To foldable living mediums for bacteria to lie down. See the living middles. Apply differential-diagnostic media
Microbiological research - the observation of pure cultures of microorganism, cultivation and cultivation of their dominance. Cultures are called pure if they are microorganisms of the same species. The stench is needed in the diagnosis of infectious diseases, the identification of species and typical affiliation of microbes, in the following robots, the elimination of the products of the life of microbes (toxins, antibiotics, vaccines, etc.).
For the cultivation of microorganism (cultivation in piece minds in vitro), special substrates are necessary - life-giving medium. On the middles of the microorganism, all life processes are carried out (live, die, multiply thinly), so they are also called the middles for cultivation.
Live in the middle
The life of the medium is the basis of microbiological work, which often indicates the results of all research. The middle part of the guilt is to create the optimal (best) wash for the life of microbes.
Vimogi to the middle
The middle ones are responsible for such actions:
1) be alive, so that you can take away from the easily conquered person all the speeches, the necessary satisfaction of food and energy needs. They are the source of organogens and mineral (inorganic) speeches, including microelements. Mineral speech is not only included in the structure of clitinum and activates enzymes, but also determines the physical and chemical power of the media (osmotic pressure, pH and in.). When cultivating low micro-organisms in the middle, add growth factors - vitamins, active amino acids, such as clitin, which is impossible to synthesize;
Respect! Microorganisms, like all living beings, will require a large amount of water.
2) mothers the optimal concentration of water ions - pH, so that only with the optimal reaction of the middle, which contributes to the penetration of the tunic, microorganisms can conquer the vitality of the speech.
For a large number of pathogenic bacteria, a weakly lumen medium is optimal (pH 7.2-7.4). Vignatok to become cholera vibrio - its optimum is found in the puddle zone (pH 8.5-9.0) and the vibrator of tuberculosis, which requires a slightly acidic reaction (pH 6.2-6.8).
Sob the hour of growth of microorganism in acidic or puddle products of their life did not change the pH, the medium is responsible for buffering, so that speech can be avenged, so that the products are neutralized; exchange;
3) be isotonic for microbial cells; so the osmotic grip of the middle can be the same as the middle of the cells. For a large number of microorganisms, the middle medium is optimal, which contains 0.5% difference to sodium chloride;
4) be sterile, shards of third-party microbes change the growth of the existing microbe, signifying its power and changing the power of the medium (warehouse, pH, etc.);
5) main middleware due to butyvology and optimal consistency for microorganism;
6) mother pevny okislyuvalno-vydnovny potential, that spіvvіdnoshnja rechovins, scho to give and receive electrons, which is expressed by the index RH 2 . This potential shows the increase in the middle of the sourness. For some microorganisms, the required high potential, for others - low. For example, anaerobes multiply at RH 2 not higher than 5, and aerobes - at RH 2 not lower than 10.
7) be unified as far as possible, so as to increase the quantity of other ingredients. Thus, the medium for the cultivation of more pathogenic bacteria is responsible for 0.8-1.2 g / l of amino nitrogen NH 2 to total nitrogen of amino groups of amino acids and lower polypeptides; 2.5-3.0 g/l of nitrogen nitrogen N; 0.5% of chlorides in the pererahunka for sodium chloride; 1% peptone.
Bazhano, so that the environments were clear-sighted - it is better to follow the growth of cultures, it is easier to remember the straying of the environment by third-party microorganisms.
Classification of the middle
The need for living speeches and power of the medium in various types of microorganism is not the same. Tse unstoppable creation of the universal medium. In addition, on the choice of that chi of the middle, add the number of achievements.
In this hour, the greatness of the number of mediums has been proponated; such signs are laid as the basis for the classification of them.
1. External components. By vihіdnim components distinguish between natural and synthetic media. Natural mediums are prepared from the products of the botanical and growing plant. In Denmark; an hour of rotting of the middle, in some varieties of grub products (meat and other) are replaced by non-harvested ones: cystic and ribnim boroshny, fodder drizzles, blood clots and іn. Irrespective of the fact that the warehouse of living middles from natural products is also foldable and changes in fallowness in the form of wild syrovin. Synthetic mediums are prepared from chemically pure organic and inorganic compounds, taken from precisely determined concentrations and differences in two distilled waters. The priority of these middles is important for those who have a permanent warehouse (apparently, skіlki and yakі speeches are included in them), that these middles are easy to create.
2. consistency(Stupin schіlnostі). The middle ones are rіdkі, schіlnі and napіvіrіdki. These middlewares are prepared from rare ones, until they are added to add agar-agar or gelatin to the middle of the required consistency.
Agar-agar is a polysaccharide, which is taken from song varieties of seaweeds. Vіn є for microorganism with lively speech and serve only for the strengthening of the middle. In water, agar melts at 80-100°C, melts at 40-45°C.
Gelatin is the protein of the creature's journey. At 25-30 ° C, gelatinous media melt, so the cultures on them will vibrate at room temperature. The thickness of these media at a pH lower than 60 and higher than 70 changes and the stench is badly overtaken. Active microorganism vicarious gelatin like life-giving speech - for the growth of the medium rises.
In addition, as a mean heart, burn the burnt blood serum, burn the eggs, potatoes, the middle with silica gel.
3. stock. The middle parts are divided into simple and folding. Meat peptone broth (MPB), meat peptone agar (MPA), broth and Hottinger's agar, live gelatin and peptone water are added to the first ones. Folding middles are prepared, adding blood to simple middles, sirovatka, carbohydrates and other speech, necessary reproduction of these other microorganism.
4. Appointment: a) the main (half-living) media serve for the cultivation of more pathogenic bacteria. Tse vishchezgadani MPA, MPB, broth and Hottinger agar, peptone water;
b) special media serve to see and grow microorganisms, like growing on simple media. For example, for the cultivation of streptococcus, add tsukor to the middles, for pneumo-i meningococci - blood serum, for a cough cough - blood;
c) selective (selective) mediums serve to see the singing type of microbes, the growth of such stinks to saturate, suffocating the growth of accompanying microorganisms. Thus, salts of zhovchnyh acids, aggravating the growth of intestinal coli, make the medium selective for the causative agent of typhoid fever. The middle becomes selective when added to them singing antibiotics, salts, pH changes.
Some elective media are called the accumulation media. The butt of such a medium is peptone water with a pH of 8.0. At this pH, cholera vibrio actively reproduces on it, other microorganisms do not grow;
d) differential-diagnostic media allow to distinguish (differentiate) one type of microbes with other enzymatic activity, for example, the Hiss medium with carbohydrates and an indicator. With the growth of microorganisms, which are split into carbohydrates, the color of the medium changes;
e) conserving media are designated for primary sowing and transportation of subsequent material; they have a preference for pathogenic microorganisms and neglect the development of saprophytes. The butt of such a medium is glycerin sum, which is victorious for the selection of vigor during follow-up, which is carried out with the method of detecting low intestinal bacteria.
The recipes for the preparation of the deaky middles are suggested for the example of an offensive division and that of the other part of the handkerchief.
Control nutrition
1. What kind of vimog may be satisfied with the living quarters of the middle?
2. How to classify the middle components by external components?
3. What speeches are for strengthening the middle?
4. What are the middles?
5. What are the middle parts called collapsible, what is their basis?
6. How do the mediums allow you to take into account the more important growth of some microbes while strangling others for an hour?
7. Which media show the enzymatic activity of microbes?
manager
Fill in the form, indicating which groups add the middle.
Preparation of mediums
Utensils for preparing mediums are not guilty of revenge of third-party speeches, for example, meadows, which are seen by some sorts of skla, or oxidized halls, which can be consumed on Wednesday when cooking її in rusty pots. Let's curse the best, enamel or aluminum utensils. Large quantities of medium (tens and hundreds of liters) are prepared in special digesters or reactors (small 14). Before implantation, the dishes must be thoroughly washed, rinsed and dried. Boil new glassware in advance for 30 mins in 1-2% chlorine-hydrochloric acid, or zanyruyut at the same time for the night, after which it is rinsed with running water for a long time.
Respect! Utensils intended for the preparation of mediums cannot be used for other purposes, for example, for the storage of chemical reagents, or for decontamination, which can be disinfected - navit traces of these speeches can cause the growth of microorganisms.
Vihіdnoy syrovina for the preparation of a large number of mediums, serve the products of a creature's aborosline trip: meat and yogo zaminniki, milk, eggs, potatoes, soybeans, corn, yeast and other.
The main livestock broths are cooked in meat water or digestions, taken away with acid or enzymatic hydrolysis of the syrovin. Bouglioni with digestion in 5-10 times economical, less with meat water. Seredovishcha on the digests rich in amino acids, otzhe, pozhivnishі; can be more buffered, so that a more stable pH value can be obtained. In addition, digest can be prepared from meat substitutes (blood clots, placenta, casein too).
At this hour, the laboratories were supplied with meat water and centralized digestions. Most often they are crusted with Hottinger's pancreatic digest, casein hydrolysates or fodder yeasts. From tsikh napіvfabrikativ for singing recipes prepare the necessary middleware.
Steps of cooking middle: 1) variant; 2) setting the optimal pH value; 3) lighting; 4) filtration; 5) bottling; 6) sterilization; 7) control.
boil mediums on hot fire, water baths, in autoclaves or boilers, which are heated in pairs.
pH setting the middle ones are orientated for the help of indicator papers. For accurate determination of pH, they are cored with a potentiometer, zastosovuyuschie glass of electrodes is connected to the instructions of either the comparator (Mikhailis apparatus), which consists of a tripod with sockets for test tubes (Fig. 15) and a set of pH standards. When preparing the middles, they sound like the indicator methanitrophenol, which changes the color in the range of 6.8-8.4.
To determine the pH of the medium of test tube 4, the diameter and color of the flasks, which do not match the test tubes with standards, are placed at nests 1, 2, 3 and 5 (div. Fig. 15). In the 1st and 3rd tubes pour 5 ml of distilled water; in 5-y - 7 ml; in the 2nd - 4 ml of water and 1 ml of the indicator. In nest 4 and 6, set the standard for the required pH. At the 1st, 2nd and 3rd test tubes, pour 2 ml of the cooled medium. Instead of test tubes, mix.
The color of ridins in test tubes is equal to the light that passes through, closing the rear opening with a filter (matte or blue, as if it were intensely yellow). The pH of the sample tested varies according to the pH standard, from the color of which the color is chosen.
Preparing the medium by setting the pH, in nests 4 and 6 set the standards, the pH of which is close to necessary, and in the 2nd test tube with the test medium and indicator, add from the burette the same quantity of the meadow, as well as the light in the 2nd test light for the standard, or the difference in acidity is like a light standard. The puddle (or acid) is added until it is quiet, until the color of the other sample does not match the color of the standards. Kіlkіst lulu (abo acids), add up to 2 ml of the medium in 2 samples, pereahovuyut for the entire length of the prepared medium. For example, 2 drops (0.1 ml) of 0.05 n. a meadow, then for the treatment of 1 liter it is necessary 500 times more, tobto 50 ml of 0.05 n. or 2.5 ml 1n. open up the meadow.
During sterilization, the pH of the mediums decreases by 0.2 to that for the removal of the medium with a pH of 7.2-7.4, and the cob is prepared with a pH of 7.4-7.6.
Lighting the middles vibrate, as if when cooking, the stench kalamutnіyut or darken. For lightening on Wednesday, warm up to 50 ° C, pour in the white of a chicken egg, beat with water, stir and boil. Zgortayuchis, squirrel zahoplyuє in the siege of the call of the middle part. In the same way, it is possible to replace egg white with vicarious blood serum (20-30 ml per 1 liter of medium).
Filtration bath rare and melting gelatinous media are vibrated through paper water or through mater'yanі filters. Filtration of agar mediums is difficult - the stench catches quickly. Sound them through a cotton-gauze filter (a gauze servlet is placed near the virva and a breast of cotton wool is written on it). It is possible to vikoristovuvaty paper or mater'yanі filtry, so carry out filtration in a hot autoclave or in virvah with pidgrivom.
Filtration of agar media can be replaced by circumstances. The middle is poured near a tall cylindrical vessel and melted in an autoclave. In the event of sufficient cooling of the middle of the winding device, the air in it often settles to the bottom. On the coming day, the agar clot is removed from the vessel (for which the vessel is temporarily placed near hot water) and the lower part of the siege is cut with a knife, which has accumulated. The upper part is melted and poured at the top of the container.
bottling middles in test tubes (3-5 ml or 10 ml each), vials, flasks, mattresses and balls no more than 2/3 of the capacity, so that during sterilization, the corks can get wet and the middles lose sterility.
The middle, like sterilized at temperatures above 100 ° C, poured into clean, dry dishes. Mediums that are sterilized at low temperatures are poured obov'yazkovo near sterile dishes.
Pouring out the middle for the help of a watering can, on the end of which a gum pipe is dressed up from the zatiskach Mora. For peaceful bottling, put beakers, burettes, dispensers, syringes, pipettes thinly (small 16).
The utensils from the middle are closed with cotton-gauze stoppers, over which paper caps are put on. It is important that when pouring, the middle did not wet the edges of the dishes, otherwise corks may stick to them. Attach a label with the name of the place of residence and the date of preparation to the skin vessel.
Sterilization. The mode of sterilization should be deposited in the warehouse of the medium of residence and indications for this recipe. Zrazkova scheme for the mode of sterilization of the media is shown in Table. eight.
1 (Some mediums with carbohydrates, proteins and vitamins are more likely to be sterilized for additional bacterial filters.)
The control prepared media: a) to control the sterility of the media, put it in a thermostat for 2 days, after which it is reviewed. However, there are no signs of growth in the environments, they are considered sterile and transferred for chemical control of the amount of skin lesions; b) chemical control: residually restore the pH, instead of total and amino nitrogen, peptone, chlorides (their quantity may be the same as indicated in the recipe).
Chemical control of the media is performed in the chemical laboratory; c) for biological control, a sprat of seeds of the medium is planted with specially selected cultures of microorganism, and for their growth to judge the life (growth) of the power of the medium. A label and a passport are added to the finished medium, and the name of the warehouse of the medium, the results of control and that in.
They take care of the medium for the room temperature in the wardrobes, it is especially reserved for them. Deyakі seredovishcha, for example, seredovischa with blood and vitamins, take it from the refrigerator.
Recipes for the preparation of simple (basic) mediums and isotonic sodium chloride
Isotonic solution of sodium chloride. To 1 liter of distilled water, add 9 g of sodium chloride. Rozchin filter, set pH і tasks, as required, sterilize at 120 ° for 30 minutes.
Meat pepton broth (MPB). Add 1% peptone and 0.5% x to meat water. including sodium chloride, boil on low heat for 10-15 minutes to open the mouths, restore the required pH and boil again for 30-40 minutes until the siege is over. Filter, add to the primary volume with water and sterilize for 20 minutes at 120°.
Bouillon Hotinter. Digest Hottinger's digest with water 5-6 times fallow, depending on how much amino nitrogen can be removed and how much can be in the broth (indicated in the passport of the digest and the recipe of the medium). For example, for the preparation of a medium with 1.2 g / l of amino nitrogen, it was digested to 9.0. g/l must be diluted 7 5 times (9.0:1.2). Add 0.5% sodium chloride to the diluted digest and boil on low heat until the salt is clear.
Mesopeptone agar (MPA). To the finished broth (before sterilization or after it), add 2-3% of the refined agar-agar and boil, stirring, on a weak fire until the agar is fully melted. MPA can be cooked in an autoclave or a Koch apparatus. The middle is ready, as it is necessary, light, filter and sterilize for 20 hours at 120°C.
Napіrіdky agar smite 0.4-0.5% agar-agar.
Zhivilny gelatin. Add 10-15% gelatin to the ready broth, heat it up until it melts (do not boil!), pour it into sterile dishes and sterilize with a plain steam.
Recipes for making foldable cores
Medium with carbohydrates. To the main broth of melted agar, add the required quantity (0.1-2%) of a pure carbohydrate (for example, glucose). After this, they are poured into sterile dishes and sterilized with a straight pair. Shards in carbohydrates are often destroyed in this mode of sterilization, most importantly 25-30% of the differences in carbohydrates, sterilizations through a bacterial filter, add in the necessary asepsis to the sterile main media - after the control of sterility, the core is ready for implantation.
Middle of the blood prepare 3 sterile simple media, supplementing in aseptic tanks (preferably in boxes) with up to 30% (say 5%) of sterile defibrinated blood. The agar middles are melted and cooled to 45 ° C before the cym. At the required temperature, it can be tolerated as hot, but not opiku. After the addition of blood, until the middle is overtaken, instead of the judge, they resolutely mix and pour cups or test tubes.
Respect! You can't spit in the middle with blood - the blood can change your authority.
Among the gray blood prepare just like that, like a middle of the blood. To the main middles add 10-20% of the serum, so as not to avenge the preservative and forward inactivated at 56 ° with a stretch of 30 minutes in a water bath or in an inactivator. When inactivated, the speech (complement) collapses, as a detrimental effect on microbes.
In the middle of life. To simple mediums, add jovch at a quantity of 10-40% of the medium, restore the required pH and sterilize for 20 minutes at 120 ° C. You can add sterile jovch to a sterile medium in aseptic pits.
Bottling of agar mediums at the Petri dish. Before bottling, the middle is melted in a water bath and cooled to 45-50 ° C. For a cup with a diameter of 9 cm, add 15-20 ml of the middle (ball height 0.25-0.3 cm). As the ball is bigger, the colonies look at the new smaller one in contrast. At the arch of a thin ball, a handful of mortal speeches and vologs (the middle is shvidko pendulous) are sharply bordered - the cultivation of the mind is dwindling.
Pour the middles into sterile cups in aseptic tanks. Put the cups with a lid, burn down. Take the vessel with the middle from the right hand, trimming the flame of fire. With the left hand, they wring the cork, squeezing it with the little finger and that side. Burn the neck of the judge and with two fingers of the left hand lightly make a cover. Introduce under the neck of the vial without sticking to the edge of the cup. Pouring the middle, stitch, so that it evenly rose along the bottom of the cup. Like when pouring on the surface of the middle, the bulbs are set up again, before them, like the middle is caught, bring half a day of sirnik or a palnik - the bulbs will peel. Then we close the cup and let the middles be caught. Yakshcho sivba spend on the day of bottling, the middle is necessary to dry. For this cup in a thermostat, carefully open and install the lids of that cup with the open side down for 20-30 minutes. How to spend the coming day after bottling, the cups, not drying, burn at the same paper, which they sterilized, and place them at the refrigerator.
Agar slant preparation. Test tubes with 4-5 ml of sterile molten agar medium are placed in a frail position (approximately 20 ° below the cut) with such a gap so that the middle does not go beyond 2/3 of the test tube, otherwise it can wet the cork. After that, as the middle is caught, put the test tubes vertically - to give the drain to the condensate. It is better to get used to fresh cut agar.
Respect! It is not possible to coristuvatysya with the middle, for which there is no condensate. Її I next again melt the roses in the water bath and mow.
Dry medium
Vitchiznyana promyslov_st vpuskaє dry medium of different recognition: simple, elective, differential-diagnostic, special. Tse powders in bottles with caps that will screw up. They take away the dry medium from the dark mist and close it - the stench is hygroscopic. At the laboratory, powders are used to prepare the middle books on labels.
The advantage of dry mediums in pure mediums prepared in the laboratory is standard (they are produced in large batches), ease of preparation, so that they can be made accessible in any (navit) minds, stability, economy. It is important that they can be prepared from meat substitutes: casein hydrolyzate, fibrin, sprats, and to induce protein fractions of microbial cells (sarcin).
Control nutrition
1. How can I use the pH of the medium for cultivating more pathogenic microbes before sterilization and why?
2. At what temperature do agar mediums melt and catch?
3. How is it to blame for the preparation of dishes, in which mediums are poured with carbohydrates and proteins?
manager
1. Prepare MPB, MPA, broth and Hottinger agar pH 7.2-7.4, pour into vials and test tubes; sterilize.
2. Prepare from dry powders of the Hess medium, pour into test tubes of 4-5 ml and sterilize.
3. Prepare blood agar and pour yogo into Petri dishes.
4. Prepare from dry powders of the middle of Endo, EMC, Ploskireva and pour them in a Petri dish.
5. Prepare agar slants.
Methods of Posiviv
An important step in bacteriological follow-up is posting. It is necessary to study the nature of the sowing material and the medium of vicorous vicorous in different methods of sowing. All stinks include the binding method: protection against third-party microbes. To this, practice the next fast, but without sharp rukhivs, which will help the colivanna every time. It is not possible to tell about the hour of posіvіv. Work better at boxing.
Respect! Do not forget to follow the rules of special safety for an hour of work with the material.
Sowing from test tube to test tube. The test tube with the original material and the test tube with the middle are lightly shriveled in the left hand between the thumbs and fingers so that the edges of the test tubes are on the same level, and their bases are on top of the brush. Sound the test tube from the source material closer to you. At the right hand, like a writing pen, they trim the bacterial loop, and sterilize it, trimming vertically in a half moon. With the little finger of that edge of the palm of the right hand, offend traffic jams at once. Shoot the corks not with a rivk, but smoothly - with light twisted moves. Winking corks, the edges of the test tubes are scorched by a half-moon of a palnik. Introduce the fried loop through a half-moon of the palnik into the test tube with the middle material, cool it down and, having collected a little of the material, carefully transfer it to the test tube from the middle.
When planting the middle, the last material is rubbed on the walls of the test tubes over the motherland and streaked with the middle.
When planting on a regular medium with a tampon, yoga should be washed in the middle for 3-5 seconds, rinsed with it. When sivbі on the core of the middle, the material is rubbed at the surface, wrapping a tampon, after which the tampon is not infected (placed at the test tube, in the same way you deliver it to the laboratory, it is autoclaved).
Respect! Keep in mind that the middle did not wag and did not wet the cork.
When sivbі on bevels of agar, the material should be rubbed on the surface of the middle with zigzag-like roux from the bottom uphill, starting from between the condensate.
When sіvbі on schіlnі middles, poured into test tubes with a stovpchik, pierce the stovpchik with a loop with the material, vibrating so the ranks are planted with a "prick".
After sowing, the loop is taken out of the test tube, the edges of the test tubes are scorched and, after passing the plugs through the half-width of the palnik, the test tubes are closed, after which the loop is fried.
Rare material can be sown with sterile pipettes (Pasteur or graduation). After sowing, the pipettes will zanuryuyut at the disinfecting motherland.
Place at the bottles, mattresses and bottles approximately so, like at a test tube, just pick up the material on the back (with a loop or a pipette), and then open the vessel with the middle.
Judges from the planted culture are written and placed at the thermostat.
Sowing on test tubes from Petri dishes. Vivchivshi character of growing culture on the bowl, from the side of the bottom, with a wax olive tree, the necessary planting of a delyanka. Put a cup with a rich material in front of you with a burnt lid. Open the lid with the left hand and insert the scorched loop under it. Having cooled the loop, collect supplies, material from the appointed plot. We make a loop, close the cup and take a test tube from the middle of the left hand. Sowing is carried out the same way, like from test tube to test tube. After I put the cup down, turn it upside down.
Sowing on agar near a Petri dish. Sowing with a spatula. The spatula is a glass or metal tube, the end of which is bent at the look of the tricutnik. The spatula can be worked with a pasteurist's pipette, zignuvshi under the kutom її thin kіnets, in front of it in the half-moon of the palnica.
Lightly open the lid with the left hand, stroking it with a great and impressive finger. With a loop, with a pipette or with a glass stick, apply the covering material to the surface of the middle, after which you carefully rub it with a circular spatula doti, do not stop moving the spatula along the surface of the middle, with your left hand, with it, trim the lid and immediately wrap the cup. Upon completion of sowing, the spatula is removed from the cup and the lid is closed. A squeaky spatula is placed near a disinfectant, and a metal spatula is fried near a half-burner.
Sow a loop. A small amount of planting material (even if it is mixed in front with a sterile isotonic rosette or broth) is rubbed with a loop into the surface of the middle of the cup edge, the sprat is once passed through the loop from the side to the beak. Let's wait until the end of that month, when the strokes are over, pierce the agar with a loop, knowing too much seed material. The remaining material, which is left on the loops, is spread with zigzag-like ruffles along the entire surface of the middle. After finishing the sowing, close the cup and weed the loop.
Sow a loop on the sector. The cup from the side of the bottom is creased into sectors. The sowing is swayed with zigzag-like waves from the edge of the cup to the center. It is necessary to keep the stitches so that the strokes do not enter the land sector.
Sowing with a tampon. A tampon with additional material should be brought into the cup at the troch and rubbed with circular ruhs on the surface of the middle, wrapping around the tampon and the cup.
Sowing a lawn. Approximately 1 ml (20 drops) of a native culture (such as a culture from the stomach medium, which can be mixed with a sterile isotonic variety or broth) is applied to the surface of the agar and carefully spread the stool over the surface of the medium. The cup is lightly healed and with a pipette, the excess culture is removed, waving into the disinfectant. There they put a pipette.
Sowing agar with a comrade. Culture grown on a rare medium, or emulsified material should be added to a vessel with melted and cooled to 45 ° C agar, mixed and wiggled in a sterile Petri dish. You can add additional material to an empty cup and pour 15-20 ml of agar cooled to 45 ° C. For mixing in place of the cup, the trochs are stolen and wrapped. The cups are left on the table until the middle is caught.
Lighting cups are signed from the side of the bottom and placed at the thermostat with the bottom up.
Control nutrition
1. What do you need aseptic wash for a day of sivby? Round it up.
2. How is it necessary to complete the work space after planting?
Methods of cultivation
For successful cultivation, krіm correctly selected medium and correctly cultivated sowing, you need optimal mind: temperature, moisture content, aeration (patch repeat). As a rule, mindful minds are allowed to create, resolutely mindful of the natural environment.
Temperature. The optimal temperature for the cultivation of most pathogenic microorganisms (37 ° C) is determined at the thermostat (Fig. 17). This is an attachment made of underhanging walls, which can be known again and again, or water, which is heated by electricity. Vіn zabezpecheniy with a thermostat, which automatically adjusts the required temperature, and a thermometer to control the temperature.
Test tubes with crops in racks, dart nets or jars are installed on thermostat police. The cups in the thermostat are turned upside down. So, repeating in the thermostat, it circulated freely and the heating was equal, the police in the thermostat blew with cuts and did not overwhelm. In order not to cool the culture, the thermostat does not deprive it of fresh water.
The laboratory assistant of goiters can register the temperature in the thermostat and improve the cleanliness in the accessories, and in case of a malfunction, call the master.
Light The most important microbes (all pathogens can be seen before them) are not needed - they are cultivated in dark trees. However, for the cultivation of pigmentation, as it is more active on a pink light, the culture after the thermostat is vibrated for 2-3 days in a room light.
Respect! The next unique hit of direct sony exchanges, which is detrimental to culture.
Water content. The life of microbes is impossible without vology - the life of speech penetrates the clitin less than the rozchinennym look. It is necessary to protect when cultivating on open mediums: pour them in a cup and mow them in test tubes more often than a day of sowing. When cultivating microbes that are especially sensitive to the presence of water, for example gonococci, put a thermostat in a vessel filled with water.
Terms of cultivation. Most pathogenic microbes are cultivated for 18-24 years, but see that it grows properly (up to 4-6 days). In order to save the water in them, cotton plugs should be replaced with sterile humic ones, or humic pads should be put on them.
Respect! The gum corks are sterilized in an autoclave, burnt in paper.
Aeration. For the consumption of microbes in a free sour, they can be divided into aerobes and anaerobes. Offensive groups will require different minds of cultivation.
Supplementation of sour, necessary for the cultivation of aerobes and facultative anaerobes, develops with passive and active aeration.
Passive aeration - ce cultivation on thin and rare mediums in vessels, closed with cotton or cotton-gauze stoppers, or Petri dishes. With such a cultivation, microbes keep the sour, there are differences in the middle, which is found in the vessel above the middle and comes through the crust. Passively aerated cultures can be grown on the surface or on a thin ball of the middle, where the sour air penetrates.
Active aeration is stagnant with glibin cultivation of microbes, if they grow in the great obsyagi of the medium. In order to ensure enough sourness of such cultures, they are placed at a special goydalka - a constant remixing of cultures to ensure the safety of repetitions. When cultivating in obsyagi rіdini, which reach tens and hundreds of liters, which are carried out in attachments, they are called reactors or fermenters, they are again blown through the culture for the help of special outbuildings.
Cultivation of anaerobes folded, lower aerobic, scallops are necessary to allow access to a free air. For whom, you need to repeat the life-giving medium in different ways.
Cultivation of actinomycetes, fungi, mycoplasmas, L-forms, spirochetes and the simplest. Cultivation of these microorganisms is importantly similar to the cultivation of microbes. For them, special media and selected regimes have been developed that meet their needs.
A pure culture is called a collection of microbes of the same species on an alkaline or rare living medium.
We use a number of methods to see pure culture fallow in terms of the power of the material, which is being cultivated, and meti dosledzhennya. Sound pure cultures from the isolated colonies - the concentration of microbes on the alkaline medium. It is important to note that most of the colony develops from one microbial cell, so that it is a pure culture of that microorganism.
Stages of seeing pure culture:
1st day - removal of isolated colonies. Drop the finished material with a loop, apply with a pipette or a glass stick on the surface of the agar in Petri dishes. Use a spatula to rub the material on the surface of the core; without proplyuyuchi and without turning over the spatula, spread the seed on the 2nd, and then on the 3rd bowl. With such a spread, the 1st cup has a lot of material and a lot of microbes, the 2nd cup less and the 3rd cup even less.
You can remove the isolation of the colonies with a sib loop. For this purpose, the material is emulsified in broth or isotonic sodium chloride.
2nd day - microbes grow on cups. At the 1st cup, sound more sucily growing - you can’t see the isolated colony. Isolated colonies grow on the surface agar in the 2nd and 3rd plates. They wiggle with an indefatigable eye, for an extra help, with a small increase in the microscope and in the case of a stereoscopic microscope (div. division 31). A colony is taken from the side of the bottom of the dish and transferred to agar bevels. Posivi to put at the thermostat.
Respect! It is possible to peresivate less isolated colonies.
3rd day - cultivating the nature of the growth on a slanted agar. Robbing a smear, farbuyut yogo i, having changed, that the culture is pure, proceed to її vyvchennya. Whose vision of pure culture will come to an end. Seen from the singing dzherel, that cultivated culture is called a strain.
When you see a pure blood culture (hemoculture) in front of the rare medium: 10-15 ml of sterile taken blood is inoculated in 100-150 ml of the rare medium. So shy to the one who has few microbes in the blood. Spivvіdnoshenie krovі, shо sіvaєєєє, i zhivotnoe sredovischa 1:10 not vypadkovo - so reach rozvedennâ blood (unblended blood zhubno іє іє on mikroorganіzmi). Colby should be placed at the thermostat. Through dobu (and after more than an hour fallow culture, which is seen) from instead of flasks, drop hangings onto dishes for isolating colonies. If necessary, repeat hanging at intervals of 2-3 days.
When you see pure culture from the section, washing waters of the drain and other rodins, they are centrifuged forward in aseptic tanks and sow the siege. Farther away, the vision of pure culture is vibrated in an extraordinary way.
To see a pure culture, selective media are widely planted.
A number of methods for developing pure vicory cultures have biological features of the microbe that are seen. For example, when seeing spore-forming bacteria, heat up at 80 ° for 10 minutes, driving in the vegetative forms; when you see a tuberculosis bug, resistant to acids and lunivs, for the help of these speeches, the material is reduced in the form of accompanying flora; to see pneumococcus and plague sticks, the material should be administered to more mice - in that organism, which is highly sensitive to these people, microbes multiply more rapidly than others.
In scientifically advanced robots, especially for genetic studies, it is necessary to take cultures from one cell. Such a culture is called a clone. For її otrimannya, they are most often peeled with a micromanipulator - an attachment, with tools (needles, pipettes) of microscopic expansions. For the help of trimach under the control of a microscope, they should be injected with the drug "hanging droplet", take the clitina (one) and transfer it to the life-giving medium.
Seeing cultures
Vivchennya morphologists, collapses, tinktorial power of power (div. Rodil 3), the nature of the zrutanni on the core (cultural power), enzymatic actively the low of the visible mirobs, the yogo taxis, the Tobto is the Type of the classy, , variety. It is called identification. Identification of microorganism is also important in the diagnosis of infections, insertion of dzherel and channels in її transfers and at the lowest scientific and practical results.
Cultural authorities
Different types of microorganism grow in different ways on the media. Tsі vіdminnosti serve їх differentiations. Some are good to grow on simple mediums, others are strong and grow only on special ones. Microorganisms can give lighter (pishne) growth, peace or miser. Cultures can be bezbarvnimi, siruvatimi, siro-blakitnimi. Cultures of microorganism, which make the pigment, may be different infestation: white, yellow or golden in staphylococcus, chervone - in the wonder stick, blue-green - in the blue-green stick, the pigment is different in the water, the serpentine is not only .
On schіlnyh Among the microorganisms in the fallow, depending on the amount of planting material, either sucrose pour ("lawn"), or isolated colonies are approved. Cultures are rough and low, gaps and opaque, with a matte surface, glossy, smooth, short, dry, bumpy.
Colonies can be large (4-5 mm in diameter and larger), medium (2-4 mm), small (1-2 mm) and dwarf (less than 1 mm). The stench is differentiated by shape, rotting on the surface of the middle (opukli, flat, dome-like, depressed, round, rosette-like), the shape of the edges (riven, frizzy, torn).
Rare The middle microorganism can make a smooth calamute, give a siege (granular, saw-like, plastic-like) or spit (lower, rough, wrinkled).
For example when planted with a prick, the microbes grow cloudy, the middle ones grow cloudy, the middle ones grow only after the “prick”, blocking the opening of the gap.
Cultural powers signify, emphasizing the nature of the growth of culture, with a simple eye, for the help of a magnifying glass under a small magnification of a microscope, or a corrosive stereoscopic microscope. The size and shape of the colonies, the shape of the edges and the transparency are seen in the light that passes through, looking at the cups from the side of the bottom. In the broken light (from the side of the lid), the character of the surface, the furring, is determined. The consistency is determined by the dot of the loop.
Morphological power
The study of morphology of microbes can serve as their differentiation. The morphology is observed in the treated preparations. Restore the shape and size of the clitin, its expansion in the preparation, the presence of spores, capsules, flagella. In farbovanized preparations, it is necessary to bring microbes to farb (tinctorial power) - it’s good or bad to take farb, as it can be brought to differential zabarvlen (in such a color, it’s zabarvlyuєtsya for Gram, Tsil - Nielsen and іn.). Vital (living) zabarvlennya allows you to install frivolity, to differentiate living and dead cells, to follow their podil. Subdivision and friability can be treated in native (uncontaminated) preparations (div. section 3).
Enzymatic activity
Enzymatic activity of microorganism in bagat is different. It can be used as a species and type belonging to a microbe, and it can be used to designate its variant (the so-called biovariant). Let's take a look at the main enzymatic power of that yoga yakіsne appointment.
Breakdown into carbohydrates(saccharolytic activity), so that the ability to decompose the ring and rich atomic alcohol with dissolved acid and acid gas, grows on the middles of Hiss, yakі avenge that which is the other carbohydrate and indicator. Under the action of acid, which is satisfied when splitting into carbohydrate, the indicator changes the contamination of the medium. That is why the middle is called "string row". Microbes, which do not ferment into carbohydrates, grow on the medium, without changing it. The presence of gas is established for the closure of bulbs in the middles with agar, or for the bunching of yoga in the "floats" on rare middles. "Float" - a glass tube with a soldered glass, an animal uphill, so that before sterilization they place a test tube from the middle (Fig. 18).
Rice. 18. Vivchennya tsukrolytic activity of microorganism. I - "string row": a - middle middle with carbohydrates and Andrede's indicator; b - similar to the middle with the BP indicator: 1 - microorganisms do not ferment into carbohydrate; 2 - microorganisms ferment into carbohydrates from acid solutions; 3 - micro-organisms ferment into carbohydrates with dissolved acid and gas; II - colonies of microorganism that do not lay out (bezbarvnі) and lay out lactose
In addition, saccharolytic activity is shown on the media of Endo, EMC, Ploskireva. Microorganisms, fermenting lactic acid (lactose) to acidity in their mediums, make the colony infected - acid changes the color of the indicator. Colonies of microbes that do not ferment lactose are barren (div. Fig. 18).
Milk with the growth of microbes, which ferments lactose, is swallowed.
With the growth of microorganism that utvoryuyut amylase, on the middle of the starch, it is decomposed. They know about it, having added Lugol's sprat to culture, the color of the medium does not change. Non-decomposing starch is given from cym rozchinom blue farbuvannya.
Proteolytic power(For this purpose, the ability to split proteins, polypeptides, etc.) grows on the media with gelatin, milk, sirovatka, peptone. When growing on the gelatinous medium of microbes, which ferment gelatin, the medium grows. The nature of the distribution, which is called by different bacteria, is different (Fig. 19). Microbes that break down casein (milk protein), call for peptonization of milk - it looks like milky milkweed. When splitting peptones, indole, circulatory water, ammonia can be seen. The light is being installed for the help of indicator papers. The filter papier is lately oozing out with singing roses, hanging, streaked with narrow females with a dozhina of 5-6 divs, and after planting the culture on the MPB, a stopper is placed between it and the wall of the test tube. After incubation at the thermostat, the result is guaranteed. Ammiac turns blue on litmus papyrus; when you see sirvodnya on paprika, leaked 20% lead acetate and sodium bicarbonate, lead sulfate is dissolved - black paprika; indole viklikaє black paprika, leaked with oxalic acid (div. fig. 19).
Krіm zaznachenih sredovishch, zdatnіst mikroorganіzmіv rasplyuvatі vіznі zhivnі substrates vyznachayut for the help of paper disks, leaking songs reagents (indicator systems of paper "SIB"). The discs are lowered near the test tube with the final culture and after 3 years of incubation in a thermostat at 37 ° C, change the color of the discs to judge the distribution of carbohydrates, amino acids, proteins, etc.
Hemolytic power (development of erythrocytes) grows on the middle of the blood. Some of the middles become clear in their own, but in the narrow middles of the colony there is a clear zone (Fig. 20). When approved by methemoglobin, the middle is green.
crop conservation
Vidileni and vyvchenі kultury (shtami), scho to become valuable for science or virobnitstva, are taken from museums of living cultures. The Sovereign Union Museum is located at the Sovereign National Institute of Standardization and Control of Medical Biological Preparations. L. A. Tarasevich (DIBK).
Zavdannya zberіgannya - pіdtremati zhittєzdatnіst microorganіzmіv і ahead їх іх мілівії. For whom you need to relax, or add an exchange in microbial cells.
One of the most advanced methods of trivial conservation of crops is lyophilization - hanging in a vacuum from a frozen mill allows the establishment of an anabiosis camp. Hanging out is carried out at special devices. Store cultures in sealed ampoules at a temperature of 4 ° C, preferably -30-70 ° C.
Reinvention of dried crops. Strongly heat the tip of the ampoule in a half-moon and stick it with a new cotton swab, lightly soak it with cold water, so that microcracks are created on the folds, through which it is possible to penetrate the middle of the ampoule. With this, passing through the cracked edges of the cracks, they will be sterilized again.
* (If there is too much water on the tampon, you can drink it into the ampoule and destroy the sterility of the culture: it gets wet through the microcracks that have been established, so there is a vacuum in the ampoule.)
Respect! Do not forget that the sealed ampoules have a vacuum. As soon as you drink it in it through the great opening, the culture that is found in the ampoule may spread, and it will come out.
Letting you see in the air, with a pair of tweezers break and remove the top of the ampoule. Lightly scorch the opening and pasteurized with a sterile pipette, or add a broth or an isotonic broth into the ampoule with a syringe. Mix the ampoules together and sow on the medium. The growth of new crops in the first crops can be more common.
Long-term conservation of culture can also be done with rare nitrogen (-196 ° C) with special accessories.
Methods of non-trivial conservation of crops are as follows: 1) subcultivation (periodic replanting on fresh mediums) at intervals to lie in the dominance of the microorganism, the medium and minds of cultivation. Intermediate cultures are taken at 4°; 2) savings under the ball of olії. The culture is grown in an agar stand 5-6 cm high, poured with sterile vaseline oil (olive ball approximately 2 cm) and stored vertically in the refrigerator. The terms of the preservation of different microorganism in different, to that from test tubes periodically hang the culture, in order to check the life; 3) storage at -20-70 ° С; 4) collection from sealed test tubes. If necessary, the material is taken and hung on a fresh center.
Control nutrition
1. What should be included in the understanding of "bacteriological research"?
2. What kind of culture can be for such a follow-up?
3. What is a bacterial colony, culture, strain, clone?
4. What is the understanding of the "cultural power of microbes"?
manager
1. Learn and describe the sprat of colonies. Reseed them for agar slant per sector.
2. Vivechit and describe the nature of growth - culture on slant agar. Consider the purity and morphology of the culture in the inoculated preparation.
3. Transfer culture from agar slant to broth and differential diagnostic medium. Check and write down in the protocol the nature of the culture growth on these media and enzymatic power.
M'yasopeptonny broth and m'yasopeptonny agar are the simplest living mediums added to most bacteria. The main material for the preparation of these and other living mediums is meat water, which is the yellow of the native land with an acidic reaction. M'yasna water to avenge white speech (albumine), extractive speech and active salt.
Meat water and concentrations of meat-pepton broth (according to GOST 10444-63)
M'yaso yaloviche or kіnské after the removal of brushes, fat and tendon are passed through a meat grinder, and minced meat is poured with cold tap water, pouring 500 g of minced meat into 1 liter of water on the skin. After mixing the sum, they heat it up to boiling point, and then warm it up for 1.5 years. A small amount of summish minced meat with water (up to 5 l) can be boiled on hot fire, stirring often, so that the dry particles of meat do not burn.
A large quantity is better than boiling in cauldrons with a steam shirt. The readiness of meat water is due to filtering a small amount of them in a test tube through a paper filter. Yakshcho filtrate prozory, meat water is ready. If the filtrate comes out kalamutny, then the next step is to doti, the docks do not see the prozory filtrate. After the end of the cooking, the homeland is boiled through the canvas or double-folded gauze, the whole sik and otriman homeland is brought out of the boiled meat to bring boiled water to the cob volume. It is not necessary to add a large amount of water, because, as a result of boiling meat, water will pass a lot of juice from meat. Only in the case of a too tempestuous variant, that arc of a boisterous boil, there is a great evaporation of the radiny.
Otriman's meat water is poured into glass jars, then it is boiled and sterilized in an autoclave for 20 minutes at a normal temperature of 120 °C. From the meat water harvested by such a rank, if necessary, prepare the necessary life-giving medium.
To reduce the concentration of meat peptone broth (MPB), add 10 g of peptone and 5 g of sodium chloride to 1 liter of meat water. Bring the reaction of the medium to pH 7.0-7.2, boil, filter through a paper filter, pour into clean test tubes and close with cotton plugs, sterilize in an autoclave for 20 minutes at a temperature of 120 °C.
Breeding m'yasopeptonny broth
The preparation of meat water is carried out in the same way, as for concentrated broth, and instead of 500 g of minced meat, 250 g of yogo per 1 liter of water is taken. It is possible to dilute the prepared concentrated meat water into vdvіchі. Add 5 g of peptone, 2.5 g of sodium chloride to 1 liter of diluted meat water, adjust the pH to 7.0-7.2, and let it simmer the same way, as when preparing a concentrated meat-peptone broth.
ribopeptone broth
In factories that make canned fish, meat water for the preparation of mediums can be cured with fish water. Ribna water should be prepared from the great thin ribi. Best for cієї met pike perch, cod and pike. Zvіlnenu vіd kіstok and fat riba is passed through a meat grinder and poured with cold tap water from a rosemary 1 liter of water per 500 g of minced fish. All further operations for the preparation of fish water are similar to those for the preparation of meat water.
Ribna water is vicorated for the preparation of ribopeptone broth. For 1 liter of fish water, add 10 g of peptone, 5 g of kitchen salt, neutralize to pH 7.0-7.2 and sterilize as it is, like meat-peptone broth.
Pouring the broth into test tubes (before sterilization) is carried out through a glass of virva, on the end of which a gum tube is put on with a tip and Mora's grip (Fig. 52). Curl the tip of the sprat when pouring into a test tube, so that the edges of the test tube are not wetted with broth, otherwise the cotton plug is dry to the point and may be germinated by microorganisms.
Installed reactions of life-giving mediums
With microbiological findings, it is necessary to keep the acidity and power of the living environments alive. Active acidity of the living substrate may, as it was intended, be of great importance in the life processes of microbes: they add their growth, morphological and physiological power. For most bacteria, a medium with a pH of 7.2-7.4 is needed, for yeasts and a plethora - with a pH of 4.5-5.5.
The life of the middle, which is prepared for the manifestation of the biochemical powers of microbes, is due to the mother of the optimal, strictly assigned reaction for this microbe. Set the pH of the prepared medium in the microbiological laboratory can be either electrometric or colorimetric methods. Electrometrically, pH is determined with the help of a laboratory pH meter (for example, LP-58 - Fig. 53) or ionomir (KFV-I1 - Fig. 54).
In order to measure the pH value of the added revitalizing medium for an additional ionomer, pour about 40 ml of the medium into the flask, fill it with a cartridge with a chlorine-silver element and add an electric attachment. The arrows of the galvanometer move and show the pH value.
When using a laboratory pH meter (LP-58), it is easier to stop the glass and calomel electrode. A bag of a glass electrode can be even thin walls - 0.03-0.05 mm, so when working with it, you need to be careful. Before zastosuvannya sklyany electrode should be kept in distilled water for at least 1-2 years, and in case of frequent operation, it is necessary to gradually save yoga in distilled water, periodically replacing it with fresh water.
Before adjusting the pH with a glass electrode, correct the pH scale of the buffer. Rosemary The pH of the buffered range may be close to the pH of the specified range. The temperature compensator of the attachment is set to the temperature of the buffer range and adjusts the adjustment and potentiometric part of the attachment behind the normal element. Then, after rinsing the electrode with distilled water and vimiryuvalny vessel, saturate it with the remaining medium and moderate the pH of the medium; setting the temperature compensator to the temperature of the medium and pressing the button for turning on the accessory on the front doshtsi of the pH meter, mark the indication of the galvanometer arrows on the pH scale. Detailed rules for corystuvannya, nalashtuvannya and watch over the accessories are described in the instructions on how to add to the accessories. Trivality of analysis 3-5 min. Report the results accurately.
Electrometrically for the help of a pH meter and ionomir, you can also manually check the pH of the prepared media. When preparing living substrates for bacteriological analyses, it is necessary to adjust the pH level of the medium, and to bring the reaction to the highest value, then the practical colorimetric method will prove to be effective.
The basis of the colorimetric method is based on the power of indicators to change their concentration by changing the concentration of H-ion ions. For a skin indicator, its characteristic range of pH, in the range of any one color changes. According to GOST 10444-63, for the establishment of the pH of living mediums, 0.04% of bromthymol blue should be added. The range of bromthymol blue is 6.0-7.6. In sour mediums, vines are filled with yellow zabarvlennya, in puddles - blue. At pH 7.1, it gives a light green-green infestation.
For the purpose of the reaction of the medium or canned products, after the development of microorganisms in them, 0.04% of the bromocresolpurpur compounds are added. Bromocresolpurpur changes contamination in the pH range of 5.2-6.3. In sour mediums, the wines are pale yellow, in neutral ones - red-violet, and at pH 6.3 it gives green with a purple tint of zabarvlennia.
Indicators are prepared in the coming rank: 0.1 g of a viable indicator, named on analytical tereses, is rubbed at the hub (bagzhano agate) from 1/20 n. caustic soda. To dispense 0.1 g of bromthymol blackite, take 3.2 ml of 1/20 n. the difference of NaOH; for dispensing 0.1 g bromcresolpurpur - 3.7 ml 1/20 n. open up the meadow. Add 250 ml of distilled water to the finished puddle solution and add 0.04% of the indicator solution. Preparations for the development of the indicator should be taken in the cold from the glass vessels with a ground cork.
Before sterilization, the pH of the medium should be adjusted to equal (7.0-7.2). For this, a large flask (2-3 l) is poured into 1.5 l of the living medium. From the burettes in it, start pouring drop by drop 10% of the soda bicarbonate solution or a weak solution of the meadow (for example, 0.1 N. of the sodium hydroxide solution), all the time pereveryayuschie reaction of the medium according to the indicator (in this case, according to bromthymol blue). For this purpose, on a white porcelain tile (it is possible to make a cachelna), apply a sprat of specks of the middle, to which add a speck of indicator. If bromthymol blue, when you give it a zhovte zabarvlennya, then the soda infusion or the meadow needs to continue doti, the midday docks in the drops of the indicator do not get bogged down in light green colors. If a green-blue tone appears in the droplets of the remaining medium when the indicator is added, then the puddle coloration, when titrated, inflows in excess and into the flask with the middle, should be added fresher, not yet titrated, the life-giving medium and the reaction will be rediscovered.
Perfectly proving itself in practice for the help of a comparator and the standard Michaelis scale. This method should be accurate - the reaction of the medium varies up to 0.1 pH units. To determine the pH of the medium for the additional accessory of Michaelis, it is necessary to override the reaction of the medium for the supplementary litmus paprika, so to know, as an indicator of such a number of ampoules from the accessory of Michaelis, it should be quickened. Since for most living media a neutral and low-lumen reaction (pH 7.0-7.2) is necessary, it is necessary to take the indicator methanitrophenol and a number of ampoules with a pH of 6.8-8.2. During sterilization, the pH of the substrate should decrease by 0.2 units, so for the creation of optimal minds for the growth of microorganisms before sterilization, the middle, which is prepared, is the mother's fault pH 7.2-7.4.
After rechecking the reaction of the medium according to litmus and choosing an indicator, take a comparator, install standard ampoules with the correct pH in a new test tube (Fig. 55). At another test tube of the first row (No. 2), at the comparator, 2 ml of the last revitalizing medium is poured; here, add 4 ml of distilled water and 1 ml of a 0.3% solution of the selected indicator (called, as already mentioned, methanitrophenol). At the two extreme test tubes of the first row No. 1 and 3, 2 ml of the revitalizing medium and 5 ml of distilled water are poured. At the middle tube of another row No. 5, place only 7 ml of distilled water. At the two extreme sockets of the other row of the comparator No. 4 and 6, insert ampoules with a standard range of pH indicators, in the interval between which the reaction of the medium is restored.
Looking at the test tubes of light through the lower opening in the comparator on a white matte plate, poring over the zabarvlennya dosledzhuvannya rodini from the zabarvlennyam of the indicator. If the contamination in the sample with the medium is taken from the contamination of the indicator in one ampule, then the pH value of the medium is equal to the pH value of this standard ampoule. If the color of the finished medium (in another sample) appears lighter, lower in the standard ampoule, then the test tube with the medium is added drop by drop with 10% of the amount of bicarbonate of soda or 0.1 n. rozchin їdkogo natra, vikoristuuuuchi microburette for this. The infusion of variations, which neutralize, drop by drop dots, until the color of the sample does not match the color in one ampoule. For a quantity of milliliters of soda abolugu, streaked for neutralization 2 ml of the medium (placed in test tube No. 2), it doesn’t matter, you need a few milliliters of a neutralizing ratio to set the pH level in all obligatory preparation of a living organism.
In highly acidic environments, the pH value is assigned to the additional indicator para-nitrophenol. The rozchini іndikatorіv prepare the coming rank.
1. 0.3 g of meta-nitrophenol is mixed in 100 ml of distilled water (pH value range from 6.8 to 8.4).
2. 0.1 g of para-nitrophenol is dissolved in 100 ml of distilled water (pH value range from 5.4 to 7.0).
You can also set an approximate pH value of the medium with the help of a universal indicator. For this, pour 2-3 ml of the last medium into a porcelain cup and add 2-3 drops of a universal indicator. Having mixed the sum with a glass stick, smoky the color of the swarm of swarms on the paper color scale. The pH of the finished medium will be the same as indicated in the equal-farmed female of the color scale. The degree of accuracy of pH assignment by a universal indicator is close to 0.5.
Preparation of meat peptone agar (MPA)
Agar (in Malay “jelly”) is a foldable organic speech, which is taken from sea algae. Behind the chemical warehouse of wines there is a sum of carbohydrates, which can be seen up to polysaccharides - similar galactose - and may wish for building. Additions to the rare medium agar give such a thickness that the melting is more than boiling temperature. Agar melts in water at about 80-86 °C, harder at 36-40 °C. Zavdyaki tsoma on agar mediums can be cultured microbes for any temperature that allows them to live.
Meat peptone agar is prepared from concentrated meat peptone broth. In a large Erlenmeyerivsk flask with a refractory layer, the capacity is up to 2 liters, sealed with a cotton stopper, pour in concentrations of m'yasopeptonny broth, add 2 to 4% (deposit variety) of finely chopped agar, without curving with a stopper, put on a weak stopper.
Meat pepton broth must be taken with a medium reaction of 7.4, so that during sterilization the pH will decrease by 0.2. The reaction of m'yasopeptone agar can be installed if it appears outside of the melt. If the agar is of good viscosity and does not precipitate, then after the pH is set, boil for 12-15 minutes, filter through cotton wool and, pour into the flask or test tubes (deposit for consumption), sterilize in an autoclave for 20 minutes at 120 °C.
If agar is not enough purification, you can give a siege. In any case, before sterilization, m'yasopeptone agar should be clarified. For this, until melted and cooled to 50 ° C, meat peptone agar, add egg white, mix with 30 ml of water and beat until I become a pin. For 1 liter of medium, take the protein of one chicken egg. Agar should be taken seriously with the introduction of protein and put in an autoclave for 10 minutes at 120°C. Under the hour of boiling, the whites burn and choke all the important parts. After autoclaving, the agar is filtered through a cotton-gauze filter. For whom, take a glass of virva of great diameter, pull gauze over it, and put a thin ball of cotton wool on the animal. Filter boiling agar as needed quickly and filtrate until it is caught pouring into test tubes of 5-10 ml. Sterilization of the middle is carried out in an autoclave, as indicated above.
After sterilizing the test tubes with the middle, which should be replaced by 5 ml of agar, put at the sickly camp so that the agar does not stick out of the cork. After being caught, they gain "oblique" (or beveled) agar. Test tubes with 10 ml of agar are placed on a tripod and allowed to catch, removing the middle, pour on a “high table”. Skew agar is not recommended for more than 4-7 days, shards of wine hang.
Meat pepton agar is the main life-giving medium, which is vicorated during bacteriological analyzes in microbiological laboratories. You can beat it like a “simple agar”, like this: after adding carbohydrates, making a complex differential-diagnostic medium.
Live in the middle in microbiology, they name mediums, which are different from the folding side of a simple warehouse, like zastosovuyutsya for the reproduction of bacteria and other microorganisms in laboratory industrial minds.
Ready for life in the middle from the products of the creature's number of growing adventures. Great significance in the life-giving medium of growth factors that catalyze the metabolic processes of microbial cells (vitamins of group B, nicotinic acid, etc.).
Piece media prepare for singing recipes from various infusions or from the brine and aboroslyne trip to the addition of inorganic salts, carbohydrates and nitrogenous speeches.
In bacteriological practice most often vicarious are dry life-giving mediums, which are obtained on the basis of the current biotechnology. Для їх приготування використовують економічно рентабельну нехарчову сировину: що втратили термін придатності кровозамінники (гідролізин-кислотний гідролізат крові тварин, амінопептид - ферментативний гідролізат крові; продукти біотехнології (кормові дріжджі, кормовий лізин, виноградне борошно, білколізин). часу, зручні при транспортуванні та мають visible standard warehouse.
For consistency life-giving centers can be rare, napіrіdkie, schіlniy. Other mediums are prepared with a path to add 1.5-2% agar to a rare medium, for example - 0.3-0.7% agar. Agar is a product of the processing of a special type of seaweeds, it melts at a temperature of 80-86 ° C, it is harder at a temperature of close to 40 ° C and in a frozen state there is a medium thickness. In some cases, for the extraction of the scleral life-giving medium, gelatin (10-15%) is used. A number of natural living mediums (burnt blood serum, burnt egg white) are scallops.
For the purpose of recognition middles are subdivided on the main, selective and differential-diagnostic.
Up to the main ones one can see the middle ones, which zastosovuetsya for the growth of rich bacteria. These are tryptic hydrolysates of meat, fish products, blood creatures or casein, from which they prepare a rare medium - a lively broth and a lively agar. Such middles are the basis for the preparation of folded fatty middles - bloody, bloody ones that satisfy the food needs of pathogenic bacteria.
Elective life-giving mediums are recognized for vibrating vision and accumulation of microorganisms of a singing type (or a singing group) from materials, in order to avenge a different third-party microflora. When the selective living organisms are combined, they emerge from the biological features, which are similar to the microorganism data from the greater number of others. For example, vibratory growth of staphylococcus aureus is associated with increased concentration of sodium chloride, vibrio cholerae - in the puddle medium thinly.
Differential-diagnostic life-giving mediums are vicorous for demarcation of four species (or groups) of microorganisms. The principle of encouraging these media is based on the fact that different bacteria can be distinguished among themselves for biochemical activity due to an unequal set of enzymes.
A special group is made up of synthetic and non-synthetic life-giving mediums. The warehouse of synthetic media includes chemically pure speech: amino acids, mineral salts, carbohydrates, vitamins. In the synthetic medium, additionally include peptone, yeast extract and other living speech. These middle ones are most often used by scientifically advanced robots and in microbiological industry with the selection of antibiotics, vaccines and other preparations.
In the rest of the world, with the method of saving the dead, the accelerated identification of certain microorganisms (enterobacteria, staphylococci, streptococci, etc.) will be called the microtest system (MTS). The stench is polystyrene plates with holes, in which sterile differential-diagnostic media are placed. Sterilization of MTS is carried out by UV testing. Microtest systems are especially effective in case of massive bacteriological studies in practical laboratories.
Wimogi to the living middle
Whether it be a life-giving medium, it can be reassured by advancing vimog: revenge everything necessary for the reproduction of microorganism of speech in a form that is easy to conquer; mothers have optimal water content, viscosity, pH, but are isotonic and can be transparent. The skin life-giving medium is sterilized in a singing way in a fallow manner in the warehouse.
When preparing life-giving mediums, it is necessary to protect the need for microorganisms, which are cultivated, in various life elements. Іsnuyut different classifications of living mediums.
Classification of living mediums behind the warehouse:
1. Forgive the middle(MPB, MPA, gelatin, peptone water). Meat-pepton broth (MPB) is the protein base of all mediums.
Іsnuє kіlka ways to prepare MSB:
a) on meaty water from adding finished peptone;
b) on the digests of the products of hydrolysis of free syrup with additional enzymes.
M'yaso-peptonny agar (MPA) otrimuyut a way to add agar-agar (1.5-3%) to MPB. For example, MPA divisions along the diagonal of the test tubes and vials are the same as bevels of agar. Like the middle part, the sample is vertically 5-7 cm high, with an agar stand. MPA, caught in Petri dishes like shtastshki - plate parts of agar. Like the middle one, there is a vertical ball 2-3 cm high and a diagonal ball of the same size, the price of agar agar.
2. Foldable middle prepared on the basis of simple sing additives (carbohydrates, blood, zhovch, eggs, sirovatka, milk, salt, factors for growth too)
Classification of life-giving media for external components:
1. Natural life centers- It's a natural product of a creaturely or a growing plant.
Can buti:
Roslinnі (external products - soybeans, peas, potatoes, carrots).
Creatures (external products - meat, fish, eggs, milk, animal tissues, jovch, blood serum).
Zmishani (MPA, the middle of Levenshtein - Jensen toshcho).
2. Piece media to take away processed natural products (meat water, digest), speech, otrimanі z tsikh produktіv (peptone, yeast and corn extracts) and various additives. The most important and most varied group of mediums behind the warehouse is the most stagnant. They are prepared for singing recipes from various infusions of chi in the wild number of the plant, with the addition of inorganic salts, carbohydrates and nitrogenous speeches.
3. Synthetic media(in the home chemical warehouse) are formed from chemically pure stocks at exactly the right concentrations (with the addition of carbohydrates, salts, amino acids, vitamins, etc.). On the basis of these middles, adding natural or pieced middles to them, they acquire synthetic middles.
Classification of living media for consistency: middles buvayut rare(media without agar), like(With agar up to 1%), schіlnі(Agarovі - 1.5-2.5%). Rare mediums are most often used for the cultivation of physiological and biochemical characteristics of microorganism, for the accumulation of biomass and products of exchange. Napіvridkі seredovishcha sing vicorist іz zberіgannya cultures, shіlnі — vіdіlennya mikroorganіzmіv, vyvchennya morphologiiї colіnіy, dіnostichnyh іlіy, kolіkіsny іnіku, vznаchennya antagonistіchnyh vіlnostіh іnshih.
Classification of the living mediums for the purposes of recognition: universal (spread) and special.
Universal (basic) mediums. Vicorous mediums are used for cultivating more nevibagly microorganisms, or as a basis for preparing special mediums, adding to them blood, zukor, milk, buttermilk and other ingredients necessary for the reproduction of that microorganism. To tsієї groups lie: MPB - m'yaso-pepton broth, MPA - m'yaso-pepton agar, MPZH - m'yaso-pepton gelatin thinly.
Special media. Appointed vidilennya and selective cultivation of singing species of microorganism, yakі grow on simple mediums.
There are such types of special media: enrichment media, elective, differential-diagnostic, conservative and accumulation media.
Middle wealth. A lot of microorganism does not grow on natural mediums, to increase the living value of the medium, it is added to it in carbohydrates (sucrose broth or agar) or proteins (serovatkovy agar and broth, blood agar and broth). Blood agar or blood broth is taken with a path to add 5-10% of sterile defibrinated blood of a sheep, rabbit, horse, human being to the life-giving medium. The middle vicorist is used for the observation of streptococci, pneumococci and other bacteria, as well as the development of hemolytic activity. Sirovatkovy broth or grayish agar is taken by the way to add to simple mediums 15-20% of Kіnskoy or bichachoї syrovatka.
2. Elective (selective) middles. These mediums are recognized for vibrating vision and the accumulation of microorganisms of the same type from the material, which is to avenge the appearance of microbes. When posting on them the material, which will avenge the sum of different microorganism, we will show the growth of that kind, for which given medium will be selective. The vibrancy of the medium is reached by the way of the creation of minds, optimal for the cultivation of singing microbes (pH, Eh, salt concentration, storage of living speeches), tobto. positive selection. Abo way to add to the middle of the speeches, which ignoring other microorganisms (zhovch, high concentrations of NaCl, antibiotics, etc.), tobto. negative selection. To tsієї group to lie:
Selenite middle- є the best middle ground for salmonella and dysentery microbes in Sonne. Sodium selenium, which is found in the middle, stimulates the growth of these bacteria and suppresses the growth of the accompanying flora.
Bismuth-sulfite agar avenge the salt in the mud, the diamond green. Salmonella grow on the middle of the medium that looks like black color colonies. Otherwise, do not give bacteria on the middle growth.
Zhovtkovo-salting agar (JSA) the middle ground for the observation of staphylococci is up to 10% sodium chloride, which takes into account the greater number of bacteria that are contained in the material. In addition, the middle is also differentially diagnostic, since the presence of egg yolk allows the enzyme lecithinase (lecitovitelase) to be detected, which kills pathogenic staphylococci. Lecithinase decomposes lecithin into phosphorcholine and non-reactive water fatty acids, which is why the middle of the calamity-positive lecithinase-positive colonies appears as an opalescent zone in the appearance of a "radius vein".
beef broth selective for salmonella, the reproduction of such stimulants is added 10% zhovch, which at once galmues the growth of accompanying microorganisms.
puddle agar or puddle peptone water selective for cholera vibrio, puddle reaction of the medium (pH 9.0) does not change the growth of cholera vibrio, but galmui growth of other microorganisms. 3-5 deb. F
3. Differential-diagnostic media. Differential-diagnostic media should be used for differentiation of one type of microorganism with a different character of their enzymatic activity. The warehouse of these mediums should be selected with such a rosary, in order to clearly reveal the most characteristic dominance of the singing type of microorganism, outlining the peculiarities of its exchange of speech.
The middle of the manifestation of proteolytic and hemolytic zdatnosti microbes, revenge in your warehouse of protein speech: blood, milk, gelatin thin. The most wide-ranging mediums are meat-peptone gelatin (MPZ), which has been burned, milk and blood agar (KA).
Mediums for the development of glycolic powers include three main components: a life-giving base (broth, agar), a substrate (monota of disaccharide, rich alcohol) and an indicator for the manifestation of certain enzymes. Enzymatically cleave substrates to produce a pH value that changes the concentration of the medium. The widest range of colors of the medium with various carbohydrates (for example, from bromthymol blue, indicator BP). Also wider is the middle of Giss, in which they protect the diversity and the ability to ferment different carbohydrates with acid solutions, acid and gas.
For the differentiation of enterobacteria, saturate peptone water with a set of various carbohydrates, Andrede's indicator and floats, which make it easier to detect gasification and help to visually determine the change in pH, which is characteristic of various microorganisms. Zokrema, crushed in the sour side, turns reddening of the medium with Andride's reagent, or pozhovtinnya with vicarious medium with bromthymol blue, the same way when tinned with Andrede's indicator and blue bromthymol, do not change the color of the medium. For example, the observation of pathogenic bacteria from the intestines infects the medium, which allows the differentiation of pathogenic microorganisms from the post-intestinal inhabitants - microorganisms that decompose lactose.
Such a middle is the middle of Endo. The main components of the medium Endo are MPA, lactose and basic fuchsin, sodium sulfite. In the middle of the life-giving place, the center was turned into a light erysipelas color. When fermenting lactose, acetaldehyde is dissolved, which reacts with sulfite i, which vibrates with coma, fuchsin turns colonies into a bright red color. For this, the intestinal stick, as it ferments lactose, when growing on this medium, it establishes red colonies with a metal blisk, and salmonella and shigels without a barrel, for that stench does not ferment lactose.
4. The middle of accumulation, in some of them, there is an increase in the number of singful species of microorganisms.
5. Preservative (transport) media. Appointed for the preservation of microorganism during the next hour of transportation to the month of delivery. These mediums should take care of additives, which prevent the reproduction and death of microbes, which saves their lives. Glycerin sum (Tiga's medium), phosphate-buffer sum and Kari-Bleyr's, Amies's (with activated vugillia and without activated vugillya), Stewart and in.
Sterilization of life-giving media.
All life-giving mediums are poured independently from their sign in clean dishes and sterilized. Most mediums are sterilized by autoclaving, but under different conditions, fallow in their warehouse.
1. Synthetic media and all agar media, which should not be kept in one's own warehouse of native protein and carbohydrates, are sterilized for 15-20 minutes in an autoclave at a temperature of 115-120°C and a pressure of 1-1.5 atmospheres.
2. Seredovishcha with carbohydrates and milk (up to the level of lactose), live gelatin is sterilized with a straight steam at a temperature of 100 ° C fractionally or in an autoclave at 112 ° C and with a pressure of up to 1 atmosphere.
3. Seredovishcha, to the warehouse of which enter white speech (blood serum, ascitic motherland), fluctuate with tyndalization or filtration.
4. For the sterilization of life-giving mediums, which are kept in their own warehouse, native proteins are cured by filtration through the Seitz membrane filters.
To control the sterility of the medium after sterilization, place a thermostat at 37°C for 3-5 dB. Some of the middle parts of the guilty are left open, and on the surface in the comrades of the living middles of the innocent are signs of growth. For the control of sterility, the chemical control of the prepared media is determined, which affects the fact that pH, the amount of total and amine nitrogen and chlorides are determined in decals of the skin series.
Іsnuє also biological control of media. In each case, a sprat of sprouts of the middle is planted with a laboratory culture of that microbe, for which the middle is prepared, and the character of the yogo growth is cultivated. Only after that, as the middles showed control, they can be victorious for confessions.
For recognition of living quarters, the middle ones are subdivided into such main categories.
Universal- Medium, on which good growth is rich in species of pathogenic and non-pathogenic bacteria. Before them you can see: meat-pepton broth (MPB = meat water + 1% peptone + 0.5% NaCl), meat-pepton agar (MPA = MPB + 2-3% agar).
Differential and diagnostic- media, which allow one species of bacteria to be treated with other types of enzymatic activity or cultural manifestations. Before them one can see the middles of Endo, Levina, Ploskirev, Gissa and many others.
selective(synonyms: selective, selective, zbagachuvalnі) - middleware, which avenge speech, which are vicarious by microorganisms of the first species and do not take, or cause a change in the growth of other microorganisms. Selective media allow direct selection of bacteria from the studied material. Among these are Muller, Selenitov, Rapoport, 1% peptone water and other.
Differential selective- middles, which will gain the power of differential-diagnostic and selective middles. The stench vikoristovuyutsya, zokrema, for the early identification of that іdentification of bacteria, which can be seen to a large number of widely widened species of enterobacteria and pseudomonads (Syvolodsky's middle).
Special- Mediums, specially prepared for the growth of silent bacteria, so that they do not grow, or else it is nasty to grow on universal mediums. Before them, one can see the middle of McCoy-Chapin (for the prevention of the growth of the tularemia), the blood of the MPA (for the prevention of the growth of pathogenic streptococci), the middle of the Levenshtein-Jensen (for the observation of the tuberculosis of the tuberculosis) and іn.
Synthetic- The middle of a savory chemical warehouse, which is a source of inorganic salts with additions of chemical fields, yak serve as a dzherel of coal or nitrogen. The butt of such a synthetic medium is the minimum medium M-9, in which energy and carbon is glucose, and nitrogen is NH4C1. Synthetic mediums can be more foldable with various amino acids, bases and vitamins included.
Nap_synthetic- synthetic media, to which one can be added, whether it be a natural product, for example, blood serum. There are a lot of different options for living media, designed to meet the needs of different types of bacteria and diagnostic purposes.
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