywhere: in soils, water bodies, on the surfaces of various objects, people and animals are inhabited by them. All this can serve as sources of microbial contamination of food, drugs, and production lines. The cultivation of bacteria is necessary to study their properties, needs, and characteristics. This, in turn, is an important step in the development of various drugs, laboratory diagnosis of diseases, the calculation of production reactors and much more.
General concepts
Cultivation of bacteria in microbiology refers to the cultivation of microorganisms carried out in the laboratory. In turn, microbes that have grown on a selected nutrient medium are called a culture. Cultures can be mixed if they are formed by different types of microorganisms, and pure if they are represented by only one type of bacteria.
If nutritionalOnly one cell is placed in the medium, and a group of individuals is obtained as a result of its reproduction, then this set of microorganisms is called a clone. When a clone develops to the point where it becomes visible to the naked eye, this collection of bacteria is called a colony.
Usually cultivation of bacteria isolated from different sources is carried out separately from each other. Each such separately grown group of microbes is called a strain. So, if one type of staphylococcus is isolated from three sources (or different portions of the same product, different people), they talk about three strains of this type of staphylococcus.
Bacteria Growth Factors
These include various amino acids, lipids, purine bases and other compounds necessary for the development of microorganisms. Some microbes can independently produce the substances they need, while others need to receive them in finished form. According to the needs of microorganisms in certain growth factors, identification and differentiation of bacteria are carried out. Also, this parameter is important for the correct preparation of a nutrient medium for laboratory and biotechnological work:
- Amino acids. Bacteria may require one particular amino acid or group of acids. So, clostridia need leucine and tyrosine, streptococci need leucine and arginine. Microorganisms that require amino acids from outside to grow are called auxotrophs.
- Purine and pyrimidine bases, as well as their derivatives (adenine, guanine and others). They are an important factor in the growth of manyStreptococcus species.
- Vitamins. They are part of the coenzymes required by bacteria. So, nicotinic acid, as well as its amide, which are part of NAD and NADP, is needed by diphtheria and shigella corynebacteria. Thiamine, as an integral part of pyrophosphate, is required by Staphylococcus aureus, pneumococcus, brucella. Pantothenic acid, which is part of the CoA coenzyme, is required by tetanus bacilli and certain types of streptococcus. Cytochromes, and therefore the folic acid, hemes and biotin that form them, are necessary for Mycobacterium tuberculosis and Haemophilus influenzae.
Environment Requirements
Conditions for culture media for culturing bacteria:
- Nutrition. They must contain substances, moreover, in an easily digestible form, necessary for microorganisms to feed and replenish energy. These include organogens and minerals. Some microorganisms additionally require vitamins and amino acids that they cannot synthesize.
- Optimal pH level. It affects the permeability of the cell membrane and, accordingly, the ability to absorb nutrients by the bacterium. Most often, the pH value should be at the level of 7, 2–7, 4. Many microorganisms in the course of their life produce products with acidic or alkaline reactions, and in order for the pH of the nutrient medium to not change, it must be buffered.
- Isotonic. The osmotic pressure in the nutrient medium for the cultivation of bacteria should have the same values asinside microbial cells. It usually corresponds to a 0.5% NaCl solution.
- Sterility. This is due to the fact that the appearance of foreign bacteria will distort the results of the study of the analyzed strain.
- Humidity level. This indicator, along with the consistency of the medium, should have optimal characteristics for a particular type of bacteria.
- Redox potential (RH2). It shows the ratio of substances that donate and accept electrons, as well as the level of oxygen saturation of the nutrient medium. For aerobes and anaerobes, the conditions for cultivating bacteria differ somewhat in this indicator. Anaerobic microorganisms reproduce best at RH2 values below 5 and aerobic microorganisms at least 10.
- Uniformity. It is important that the culture medium contains constant amounts of its individual ingredients. In addition, clear solutions are preferred, which make it easier to monitor crop growth or notice contamination.
Types of culture media
The choice of a particular medium for growing microorganisms is influenced by many factors, among which are the characteristics of their nutrition and the purpose of the study. The main features underlying the classification of nutrient media are:
1. Components. According to the initial substances used to create the substrate, they distinguish:
- natural, which are prepared from animal or vegetable products (e.g. meat, milk, fruits) and are suitable for growing mixedcrops;
- semi-synthetic, in which expensive natural food products are replaced by non-food products (for example, bone meal, blood clots), and which are optimal for cultivating certain types of bacteria or isolating their metabolic products from the environment;
- synthetic, which are prepared from precise amounts of chemical compounds, have a known constant composition and are easily reproducible.
2. Consistency (density). Distinguish environments:
- liquid;
- dense;
- semi-liquid.
The last two are prepared from special solutions or liquid substances with the addition of agar-agar or gelatin to create the required density. In addition, a dense environment for the growth of bacteria is clotted blood serum, potatoes, silica gel media, carrageenan.
3. Compound. On this basis, the environments are:
- simple, the list of which is short is Meat Peptone Broth (MBB), Hottinger Broth and Agar, Meat Peptone Agar (MPA), nutrient gelatin and peptone water.
- complex, prepared from simple ones with the addition of blood, whey, carbohydrates and other substances.
4. Appointment. The following nutrient media are distinguished:
- main are used to grow many pathogenic microbes (usually simple composition);
- special ones are used to isolate and cultivate bacteria that do not grow on simple substrates;
- selective (they are also selective) are suitable for isolating a specific type of bacteria and inhibit the growth of associated microbes (selectivitycreated by adding certain substances to the media, such as antibiotics or s alts, or by adjusting the pH);
- differential diagnostics make it possible to distinguish one type of bacteria from another by assessing the enzymatic activity, for example, of the medium;
- preservatives are needed for initial inoculation with subsequent transport of samples, as they prevent the death of microorganisms, as well as inhibit the growth of other bacteria.
Media preparation
The most important step in the cultivation of anaerobic bacteria is the preparation of a suitable nutrient medium. After the optimal parameters are selected, proceed to the following stages:
- weighing, by selecting a sample of components on an analytical balance;
- dissolution carried out in distilled water heated to 70 ° C, and phosphates, micro- and macros alts are separately dissolved;
- boiling in a water bath for two minutes;
- pH determination by indicator paper or potentiometer;
- filtration through wet cloth or paper filters for liquid as well as molten dense media, and through a cotton-gauze filter for agar media;
- bottling performed on 3/4 capacity;
- medium dependent sterilization;
- control for sterility is carried out by settling for two days in a thermostat, followed by viewing;
- chemical control to establish the pH and content of the necessaryelements;
- biological control by trial inoculation.
Sterilization of glassware and media
One of the basic principles of bacterial cultivation is sterility. The growth and development of foreign microorganisms can affect the characteristics of the nutrient medium by changing its chemical composition and pH. Sterilization is the main condition for growing pure cultures. In practice, this term means the methods of destruction of absolutely all life forms on the surface and in the volume of sterilized objects. Dishes, instruments used, media, and other items used during the study are sterilized.
Some types of sterilization:
- Ignition. Sterilization of loops and needles for seeding, glass slides, some instruments can be performed using a burner or spirit lamp.
- Boiling. Good for syringes, needles, food, but does not kill bacterial spores.
- Dry heat sterilization. It is carried out in a special drying cabinet and is suitable for processing flasks, test tubes and other laboratory glassware.
- Steam sterilization. Carried out in an autoclave, this method is highly effective. But it is not suitable for nutrient media containing proteins or any other compounds that break down at high temperatures. More sparing can be called tyndalization. It is carried out in the Koch Boiler and combines the germination of spores with their destruction.
- Pasteurization. It is used for media that change their properties when boiled (for example, milk, wine, beer), capable ofrid them of non-spore-bearing microorganisms. The processing temperature is only 50-60 ° C for fifteen to thirty minutes. In some cases, cold sterilization is used, carried out using filters or UV rays.
Bacteria cultivation conditions
The growth and development of bacteria is possible only under certain factors and the values of each of them:
1. Temperature. There are three groups of bacteria that differ in temperature preferences:
- thermophiles, or heat-loving microbes, grow at 45-90°C, which means they do not multiply in human and animal organisms;
- psychrophiles, or cold-loving microorganisms, prefer temperatures in the range of 5-15 ° C and are grown in cold stores;
- mesophiles, develop at a temperature of 25-37 ° C, they include the bulk of bacteria.
2. Light. It is a feature of the cultivation of phototrophic bacteria, since they carry out the photosynthetic process. But for most microbes, lighting is not a prerequisite. And even vice versa, solar ultraviolet can suppress their development.
3. Water. All microorganisms need water in an accessible (liquid) form. That's why frozen food has little to no bacteria growth.
4. Acidity of the environment. This principle of cultivating bacteria has already been discussed in detail above.
5. Aeration. Oxygen, as a chemical element, is an integral part of water and a considerable number of compounds used forcultivation of microorganisms. Gaseous oxygen can also be contained in water and other liquids in dissolved form. A significant part of bacteria needs a constant supply of oxygen molecules. But for a number of microorganisms, it is unnecessary, or, worse, gaseous oxygen is toxic to them, since they do not have catalase and peroxidase, which destroy toxic respiratory products. Therefore, the most important step in the cultivation of anaerobic bacteria is the removal of O2 molecules from the nutrient medium.
6. Cultivation of microorganisms. The cultivation of aerobic and anaerobic bacteria is carried out in different layers of the environment and in different modes.
Cultivation of aerobic microorganisms
Cultivation of aerobic bacteria requires molecular oxygen. To obtain pure cultures of aerobes that can be successfully used in medicine and the food industry, the following methods are used:
- surface growing on dense media or in liquid media (their thin layer) when oxygen comes directly from the air;
- deep cultivation in liquid media, when an increase in the amount of oxygen dissolved in them is achieved by constant aeration.
Cultivation of anaerobic microorganisms
The basic principle of cultivating bacteria of this type is their minimal contact with atmospheric oxygen. Providing conditions for their growth is much more difficult than for aerobes. The following methods are used to isolate anaerobes from molecular O2:
- Physical. This method of cultivation of anaerobic bacteria is reduced to their cultivation in a special vacuum apparatus - a microanaerostat. The air in it is replaced by a special gas mixture of nitrogen with the addition of 10% hydrogen and 5% carbon dioxide.
- Chemical. These include: use of absorbing agents (e.g. Fe, Na2S2O4, CuCl) or reducing agents (such as ascorbic acid).
- Biological. It comes down to the co-cultivation of aerobes and anaerobes in a closed system. This method of cultivating bacteria involves seeding one half of a Petri dish with some of the aerobic species of bacteria, and the other half with the studied anaerobe. Its development will begin at the moment when all the oxygen is used up.
The following seeding methods are suitable for cultivating anaerobic bacteria:
- in the surface layer;
- in the surface layer filled with sterile paraffin;
- in the thick of a dense nutrient medium;
- in deep layers of viscous media.
Obtaining pure culture
Microbiologists usually work with samples inhabited by many different types of microbes. However, to determine the systematic position of microorganisms (family, genus, species), as well as to study their characteristics, it is necessary to isolate them and grow a pure culture. They are of great importance in many food industries, for example, cheese, bread, kvass, wine, etc. The cultivation of lactic acid bacteria makes it possible to obtainan essential component for the production of fermented milk products, dough, cocoa, silage and even plastic.
The method of isolating a pure culture in a dense medium is based on the mechanical separation of microorganism cells with their subsequent isolated cultivation. The sample is transferred into a sterile volume of water or saline (volume 10-100 ml) and then shaken for two minutes. In order to extract microorganisms located in the thickness of the material under study (for example, sausages or cheese), first rubbing the sample pieces with sterile instruments with sand is performed. The material that has undergone preliminary preparation, weighing 1 g or a volume of 1 ml, is diluted with sterile water by 10, 100, 1000, etc. times. Choose the degree of dilution that gives the concentration of cells corresponding to the capabilities of the method.
The subsequent cultivation of microorganisms is to prepare a nutrient medium. Usually a dense medium (MPA) is chosen. It is first melted and cooled to 45-50 °C, and only then it is poured into several Petri dishes (three to five pieces), on the bottom of which swabs from the test substance of various concentrations are placed. Next, mixing of the still not frozen nutrient medium and the material introduced into it is carried out. This is how cells are fixed at various points in the volume of the substrate.
Next, Petri dishes are placed in a thermostat for 2 days at 22 °C. During this time, the cells multiply to such an extent that the colony formed by each of the cells becomes visible to the naked eye. Each of them is a pure culture of the type of bacteria from whose cells itrose.
After that, from Petri dishes, microorganisms are subcultured into separate test tubes filled with a nutrient medium. In this way, pure cultures are isolated from a mixed sample. This method bears the name of its developer - R. Koch. It is also commonly called the cup method, or depleting sowing. After obtaining pure cultures of various types of bacteria, their shape, spores, and families are determined.
All work must be carried out according to the principles of asepsis. To avoid premature development of microorganisms, the study should be carried out immediately after sampling. Tap water is analyzed after draining the first portions, since they may contain microbes accumulated in pipes and taps. The microflora of fruits, berries and vegetables is mainly located on the surface (peel), therefore, washings are performed from it. To do this, place the fetus in a sterile container and fill it with the required amount of water. Then they are shaken quite vigorously and the water is poured into another container. Crops from cloth products are also obtained by swabs, but beforehand, pieces of a given size are cut out of them.
