The study of bacteria is of great practical importance to humans. To date, a large number of prokaryotes have been discovered, which differ from each other in pathogenicity, area of distribution, shape, size, number of flagella and other parameters. In order to study this strain in detail, a bacteriological research method is used.
What are the methods for analyzing bacterial cells?
To determine if bacteria are pathogenic, culture studies are carried out in various ways. Among them:
1. Bacterioscopic method.
2. The bacteriological method.
3. The biological method.
Bacterioscopic and bacteriological research methods are based directly on working with prokaryotic cells, when biological analysis is required to study the effect of such cells on the living organism of experimental animals. According to the degree of manifestation of certain signs of the disease, the scientist can conclude that there are pathogenic bacteria in the sample or not, and naturally reproduce them in the animal’s body to obtain their culture and use in other works.
The bacteriological research method differs from the bacterioscopic one. In the first, a specially prepared culture of living prokaryotes is used for analysis, when in the second, work is performed with dead or living cells on a glass slide.
Stages of the bacteriological research method. Microbiology
The principle of studying the properties of a bacterial culture can be useful both for microbiologists who set a goal to study prokaryotic cells, and for laboratory assistants whose task is to establish the pathogenicity or pathogenicity of bacteria, and then the patient’s diagnosis.
The methodology for studying bacteria is divided into three stages:
1. Isolation of bacteria from the initial sample.
2. Sowing bacteria and growing a pure culture, the study of its properties.
3. A detailed study of bacterial cells.
First stage
A sample, or smear, is taken from the free surface of the medium or from the patient. Thus, we get a “cocktail” of many types of bacteria that must be sown on a nutrient medium. Sometimes it becomes possible to isolate immediately the necessary bacteria, knowing their foci of distribution in the body.
After two or three days, the necessary colonies are selected and plated on solid media of Petri dishes using a sterile loop. Many laboratories work with test tubes where solid or liquid growth media may be present. So the bacteriological research method in microbiology is carried out.
Second phase
After obtaining individual colonies of bacteria, a direct macro- and microanalysis is carried out. All parameters of the colonies are measured, the color and shape of each of them is determined. Often, colonies are counted on a Petri dish, and then in the starting material. This is important in the analysis of pathogenic bacteria, the degree of the disease depends on the number of which.
A bacteriological research method, the 2nd stage of which is the study of individual colonies of microorganisms, can be coupled with a biological method for analyzing bacteria. Another goal of the work at this stage is to increase the amount of source material. This can be done on a nutrient medium, or you can conduct an experiment in vivo on living experimental organisms. Pathogenic bacteria will multiply, and as a result, the blood will contain millions of prokaryotic cells. From the taken blood it is easy to prepare the necessary working material of bacteria.
Third stage
The most important part of the study is the determination of the morphological, biochemical, toxigenic and antigenic properties of the bacterial culture. The work is carried out with pre-"purified" cultures on a nutrient medium, as well as with preparations (often stained) under a microscope.
To establish the affiliation of pathogenic or conditionally pathogenic bacteria to one or another systematic group, as well as to determine their resistance to drugs, allows the bacteriological method of research. Stage 3 - antibiotics, i.e., analysis of the behavior of bacterial cells in the conditions of the content of drugs in the environment.
The study of culture resistance to antibiotics is of great practical importance when it is necessary to prescribe the necessary, and most importantly, effective drugs for a particular patient. This is where the bacteriological research method can help.
What is a culture medium?
For development and reproduction, bacteria must be in previously prepared nutrient media. By consistency, they can be liquid or solid, and by origin - plant or animal.
Basic requirements for nutrient media:
1. Sterility.
2. Maximum transparency.
3. Optimal indicators of acidity, osmotic pressure, water activity and other biological quantities.
Obtaining isolated colonies
1. The Drigalski method. It lies in the fact that a smear with various types of microorganisms is applied to the bacterial loop. This loop is carried out on the first Petri dish with a nutrient medium. Further, without changing the loop, the method of residual material is carried out on the second and third Petri dishes. So, on the last samples of the colony, the bacteria will not be seeded too tightly, thereby simplifying the ability to find the bacteria necessary for the work.
2. The Koch method. It uses tubes with molten nutrient medium. A loop or a dropper with a smear of bacteria is placed there, after which the contents of the tube are poured onto a special plate. Agar (or gelatin) freezes after some time, and in its thickness it is easy to detect the desired cell colony. It is important to dilute the mixture of bacteria in test tubes before starting work so that the concentration of microorganisms is not very large.
The bacteriological research method, the stages of which are based on the selection of the desired bacterial culture, cannot do without these two methods of finding isolated colonies.
Antibioticogram
Visually, the reaction of bacteria to drugs can be seen in two practical ways:
1. The method of paper disks.
2. Breeding bacteria and antibiotic in a liquid medium.
The paper disc method requires a culture of microorganisms that have been grown on solid nutrient media. On such an environment, several round-shaped papers impregnated with antibiotics are placed. If the drug successfully copes with the neutralization of bacterial cells, after such treatment there will remain a site devoid of colonies. If the reaction to the antibiotic is negative, the bacteria will survive.
In the case of using a liquid nutrient medium, first prepare several tubes with a culture of bacteria of different degrees of dilution. Antibiotics are added to these tubes, and the process of interaction of the substance and microorganisms is observed during the day. In the end, a high-quality antibioticogram is obtained, by which one can judge the effectiveness of the drug for this culture.
The main objectives of the analysis
The goals and stages of the bacteriological research method are listed here according to the points.
1. Get the source material that will be used to isolate the colonies of bacteria. This can be a smear from the surface of any object, mucous membrane or cavity of a human organ, a blood test.
2. Cultivation of the culture on a solid nutrient medium. After 24-48 hours, colonies of bacteria of various types can be found on the Petri dish. We select the necessary one according to morphological and / or biochemical criteria and carry out further work with it.
3. Reproduction of the resulting culture. The bacteriological research method can be based on a mechanical or biological method of increasing the number of bacteria cultures. In the first case, work is carried out with solid or liquid nutrient media, on which bacteria multiply in the thermostat and form new colonies. The biological method requires natural conditions to increase the number of bacteria, therefore, an experimental animal is infected with microorganisms here. After a few days, many prokaryotes can be found in a blood sample or smear.
4. Work with a refined culture. To determine the systematic position of bacteria, as well as their belonging to pathogens, it is necessary to conduct a thorough analysis of cells according to morphological and biochemical characteristics. In the study of pathogenic groups of microorganisms, it is important to know how effective the action of antibiotics is.
This was a general characteristic of the bacteriological research method.
Features of the analysis
The main rule for conducting bacteriological studies is maximum sterility. If work with test tubes is in progress, bacteria should be seeded and reseeded only on a heated spirit lamp.
All stages of the bacteriological research method require the use of a special loop or a Pasteur pipette. Both tools must be pretreated in the flame of an alcohol lamp. As for the Pasteur pipette, then before thermal sterilization it is necessary to break off the tip of the pipette with tweezers.
The technique of sowing bacteria also has its own characteristics. First, when plating on solid media, a bacterial loop is carried out along the surface of the agar. The loop, of course, should already have a sample of microorganisms on the surface. Sowing inside the nutrient medium is also practiced , in which case the loop or pipette should reach the bottom of the Petri dish.
When working with liquid media, test tubes are used. It is important to ensure that liquids do not touch the edges of the laboratory glassware or cork, and the instruments used (pipette, loop) do not touch foreign objects or surfaces.
The value of the biological research method
Bacterial sample analysis has its practical application. First of all, the bacteriological research method can be used in medicine. For example, it is necessary to study the microflora of the patient in order to establish the correct diagnosis, as well as to develop the correct course of treatment. An antibioticogram helps here, which will show the activity of drugs against the pathogen.
Bacterial analysis is used in the laboratory to identify dangerous diseases such as tuberculosis, relapsing fever, or gonorrhea. It is also used to study the bacterial composition of tonsils, organ cavities.
The bacteriological research method can be used to determine the environmental pollution. According to the quantitative and qualitative composition of the smear from the surface of an object, the degree of microorganism occupation of a given environment is determined.