29. Basic principles of bacterial cultivation. Factors influencing the growth and reproduction of bacteria. Cultural properties of bacteria.
A universal tool for the production of crops is a bacterial loop. In addition to this, a special bacterial needle is used for inoculation by injection, and metal or glass spatulas are used for inoculation on Petri dishes. For inoculation of liquid materials, Pasteur and graduated pipettes are used along with the loop. The first ones are pre-made from sterile low-melting glass tubes, which are drawn on a flame in the form of capillaries. The end of the capillary is immediately sealed to maintain sterility. For Pasteur and graduated pipettes, the wide end is covered with cotton wool, after which they are placed in special cases or wrapped in paper and sterilized.
When reseeding a bacterial culture take the test tube in your left hand, and with your right hand, grasping the cotton plug with fingers IV and V, take it out, passing it over the burner flame. Holding the loop with the other fingers of the same hand, use it to collect the inoculum, and then close the test tube with a stopper. Then, a loop with inoculum is introduced into the test tube with the slanted agar, lowering it to the condensate in the lower part of the medium, and the material is distributed in a zigzag motion over the slanted surface of the agar. Having removed the loop, burn the edge of the test tube and close it with a stopper. The loop is sterilized in a burner flame and placed on a tripod. Test tubes with cultures are written above d, indicating the date of sowing and the nature of the inoculum (test number or name of the culture).
Sowing with a lawn produced with a spatula on nutrient agar in a Petri dish. To do this, open the lid slightly with your left hand and apply seed material to the surface of the nutrient agar using a loop or pipette. Then pass the spatula through the burner flame, cool it on the inside of the lid and rub the material over the entire surface of the medium. After incubation of the inoculation, uniform continuous growth of bacteria appears.
In order for a culture of microorganisms to grow, multiply and carry out the biosynthesis of any substance normally, favorable environmental conditions are necessary. Under unfavorable conditions, the properties of microorganisms change, their vital activity is suppressed or death occurs. Under unfavorable conditions, the properties of microorganisms change, their vital activity is suppressed or death occurs.
Physical– temperature, environmental humidity, nutrient concentration.
To chemical factors that affect the life activity of microorganisms include: pH of the environment, redox potential (rH2) and the presence of toxic substances in the environment.
Biological factors – come down to the relationship between microorganisms that come into contact during their life activity.
Cultural properties of bacteria– nutritional needs, growth conditions and growth patterns of bacteria on bacteria. environments In nutritional, nitrogen and growth factors, the ability of bacteria to grow on certain nutrient media, in growth conditions - pH, Eh, O2 concentration, density, osmotic pressure of the medium, growth temperature; in the nature of growth - the growth rate (fast, slow), the appearance of the product in liquid, solid and semi-liquid media, the changes that occur in the medium or its individual components during the growth of microbes. Information about K.s. used when choosing cultivation methods and when identifying the isolated plant
30. Principles and methods for isolating pure cultures of aerobic and anaerobic bacteria.
A pure culture is a population of bacteria of one species or one variety grown on a nutrient medium. Many types of bacteria are divided according to one characteristic into biological variants - biovars (syn: biotypes). Biovars that differ in biochemical properties are called chemovars, in antigenic properties - serovars, and in sensitivity to phage - phagevars. Cultures of microbes of the same species, or biovar, isolated from different sources or at different times from the same source are called strains, which are usually designated by numbers or some symbols. Pure cultures of bacteria in diagnostic bacteriological laboratories are obtained from isolated colonies by subculture with a loop into a test tube with a solid or, less commonly, liquid nutrient medium.
A colony is an isolated accumulation of bacteria of one species, or biovar, grown on a dense nutrient medium as a result of the multiplication of one or more bacterial cells. Colonies of bacteria of different species differ from each other in their morphology, color and other characteristics.
A pure culture of bacteria is obtained for diagnostic studies, which consist of identification, i.e., determining the genus and species of the isolated bacteria. This is achieved by studying their morphological, cultural, biochemical and other characteristics (see Scheme 1).
Morphological and tinctorial signs of bacteria are studied by microscopic examination of smears stained by different methods and native preparations.
Cultural properties characterize the nutritional needs, conditions and type of growth of bacteria on solid and liquid nutrient media. These properties are established by the morphology of the colonies and the growth characteristics of the culture.
The biochemical characteristics of bacteria are determined by a set of constitutive and inducible enzymes inherent in a particular genus, species, or variant. In bacteriological practice, saccharolytic and proteolytic characteristics of bacteria, which are determined on differential diagnostic media, are most often of taxonomic importance.
To identify bacteria to genus and species, pigments are important, coloring colonies and cultures in a variety of colors. For example, the red pigment is produced by Serratia marcescens (wonderful blood stick), the golden pigment by Staphylococcus aureus (staphylococcus aureus), and the blue-green pigment by Pseudomonas aeruginosa (blue-green pus stick).
To establish the biovar (chemovar, serovar, phage type), additional studies are carried out to perform the corresponding marker - determination of the enzyme, antigen, sensitivity to phages.
31. Microflora of soil, water, air. Pathogenic species that persist in the external environment and are transmitted through soil, water, food, and air.
The soil. Depending on the depth of the soil layer, the composition of its microflora also changes. In the upper layers, rich in plant and animal remains and well supplied with air, aerobic microorganisms predominate, capable of decomposing complex organic compounds. Deeper soil layers contain less organic compounds and air, resulting in a predominance of anaerobic bacteria.
The soil serves as a habitat for spore-forming rods of the genera Bacillus and Clostridium. Non-pathogenic bacilli (Bac. megatherium, Bac. subtilis, etc.), along with pseudomonads, Proteus and some other bacteria, are ammonifying, forming a group of putrefactive bacteria that mineralize proteins. Pathogenic bacilli (the causative agent of anthrax, botulism, tetanus, gas gangrene) can persist in the soil for a long time.
There are also numerous representatives of fungi in the soil. Fungi participate in soil-forming processes, transformations of nitrogen compounds, and release biologically active substances, including antibiotics and toxins. Toxin-forming fungi, when they get into human food, cause intoxication - mycotoxicosis and aflatoxicosis.
Microflora of water reflects the microbial composition of the soil, since microorganisms mainly enter the water with its particles. Certain biocenoses are formed in water with a predominance of microorganisms that have adapted to the conditions of location, illumination, degree of solubility of oxygen and carbon dioxide, content of organic and mineral substances.
Various bacteria are found in the waters of fresh reservoirs: rod-shaped (pseudomonas, aeromonas), coccoid (micrococci) and convoluted. Water pollution with organic substances is accompanied by an increase in anaerobic and aerobic bacteria, as well as fungi. The microflora of water plays the role of an active factor in the process of self-purification from organic waste, which is utilized by microorganisms. Representatives of the normal microflora of humans and animals (Escherichia coli, Citrobacter, Enterobacter, Enterococcus, Clostridia) and pathogens of intestinal infections (typhoid fever, paratyphoid fever, dysentery, cholera, leptospirosis, enteroviral infections) enter with wastewater. Thus, water is a factor in the transmission of pathogens of many infectious diseases. Some pathogens can even multiply in water (Vibrio cholera, Legionella).
Air microflora interconnected with the microflora of soil and water. Microorganisms also enter the air from the respiratory tract and with drops of saliva from humans and animals. Sun rays and other factors contribute to the death of air microflora. Coccoid and rod-shaped bacteria, bacilli and clostridia, actinomycetes, fungi and viruses are found in the air. Many microorganisms are contained in the air of enclosed spaces, the microbial contamination of which depends on the degree of cleaning of the room, the level of illumination, the number of people in the room, the frequency of ventilation, etc. The number of microorganisms in 1 m3 of air (the so-called microbial number, or air contamination) reflects the sanitary and hygienic air condition, especially in hospitals and children's institutions. Indirectly, the release of pathogenic microorganisms (causative agents of tuberculosis, diphtheria, whooping cough, scarlet fever, measles, influenza, etc.) when talking, coughing, sneezing of patients and carriers can be judged by the presence of sanitary indicator bacteria (Staphylococcus aureus and streptococci), since the latter are representatives of the microflora of the upper respiratory tract and have a common route of excretion with pathogenic microorganisms transmitted by airborne droplets.
32. Sanitary indicator microorganisms. If - titer, if - index, methods of determination.
Sanitary indicators are microorganisms that can be used to indirectly and with an even greater degree of probability judge the possible presence of pathogens in the external environment.
Their presence indicates that the object is contaminated with human and animal secretions, since they constantly live in the same organs as pathogens and have a common route of release into the environment. For example, pathogens of intestinal infections have a common route of excretion (with feces) with such sanitary-indicative bacteria as bacteria of the Escherichia coli group - (the group includes bacteria of the genera Citrobacter, Enterobacter, Klebsiella with similar properties), enterococci, and clostridia perfringens. The causative agents of airborne infections have a common route of excretion with bacteria (cocci) that constantly live on the mucous membrane of the upper respiratory tract and are released into the environment (when coughing, sneezing, talking), therefore, hemolytic bacteria have been proposed as sanitary indicator bacteria for indoor air streptococci and Staphylococcus aureus. Sanitary indicative microorganisms must meet the following basic requirements:
1. must live only in the body of people or animals and are constantly found in their secretions;
2. must not reproduce or live in soil and water;
3. their survival time and resistance to various factors after release from the body into the environment must be equal to or exceed those of pathogenic microbes;
5. methods for their detection and identification must be simple, methodologically and economically accessible;
6. must be found in the environment in significantly larger quantities than pathogenic microorganisms;
7. There should be no closely similar inhabitants - microorganisms - in the environment.
Coli index- the number of Escherichia coli found in 1 liter (for solids of 1 kg) of the object under study; determined by counting colonies of Escherichia coli grown on a solid nutrient medium when sowing a certain amount of the test material, followed by recalculation per 1 liter (kg). Coli index is a value proportional to the actual content of Escherichia coli in the substrate under study.
Coli titer- this is the smallest amount of test material in milliliters (for solids - in grams), in which one E. coli was found. To determine the coli titer, tenfold decreasing volumes of the test material are separately inoculated onto liquid media (for example, 100; 10; 1; 0.1; 0.01; 0.001 ml).
To convert the coli titer to the coli index, divide 1000 by the number expressing the coli titer; To convert the coli index to coli titer, divide 1000 by the number expressing the coli index.
33. Microflora of the human body at different age periods. The role of microbes - permanent inhabitants of the human body in physiological processes. The concept of dysbiosis, its classification, manifestations and methods of treatment.
Microflora is located only on the skin and on the mucous membranes of cavities communicating with the external environment (except for the uterus and bladder). All body tissues are normally completely free of germs.
The natural automicroflora of the body is a single natural complex consisting of a set of heterogeneous microbial communities in various parts of the human body.
Before birth, the human body is sterile; in the womb, the embryo is protected from microbial invasion by the placental and other barriers.
The microflora of the digestive tract is the most numerous and most significant for maintaining human health. Its role is especially great in the developing child’s body.
There are two critical moments in the process of formation of intestinal microbiocenosis. The first is at the birth of a child, when colonization of the sterile intestine begins during the first day, the second is when the child is weaned from breastfeeding.
During childbirth, the baby’s skin and mucous membranes come into contact for the first time with the microflora of the mother’s birth canal, the air, and the hands of medical personnel. As a result, the intestinal microflora of the first days of a child’s life is represented by an association of aerobes (mainly facultative anaerobes) - micrococci, enterococci, clostridia, staphylococci. By the 4-5th day of life, the species composition of fecal microflora becomes more diverse, associations of non-spore-forming anaerobes (bifidobacteria, propionibacteria, peptococci, peptostreptococci, bacteroides and fusobacteria) appear. However, aerobic bacteria still dominate - lactobacilli, cocci, and yeast fungi.
Further formation of automicroflora of the gastrointestinal tract mainly depends on the type of feeding. When breastfeeding healthy full-term babies, already at the end of the first - beginning of the second week of life, the anaerobic component clearly predominates (more than 95%) in the microbial community of the large intestine due to accelerated growth rates. The remaining part (about 4-5%) is represented by a variety of facultative aerobes: lactobacilli, Escherichia, enterococci, epidermal staphylococcus, yeast fungi.
The role of microbes - permanent inhabitants of the human body in physiological processes
Microbial biocenoses support normal physiological functions and play a certain role in immunity. Disturbances in microbial biocenoses in many cases can lead to the occurrence of pathological processes in the corresponding organs.
The microflora of the colon plays an important role. It has pronounced antagonistic properties (especially anaerobic microbes) and prevents the development of pathogenic bacteria that can enter the intestines with food and water, as well as putrefactive bacteria. Microbes - permanent inhabitants of the intestines - produce bacteriocins, antibiotics, lactic acid, alcohols, hydrogen peroxide, fatty acids, which suppress the reproduction of pathogenic species. Thus, intestinal anaerobes are involved in ensuring colonization resistance, as they prevent the colonization (colonization) of the mucous membranes by foreign microorganisms.
Intestinal microbes also participate in the processes of digestion, water-salt, protein, carbohydrate, lipid metabolism, form a protective film on the intestinal mucosa, contribute to the formation and development of the immune system, participate in the neutralization of toxic compounds, synthesize biologically active substances (vitamins, antibiotics, bacteriocins ).
Of great importance is E. coli, which has high enzymatic activity, synthesizes vitamins B1, B2, B12, B5, K, and has antagonistic properties against pathogenic representatives of the family Enterobacteriaceae, against staphylococci and fungi p. Candida.
The concept of dysbiosis, its classification, manifestations and methods of treatment.
Dysbacteriosis(dysbiosis) is a condition that develops as a result of the loss of normal microflora functions. In this case, there is a violation of the existing balance between the types of microbes, as well as between them and the human body, i.e. the state of eubiosis is disrupted. With dysbacteriosis, qualitative and quantitative changes in the bacterial microflora occur. With dysbiosis, there are changes among other microorganisms (viruses, fungi). Dysbacteriosis is caused by various endogenous (internal) and exogenous (external) factors. Most often, intestinal dysbiosis develops.
Type of dysbacteriosis by pathogen:
staphylococcal
Proteaceae
yeast
associated (staphylococcal, proteaceous, yeast)
compensated - there may be no clinical manifestations;
subcompensated - manifestations of dysbiosis sometimes occur with dietary disorders, for example;
decompensated - adaptive mechanisms are exhausted, it is difficult to cure dysbiosis.
BASIC PRINCIPLES OF CULTIVATION OF MICROORGANISMS
Growing microorganisms on nutrient media is called cultivation
(from Lat. c ultus– cultivation), and the resulting microorganisms – culture.
When developing in a liquid medium, cultures form suspension, sediment or film, and when developing on a dense medium – colonies. Culture can be clean
– contain the offspring of cells of only one type and cumulative
– consist predominantly of cells of one type of microorganism.
The introduction of microorganism cells (seed material - inoculum) into a sterile nutrient medium to obtain a pure or enrichment culture is called sowing
. The transfer of already grown cells from one environment to another (sterile) is called reseeding
, or passivation
.
Typically, microorganisms are grown at a certain constant temperature in thermostats (wooden or metal cabinets) or thermostatic rooms. In both, a constant temperature is maintained using thermostats.
Cultivation at a certain temperature is called incubation
, or incubation
.
Microorganisms are grown in glass containers: test tubes, flasks or Petri dishes. To do this, unused glassware is cleaned of alkali by boiling in a solution containing potassium dichromate K2Cr2O7 (6%) or concentrated sulfuric acid H2SO4 (6%).
In test tubes, microorganisms are cultivated in both liquid and solid media. Usually 1/3 of the tube is filled with liquid medium for aerobic cultures, and 2/3 for anaerobic cultures. If the dense medium in the test tubes is intended for the subsequent cultivation of microorganisms, in preparation for sterilization it is poured into 1/3…1/4 of the volume of the test tubes.
After sterilization, the tubes with the medium that has not yet solidified are laid out on a flat table surface in an inclined (slight angle) position to obtain a beveled agar surface. These are the so-called jambs
– oblique or sloping media.
A dense medium frozen when the test tube is in a vertical position is called column
. Columns of nutrient medium, occupying from 1/3 to 1/2 of the volume of the test tube, are used for sowing the culture injection
. Columns of nutrient medium, occupying 2/3 of the volume of the test tube, after sterilization, are used to fill sterile Petri dishes intended for microbiological inoculation.
Test tubes with media and cultures are placed in racks during operation; test tubes with media prepared for sterilization are placed in wire baskets or metal buckets with holes; test tubes with cultures during incubation or storage - in cardboard boxes.
When cultivating microorganisms in flasks, only liquid nutrient media are used. For aerobic microorganisms, the medium is poured into a thin layer (for example, 30 ml into a 100 ml Erlenmeyer flask); for anaerobic microorganisms, the flask is filled to 2/3 of the volume.
In Petri dishes, microorganisms are cultivated only on solid media. The height of this dish is about 1.5 cm, the diameter is from 8 to
10 cm, and the diameter of the upper cup (it serves as a lid) is slightly larger than the diameter of the lower one.
2.1 Technique for sowing and reseeding microorganism cultures
Sowing (and reseeding) of microorganisms is carried out subject to certain sterility rules, which must be followed in order to protect the culture under study from contamination by foreign microorganisms and not to pollute the environment with the studied cultures of microorganisms.
2.1.1 Sowing on solid media in Petri dishes
Sowing in Petri dishes is carried out by surface and deep methods.
2.1.1.1 Surface inoculation method A sterile solid nutrient medium is melted in a water bath in a flask and cooled to a temperature of 50°C.
Remove the Petri dishes from the paper in which they were sterilized and place them on a flat horizontal surface.
Take a flask with a nutrient medium cooled to a temperature of 50°C, remove the cotton plug, burn the edges of the test tube on a burner flame and hold it in an inclined position.
Open the lid of the Petri dish with your left hand, and with your right hand pour the medium into the bottom of the Petri dish, filling its entire surface (Figure 1).
Figure 1 – Filling a Petri dish with agar
Leave the Petri dish on the table until the medium completely hardens, then put it in a thermostat for 15...20 minutes to dry.
Inoculation onto agar plates is done by streaking using a loop or rubbing with a glass or metal spatula.
When sowing with a loop, a small amount of inoculum is captured and easily passed along the surface of the agar, drawing a series of parallel lines or a wavy line (stroking).
When sowing with a spatula, it is removed from the paper and taken in the right hand. Open the lid of the Petri dish with your left hand and insert a spatula into it.
Smear a drop of inoculum (previously added with a pipette or bacteriological loop) with a spatula using rotational movements on the surface of the agar plate (Fig.
nok 2). You should not press the spatula onto the solid medium, as this can damage it. 
Figure 2 - Sowing with a spatula
2.1.1.2 Deep sowing method
Open the sterile Petri dish slightly and place a drop of inoculum onto the bottom of the dish using a loop or pipette.
Melt the agar nutrient medium in a test tube or flask and cool it to a temperature of 45°C.
Burn the edges of the test tube or flask in a burner flame and pour the medium into a Petri dish with added inoculum, observing the rules of sterile work.
The seed material is evenly distributed in the nutrient medium, for which purpose the Petri dish is carefully moved in a circular motion over the table surface.
Leave the Petri dish on the table until the medium completely hardens.
Make an inscription on the Petri dish (number, name of the microorganism). All crops performed using the described methods are placed in a thermostat for growing microorganisms at a temperature favorable for their growth.
2.1.2 Sowing by injection into a column of agar or gelatin
The test tube with agar or gelatin is held bottom up. The material to be inoculated is taken with a platinum needle, which is stuck vertically into the surface of agar or gelatin and moved along the axis of the
tags all the way to the bottom. The needle is then removed, fired and the tube is capped (Figure 3). 
Figure 3 – Sowing by injection
2.1.3 Subculture from tube to tube
2.1.3.1 Subculture onto agar slant
Light the burner. Reseeding is carried out over the burner flame so that the warm air prevents the deposition of microorganisms from the surrounding air and partially destroys them (Figure 4).
a, e – loop sterilization; b – sterilization of the edges of the test tube;
c, d – taking and sowing material; e – closing the test tubes with stoppers
Figure 4 – Reseeding of microorganisms from test tube to test tube
Take a bacteriological loop in your right hand, with the help of which you carry out reseeding (hold the loop like a pencil).
Sterilize the bacteriological loop in a burner flame, calcining the wire red-hot, and at the same time burn the part of the holder adjacent to the loop, which will be inserted into a test tube with a culture of microorganisms. When calcining, the loop is held in the flame almost vertically so that the entire wire is red-hot.
Both test tubes, i.e. the one from which reseeding is being done and the one to be sowed are taken together and held between the thumb and index and middle fingers of the left hand. Moreover, the test tube with a sterile medium is placed further away from you, and with a culture of microorganisms closer to you.
Without releasing the bacteriological loop from the right hand, use the little finger and ring finger of the right hand to press the outer ends of the cotton plugs to the palm and remove the plugs from the test tubes. You cannot place corks on the table.
Lightly burn the edges of the open test tubes in the flame of the burner.
A loop is inserted into a test tube with a culture of microorganisms. In order not to damage the cells of microorganisms, the loop is first cooled by touching the inner surface of the test tube or a nutrient medium free of microbial cells, and only then a small amount of microbial mass is removed.
Remove the loop and insert it into a test tube with a sterile nutrient medium, avoiding touching the walls of the test tube.
Draw a loop from the bottom up in a zigzag or straight line
line-stroke, lightly touching the surface of the agar.
Burn the cotton plugs and the edges of the test tubes simultaneously in a flame and close both test tubes.
Burn the loop in the flame of the burner.
2.1.3.2 Reseeding microorganism cultures into a liquid medium
A graduated sterile pipette is removed from sterile paper by the upper end, the pipette is taken with the middle and thumb of the right hand, without touching the surface of that part of the pipette that will be inserted into the vessel with the liquid medium.
Take a test tube (or flask) with a culture of microorganisms grown in a liquid medium in your left hand and hold it in a vertical position so as not to soak the stopper.
Open the stopper, observing all the rules of sterility described above, and insert the pipette into the test tube.
Pipette a suspension of microorganisms, cap the test tube (or flask), add a certain amount of the suspension to a fresh sterile nutrient medium, observing the precautions already described.
The pipette is placed in a vessel with a disinfectant solution (0.5...3% aqueous solution of chloramine or 3...5% aqueous solution of phenol), without touching surrounding objects.
If reseeding is carried out using a bacteriological loop, then the material added to the liquid nutrient medium for inoculation is ground on the wall of the test tube closer to the liquid and shaken in it.
In laboratory conditions, microorganisms are grown on nutrient media, which must be sterile, transparent, moist, contain certain nutrients (proteins, carbohydrates, vitamins, microelements, etc.), have a certain buffering capacity, have an appropriate pH, and redox potential. Nutrient media are classified by consistency - liquid, semi-liquid, dense (solid); origin - animal or plant origin and synthetic media prepared from certain chemically pure compounds in precisely specified concentrations; by purpose - commonly used (universal), differential, elective and enrichment media, special.
Conventional (simple) media are suitable for cultivating many types of pathogenic and non-pathogenic bacteria. These include meat-peptone broth (MPB), meat-peptone agar (MPA), meat-peptone gelatin (MPG). Meat-peptone agar is prepared from meat-peptone broth by adding 1-2% factory agar, which gives the nutrient medium the consistency of a dense jelly when cooled. Agar is obtained from certain algae.
Differential media make it possible to distinguish bacteria of different species and genera according to their cultural and biochemical properties. These include meat-peptone gelatin, Hiss, Endo, blood agar, Ploskirev’s bactoagar (Bactoagar Zh), etc.
Elective (selective) environments and enrichment environments that favor the growth of certain types of bacteria and suppress the growth of other microbes. These include Petragnani and Gelberg egg media for growing mycobacterium tuberculosis, Dube-Smith media modified by A.P. Alikaeva for growing the causative agent of paratuberculosis, etc.
Special media are the most optimal for growing bacteria that do not reproduce on commonly used media. These include blood agar, serum agar, whey broth, Kitt-Tarozzi medium (MGSHB), Sabouraud medium, etc.
On solid nutrient media, microbes form colonies of different shapes and sizes, which are visible clusters of individuals of the same type of microorganisms, formed as a result of reproduction from one or several cells.
Colonies are characterized by size - large (up to 4 mm), medium (2-4 mm), small (1-2 mm); round, ellipsoidal, bubble-shaped, branched in shape (it can vary depending on nutritional conditions and other environmental influences); surface - shiny, matte, uneven, wrinkled, folded, brain-shaped, smooth, striated; transparency - transparent, cloudy, opalescent; consistency - mucous, viscous, crumbly, mealy, horn-like; edges - smooth, rugged, fringed, uneven, lobed, curl-shaped, bay-shaped, corroded, blurry; profile or relief - flat, raised, convex, depressed, dome-shaped; structure - homogeneous (homogeneous), granular; pigment - no, yes, what color; smell - absent, sharp, which reminds. The study is carried out macroscopically (size, shape, color, transparency) and microscopically (structure and edges of the colony).
In crops grown on liquid nutrient media, surface growth is studied (wall ring, film, flakes, their character); turbidity - weak, moderate, strong, persistent, passing; sediment - dense, cottony, granular, in the form of a piece of cotton wool; its quantity is abundant, meager; color I
Peculiarities of reproduction of various microorganisms. For the cultivation of protozoan spirochetes, nutrient media containing native proteins (serum, blood), pieces of fresh organs and tissues (rabbit kidneys, chicken brain tissue), and synthetic nutrient media consisting of certain amino acids are used.
For the cultivation of pathogenic fungi, as a rule, selective media of a weakly acidic or acidic reaction (pH 6.8-4.5) are used. Selectivity is achieved by selecting nutrients and adding antibiotics or dyes to the media to suppress the growth of bacterial flora. The optimal cultivation temperature is 30-33 "C. Solid Sabouraud media, beer wort agar, etc. are widely used. Among liquid media, sugar broth, beer wort, Czapek-Dox medium, pH 6-6.8, have proven themselves well.
Mycoplasmas, due to their structural characteristics, poorly adapt to nutrient media. Some strains cause turbidity in the medium, others form a light film; some grow in the upper layer of the nutrient medium, others in the bottom part. On solid nutrient media, mycoplasmas form characteristic colonies that resemble fried eggs. At the same time, in primary crops, growth begins on the 3-7th day, while adapted strains grow much faster.
Synthesis of microbial pigments, phosphorescent and aroma-forming substances. Microorganisms, in the process of life activity, synthesize dyes - pigments that give colonies of bacterial cultures a variety of colors and shades, which is taken into account when differentiating microorganisms. There are red pigments (actinomycetes, yeast, fungi, “wonderful stick” - Bact. prodigiosum), yellow or orange (mycobacterium tuberculosis, sarcina, staphylococci), blue (pseudomonas aeruginosa - Pseudomonos aeruginosa, blue milk bacterium - Bact. syncyaneum), purple (Chromobacterium violaceum), black (some types of fungi, yeast, soil microbes). Pigment formation occurs in the presence of oxygen at room temperature and low light. Microorganisms, developing on food products (milk, cheese, meat, fish, butter, cottage cheese), change their color.
There are pigments that are soluble in water (pseudomonas aeruginosa, blue-green milk bacteria - pyocyanin, syncyanin), in alcohol (pigments of the “wonderful” bacteria, staphylococci and sarcin - red, golden, lemon yellow and yellow), insoluble in water , nor in alcohol (black pigments of yeast, fungi, azotobacter), released into the environment (chromonary), remaining in the body of microorganisms (chromophoric).
The physiological significance of pigments in the life of microorganisms has not been fully studied. It is well established that pigment-forming microorganisms are more resistant to the action of physicochemical and biological factors.
Luminous microorganisms (photobacteria), due to oxidative processes in the bacterial cell, have the ability to glow (luminescence). Photobacteria are strict aerobes; when oxygen supply is cut off, their glow stops. The glow of rotten insects, old trees, meat, fish scales, luminous termites, ants, spiders, and other objects observed in nature is explained by the presence of photobacteria in them. Among them are cocci, vibrios, some fungi and bacteria. They develop well on ordinary nutrient media, on fish and meat substrates at temperatures from 15 to 37 ° C. A typical representative of photobacteria is Photobacterium phosphoreum. No pathogenic photobacteria were found.
Flavor-producing microbes have the ability to produce volatile aromatic substances, for example, ethyl acetate and amyl acetate esters, which impart aromatic properties to wines, beer, lactic acid products, hay, soil, etc. A typical representative of aroma-producing bacteria is Leuconostoc cremoris, which is widely used in the production of lactic acid products.
To isolate a pure culture of microorganisms, study their biological properties for the purpose of identification, and also to obtain biomass, it is extremely important to propagate microorganisms in a laboratory. Cultivation, or growing, of microbes is possible only when certain conditions are created for their life activity. Most bacteria, yeasts, and molds are cultivated in artificial nutrient media. Viruses and rickettsiae reproduce only in living cells, tissue culture, chicken embryos, or in the animal body.
Artificial media used for the cultivation of microorganisms must meet certain requirements: be easily digestible, with the necessary composition of nitrogen and carbohydrate substances, vitamins, an extremely important concentration of salts, with a certain pH value (medium pH); have buffering properties ; have an optimal redox potential.
Nutrient media must also contain a sufficient amount of water and must be sterile, i.e., free of microorganisms before sowing. The source of nitrogen in media are various organic, and rarely inorganic compounds. Peptone, which is a product of incomplete protein hydrolysis, is often added to protein-free media. Proteolytic microorganisms can use gelatin (“animal jelly”) as a nitrogenous substance. The source of carbon in nutrient media is often carbohydrates, alcohols, and some organic acids.
To prepare artificial nutrient media, you can use various natural products: milk, blood, whey, meat, chicken egg yolk, potatoes and other organic substances and mineral salts.
Artificial nutrient media are divided into four main groups according to their intended purpose: universal, special, selective (elective) and differential diagnostic.
Universal media include meat-peptone broth and meat-peptone agar, on which many types of pathogenic and non-pathogenic bacteria grow.
Special media are used to grow bacteria that do not grow on universal media. Special foods include foods with milk, blood serum, with the addition of animal blood, glucose, etc. Lactic acid bacteria, pathogenic and other microorganisms are grown on them.
In selective (elective) environments, only bacteria of certain species develop well. Such environments include enrichment environments in which the species of interest to the researcher grows faster than the accompanying bacteria. For example, Kessler's medium, containing gentian violet and cattle bile, is selective for gram-negative E. coli that are resistant to these substances and at the same time selective for sensitive gram-positive
bacteria.
Differential diagnostic media are used to differentiate certain types of bacteria according to their cultural and biochemical properties. These include:
media for determining proteolytic activity (meat peptone gelatin - MPG, milk agar, etc.);
media for determining the fermentation of carbohydrates (Gissa, Eido, Ploskirev media, etc.);
media for determining hemolytic capacity (blood agar and other media with the addition of animal blood);
media for determining the reducing (reducing) ability of microorganisms (Wilson-Blair medium);
selective media used to differentiate prototrophic and auxotrophic bacteria.
In terms of consistency, nutrient media can be solid, semi-liquid or liquid. To obtain media with a dense consistency, add 2-2.5% agar or 10-20% gelatin to liquid media. Semi-liquid media are obtained by adding 0.5-1.0% agar. Agar (in Malay “jelly”) is a dense fibrous substance obtained from red algae and forms a dense gel (jelly) in aqueous solutions. It consists mainly of polysaccharides (70-75%). The main components of agar are high-molecular substances agarose and agaropeptin, which are not broken down or absorbed by microorganisms. In this regard, agar is not a nutrient substrate; it is added to media solely to obtain a dense consistency. Agar melts in water at 100 °C and hardens at 40-43 °C. It is produced in the form of yellowish plates or grayish-white powder.
The osmotic conditions necessary for the life of microbes are created in the nutrient medium by adding sodium chloride or a certain combination of sodium phosphate and potassium phosphate salts. For the life of microorganisms, the reaction of the medium is of great importance - the pH value, which is determined by the ratio of hydrogen (H +) and hydroxyl (OH) ions. It is the logarithm of the number of absolute concentrations of hydrogen ions.
The hydrogen index of a neutral reaction corresponds to 7.0. In this case, the number of hydrogen ions is equal to the number of hydroxyl ions. A reading below 7.0 indicates an acidic reaction, and a reading above 7.0 indicates an alkaline reaction. Microorganisms have adapted to develop in conditions with an extremely wide pH range - from 2.0 to 8.5. Most saprophytic and pathogenic microorganisms are cultivated in a slightly alkaline reaction medium with a pH of 7.2-7.4. For the cultivation of lactic acid bacteria, yeast and molds, an acidic reaction environment is required, pH 5.0-6.5.
Today, many nutrient media are produced in the form of ready-made dry semi-finished media containing all the ingredients necessary for the life of microorganisms. To prepare the nutrient medium, the powder is diluted with water, the resulting mixture is boiled, the pH value is adjusted to an extremely important value and sterilized.
Temperature conditions are of great importance for the growth and reproduction of microorganisms on artificial nutrient media. In relation to the temperature regime, all microorganisms are divided into three groups: psychrophilic (cold-loving), mesophilic (average), thermophilic (heat-loving). The temperature limits of reproduction for psychrophiles range from 0 to 20 °C, for mesophiles - from 20 to 45 °C, for thermophiles - from 45 to 70 °C.
When growing aerobes, the crops are cultivated in thermostats with access to atmospheric oxygen, i.e. under normal conditions. For the cultivation of anaerobes, oxygen-free conditions are created, which can be achieved by physical, chemical and biological methods. Anaerobic thermostats are also used.
Physical methods are based on creating a vacuum in special anaerostat apparatus or in vacuum desiccators, in which the crops are first placed, and then a vacuum is created in the apparatus.
Sometimes the air in anaerostats is replaced with carbon dioxide, nitrogen or other inert gas. The access of oxygen to the nutrient medium can be hampered if anaerobes are cultivated deep in a column of nutrient agar or inside sealed glass tubes. Anaerobic conditions can be created in simpler ways: using a layer of agar poured over the crops on a dense nutrient medium, or using vaseline oil, which is used to cover a liquid nutrient medium (Kitta-Tarozzi medium). Chemical methods consist of using a desiccator With Chemical substances are placed in the crops, for example pyrogallol and alkali, the reaction between which occurs with the absorption of oxygen.
Biological method is based on the simultaneous cultivation of aerobes and anaerobes on solid nutrient media in hermetically sealed Petri dishes. In this case, oxygen is absorbed by growing aerobes seeded on one half of the medium, after which the growth of anaerobes begins, seeded on the other half.
To isolate a pure culture of microorganisms, study their biological properties for the purpose of identification, and also to obtain biomass, it is necessary to propagate microorganisms in a laboratory. Cultivation, or growing, of microbes is possible only if certain conditions are created for their life activity. Most bacteria, yeasts, and molds are cultivated in artificial nutrient media. Viruses and rickettsiae reproduce only in living cells, tissue culture, chicken embryos, or in the animal body.
Artificial media used for the cultivation of microorganisms must meet certain requirements: be easily digestible, with the required composition of nitrogenous and carbohydrate substances, vitamins, the required salt concentration, with a certain pH value (medium pH); have buffering properties; have an optimal redox potential.
Nutrient media must also contain a sufficient amount of water and must be sterile, i.e., free of microorganisms before sowing. The source of nitrogen in media can be various organic, and rarely inorganic compounds. Peptone, which is a product of incomplete protein hydrolysis, is often added to protein-free media. Proteolytic microorganisms can use gelatin (“animal jelly”) as a nitrogenous substance. The source of carbon in nutrient media is often carbohydrates, alcohols, and some organic acids.
To prepare artificial nutrient media, you can use various natural products: milk, blood, whey, meat, chicken egg yolk, potatoes and other organic substances and mineral salts.
Artificial nutrient media are divided into four main groups according to their intended purpose: universal, special, selective (elective) and differential diagnostic.
Universal media include meat-peptone broth and meat-peptone agar, on which many types of pathogenic and non-pathogenic bacteria grow. Special media are used to grow bacteria that do not reproduce on universal media. Special media include milk, blood serum, with the addition of animal blood, glucose, etc. Lactic acid bacteria, pathogenic and other microorganisms are grown on them.
In selective (elective) environments, only bacteria of certain species develop well. Such environments include enrichment environments in which the species of interest to the researcher grows faster than the accompanying bacteria. For example, Kessler's medium, containing gentian violet and cattle bile, is selective for gram-negative Escherichia coli resistant to these substances and at the same time selective for sensitive gram-positive bacteria.
Differential diagnostic media are used to differentiate certain types of bacteria according to their cultural and biochemical properties. These include:
media for determining proteolytic activity (meat peptone gelatin - MPG, milk agar, etc.);
media for determining the fermentation of carbohydrates (Hiss, Endo, Ploskirev media, etc.);
media for determining hemolytic ability (blood agar and other media with the addition of animal blood);
media for determining the reducing (reducing) ability of microorganisms (Wilson-Blair medium);
selective media used to differentiate prototrophic and auxotrophic bacteria.
The consistency of nutrient media can be solid, semi-solid and liquid. To obtain media with a dense consistency, add 2-2.5% agar or 10-20% gelatin to liquid media. Semi-liquid media are obtained by adding 0.5-1.0% agar. Agar (in Malay “jelly”) - dense a fibrous substance obtained from red algae and forms a dense gel (jelly) in aqueous solutions. It consists mainly of polysaccharides (70-75%). The main components of agar are high-molecular substances agarose and agaropeptin, which are not broken down and not absorbed by microorganisms. therefore, agar is not a nutrient substrate, it is added to media solely to obtain a dense consistency.Agar melts in water at 100 ° C and solidifies at 40-43 ° C. It is produced in the form of yellowish plates or grayish-white powder.
The osmotic conditions necessary for the life of microbes are created in the nutrient medium by adding sodium chloride or a certain combination of sodium phosphate and potassium phosphate salts.
For the life of microorganisms, the reaction of the environment is of great importance - the pH value, which is determined by the ratio of hydrogen (H +) and hydroxyl (OH -) ions. It is the logarithm of the number of absolute concentrations of hydrogen ions.
The hydrogen index of a neutral reaction corresponds to 7.0. In this case, the number of hydrogen ions is equal to the number of hydroxyl ions. A reading below 7.0 indicates an acidic reaction, and a reading above 7.0 indicates an alkaline reaction. Microorganisms have adapted to develop in conditions with an extremely wide pH range - from 2.0 to 8.5. Most saprophytic and pathogenic microorganisms are cultivated in a slightly alkaline reaction medium with a pH of 7.2-7.4. For the cultivation of lactic acid bacteria, yeast and molds, an acidic reaction environment is required, pH 5.0-6.5.
Currently, many nutrient media are produced in the form of ready-made dry semi-finished media containing all the ingredients necessary for the life of microorganisms. To prepare the nutrient medium, the powder is diluted with water, the resulting mixture is boiled, the required pH value is adjusted and sterilized.
Temperature conditions are of great importance for the growth and reproduction of microorganisms on artificial nutrient media. In relation to the temperature regime, all microorganisms are divided into three groups: psychrophilic (cold-loving), mesophilic (average), thermophilic (heat-loving). The temperature limits of reproduction for psychrophiles range from 0 to 20 °C, for mesophiles - from 20 to 45 °C, for thermophiles - from 45 to 70 °C.
When growing aerobes, crops are cultivated in thermostats with access to atmospheric oxygen, i.e. under normal conditions. For the cultivation of anaerobes, oxygen-free conditions are created, which can be achieved by physical, chemical and biological methods. Anaerobic thermostats are also used.
Physical methods are based on creating a vacuum in special anaerobic apparatus or in vacuum desiccators, in which crops are first placed, and then a vacuum is created in the apparatus.
Sometimes the air in anaerostats is replaced with carbon dioxide, nitrogen or other inert gas. The access of oxygen to the nutrient medium can be hampered if anaerobes are cultivated deep in a column of nutrient agar or inside sealed glass tubes. Anaerobic conditions can be created in simpler ways: using a layer of agar poured over crops on a dense nutrient medium, or using petroleum jelly to cover a liquid nutrient medium (Kitta-Tarozzi medium).
Chemical methods consist in placing chemicals, such as pyrogallol and alkali, into a desiccator with crops, the reaction between which occurs with the absorption of oxygen.
The biological method is based on the simultaneous cultivation of aerobes and anaerobes on solid nutrient media in hermetically sealed Petri dishes. In this case, oxygen is absorbed by growing aerobes sown on one half of the medium, after which the growth of anaerobes begins, sown on the other half.
FORMATION OF PIGMENTS AND AROMATIC SUBSTANCES BY MICROORGANISMS. GLOW OF MICROBES
Pigments. Some types of bacteria and fungi that live in soil, water and air are capable of producing coloring substances called pigments.
Pigments are divided into water-soluble, alcohol-soluble, water-insoluble and alcohol-insoluble. There are also chromopair pigments that enter the external environment, and chromophoric pigments located in the cytoplasm, vacuoles and membrane.
The formation of pigments occurs with good access to oxygen, in most species in diffuse sunlight and an optimal temperature of 20-25 ° C.
Microorganisms secrete various pigments, the color of which is determined by the color of colonies on a solid nutrient medium, and sometimes by the color of a liquid nutrient medium. The water-soluble blue pigment pyocyanin is produced by Pseudomonas aeruginosa. The pigment causes defects in milk, turning it blue. The green water-soluble pigment fluorescein is produced by fluorescent rods (Ps. fluorescens); The red, alcohol-soluble pigment prodigiosin is produced by the miracle stick (Serratia marcescens). Red pigments can also be produced by actinomycetes and yeast, pink pigment by yeast and pink micrococcus. Staphylococci produce golden, white and yellow pigments. Sarcin colonies are yellow, lemon or golden in color. Molds produce predominantly water- and alcohol-insoluble pigments of black, green, brown, and chocolate brown colors. Brown pigment is produced by some strains of spore-forming putrefactive aerobes (mushroom sticks, cabbage sticks).
In the absence of favorable conditions, pigment-forming microorganisms do not produce pigments and form colorless (grayish-white colonies).
Pigment formation in microbes has a certain physiological significance. Pigments provide cell protection from natural ultraviolet radiation, participate in biochemical reactions, and have an antibiotic effect.
Aromatic substances. Some microorganisms, during their life processes, produce volatile aromatic substances that give dairy products (butter, cheese) a pleasant specific smell and taste. Of these substances, the most important are diacetyl, volatile acids, ethyl alcohol, ethyl acetate and amyl acetate ethers, etc.
Among lactic acid bacteria, aroma formation is most intense in heterofermentative lactic acid streptococci Lactococcus diacetylactis, Leuconostoe cremoris, Leuconostoc dextranicum.
The intensity of aroma formation is influenced by the milk fermentation temperature, reaction and redox conditions of the environment. The optimal conditions for aroma formation for lactic acid streptococci are: temperature 23-25 ° C; pH of the environment is about 5.0; redox potential Eh 6; periodic stirring of the starter to enrich it with oxygen.
During long-term storage of the product, aromatic substances are destroyed, especially at high temperatures above zero.
Glow microorganisms. Glow (luminescence) is a unique form of energy release during oxidative processes. Glowing microorganisms can cause various foods (meat, fish, cheese, etc.) to glow. They penetrate the body of small crustaceans, causing these animals to glow brightly at night near the seashore. In some fish, luminous bacteria are permanent symbionts (cohabitants), serving as a source of light. Some mushrooms that live in old stumps and tree roots glow.
Glowing bacteria are called photobacteria. These include some cocci, vibrios, and rods that stain negatively on Gram and do not form spores.
Most types of luminous bacteria are aerobes, they do not cause decay, grow on fish and meat substrates, and are cultivated in ordinary environments. The optimal growth and glow temperature is 15-18 ° C, sodium chloride content is about 3%. A typical representative of photogenic microbes is Fotobacterium phosphoreum - a motionless coccoid rod that develops at 28 °C; growth stops at temperatures above 30 °C. Proteolytic properties are not expressed, gelatin does not liquefy.
The development of photogenic microbes is suppressed by reducing the concentration of salts in the environment, under the influence of sulfonamide and other chemicals, sound vibrations, mechanical rubbing, extraction with various solvents, slow autolysis, etc.