Bacteriological method of research: stages, goals, characteristics

The study of bacteria is of great practical importance for man. To date, a large number of prokaryotes have been discovered, which differ from each other in pathogenicity, distribution, shape, size, number of flagella and other parameters. To study this strain in detail, a bacteriological method of investigation is used.

What are the methods for analyzing bacterial cells?

To determine whether the bacteria are pathogenic, the culture is tested in various ways. Among them:

1. Bacterioscopic method.

2. Bacteriological method.

3. Biological method.

Bacterioscopic and bacteriological methods of research 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 or do not pathogenic bacteria in the sample, and also naturally multiply them in the animal's body to obtain their culture and use in other works.

The bacteriological method of research differs from the bacterioscopic method. In the first one, a specially prepared culture of living prokaryotes is used for analysis, when the second one is working with dead or living cells on a slide.

Stages of the bacteriological method of investigation. Microbiology

The principle of studying the properties of bacterial culture can be useful both for microbiologists who have set the goal of investigating prokaryotic cells and for laboratory assistants whose task is to establish the pathogenicity or non-pathogenicity of bacteria, and then diagnose the patient.

The method of studying bacteria is divided into three stages:

1. Isolation of bacteria from the original sample.

2. Sowing of bacteria and cultivation of pure culture, study of its properties.

3. Detailed study of bacterial cells.

First step

The sample, or smear, is taken from the free surface of the medium or the patient. Thus, we get a "cocktail" from a variety of species of bacteria that must be sown to a nutrient medium. Sometimes it becomes possible to identify immediately the necessary bacteria, knowing their foci of distribution in the body.

After two to three days, the required colonies are selected and plated on the solid media of Petri dishes with the help of a sterile loop. Many laboratories are working with test tubes, where a solid or liquid nutrient medium may be present . So is the bacteriological method of research in microbiology.

Second phase

After obtaining individual colonies of bacteria, a direct macro and microanalysis is carried out. All the parameters of the colonies are measured, the color and shape of each of them are determined. It is often done by counting colonies on a petri dish, and then in the starting material. This is important in the analysis of pathogenic bacteria, on the number of which the degree of disease depends.

The bacteriological method of investigation, the second stage of which is to study individual colonies of microorganisms, may be associated with a biological method for the analysis of bacteria. Another goal of the work at this stage is to increase the amount of source material. It can be done on a nutrient medium, and it is possible to conduct an experiment in vivo in living experimental organisms. Pathogenic bacteria will multiply, and as a result, the blood will contain millions of prokaryotic cells. From the blood it is easy to prepare the necessary working material of bacteria.

The 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 previously "purified" crops on a nutrient medium, as well as with preparations (often colored) under a microscope.

To identify the belonging of pathogenic or conditionally pathogenic bacteria to a particular systematic group, as well as to determine their resistance to drugs, the bacteriological method of investigation allows. Stage 3 - antibiotics, i.e. analysis of the behavior of bacterial cells in conditions of medicinal products in the environment.

The study of the resistance of culture to an antibiotic is of great practical importance when it is necessary to prescribe for a particular patient the necessary, and most importantly, effective drugs. Here and the bacteriological method of research can help.

What is a nutrient medium?

For development and reproduction, bacteria should be in pre-prepared nutrient media. By consistency, they can be liquid or solid, and by origin - vegetable or animal.

Basic requirements for nutrient media:

1. Sterility.

2. Maximum transparency.

3. Optimum parameters of acidity, osmotic pressure, water activity and other biological quantities.

Preparation of isolated colonies

1. Drigalsky's method. It consists in applying a smear with various kinds of microorganisms to the bacterial loop. This loop is carried out on the first Petri dish with 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 making it easier to find the bacteria necessary for the work.

2. The Koch method. It uses tubes with a molten nutrient medium. There, a loop or a pipette with a smear of bacteria is placed, after which the contents of the tube are poured onto a special plate. Agar (or gelatin) freezes after a while, and in its thickness it is easy to find the necessary colonies of cells. It is important before starting to dilute the mixture of bacteria in test tubes, so that the concentration of microorganisms is not very large.

The bacteriological method of investigation, the stages of which are based on the isolation of the desired bacterial culture, does not 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 discs.

2. Breeding of bacteria and antibiotic in a liquid medium.

The method of paper discs requires the presence of a culture of microorganisms that have been grown on a solid nutrient medium. On this medium, several round-shaped papers are placed, impregnated with antibiotics. If the drug successfully copes with the neutralization of bacterial cells, after such treatment remains 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, several tubes with a culture of bacteria of different degrees of dilution are first prepared. Antibiotics are added to these tubes, and during the day they observe the process of interaction of the substance and microorganisms. Ultimately, a qualitative antibioticogram is obtained, from which one can judge the effectiveness of the drug for a given culture.

The main tasks of the analysis

The goals and stages of the bacteriological method of investigation are listed here.

1. Obtain the source material that will be used to isolate bacterial colonies. It can be a smear from the surface of any object, mucous membrane or cavity of the human body, a blood test.

2. Cultivation of culture on a solid nutrient medium. After 24-48 hours on a petri dish, you can find colonies of bacteria of different species. We select the required morphological and / or biochemical criteria and carry out further work with it.

3. Reproduction of the obtained culture. The bacteriological method of research can be based on a mechanical or biological method of increasing the number of bacterial cultures. In the first case, work is carried out with solid or liquid nutrient media on which the bacteria multiply in a thermostat and form new colonies. The biological method requires natural conditions for an increase in the number of bacteria, so a test animal is infected with microorganisms here. A few days later, a lot of prokaryotes can be found in a blood sample or smear.

4. Work with the purified culture. To determine the systematic position of bacteria, as well as their belonging to pathogens, it is necessary to conduct a thorough analysis of the cells according to morphological and biochemical characteristics. When investigating pathogenic groups of microorganisms, it is important to know how effective the action of antibiotics is.

This was a general characteristic of the bacteriological method of investigation.

Features of the analysis

The main rule of conducting bacteriological research is maximum sterility. If work is in progress with test tubes, crops and bacteria must be replicated only over the heated spirit.

All stages of the bacteriological method of investigation require the use of a special loop or a Pasteur pipette. Both instruments must be pretreated in the flame of the spirit lamp. As for the Pasteur pipette, then before thermal sterilization it is necessary to break the tip of the pipette with tweezers.

The technique of sowing bacteria also has its own characteristics. Firstly, when sowing on solid media, carry out a bacterial loop over the surface of the agar. The loop, of course, should already have a sample of microorganisms on the surface. It is also practiced to sow inside the culture medium, in which case the loop or pipette should reach the bottom of the Petri dish.

When working with liquid media, tubes are used. It is important to ensure that liquids do not touch the edges of laboratory dishes or cork, and the instruments (pipette, loop) used do not touch foreign objects and surfaces.

The importance of the biological method of research

The analysis of a sample of bacteria has its practical application. First of all, the bacteriological method of research can be used in medicine. For example, it is necessary to study the patient's microflora in order to establish the correct diagnosis, and also to work out the correct course of treatment. Here an antibioticogram helps, which will show the activity of drugs against the causative agent of diseases.

The analysis of bacteria is used in the laboratory to determine such dangerous diseases as tuberculosis, recurrent typhoid or gonorrhea. It is also used to study the bacterial composition of the tonsils, the cavities of the organs.

The bacteriological method of investigation can be used to determine the contamination of the environment. According to the quantitative and qualitative composition of the smear from the surface of an object, the degree of population of a given medium is determined by microorganisms.