Elektivnі zhivilnі middle butt. Live in the middle. Czapek-Dox sugar agar
Elective media were introduced into microbiological practice by S.M. Vinogradsky and M. Beyerink. Therefore, life-giving mediums, in some ways adding one or more chemical fields, create optimal minds for the growth and reproduction of one type of microorganism (or a group of sporadne microorganism) and unacceptable - for the solution. Such media are the main rank of seeing a pure culture of microorganism from the area of their natural habitation and accumulation of a mass of cultures (the chemical method of seeing a pure culture). For example, a life-giving medium, as it is a burnt kіnsk sirovatka, an elective medium for diphtheria bacteria, peptone puddle water - for cholera vibrio, beetroot broth - for typhoid fever, liver broth - for brucella, etc.
Accumulation of microbes in selective living mediums in bugs to serve as an important first step when seeing pure cultures from other secondary materials (for example, cholera vibrio or typhoid bacteria from succumbing to ailments in general).
What do you understand about the difirincially life-giving medium?
Differentially - diagnostic media - these are the same media, up to the warehouse of some kind of cream of speeches, which ensure the growth and development of microorganisms, enter the speech, which zastosovyvaetsya as a substrate for singing enzymes. Behind the acid snake substrate, the presence of that other enzyme is indicated (it is assessed for the help of an indicator that reacts to the presence of a life-giving medium in the breakdown of the substrate).
The skin type of microorganism is characterized by a stable set of enzymes. Designation of a set of enzymes with additional differentially diagnostic media allows differentiation of microorganism species. For example, blood agar allows the enzyme hemolysin to be detected, the middle Hisa - tsukrolytic enzymes (carbohydrase), gelatin vicory to overcome the proteolytic powers of microbes too.
Blood agar. The presence of the enzyme hemolysin is judged by the destruction of erythrocytes and the established light zone around the microbes that grew on blood agar.
Middle His. About the presence of enzymes - carbohydrase, which decompose into carbohydrates to acidity, change the pH of the medium to the acid side and change the color of the vital medium. Vicoristan can be used in a set of enzymes for rechecking the purity of a culture, which is seen, as well as for the rapid differentiation of one species from others in the first case of inoculation of an infectious material.
Chemical reagents
1. What kind of chemical reactive is that for which the stench zastosovuetsya?
Reactive chemicals- Speech, which are victorious in laboratory practice for the development of various chemical reactions.
Most of the chemical reagents are individual speeches, prote often stink in warehouses. There is no globally accepted classification of chemical reagents, most often they are divided into analytical chemical reagents otherwise.
2. At the veterinarian's, with what method does the stink of vicory stink?
In veterinary chemistry, reagents are vicorated for analytical and diagnostic purposes in clinical, veterinary and sanitary, hygienic, expert, biochemical and other laboratory studies. Methods of research, which are developed and developed by biological and clinical practice, require a great assortment of chemical reagents, which are to the satisfaction of the most demanding scientists. For example, for clinical and biochemical studies, highly purified substrates for enzymes, enzymes themselves, reagents for specific groups (SH, NH3, COOH - groups and іn) are thin. For carrying out inorganic and organic syntheses, as well as for acidic and acidic analyses, incl. during veterinary and sanitary control in various tests, analyses) of medical tests, during veterinary, sanitary and hygienic analyzes of grubs, food, water, etc. vikoristovuyut a great number of the most versatile chemical reagents of a high degree of purification.
Zukrovy broth and tsukrovy agar cook in a way to add to the perfect broth or melted simple agar of the same amount of carbohydrate in the amount of 0.5 to 1%. The carbohydrate is dispersed in a small amount of water, and then added to a life-giving medium. Sterilization of the chicken broth and agar is vibrated in the Koch apparatus for 3 days after 1 year.
Agar with blood, grayish or with ascitic homeland prepare with dotrimann suvoroy asepsis. To the ready slightly puddle melted and chilled to 45° agar, poured into test tubes or bottles, add 20-25% sterile syrup or ascitic rіdini or 5-25% sterile defibrinated blood. After a repetitive mixing (to uniquely dissolve pukhiriv and stumps), the agar is cut off in test tubes, and instead of bottles it is poured into Heidenreich cups. Broth from grayish, ascitic homeland or blood cook in this very way, like agar, but without heating. To control the sterility of the broth, set the thermostat for 48 years at 37°.
Differential-diagnostic life-giving medium. The warehouse is recognized.
Middle Endo. Get ready without a hitch before living. Up to 100 ml of meat-peptone agar or agar on Hottinger broth (pH 7.6) sterile add 1 g of chemically pure lactose, dissolved in 5 ml of sterile water, cool to 70 ° and add the sum of 0.5 ml of the main 1 25 ml of freshly prepared 10% sodium hydroxide solution, mix and pour into cups. For podsushuvannya vіdkritі cups placed on 30 minutes of thermostat at 37 °.
On the middle of the Endo intestinal stick gives a colony of red color with a metal tone, and bacteria of the typhoid-paratyphoid and dysenteric group give colonies without a bark.
Structural row of carbohydrates (Hissa's middle). Up to 100 ml of water, add 1 g of peptone, 0.5 g of kitchen salt. Peptone and strength are dispersed in hot water with a thin layer of thinner and filtered through a paper filter (we can see the difference more clearly). Restore pH 7.0. At the designated medium, 1% of one of the stagnant carbohydrates is added, floats are lowered to the bottom of the test tubes to trap the gas (which indicates about the deep splitting of the tsukrіv) and an indicator is added. As an indicator of vikoristovuyut:
a) litmus tincture - 5 ml per 100 ml of the medium. In sour medium, litmus tincture shows redness, in puddle - blue;
b) azolithmin, which can be purified with litmus, and potassium salt (to add 0.25 g of azolithmin to 1 liter of the medium);
c) bromothymol blue - per 1 liter of the medium 1 ml of 1.6% alcoholic indicator (the middle becomes green in color, blue with a settled meadow, yellow with a settled acid);
d) Andrede's indicator, which consists of 1 g of acid fuchsin, 400 ml of distilled water and 64 ml of normal sodium hydroxide. Add 1% of the indicator to the revitalizing medium. The middle part of the sample may have a strawy yellow color without an erysipelatous color. In a sour medium, the color of the medium changes to red.
The indicator should be given in order to determine the change in the reaction of the revitalizing medium, which is observed under the influx of fermentation into carbohydrates, i.e., the manifestation of the biochemical activity of microbes. The rest of May is of great importance for microbiological diagnostics of low infectious diseases (typhoid fever, dysentery, cholera and other). The medium with carbohydrates and an indicator is poured into sterile test tubes and sterilized with a shot at 100 ° for 3 days.
Short row. Changes in the growth of E. coli, S. typhi.
Methods of visualization of pure cultures of aerobics.
Drigalsky method
View of pure culture of anaerobes.
Div praknaviki
Virological methods. Appointment, principle.
Virological method
Main stages:
1. Collection of the finished material.
2. Select for the principle of cytotropism and the selection of a sensitive test system, designed for life.
3. Infection of the selected system.
4. Indication of the virus on the basis of the detection of iogo nucleic acid, antigens, hemagglutinin, CPD, inclusive.
5. Identification and titration of the virus to be carried out on a base:
a) designation of antigens in the virus for additional immunological reactions (RIF, IFA, RPHA, RSK, RN, WIEF and other); b) pathological findings of organs and tissues; c) CPD; d) clinical symptoms, biological tests (keratoconjunctival and other).
Virus detection methods
In case of virus-infected clitin cultures, it is possible to eliminate different visible manifestations of the virus:
1. Cytopathic to the virus on the culture of clitin (CPD)– viniknennya in her visible morphological degenerative changes;
2. The arrival of a bathhouse infected with a culture of clitin haemadsorption- to the adsorption of erythrocytes on the surface of the clitin ball;
3. Illumination in infected clitin culture under a special agar-covered ball of characteristic plaques, which are “negative colonies” of viruses;
4. Internal inclusions
Sobi for the cultivation of anaerobic microbes.M'yasopeptonny broth liver Kita - Taroztsі (MPPB). Fresh or frozen liver (rather than great horned thinness) is cut into small pieces, poured with equal mass of water supply water, boiled for a long time, filtered through cotton wool and add up to 1 part of the removed extract to 3 parts of m'yasopeptone broth. Sumish is heated to a boil, add chemically clean kitchen water (per 1 liter 1.25 g) and bring the pH to 7.6-7.8, after which boil for 15 minutes and filter through paper or cotton wool. To the filtered broth, add finely sliced \u200b\u200bsmachki (1.5-2 g each) of the liver, 100 g of the liver for 1 liter of broth (the liver is cleaned in front of the plіvok and washed with water). Place a sprat of such shmatochkiv near the test tube, pour 7-10 ml of broth with a high glass, put vaseline or paraffin oil on the surface of it.
The broth with liver cloves is sterilized under an overworld vice of 0.1 MPa in 30 min. For removal from the test tubes, before sowing, boil the middle of the day for 10 minutes and cool it down with water.
Nap_vridky agar for anaerobes. Add 0.25-0.75% agar-agar and 1% glucose to the MPB; medium pH 7.4. The middle is poured with high stacks at the test tube. Sterilize with shotgun pair for 15-20 mins for 3 days.
Sobi for the cultivation of lactic acid bacteria.Milk (cilisne). Heat up to a boil. Pour a flask at the tube and set it for 10-20 years in a cold place for the tops. After that, through the tap of the tube, pour the low-fat part of the milk into test tubes, close with cotton plugs. Sterilize the shot at 100 ° C for three days for 20 minutes each, or at 112 ° C once for 30 minutes.
skimmed milk. For the extraction of skimmed milk, non-selected milk is separated and removed in the same way, as in the case of non-selected non-selected milk.
Hydrolyzed milk (for Bogdanov). Take 1 liter of boiled і cooled to 45 ° C skimmed milk, restore pH 7.6-7.8, add 0.5 g of pancreatin powder (dissolved in front in a small amount of warm water) or 2-3 g of podshlunkovoy slough and 5 ml of chloroform through sprat. After this salt, it is strongly shaken, strongly closed with a cork stopper and placed for 3 doses in a thermostat at a temperature of 40 ° C with a slight strushuvanni r_dini. After the end of the designated term, with the method of removal, sulphate chloroform is removed, the homeland is filtered and diluted 2-3 times with tap water. Bring the pH of the medium to 7.0-7.2 and sterilize.
Agar from hydrolyzed milk. Add 1.5-2% agar to hydrolyzed milk, melt it, pour it into test tubes and sterilize it under an overpressure of 0.1 MPa in draw 15 min. On this medium, lactic acid sticks grow well.
Sirovatkovy agar. For 100 ml of tap water, take 7.5 g of agar, boil until full, add water to the volume of the cob (that is, in volume, equal to the volume of water that was boiled), add 400 ml of the distance of the prepared milk serum , give a fluent bet in the 30s, filter through a ball of cotton, pour after test tubes і sterilize under a vice of 0.05 MPa with a stretch of 30 min.
Cabbage middle. 200 g of chopped cabbage (or alfalfa) is poured into 100 ml of water and boiled in a saucepan for 10 minutes, squeezed through a ball of gauze. Otriman native is filtered and diluted 2 times with tap water. Add 2% glucosit and 1% peptone, pour into test tubes and sterilize with three overworld vise 0.05 MPa for 15 minutes.
Medium for growing osmophilic yeasts. Approximately 1 liter of distilled water, add 200 g of honey, 1 g of potassium diphosphate, 0.5 g of magnesium sulfate, 0.5 g of ammonium tartrate, 0.1 g of sodium chloride and 0.1 g of potassium chloride. Mustache components change and sterilize under an oversized vice of 0.1 MPa with a pull of 20 minutes.
Medium for growing halophiles. Vykoryvouyut zvichayny m'yasopeptoni with additions of 10-15 to 20-30% kitchen salt. In addition, with the preparation of lively fatty media, a percentage of the agar is added. Sterilization is carried out under an oversized vice of 0.1 MPa with a pull of 20 minutes.
The middle of the enrichment. Muller's middle. Up to 4.5 g of chemically pure creedy, sterilized in front with dry heat, add 90 ml of MPB and sterilize under an excess vice of 0.1 MPa with a pull of 20 minutes. Prepare: a) dispensing hyposulfite (50 g of pure crystalline hyposulfite is poured up to 100 ml with distilled water, sterilized with a plain steam for 30 minutes); b) distillation of iodine (20 g of metal iodine and 25 g of potassium iodide are poured up to 100 ml with distilled water). Before sowing, sterilely add 10 ml of hyposulfite solution and 2 ml of iodine solution to the broth with a crade. When adding a skin ingredient, sumish is needed. Pour into sterile test tubes and flasks.
Wednesday Killian. Up to 100 ml of pure BCH, sterilely add 1 ml of an aqueous solution (1:1000) of diamond green in front of the whiskers.
Healthy media are the basis of bacteriological studies. The stench is to serve as a vision of the finished material of pure cultures of microbes, the cultivation of their powers. On the life-giving mediums, the optimal minds are created for the reproduction of microorganisms. To the warehouse of the middle parts, speech is included, necessary for all the components of the cytoplasm, tobto. all dzherela growth of a living organism. Here in front of us we can see the nitrogen, the charcoal, the water and the sourness.
Dzherelo water that sour in the middle of the living - water. Dzherelom nitrogen є organic half, yakі otrimuyut z meat, ribs, placenta, milk, eggs, blood. As a result of hydrolysis with pancreatin or trypsin, so-called. hydrolyze, which will avenge the great number of amino acids and peptones, so that the majority of microorganisms will be well conquered. Native protein is conquered by less than the same microorganism that exoproteases can. Hydrolyzates are the basis for the preparation of mediums rich in microorganisms.
Dzherelom vugletsyu for pathogenic microbes - the main rank of difference in carbohydrates: mono- and disaccharides, rich alcohols, organic acids and their salts.
Crimean organogens, bacteria need inorganic substances to protect phosphorus, potassium, sulfur, sodium, magnesium, zinc, as well as microelements: cobalt, iodine, manganese, boron, zinc, molybdenum, copper and other.
The needs of microorganism in inorganic soils are satisfied with the addition to the life-giving medium of KH2PO4 K2HPO4 and other salts. A charge from the listed organic elements, a lot of microorganism will require growth officials, tobto. in speeches, like stinks cannot be synthesized. Increasing factors must be added to the living quarters at the finished look. To the factors of growth, there are various vitamins, which are the core of life in the middle of life, to add to the life-giving medium of the product of the growth and the creature's journey, which should be avenged at its warehouse for nicotinic, pantothenic, parabenzoic acid, vitamins A, B, C and іn.
Lifetime speech by microbes can be conquered only for the singing reaction of the middle, tk. the penetration of the membranes of microbial clitin changes in the fallow time depending on the pH of the medium.
Wimogi to the middle of life.
1. Life of the middle ground of guilt and the necessary life of microbes of life of speech.
2. Mother pH reaction, optimal for the type of microbe that grows. -
3. Live the middle of the guilty mother enough moisture and viscosity, tk. microbes are hungry for the laws of diffusion and osmosis.
4. Mother isotonicity and mother oxide-water potential (gH2).
5. Liver mediums can be sterile, ensuring the possibility of growing pure cultures.
The need for living speeches and physical minds in different types of microbes is not the same, and the possibility of creating a universal life-giving medium is turned off.
According to the consistency, they distinguish small and rare life-giving mediums. Schіlnі prepare on the basis of rare for additional addition to them glue speeches: agar-agar or gelatin! Agar-agar (in Malay - jelly) is a product of the growing season, obtained from sea algae. In water, agar-agar differs at a temperature of 80-86°C, it is harder at 36-40°C, and it vicorates to strengthen the vital media for the growth of various groups of microorganisms at the optimum temperature for them.
Classification of living mediums is carried out for their warehouse and recognition
1. According to the warehouse of the living medium, it is divided into simple and folding
Distinguish a group of mediums of infamous confession - the simplest. Meat-peptone broth (simple live broth), meat-peptone agar (simple live agar), live gelatin are added to the group. Qi media zastosovuyutsya growing rich pathogenic microbes. The middles of the sacral recognition, or simply the life-giving middles, are prepared to sound from the hydrolyzates with the addition of peptone and sodium chloride. Їх vicorist is also the basis for the preparation of folding cores.
2. The other group includes the middle elective, special and differential-diagnostic.
The middle ones are elective (selective, vibratory, accumulated, enriched). The principle of the creation of selective living mediums of foundations on the satisfaction of the main biochemical and energy needs of that type of microbe, for the cultivation of which stench is recognized, or the addition of inhibitors, which the growth of the suputnio microflora is considered. Pevny storage and concentration of living things, microelements, growth factors at a high pH value, or adding ingibіtorіv, will ensure the optimal mind for growing one or more species of microorganism. When you plant material on them, which will avenge the sum of various microbes, we will see the growth of that kind, for which the medium will be selective. The butt of elective media is zhovtkovy broth, selenitovy broth, Ploskireva's middle - for growing microbes of the intestinal family, a pool of peptone water - for cholera vibrio.
Zhovtkovy broth. To MSL add 10-20% bichacho zhovchi. Zhovch prinіchuє rіst kokіv and poіtryanoї flori, ale is susceptible to the reproduction of salmonella.
Selenium broth. It is combined with phosphate broth with addition of sodium salt selenite, as an inhibitor of the growth of cocoa flora, Escherichia coli, and does not inhibit the growth of salmonella.
Wednesday Ploskirev. A strong middle ground that can avenge intestinal coli, cokiv, but is more friendly to the growth of shigell and salmonella, the reproduction of which is not galled by diamond greens and gall salts.
Peptone water. Brush with 1% peptone and 0.5% sodium chloride. The middle is selective for chlorine vibrions, because the stench is more likely for other bacteria to multiply on the "hungry middle", especially with a puddle reaction, which they themselves see as acidic products of life.
Special media. Necessary for cultivating bacteria that do not grow on simple living mediums. For some organisms, it is necessary to add carbohydrates to simple living mediums, blood and other living things. Butts of simple living media - tsukrovy broth and tsukrovy agar for streptococcus (prepare in the form of MPB and MPA, to which 0.5-2% glucose is added).
For pneumococci and meningococci, a special medium is gray-wool broth and gray-wool agar (to prepare gray-wool broth, mix 1 part of MPB with 2 parts of fresh sirowort, for tasting gray-wool agar to molten MPA, add 10-25% of stubble)
Differential-diagnostic mediums of vicorous vicarious according to the species affiliation of the studied microbe, depending on the peculiarities of this exchange of speeches. According to their recognition, differential-diagnostic media are subdivided as follows:
1. The middle of the manifestation of the proteolytic zdatnost of microbes, which is necessary to take milk, gelatin, blood, etc. in your warehouse.
2. Mediums with carbohydrates and rich atomic alcohols for
detection of various cucrolytic enzymes.
To the stock of differential-diagnostic media, which are used to reveal tsukrolytic authorities and oxide-hydric enzymes, introduce indicators: neutral chervone, sour fuchsin, bromthymol blue, aqueous blakytny with erysipelas (BP). Changing its contamination for different pH values, the indicator indicates the presence of the enzyme and the splitting of the ingredient introduced into the medium.
Apply differential-diagnostic media:
Middle Endo. It is stored with MPA with the addition of 1% lactose and sodium-free sodium sulfite basic fuchsin (indicator). The middle Endo has a weak erysipelas color. Vykoristovuetsya in the diagnosis of intestinal infections for the differentiation of bacteria, so that lactose is laid out with the adoption of acidic products, in the form of bacteria, so as not to worry about the health of the population. Colonies of lactose-nosing microbes (intestinal coli) have a reddish color, inherited from fuchsin. Colonies of lactose-negative microorganisms - salmonel, shigel and in-barbaric.
Before the differential-diagnostic mediums, there is a short and open row of strings. Wines are made up of carbohydrates (Hissa middles), MSB, milk, meat-peptone gelatin.
Gissa middles are prepared on the basis of peptone water, to which chemically pure mono-, di-or polysaccharides (glucose, lactose, starch and other) are added.
Add an indicator to reveal the change in pH as a result of the absorption of acids and the breakdown of carbohydrates. With a deep splitting of carbohydrates, gas-like products (CO2, CH4 and others) are dissolved, they are caught by additional floats - small tubes, lowered by the middle of the bottom. Mediums with carbohydrates can be prepared and alkaline - with the addition of 0.5-1% to agar-agar. Todi gasification is caught for the closure of the bulbs (opening) at the center of the middle.
On the BCH, which are included in the row of lines, products are revealed that are dissolved when amino acids and peptones are decomposed (indole, sirvoden). Circumnavigation seems to be a route for the introduction of MPB after sowing the culture of filter paper, leaked with octate lead. With the splitting of amino acids, to avenge the sirk, one sees a sirkovodny, papriets black rahunok of the adoption of sulphurous lead. For the indication of indole, a folding indicator can be used. Indole dissolves when tryptophan is degraded, and it can be detected when added to a culture grown with MPB, which is an indicator. For the presence of the indole, the MPB zabarvlyuetsya near the greens and the blue color.
Dry middle.
Living agar, and navit the main differential-diagnostic media, are released as dry preparations, yakі mіstat uѕі nebhіdnі warehouses. Before such powders, it is necessary to add more water and boil it, and then, after pouring, sterilize it.
