METHODS OF CULTIVATION OF MICROORGANISMS. STUDY OF CULTURAL AND BIOCHEMICAL
PROPERTIES
Cultivation, that is, the cultivation of microorganisms in the laboratory, is used to study their properties and to obtain biomass. Bacteria, fungi, actinomycetes, spirochetes and some protozoa are cultivated on nutrient media. Chlamydia, rickettsia, viruses and some protozoa can reproduce only in the body of an animal or in living cells.
The cultural properties of this type of microorganisms are: 1) the conditions necessary for reproduction, and 2) the nature of growth on nutrient media. Cultural properties are one of the characteristics that are taken into account when identifying (determining the type) of microorganisms.
Culture media
Culture media must meet certain requirements. They must contain all the nutrients necessary for the reproduction of this type of microbes. Some pathogenic microorganisms grow on simple nutrient media, while others need the addition of blood, blood serum, and vitamins for their reproduction.
In culture media, certain conditions must be created by adding sodium chloride or buffer solutions. For most bacteria, a nutrient medium containing 0.5% sodium chloride is favorable. The reaction of the nutrient medium, favorable for most pathogenic bacteria, is slightly alkaline, which corresponds to pH = 7.2-7.4. Vibrio cholerae grows at pH = 7.8-8.5, mushrooms - at pH = 5-5.5. The culture media should be moist, that is, contain a sufficient amount of water, be as transparent and sterile as possible, that is, before sowing, do not contain microbes.
In terms of composition and origin, nutrient media are natural, artificial and synthetic. Natural culture media are natural products such as potatoes and other vegetables. Artificial nutrient media are prepared according to a specific recipe from products with the addition of organic and inorganic compounds. Synthetic media contain certain chemical compounds in known concentrations.
By consistency, nutrient media are liquid, semi-liquid, dense. Agar-agar-polysaccharide isolated from seaweed is usually used as a sealant. Agar-agar is not used by microorganisms as a nutrient; it forms a gel in water that melts at 100 ° C and solidifies at 45 ° C.
To obtain a dense nutrient medium, agar-agar is added at a concentration of 1.5-2%, for semi-liquid - 0.5%.
According to their intended purpose, culture media can be divided into ordinary (simple), special, elective, differential diagnostic.
Conventional (simple) nutrient media are used for the cultivation of most microorganisms, this is mesopatamia broth (MPB), mesopatamia agar (MPA).
Special nutrient media are used for the cultivation of microorganisms that do not grow on simple media. For example, blood agar and sugar broth for streptococcus, serum agar for meningococcus and gonococcus.
Elective culture media are used to isolate one species from a mixture of different bacteria. This type of bacteria grows on this environment faster and better than others, outstripping them in its growth; the growth of other bacteria is delayed on this medium. For example, coagulated serum for diphtheria bacillus, alkaline peptone water for cholera vibrio, bile broth for typhoid bacillus, saline media for staphylococcus.
Differential diagnostic culture media are used to distinguish some types of bacteria from others by their enzymatic activity (see the corresponding section).
Cultivation and isolation of pure cultures of aerobic bacteria
For the cultivation of microorganisms, certain conditions are necessary: temperature, aerobic or anaerobic conditions.
The temperature should be optimal for the species. Most pathogenic bacteria thrive at 37 ° C. However, for some species, a lower temperature is optimal, which is associated with the peculiarities of their ecology. So, for the plague bacillus, the natural habitat of which are rodents during hibernation, the optimum temperature is 28 ° C, as for leptospira, for the botulism bacillus - 28 ° C-35 ° C.
In addition to the optimum temperature, for the cultivation of microorganisms, depending on the species, aerobic or anaerobic environment is required.
In order to study the morphology, cultural, biochemical and other properties of microbes, it is necessary to obtain a pure culture. Usually a culture of microbes is called their accumulation on a nutrient medium in the form of turbidity, near-bottom (wall) growth or a film on the surface of a liquid medium or colonies on a dense medium. A single colony is formed from one microbial cell. A pure culture is a culture of microbes of the same species obtained from a single colony. In laboratories, certain known strains of microbes are used for various studies. A strain is a pure culture of microbes obtained from a specific source, at a specific time, with known properties. Typically, microbial strains are designated by a specific number. For example, the Staphylococcus aureus 209P strain is used to determine the activity of penicillin.
Isolation of pure cultures of aerobes usually takes three days and is carried out according to the following scheme:
1st day - microscopy of a smear from the test material, stained (usually by Gram) - for a preliminary acquaintance with the microflora, which can be useful in choosing a culture medium for inoculation. Then inoculation of the material on the surface of the frozen nutrient agar to obtain isolated colonies. Sieving can be performed according to the Drygalsky method into three Petri dishes with a nutrient medium. A drop of material is applied to the first cup and spread over the entire cup with a spatula. Then, with the same spatula, distribute the remaining culture on it on the second cup and in the same way on the third. The largest number of colonies will grow on the first plate, the least on the third. Isolated colonies will grow on one of the plates, depending on how many microbial cells were in the test material.
The same result can be achieved by sieving on one cup. To do this, divide the cup into four sectors. The material under study is inoculated with a bacteriological loop with strokes on the first sector, then, after calcining and cooling the loop, the inoculation is distributed from the first sector to the second and in the same way sequentially into the third and fourth sectors. Isolated colonies are formed from individual microbial cells after daily incubation in a thermostat.
2nd day - study of colonies grown on plates, description of them. Colonies can be transparent, translucent or opaque, they have different sizes, rounded regular or irregular outlines, convex or flat shape, smooth or rough surface, smooth or wavy, jagged edges. They can be colorless or white, golden, red, yellow. Based on the study of these characteristics, the grown colonies are divided into groups. Then an isolated colony is selected from the study group, a smear is prepared for microscopic examination in order to check the homogeneity of microbes in the colony. The same colony is inoculated into a test tube with a slant nutrient agar.
3rd day - checking the purity of the culture grown on the agar slant by smear microscopy. With the homogeneity of the studied bacteria, the isolation of a pure culture can be considered complete.
To identify the isolated bacteria, cultural traits are studied, that is, the nature of growth on liquid and solid nutrient media. For example, streptococci on sugar broth form a bottom and parietal sediment, on blood agar - small, pinpoint colonies; cholera vibrio forms a film on the surface of alkaline peptone water, and transparent colonies on alkaline agar; the plague bacillus on nutritive agar forms colonies in the form of "lace handkerchiefs" with a dense center and thin wavy edges, and in a liquid nutrient medium - a film on the surface, and then filaments extending from it in the form of "stalactites".
Cultivation and isolation of pure cultures of anaerobic bacteria
For the cultivation of anaerobes, it is necessary to lower the oxidation-reduction potential of the medium, to create anaerobiosis by removing oxygen by physical, chemical or biological methods.
Physical methods include:
1) mechanical removal of air by means of a pump from anae-rostat, in which dishes with inoculations are placed. At the same time, you can replace the air with an indifferent gas: nitrogen, hydrogen, carbon dioxide.
2) growing in a medium containing reducing substances. Kitta-Tarozzi Wednesday is a sugar broth with pieces of liver or meat. Glucose and pieces of organs have a reducing ability. The medium is poured on top with a layer of vaseline oil to block the access of air oxygen.
3) The simplest, but less reliable method is to grow deep in a tall column of sugar agar.
Chemical methods consist in the fact that dishes with crops of anaerobes are placed in a hermetically sealed desiccator, where chemicals are placed, for example, pyrogallol and alkali, the reaction between which proceeds with the absorption of oxygen.
The biological method is based on the simultaneous cultivation of anaerobes and aerobes on solid nutrient media in Petri dishes, hermetically sealed after inoculation. First, oxygen is absorbed by the growing aerobes, and then the growth of anaerobes begins.
Isolation of a pure culture of anaerobes begins with the accumulation of anaerobic bacteria by inoculation on Kitta-Tarozzi medium. In the future, isolated colonies are obtained in one of two ways:
1) the material is inoculated by mixing with melted warm sugar agar in glass tubes. After the agar has solidified, isolated colonies grow in its depths, which are removed by cutting the tube and subcultured on Kitt-Tarozzi medium (Weinberg's method);
2) inoculation of the material is carried out on plates with a nutrient medium and incubated in an anaerostat. Isolated colonies grown on a plate are subcultured on Kitt-Tarozzi medium (Zeissler method).
Cultivation of other microorganisms
Cultivation of mycoplasmas
Mycoplasmas are cultured on nutrient media supplemented with serum and carbohydrates. Since mycoplasmas lack a cell wall, they grow only in isotonic or hypertonic environments. On solid nutrient media, for several days, very small colonies are formed, resembling fried eggs - with a convex center and a flat translucent periphery. Mycoplasmas can also be grown in chicken embryos or cell culture.
Cultivation of rickettsia and chlamydia
Rickettsiae and chlamydiae are obligate intracellular parasites. For their cultivation, cell cultures, chicken embryos and animal infection are used.
Mushroom cultivation
For the cultivation of mushrooms, dense and liquid nutrient media are used: most often Sabouraud's medium, as well as media containing beer wort. Mushrooms grow more slowly than bacteria, they form visible growth within a few days. The cultivation temperature is lower than that of bacteria - 22-30 ° C.
Cultivation of spirochetes and protozoa
Among spirochetes, it is most easy to grow leptospira, for which water mixed with rabbit blood serum can serve as a nutrient medium.Borrelia and treponema are cultivated under anaerobic conditions on more complex nutrient media containing serum, pieces of animal tissue.
Among the protozoa, dysentery amoeba, lamblia, Trichomonas, leishmania, trypanosome, balantidia are cultivated on nutrient media. Toxoplasma is cultivated in chicken embryos and tissue cultures. Cultivation methods for malaria plasmodia are under development.
Methods for studying enzymatic activity (biochemical properties)
In microbiological practice, the study of enzymatic activity is used to identify microorganisms, since each microbial species has a certain set of enzymes.
To determine the proteolytic activity, microbes are inoculated with an injection into a column of gelatin, and after 3-5 days of incubation at room temperature, the character of gelatin liquefaction is noted: in the form of a funnel, a nail, a stocking or in the form of an overturned Christmas tree. Proteolytic activity is also determined by the formation of protein decomposition products: indole, hydrogen sulfide, ammonia. To determine them, microorganisms are inoculated into meat-peptone broth, and indicator papers are placed between the neck of the test tube and a cotton stopper, excluding their contact with the medium. When indole is formed, paper impregnated with a saturated solution of oxalic acid turns pink; in the presence of hydrogen sulfide, paper impregnated with lead acetate turns black; when ammonia is formed, the red litmus paper turns blue.
To determine the saccharolytic properties of microbes, differential diagnostic media are used, such as Giss media, Olkenitsky's medium, Endo's medium, Levin's medium, Ploskirev's medium.
Media Endo, Levin, Ploskirev in Petri dishes are used to differentiate bacteria of the intestinal group by their ability to ferment lactose. These media contain nutrient agar, lactose and an indicator that changes color in an acidic medium - a pH indicator. If you sow bacteria that ferment lactose, such as E. coli, on such an environment, acid is formed as a result of fermentation of lactose, and the indicator will change color in an acidic environment. Therefore, colonies of Escherichia coli on such media will be colored according to the color of the indicator: on Endo's and Ploskirev's medium - in red, on Levin's medium - in black and blue. Colonies of bacteria that do not ferment lactose, such as salmonella and dysentery sticks, will be colorless.
Giss's media (“variegated range” media) are prepared on the basis of peptone water or semi-liquid meat-peptone agar. Contain any one carbohydrate or polyhydric alcohol and an indicator. When a microbe grows on Giss's medium, fermenting this substrate with the formation of acid and gas, the medium will change color, in a semi-liquid medium, bubbles and ruptures will appear in the thickness of the agar, in a liquid medium - a gas bubble in a glass float. When the substrate is fermented only to acid, only a change in the color of the medium occurs.
Combined media containing not one carbohydrate, but two or three are also used, for example, Olkenitsky's medium. One tube of this medium replaces agar slant and Giss media with lactose, glucose and sucrose. After sterilization in the molten state, the medium in the test tube is beveled so that a column and a beveled part are obtained. Sowing is done by a stroke on the beveled part and a prick in a column. When lactose or sucrose is fermented, the color of the entire medium changes; when one glucose is fermented, only the color of the column changes. Gas formation is indicated by the presence of bubbles in the agar column. When microbes release ammonia, the color of the medium does not change. The formation of hydrogen sulfide is manifested by blackening in the agar table
For an express method for determining the enzymatic activity of bacteria, microtest systems and an indicator paper system (NIB) are used
The microtest system is a container made of transparent polystyrene, consisting of several cells. The cells contain dried nutrient media with carbohydrates and pH indicators. A suspension of a culture of bacteria of a certain density is inoculated into each cell. A saline solution is poured into the control cells. colors
indicator
Indicator paper systems (NIB) for the identification of the enterobacteriaceae family are disks or strips of chromatographic paper, covered with a protective film and containing a specific substrate and an indicator. by changing the color of the indicator To determine the hydrogen sulfide, the disc is placed on the surface of the MPA, seeded with an injection, which allows you to simultaneously determine the mobility
In all test tubes, the preliminary result on the same day and the final result on the next day are taken into account.
Oxidase activity is determined by grinding the culture on an indicator paper. The result is taken into account after a minute.
INSULATED TISSUE AND PLANT CELLS
Cultivation of isolated cells and tissues on artificial nutrient media under sterile conditions (in vitro) received the name of the method of culture of isolated tissues.
Due to the fact that seed plants are of the greatest importance in human life, methods and conditions for their cultivation have been developed better than for gymnosperms or algae, the cultivation of which in sterile conditions causes certain difficulties. However, regardless of the belonging of plants to a particular taxonomic group, there are general requirements for the cultivation of objects in culture. in vitro.
Asepsis. First of all, the cultivation of fragments of tissue or plant organ - explants, and even more so of individual cells, requires complete asepsis. Microorganisms that can enter the nutrient medium release toxins that inhibit cell growth and lead to the death of the culture. Therefore, for all manipulations with cells and tissues during cultivationin vitro observe certain rules of asepsis in a laminar box or in aseptic rooms. In the first case, asepsis is achieved by supplying filtered sterile air directed from the laminar box outside to the worker. Aseptic rooms are sterilized using ultraviolet lamps, and they work in such rooms in sterile clothing. The working surface of tables in aseptic rooms and tools are additionally sterilized with alcohol before work.
Clean dishes previously wrapped in paper or foil, tools, paper, cotton wool are sterilized by dry heat in an oven at 160 ° C for 1.5-2 hours. Culture media are sterilized in an autoclave at 120 ° C and high pressure in within 15 - 20 minutes. If the culture media contain substances that decompose during autoclaving, they should be sterilized by filtration through a bacterial filter. Then sterile filtered components are added to a pre-autoclaved medium cooled to a temperature of 40 ° C.
Plant tissues themselves can serve as a serious source of infection, since epiphytic microflora is always located on their surface. Therefore, surface sterilization is required, which is carried out as follows. Previously, the part of the plant from which the explant will be extracted is washed with soap and water and rinsed with clean water. Then the plant material is sterilized in solutions of disinfectants. Some of these substances, as well as the sterilization time are presented in table. 6.1.
|
Table 6.1 Sterilization of the original plant material (according to R.G.Butenko, 1999) An object |
Sterilization time, min |
||
|
diacid 0.1% |
mercuric chloride 0.1% |
hydrogen peroxide, 10-12% |
|
|
Seeds are dry |
15-20 |
10-15 |
12-15 |
|
Seeds are swollen |
6-10 |
6-8 |
6-8 |
|
Stem tissue |
20-40 |
20-25 |
— |
|
Leaves |
1-3 |
0,5-3 |
‘ 3-5 |
|
Apexes |
1-10 |
0,5-7 |
2-7 |
After keeping the explants in a disinfectant solution, they are washed several times in distilled water and the outer layer of cells on the slices of the explants is removed with a scalpel, since it can be damaged during sterilization.
Microorganisms can also be found inside plant tissue. Internal infection is most common in tropical and subtropical plants. Therefore, in addition to surface sterilization, sometimes it is necessary to use antibiotics, which kill the microbial flora inside the tissue. However, it should be noted that such treatment does not always lead to sterilization of internal tissues, since it is difficult to choose a targeted antibiotic.
Culture media. Isolated cells and tissues are cultured on multicomponent nutrient media. They can differ significantly in their composition, however, the composition of all media necessarily includes macro- and microelements necessary for plants, carbohydrates, vitamins, phytohormones and their synthetic analogues. Carbohydrates (usually sucrose or glucose) are included in any nutritional formula at a concentration of 2-3%. They are necessary as a nutritional component, since most callus tissues lack chlorophyll and are not capable of autotrophic nutrition. Therefore, they are grown in diffused lighting conditions or in the dark. The exception is the callus tissue of mandrake, amaranth and some other plants.
The indispensable components of culture media should be ^ auxins, which cause dedifferentiation of the explant cells, and cytokinins, which induce cell division. When the ratio between these phytohormones changes or when other phytohormones are added, different types of morphogenesis can be caused.
The high content of nitrates, ammonium ions, potassium, phosphate promotes rapid cell growth. Depletion of the environment significantly reduces the growth and processes of secondary metabolism. However, the initially low content of phosphates in the nutrient medium can stimulate the synthesis of secondary metabolites. It was found that the cultivation of licorice calli on a medium with half the concentration of nitrogen and phosphorus in the dark increases the content of phenolic compounds by 1.6 times compared with calli growing on a complete medium. Endosperm of immature embryos (coconut, horse chestnut, etc.), sap of some trees, various extracts (malt, yeast, tomato juice) can be added to the medium. Introducing them to the environment. do gives interesting results, but such experiments are difficult to reproduce, since the active ingredient is usually not known exactly. For example, adding separate fractions of coconut milk to the nutrient medium did not give any results, while unfractionated endosperm caused cell division.
When preparing solid nutrient media for the surface cultivation of callus tissues, purified agar-agar, a polysaccharide obtained from seaweed, is used. As examples in table. 6.2 shows the compositions of the most common culture media.
The environment of Murashige and Skoog is the most versatile. It is suitable for the formation of calli, the maintenance of disorganized callus growth, and the induction of morphogenesis in most dicotyledonous plants. Thus, a change in the ratio of auxin and kinetin leads to the formation of either roots (predominance of auxin), or; stem crops (predominance of kinetin).
Medium Gamborg and Eveleg is well suited for the cultivation of cells and tissues of legumes and cereals, White's medium provides * rooting of shoots and normal growth of the stem after regeneration, and Nitsch and Nitsch medium is suitable for the induction of andro-igenesis in anther culture.
Physical factors. For the growth and development of plant tissues; in vitro physical factors - light, have a great influence; temperature, aeration, humidity. |
Light. Most callus tissues can grow in low light or dark conditions, since they are not capable of photography *; synthesize.At the same time, light can act as a factor providing morphogenesis and activating processes for the second time.
|
!Composition of nutrient media used in the cultivation of cells and tissues (after R. G. Butenko, 1999) |
Concentration of culture media, mg / l |
|||
|
Media component |
Murashige and |
Gamborg and |
White, |
Nich and Nich, |
|
Skoog, 1962 |
Evelega, 1968 |
1939 |
1974-1975 |
|
|
KN03 |
1900 |
3000 |
81 |
950 |
|
NH4NO3 |
1650 |
— |
— |
720 |
|
Ca (N03)2 |
— |
— |
142 |
— |
|
Ca (N03)2-4H20 |
— |
— |
— |
— |
|
(NH4)2S04 |
— |
134 |
— |
— |
|
MgS04-7H20 |
370 |
500 |
74 |
185 |
|
CaCl2H20 |
— |
— |
166 |
|
|
CaClr2H20 |
440 |
150 |
— |
— |
|
KC1 |
— |
65 |
— |
|
|
KH2P04 |
170 |
— |
12 |
68 |
|
NaH2P04H20 |
— |
150 |
— |
— |
|
MnS04H20 |
— |
10 |
— |
— |
|
MnS0 „-4H20 |
22,3 |
— |
— |
25 |
|
ZnS04-4H20 |
8,6 |
— |
— |
— |
|
ZnS04-7H20 |
— |
2 |
— |
10 |
|
H3B04 |
6,2 |
3 |
— |
10 |
|
CuS04-5H20 |
0,025 |
0,075 |
— |
0,025 |
|
Na2Mo04-2H20 |
0,25 |
0,25 |
— |
0,25 |
|
CoCl2-6H20 |
0,025 |
— |
— |
— |
|
FeS04-7H20 |
27,8 |
— |
— |
27,8 |
|
Na EDTA-2H20 |
37,3 |
— |
— |
37,3 |
|
Sequestrene 330-Fe |
28 |
— |
— |
|
|
Mesoinositis |
100 |
— |
— |
200 |
|
Ascorbic acid |
— |
— |
— |
3 |
|
Thiamine-HCl |
0,5 |
— |
— |
3 |
|
Pyridoxine-HCl |
0,5 |
— |
— |
1 |
|
A nicotinic acid |
0,5 |
— |
— |
— |
|
Sucrose |
30 000 |
20000 |
2000 |
60 000 |
|
Agar "Difco", gel |
— |
— |
— |
7000 |
|
rith, agarose |
||||
th synthesis. Fluorescent lamps are used as a light source. For most herbaceous plants, the optimum illumination is around 1000 lux. Too low (300 lux) or high (3000-10,000 lux) illumination inhibits growth. Lighting can affect the metabolism of callus cells. Thus, in tea plant cultures, the biosynthesis of polyphenols increased under the influence of light. On the contrary, in cell culture Scopolia parvi- flora light suppressed the formation of alkaloids. In addition to the intensity of illumination, the quality of light affects the tissue culture and its physiological characteristics. Thus, more than 20 flavones and flavonol glycosides are formed in parsley cell cultures after illumination of it with continuous luminescent light "cold white". At the same time, the synthesis of flavone glycosides is activated by successive irradiation with ultraviolet light, and then by light lying in the region of "red-long-wave red"
Temperature. For most callus cultures, the optimum temperature is 26 ° C. At the same time, calli and cell cultures of diosorea deltoid grow well even at a temperature of 32 ° C. IN Unlike the growth of cell and tissue cultures, the induction of their morphogenesis requires lower temperatures (18 - 20 ° C). Effect of temperature on cell metabolism in vitro poorly studied. There is evidence that in callus cultures the maximum formation of alkaloids was observed at a temperature of 25 ° C, and with an increase in temperature, it sharply decreased. In suspension cell cultures Ipomoea the content of fatty acids increased significantly if they were grown at suboptimal growth temperatures (15 ° C) Therefore, when growing a culture in vitro it is necessary to carefully study the influence of all abiotic factors, including temperature, on the growth and metabolism of cells.
Aeration. For the cultivation of suspension crops, aeration is of great importance. It is especially important to supply the cultured cells with air in large volumes of fermenters.
When comparing different types of fermenters, it was shown that the synthesis of secondary metabolites in suspension culture was greatest when air was supplied from below. When cells are grown in small volumes (in flasks), normal aeration is achieved with constant stirring of the suspension.
Humidity. The optimum humidity in the room where I grow! Crops should be 60 - 70%.
Thus, the cultivation of cells and tissues depends on many factors of the external environment, and their action is not always good? stno. Therefore, when introducing a new plant species into culture, it is necessaryT First of all, it is necessary to carefully study the influence of physical factors on the growth and physiological characteristics of this culture.
The cultivation of isolated cells and tissues on artificial nutrient media under sterile conditions (in vitro) is called the method of culture of isolated tissues.
Due to the fact that seed plants are of the greatest importance in human life, methods and conditions for their cultivation are better developed than for gymnosperms or algae, the cultivation of which under sterile conditions causes certain difficulties. However, regardless of the belonging of plants to a particular taxonomic group, there are general requirements for the cultivation of objects in culture in vitro.
Asepsis. First of all, the cultivation of fragments of tissue or organ of a plant - explants, and even more so for individual cells, requires complete asepsis.Microorganisms that can enter the nutrient medium release toxins that inhibit cell growth and lead the culture to death, therefore, during all manipulations with cells and tissues during in vitro cultivation, certain aseptic rules are observed.
Culture media. Isolated cells and tissues are cultured on multicomponent nutrient media. They can differ significantly in their composition, however, the composition of all media necessarily includes macro- and microelements necessary for plants, carbohydrates, vitamins, phytohormones and their synthetic analogues. Carbohydrates (usually sucrose or glucose) are included in any nutrient mixture at a concentration of 2-3%. They are necessary as a nutritional component, since most callus tissues lack chlorophyll and are not capable of autotrophic nutrition. Therefore, they are grown in diffused lighting conditions or in the dark. Mandatory components of culture media should be auxins that cause dedifferentiation explant cells, and cytokinins that induce cell division. When the ratio between these phytohormones changes or when other phytohormones are added, different types of morphogenesis.
Physical factors. The growth and development of plant tissues in vitro is greatly influenced by physical factors - light, temperature, aeration, humidity. Most callus tissues can grow in low light or dark conditions because they are unable to photosynthesize. At the same time, light can act as a factor providing morphogenesis and activating the processes of secondary synthesis. For most callus cultures, the optimum temperature is 26 ° C. For the cultivation of suspension crops, aeration is of great importance. It is especially important to supply the cultured cells with air in large volumes of fermenters. The optimum humidity in the room where crops grow should be 60-70%. Thus, the cultivation of cells and tissues depends on many environmental factors, and their effect is not always well known. Therefore, when introducing a new plant species into a culture, it is necessary, first of all, to carefully study the influence of physical factors on the growth and physiological characteristics of this culture.
Date Added: 2016-06-02; views: 493;
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Growing process cells and fabrics on artificial nutrient media under sterile conditions (in vitro) is called a method of obtaining a culture of isolated tissues. Isolated tissue culture is usually callus and much less often tumor tissues (fig. 3.1. and 3.2).
Cultures of isolated cells and tissues.
Using a culture of isolated cells and tissues.
Isolated tissue cultures are widely used in agriculture and industrial production. They possess advantages compared to traditional plant materials (wild and plantation-grown plants), namely:
1. Independence from external conditions.
2. Optimization of growing conditions.
3. Automation and computerization of processes.
4. The ability to grow endangered plant species.
5. Possibility of mass clonal micropropagation of fruit and vegetable and ornamental plants.
6. Recovery from viral and other infections.
7. Opportunity through culture in vitro, expanding the areas of breeding work, that is, to get clones cells, then plants with programmed properties.
Due to the ability of cells to synthesize in culture secondary metabolites, a new branch of industry arose, carrying out the biological synthesis of substances necessary for man.
Conditions for the cultivation of tissues and cells.
Cultivation of fragments of tissue or one plant - explants, and even more so for individual cells, requires complete asepsis.
Microorganisms that can enter the culture medium secrete toxinsinhibiting the growth of cells and leading the culture to death. Therefore, all manipulations with cells and tissues during cultivation in vitro carried out in compliance with certain rules of asepsis in laminar box or in aseptic rooms. Asepsis is achieved by supplying sterile air passed through the filters, directed from the laminar box to the outside. On the surface of plant tissues there is always epiphytic microflora. Therefore, surface sterilization is necessary, which is achieved by treating areas of plants from which they will isolate explants, disinfectants (hydrogen peroxide, mercuric chloride).
To work with biological material, the following components are required:
- clean dishes and bioreactor (fermenter);
- sterile nutrient medium;
- optimal lighting;
- optimal temperature;
- optimal humidity;
- aeration.
Clean dishes and fermenter. Instruments and cotton wool previously wrapped in paper or foil are sterilized by dry heat in drying cabinet at a temperature of 160 ° C for 1.5-2 hours.
When using different types of fermenters, it was found that the physiological properties of cultures can change; therefore, the synthesis of secondary metabolites in a suspension culture should be carried out with air supply from below. Under these conditions, the synthesis proceeds much more actively (Fig. 3.3).
When growing cells in small volumes, for example, in flasks, normal aeration is achieved with constant stirring of the suspension.
Culture media... Cultivation of isolated cells and tissues takes place in multicomponent nutrient media... They can differ significantly in their composition, however, the composition of all media necessarily includes a component the foundation, to which the groups of substances necessary for plants are added: macro- and microelements, carbohydrates, vitamins and phytohormones. Such a component base is agar-agar - a polysaccharide of seaweed, capable of forming gels in water that solidify at 45 ° C. Can be added to the culture medium endosperm immature embryos (coconut, horse chestnut), syrup some trees, different extracts (malt, yeast), tomato juice. Their introduction into the environment gives a positive result. However, each active component has technological characteristics inherent only to it.
Rice. 3.3. Bioreactor (fermenter) for growing microorganisms, cells or tissues
Dedifferentiation is the basis of the callus formation process.
Dedifferentiation is the return of specialized cells to the meristematic state when they gain the ability to divide.
Organs and tissues of plants serve as the starting material for obtaining cultures of cells and tissues; however, plant leaves are more often used for this.
The previously worked sterile leaf fragment is placed on a solid nutrient medium. Leaves consist of different tissues, the cells of which are differentiated and synthesize various molecules of proteins, carbohydrates, lipids, and other substances characteristic of these cells. These differentiated cells do not have the ability to divide.
In the process of mechanical damage to the sheet the explant is released, and under the influence of plant hormones added to the environment, auxins and cytokinins the cell is dedifferentiated. It loses storage substances - starch, proteins, lipids, specialized organelles, chloroplasts, etc. As a result, all cells acquire new close morphological and physiological properties, i.e. become callus (dividing) cells. Callus tissue is formed on the leaf explant.
Types of cell cultures. Depending on the method and conditions of cultivation, there are several types of cell and tissue cultures:
- callus culture;
- suspension culture;
- single cell culture.
If cultivation happens superficially on the agar nutrient medium, then callus tissue is formed. It does not have a clear structure, but it can vary in density. The origin and growing conditions determine whether the callus tissue is loose, medium density, or dense.
General characteristics of callus cells. The callus cell has a number of properties in common with all plant cells... This is the ontogeny of cells, resistance to certain unfavorable environmental factors and other properties. At the same time, during their cultivation, callus cells develop their own individual characteristics, such as physiological asynchrony, genetic heterogeneity, and some others.
Physiological asynchrony lies in the fact that at each moment of time the cells are in different phases of growth: some are dividing, others are growing, and still others are already aging. Therefore, the general physiological aging of the entire population of callus cells is usually assessed by the state of most cells (Fig. 3.4).
Rice. 3.4. Callus cells inRice. 3.5. Protoplast exit through
Date Added: 2016-05-28; views: 1915;
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