What kind of microscope is used to see the shape of microbial cells
A collective term for all tiny organisms that are difficult for individuals to observe with the naked eye. Microorganisms include bacteria, viruses, fungi, and a few algae. (However, some microorganisms are visible to the naked eye, such as mushrooms belonging to fungi, Ganoderma lucidum, etc.) Viruses are a type of "non-cellular organisms" composed of a few components such as nucleic acids and proteins, but their survival must depend on living cells . According to the different environments that exist, they can be divided into prokaryotic microorganisms, space microorganisms, fungal microorganisms, yeast microorganisms, marine microorganisms, etc.
The role and harm of microorganisms:
One of the most important impacts of microorganisms on humans is the prevalence of infectious diseases. 50% of human diseases are caused by viruses. The history of microbes causing human disease is the history of human beings' constant struggle with them. Human beings have made great progress in the prevention and treatment of diseases, but new and reappearing microbial infections continue to occur, such as a large number of viral diseases that lack effective therapeutic drugs. The pathogenic mechanism of some diseases is not clear. The abuse of a large number of broad-spectrum antibiotics has caused strong selection pressure, causing many strains to mutate, leading to the emergence of drug resistance, and human health is threatened by new threats. Some segmented viruses can mutate through recombination or reassortment. The most typical example is influenza virus.
After knowing the specific definition of microorganisms, what type of microscope should the experimenter use when studying microorganisms to see, and what microscope can be used to see better, and to observe and analyze common microbial forms.
The invention of the microscope is to be able to see smiling objects that cannot be seen by the naked eye. The size of microorganisms is very small, so they must be magnified and observed with the help of a microscope. In addition, there are many types of microorganisms, so basically most of the optical microscopes can To observe microorganisms, the next question is what type of microscope should be used for observation and analysis of microorganisms. Common microscopes for microbial morphology observation include biological microscopes, phase contrast microscopes, inverted microscopes, fluorescence microscopes, and confocal microscopes. Microscope and so on.
The following describes the various microscopes used to observe microorganisms:
1. Ordinary light microscope
Natural light or light is used as the light source, and its wavelength is about 0.4 μm. The resolution of the microscope is one-half of the wavelength, that is, 0.2 μm, and the smallest image visible to the naked eye is 0.2 mm. Therefore, using an oil (immersion) mirror to magnify 1000 times can enlarge the 0.2μm particles into 0.2mm visible to the naked eye. Ordinary optical microscopes can be used for the observation of bacteria, actinomycetes and fungi.
2. Darkfield microscopy is commonly used to observe unstained microbial morphology and movement. After the dark field condenser is installed in the ordinary microscope, the light cannot penetrate directly from the middle, and the field of view is dark. When the specimen receives oblique light from the edge of the condenser, it can be scattered, so bright microorganisms can be observed in the dark field background such as bacteria or spirochetes.
3. Phase contrast microscope Phase contrast microscope uses the light effect of the phase difference plate to change the light phase and amplitude of direct light, and convert the difference of light phase into light intensity difference. Under a phase-contrast microscope, when light passes through an unstained specimen, the difference in light phase is caused by the inconsistency of the density of different parts of the specimen, and the morphology, internal structure and movement mode of microorganisms can be observed.
4. Fluorescence microscope Fluorescence microscope is basically the same as ordinary optical microscope, the main difference is the light source, filter and condenser. At present, most of them use epi-light devices, and high-pressure mercury lamps are commonly used as light sources, which can emit ultraviolet or blue-violet light. There are two kinds of filters: excitation filter and absorption filter. In addition to general bright-field condensers, dark-field condensers can also be used in fluorescence microscopes using blue light to enhance the contrast between fluorescence and background. This method is applicable to the detection or identification of bacteria stained with fluorescent pigments or combined with fluorescent antibodies.
5. Electron microscopes use electron flow as the light source. Compared with visible light, the wavelength is tens of thousands of times different, which greatly improves the resolution. The magnetic coil is used as the optical amplification system, and the magnification can reach tens of thousands or hundreds of thousands of times. It is often used in virus particles. and the observation of bacterial ultrastructure.
Observation of unstained microbial specimens:
Unstained specimens can generally be used to observe bacterial morphology, power and movement. Bacteria are colorless and transparent when unstained, and are observed under a microscope mainly by the difference between the refractive index of the bacteria and the surrounding environment. Bacteria with flagella move vigorously, while bacteria without flagella show irregular Brownian motion. Viable bacteria such as Treponema pallidum, Leptospira, and Campylobacter have distinctive shapes and movement patterns, which are of diagnostic significance. Commonly used methods are pressure drop method, pendant drop method and capillary method.
1. Hanging drop method Apply vaseline around the concave hole of the clean concave glass slide, take a ring of bacterial suspension with an inoculation loop and put it in the center of the cover glass, then align the concave hole of the concave glass slide with the droplet in the center of the cover glass and Put the cover on, then turn it over quickly, lightly press the cover slip to make it stick tightly to the vaseline on the edge of the concave hole, and then observe under a high-power microscope (or dark field).
2. Take a ring of bacterial suspension with an inoculation loop and place it in the center of a clean glass slide by pressure drop method, and gently cover the bacterial suspension with a cover glass, taking care to avoid the generation of air bubbles and prevent the bacterial suspension from overflowing. Brightfield (or darkfield) observation under a high-power lens.
3. The capillary method is mainly used for the examination of the kinetics of anaerobic bacteria. Usually choose 60~70mm long. After siphoning the anaerobic bacteria suspension through a capillary with an aperture of 0.5-1.0 mm, seal the two ends of the capillary with a flame. The capillary was fixed on the glass slide with plastic paper, and observed under a high-power lens in dark field.
Observation of stained microbial specimens with a microscope:
After the bacterial specimen is stained, due to the sharp contrast in color between the bacteria and the surrounding environment, the morphological characteristics of the bacteria (such as the size, shape, arrangement, etc.) of the bacteria and some special structures can be clearly observed under an ordinary optical microscope (such as capsules, flagella, spores, etc.), and the bacteria can be classified and identified according to the staining reactivity.
(1) General procedure of bacterial staining The general procedure of bacterial staining is: smear (drying)—fixation—staining.
1. Smear Preparation of blood, secretions, excretions, puncture fluid and liquid culture, and direct thin film smears on glass slides; autopsy or infected animal tissues, smear the lesion with a cotton swab for sampling. For the preparation of bacterial colonies or lawns on solid medium, first use an inoculation loop to take a ring of normal saline and put it in the center of the slide, then use a sterile inoculation loop to take a small amount of culture and grind it evenly in normal saline, and spread it to 1cm2 Large or small painted surfaces, let it dry naturally at room temperature or dry slowly at a distance.
2. The purpose of fixation is to kill bacteria, coagulate bacterial protein and structure, and facilitate staining; promote bacteria to adhere to the slide to avoid being washed away by water during washing; change the permeability of bacteria to dyes, which is beneficial to the structure of bacteria cells of staining. It is usually fixed by heating with a flame, and the dried smear is quickly passed through the flame for 3 times. It is better not to burn the skin on the back of the hand when it touches the slide.
3. Dyeing According to different inspection purposes, choose different dyeing methods for dyeing. When dyeing, add the dye solution dropwise to increase the coverage.
4. Mordant Any substance that can enhance the affinity between the dye and the dyed object, fix the dye on the dyed object and cause a change in the permeability of the cell membrane is called a mordant. Commonly used are alum, tannic acid, metal salts and iodine, etc., and heating is also used to promote coloring. Mordants can be used between primary staining and counterstaining, and can also be used after fixation or contained in fixative and staining.
5. Decolorization Any chemical agent that can remove the color of the dyed object is called a decolorizer. Ethanol, acetone, etc. are commonly used as decolorizers. The decolorizing agent can detect the degree of stability of the combination of bacteria and dyes, which can be used for differential staining.
6. Counterstaining Bacteria or their structures that have been decolorized are often counterstained with a counterstain solution for easy observation. The color of the counterstaining solution is different from that of the primary dyeing solution to form a sharp contrast. The counterstaining should not be too strong, so as not to cover up the color of the initial staining.
