Bacteriological techniques

Bacteriological equipment - a set of methods and techniques used bacteriological examination. Bacteriological techniques include detection methods, cultivation and selection of bacteria from the investigated material with their subsequent identification.
The main methods of bacteriological tests. One of the common methods of bacteriological technology is making preparations for microscopy (see). Fat-free glass evenly distribute drop of the suspension, containing the material. Dense material is put on a glass slide, cover with a second glass (Fig. 1), then glass pushed to the side, getting two stroke; liquid objects (blood and so on) put on the middle line of glass, polished glass down to the drop angle (Fig. 2) and, after a drop will be spread, moving from right to left. Smear dried at room temperature or in the thermostat, then fixed to the surface of the glass in the flame of the burner; for that glass of smear-up threefold carried out through the flames. When studying the details of the structure of bacteria, blood smears, prints agencies use locking liquids (alcohol, mixture Nikiforova, liquid, Carnoy and others).
For colouring use the following dyes: red - basic fuchsin, Magenta sour, safranin, neutral red, Congo red; blue methylene blue, toluidine blue, opal blue, violet - gentian violet, crystal violet. Simple methods of colouring bacteria - colouring by water solution of methylene blue, alkaline blue the Loeffler and Magenta of Pfeifer. Sophisticated methods of colouring: gram, Zn Nelsen, Romanovsky - Giemsa, Nassera and others,are used for differentiation of bacteria; to study the structure of the bacterial cell - Bennetti method (see), Gypsum method (see), Leffler method, etc. After coloring the drug is washed with water and dried.
The main elements of bacteriological techniques - crops and subcultures bacterial cultures. Test tubes with the culture and the early years of the environment, taking in the left hand; in the right hand take bacterial loop and calcined in the flame of the burner. Then plug from the tubes are removed, capturing them with the little finger of the right hand. Burn the edges of open tubes, enter sterilized loop in a test tube culture, cool loop of the tube wall and touched it to the surface of bacterial growth, a slight movement gaining culture, quickly transfer loop in the early years tube and produce sowing (Fig. 3). Over the flame close tubes tubes and sterile loop roasting. When planting in liquid media using the Pasteur pipette. Sometimes produce a crop prick in thickness of environment, piercing bacterial loop or a special needle nutrient medium to the bottom (Fig. 4).
Isolation of pure cultures. For research and practical purposes it is necessary to work with a pure bacterial culture (see). There are methods to obtain pure cultures of biological and based on the mechanical principle of separation of microbes. Method sieving surface dense environment: a drop of the investigated solution put on a solid growth medium in a Petri dish, cover them slightly open (Fig. 5); then drop rubbed shpatelem; remaining on him, the material is transferred sequentially in the second and third cups. Sown Cup placed in the thermostat.
If necessary, count the number of bacteria in the test material used methods of screening in the depth of the layer. Make three consecutive breeding material in test tubes with a certain amount of melted and cooled agar, which is then poured into a Petri dish (Fig. 6). When high concentrations of bacteria in the source material it first bred in the physiological solution. On each successive Cup of the number of bacterial colonies decreased respectively breeding (Fig. 7). The counting of colonies produced in the chamber of Wolfhugel.
Biological methods obtain pure cultures based on the properties of allocated microbe: 1) allocation of net culture motile bacteria method Shukevich: the tested material sow loop in the condensation water oblique plate, avoid touching the surface of the environment; motile bacteria grow not only in the condensation water, but also on the surface of the agar; 2) allocation of net culture through the use of different sensitivity of bacteria to low and high temperatures. This method is used to obtain pure cultures spore-forming bacteria. To obtain pure cultures of some bacteria can use the body of the animal, susceptible to this infection.
The cultivation of bacteria. All the bacteria in their relation to temperature fall into three main groups: psychrophilic (temperature optimum growth 5-20 degrees), mesophilic (most pathogenic bacteria; optimum temperature 34-37 degrees) and thermophilic (temperature optimum growth 65-70 degrees). Duration of cultivation depends on the type of bacteria and ranges from 24 hours. to 2-3 weeks. To create the required temperature conditions apply thermostats. Deep cultivation of bacteria - see Aeration in Microbiology; cultivation anaerobov - see Anaerobes.
Cm. also Bacteriological examination.

Fig. 1. Preparation smear.
Fig. 2. Preparation of a blood smear.
Fig. 3. Inoculation of bacteria on the beveled agar.
Fig. 4. Inoculation prick.
Fig. 5. Sowing on a Petri dish.
Fig. 6. Bottling agar culture for growing deep in the environment.
Fig. 7. The growth of bacteria in Petri dishes with successive planting.