Study of microorganisms. Methods for studying microbes in culture
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The smallest sizes of microorganisms determine the use of precise optical instruments - microscopes - to study the morphology of bacteria. The most commonly used microscopies are bright-field microscopy, dark-field microscopy, phase contrast microscopy, and fluorescence microscopy. For special microbiological studies, electron microscopy is used.
Bright field microscopy
Bright-field microscopy is carried out using a conventional light microscope, the main part of which is the lens. The magnification is indicated on the lens frame: 8, 10, 20, 40, 90.When studying microbes, an immersion system (lens) is used. The immersion lens is immersed in a drop of cedar oil applied to the preparation. Cedar oil has the same refractive index as glass, and this achieves the least dispersion of light rays (Fig. 1.12).
Rice. 1.12. Path of rays in an immersion lens
The image obtained in the lens is magnified by an eyepiece consisting of two lenses. Domestic microscopes use eyepieces with magnifications of 7, 10, 15 (Fig. 1.13). The overall magnification of a microscope is determined by the product of the objective magnification and the eyepiece magnification. In microbiology, magnifications of 900-1000 times are commonly used. The quality of a microscope depends not on the degree of magnification, but on its resolution.
Rice. 1.13. Diagram of a complex light microscope for observation in bright field, adjusted for Köhler illumination
By this we must understand the smallest distance between two points of the preparation, at which they are still clearly distinguishable under a microscope. The resolution of conventional light microscopes with an immersion system is 0.2 microns.
Dark-field microscopy
Dark field microscopy is based on the following principle (Fig. 1.14). The rays illuminate the object not from below, but from the side and do not enter the observer’s eyes: the field of view remains dark, and the object against its background appears luminous. This is achieved using a special condenser (paraboloid) or a regular condenser, covered in the center with a circle of black paper.
Rice. 1.14. Diagram of a microscope for observation in a dark field.
Preparations for dark-field microscopy are prepared using the “hanging” and “crushed” drop type. When preparing the preparation, a “crushed” drop of the test material (bacterial culture in physiological solution) is applied to a glass slide, which is covered with a coverslip. A drop of material fills the entire space between the cover glass and the slide, forming an even layer. To prepare a hanging drop, it is necessary to use special glass slides with a depression in the center and cover slips.
The material to be tested is applied to the middle of the cover glass. The edges of the recess on the slide are smeared with Vaseline, and a cover glass is covered with it so that the drop is located against the center of the recess. Then turn the preparation over with the cover glass facing up. Dark-field microscopy is used to study living, unstained microorganisms.
Phase contrast microscopy
When a beam of light passes through an unpainted object, only the oscillation phase of the light wave changes, which is not perceived by the human eye. In order for the image to become contrasty, it is necessary to convert the phase changes of the light wave into visible amplitude ones. This is achieved using a phase-contrast condenser and a phase objective (Fig. 1.15).
Rice. 1.15. Diagram of a phase contrast microscope.
A phase contrast condenser is a regular lens with a revolver and a set of annular diaphragms for each lens. The phase lens is equipped with a phase plate, which is obtained by applying salts of rare earth elements to the lens. The image of the annular diaphragm coincides with the phase plate ring of the corresponding lens.
Phase contrast microscopy significantly increases the contrast of an object and is used to study native preparations.
Fluorescence microscopy
Luminescence microscopy is based on the ability of some substances, under the influence of light incident on them, to emit rays with a different (usually longer) wavelength (fluoresce). Such substances are called fluorochromes (acridine yellow, rhodamine, etc.). An object treated with fluorochrome, when illuminated with ultraviolet rays, acquires bright color in a dark field of view.The main part of a fluorescent microscope is an illuminator that has an ultraviolet lamp and a filter system for it (Fig. 1.16). It is very important to use a non-fluorescent immersion oil.
Luminescent microscopy in practical microbiology is used for indication and identification of pathogens infectious diseases.
Rice. 1.16. Schematic representation of a fluorescent microscope: 1 - arc lamp; 2 - quartz collector; 3 - cuvette filled with a solution of copper sulfate; 4 - front part of the collector; 5 - ultraviolet filter; 6 - prism; 7 - uranium glass plate; 8 - eyepiece filter, absorbing
ultra-violet rays.
Electron microscopy
The capabilities of optical microscopes are limited by the too long wavelength of visible light (6000 A). Objects smaller than this value are beyond the resolution of a light microscope. In an electron microscope, instead of light waves, electron beams are used, which have an extremely short wavelength and high resolution (Fig. 1.17).
Rice. 1.17. Scheme of a transmission electron microscope.
An electron gun is used as a source of electron beams, the basis of which is a heated tungsten filament electric shock. There is an electric field between the tungsten filament and the anode in the path of electrons high voltage. The electron flow causes the phosphorescent screen to glow. When passing through an object whose parts have different thicknesses, electrons will be delayed accordingly, which will appear on the screen as dark areas. The object gains contrast.
Preparations for electron microscopy are prepared on the thinnest colloidal films; objects are examined after drying them (“native preparations”), sputtering using heavy metals, ultrathin sections of the replica method, etc.
Electron microscopy can detect the smallest structures, achieve magnification up to 200,000 and see objects as small as 0.002 microns.
L.V. Timoschenko, M.V. Chubik
To study microbes, appropriate laboratory settings and equipment are required. The laboratory premises are spacious, bright, clean and isolated. Working in a laboratory requires special care because you have to work with infectious material. Microscoping. Due to their very small size, microorganisms are studied using special equipment - microscopes.
The microscope consists of two parts: mechanical and optical. The mechanical part of the microscope consists of a tripod, tube 7 (Fig. 6), “revolver” 2, stage 4, micrometric 10 and macrometric 11 screws. The optical part includes lenses 3, eyepieces, mirrors 6, lighting apparatus 5 (condenser). The optical part is the most important part of the microscope. Under the slide there is a mirror and condensers. The mirror serves to reflect (???) the direction of light rays through the condenser into the lens. The condenser consists of several lenses that collect rays reflected from the mirror at the level of the object being examined. An iris diaphragm is mounted on the lower surface of the lighting device, with which you can reduce or increase the illumination of the object being studied. The lens consists of several lenses enclosed in a common metal frame, on which a number is applied indicating the magnification. The eyepiece consists of two lenses and magnifies the image that is obtained (???) from the lens. The eyepiece also has a number indicating magnification. The total magnification of the microscope is equal to the product of the objective magnification and the eyepiece magnification.
The resolution of a microscope is limited by the wavelength of light.
There are microscopes of more advanced designs. Thus, in binocular microscopes, objects are viewed with both eyes, resulting in a more prominent image of objects. Ultramicroscopes have been designed to examine objects with dimensions less than 0.2 microns. Objects in these microscopes are illuminated not by transmitted rays, as in a conventional microscope, but by side rays emanating from a strong light source.
The electron microscope, which provides magnification of 20,000 to 200,000 times or more, was invented in 1932. With its help, you can study microorganisms such as viruses that are several millimicrons in size. In these microscopes, a stream of fast-moving electrons is passed through the object being studied, and the image is obtained on a special screen.
IN last years In addition to those described above, luminescent phase-contrast microscopes also began to be introduced, the use of which expanded the possibilities of studying microorganisms. Thus, with fluorescent microscopy, the object being studied is illuminated with ultraviolet rays from a special source. In this case, some microbes that absorb energy can then produce visible colored (green, yellow, violet) radiation. Thus, unlike conventional microscopy, a fluorescent microscope examines objects in the light they emit.
In a phase-contrast microscope, the internal structure of living cells during life and the function of movements are more clearly studied. This is achieved using specially designed phase (ring) lenses and a condenser. They change the phase of the wave of transmitted light, dramatically increasing the contrast of the image.
Rice. 6. Microscope:
1 - tube; 2 - “revolver”; 3 - lens; 4 - object table; 5 - lighting apparatus; 6 - mirror; 7 - leg; 8 - hinge; 9 - column; 10 - micrometric screw; // - macrometric screw; 12 - eyepiece.
Nutrient media. To study the various properties of microbes, they are grown on nutrient media. In order for microbes to multiply, such an environment must contain a sufficient amount nutrients, water, mineral salts and sources of nitrogen and carbon. Special attention make sure that the environment for growing microbes is sterile, since contamination of the nutrient medium makes it unsuitable for use.
There are natural and artificial nutrient media. Milk, bile, potatoes, carrots, eggs, etc. are used as natural nutrient media. Artificial nutrient media are prepared mainly from meat or plant infusions, adding various nitrogenous products, carbohydrates and salts.
Experimental animals. The role of individual microbes in the occurrence of diseases, the study of the nature of the infectious process, the method of treatment and prevention of many infectious diseases have been clarified thanks to the widespread use in microbiology of the method of experimental infection of experimental animals.
Of the laboratory animals in microbiological practice, the most widely used are guinea pigs, rabbits, white mice, white rats, sometimes monkeys, small and cattle, cats, dogs and rarely birds (pigeons, chickens). The choice of one or another animal for research depends on two conditions: firstly, the animal must be susceptible to a given infection, and secondly, under natural conditions it should not have this infection. Therefore, to study each infection they use separate species animal. For example, when studying tuberculosis and diphtheria, the experimental subjects are Guinea pigs, when studying rabies - rabbits, etc.
The number of bacteria living in the body of the average healthy adult exceeds the number of body cells by 10 times. Changes in these microbial communities can lead to digestive disorders, skin diseases, gum disease and even obesity. Despite their vital importance to human health and disease, the microorganisms that live within us remain virtually unexplored. It is only now that microbiologists around the world, having realized the importance of the body's bacteria, are attempting to conduct collaborative research efforts to better understand how they work.
Microbes and bacteria in the body
This may be the basis of a completely new way of looking at disease. In order to understand how bacteria influence changes in the normal bacterial population, it is necessary to first establish what the normal level should be.
Researchers have long suspected the role of the microbial community within people, known as the human microbiome. Molecular technologies have now reached the point where it is actually possible to begin to identify and characterize all the species that make up the human microbiome.
Scientists have identified various microorganisms that live on human skin and help form a protective barrier on the outside. It is already known that no less than 100 various types bacteria live on the skin. Using relatively new DNA sequencing methods, it was possible to identify bacterial species on the forearm of healthy subjects. Other bacterial species live on other human organs, where the number of different bacterial species living on the skin may approach 500. It is quite possible that each species may have a unique bacterial DNA species or a unique fingerprint.
Initial studies of patients with psoriasis show, for example, differences in the skin bacterial populations of patients who have the disease.
The role of bacterial communities in the human digestive tract is especially important in inflammatory bowel diseases. Microbial communities of ecosystems are being studied in people with Crohn's disease, inflammation of the gastrointestinal tract, ulcerative colitis, and E. coli.
The task of microbiologists is to see changes in microbes in the intestine as a whole and how this can affect the disease. Considering a specific organism with inflammatory diseases gut, shifts in microbial populations are visible between healthy and sick individuals, examining the loss of protective bacterial populations.
Bacteria in gastrointestinal tract may also play a role in obesity. Several years ago it was discovered that obesity was associated with changes and significant appearance certain types of bacteria in the digestive tract. This speaks to the fact that their by-products play a potential role in health and disease, that mapping and understanding the human microflora is essential to understanding human health, as is mapping and understanding the human genome. In any case, given the complexity of the system, it is definitely difficult.
New, complex laboratory technologies are now being used to characterize microbial communities that cannot be grown in laboratory conditions. Samples are collected from five areas of the body known to harbor microbial communities: the digestive tract, oral cavity, skin, nose, and female genitourinary tract. This will allow researchers to correlate the relationship between changes in the microbiome of a particular organ to a specific disease.
Initially, looking at small living creatures through a microscope was a kind of fun for inquisitive minds. It took a long time before the study of bacteria was put on a scientific basis. Thanks to this, scientists were able to link the presence of living microorganisms with the occurrence of diseases and epidemics.
Nowadays, the development of science in general and medicine in particular can no longer be imagined without microbiology. Serious Scientific research carried out in laboratories using special equipment, but some experiments can be repeated at home.
Every student now knows about the existence of bacteria. primary school, but this was not always the case. The scientist from the Netherlands, Antonie van Leeuwenhoek, was able to see bacteria for the first time in 1674. In order to conduct research and study of bacteria, he had to independently develop and create the first microscope in human history.
A little later, in 1828, the name “bacterium” appeared (from the Greek “small stick”). The word was introduced into use by the German scientist Christian Ehrenberg.
Even later, the Frenchman Louis Pasteur and the German Robert Koch, continuing their work on the study of microorganisms, linked the occurrence of diseases with the presence of bacteria in the human or animal body. For the creation of the bacteriological theory of the occurrence of diseases, Robert Koch was awarded the Nobel Prize in 1905.
In the 19th century, the world already understood the danger pathogenic bacteria posed, but people did not immediately learn to fight them in an organized manner. It was not until 1910 that Raphael Ehrlich created the first antibiotic.
Why microbial research is needed?
The study of living microorganisms is necessary to detect and identify the causative agent of a disease in a person, animal or environment. The microbiological laboratory studies pathogenic bacteria, determines their type and tests for resistance to antimicrobial drugs.
Microbiological examination is necessary not only to establish an accurate diagnosis (blood, urine, feces, mucus tests), but also to determine safety for humans environment. For example, the sanitary and epidemiological service is required to examine products intended for sale to the public.
Sampling for research
To get an idea of the state of a person, animal or environment, samples of material (samples) are needed, with which the laboratory will work. For humans and animals, this will be various tests (blood, urine, feces) or smears (mucus), and for the study of products or the environment, a small amount of the product itself (meat, milk and dairy products) or environment is used.
Samples for each type of research are taken according to a specific method, but there are several general rules. Sterile containers should be used and, if possible, sampling should be carried out under aseptic (disinfected) conditions. Samples are delivered to the laboratory as quickly as possible, if necessary in refrigerated boxes. Compliance with these conditions is especially necessary in medicine.
Some samples may be hazardous to health, so it is especially important to properly prepare the accompanying documentation.
Methods for studying microorganisms
So, samples are taken and delivered to the laboratory. Do you think that now it’s enough to look into a microscope to figure out what’s what? In reality, everything is much more complicated. There are several basic methods for determining living bacteria.
Bacteriological is a method of studying bacteria (seeding) in various biological samples - material from a sick person or animal, samples external environment, feed, meat, milk, etc.
Microscopy, i.e. studying a laboratory sample under a microscope makes it possible to determine total number microorganisms, their shape, size and structure (their morphology).
But you can’t just stick a tube of milk or urine under a microscope. To study living (unfixed) bacteria, use preparations prepared by one of two methods:
- “Crushed drop” method. A drop of material is placed on a glass slide and covered with a coverslip. The liquid should be distributed over the entire surface, but not extend beyond the edge of the coverslip.
- The hanging drop method is used for living microorganisms where colony growth is possible. With this method, you can observe the object for several days. The test material is dripped onto the cover glass, quickly turned over, drop side down, and carefully placed on a prepared glass slide with a hole in the middle. The edges of the well are pre-smeared with Vaseline to completely isolate the sample. Then the glasses are turned over again and a freely hanging drop is obtained.
To study pathological (hazardous to health) material, fingerprint smears (from organs, tissues) or thin smears from other material are used. The samples are dried, fixed (most often by passing the sample over a burner) and stained.
Sediment microscopy
In some research methods, not only the laboratory material itself is studied, but also the precipitate that falls out. This method is used when performing urine analysis.
A general urine test is needed to diagnose and control many diseases. Morphological examination of urine sediment is carried out as follows: 10-12 ml of urine is poured into a test tube, placed in a centrifuge (speed 1500-2000 rpm) for 10-15 minutes. The remaining urine is drained and the sediment is mixed.
When conducting microscopy of urine sediment, the presence of cellular elements in it is determined - red blood cells, leukocytes, casts, salts and epithelial cells.
Growing cultures of microorganisms
A bacterial culture is a collection of microbes of the same species. To grow bacterial cultures, the material is inoculated onto a nutrient medium. For example, the diphtheria bacillus was discovered and grown in pure culture 100 years ago.
For different types of bacteria, there are certain comfortable conditions (nutrition, temperature, humidity, etc.), in which the main bacteria reproduce well, but foreign microbes reproduce much worse.
Inoculated laboratory dishes and test tubes are sent to a thermostat, where they are kept at the required temperature for one to two days, and sometimes (tuberculosis) up to three to four weeks. The morphology is then compared with known characteristics of bacteria described in classification schemes or microbial guides.
Is it possible to grow bacteria at home?
Children will be curious to try growing their own colonies of bacteria at home. In addition, such experience will help them in biology classes at school.
Bacteria are everywhere, on all surfaces, in water, air, soil. The easiest way to use microorganisms at home is that living on kitchen surfaces or in the toilet. To do this, you need a Petri dish, a nutrient medium (agar-agar or meat broth) and a cotton swab.
The Petri dish must be thoroughly washed and a small amount of agar-agar or a few drops of meat broth placed in it. Use a cotton swab to wipe any surface of your choice and dip the swab into the nutrient medium. Cover the Petri dish tightly and place it in a warm place, where you leave it for 2 to 3 days. Every day, observe what is happening, you can make drawings or photographs. Show your children that interesting scientific experiments can be done at home!
Pasteurization of milk
This is also an interesting experiment that can be done at home, only aimed at destroying bacteria.
The world owes the appearance of shelf-stable milk (pasteurized) to the Frenchman Louis Pasteur. This scientist developed a process to kill microorganisms found in liquids. True, Pasteur processed wine and beer, not milk.
Pasteurization of milk involves heating it to a temperature close to its boiling point and maintaining it under such conditions. When pasteurizing milk, unlike boiling, its taste, smell and consistency do not change. This is a simple and cheap way to disinfect milk. Besides, everything dairy products now also made from pre-pasteurized milk.
In a regular kitchen you can easily pasteurize milk. To do this, place the container with milk in a steam bath (in a saucepan with hot water) and with constant stirring bring to a temperature of 63 - 65⁰C. After half an hour, the container with milk is transferred to cold water to reduce the temperature faster.
Carriers of bacteria
In addition to the harmless microorganisms that live next to us, there are also hidden enemies. Microbes, which we do not know about, like a time bomb, live in our body and can “explode” at any minute.
Pathogenic bacteria and the human body are in balance for some time, which can be disrupted by strengthening or weakening of immunity. In the first case, the body’s defense system defeats the disease, and carriage as a process stops. Otherwise, weakened immunity leads to illness.
Types of carrier:
- Healthy carrier status. Pathogenic bacteria exist in the cells of an apparently healthy person. As a rule, this process does not last long and is accompanied by a small amount of pathogenic bacteria - most often diphtheria bacillus, the causative agents of scarlet fever and dysentery.
- Incubation carriage is observed in all infectious diseases, but does not always mean that the pathogen is released into the environment.
- Acute carriage is called when the release of pathogenic microbes continues from several days to several weeks after the person has suffered the disease. If the process lasts longer than the established period, the carriage is considered chronic.
Carriage can only be determined by laboratory research methods, isolating pathogens from urine, blood, mucus, and feces. Carriers are treated in a hospital with antibiotics and vaccines.
Diphtheria bacillus
One of the pathogens transmitted by the carrier is diphtheria bacillus. This microbe has many forms but is easily identified by staining with aniline dye.
Diphtheria bacillus
Diphtheria bacteria grow with free access to oxygen and temperatures from 15 to 40⁰C. They reproduce well in an environment containing blood. That is, the human body has everything the necessary conditions for the growth of diphtheria bacilli.
The diphtheria bacterium is also spread by airborne droplets and poses a great threat to health. With diphtheria, acute inflammation of the upper respiratory tract and poisoning of the body with toxins secreted by the diphtheria bacillus occurs. This last circumstance leads to serious damage to the cardiovascular and nervous systems.
To carry out bacterioscopy, mucus and films are taken from the pharynx using dry cotton swabs. The test must be delivered to the laboratory in three hours or less. If this is not possible, a Petri dish is inoculated on site and sent for examination. The result appears after 24 or 48 hours.
The process of carriage of the diphtheria bacillus maintains the circulation of the disease and maintains the threat of an epidemic. Active immunization remains the main way to curb the growth of diphtheria pathogens.
The world of bacteria is huge and amazing. By studying microorganisms, we get the opportunity to reveal many of the secrets of nature, take care of our health and keep the environment clean.
