Microscopic research methods in microbiology

представляют собой способы изучения разнообразных объектов с использованием специального оборудования. Microscopic research methods are ways to study a variety of objects using special equipment. It allows us to consider the structure of substances and organisms, the magnitude of which lies beyond the limits of the resolving power of the human eye. In the article we will make a brief analysis of microscopic methods of investigation.

General information

используют в своей практике разные специалисты. Modern methods of microscopic research are used by different specialists in their practice. Among them, virologists, cytologists, hematologists, morphologists and others. The main methods of microscopic research have been known for a long time. First of all, this is the light mode of viewing objects. In recent years, other technologies have been actively introduced into practice. . So, popularity acquired a phase-contrast, luminescent, interference, polarization, infrared, ultraviolet, stereoscopic method of investigation . They are all based on a variety of properties of light. . In addition, electron microscopic methods of investigation are widely used. These methods allow you to display objects using a directed flux of charged particles. It should be noted that such methods of studying are used not only in biology and medicine. в промышленности. A microscopic method for studying metals and alloys in industry is quite popular. Such a study allows us to evaluate the behavior of compounds, develop technologies to minimize the probability of destruction and strengthening strength.

Light methods: characteristic

и других объектов базируются на различной разрешающей способности оборудования. Such microscopic methods of studying microorganisms and other objects are based on different resolution of equipment. Important factors in this case is the direction of the beam, the features of the object itself. The latter, in particular, can be transparent or opaque. In accordance with the properties of the object, the physical properties of the light flux - brightness and color, caused by the amplitude and wavelength, the plane, phase and direction of wave propagation - change. . On the use of these characteristics, different microscopic methods of investigation are constructed.

Specificity

To study light, objects are usually painted. This allows us to identify and describe some of their properties. It is necessary that the tissues are fixed, since the color will reveal certain structures exclusively in the killed cells. In living elements, the dye is released as a vacuole in the cytoplasm. It does not paint the structure. But with the help of a light microscope, you can also explore living objects. For this, a vital method of learning is used. In such cases, a dark-field condenser is used. It is built into a light microscope.

Study of unpainted objects

It is carried out by means of phase-contrast microscopy. This method is based on the diffraction of the ray in accordance with the features of the object. In the process of exposure, the phase and wavelength changes are noted. A translucent plate is present in the lens of the microscope. Live or fixed, but not colored objects because of their transparency almost do not change the color and amplitude of the beam passing through them, provoking only the shift of the wave phase. But while passing through the object, the light flux deviates from the plate. As a result, between the rays passed through the object, and entering into the light background, there appears a difference in wave length. At a certain value, a visual effect occurs - a dark object will be clearly visible on a light background or vice versa (in accordance with the features of the phase plate). To obtain it, the difference should be not less than 1/4 of the wavelength.

An anthropic method

It is a kind of phase-contrast method. An anthropic method involves the use of a lens with special plates that change only the color and brightness of the background light. This greatly expands the possibilities of studying unpainted living objects. , паразитологии при изучении растительных и животных клеток, простейших организмов. A phase-contrast microscopic method is used in microbiology , parasitology in the study of plant and animal cells, protozoa. In hematology, this method is used to calculate and determine the differentiation of blood and bone marrow elements.

Interference receptions

решают в целом те же задачи, что и фазово-контрастные. These microscopic methods of research solve in general the same problems as phase-contrast ones. However, in the latter case, specialists can observe only the contours of objects. методы исследования позволяют изучать их части, выполнять количественную оценку элементов. Interference microscopic research methods allow you to study their parts, perform a quantitative evaluation of the elements. This is possible due to the bifurcation of the light beam. One stream passes through a particle of the object, and the other passes by. In the eyepiece of the microscope, they converge and interfere. The resulting phase difference can be determined by the weight of different cellular structures. If it is measured sequentially with the given refractive indexes, it is possible to establish the thickness of non-fixed tissues and living objects, the content of proteins in them, the concentration of dry matter and water, etc. In accordance with the data obtained, specialists are able to indirectly assess membrane permeability, enzyme activity, and cellular metabolism.

Polarization

It is carried out with the help of Nicolas prisms or filmy polaroids. They are placed between the preparation and the light source. позволяет изучать объекты с неоднородными свойствами. Polarization microscopic method of investigation in microbiology allows to study objects with inhomogeneous properties. In isotropic structures, the speed of propagation of light does not depend on the chosen plane. In anisotropic systems, the velocity varies in accordance with the direction of light along the transverse or longitudinal axis of the object. If the refractive index along the structure is greater than along the transverse, a double positive refraction is created. This is characteristic of many biological objects, in which a strict molecular orientation is detected. They are all anisotropic. This category, in particular, includes myofibrils, neurofibrils, cilia in the ciliated epithelium, collagen fibers and others.

The polarization value

Comparison of the nature of the radiation refraction and the anisotropy index of the object makes it possible to evaluate the molecular organization of the structure. The polarization method acts as one of the histological methods of analysis, is used in cytology, etc. In the light it is possible to study not only colored objects. The polarization method makes it possible to investigate unpainted and non-fixed - native - preparations of tissue sections.

Fluorescent techniques

They are based on the properties of some objects to give a glow in the blue-violet part of the spectrum or in the UV-rays. Many substances, for example proteins, some vitamins, coenzymes, medicines, are endowed with primary (own) luminescence. Other objects start to glow with the addition of fluorochromes - special dyes. These additives are selectively or diffusely distributed to individual cellular structures or chemical compounds. This property formed the basis for the use of luminescence microscopy in histochemical and cytological studies.

Areas of use

Using immuno-fluorescence, specialists detect viral antigens and establish their concentration, identify viruses, anti bodies and antigens, hormones, various metabolic products and so on. In this regard, in the diagnosis of herpes, mumps, viral hepatitis, influenza and other infections luminescent methods of materials research are used. иммуно-флуоресцентный способ позволяет распознавать опухоли злокачественного характера, определять ишемические участки в сердце на ранних этапах инфаркта и пр. Microscopic immuno-fluorescent method allows to recognize tumors of malignant character, to determine ischemic patches in the heart at early stages of infarction, etc.

Use of ultraviolet light

It is based on the ability of a number of substances included in living cells, microorganisms or fixed, but unpainted, transparent in visible light to absorb UV rays of a certain wavelength. This is typical, in particular, for high-molecular compounds. These include proteins, aromatic acids (methylalanine, tryptophan, tyrosine, etc.), nucleic acids, pyramidal and purine bases, and so on. Ultraviolet microscopy makes it possible to clarify the localization and the number of these compounds. When studying living objects, specialists can observe changes in the processes of their life activity.

Additionally

Infrared microscopy is used in studies of objects opaque to light and UV rays by absorbing them by flow structures with a wavelength of 750-1200 nm. To apply this method, it is not necessary to pre-chemically treat the preparations. As a rule, the infrared method is used in anthropology, zoology and other biological branches. As for medicine, this method is used mainly in ophthalmology and neuromorphology. The study of volumetric objects is carried out using stereoscopic microscopy. The design of the equipment makes it possible to observe the left and right eye at different angles. Opaque objects are investigated at a relatively small increase (not more than 120 times). Stereoscopic methods are used in microsurgery, pathomorphology, in forensic medicine.

Electron microscopy

It is used to study the structure of cells and tissues at the macromolecular and subcellular levels. Electron microscopy has made it possible to make a quantum leap in the field of research. This method is widely used in biochemistry, oncology, virology, morphology, immunology, genetics and other branches. A significant increase in the resolving power of the equipment is ensured by the flow of electrons that pass through the electromagnetic fields in a vacuum. The latter, in turn, are created by special lenses. Electrons have the ability to pass through the structure of the object or to be reflected from them with deviations from different angles. As a result, a display is created on the device's fluorescent screen. With transmission microscopy, a flat image is obtained, with a scanning image, respectively, a volume image.

The necessary conditions

It is worth noting that before the electronic microscopic examination, the object is subjected to special training. In particular, physical or chemical fixation of tissues and organisms is used. The sectional and biopsy material, in addition, is dehydrated, embedded in epoxy resins, cut with diamond or glass knives into ultra-thin sections. Then they are contrasted and studied. In the scanning microscope, the surfaces of objects are examined. To do this, they are sprayed with special substances in a vacuum chamber.