GENERAL AND FOOD MICROBIOLOGY PART I - L. V. Krasnikova - 2016

1. MICROSCOPY

The Study of microorganism Cells, which are invisible to the naked eye, is only possible using microscopes (from the Greek micros meaning small, and skopeo meaning to look). These instruments make it possible to obtain images of the studied objects magnified hundreds of times (light microscopes) and tens to hundreds of thousands of times (electron microscopes).

1.1. Structure OF THE Light Microscope

Purpose of the work: to familiarize students with the design of a light microscope, its specifications, and the rules for operating it.

A microscope is called a light microscope because it allows objects to be studied in transmitted light. The Main Components of modern light microscopes are the mechanical and optical systems (Fig. 1.1).

Class="center">Fig. 1.1. Microscope structure: 1 - base; 2 - limb (arm); 3 - body tube; 4 - eyepiece; 5 - revolving nosepiece; 6 - objective lens; 7 - stage; 8 - specimen clips; 9 - condenser; 10 - condenser bracket; 11 - condenser adjustment knob; 12 - swing-out lens; 13 - mirror; 14 - coarse adjustment knob; 15 - fine adjustment knob; 16 - micrometric focus mechanism housing; 17 - HEAD for attaching the body tube and nosepiece; 18 - head mounting screw

The mechanical parts include the stand, body tube, revolving nosepiece, fine focus housing, stage, as well as the coarse and fine adjustment knobs.

The stand consists of two parts: the base and the limb (Column). The base of the microscope is rectangular and features four support pads on the underside, ensuring a stable position on the laboratory workbench.

The limb is connected to the base and can be moved vertically using the coarse and fine adjustment knobs. Turning the knobs clockwise lowers the limb, while turning them counterclockwise raises it away from the specimen. The upper part of the limb features a head with a socket for a monocular (or binocular) head and a guide rail for the revolving nosepiece. The head is secured with a screw.

The body tube is the microscope cylinder that maintains a fixed distance between the main optical components: the eyepiece and the objective lens. The eyepiece is inserted into the top of the tube. Modern microscope models feature an inclined body tube.

The revolving nosepiece is a concave disk with several sockets into which 3-4 objective lenses are screwed. By rotating the nosepiece, any objective lens can be quickly brought into the working position beneath the tube opening.

The fine mechanism housing holds the guide rail for the condenser bracket on one side and the guide rail for the limb on the other. Inside the housing is the microscope focusing mechanism, which is a system of gear wheels.

The stage is designed to hold the specimen or other object of study. The stage can be square or round, and either movable or fixed. A movable stage can be translated horizontally using two lateral knobs, allowing the specimen to be viewed across different fields of view. On a fixed stage, the specimen is moved manually to examine different fields. A central aperture is located at the bottom in the middle of the stage to let through illumination light directed from the light source. Two spring clips are mounted on the stage to secure the specimen. Some microscope systems are equipped with a mechanical stage (stage clip assembly), which is essential when examining The surface of a specimen or counting cells. The mechanical stage allows the specimen to be moved in two mutually perpendicular directions. It features a system of vernier scales that make it possible to determine the coordinates of any point on the studied object.

The coarse adjustment knob (coarse focus) is used for preliminary, rough positioning of the image of the object being viewed. Turning the coarse knob clockwise lowers the microscope tube, while turning it counterclockwise raises it.

The fine adjustment knob (fine focus) is used to precisely focus the image of the object. The fine adjustment knob is one of the most fragile PARTS OF THE microscope and must be handled with care: to avoid spontaneous lowering of the tube, do not turn this knob for rough focusing. A full revolution of the fine adjustment knob moves the tube by 0.1 mm.

The Optical System of the microscope consists of the primary optical components (the objective lens and the eyepiece) and the auxiliary illumination system (the mirror and the condenser).

Objective lenses (from the Latin objektum meaning object) are the most important, valuable, and delicate part of the microscope. They consist of a system of lenses enclosed in a metal mount, which indicates the magnification level and numerical aperture. The outer lens, facing the specimen with its flat side, is called the front lens. It is this lens that provides the magnification. The remaining lenses are called correction lenses and serve to eliminate optical aberrations that occur when viewing the studied object.

Objective lenses can be dry or immersion lenses. A dry lens is one where air is present between the front lens and the object being viewed. Dry lenses typically have a long focal length and a magnification of 8x or 40x. An immersion lens is one where a special liquid medium is placed between the front lens and the specimen. Due to the difference in refractive indices between Glass (1.52) and air (1.0), a portion of the light rays is refracted and fails to enter the observer's eye. As a result, the image becomes blurred and finer structures remain invisible. Light scattering can be avoided by filling the space between the specimen and the front lens of the objective with a substance whose refractive index is close to that of glass. Such substances include glycerin (1.47), cedar oil (1.51), castor oil (1.49), linseed oil (1.49), clove oil (1.53), anise oil (1.55), etc. Immersion objectives are marked on their mounts with designations such as: I - immersion, HI - homogeneous immersion, OI - oil immersion, and MI - oil immersion (Russian abbreviation). Currently, synthetic products with optical properties matching cedar oil are most commonly used as immersion liquids.

Objective lenses are distinguished by their magnification. The magnification value is marked on their mount (8x, 40x, 60x, 90x). In addition, each objective is characterized by a specific working distance. For an immersion objective, this distance is typically 0.12 mm, while for dry objectives with 8x and 40x magnification, it is 13.8 mm and 0.6 mm, respectively.

The eyepiece (from the Latin ocularis meaning pertaining to the eye) consists of two lenses—the eye lens (upper) and the field lens (lower)—enclosed in a metal mount. The eyepiece serves to magnify the image produced by the objective lens. The eyepiece magnification is marked on its mount. Eyepieces with working magnifications ranging from 4x to 15x are available.

When working with a microscope for extended periods, a binocular head should be used. The housing of the binocular head can be adjusted within 55–75 mm depending on the interpupillary distance of the observer. Binocular heads often feature their own magnification (around 1.5x) and correction lenses.

The condenser (from the Latin condenso meaning to thicken or condense) consists of 2–3 short-focus lenses. It collects the rays coming from the mirror and directs them onto the object. Using a lever located beneath the stage, the condenser can be moved vertically, which increases the illumination of the field of view when raised and decreases it when lowered. To regulate light intensity, the condenser is equipped with an iris Diaphragm consisting of steel crescent-shaped blades. It is recommended to view stained specimens with the diaphragm fully open, and unstained specimens with a reduced aperture. Located beneath the condenser is a swing-out lens in a mount, which is used when working with low-magnification objective lenses, such as 8x or 9x.

The mirror has two reflecting surfaces: flat and concave. It is mounted on gimbals at the Base of the stand and can be easily rotated. For artificial lighting, it is recommended to use the concave side of the mirror, and for natural lighting, the flat side.

The illuminator serves as an artificial light source. It consists of a low-voltage incandescent lamp mounted on a stand and a step-down transformer. The transformer housing features a rheostat knob to adjust lamp brightness and a toggle switch to turn the illuminator on and off.

In many modern microscopes, the illuminator is built directly into the base.

1.2. Key CHARACTERISTICS OF THE Light Microscope

The numerical aperture A (from Latin apertura — opening) of an objective lens determines its light-gathering capacity and is calculated by the formula

А = n sin 1/2 α,

where n is the refractive index of the light ray passing through the microscope slide into the medium between the front lens of the objective and the slide; α is the angle whose one side coincides with the optical axis and the other is formed by the line connecting the exit point of effective rays from the objective with the edge of the working aperture of the objective; 1/2 α is the semi-aperture angle of the objective.

It is essential that the value of n be as high as possible. This can be achieved by introducing a substance into the space between the front lens of the objective and the slide with a refractive index close to that of glass, as noted above. It is equally important that the value of sin 1/2 α be maximized. The upper limit of this value depends on the curvature of the immersion objective's front lens and the numerical aperture of the condenser. Note that the value of sin 1/2 α when using immersion objectives can be increased by raising the condenser to its highest position, which relies on its light-gathering function. If the condenser is lowered, this function is effectively compromised.

The numerical aperture and magnification of the objective are marked on its mount: 8x0.2; 40x0.65; 90x1.25.

The magnifying power of a microscope (D) is defined as the product of the eyepiece magnification (K) and the objective magnification (V):

D = KV.

Theoretically, a microscope can provide a magnification of x2000 or more. However, a distinction must be made between useful and empty magnification. Useful magnification is defined as the magnification that allows the object to be viewed at the limiting angle of resolution. In conventional light microscopes, the limit of useful magnification reaches 1400. Exceeding useful magnification leads to diffraction and other phenomena caused by the wave nature of light. Specifically, for an objective with 40x magnification and a numerical aperture of 0.65, the useful magnification ranges from 325 to 650. This magnification reveals all structural details resolvable by that particular objective. Therefore, to achieve resolution within the useful limit when using a 40x objective, a 15x eyepiece should be used. Employing higher-power eyepieces will not reveal finer details.

If the objective has a magnification of 90x (numerical aperture A = 1.25), its useful magnification is 1250. Consequently, in this case as well, eyepieces with a magnification exceeding 15x should not be used in order to stay within the limits of useful magnification.

The resolving power of a microscope. While the magnifying power depends on both the objective and the eyepiece, the resolving power is determined primarily by the objective and the condenser. Resolving power is calculated using the formula

d = λ / 2А,

where λ is the wavelength of light perceived by the human eye (0.4–0.7 µm, with an average of 0.55 µm); A is the numerical aperture of the objective.

The maximum resolving power of a light microscope is 0.2 µm. The resolving power of a microscope improves as the absolute value of d decreases.

1.3. Microscope Operating Guidelines

1. Hold the microscope with one hand around the limb (arm) and support the base with the other. Lifting or carrying the microscope by any other parts is strictly prohibited.

2. Place the microscope on the workbench with the arm facing toward you. Before starting work, gently wipe dust off the optical components using a soft, dry cloth, taking care not to Touch the lenses with your fingers.

3. Select the desired objective using the revolving nosepiece. A distinct click from the internal detent indicates that the objective is properly centered. Keep in mind that the lower the magnification of the objective, the longer its working distance. When using the 8x objective, the distance between the specimen and the objective is approximately 9 mm; with the 40x objective, it is about 0.6 mm; and with the 90x objective, it is approximately 0.15 mm.

4. Place the microscope slide onto the stage and secure it with the stage clips.

5. Carefully lower the microscope tube using the coarse adjustment knob while observing the objective from the side, bringing it close to the specimen (without touching it) to a distance less than the working distance. Then, looking through the eyepiece, slowly turn the coarse adjustment knob to raise the tube until the image of the specimen comes into view.

6. Adjust the fine adjustment knob to bring the specimen image into sharp focus.

7. When working with an immersion objective, place a drop of cedar oil on the slide. While watching from the side, carefully lower the tube using the coarse adjustment knob until the front lens of the objective is immersed in the oil. Then, looking through the eyepiece, slowly turn the coarse adjustment knob upward until the image appears. Use the fine adjustment knob for precise focusing, turning it within a single revolution limit.

Caution! Never attempt to locate the specimen image using the fine adjustment knob.

8. Examine the preparation across several fields of view by moving the mechanical stage using the lateral adjustment knobs, or by manually shifting it on the stage. Select the most suitable field of view in an area where microorganisms are clearly visible and present in sufficient numbers for observation, and sketch the microscopic view.

9. When changing objective lenses, adjust the illumination intensity of the object under study. The desired degree of lighting is achieved by lowering or raising the condenser.

10. Upon completion of the work, raise the body tube, remove the slide from the stage, and clean the oil from the front lens of the immersion objective using lens paper moistened with gasoline. Then, rotate the nosepiece to set the 8x objective, place a small piece of clean gauze on the stage, and lower the body tube.

1.4. Dark-Field Cell/15.html">Microscopy

Dark-field microscopy increases the resolving power of an objective by approximately 10 times, making it possible to observe objects whose sizes lie beyond the limits of conventional light microscopy. This technique relies on illuminating the specimen with oblique light rays that do not enter the objective and remain invisible to the naked eye (the Tyndall effect), causing the field of view to appear completely black. If the preparation contains particles, such as microorganisms, the oblique rays directed at a specific angle are diffracted and reflected off their surfaces, deviating from their initial path enough to enter the objective. Because the light rays originate directly from the object, the observer sees a distinct luminous image of microbial cell contours or other particles against a dark Background. A dark field is achieved by using a specialized dark-field condenser instead of the standard bright-field microscope condenser. However, this dark-field effect can only be attained if the numerical aperture of the condenser exceeds that of the objective by 0.2–0.4 units.

Dark-field microscopy is utilized to study living microbial cells. It is particularly valuable for observing microbial motility and detecting the causative agents of specific diseases (such as leptospirosis). Nonetheless, the dark field does not permit a detailed examination of the internal structure and precise shape of microbial cells.

When observing Yeast cells, the Cytoplasm exhibits a faint and uniform opalescence, against which dark vacuoles and highly refractive liposome granules stand out clearly. The protoplast of dying cells takes on a milky-white color.

1.5. Phase-contrast microscopy

Phase-contrast microscopy enables the examination of live specimens without The Need for staining or fixation. The human eye responds to changes in light wavelength (color) and amplitude (intensity, contrast) but is incapable of perceiving phase differences. Phase-contrast microscopy was developed specifically to observe transparent specimens that transmit light rays of identical wavelength and amplitude but shift their phase. The magnitude of this phase shift depends on the thickness and refractive index of the cellular structures. A phase-contrast device converts these phase alterations of light waves—caused by passing through transparent structures—into amplitude variations, thereby rendering the details of the examined objects clearly visible and high in contrast.

To perform these examinations, a phase-contrast attachment must be used in conjunction with a standard light microscope. The most widely used model is the KF-4, which consists of an auxiliary focusing telescope, specialized phase objectives, and a condenser equipped with a set of annular diaphragms, each corresponding to the phase plate of a specific objective.

The auxiliary telescope is installed by replacing the standard microscope eyepiece. To achieve phase contrast, a specialized phase plate is integrated into the objective; this consists of a thin disk coated with rare-metal salts on one of the inner lenses of the objective. It alters the phase of the transmitted light wave by 1/4 λ, thereby converting phase differences into amplitude differences. Phase objectives feature specific markings on their mounts: Ф10, Ф20, Ф40, and Ф90. The annular diaphragms are housed in a rotating disk beneath the condenser and can be rapidly switched by turning the disk. Each annular diaphragm allows only a ring of light to pass through the condenser into the plane of the preparation. The phase-contrast effect is attained by precisely aligning the ring of the phase plate with the projected image of the annular diaphragm.

1.6. Fluorescence Microscopy

Fluorescence microscopy is based on the property of certain biological specimens to emit light when irradiated with short-wave rays invisible to the human eye (blue-violet light with a wavelength around 460 nm or ultraviolet light at 300-400 nm). This phenomenon occurs because a portion of the incident light energy is absorbed, causing the illuminated object to emit rays of a longer wavelength than the exciting light. Consequently, the cells glow with a yellow-green or orange light (they fluoresce). This is known as primary or autofluorescence.

Primary fluorescence is exhibited by plant and algal cells due to the presence of chlorophyll, as well as by certain pigment-producing Bacteria. However, the majority of microbial cells display only weak primary fluorescence.

Secondary (or induced) fluorescence in specimens lacking autofluorescence is achieved by treating them with specialized Dyes known as fluorochromes. The most commonly used fluorochromes belong to the acridine and thiazole groups (such as acridine orange and yellow, auramine, uranin, rhodamine, thioflavin, primuline, and fluorescein). Specifically, acridine orange stains the cytoplasm green-yellow, metachromatin bright red, vacuoles pink, and The Nucleus light green.

Fluorescence microscopy is performed in a darkened room using a specialized fluorescence microscope, such as the ML-2 model.

Specialized quartz lamps serve as the ultraviolet radiation source in fluorescence microscopes. Before reaching the specimen, the lamp rays pass through a series of light filters that transmit only a specific portion of the ultraviolet spectrum where λ = 360-380 nm.

A more accessible method for exciting fluorescence involves utilizing the short-wave portion of visible light—blue-violet rays at λ = 460 nm. In this case, observations can be carried out not only with a dedicated fluorescence microscope but also using a standard light microscope by placing a blue glass or liquid light filter in the optical path. Excess blue rays are filtered out using a yellow barrier filter placed over the microscope eyepiece. As a result, fluorescent objects become visible against a dark background in the preparation.

Fluorescence microscopy makes it possible to observe morphological features that remain invisible under conventional light microscopy. Furthermore, it enables the differentiation of microbial species based on their fluorescence characteristics and facilitates the study of structural changes across various physiological states of The Cell. This method is also valuable for examining the antigenic structure of bacteria and detecting infectious disease agents through The Use of fluorochrome-labeled immune sera.

1.7. Electron microscopy

An Electron microscope utilizes a beam of moving electrons instead of light waves, which increases the instrument's resolving power by 100-fold or more. The high resolving power of modern electron microscopes allows researchers to observe and study objects that are invisible under a light microscope, including Viruses, Bacteriophages, Mycoplasmas, and the fine ultrastructure of prokaryotic and Eukaryotic cells, along with their macro- and microstructural components.

An electron microscope consists of several complex units:

1. A column housing the electron gun, electromagnetic focusing lenses for the electron beam, the condenser lens, the specimen chamber (containing the stage, objective, intermediate, and projector lenses), a fluorescent screen, and a camera.

2. A Vacuum system designed to maintain a high vacuum within the column.

3. A control panel.

4. Power supply unit housed in a metal cabinet behind the column.

5. Auxiliary devices.

The illumination system of an electron microscope consists of an electron gun and a condenser lens. An electromagnetic field is used to focus the electron beam (glass lenses cannot be used because glass is impermeable to electrons). Since air impedes the movement of electrons, a high vacuum must be maintained inside the microscope.

The technique for preparing specimens for electron microscopy differs significantly from that used for light microscopy. Specimens for electron microscopy are prepared on an extremely thin, electron-permeable substrate. Films made of collodion, quartz, or other Materials with a thickness of about 1 µm are typically used as substrates. Because these films are very thin and fragile, they are placed on special fine-mesh metal grids. The total thickness of the specimen and substrate must not exceed 0.25 µm. To enhance the contrast of the studied specimens, they undergo additional Processing under high vacuum conditions by evaporating an extremely thin layer of chromium, gold, platinum, or palladium onto them, or by using various contrasting agents (such as uranyl acetate, uranyl nitrate, or phosphotungstic acid). The specimen is placed on a stage located between the condenser and the objective lens. As the electron beam passes through the specimen, it is scattered and focused by the electromagnetic refracting field of the objective lens. This produces the first magnified real image of the object (enlarged 40,000–50,000 times), which can be viewed through the viewing window. Next, the electron stream enters the electromagnetic field of the projection lens, which condenses the passing electrons and focuses them onto a fluorescent screen, yielding a final magnified image of the object enlarged 200,000–300,000 times.

A camera is located beneath the fluorescent screen. By raising the screen with a special lever, the electron beam is allowed to pass onto a photographic plate to capture an image of the specimen.

Review Questions

1. What are the basic safety and operational rules in a microbiological laboratory?

2. Name the main mechanical components of a microscope.

3. Name the main optical components of a microscope.

4. What is the numerical aperture of a microscope?

5. What is the difference between dry and immersion objectives?

6. What is the purpose of the coarse and fine adjustment knobs?

7. What is the function of the revolving nosepiece?

8. How do you determine the magnifying power of a microscope?

9. How is the illumination intensity of a specimen adjusted?

10. What are the Main characteristics of a light microscope?

11. What is the underlying principle of dark-field microscopy?

12. Explain the working principle of a phase-contrast microscope.

13. Explain the working principle of a fluorescence microscope.

14. What are the specific features and Procedures of working with an electron microscope?