EntranceMethods for sowing and cultivating bacteria. Koch plate method Koch method microbiology isolation of pure cultures
Pasteur method Koch method Biological Physical
(has historical (lamellar)
meaning) wiring) Chemical Method Shchukevich
Modern
Sowing with a loop Sowing with a spatula
(Drigalski method)
Methods for isolating pure cultures (Scheme 11):
1. Mechanical release methods are based on the separation of microbes by sequential rubbing of the test material over the surface of an agar.
A) Pasteur's method- It has historical meaning, provides for the sequential dilution of the test material in a liquid nutrient medium by the rolling method
b) Koch method– plate method – based on sequential dilution of the test material with meat-peptone agar, followed by pouring test tubes with the diluted material into Petri dishes
V) Drigalski method– when sowing material richly contaminated with microflora, use 2-3 cups for sequential sowing with a spatula.
G) Sowing with a loop in parallel strokes.
2. Biological methods based on the biological properties of pathogens.
A) Biological– infection of highly sensitive animals, where microbes quickly multiply and accumulate. In some cases, this method is the only one that allows isolating a culture of the pathogen from a sick person (for example, with tularemia), in other cases it is more sensitive (for example, isolating pneumococcus in white mice or the causative agent of tuberculosis in guinea pigs).
b) Chemical– based on the acid resistance of mycobacteria. To free the material from accompanying flora, it
treated with acid solution. Only tuberculosis bacilli will grow, since acid-resistant microbes died under the influence of acid.
V) Physical method based on the resistance of spores to heat. To isolate a culture of spore-forming bacteria from
mixtures, the material is heated at 80°C and inoculated on a nutrient medium. Only spore bacteria will grow, since their spores remained alive and gave rise to growth.
G) Shchukevich method– is based on the high mobility of Proteus vulgaris, capable of producing creeping growth.
Method of reseeding from colonies onto slanted agar and MPB:
A) Transferring from colonies to agar slant
Open the lid of the dish slightly, remove part of a separate colony with a calcined, cooled loop, open a test tube with sterile slanted agar, holding it in your left hand in an inclined position, so that you can observe the surface of the medium. Transfer the loop with the culture into the test tube without touching the walls, rub it over the nutrient medium, sliding along the surface from one edge of the test tube to the other, raising the strokes to the top of the medium - streak seeding. The test tube is closed and, without letting go, the name of the inoculated microbe and the date of inoculation are signed.
b) Transferring from the colony to meat-peptone broth
The technique for reseeding on MPB is basically the same as when sowing on solid media. When sowing on the MPB, the loop with the material on it is immersed in the medium. If the material is viscous and cannot be removed from the loop, it is rubbed on the wall of the vessel and then washed off with a liquid medium. The liquid material, collected with a sterile Pasteur or graduated pipette, is poured into the nutrient medium.
As a result independent work the student must know:
1. Methods for isolating a pure culture of microorganisms
2. Methods for cultivating microorganisms
Be able to:
1. Skills in complying with the rules of the anti-epidemic regime and safety precautions
2. Disinfect the material, disinfect hands
3. Prepare preparations from bacterial colonies
4. Microscopy colonies
5. Gram stain microorganisms
LESSON 8
SUBJECT. Methods for isolating pure cultures (continued). Enzymatic activity of bacteria and methods for studying it.
widely used to determine the number of viable microorganisms in soil and other natural substrates. Its use allows not only to take into account the number of microorganisms, but also to evaluate their diversity based on the morphology of the colonies.
Soil samples are taken using a sterile spoon, and the study is carried out on the day the samples are taken. The essence of the method is to sow the soil sample under study onto a dense medium in Petri dishes and then count the grown colonies. It is believed that each colony is the result of the reproduction of one cell. The work is carried out in three steps: preparing dilutions, sowing in dishes, and counting the grown colonies.
Inoculation is done from dilutions of the suspension, depending on the expected number of microorganisms in the substrate under study. Dilutions are made in sterile tap water or isotonic solution sodium chloride. During the experiment they use constant coefficient breeding. Most often, decimal dilutions are made.
A sample of the soil to be analyzed (1-10 g) is placed in a flask with 100 ml of sterile water and shaken. Then transfer 1 ml of the test material with a sterile pipette into a test tube with 9 ml of sterile water. If the test material has already been diluted 100 times, a dilution of 1:1000 is obtained. The suspension of this dilution is thoroughly mixed by taking the resulting suspension into a pipette and releasing it from it. Then, with the same pipette, take 1 ml of the resulting dilution and transfer it to a second test tube - a dilution of 1:10000 is obtained. Subsequent dilutions are prepared in the same way. The degree of dilution is determined by the estimated number of microorganisms in the sample: the more microorganisms in the original substrate, the greater the number of dilutions.
Inoculation is carried out on agar media in Petri dishes. To determine the total number of microorganisms, meat-peptone or fish-peptone agar (MPA, RPA) is used; to determine the content of fungi in the soil, wort agar (SA) is used; to determine the number of various physiological groups and sanitary-indicative microorganisms, appropriate nutrient media. Agar medium melted in a water bath is poured into sterile Petri dishes, 20-30 ml each. The cups are left on horizontal surface until the agar hardens. Using a sterile pipette, apply a certain volume (usually 0.1-0.5 ml) of the appropriate dilution, previously thoroughly mixed, onto the surface of an agar plate in a Petri dish. This volume is distributed over the surface of the medium with a sterile spatula. Then this spatula is passed over the entire surface of the medium in the second and third cups, where the inoculum was not added (exhaustive inoculation method).
From each dilution, 4-6 parallel seedings are made. When inoculating the same dilution in parallel, you can use one sterile pipette and one spatula. Cups with inoculated media are placed in a thermostat adjusted to a temperature favorable for the development of the organisms being detected. Bacteria are counted during cultivation at a temperature of 30 °C after three days, at room temperature - after seven days. Counting yeast and mushrooms - at room temperature after 310 days (at a temperature of 25 ° C, the period of observation of mushrooms can be reduced to 2-3 days).
The number of colonies grown in a Petri dish is counted and recalculated per 1 g. The results of parallel seedings are summed up and the average number of colonies grown when seeded from this dilution is calculated. Colonies are counted without opening the Petri dishes.
The accuracy of the method depends on the number of colonies counted, not on the number of replicates. The best breeding is considered to be one that produces from 50 to 100 colonies when sown on a solid nutrient medium. If the number of grown colonies is less than 10, then these results are discarded and are not used to calculate the number of cells in the original substrate. It is desirable that the total number of colonies counted when sown from a given dilution is at least 300.
The number of microorganisms in 1 g (1 ml) of the initial substrate is calculated using the formula:
T = a x b x c / d,
where T is the number of microorganisms in 1 g, a is the number of colonies counted, b is the dilution from which the seeding was made, c is 10 (if 0.1 ml of suspension was sown onto the dishes), d is the mass of the substrate (soil) taken for analysis
Statistical processing of results is possible only with minimal technical error, so the cup method requires great cleanliness and accuracy when performing all operations. It is necessary to carefully protect pipettes and media from contamination by foreign microorganisms, since an accidentally introduced cell can overestimate the number of microorganisms in the test suspension. Preparation of dilutions and seedings should be done in a box.
The described method is applicable to counting aerobes and facultative anaerobes. To account for strict anaerobes, Petri dishes are placed under anaerobic conditions after inoculation.
Ecological methods for studying soil microorganisms
Relatively few methods are known for isolating bacteria as pure cultures. This is most often done by isolating individual cells on a solid culture medium, using the streak plating method, or by pouring a small amount of liquid culture into plates ( limiting dilution method). However, obtaining a separate colony does not always guarantee the purity of the culture, since colonies can grow not only from individual cells, but also from their clusters. If microorganisms form mucus, then foreign forms are often attached to it. For purification, it is preferable to use a non-selective medium (NSM), since contaminating microorganisms grow better on it and are easier to detect.
Obtaining isolated colonies on a solid nutrient medium is achieved either by sieving a suspension of microorganisms with a spatula ( Koch method), or using a bacteriological loop ( exhaustion stroke method). As a result of mechanical separation of microorganism cells, each of them can give rise to an isolated colony of one type of microbe.
Sieving with a spatula (Koch method) produced in the following sequence:
1) a drop of an enrichment culture is applied to the surface of the nutrient medium in dish No. 1 with a sterile pipette and distributed with a sterile spatula;
2) take out the spatula, quickly close the cup and transfer the spatula to cup No. 2 without sterilizing it. Simulate the distribution of the culture over the entire surface of the medium by touching its surface with the same side of the spatula that was previously used to distribute the sample;
3) exactly the same actions are carried out in cup No. 3, after which the spatula is sterilized;
4) the seeded dishes are placed in a thermostat and incubated at the optimal temperature.
After a certain time, the cups are removed from the thermostat and the growth of microorganisms is studied. Usually, continuous growth of bacteria is observed in cup No. 1, and colonies are noted in subsequent cups.
Loop sieving (draining streak method) involves sowing a bacteriological loop from an enrichment culture onto the surface of an agar medium in Petri dishes. At the first stage, a series of parallel strokes are applied to the agar medium using a loop with the culture (Figure 4.2, A). The loop is sterilized, cooled on the uninoculated part of the agar medium and a series of strokes are made in the direction perpendicular to the first ones (Figure 4.2, B). Then the loop is sterilized again, cooled and strokes are applied in the direction IN(Figure 4.2), and after the next sterilization - in the direction G(Figure 4.2). The cups are placed in a thermostat and the results are taken into account after a certain time. Usually on the strokes A And B grows up big number colonies (sometimes continuous growth), while on streaks IN And G isolated colonies are formed.
Figure 4.2 – Scheme of streak sieving of bacteria to obtain isolated colonies
Serial dilutions in solid medium- the simplest method of sowing plates, which consists in the fact that after inoculating the sample into a test tube with sterile melted and cooled agar, the medium is mixed, poured into a Petri dish and allowed to harden. To obtain well-isolated colonies, prepare a series of successive tenfold dilutions and add 1 ml of samples directly to the cup, add 15–20 ml of molten agar medium and mix by shaking the cup. Sometimes individual colonies end up immersed in agar and can only be removed mechanically. It is also bad that the bacteria spend some time in an environment at the temperature of molten agar.
8. Methods for isolating pure cultures of microorganisms
The cultivation of microorganisms, in addition to the composition of the nutrient medium, is highly dependent on physical and chemical factors (temperature, acidity, aeration, light, etc.). Moreover, the quantitative indicators of each of them are not the same and are determined by the metabolic characteristics of each group of bacteria. There are methods for cultivating microorganisms in solid and liquid nutrient media under aerobic, anaerobic and other conditions.
Methods for isolating pure cultures of aerobic microorganisms. In order to obtain isolated colonies, during application the material is distributed so that the bacterial cells are distant from each other. To obtain a pure culture, two main groups of methods are used:
a) methods based on the principle of mechanical separation of microorganisms;
b) methods based on the biological properties of microorganisms.
Methods based on the principle of mechanical separation of microorganisms
Sieving with a spatula according to Drigalski. Take 3 Petri dishes with nutrient medium. Apply a drop of the test material to the 1st cup with a loop or pipette and rub it with a spatula over the entire surface of the nutrient agar. Then the spatula is transferred to the 2nd cup and the culture remaining on the spatula is rubbed into the surface of the nutrient medium. Next, the spatula is transferred to the 3rd cup and sowing is done in the same way. On the 1st plate the maximum number of colonies grows, on the 3rd plate the minimum number grows. Depending on the content of microbial cells in the material under study, individual colonies grow on one of the dishes, suitable for isolating a pure culture of the microorganism.
Pasteur's method (dilution method). A series of successive, usually tenfold serial dilutions are prepared from the material being studied in a liquid sterile medium or physiological solution in test tubes. Next, the material is sown with a lawn of 1 ml from each tube. It is assumed that in some of the test tubes there will remain a number of microorganisms that can be counted when sown on plate media. This method makes it possible to calculate the microbial number in the material under study. (Microbial count is the number of colonies on the last microbial growth plate multiplied by the dilution rate of the material).
Obtaining a pure culture by sieving in the depths of the medium Koch method (filling method). The test material in a small amount is added to a test tube with melted MPA and cooled to 45-50°C, mixed, then a drop of the nutrient medium with the diluted material is transferred to a second test tube with molten MPA, etc. The number of dilutions depends on the expected number of microorganisms in the material under study. Prepared dilutions of microbes are poured from test tubes into sterile Petri dishes, marked with numbers corresponding to the numbers of the test tubes. After the medium with the test material has gelled, the cups are placed in a thermostat. The number of colonies in the culture medium plates decreases as the material is diluted.
Loop sowing (stroke sowing). Take one Petri dish with nutrient agar and divide it into 4 sectors, drawing demarcation lines on the outside of the bottom of the dish. The test material is inserted into the first sector using a loop and parallel lines are drawn along the entire sector at a distance of about 5 mm from one another. Using the same loop, without changing its position in relation to the agar, draw the same lines on other sectors of the dish. In the place where a large number of microbial cells have fallen on the agar, the growth of microorganisms will be in the form of a continuous streak. In sectors with a small number of cells, individual colonies grow. Alternatively, diluted mixed culture solutions can be poured onto the surface of solid media in plates.
Filtering method. It is based on passing the material under study through special filters with a certain pore diameter and separating the contained microorganisms by size. This method is used mainly for the purification of viruses from bacteria, as well as for the production of phages and toxins (in the filtrate - pure phage, purified toxin).
Methods based on the biological properties of microorganisms
Creating optimal conditions for reproduction
Creation of an optimal temperature regime for selective suppression of the reproduction of accompanying microflora at low temperatures and obtaining cultures of psychrophilic or thermophilic bacteria. Most microbes develop well at 35-37°C, Yersinia grows well at 22°C, Leptospira is cultivated at 30°C. Thermophilic bacteria grow at temperatures outside the temperature ranges of other associated bacterial species (for example, Campylobacter is cultivated at 42°C).
Creating conditions for aerobiosis or anaerobiosis. Most microorganisms grow well in the presence of atmospheric oxygen. Obligate anaerobes grow in conditions that exclude the presence of atmospheric oxygen (causative agents of tetanus, botulism, bifidumbacteria, bacteroides, etc.). Microaerophilic microorganisms grow only at low oxygen content and high CO 2 content (Campylobacter, Helicobacter).
Enrichment method. The material under study is inoculated on selective nutrient media that promote the growth of a certain type of microorganism.
Shukevich method. The test material is inoculated into the condensation water of the agar slant. During reproduction, mobile forms of microbes from condensation water spread throughout the agar, as if “crawling” onto its surface. By sifting the upper edges of the culture into the condensation water of freshly cut agar and repeating this several times, a pure culture can be obtained. Thus, to isolate the culture of Proteus vulgaris, Clostridium tetani, the material is inoculated into condensation water at the bottom of a test tube with a slanted dense medium, without touching the surface of the medium. These microorganisms are capable of creeping growth (swarming) on the surface of the medium. Associated microbes grow in the lower part of the nutrient medium, and the proteus and tetanus microbes in the form of a film spread upward and occupy the entire slanted part of the agar.
Warming up method. Allows you to separate spore-forming bacilli from non-spore forms. Heat the test material in a water bath at 80°C for 10-15 minutes. In this case, the vegetative forms die, and the spores are preserved and germinate when sown on an appropriate nutrient medium.
Bacteriostatic method (inhibition method). Based on the different effects of certain chemicals and antibiotics on microorganisms. Certain substances inhibit the growth of some microorganisms and have no effect on others. For example, small concentrations of penicillin inhibit the growth of gram-positive microorganisms and do not affect gram-negative ones. A mixture of penicillin and streptomycin allows you to free filamentous fungi and yeast from bacterial flora. Sulfuric acid (5% solution) quickly kills most microorganisms, and the tuberculosis bacillus survives under these conditions. It is necessary to take into account that selective factors are often included in the medium in bacteriostatic concentrations, so the accompanying microorganisms remain viable and when transferring colonies of the culture under study to conventional media, they can be the reason for obtaining a mixed culture.