- Optical laboratory microscope for biological purposes: structure, major components, working principle.
- Structure, basic components and operating principle of transmission electron microscopes and scanning (SEM).
- What is the resolution of the microscope?
- Compare the structure and working principle optical and electronic (TEM / SEM) microscopes
Laboratory work № 1.4.
Study of the main features of unicellular eukaryotic and prokaryotic cells. Electron microscopy.
The development of methods of preparation of lifetime and regular medication. Task
Staining of living and fixed cells
Staining Gram (Gram or method) - empirical method derived differentiation of bacteria by staining them in a certain method into two major groups: Gram-positive and Gram-negative), different chemical and physical properties of their cell walls.
Gram Staining refers to complex staining method, when the stroke affected the two stains, one of which is the principal, and the other - additional. Besides coloring substances in complex ways color is used stain-removing substances: alcohol, acid, etc.. For Gram staining is more commonly used stains trifenilmetane groups: hentsyan-violet, methyl violet or kristallviolet. Gram-positive (Gram (+)) bacteria give strong connection with these stains and iodine. However, they are not discolouring exposed to alcohol, resulting in an extra color magenta Gram (+) bacteria do not change initially adopted purple.
Gram-negative (Gram (-)) microorganisms form with basic stains and iodine compounds that easily destroyed by alcohol. As a result of microbes obezbarvlyuyutsya and then colored magenta, getting red
Considering the characteristics of the light microscope, we can see that the only way to increase the optical system will permit the use of the light source that emits waves with the smallest length. This source can be a hot filament, which emits an electric field flow of electrons, the latter can be focused by passing through a magnetic field. This was the basis for the creation of an electron microscope, which has now reached a resolution of 1 A (0.1 nm). By the principle of electron microscope design is very similar to the optical: it is the light source (cathode electron gun), condenser system (condenser magnetic lens), the lens (objective magnetic lens) glasses (magnetic projection lens), but instead of retinal eye electrons fall on lyumynestsyruyuschyy screen or photographic plate The main part of the microscope is a hollow cylinder (column microscope), which pumped air in order to avoid the interaction of electrons with molecules of gases and oxidation of tungsten filament in the cathode electron gun. Between the cathode and the anode is a high voltage (50 to 200-5000 kW), which causes the acceleration of electrons. In the center of the anode has a hole passing through which electrons form a beam that runs down the column of the microscope. Electron microscope lenses are electromagnets field which can change the way electrons (as glass lenses change the way photons). In the condenser lens beam of electrons is fixed and falls on the object with which the electrons interact rejected, scattered, absorbed or are unchanged. Electrons passing through the object focus of the objective lens, which forms a larger primary image of the object. Just as in the light microscope, the objective lens determines its key figures. Initial increases image projection lens and projected onto a screen covered with a layer of luminescent, glowing in contact with him electrons. Instead glowing screen image can be placed on a photographic plate and get the shot. The voltage used to accelerate electrons in most translucent (transmission) electron microscopes reaches 50-150 kV. When la voltage of 50 kV electron has a wavelength of 0.05 A, in this case). theoretically could get permission to 0.025 A (d ~ 0,5 However, in modern designs electron microscopes achieved resolution of about 1 A due to insufficient stability of the voltage stability of AC lenses, metal inhomogeneity of magnetic lenses and other imperfections device (even theoretically possible to increase the resolution of the electron microscope 100 times.) But the resolution achieved enormous (remember that the value of O-H bond in the molecule of water is 0 99 A): it is already 106 times higher resolution eyes! The screens and plate electron microscopes can be increased to 50 000 times further at photo printing, you can get another 10-fold increase, so that the final increase at which the maximum resolution is implemented, can reach 106 times (for example, if 1 mm increase in 106 times then it reaches a length of 1 km). Currently, electron microscopy images of fluorescent screen with a digital camera is transmitted directly to the computer where on the screen it can be processed in different ways (change magnification, contrast, apply densitometry, plans and morphometry of individual components). By using the printer, you can get prints of images
Equipment, materials and methods. Microscope objective lenses, glasses with hole, glass rods, hinges, cover lenses. set of paints and chemicals for coloring preparates
Bacteria, lower plants - algae, moss, fern.
The order of performance and recommendations
The process of staining
1. At fixed smear poured one of basic stains for 2-3 minutes. To avoid precipitation paint through a filter paper. 2. Drained paint, carefully remove the filter paper. Smear pour for 1-2 minutes a solution of iodine or iodide solution Gram (aqueous solution of potassium iodide and iodine crystal in the ratio 2:1) for 1-2 minutes to blackening of the preparate. 3. The solution is drained, rinsed stroke 96 ° ethanol or acetone, pouring and merging it as a smear and not obezbarvytsya and flowing liquid becomes clear (about 20-40-60 seconds). 4. Thoroughly wash slides in running water or distillatory 1-2 minutes. 5. For detection of gram-negative bacteria preparations additionally paint fuchsin or safranin (2-5 minutes). 6. Wash in running water and dried filter paper.
Control questions - How large a resolution of optical microscopes and 2 types of electron microscopes (TEM/SEM) ?
- What factors can explain the resolution limits for different types of microscopes? - What kinds of optical microscopes do you know? - The structure of the living cell
Laboratory work № 1.5. Study of plant cells characteristics.
Purpose Study characteristics of plant cells.
Task Independently dissect and examine micropreparations. Theoretical information Preparation of material for painting The study material is distributed over the surface of a thin layer of good fat glass. Prepared smear dried in air and then dry completely fixed. Fixation When fixing the smear is fixed on the glass surface, and therefore the further stained microbial cells not washed. Furthermore, killed microbial cells stain better than alive. There are physical method of fixation, which is based on the action of high temperature on microbial cell, and chemical methods involving the use of chemical agents, daring coagulation proteins cytoplasm.
The order of performance and recommendations The physical method of fixation: Glass with preparate taking tweezers and smooth motion carried 2-3 times above the upper part of the flame. The entire process of fixing should take no more than 2 seconds. Reliability fixation check the following techniques: free from smear the glass surface is applied to the back surface of the left hand. When properly fixed smear glass should be hot, but not cause a feeling custody (70-80 ° C). Chemical method of fixing: For fixing smears used methanol, acetone, Nikiforov mixture (a mixture of ethanol 96% and ether anesthesia in the ratio 1:1), Karnua liquid (ethanol 96% - 60%, 30% chloroform, glacial acetic acid, 10 %). Glass with dried smear is immersed in a glass with a fixing agent for 10-15 minutes and then dried in air.
Control questions - Methods for fixation of cells and tissues? - What are the main fixative mixture. - How to dehydrate the material?
2. Module number 2 " CELL FUNCTIONAL SYSTEMS "
Laboratory work № 2.1.
Cell wall, membrane and bacterial spores. Photosynthetic prokaryotes. Plants Purpose Practical assimilation of theoretical ideas about the structure of the functional systems of living cells. Membrane, cell wall, transport of substances.
Task Microscopy of native and stained preparate. Theoretical information staining with hematoxylin and eosin kernel perceived mainly hematoxylin and stained in blue and purple and cytoplasm becomes pink, contacting its major radicals with acid stains - eosin. Widely used (especially for painting blood cells) mixture Azur and eosin. Azur as a basic stain, paints acidic components of cells, especially nucleic acids, acidic proteins and basophilic granularity of mast cells and basophils blood and eosin - cytoplasmic basic proteins and oxyphilic granular eosinophils. Basophilia - affinity cellular structure to the basic stain. So basophilia characteristic acidic components of the cell. Oksyfhliya - affinity with acid stain. Oxyphilic cells are alkaline components.
The method of scanning (scanning) electron microscopy allows us to study a three-dimensional picture of the cell surface. In scanning electron microscopy, thin electron beam (probe) runs along the surface of the object and the information is passed on cathode-ray tube. Images can be obtained in the reflected or secondary electrons. With this method, fixed and dried in a special way the object is covered with a thin layer of ablated metal (usually gold), reflected from which electrons enter the receiving device that transmits a signal to the cathode-ray tube. Because of the tremendous depth of focus scanning microscope, which is much more than the transmission, comes almost three-dimensional image of the investigated surface. Resolution of this type of instrument is slightly lower than in the transmission electron microscope, but now produced devices with a resolution of 3-5 nm Using scanning electron microscopy can provide information about the chemical composition in different parts of the cell. Thus, the method of X-ray microanalysis is based on the identification and quantitative estimation of chemical elements in the spectra of x-ray radiation produced by the interaction of primary electrons with the atoms of the object. To obtain such information, of course, objects should not be covered with a layer of metal, as in the ordinary method of scanning electron microscopy. Moreover, the object should be prepared so that there was no loss or addition of elements. For this purpose, quickly frozen and dried in a vacuum objects. Equipment, materials and methods. Microscope objective lenses, glasses with hole, glass rods, hinges, cover lenses. Lower plants - algae, moss, ferns, single-celled animals - ciliates, amoebas. The order of performance and recommendations - Obtain samples of living cells and tissues. - Prepare for microscopy native (unstained preparates) types "hanging drop" and "crushed drop." - Prepare smears and other preparations for painting. - Fix samples chemically and thermally - Conduct coloring technique. - These preparates microscopy. To fix the image on all available degrees of increase, from less to most. - Compare appearance of cells fixed and stained and native. - Set clear image of moving the condenser.
Control questions - What resolution with optical microscopes and 2 types (TEM / SEM) e? - What factors explain the resolution limits for different types of microscopes? - What kinds of optical microscopes, you know? - The structure of the living cell.
Laboratory work № 2.2.
Cytoplasmic membrane. Intercellular connection plants. Purpose Studies of lifetime and regular medication.
Task Document microscopic images comparable size. Theoretical information If you select any cell from an animal and put it in water, then a short time after cell swelling burst, Lisa. This is because through the plasma membrane water will flow into the cytoplasm, a zone with a high concentration of salts and organic molecules. This will increase the internal volume of the cell as long as the plasma membrane is maintained. In the animal organism that does not happen, because the cells of the lower and higher animals are surrounded by fluid internal environment, the concentration of salts and substances which is close to that in the cytoplasm. Free-living in fresh water unicellular protozoa not lysed (in the absence of cell wall), because they are constantly working cell pump, pump water from the cytoplasm - contractile vacuole. If we put water in the cells of bacteria or plants, they will not be lysed as long as the whole of their cell wall. The influence of a set of different enzymes, these walls can be dissolved. In this case, immediately going swell and rupture, lysis of cells. Thus, in vivo cell wall prevents this destructive process cells. Moreover, the presence of cell walls is one of the main factors that regulate the flow of water into the cell. The cells of bacteria and plants live mainly in hypotonic aqueous environment, they do not have contractile (excretory) vacuoles to pump out the water, but tough cell wall protects them from extreme swelling. As the flow of water in the cell there is internal pressure, turgor, which prevents further flow of water. Interestingly, in many lower plants, such as green algae cells have well established cell membrane, but sexual reproduction when formed motile zoospores, to lose cell membrane and they appear pulsating vacuoles. The cell wall of plants is formed with the plasma membrane and is ekstrakletochnoy (extracellular) multilayer formation, which protects the cell surface, officers like exoskeleton plant cell (Fig. 158). The cell wall of plants consists of two components: an amorphous plastic gel matrix (base) with a high water content and supporting fibrillar system. Often giving properties of hardness, etc. nesmachivaemost the shell includes additional polymeric substances and salt. Chemically, the main components of plant membranes related to structural polysaccharides. The structure matrix membranes of plants are heterogeneous group of polysaccharides dissolved in concentrated alkali, hemicellulose and pectin. Hemicellulose is a branched polymer chains consisting of different hexoses (glucose, mannose, galactose, etc.), pentoses (xylose, arabinose) and uronic acids (glucuronic and galacturonic acid). These components hemicelluloses are combined together in different quantitative relationships and form various combinations. Chains hemitsellyuloznyh molecules not crystallize and form elementary fibrils. Because of the presence of polar groups of uronic acids are highly hydrated. Pectic substances - heterogeneous group, which includes branched heavily hydrated polymers that carry negative charges through a lot of galacturonic acid residues. Due to the properties of its components matrix is a soft plastic mass, fortified with fibers Equipment, materials and methods. Microscope objective lenses, glasses with hole, glass rods, hinges, cover lenses. Single-celled animals - ciliates, amoebae, single-celled algae, yeast, blood cells of man and animals.
The order of performance and recommendations Preparation of blood smears and staining them with hematoxylin and eosin. A drop of fresh blood is applied to a very clean, skim slide near its edge and make a smear. For this to drop on a slide edge put another substantive (or the cover) glass with sanded face. The angle between the two panes should be about 30 °. When faced with a drop last glass spreads on the border of two glasses. The rapid movement of polished glass carried by the object glass and drop smeared flat strip. Smear slightly dried in air and fixed 10 -20 min in methanol. Then smear stained with hematoxylin first Böhmer or Karachchi, and 0.1% m aqueous eosin. Good results are obtained by staining of blood smears Azur II eosin. While painting Azur 11-eosin stained nuclei of cells in the blue-violet color, grain eosinophils - in red and orange, and basophils - in purple-cherry color. The cytoplasm of lymphocytes and monocytes - blue-blue, red - orange.
Control questions - Specify the size of the monitoring cell - Describe the process of making cuts on microtomes. - Tell us about the technique of placing the preparate under the cover glass. - What are the methods of staining sections and smears fixed tissues?
Purpose Studying the functions of ultrastructural components of cells. Task Studies constant micropreparations
Theoretical information For all vakuolyarnoyi system characterized cooperativity of its functioning, the relationship and sequence of stages of education, restructuring, transport and export of synthesized proteins. Briefly functions of individual components are as follows: 1. Granular endoplasmic reticulum: kotranslyatsionnyy synthesis of soluble proteins vnutrivakuolyarnyh (secretory proteins, lysosomal hydrolases, etc.) kotranslyatsionnyy synthesis of insoluble proteins that are part of all membranes vakuolyarnoyi system, the primary modification of soluble and insoluble (membrane) proteins and their connection with oligosaccharides - glycosylation synthesized proteins, glycoproteins education, synthesis of membrane lipids and their embedding in the membrane - "a collection of membranes". 2. Department vacuoles containing newly created products and their transition in cis-Golgi apparatus zone (ER-AG complex). 3. Cis-Golgi apparatus zone: secondary modification of glycoproteins, synthesis of polysaccharides (hemicellulose plants) and heksozaminohlikanov. 4. The intermediate region of Golgi apparatus: additional modification of glycoproteins, transhlikozilirovanie. 5. Trans-Golgi network: sorting of secretory and lysosomal proteins; department vacuoles. 6. Exocytosis (secretion). 7. Exocytosis constant. 8. Department of primary lysosomes with hydrolases. 9. Endocytosis. 10. Secondary lyzosoma. 11. Recyclisation receptors hydrolases. 12. Recyclisation plasma membrane receptors. 13. Smooth endoplasmic reticulum: synthesis and condensation lipid deposition ions Ca2 +, synthesis and glycogen resorbtsyya etc. 14. Transport in the area Golgi apparatus. 15. Transport from the Golgi apparatus to the endoplasmic reticulum.
Mitochondria as ATP synthesis organelles characteristic, with a few exceptions, all eukaryotic cells as autotrofnymi (photosynthetic plants) and heterotrophic (animals, fungi) organisms. Their main function is associated with the oxidation of organic compounds and the use released by the decay of these compounds in the synthesis of the energy molecule ATP. Because mitochondria are often called the power plants of the cell. General morphology of mitochondria or hondriosomy (from the Greek. Mitos-thread, chondrion-grain, soma-corpuscle) are granular or threadlike organelles present in the cytoplasm of protozoa, plants and animals. Mitochondria can be observed in living cells, as they have a very high density. In living cells, mitochondria can move, move, merge with each other. Especially good are the mitochondria in preparations stained in various ways after osmiyeva fixation, which is well stabilizes lipids. The most widely used method for coloring by Altman, who described these cell organelles at the end of the last century, calling them "bioblasts." Dimensions mitochondria are highly unstable in different species, as well as their shape variable Yet in most cells the thickness of these structures is relatively constant (about 0.5 microns), and the length varies, reaching into filamentous forms to 7-60 microns. I must say that the study of mitochondrial size - not a simple matter. In the light microscope on stained preparations is not always possible to trace the actual size of mitochondria (Fig. 200, two hundred and first). Studying mitochondria in the electron microscope on ultrathin sections, it is difficult to decide on the true length of the mitochondria, as the slice gets only a small amount of mitochondria. Moreover, cut one twisted mitochondrion can be presented in several sections (3-5), and only the spatial three-dimensional reconstruction, based on a study of serial sections, can decide whether we are dealing with 3-6 individual mitochondria or with a curved or branched. Dedicated mitochondria are usually damaged and fragmented, which also limits the use of this method to solve the question of size and number of mitochondria Equipment, materials and methods. Microscope objective lenses, glasses with hole, glass rods, hinges, cover lenses. pipette graduated to 2 ml, 5 ml, 10 ml, Goryaev camera , test tubes, Petri dishes, flasks of 100 - 200 ml Ciliates, amoebae, single-celled algae, blood cells of humans, animals, yeast.
Laboratory work № 2.4.
Ultrastructural organization of mitochondria, plastids. Purpose Studying the functions of ultrastructural components of cells Task Study of chloroplasts in cells Theoretical information
The main functional loads plastids and mitochondria are the processes of nature's energy, leading to the synthesis of specific molecules of adenosine triphosphate (ATP), a donor of energy to any cellular processes. In mitochondria, chloroplasts, as in bacteria, ATP is synthesized by the same method: using the energy given to electrons in moving them elektronnotransportnoy chain inner membrane proteins, there is a hyphen, "pumping" protons from the inside to the outer membrane. The resulting electrochemical proton gradient, whose energy through other proteins used for the synthesis of ATP. In the chloroplasts of plants, in addition, by using energy of ATP formed by phosphorylation is an essential biological process - binding CO2 and synthesis of carbohydrates.
Lysosomes as intracellular membrane particles were discovered biochemist (Where Dyuv, 1955). In the study of light podfraktsii macro from rat liver homohenatov found that this podfraktsiya (unlike the main fraction macro - mitochondrial fraction) is a group of acidic hydrolytic enzymes (hydrolases), break down proteins, nucleic acids, polysaccharides and lipids. Impression that these enzymes are contained in a special kind of cytoplasmic particles and lysosomes. It was found that the enzymes isolated lysosomes exhibit their activity only if previously caused damage themselves lysosomes, or the influence of osmotic shock or detergents, or freezing and melting agents. On this basis it was concluded that lysosomes are surrounded by a lipoprotein membrane that prevents access are outside of substrates to enzymes inside lysosomes. A characteristic feature of lysosomes is that they contain about 40 hydrolytic enzymes: proteases, nucleases, hlikozidazy, phosphorylase, phosphatase sulfitazy, optimum action which is carried out at pH 5. In acidic lysosomes value environment created by the presence in their membrane H + pump-dependent ATP. In addition, lysosomal membrane proteins embedded vectors for transport of lysosomes in hialoplazmu hydrolysis products: monomers split molecules - amino acids, sugars, nucleotides and lipids. In reading the work of lysosomes, the question always arises, why these membrane Education not digest themselves? Likely that lysosomal membrane elements are protected from the effects of acid hydrolases oligosaccharide areas that are either not recognizable lysosomal enzymes, or simply prevent hydrolases interact with them. Anyway lysosomal membrane components are very resistant to hydrolases contained within lysosomal vesicles. Having some hydrolases can be detected by histochemical methods. So one of the typical hydrolases that are both light and electronic microscope is acid phosphatase, the presence of which can be clearly identified, there is a particular vesicle membrane phospholipids. Under the electron microscope shows that the fraction of lysosomes consists of very colorful bubbles class size 0.2-0.4 mm (for liver cells), limited single membrane (thickness of about 7 nm), with a very diverse content inside (Fig. 187, 188). In fractions of lysosomes are vesicles with homogeneous, unstructured content found bubbles filled with a dense substance that has in turn vacuoles, membranes and dense clusters of homogeneous particles, can often be seen in lysosomal membranes not only land, but also fragments of mitochondria and ER. In other words, the fraction of morphology was extremely heterogeneous, despite the presence of hydrolases stability. Similar to the morphology of the particles were described previously in various tissues of many animals. However cytologist could not figure out the functional significance of these polymorphic particles. Only a combination of biochemical, cytochemical and electron microscopic research methods allowed sufficient detail to understand the structure, origin and functioning of cellular lysosomes Equipment, materials and methods. Microscope objective lenses, glasses with hole, glass rods, hinges, cover lenses. pipette graduated to 2 ml, 5 ml, 10 ml, Goryaev camera , test tubes, Petri dishes, flasks of 100 - 200 ml Mushrooms. Yeast, mold spores, unicellular animals, plants. The order of performance and recommendations - Obtain samples of living cells and tissues. - Prepare for microscopy native (unpainted preparates) types "hanging drop" and "crushed drop." - Prepare smears and other preparations for painting. - Fix samples chemically and thermally - Conduct coloring technique. - These preparates microscopy. To fix the image on all available degrees of increase, from less to most. - Compare appearance of cells fixed and painted and native. - Set clear image of moving the condenser.
Control questions - Sub-cellular elements. - Preparation of samples for microscopy in cell biology: basic approaches. - What microtome? - Stains for micropreparations, contrast, selectivity.
3. Модуль № 3 "Система зберігання, відтворення та реалізації генетичної інформації"