BACTERIOLOGICAL TECHNIQUES — set of the methods and techniques which are applied to detection and allocation of pathogenic or conditionally pathogenic bacteriums from patients, carriers or from objects of external environment.
B. m were improved and enriched during historical development of microbiology and its separate industries. B.'s character of m is defined by a research objective, conditions of dwelling of microbes in the studied object, them biol, properties, and also a pathogeny of a disease, at Krom conduct a bacteriological research. E.g., at the diseases which are followed by bacteremia, the best method of detection of the activator are crops of blood of the patient. In the presence of the expressed focal lesions (diphtheria, dysentery, gonorrhea, etc.) make crops separated or allocations from body, in Krom bacteria are localized. Whatever tasks faced the researcher, they are generally carried out by means of the same B. of the m applied in a certain sequence to allocation and definition of microorganisms.
Allocation of bacteria from the studied material in many respects depends on correctness of its collecting. Character and a technique of capture and transfer of material at various infectious diseases are not identical, but there are general provisions, to-rye it is necessary to consider irrespective of an object of a research and properties of material.
1. The studied material is, whenever possible, collected in aseptic conditions in sterile ware, being guided at the same time by the purposes and problems of this research. E.g., at a research on anaerobe bacterias material is taken from deep layers of fabrics, at diagnosis of diphtheria it is necessary to watch that the tampon made a smear from almonds, but not superficial hiting at by it.
2. At capture of the studied material the relevant document shall be made, in Krom it is necessary to specify date of capture, character of material, a source and precisely to define a research objective.
3. It is necessary to bring the studied material to laboratory, whenever possible, quickly. Long-term storage of material leads to death of bacteria and reduces probability of their detection.
4. If necessary before transfer to laboratory the material which is subject to a research is stored in the cool place.
5. The studied material brought to laboratory is subject to a bystreyshy research.
Bakterioskopichesky research (bacterioscopy) of the studied material — one of widespread B. of m. In laboratory practice apply microscopy of the native and fixed drugs. In the first case drugs are prepared from the fresh raw material, to-ry applied on a pure slide plate, covered with cover glass and mikroskopirut. Slide plates for drugs shall be transparent, pure and fat-free, to have thickness of 1 — 1,2 mm. Processing and purification of glasses are carried out by various ways; one of them is described below. At first glasses boil in 1% soda solution, then wash out water, salt to - that and in conclusion once again carefully water. Glasses store in 95% alcohol in bank with a ground stopper or wiped dry in the closed vessels. During microscopy of the native drugs prepared from pure bacterial growths it is possible to identify at bacteria a number of morphological structures on their histochemical properties, using for this purpose vital coloring, napr, existence of the capsule during the coloring by divorced solutions of methylene blue. The intravital microscopy of bacteria is carried out also in drugs — hanging drop (see).
The bacterioscopy of the fixed drugs is more widely applied. Apply a drop of sterile water on the pure fat-free glass, in to-ruyu bring the calcinated and cooled bacterial loop the studied material.
The drop with material is distributed on glass a uniform thin coat. The rest of material on a loop is destroyed by calcinating. The dense studied material (a phlegm, pus, etc.) is applied on a slide plate and covered with the second glass so that the ends remained free. Having densely pressed glasses to each other, crush material and move apart glasses in the parties, receiving at the same time two uniform smears (fig. 1). Blood and other liquid objects put on the centerline of glass closer by one of the ends. Glass is placed horizontally, holding for his edges. Polished subject (or cover) glass is brought to a drop at an angle in 45 °; after the drop spreads, it is moved on a slide plate (fig. 2). The received smear shall be already, than a slide plate, uniform on density, with smooth edges.
From bodies or bacterial colonies and other objects prepare also contact preparations. The piece of the body fixed by tweezers is put a cut surface to a slide plate on the centerline, and prints of bacterial clumps do on cover glass.
The prepared drugs dry up at the room temperature or in the thermostat. Unstable drug is infectious therefore during the work with it it is necessary to follow carefully rules of precaution, not to touch the surface of glass with hands. The dried-up drug is fixed to the surface of glass. During the fixing of drug there is at the same time its disinfecting. In bacteriological practice the most widespread method of fixing is flaming — fixing by heat in a flame of a torch. For this purpose drug by a smear is carried three times out through a flame up. Action of a flame shall continue on average 2 — 3 sec. Long fixing by heat reduces quality of drug, and nedofiksirovanny drugs at after-treatment are washed away and conceal in themselves danger of infection. During the studying of nek-ry details of structure of bacteria, and also blood smears, prints of bodies use the fixing liquids applied in histologic practice (methyl alcohol, alcohol, spirtformol, Nikiforov's mix, liquid Carnoy, etc.). After that drugs paint (see. Colouring of microorganisms ) also study by means of a microscope.
Widely use method of a bacterioscopy for bacteriological diagnosis of tuberculosis, gonorrhea, a typhinia and other infectious diseases.
Crops and resowings of bacterial cultures are one of basic elements of B. of m. Technology of resowing of bacteria on a slant is as follows. Test tubes with culture or other inoculum and not sowed environment take in the left hand so that the test tube was closer to culture to working, but not sowed with Wednesday — is farther from it. Surfaces of an agar shall be turned up. Take a bacterial loop in the right hand and calcinate it in a flame of a torch, carrying out through a flame and a part of the handle of a petlederzhatel (see. Loop bacterial ). Watch that the calcinated loop did not touch anything. After that cotton stoppers of test tubes take under a little finger of the right hand, take out and hold so that during crops they also did not touch anything. Burning edges of open test tubes in a flame of a torch, the sterilized loop is entered into a test tube with culture. Touch with a loop the site of not sowed agar and if it at the same time does not melt, then it means that the wire is rather cooled. If the agar melts, then a loop, without taking out from a test tube, cool. Touch with the cooled loop the surface of bacterial growth, the easy sliding movement gain culture on a loop and quickly transfer it to not sowed test tube. At transfer it is impossible to touch with a loop edges and walls of both test tubes. The infected loop is lowered in water of condensation and make crops, pounding material on a surface of an agar (fig. 3). Having taken out a loop, burn edges of test tubes and over a flame close them traffic jams. The loop will be sterilized calcinating in a flame of a torch and put in a support. Technology of crops on liquid mediums generally same, as well as at crops on firm environments. During the work with fluid mediums it is necessary to watch that liquid did not pour out from test tubes and did not moisten their edges and cotton stoppers therefore mediums and cultures can never be held in horizontal position.
In nek-ry cases (cultivation of anaerobe bacterias, detection of proteolytic properties) make stab culture in thickness of a medium — an agar or gelatin a column for what puncture with a bacterial loop or a special needle with the sowed culture a medium to the bottom. At the same time a test tube it is possible to hold a bottom up (fig. 4). After crops burn edges of a test tube and at the same time over a flame close it a stopper. The needle is calcinated and put in a support.
Crops of liquid materials on mediums can be made also pasterovsky pipette (see. Pasteur pipette ). At a sterile pipette tweezers break off the soldered end, 2 — 3 times through a flame of a torch carry out it, allow to cool down and, following the rules stated above, gather a small amount of an inoculum, to-ry bring in a test tube with the fresh environment. For the rest work as well as at crops with a loop. The used pipette is placed in a vessel with disinfecting solution.
Test tubes with Wednesday, in to-rye crops were made, it is necessary to inscribe immediately. In a text specify date of crops and character of an inoculum. The sowed and inscribed test tubes place in the thermostat for cultivation of bacteria.
Release of pure growths. Under natural conditions bacteria in most cases are not separately, and in the form of bacterial mixes. Therefore at a research of this or that object it is necessary to consider a possibility of presence of several species of microbes. At the same time for a research and the practical purposes it is necessary to work with pure bacterial culture (see), i.e. with the population containing bacteria of one look. All methods of release of pure growths from microbic mixes can be divided into two basic groups: 1) the methods based on the mechanical principle of division of microbes; 2) biological. Methods of the first group in turn subdivide: a) on methods, unmixing on separate bacterial cells on the surface of nutrient medium, and b) methods of isolation of separate bacterial cells in the micromanipulator under control of an eye is the basis to-rykh. Release of pure growth on solid nutrient mediums can be carried out by sowing of the examinee of material on a surface or in the thickness of Wednesday. Most often use the first way. In either case try to obtain such cultivation of material that at its subsequent sowing on the surface of dense nutrient medium separate bacteria appeared enough far apart on the isolated sites where they breed. At the same time the isolated bacterial colonies during the resowing form to-rykh and pure growth turns out.
Method of sowing on a surface of the dense environment. Take three Petri dishes (see. Petri cup ) with a sterile dense medium. The drop of the studied material with a loop or a pasterovsky pipette is applied on a medium in the first cup, and then the glass or platinum pallet carefully smeared on all surface (fig. 5). Without burning the pallet, the material which remained on it is transferred consistently to the second and third cups and smeared on the surface of nutrient medium. Sowing of material can be done also by means of a loop. At introduction of material it is not recommended to uncover completely a cup, and only slightly to slightly open it. After sowing of a cup overturn upside down and place in the thermostat.
The method of sowing in the depth of Wednesday has more limited value, than previous, and is applied only if it is necessary to count quantity of bacteria in a certain rather large volume of the examinee of material (water, pus and so forth). At this method apply only to gelatin and an agar. In the water bath in three test tubes Wednesday is kindled, and then cooled to t ° 45 °. Bring a certain volume of the studied material in the first test tube; having carefully stirred it in a liquid agar, the same volume from the first test tube transfer to the second, and from it the same volume in a third, every time carefully stirring contents of a test tube. Then contents of each test tube are poured out in a Petri dish (fig. 6). When the agar in cups hardens, they are overturned upside down, inscribed and placed in the thermostat.
In both described methods on the first cup where the concentrated material was sowed, bacterial cells can appear so closely to each other that at the subsequent cultivation separate colonies will merge and will give acervate lawn growth. In the second and third cups, in to-rykh quantity of the bacteria which got there it is much less, separate colonies (fig. 7) are formed. If it is supposed that concentration of bacteria in a starting material is so big that in the second and third cups lawn growth will be created, then the starting studied material before crops needs to be dissolved with normal saline solution or a liquid medium. After stay of cups with crops in the thermostat during the certain time depending on a species of bacteria start release of pure growth. At the same time recognize situation that each isolated colony represents posterity of one bacterial cell which got on the certain site of a medium. Release of pure growth from colony consists in its research and crops on a fresh medium.
Biological methods of release of pure growths are based on the accounting of this or that property of the allocated microbe distinguishing it from others, being with it in the studied material. For release of pure growths it is possible to specify the following as examples of use of biological features of bacteria.
1. Release of pure growth of mobile bacteria by Shukevich's method. The examinee material, from to-rogo is allocated a mobile microbe, sowed in water of condensation of a slant agar; at the same time it is necessary to watch that the loop with material did not concern a surface of an agar over water of condensation. The bacteria having mobility grow not only in water of condensation, but also out of it by surfaces of an agar. From an upper part of growth seeding in water of condensation of a fresh medium is made. Having made thus several resowings, eventually receive pure growth of a mobile bacterium.
2. Release of pure growth by use of various sensitivity of bacteria to high and low temperatures. Nek-ry bacteria form the disputes steady against high temperatures (80 — 100 °); it can use for release of pure growth of the spore-forming bacterium which is in mix with asporous. For this purpose the studied material is warmed up within 10 min. at t ° 80 ° or 2 — 3 min. at t ° 100 °, and then make seedings on cups with an agar. After an incubation in the thermostat on cups colonies of spore-forming bacteria are formed.
3. For simplification of release of pure growths of nek-ry bacteria it is possible to use that they grow at various temperature optimum. E.g., for release of colibacillus from water cultivation is carried out at t ° 45 °; at this temperature the accompanying microbes breed very slowly.
Allocation of a plague microbe from the contaminated material is carried out by cultivation at t ° 20 ° and even 5 °. In the latter case growth though is late, but in much smaller degree, than the accompanying bacteria.
4. Release of pure growths on the basis of various biol, properties of bacteria is widely carried out on so-called selective (elective) mediums (see. Differential and diagnostic environments ).
5. For release of pure growths of some bacteria (a pneumococcus, a tulyaremiyny stick, a stick of a malignant anthrax, etc.) it is possible to use method of infection of an animal sensitive to this infection. E.g., for allocation of a pneumococcus from a phlegm infect a white mouse, edges also the rezistentna to other bacteria which are in a phlegm is very sensitive to this microbe. The pneumococcus quickly breeds in an organism of a mouse, and other microbes perish. In 18 — 20 hours after infection a mouse hammer also the blood taken from heart sow on a medium. Since blood contains only a pneumococcus, on a medium pure growth grows.
Cultivation of bacteria. All bacteria in relation to temperature can be subdivided into 3 basic groups: psychrophile, mesophilic and thermophilic.
The microorganisms allocated from water of cold sources, lakes, the seas and oceans usually concern to group psychrophile. Optimum temperature for them 20 °, and for nek-ry even 5 °. The temperature optimum of mesophilic bacteriums, to the Crimea belongs the majority of pathogenic, lies within 34 — 37 °. And, at last, the optimum of growth of thermophilic bacteria is above an optimum of mesophylls. Nek-ry representatives of this group grow at the high temperatures reaching 65 — 70 °.
Use special devices to creation of optimal heating environments — thermostats (see. Thermostat ), in to-rykh also grow up bacteria. In the conditions of big laboratories use the equipped thermostately rooms. Duration of cultivation depends on a species of bacteria. The majority of bacteria is grown up at optimum temperatures during 24 hours Nek-ry types are incubated much more long time: the causative agent of whooping cough during 2 — 4 days, and a tubercular stick even of 2 — 3 weeks. Duration of an incubation of this or that microbe is defined by dynamics of growth and reproduction of this bacterium and depends on quality of a medium.
The following element B. of m is identification of the microbes which grew in the thermostat (see. Identification of microbes ).
The data received as a result of a research of microorganisms shall be noted in the magazine. Only comparison of all signs of the bacteria revealed during the studying their morphological, biochemical, serological and where it is necessary and biol, properties, can form the basis for identification. The found deviations from the typical characteristic are estimated in connection with the importance of atypical properties for identification of this microbe. Detailed records allow to compare also the microbes allocated under homogeneous conditions or at the same patient. At last, the material collected in the current researches of practical laboratory can be subjected to scientific processing and generalization if it is carefully recorded. The magazine for recording shall contain columns for record of an origin of the studied material, conditions of its collecting, the short characteristic of outward, results of primary crops, studying of morphology of the allocated microbe, its enzymatic activity, results of a serological research and, at last, for the indication of destiny of culture (it is destroyed, left for studying, it is transferred). It is also necessary to enter the column for the answer on a research.
See also Bacteria .
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