INSTITUTIONS OF HIGHER EDUCATION
V. N. KISLENKO
WORKSHOP
ON VETERINARY
MICROBIOLOGY
AND IMMUNOLOGY
Approved by the Ministry Agriculture RF in
quality study guide for university students
educational institutions students in the specialty
"Veterinary"
MOSCOW "Colossus" 2005
UDC 619: 579 (075.8) BBK 48ya73 K44
Editor T.S. Moyaochaeva
Reviewers: Doctor of Veterinary Sciences, Professor V. I. Pleshakova(Veete Institute
rhinary medicine of the Omsk State Agrarian University); doctor of veterinary sciences, professor IT. I. Ba
Ryshnikov(Institute of Veterinary Medicine of the Altai State Agrarian University)
Kislenko V. N.
K44 Workshop on veterinary microbiology and immunology. - M.: KolosS, 2005.- 232 p. l .: ill. - (Textbooks and textbooks for students of higher educational institutions). ISBN 5-9532-0332-2
The workshop consists of two sections. The section "General Microbiology" contains information about the rules of work in a bacteriological laboratory, a description of the main microbiological, genetic and immunological methods for studying microorganisms. In the section "Pathogens of infectious diseases" methods of laboratory diagnostics, differentiation of pathogens are listed and a list of biological preparations used is given.
Guidelines for conducting practical classes for teachers are given.
Attached are sets of tests on electronic media (CD-disk) for general and particular microbiology, immunology, as well as for a theoretical course.
For students of higher educational institutions in the specialty "Veterinary".
INTRODUCTION
Veterinary laboratories are institutions of the state veterinary service, whose activities are aimed at ensuring welfare in animal husbandry, preventing and eliminating diseases and death of animals, as well as protecting the population from diseases common to animals and humans. By appointment, veterinary laboratories are district, inter-district (zonal), regional (territorial) and republican.The main task of veterinary laboratories is to establish an accurate diagnosis of diseases of farm animals, including birds, fur-bearing animals, fish and bees, as well as to conduct an examination of meat, milk and other food products of animal and vegetable origin. The laboratories also perform scientific work, carry out the production of some biostimulants, antibiotics, etc.
The material for laboratory research is blood, urine, sputum, milk, feces, the contents of abscesses (pus) obtained during the life of the animal; pieces of parenchymal organs or other tissues after their death, samples of environmental objects (water, air, soil, feed, plants, washings from care items). The material in the laboratory is examined by bacteriological, serological, histological, biochemical, mycological and toxicological methods, for which the necessary conditions(specially designated premises, equipment, microclimate, etc.).
The laboratory occupies a separate building, located away from the roads. It should have admission department, pathoanatomical, bacteriological, serological, biochemical and virological departments, as well as special rooms for thermostats, washing dishes, autoclave. In the dishwashing room there are tables, sinks, hot and cold water supply, a gas or electric stove, racks for washed dishes, a fume hood, enamel baths, basins and other containers, an acid solution in glass vessels for disinfecting pipettes, slides and other utensils . A separate room is allocated for a bacteriological kitchen (environment-rock), where nutrient media are prepared for cultivating microorganisms, and dishes are prepared for sterilization. Here in the cabinets they store sterile dishes and well-packed chemical substances, Components culture media.
To perform work in aseptic conditions, special isolated rooms are equipped - boxes(English box - box), consisting of the box itself and the pre-box. They also use desktop boxes, objects and air in which are disinfected with UV lamps, and laminar flow cabinets, where active air removal is also used.
Laboratory animals (white mice, guinea pigs, white rats, rabbits, etc.) are placed in vivariums. As a rule, the vivarium also contains healthy donor sheep, whose blood is used for the complement fixation reaction (CFR) and the preparation of nutrient media. Infected laboratory animals are kept in an isolated room.
In addition, the building has rooms for specialists, service personnel, the manager's office, a library, a weighing room, a locker room, a warehouse, etc.
To maintain proper cleanliness, the floor in the rooms is covered with linoleum or tiles. Walls and ceilings, as a rule, are smooth (without cornices and moldings), with rounded corners, painted in light colors with oil paint. Ceilings can be whitewashed with lime. It is desirable to veneer the walls with plastic or tiles from floor to ceiling.
The laboratory should have hot and cold water, sewerage, foot-operated trash cans that are emptied, washed and disinfected daily, towels, soap and disinfectant solution. The rooms contain only the most necessary equipment: tables, a cabinet for storing small equipment, paints, reagents, dishes, tools, etc. Tables are usually installed in front of windows. They should be stable, comfortable, 80 cm high, with a rim. The surface of the tables is covered with plastic or linoleum, glass or white special paint. A microscope is placed on the table, as well as the necessary items for bacteriological work.
The bacteriological research method, as a rule, includes microscopy, isolation and study of the properties of a pure culture of the pathogen and infection of laboratory animals (biological sample). The results of bacteriological analysis, signed by the head of the department or the director of the laboratory, are reported only to officials: a veterinarian, a zoo engineer, a head of an enterprise.
Microbiological laboratory equipment.
To work in the laboratory, the following instruments and apparatus are required: biological immersion microscopes with additional devices (illuminator, phase-contrast device, dark-field condenser, etc.), luminescent microscopes, thermostats, sterilization equipment, pH meters, devices forobtaining distilled water (distiller), centrifuges, technical and analytical scales, filtering equipment (Seitz filter, etc.), water baths, refrigerators, an apparatus for making cotton-gauze plugs, a set of tools (bacteriological loops, spatulas, needles, tweezers and etc.), laboratory glassware (test tubes, flasks, Petri dishes, mattresses, vials, ampoules, Pasteur and graduated pipettes), etc.
The laboratory has a special place for staining microscopic preparations, where there are solutions of special dyes, alcohol, acids, filter paper, etc. Each workplace is equipped with a gas burner or spirit lamp, a jar with a disinfectant solution. For daily work the laboratory must have the necessary nutrient media, chemical reagents, diagnostic preparations and other laboratory materials. In large laboratories there are thermostatic rooms for the mass cultivation of microorganisms, setting up serological tests.
The following equipment is intended for cultivation, storage of cultures, sterilization of laboratory glassware and other purposes:
Thermostat. An apparatus that maintains a constant temperature. The optimum temperature for the reproduction of many microorganisms is 37 "C. Thermostats are air and water.
Microanaerostat. An apparatus for growing microorganisms under anaerobic conditions.
Refrigerators. Used in microbiological laboratories to store cultures of microorganisms, nutrient media, blood, sera, vaccines and other biological preparations at a temperature of about 4 °C. To store preparations below 0°C, low-temperature refrigerators are designed, in which the temperature is maintained at -20 °C and below.
Centrifuges. Used for sedimentation of microorganisms, erythrocytes and other cells, separation of inhomogeneous liquids (emulsions, suspensions). In laboratories, centrifuges with different operating modes are used.
Drying and sterilizing cabinet (Pasteur oven). Designed for air sterilization of laboratory glassware and other materials.
Steam sterilizer (autoclave). It is intended for steam sterilization under pressure. In microbiological laboratories, autoclaves of various models are used (vertical, horizontal, stationary, portable).
Rules of work in the microbiological laboratory.
The microbiologist deals primarily with pure cultures of microorganisms, which are the offspring of a single cell. Since there are always a variety of microorganisms in the air and on the surface of objects in the laboratory (on tables, instruments, instruments, as well as on clothes, hands, etc.), you should constantly take care to maintain the purity of the cultures under study. Therefore, when working in microbiological laboratory certain rules must be strictly observed, one of which is the maintenance of cleanliness, including daily hygienic cleaning of all premises.There are various methods of disinfection to kill microorganisms in the air and on surfaces.
The air in the laboratory is partially purified by ventilation. Ventilation dramatically reduces the number of microorganisms in the air, especially when there is a significant difference in temperature outside and inside the room. The duration of ventilation is at least 30 ... 60 minutes.
A more effective and most commonly used method of air disinfection is exposure to ultraviolet radiation (UVR), which has a high antimicrobial effect and causes the death of not only vegetative cells, but also spores of microorganisms. Due to the weak penetrating power, ultraviolet radiation does not pass through ordinary glass and is easily absorbed by dust particles. Therefore, for sterilization, the exposure time is from 30 minutes to several hours, depending on the degree of air pollution.
Germicidal lamps (UFL) are used as a source of UV radiation. The emitter in them is an electric arc that occurs in low-pressure mercury vapor and emits a linear spectrum in the ultraviolet region, more than 80% of the energy of which falls at a wavelength of 2.5 nm.
A bactericidal lamp is a glass tube mounted between two electrical contacts and connected to the network through a choke. The tube is made of a special glass that transmits all rays with a wavelength of 2.5 nm and delays radiation with a wavelength shorter than 2 nm. It should be remembered that UV radiation causes acute inflammation of the cornea of the eyes with characteristic lacrimation and photophobia shortly after irradiation. Therefore, in order to prevent direct or reflected ultraviolet rays from affecting the eyes, goggles are used. It is impossible to be in small rooms with the bactericidal lamp turned on.
The floor, walls and furniture in the microbiological laboratory are wiped with solutions of various disinfectants. Vacuuming removes dust and a significant part of the microflora from objects. As disinfectant solutions, 0.5 ... 3% is most often used. water solution chloramine. Particular care should be taken to disinfect the surface of the table on which work with microorganisms is carried out. It must be wiped with a disinfectant solution both before and after work. Extra items are not allowed on the desktop. All reagents and solutions must be labeled and in strictly defined places. Eating, drinking and smoking are not permitted in the laboratory. Work should be in bathrobes.
Under laboratory conditions, microorganisms are grown on solid and liquid nutrient media, which are poured into test tubes, flasks or Petri dishes. Dishes and culture media are pre-sterilized. The introduction of microorganism cells into a sterile environment is called seeding, or inoculation. Sowing (or reseeding) of microorganisms requires strict adherence to certain rules in order to protect the culture under study from contamination by foreign microorganisms.
Inoculation of microorganisms in sterile environments is best done in special rooms - boxes. Boxing is a small isolated room, divided into two parts by a partition. The main working room of the box is entered through the vestibule, which has a sliding door, which excludes a sharp movement of air and, consequently, the introduction of foreign microflora from the outside. Boxing equipment includes a table with an easy-to-clean surface, a chair, gas or alcohol burners, a bactericidal lamp mounted in a special stand or mounted on the ceiling of the box. It is convenient to have a utility table in the box, on which items necessary during work will be placed. All box equipment, its walls, floor and ceiling are periodically washed and wiped with disinfectant solutions. Before work, the box is irradiated with a bactericidal lamp for 40...60 minutes.
After sowing, test tubes or other vessels in which microorganisms are grown are placed in thermostats, where a constant temperature is maintained with the help of thermostats. Vessels containing microbial cultures to be disposed of are autoclaved to kill the cells before being washed. A disinfectant solution is poured into dishes with dense media, which is removed after a day, and the dishes are washed. Careless handling of microorganism cultures leads to the formation of a bacterial aerosol that poses a health hazard to employees.
All employees of laboratories, as well as departments of microbiology, graduate students, students who come to classes and work in scientific student circles, before starting work with infectious material (cultures of pathogenic microbes, corpses of experimentally infected animals, excretions of sick animals, blood, etc.) ) are obliged to familiarize themselves with and strictly observe the following rules of work and safety precautions in veterinary bacteriological laboratories:
enter the laboratory premises only in special clothes: in a dressing gown, a white cap or a scarf. The dressing gown should be tightly buttoned, hair tucked under a cap;
Do not bring any items or food into the laboratory. Briefcases and bags are folded in a specially designated place;
it is strictly forbidden to eat, drink and smoke in the laboratory;
each employee (student) under a certain number is allocated a workplace, a microscope and other accessories;
the workplace should have equipment only for a specific task. Usually this is a set of paints, a flask with distilled water, a drain cup, jars with clean and used glasses, a bacteriological loop, a tripod, a jar with a disinfectant solution;
before starting work, it is necessary to check the availability and serviceability of appliances, utensils, gas burners (alcohol lamps), etc. The observed shortcomings and malfunctions should be reported to the responsible person, and in the classroom - to the teacher;
in order to avoid an explosion, it is impossible to light one spirit lamp (or gas burner) from another; use only matches;
do not touch the wires and contact parts of the electrical network with metal and other objects;
students without the knowledge of the teacher or attendants should not turn on electrical appliances and equipment;
students begin to complete the task only with the permission of the teacher; the course of work must strictly correspond to the method being studied;
each: the employee and the student must observe neatness in work, keep the workplace and equipment clean;
the material used in the training sessions is taken as especially dangerous;
when unpacking the material sent for research, care must be taken: the outside of the jars is wiped with a disinfectant solution and placed only on trays or cuvettes;
in the study of the material received and when working with bacterial cultures, they observe the technical methods generally accepted in bacteriological practice, which exclude the possibility of infection of the worker;
in the process of studying pathogens of infectious diseases, students must learn the features of safety regulations when working with specific pathogens;
the autopsy of the corpses of experimental (laboratory) animals is carried out in special clothes on an equipped table using necessary tools using for this purpose a cuvette filled with wax (or paraffin). After opening, it is forbidden to put tools on the table: they are placed in a glass with disinfectant solution or burned over a burner flame;
when working with liquid infected material, use rubber balloons connected to a pipette;
if during the work the pathological material accidentally gets on the table, it is immediately removed with a swab moistened with a disinfectant solution. In case of contact with the infected material on the skin, conjunctiva, in the oral cavity, emergency measures are taken for disinfection;
at the end of the work, the pathological material, used cultures of microorganisms, instruments and the surface of the table are disinfected;
at the end of the lesson, students must hand over bacterial cultures and other material to the teacher, and put the workplace in order. It is strictly forbidden to take tubes with cultures, preparations (smears) and other items out of the laboratory;
pathological material and bacterial cultures necessary for further work are left for storage in a closed refrigerator or safe;
before leaving the laboratory, you must remove the gown, wash your hands thoroughly and treat with iodized alcohol. It is forbidden to leave the laboratory in gowns;
observance of the rules of work and safety precautions at the training sessions in microbiology is controlled by the attendants. Students get acquainted with safety precautions when working at the Department of Microbiology at the first lesson, which they sign in the journal.
Observing the above rules, an employee in the laboratory ensures the sterility of manipulations and prevents the occurrence of intra- and extra-laboratory contamination.
Maintaining laboratory records. Notebook for laboratory work serves as a document that allows you to control the correctness of the received data. It should contain information relevant to the performance of this work. The record must be kept neatly, clearly and in a certain order, for example: 1) the name of the experiment, the date of its setting and completion; 2) the object of study; 3) conditions for conducting the experiment; 4) the basic principle of the method of analysis used; 5) the results of the experiment.
The results obtained are described in detail, the digital material is summarized in tables, if necessary, in graphs, diagrams, drawings. Each laboratory work should end with its own observations and conclusions recorded in the workbook.
In the process of work, students master the technique and methods of microscopy, get acquainted with the morphology of representatives of various groups of microorganisms, master the approach to isolating pure cultures and their identification, study the influence of environmental conditions and factors on the growth and formation of various waste products of microorganisms and get acquainted with some methods of genetic research bacteria.
GENERAL MICROBIOLOGY
Federal Agency for Education
State educational institution
"Irkutsk State University»
SMALL WORKSHOP ON MICROBIOLOGY
Teaching aid
Irkutsk 2009
UDC 579(076.5)
Published by decision of the editorial and publishing council of Irkutsk State University
Zhilkin workshop on microbiology: Textbook-method. allowance for students. higher textbook institutions in the specialties "Microbiology", "Biology" and "Physiology".
6. Microbial mass should not contaminate hands, table and surrounding objects. The spilled microbial suspension is neutralized using disinfectants.
7. After the end of the work, the cultures are handed over to the teacher, the seeded test tubes and cups are placed in a thermostat.
8. Bacteriological loops, needles, tweezers and other metal objects, after contact with microorganisms, are burned in the flame of an alcohol lamp and placed in a special stand.
9. Used slides and coverslips, pipettes, spatulas, etc. are placed in a 3–5% solution of carbolic acid or other disinfectant solutions.
10. Spent cultures in test tubes, Petri dishes, etc. are neutralized with disinfectant solutions during the day, then the dishes are boiled and washed.
11. Personal hygiene should be strictly observed - after finishing work, wash your hands thoroughly with soap and water.
12. It is necessary to observe safety precautions when working with electrical appliances and chemicals.
FEDERAL AGENCY FOR EDUCATION
KARACHAYEV-CIRCASSIAN STATE TECHNOLOGICAL ACADEMY
Department of Technology for the Production of Animal Products
MICROBIOLOGY
METHODOLOGICAL INSTRUCTIONS
for laboratory and practical classes for students
agricultural institute
Cherkessk - 2010
Compiled on the basis of an exemplary and working programs on the course "Microbiology" in accordance with the State educational standard higher professional education in the specialty 110305 "Technology of production and processing of agricultural products" and 110201 "Agronomy" (2000).
Discussed at a meeting of the Department of TPPZH (minutes dated July 2, 2009)
Approved by the methodological commission of the Agrarian Institute (minutes No. 6 of 01.01.2001). Published by decision of the educational and methodological council of the Karachay-Cherkess State Technological Academy (minutes dated 01/01/2001)
Compiled by: Candidate of Biological Sciences, Associate Professor , Candidate of Agricultural Sciences, Associate Professor , assistant
Reviewers: – candidate of biological sciences
Associate Professor of the Department of Agronomy
Associate Professor of the Department "TPPZh"
Editor: Ph.D. Sciences, Associate Professor
Content
Introduction………………………………………………………………………….. 6
1. Microscopy………………………………………………………………….. 7
1.1. Light-field microscopy……………………………………………. .7
1.1.1. Microscope device………………………………………………… 7
2. Working with microorganisms ………………………………………………. 8
2.1.Methods of preparing preparations…………………………………..... 8
2.1.1. Technique for taking culture for preparations…………………………… 8
2.1.2. The study of living cells of microorganisms by the method of
crushed drop……………………………………………………….. 8
2.1.3. Fixed preparations of microorganisms……………………… 9
The lighting system is located under the stage. The mirror reflects the light falling on it into the condenser. One side of the mirror is flat, the other is concave. When working with a condenser, it is necessary to use a flat mirror. A concave mirror is used when working without a condenser with low magnification objectives. The condenser consists of 2-3 short-focus lenses, collects the rays coming from the mirror and directs them to the object. A condenser is required when working with immersion systems. The condenser has an iris (petal) diaphragm, consisting of crescent-shaped steel plates.
Stained preparations are considered with an almost fully open diaphragm, unstained - with a reduced aperture of the diaphragm.
Lens- a multi-lens system, the quality of which determines the image of the object. The outer lens is called the front lens, it provides magnification. The rest of the lenses perform the functions of correcting optical imperfections.
Lenses are dry and immersed (immersion). When working with dry lenses, there is air between the front lens of the objective and the object of study. When working with an immersion objective V = 90x, between the cover glass and the objective lenses is cedar oil, the refractive index of which is close to the refractive index of glass 1.515 and 1.52, respectively. Lenses have 8x, 40x and 90x magnification.
Eyepiece serves as a direct continuation of the "lenses" (lenses) of the human eye.
The eyepiece consists of two lenses - the upper - eye and lower - collective, enclosed in a metal frame. The purpose of the eyepiece is to magnify the image that the lens gives. The magnification of the eyepiece is engraved on its frame. The working magnification of the eyepieces ranges from 4x to 15x.
Eyepieces come in many different types, and the choice depends on the lens. When working with a microscope for a long time, a binocular attachment is used, as it improves the visibility of the object, reduces the brightness of the image and thereby preserves vision.
Working with microorganisms2.1. Preparation Methods
2.1.1. Preparation culture technique
A bacteriological needle burnt in a flame takes a small amount of microbial mass from a test tube. If the culture is liquid, then it is better to use a loop for this, in other cases - a needle.
2.1.2. The study of living cells of microorganisms by the crushed drop method
Examine the living cells of microorganisms using the crushed drop method, preliminarily stain the object with lifetime dyes - vital coloring(dyes: methylene blue, neutral red in concentrations from 0.001 to 0.0001%).
The preparations are microscoped, slightly darkening the field of view; the condenser is slightly lowered, the flow of light is regulated by a concave mirror. First, they use a low magnification - an 8x lens, after they detect the edge of the drop, install a 40x or immersion lens (90x).
In the case of the crushed drop method, a drop of tap water is applied to a clean glass slide. Culture is added to it and mixed with water. Excess water is removed with filter paper. When using an immersion lens, a drop of cedar oil is applied to a glass slide and microscoped.
2.1.3. Fixed preparations of microorganisms
Fixed preparations involve such processing of living cells, which makes it possible to quickly interrupt the course of life processes in the object, while preserving its fine structure. As a result of fixation, the cells are firmly attached to the glass and stain better. Fixation is necessary when working with pathogenic microorganisms (for safety reasons).
Smear preparation. A drop of tap water is applied to a clean, fat-free glass slide. For degreasing glass use a mixture of ethyl alcohol and sulfuric ether in a ratio of 1:1. With a calcined bacteriological needle, a small amount of microbial mass is taken from a culture tube and added to a drop of water. The drop is carefully smeared with a loop on the glass over an area of about 4 cm2.
If the suspension is thick, it is first diluted with water. To do this, a little suspension is taken with a calcined loop and transferred to a drop of water on another glass slide. A suspension of normal density is smeared with a thin layer on the glass, then the smear is dried in air at room temperature or with low heat, holding the preparation high above the burner flame. Strong heating of the drug during drying is not recommended to avoid protein coagulation, which distorts the structure and shape of cells. The dried preparation is fixed.
Smear fixation. She is being carried out over the flame of the burner or with the help of chemicalsunities. In the first case, the drug is slowly carried out three or four times with the bottom side over the burner flame, in the second case, chromium compounds are used: formalin, osmic acid, acetone. One of the common methods of fixation is the treatment of the preparation with 96% alcohol or a mixture of equal volumes of ethyl alcohol and ether (Nikiforov's liquid). To do this, the preparations are immersed for 10-30 minutes in a fixative liquid.
Staining of the drug. A few drops of dye are applied to the smear. Depending on the type of dye and the purpose of the study, the duration of staining varies from 1 to 5 minutes, in some cases taking up to 30 minutes. At the end of staining, the preparation is washed with water, water is removed with filter paper, dried in air and microscoped.
There are simple and differentiated staining methods.
At simple staining using any one dye (methylene blue, fuchsin, gentian violet), the entire cell is stained.
At differentiated staining, individual cell structures are stained with different dyes (Gram stain, spore stain).
Study of the morphology of microorganisms3.1. cell shape
3.1.1. bacteria
According to the shape, all bacteria are divided into spherical (cocci), rod-shaped and convoluted.
Spherical bacteria - cocci.
1. Micrococci - single spherical cells ( Micrococcus agilis).
2. Diplococci - spherical cocci connected in pairs. ( Azotobacter chroococcum).
3. Tetracocci- spherical cocci, connected by four.
4.streptococci- spherical bacteria connected in chains (mainly pathogenic, as well as lactic acid bacteria Lactococcus lactis).
5. Sarcins- spherical bacteria, grouped in 8 cells, arise as a result of cell division in three mutually perpendicular planes. Some types of sarcinas form large cube-shaped packages, in which there are 4 sarcinas on each side. typical representative Sarcina flava(sarcina yellow) - the most common representative of the air microflora.
All spherical bacteria except Streptococcus lactis, viewed on fixed and magenta-stained preparations.
rod-shaped bacteria. These include forms that do not form spores (genera Pseudomonas, Achromobacter, Lactobacillus etc.) and spore-forming (genera bacillus, Clostridium and etc.).
Non-spore-forming bacillus Pseudomonas stutzeri – its cytoplasm is stained evenly.
spore-forming rods Bacillus mycoides And Bacillus mesentericus. Under the microscope, they look unevenly colored. Spores do not stain like denser structures. Cells bacillus mycoides arranged in chains, these are streptobacilli.
Rod-shaped bacteria are viewed on fixed and stained preparations.
Convoluted shapes
1. Vibrios – slightly curved cells.
2. Spirilla can have one curl in the form of the Russian letter C, two curls in the form of the Latin letter S, or several in the form of a spiral.
3. Spirochetes - long and thin cells with a large number of curls; the length of the cells exceeds their thickness by 5-200 times.
It is convenient to view vibrio and spirilla on a fixed and stained preparation prepared from slurry, previously incubated for several days in a thermostat. Of the many microorganisms on such a preparation, convoluted forms are often found.
You can get acquainted with spirochetes on a fixed stained preparation of dental plaque, preparations of scraping from a carious tooth are especially successful. Dental spirochetes are very thin, hair-like, short (only 2-3 whorls).
3.1.2. actinomycetes
Actinomycetes are radiant fungi. The mycelium of actinomycetes on nutrient media is differentiated: one part of it is immersed in the substrate (substrate mycelium), the other is above the substrate (aerial mycelium).
Many representatives of actinomycetes produce pigments, therefore their aerial mycelium and especially colonies are colored blue, blue, purple, pink, brown, brown or black. Actinomycetes stain the nutrient medium in the appropriate colors.
A piece of an actinomycete colony is placed on a glass slide along with the medium. With the second glass slide, this piece is pressed tightly against the glass, crushed and smeared on the glass. The drug is dried, fixed, stained, viewed under a microscope, where mycelial unicellular filaments are partially visible.
3.1.3. Yeast
Yeasts are unicellular microscopic fungi of various shapes: ellipsoid, pear-shaped, round, cylindrical. They reproduce vegetatively and sexually.
Baker's yeast is used for laboratory studies. A small piece of yeast mass a few hours before class is placed in warm sugared water and placed in a warm place. A whitish turbid liquid is formed. A drop of this liquid is applied to a slide, covered with a coverslip, a drop of cedar oil is applied on top and the preparation is viewed with an immersion system. Budding and dividing cells are visible.
3.2. Chemical Methods research
3.2.1. Gram staining of microorganism cells
This method of differentiation of microbial cells is based on differences in the chemical composition of cell membranes. In the cells of some types of microorganisms, an alcohol-insoluble iodine compound with the main dye is formed, while in other species this compound appears temporarily and dissolves after treatment with alcohol. Microorganisms of the first group are called gram-positive second - gram-negative.
Gram stain technique. Three thin smears of different cultures of microorganisms are applied to a defatted glass slide (two of them are controls, with a known relation to Gram stain). The smears are air-dried, fixed over a burner flame, and stained for 1 min with a phenol solution of gentian violet (or crystal violet), holding the slide in a slightly tilted position. Then the dye is drained and, without washing the preparation with water, Lugol's solution is applied to it for 1 min (until the smear is completely blackened). The glass is held in an inclined position. The drug, without washing with water, is treated, continuously shaking, with 96% alcohol for 15-20 s. It is important to adhere to the discoloration time, since Gram-positive cells also become discolored if the specified time is exceeded.
After washing with water, the preparation is stained with Pfeifer fuchsin for 1 min. Gram-positive microorganisms acquire a dark purple color, and Gram-negative microorganisms stain with an additional color (magenta).
Gram stain results depend on the age of the culture: in old cultures, dead cells always stain Gram negative. Therefore, it is better to use young one-day cultures.
Yeasts are good targets for Gram staining of microbial cells. Bacillus mesentericus or Bacillus subtilis(gram-positive) and Escherichia coli (gram-negative).
Dyes and reagents for Gram staining.
1. Phenolic solution of gentian violet: gentian violet - 1 g, alcohol 96% - 10 ml, crystalline phenol - 2 g, distilled water - 100 ml.
In some cases, apply alcohol solution of gentianpurple: gentian violet (or crystal violet) - 1 g, alcohol 96% (rectified) - 100 ml, glycerin - 5 ml. The mixture is placed in a thermostat for 24 hours, then filtered.
2. Lugol's solution(potassium iodite - 2 g, crystalline iodine - 1 g, distilled water - 300 ml). First, a concentrated solution of potassium iodite is prepared in 5 ml of water, iodine is dissolved in it, then water is added to 300 ml.
3. Alcohol 96%.
4. Fuchsin Pfeiffer(aqueous solution of Ziel carbolic fuchsin): 1 ml of Ziel carbolic fuchsin and 9 ml of distilled water. It is prepared as follows: 1 g of fuchsin, 5 g of crystalline phenol, 96% alcohol - 10 ml, a few drops of glycerin, 100 ml of distilled water, fuchsin is dissolved in ethanol, phenol dissolved in water is added. The solution is stirred and left for several days. It is filtered before use.
3.2.2. Bacterial spore coloration
Bacterial spores are highly resistant to adverse environmental conditions compared to vegetative cells. They are round, oval or elliptical formations. If the diameter of the spore does not exceed the diameter of the cell in which the spore is formed, the cell is called bacillary, if it exceeds, then depending on the location of the spore in the center or at the end of the cell, this cell is called, respectively clostridial or plectridial . In a bacillary cell, the spore can be located in the center of the cell - central position at the end terminal and closer to one of the ends - subterminal position.
When observing living spore-forming bacteria, their spores can be distinguished by a stronger refraction of light rays. Spores are acid-resistant, so they are difficult to stain with dyes. This is explained by the high density of the shell, the low concentration of free water in it, and the high content of lipids in spores. In preparations stained simple ways or according to Gram, spores remain colorless (negative color).
All spore staining methods are based on single principle: first, the spores are etched with various substances: chromic, hydrochloric, sulfuric, acetic acids, ammonia, caustic soda or hydrogen peroxide, then the cell with the spore is stained when heated, and finally, the cytoplasm is discolored and additionally stained with a contrast dye.
Ziehl-Neelsen method modified by Muller. Before fixing a smear of bacteria on a flame, the preparation is prepared in the usual way. Next, a 5% solution of chromic acid is applied to the drug fixed in the flame and cooled down. After 5-10 minutes, it is washed off with water. The preparation is covered with a strip of filter paper and the paper is abundantly moistened with Ziehl carbol fuchsine. The drug is heated over a flame until vapors appear (not to a boil), then it is taken aside and a new portion of the dye is added. This procedure is carried out for 7 minutes. It is important that the dye evaporates, but the paper does not dry out. After cooling, it is removed, the preparation is washed with water and thoroughly blotted with filter paper. As a result of this treatment, cells with spores are uniformly stained.
Next, the cytoplasm of cells (but not spores) is discolored by treating with a 1% solution of hydrochloric or sulfuric acid for 15-30 s. When preparing the spore preparation bacillus mycoides or bacillus mesentericus it is recommended to decolorize the cytoplasm for 16-18 seconds (measuredly counting aloud from 21 to 37-40). If this time is exceeded, spores may also become discolored. Then the preparation is washed with water and stained with methylene blue for 2 minutes.
The color is contrasting and bright red spores stand out clearly against the blue background of the cytoplasm.
Peshkov method. Methylene blue Leffler is poured onto the preparation fixed in the flame, brought to a boil and boiled for 15-20 s, holding the glass over the flame. The smear is washed with water and stained for 30 with a 0.5% aqueous solution of neutral red. Rinse again, dry and then examine the preparation with oil immersion of the objective. The spores are stained blue or blue, the cytoplasm is pink.
For the study of spores, convenient objects can be bacillus mesentericus or bacillus mycoides at the age of 4 days.
Reagents for staining bacterial spores. 1. Carbol fuchsinTsilya(see 3.2.1).
2. Methylene blue Leffler(see 3. 2. .1).
3. Saturated aqueous solution of methylene blue. 2 g of dye and 100 ml of distilled water.
4. Chromic acid, 5% solution.
5. Salt(or sulfuric acid, 1% solution.
4. Cultivation of microorganisms
4.1. Nutrient media
4.1.1. Media preparation
Meat-peptone broth (MPB). For the preparation of meat-peptone media use meat broth, which is obtained as follows: 500 g of finely minced fresh meat is poured into an enamel pan with 1 liter of tap water heated to 50 ° C, and left to infuse for 12 hours at room temperature or 1 hour at 50-55 ° C. The meat is squeezed out, the extract is filtered through gauze with a layer of cotton wool, boiled for 30 minutes to coagulate colloidal proteins and filtered twice (the first time through gauze with cotton wool, the second time through a paper filter). The filtrate is topped up with water to 1 liter, poured into flasks, closed with cotton stoppers and sterilized at 120°C for 20 min (the stoppers of the flasks are closed from above with paper caps).
Meat stock can be used at any time to prepare media. If they are cooked immediately, then preliminary sterilization of the meat broth is not required.
To prepare MPB, add 5-10 g to 1 liter of meat broth peptone(the first product of protein hydrolysis) to increase the caloric content of the medium and 5 g table salt to create osmotic activity. The medium is heated until the peptone dissolves, stirring constantly.
A neutral or slightly alkaline reaction of the medium is established by adding a 20% solution of Na2C03 until the wet red litmus paper turns blue. It is convenient to use an indicator to check the pH of the medium. bromthymolblau: 1-2 drops of it are mixed in a porcelain cup with a drop of broth. Bromothymolblau is bottle green in a neutral environment, yellow in an acidic environment, and blue in an alkaline environment.
After the pH is established, the medium is boiled again for 5-10 minutes, and the proteins that have curdled when the reaction of the medium changes are filtered through a paper filter without clarifying the broth or clarifying it with protein. To do this, fresh egg white is beaten with double the amount of water and mixed with the broth cooled to 50 ° C. The mixture is boiled, stirring, over low heat for 10 minutes, then filtered. The transparent meat-peptone broth is poured into test tubes, closed with cotton plugs and sterilized at 120 °C for 20 minutes.
Meat peptone agar (MPA). To 1 liter of MPB add 15-20 g of agar. The medium is heated until the agar dissolves (its melting point is 100°С, its solidification temperature is 40°С), the medium is set to a slightly alkaline reaction with a 20% Na2C03 solution and poured through a funnel into test tubes (approximately 10 ml of agar in a column for subsequent pouring into Petri dishes and 5 ml each to obtain slanted agar - shoals).
When spilling agar, the edges of the test tubes must remain dry, otherwise the stoppers will stick to the glass. The tubes with the medium are sterilized in an autoclave at 120°C for 20 minutes.
4.2. Sterilization Methods
Sterilization - this is the complete destruction of microorganism cells in nutrient media, dishes, etc.
Several methods of sterilization are known. Heat sterilization is more commonly used.
4.2.1. Flaming, or roasting
You can ignite immediately before use platinum loops, needles, spatulas, small metal objects (scissors, lancets, tweezers), as well as glass rods, slides, cover slips, etc.
4.2.2. Dry heat sterilization
It is used for processing dishes and dry materials, such as starch, chalk. At the same time, the object to be sterilized is kept at 170 °C for 2 hours (from the moment the required temperature is established) in electric drying cabinets. Raising the temperature above 170 ° C is not recommended: cotton plugs and paper begin to collapse.
Before sterilization, glassware is closed with cotton plugs and wrapped in paper. Cups, test tubes, pipettes, cotton wool, gauze are wrapped in paper or placed in special cases and cases in which sterile dishes can be stored after sterilization.
At the end of sterilization, the cabinet is opened only after the temperature drops to room temperature, otherwise the glass may burst.
4.2.3. Steam Sterilization
Fluid steam (100 ° C) is used to process objects that deteriorate from dry heat, and some nutrient media that cannot withstand higher temperatures (media with carbohydrates, NRM, milk). Sterilization is carried out in a Koch boiler for 30 minutes for 3 days daily. This sterilization is called fractional.
With a single heating at a temperature of 100 ° C for 30 minutes, vegetative cells die, while spores of many microorganisms remain viable. After such heating, the medium is placed for 24 hours in a thermostat at 28–30°C. The spores preserved during the first heating have time to germinate into vegetative forms during this time, which die during subsequent heating. Then this operation is repeated 2 more times.
4.2.4. Sterilization with saturated steam under pressure
This is the fastest and most reliable method of sterilization, which kills the most resistant spores. With its help, most culture media and dishes are sterilized.
Processing with saturated steam is carried out in a hermetically sealed thick-walled boiler - autoclave. On the lid or on the side of the autoclave there is a valve for steam outlet, a pressure gauge and a safety valve. The manometer shows how much the steam pressure inside the boiler is higher than normal. To prevent an explosion, when the pressure limit is exceeded, a safety valve is activated, giving steam an outlet.
The indicator of the manometer in physical atmospheres corresponds to a certain temperature.
Reliable sterilization is achieved by heating at 120 °C and a pressure of 1 atm for 20 minutes.
Sterilization is carried out as follows. Water is poured into the autoclave, the objects to be sterilized are placed in it, the lid of the autoclave is screwed on and heating is started. The faucet is left open until all the air in the autoclave is displaced by water vapor. When the steam begins to come out of the tap in a continuous stream, the tap is closed, the steam pressure in the autoclave is brought to 1 atm and maintained at this level for 20-30 minutes. Then the heating is stopped, they wait until the pressure gauge needle drops to 0, carefully (slowly) open the tap and release the steam. Only then unscrew the lid of the autoclave. If the tap is opened before the pressure drops, the liquid in the sterilized vessels will boil and push the plugs out of them.
The autoclave is also used for fractional sterilization with flowing steam. In this case, the lid is not screwed down to allow the steam to escape freely.
4.2.5. Pasteurization
Pasteurization is an incomplete, or partial, sterilization, which means heating at 65-80°C for 30-10 minutes, respectively, followed by rapid cooling to 10-11°C. Pasteurize milk, beer, wine and other products.
Materials and equipment
BCH, agar, litmus red, bromthymolblau, porcelain plates with wells or cups, glass rods, 20% Na2C03 solution, test tubes in racks (for pouring agar), funnels, cotton wool, Petri dishes, 1 ml Mohr pipettes, paper for cup and pipette wraps, 250 ml flasks, harsh threads.
5. Accounting for the number and isolation of a pure culture of microorganisms
5.1. Methods for counting the number of microorganisms
5.1.1. Accounting for the number of microorganisms (CFU) in the soil by the method of nutrient plates in combination with the method of successive dilutions
Soil is the most favorable environment for the development of microorganisms. Due to the large heterogeneity of its composition, to account for the number of microorganisms in it, from the study area, take middle soil test.
First, suspensions are prepared (by dilution method) containing different concentrations of soil in 1 ml of water. To do this, a 1 g sample of soil is taken from a jar or bag on a sterile watch glass with a sterile porcelain spatula or an aluminum teaspoon. When weighing the soil, the watch glass is covered with another sterile watch glass.
A sample of soil, observing aseptic conditions, is transferred into a 250 ml flask with 99 ml of sterile water. The mixture is shaken for 5 minutes without wetting the stopper. 1 ml of suspension containing 10-2 g of soil is taken with a sterile pipette and transferred into a test tube with 9 ml of sterile tap water. The pipette is repeatedly washed with water in a test tube in order to wash away the cells from its walls as much as possible. With another sterile pipette, another 1 ml of suspension is taken from the flask and placed in a second flask, also containing 99 ml of sterile tap water. This pipette is washed in the same way as in the first case. Shake the test tube and the second flask for 1 min. The soil concentration in the test tube will be 10-3 g, in the second flask - 10-4 g. In the same way, with new sterile pipettes, transfer 1 ml of the suspension from the second flask into the second test tube with 9 ml and into the third flask with 99 ml of sterile tap water and prepare new suspensions containing 1 ml, respectively, 10-5 and 10-6 g of soil.
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1 Ministry of Education and Science Russian Federation Federal Agency for Education Moscow State University of Engineering Ecology Kustova N.A. LABORATORY WORKSHOP ON MICROBIOLOGY Moscow 2005
2 Laboratory workshop in microbiology is intended for students of specialties 3207 and 3302 in the discipline "Fundamentals of Microbiology and Biotechnology", as well as for students of the Department of "Environmental and Industrial Biotechnology" in the discipline "Environmental and Industrial Microbiology". The workshop consists of three sections. The first section is devoted to questions of general microbiology. In the works of this section, the morphological structure of different groups of microorganisms, methods of microscopic examination, the technique of microbiological seeding, sterilization methods, and methods for quantitative accounting of microorganisms are studied. The second section contains works on the use of microbes in biotechnology to obtain various substances of organic acids, alcohols, antibiotics, and enzymes. In the works of the third section, questions of ecological microbiology are studied. Some of the works show the role of microorganisms in global biogeochemical cycles, and the rest are devoted to the problems of biotechnological protection. environment. Each topic contains a theoretical introduction and a practical part, which describes the methods used, the order of work, the content of the report on the work, as well as control questions. 2
3 FOREWORD The laboratory workshop on microbiology is intended for 3rd year students of specialties 3207 and 3302 in the discipline "Fundamentals of Microbiology and Biotechnology", as well as for 4th year students of the Department of "Ecological and Industrial Biotechnology" in the specialization "Biotechnological Environmental Protection" in the discipline "Ecological and industrial microbiology. The laboratory workshop is based on the Guidelines for Laboratory Works, ed. P.I. Nikolaev, which have been used at the department "Processes and devices of microbiological production" since the creation of the department. Senior Researcher, Ph.D. N.V. Pomortseva. The guidelines were prepared under her guidance by the department's researchers: M.A. Boruzdina, I.E. Lomova, N.A. Kustova, T.A. Makhotkina and K.A. Change curriculum in accordance with the new specialty of an environmental engineer, it was necessary to expand the course of microbiology and supplement it with tasks on biotechnological methods of environmental protection. The laboratory workshop consists of three sections. The first section is devoted to general microbiology: the morphology of microorganisms, methods of studying it, the technique of microbiological inoculation, methods of quantitative accounting of microorganisms. The second section covers some examples of the use of microbes in industry. The third section covers the issues of ecology of microorganisms, their role in the global cycles of elements, as well as in biotechnological methods of environmental protection. The post-graduate student of the department N.V. Zyabreva and senior researcher took part in the preparation of this workshop. E.S. Gorshina. The author expresses his deep gratitude to Assoc. cafe microbiology, Moscow State University N.N. Kolotilova for valuable comments and advice, as well as senior researcher. P.P. Makeev for help in preparing the text and illustrative material. 3
4 4 GENERAL RULES OF WORK IN THE MICROBIOLOGICAL LABORATORY Rules of work and behavior in the laboratory The rules of work and behavior in the microbiological laboratory have much in common with the rules of work in chemical laboratories, but they have their own specifics. A microbiologist in most cases works with pure cultures of microorganisms, i.e. with microorganisms of any one genus, species and strain. Since there are foreign microbes on all surrounding objects and in the air, special working methods are used to avoid contamination of the microorganism culture under study or the person himself. To do this, culture media, utensils, tools are sterilized, the laboratory and workplaces are kept clean, certain rules are observed when working with microbes. There should be no unnecessary items in the laboratory. Wet cleaning should be carried out regularly. Various surfaces of laboratory premises are periodically subjected to disinfection. Disinfection is disinfection, i.e. destruction of pathogens of infectious diseases at environmental objects. To do this, use a 0.5 3% solution of chloramine or 3 5% solution of phenol (carbolic acid). The work table should be disinfected with a 70% solution of ethyl or isopropyl alcohol. Air disinfection is achieved by simple ventilation (at least min). More effective method air disinfection irradiation of the room with ultraviolet rays using bactericidal lamps. Especially often ultraviolet irradiation is used to sterilize the box. Boxing is a special small room for reseeding pure cultures, quantitative accounting of microorganisms on Petri dishes and some other work requiring especially clean conditions. Before work, the box is irradiated for min. The table is wiped with alcohol, the walls and floor are periodically washed. Instead of boxing, laboratories can be equipped with laminar cabinets (Fig. 1), which are also sterilized with germicidal lamps. At
5 work include a fan to create a laminar flow of sterile air passed through bactericidal filters. The main equipment of the microbiological laboratory includes: microscopes, thermostats for growing microorganisms, sterilization equipment (autoclave and drying cabinet), centrifuges, a distiller, a refrigerator for storing museum cultures of microorganisms, cabinets for placing glassware and reagents, necessary devices (photoelectric colorimeters, pH -meters, etc.). Each student is assigned a workplace on which they place: a microscope covered with a cover, a bacteriological loop, slides and coverslips, sterile pipettes, an alcohol burner, filter paper strips, a glass marker, a vessel with a disinfectant liquid. There should be nothing on the table that is not directly related to the work being done. In laboratory classes in microbiology, safety rules should be observed. 5
6 6 Brief information on safety in the laboratory In microbiological practice, chemical glassware is widely used. You have to be careful while working with it. Clean up broken dishes thoroughly. In the analysis, strong solutions of alkalis and acids are often used. You need to work with them with great care, as these substances have a harmful effect on the skin of the hands and clothing. If the acid is accidentally spilled, it must be covered with a large amount of soda and then rinsed several times with water. Spilled alkali must be thoroughly wiped off, and objects on which it has fallen should be treated with a weak solution of acetic acid. If acids or alkalis come into contact with human skin, they must be immediately washed off with plenty of water. In a microbiological laboratory, they deal with living microorganisms. The main work is carried out sterile, i.e. work with one culture of microorganisms, which should not be infected with foreign microbes. To prevent infection of crops, special sterilization methods are used. In addition, it is important to maintain cleanliness in the laboratory. Dishes with cultures of microorganisms should not be left open. The biomass of microorganisms, if it is not needed for analysis, is discarded only after sterilization in an autoclave. Crops of microorganisms are produced near the flame of a gas or alcohol burner, so you should beware of burns, and above all, carefully pick up long hair. The burner should only burn when needed. If a cotton plug, spirit lamp or paper accidentally catches fire during sowing, the fire is extinguished with a towel. For larger fires, fire extinguishers are used. Used pipettes, slides, coverslips, spatulas, etc. placed in a container with a disinfectant liquid. Students must constantly remember that they are dealing with microorganisms, which may not always be safe, especially in work on the isolation of microbes from environmental objects. Therefore, at the end of the lesson, students should tidy up the workplace and wash their hands with soap and water.
7 In the event of a malfunction in the electrical network, turn off electrical appliances and their centralized power supply. SECTION 1. GENERAL MICROBIOLOGY TOPIC 1. Morphology of microorganisms and methods of studying it Microbiology studies organisms that have microscopic dimensions, i.e. measured in micrometers or fractions of micrometers. One micrometer is equal to 10-6 meters and is abbreviated as micron. Microorganisms are characterized by intensive metabolism and are able to carry out various chemical transformations. Different microorganisms differ both in their structure and in the biochemical processes that they carry out. Combining them into one group is caused not only by their small size, but also by the commonality of cultivation and research methods. To study the structure of microorganisms, their appearance, shape, size, i.e. to study the morphology of microorganisms, use a microscope. The smallest particles that can be seen in modern light microscopes have a magnitude of more than 1/3 of the wavelength of light, i.e. not less than 0.2 microns, which is associated with the use of the visible part of the light having a wavelength of 0.4 microns to 0.7 microns. Microscope device In fig. 2 shown appearance common in research and educational practice microscope MBI-3. The object under consideration, the preparation, is placed on the object table and illuminated from below by light rays that come out of the illuminator, fall on the mirror, then pass through the condenser and focus on the preparation. The main parts of the microscope: eyepieces, tube, turret with objectives, stage with clips for preparations, condenser, macro and micro screws for focusing and, finally, a tripod in which all this is mounted. 7
8 Before microscopy, check the correct installation of illumination (according to Köhler). To do this, by moving the illuminator cartridge with a light bulb, a clear image of the thread is achieved 8
9 lamp incandescence on a completely closed condenser diaphragm so that this image completely fills the condenser hole. Having closed the illuminator diaphragm, the condenser diaphragm is opened and, by moving the condenser, a sharp image of the illuminator diaphragm is achieved in the field of view of the microscope. To bright light did not blind the eyes, first reduce the incandescence of the lamp filament. And, finally, the image of the diaphragm opening is set in the center of the field of view, and the aperture of the illuminator is opened so that the entire field of view is illuminated. The microscope is an optical system with two stages of magnification: the first magnification is carried out by the objective, the second by the eyepiece. The lens gives an enlarged reverse image of the object that is viewed through the eyepiece. As a result, the observer's eye sees a highly magnified inverse image of the object. Therefore, the movement of an object to the left is perceived by the eye as a movement to the right. The total magnification of the microscope, i.e. The magnification at which an object is viewed under a microscope is defined as the product of the magnifications of the objective and the eyepiece. Lenses give a magnification of 10, 40, 60, 90 times, eyepieces in times. If a binocular attachment is used, it provides additional magnification. On fig. 3 shows the circuit diagram optical system microscope. The lens O forms in the Z plane a real inverted image A of the object A. The image given by the lens is further enlarged using the eyepiece E. Since Z is in the focus of the eyepiece E, the observer sees an enlarged virtual image A in the X plane, which is usually located 25 cm from the eye , i.e. at a distance convenient for near vision. It should be borne in mind that such an idea of the mechanism of image formation is highly simplified, because it ignores the influence of diffraction and a number of other factors. In working with a microscope, students study preparations of microbes with a magnification of several times. The highest magnification given by an optical microscope is 3000x. The smallest particle size that can be viewed in such a microscope is 9
10 is equal to 0.2 microns, which is due to the wavelength of the visible part of the spectrum. Morphology of microorganisms The world of microorganisms includes a huge variety of forms that do not constitute a single systematic group. The main objects of microbiology are bacteria, but in addition to them, microbiologists also study yeast, fungi, microscopic algae, and some protozoa. On fig. 4 shows the main groups of microorganisms (with the exception of protozoa); the ratios of their sizes are preserved. All living organisms, except viruses, have cellular structure. According to their cellular organization, they are divided into prokaryotes and eukaryotes. 10
11 The main difference between eukaryotes and prokaryotes is the presence of secondary cavities that separate the nucleus and other cellular structures from the cytoplasm. It was the appearance of secondary cavities that made it possible to make a leap in the evolution of the entire living world due to an increase in the inner surface of eukaryotic membranes. This made it possible, in accordance with an increase in the diffusion rate, to simultaneously carry out a greater number of biochemical reactions occurring on the membranes. Prokaryotes are bacteria, including actinomycetes and cyanobacteria. Eukaryotes are all plants, animals, yeasts, fungi, protozoa. Among prokaryotes, a group of archaebacteria is currently distinguished, which includes methanogens, extreme halophiles (living in very salty water), extremely thermophilic bacteria that oxidize and reduce molecular sulfur, as well as thermoplasms devoid of a cell wall. A new division was made based on the comparison nucleotide sequences in small segments of ribosomal RNA. eleven
12 Archaebacteria differ in the composition of cell walls, lipids, and some other physiological and biochemical features (for example, they have a different mechanism of CO 2 fixation). Thus, in the structure of the cellular organization, 3 groups are currently distinguished: archaebacteria (according to the new nomenclature Archaea, archaea), eubacteria (according to the new nomenclature Bacteria, bacteria), and eukaryotes (according to the new nomenclature Eukarya). Work 1. Microscopic study of bacteria Morphology of bacteria Theoretical introduction This group of microorganisms is the most numerous, widespread in nature and is of great industrial importance. To name microorganisms, binary nomenclature is used, as in zoology and botany. In accordance with this nomenclature, each species has a name consisting of two Latin words. The first word means genus, and the second type. The generic name is always capitalized and the specific name is always lowercase. Most bacteria are single-celled organisms that are spherical, rod-shaped, or coiled. Among the bacteria there are a small number of filamentous forms. Bacteria are very small, the cell diameter of spherical bacteria is 1 2 microns. Bacteria reproduce by division (under favorable conditions, division occurs in minutes). Some bacteria are motile. The ability to move is associated with the presence of special flagellum organelles. The most simple in shape are spherical bacteria (cocci). They occur either in the form of single balls, or balls linked together. According to the arrangement of cells after division, spherical bacteria are divided into monococci (single cocci), tetracocci (combined by 4), sarcins (combined by 8), staphylococci (clusters), streptococci (chains of cocci). 12
13 Rod-shaped bacteria are the most numerous group of bacteria. They are cylindrical cells with rounded or pointed ends and vary greatly in length to width ratio. They can be located singly or form short or long chains. The rods can be of various lengths, usually a few microns, and a width of about 1 micron. Some rod-shaped bacteria form special spore bodies inside the cell. Each cell produces one spore, which serves to endure unfavorable conditions. The spore under appropriate conditions (temperature, humidity, nutrients) germinates, turning into a stick. The resistance of bacterial spores exceeds the resistance of any living organisms. For example, the spore of Bacillus subtilis hay bacillus can withstand temperatures of 100 ° C for 3 hours. Such spore resistance makes it difficult to fight infections. Coiled microorganisms differ in the degree of curvature of the cells and in the number of coils. They are divided into vibrios, spirilla and spirochetes. If a bacterium has one incomplete coil of a spiral, then it is called a vibrio. If a bacterium has several spiral curls, then it is called a spirilla, and microbes that have a convoluted shape with a large number of small curls are called spirochetes. Filamentous bacteria are filaments consisting of cylindrical or disc-shaped cells. The threads of some species are enclosed in a mucous membrane, which can be impregnated with iron hydroxide or manganese salts. The process of accumulation of heavy metals from solutions occurs in the cells of some iron bacteria. Large filamentous bacteria r. Beggiatoa deposit sulfur in their cells. Filamentous bacteria usually live in marine and fresh waters, are also found in decaying organic remains, in the intestines of animals. Cyanobacteria include a large group of organisms that combine the prokaryotic structure of the cell with the ability to carry out photosynthesis. Pigments of cyanobacteria cells, in addition to chlorophyll a (green), contain phycocyanin pigment 13
14 blue. For this reason, they were previously called blue-green algae. Most of them are multicellular organisms, which are long, most often unbranched filaments (trichomes). The cells in the filaments are united by a common outer wall. Sometimes they form mucous accumulations "mats". Reproduction is carried out by breaking up the thread into separate sections. Some species move by sliding (r. Spirulina). Actinomycetes (branching bacteria, radiant fungi) are a large group of prokaryotic microorganisms that form thin branching filaments several mm long and 0.5-1.5 microns in diameter. They are a peculiar group of microorganisms, which is morphologically similar to mold fungi (Fig. 5). The cells of a significant part of the representatives of this group are able to branch, which is hallmark mushrooms. However, the length of the branching filaments of actinomycetes reaches several millimeters, while the length of the fungal mycelium is several centimeters. The hyphae of fungi are usually several times thicker than the filaments of actinomycetes. According to morphology and development, actinomycetes are divided into higher and lower forms. Higher organisms include organisms with good 14
15 developed septate or non-septate mycelium and special sporulation organs. Spores are formed in the form of chains on special spore-bearing hyphae of the aerial mycelium. The structure of the sporulation organs is different in different species: long or short, straight or spiral (Fig. 6). By the presence of mycelium and the structure of the organs of sporulation, higher actinomycetes resemble mycelial fungi. Some actinomycetes have mycelium only in a young culture, which decays with age with the formation of rod-shaped and coccal cells. The lower forms of actinomycetes do not have true mycelium. The ability to form mycelium is expressed in them only in the tendency of cells to branch. The lower actinomycetes include, for example, species of the genus Mycobacterium, which have the ability to change the shape of cells with culture age (Fig. 7). This property is called pleomorphism. Among the higher actinomycetes, species of the genus Streptomyces occupy the leading place in terms of abundance in natural environments. Actinomycetes play an important role in the processes of 15
16 soil formation and creation of soil fertility. Actinomycetes destroy complex organic compounds (cellulose, humus, chitin, lignin, etc.) that are inaccessible to many other microorganisms. Almost all species of the genus Streptomyces form specific waste products with antibiotic properties. Some species are pathogens of plants, animals and humans. In addition to the main forms of bacteria, there are stalked and budding bacteria that carry outgrowths called prostek. (fig.8) 16
17 The functions of prostek are different. In some bacteria, they serve for reproduction, in others for attaching the cell to the substrate. 17
18 18 Practical part The purpose of the work is to study the morphology of representatives of bacteria. The order of work. Performing the first work, students learn how to use a microscope, look at ready-made preparations of representatives of bacteria, then independently prepare preparations of live bacteria and microscope them. First, students microscopically examine ready-made fixed preparations of bacteria. Fixed preparations are microorganisms suspended in aquatic environment dried on a glass slide and stained with aniline dyes. Preparations of some live microorganisms are prepared by students on their own. To do this, a small drop of tap water is applied to a clean glass slide, into which a small amount of microbes under study is introduced with a calcined and cooled bacteriological loop, thoroughly mixed and covered with a coverslip. Excess water is removed with filter paper. The drug is placed on the subject table and fixed with clamps. First, looking into the eyepiece and turning the macro screw, a sharp image of the object is achieved at low magnification with a 10x objective. Then the microscope is transferred to a high magnification with a 40x objective. The rotation of the screws must be done with care, as if lowered too sharply, the lens can be crushed by the lens. When microscopy, it should be borne in mind that the microscope, especially at high magnifications, does not capture the entire depth of the object, therefore, when the tube is gradually lowered with a microscrew, the object is visible first from above, and then in the optical section. 1. Viewing fixed preparations of microorganisms. Lactococcus lactis is the causative agent of lactic acid fermentation; the shape of the cells is spherical cocci; cells are connected in chains. It is used to obtain lactic acid products.
19 Lactobacillus acidophilum rod-shaped bacteria; causative agent of lactic acid fermentation. It is used to obtain lactic acid products. Acetobacter aceti are rod-shaped bacteria that oxidize ethanol to acetic acid. Used to obtain food vinegar. Streptomyces griseus actinomycete, cell shape in the form of thin branched filaments; producer of the antibiotic streptomycin. Saccacharopolyspora erythrae actinomycete, cell shape in the form of thin branched filaments; producer of the antibiotic erythromycin. 2. Viewing live preparations of Bacillus subtillis cells in the form of thin movable rods; forms disputes; producer of enzyme preparations. Spirulina platensis cyanobacteria; the shape of cells in the form of a single thread, consisting of cylindrical cells, tightly adjacent to each other; has a sliding motion. Used as a food supplement. Contents of the report 1. Latin name of the microorganism. 2. Cell morphology (give a drawing indicating the magnification of the microscope and keeping the ratio of cell sizes). 3. Use of the studied bacteria in industry. Test questions 1. Describe the structure of the microscope. 2. How to determine the magnification of a microscope? 3. What is the shape of bacterial cells. 4. Name the features of the morphology of actinomycetes. 5. What representatives of bacteria do you know and what is their practical significance? Work 2. Morphology of eukaryotic microorganisms Theoretical introduction 19
20 Eukaryotic microorganisms that are studied by microbiologists include: fungi, yeasts, microalgae and some protozoa. Morphology of fungi Fungi are chlorophyll-free microorganisms with filamentous cells. The long, branched filaments they form are called hyphae. The hyphae together form the mycelium. In terms of size, fungal hyphae are much larger than actinomycetes. The mycelium of a number of molds is divided by partitions (septate mycelium), while other types of partitions are absent. In biotechnology, mold fungi are mainly used as producers. Under the name "mold fungi" some representatives of phycomycetes, marsupials and imperfect fungi are combined. On dense substrates, molds form rounded fluffy, cobweb-like, cotton-like or powdery colonies of green, yellowish, black or white. Mold colonies consist of a large number of hyphae. Most of the hyphae develop in the air, and some in the thickness of the substrate. On hyphae, conidiophores are often formed, on which spores are formed either inside the sporangium or in the form of exospores arranged in chains. Conidiospores, or conidia, serve for asexual reproduction (Fig. 10). Conidia, getting into favorable conditions, germinate into mycelium. Mold fungi are very widespread in nature and have a powerful enzymatic apparatus. Therefore they are the main destructors organic compounds in nature. Molds are also widely used in industry for the production of organic acids, antibiotics and enzymes. 20
21 Yeast morphology Yeasts are a separate group of unicellular microscopic fungi of great practical importance. Yeast cells are large round or oval cells (Fig. 11). Some yeasts can form rudimentary mycelium called "pseudomycelium". Cell size ranges from 3 µm to 10 µm in length and 2 to 8 µm in width. Most yeasts reproduce by budding. At the same time, at 21
A small bulge of the kidney (sometimes not one, but several) appears on the surface of the cell, gradually increasing in size and, finally, separating from the cell that produces it. The bud separated from the mother cell becomes a new yeast cell. Some yeasts reproduce by fission. In the fine-grained content of live yeast (protoplasm), large vacuoles are clearly visible. Vacuoles are cavities inside the protoplasm filled with cell sap. It consists of electrolytes, proteins, carbohydrates and enzymes dissolved in water. In young yeast cells, the protoplasm is homogeneous, and at later stages of development, vacuoles appear in the protoplasm, filled with cell sap with metabolic products. When the nutrient medium is depleted, sporulation occurs in many yeasts. In some types of yeast, the spores are rounded and covered with a smooth shell. In favorable conditions, spores germinate. Yeasts are widely distributed in nature. They are found on grapes, on other berries and fruits, in milk, in water and soil, and on human skin. Many yeasts carry out alcoholic fermentation and are used to produce alcohol in baking, winemaking, and brewing. 22 Morphology of protozoa Protozoa are single-celled organisms of the animal kingdom. Among microorganisms, they are the most complex, having primitive organs characteristic of multicellular animals. Some of them have mouth and anus, contractile and digestive vacuoles. Protozoa reproduce asexually (by cell division) and sexually. The classification of protozoa is based on the methods of movement. 1. Sarcode. They move and capture food with the help of pseudopodia, or false legs. A typical representative is Amoeba. The sizes of amoebas do not exceed microns.
23 2. Flagella. They have a dense plasma membrane and move with the help of flagella. Soil forms of flagella are very small (2-5 µm), while water forms are large (up to 20 µm). A typical representative is Euglena. 3. Eyelash. The most highly organized protozoa. Cell sizes range from 20 to 80 µm. A typical representative is the Paramecium shoe ciliate, which is cultivated as food for fish fry. 4. Sporozoans. fixed forms. Many pathogens, such as the causative agent of malaria, Plasmodium. The simplest are widely distributed in nature. They are found in water bodies, in silt and soil. The value of protozoa in nature is very diverse. They live in the gastrointestinal tract of various animals, taking part in the digestion plant food, participate in the mineralization of organic residues in the soil, and are also an important part of the biocenosis in wastewater treatment plants. Feeding on bacteria and suspended solids, they contribute to water clarification. The simplest perform the function of indicators: the development of certain forms can be used to judge the quality of wastewater treatment. Thus, the predominance of amoebae and the absence of ciliates in the composition of activated sludge indicates poor performance of treatment facilities. The ciliate Tetrahymena is widely used for primary toxicity assessment. Various types of protozoa are shown in fig. 12. Morphology of algae Algae are an extensive group of plant organisms. The presence of chlorophyll common to all of them and the resulting photoautotrophic nutrition, the ability to synthesize organic substances using the energy of sunlight and carbon dioxide. In many algae, the green color of chlorophyll is masked by other pigments. Among them there are very small unicellular and multicellular forms classified as microorganisms, as well as 23
24 multicellular organisms that live in the seas and oceans and sometimes reach gigantic sizes .. The objects of study of microbiology are some algae of microscopic size. They have a variety of shapes and live both on land and in the aquatic environment (Fig. 13). 24
25 Practical part The purpose of the work is to study the morphology of representatives of various groups of eukaryotes. Order of performance of work Students independently prepare live preparations of representatives of fungi, yeast, algae and protozoa; they are microscoped with a x40 lens and sketched with an indication of the magnification of the microscope. Mold fungi Aspergillus niger form cells in the form of mycelium with partitions; on some hyphae there are unbranched conidiophores with spores. Conidiophores are unicellular, spherically swollen, on the surface of the swelling there are short pin-shaped cells (sterigmata), each of which laces off a chain of black-colored conidia. It is used to obtain organic acids and enzymes. Penicillium chrysogenum hyphae are septated; on some hyphae there are branched conidiophores with spores. Conidia are formed at the ends of whorled branched conidiophores. It is used to produce the antibiotic penicillin. 25
26 Yeast Saccharomyces cerevisiae solitary yeast with oval and rounded cells; reproduce by budding. They are used in brewing, baking, alcohol production. In nature, they are found on the surface of berries and other fruits. Saccharomyces vini cell shape is oval and round; reproduce by budding; after budding, they do not separate for some time, forming small "branches". Used in winemaking. Rhodotorula glutinis cell shape is elliptical; single cells; reproduce by budding. They are colored orange due to the content of carotenoids in the cells. Can grow in environments with oil hydrocarbons as a carbon source. Used as fodder yeast. Candida tropicalis yeast with oval and highly elongated cells, forming a "pseudomycelium". Can grow in mineral environments with hydrocarbons as a carbon source. Used as fodder yeast. On an industrial scale, they are grown on waste products from the production of alcohol and paper. Algae Chlorella vulgaris is a microscopic green algae with rounded cells (5-10 microns in diameter); reproduce by autospores, which are formed inside the mother cell in an amount of 4 to 32 and are released after the rupture of its membrane. They can be used for mass cultivation in order to obtain feed protein, as well as for air regeneration in closed systems (submarines, space stations etc.). Scenedesmus sp. belongs to the group of green algae; has an oval cell shape; outer cells often have pointed ends; cells are connected together in fours. It is used to obtain food protein. 26
27 Protozoa The preparation is prepared from the activated sludge of the aeration tank. To study the morphology of the discovered representatives of protozoa and draw them. Contents of the report 4. Latin name of the microorganism. 5. Cell morphology (give a drawing indicating the magnification of the microscope and keeping the ratio of cell sizes). 6. Use of the studied microorganisms in industry. Control questions 1. Describe the form of cells of representatives of fungi and their practical use. 2. Describe the shape of yeast cells; name representatives, and tell about their practical use. 3. Tell us about the morphology of microalgae and their practical use. 4. Name the representatives of protozoa and tell about their application in biotechnology. Work 3. Methods for studying the morphology of microorganisms The most common method for studying the morphology of bacteria is the microscopy of fixed preparations prepared from pure cultures of microorganisms or from the test sample. The study of microorganisms in a living state is used in the study of larger forms and the observation of cell motility. Preparation of fixed preparations of microorganisms To prepare fixed preparations of microorganisms, a smear is first prepared, dried, fixed, and then stained. Smears are prepared on perfectly clean glass slides. Glass can be degreased with ethyl alcohol, a mixture of equal volumes of alcohol and ether, and others.
28 liquids. The easiest and most convenient way to degrease glass is to wipe it on both sides with a piece of laundry soap. Soap is removed from the glass with a piece of dry cotton wool or a napkin. In the manufacture of a smear from a colony of bacteria grown on an agar medium, a drop of water or saline is first applied to a degreased glass. Then the bacteriological loop is calcined in the flame of the burner. After that, after cooling the loop on the inner wall of the test tube, a part of the colony without agar is captured by the loop. The loop with the culture is introduced into a drop of water or saline on the glass and 2 3 circular movements are made. Some of the bacteria are suspended in the liquid. Excess bacteria remaining on the loop is burned in the flame of a burner, heating the loop red-hot. Then a drop of the suspension is spread over the glass with a cooled loop. The smear area should be 1.5-2 cm in diameter. The smear should be thin with a uniform distribution of the material. If a smear is prepared from a culture grown on a liquid nutrient medium, then, observing the same rules of sterility, a drop of culture is collected with a loop or pipette and applied to the middle of a fat-free glass. The pipette with the rest of the culture is immersed in a vessel with a disinfectant solution. The drop is evenly distributed over the glass with a bacteriological loop. The loop is burned in the flame of the burner and placed in a tripod or in a glass. The smear is dried in air or in a stream of warm air, holding it by the longitudinal ribs high above the flame of the burner. The borders of the smear are outlined with a wax pencil with reverse side glass, and on the side of the smear at one end of the glass put the number of the drug. By these marks, you can easily navigate the location of the smear on the glass. The dried smear is fixed. Fixation has the following goals: 1) to kill microbes, which makes the drug safe for further work; 2) attach microbes to glass so that they do not wash off when painting and washing with water; 3) improve the susceptibility of paints. 28
29 The simplest, suitable for almost all microbiological objects and the most common method in practice is fixation in a burner flame. To do this, the glass slide is passed 3-4 times through the hottest upper part of the burner flame, avoiding excessive overheating of the preparation, so as not to cause protein denaturation and disrupt the structure and morphology of bacteria. Distinguish between simple and complex differential methods coloring. Simple staining is used to detect microbes in the test material, determine their number, shape and location. Simple coloring consists in applying any one aniline paint to the preparation. Most often, magenta red color is used for these purposes, as well as an alkaline solution of methylene blue (Leffler blue) blue color. Staining technique: a well-fixed preparation is placed with a smear upwards on a glass bridge above the bath. One of the indicated paints is applied to the surface of the smear with a pipette or from a dropper. Fuchsin is kept on smear 1 for 3 minutes, and blue 3 for 5 minutes. The paint is drained from the smear, the preparation is washed with water, dried in air or gently blotted with filter paper. A drop of immersion oil is applied to the dried smear, the preparation is placed on the microscope stage and microscoped with an immersion objective (90) in transmitted light. Differential methods of staining bacteria. Sophisticated staining methods have great importance in defining and differentiating various kinds microbes. They are based on the features of the physicochemical structure of a microbial cell and are used for a detailed study of the cell structure and identification hallmarks for some dyes. With these methods, the smear is stained with several paints and additionally treated with mordants or bleaching agents, alcohol, acid, etc. These methods include the most important staining method for differentiating bacteria, Gram stain. This method reveals 29
30 the ability of bacteria to retain dye or discolor in alcohol, which is related to the chemical structure of the cell wall. All bacteria are divided into two groups according to the structure of the cell wall: 1) Gram-staining Gram-positive and 2) Gram-negative non-Gram-staining. The relation to Gram stain is such an important differential feature of bacteria that it is necessarily mentioned in their characterization and serves as a taxonomic feature. 30 Gram stain technique A strip of filter paper previously impregnated with gentian violet is placed on a fixed smear. Apply 3-5 drops of tap water to the strip. After 1-2 minutes, the paper is removed with tweezers, and Lugol's solution is poured onto the preparation. After 30 sec 1 min, the Lugol solution is drained. Apply a few drops of 96 O alcohol. Discolor for 1 min until the grayish-violet streams disappear. The drug is washed with water. Fuchsin is poured onto the smear and kept for 1-2 minutes. The paint is drained, the preparation is washed with water, dried with filter paper. Microscopically with an immersion system. Gram-positive bacteria stain violet-blue (for example, butyric fermentation bacillus Clostridium pasteurianum), and gram-negative bacteria stain pink-red (Escherichia coli). In addition to this traditional Gram stain technique, there is a quick and easy method for Gram differentiation without staining. Bacterial cells (preferably 1-2-day-old) are placed in a loop in a drop of 3% KOH on a glass slide, stirred in a circular motion, and after 5-8 s, the loop is sharply lifted. The suspension of gram-negative bacteria becomes viscous and stretches behind the loop, forming mucous bands. Gram-positive bacteria are evenly distributed in a drop of alkali (as in water). The reaction is considered negative if the formation of mucous cords is not observed within 60 s. The increase in viscosity is caused by lysis of the cell walls of gram-negative bacteria in solution
31 alkali and DNA release. Gram differentiation of bacteria without staining should be used only for preliminary diagnosis, or for calculating the approximate ratio of Gram-positive and Gram-negative bacteria colonies. Identification of living and dead cells by staining with methylene blue The number of living and dead cells can be determined by staining with a solution of methylene blue. The method is based on the different permeability of living and dead cells of microorganisms. Cell permeability in dead cells is impaired, so the dye passes freely through the cytoplasmic membrane and is adsorbed by the protoplasm. Under a microscope, dead cells look blue, live ones are colorless or pale blue. Staining is carried out as follows: a drop of 2% methylene blue solution is applied to a glass slide, covered with a coverslip, excess paint is drawn off with a piece of filter paper. The drug is viewed under a microscope; in 10 fields of view, the number of living and dead cells is counted; the number of dead cells is expressed in%. Example: the average number of live cells in one field of view is 10, the average number of dead cells in one field of view is 5, total number cells in one field of view cells 100% 5 cells X X 33.3% 15 Thus, the number of dead cells in the studied suspension of microorganisms was 33.3%. Determination of cell sizes To determine the size of microorganism cells, it is necessary to have a special eyepiece with a scale (ocular micrometer) and an object-micrometer. Ocular micrometer, at 31
32 in the simplest case, it is a glass disk with a linear scale printed on it, which is inserted into the eyepiece (Fig. 14a). The object-micrometer is a glass slide, in the center of which a linear scale 1 mm long is engraved, with a division value of 10 µm. Before measurement, it is necessary to determine the division value of the ocular micrometer for each magnification of the microscope. To do this, the micrometer object is placed on the microscope stage and considered as a preparation; in this case, one of the visible divisions of the object-micrometer is aligned with the zero mark of the scale of the ocular micrometer, and the divisions of both scales coincide (Fig. 15). Count how many ocular and objective divisions fall on this segment, and calculate the value of the divisions of the ocular micrometer. If, after that, a preparation of microorganisms is placed on the object table instead of an object-micrometer and it is examined 32
33 at the same magnification, it is possible to measure the size of a microbial cell using the scale of an ocular micrometer as a ruler. For accurate measurements, a special ocular micrometer with a sliding zero mark is used, associated with a measuring drum (Fig. 14b). It allows you to determine the size of microorganisms with an accuracy of tenths of a micron. The zero mark is a double vertical line, the middle of which corresponds to the intersection of two thin lines in the form of a cross. Before measurements, it is necessary to find out the value of the scale division of the measuring drum. The reference object is a micrometer. By rotating the measuring drum, several divisions of the object-micrometer scale are circled with a zero mark. The double vertical line shows the number of complete revolutions of the measuring drum. Carrying out measurements of microorganisms, move the zero mark along the object and read the readings of the measuring drum. Practical part The purpose of the work is to master the basic methods of studying the morphology of microorganisms. 33
34 Work order 1. Prepare fixed preparations of lactic acid bacteria from biokefir and acidophilus. 2. Gram-stain Bacillus subtilis (gram+) and Escherichia coli (gram-) cells. 3. Determine the size of Saccharomyces cerevisiae yeast cells. 4. Determine the number of living and dead cells of Saccharomyces cerevisiae in the yeast suspension. 34 Contents of the report 1. Morphology of lactic acid bacteria cells (drawings of fixed preparations of lactic acid bacteria with indication of microscope magnification). 2. Drawings of fixed preparations of gram-negative and gram-positive bacteria. 3. Determination of the division value of the measuring drum of the ocular micrometer. 4. Dimensions of a Saccharomyces cerevisiae yeast cell. 5. The number of live and dead cells in the yeast suspension. Control questions 1. How to prepare a fixed preparation of bacteria? 2. What is the reason for the difference between bacteria in Gram stain? 3. What is the Gram stain method? 4. How to determine the size of microorganisms? 5. How to determine the number of living and dead cells of microorganisms by staining with methylene blue? Topic 2. Cultivation of microorganisms: principles of preparation of nutrient media; sterilization methods; methods of crops and subcultures of microorganisms. Classification and preparation of nutrient media Cultivating microorganisms means artificially creating conditions for their growth. For cultivation
35 microorganisms in the laboratory or in industry, nutrient media are used that contain all the substances necessary for the vital activity of microorganisms. From the environment, nutrients enter the cell of the microorganism, and metabolic products are removed from the cell into the environment. For the vital activity of microorganisms, water, carbon, oxygen, nitrogen, sulfur, phosphorus, potassium, calcium, magnesium, iron and trace elements are necessary. All these substances must be contained in the nutrient medium. Even without one of them, there will either be no growth at all or it will be negligible. Carbon, hydrogen, nitrogen, phosphorus and sulfur are called biogenic elements, since they account for about 90-95% of the dry mass of cells. Potassium, magnesium, calcium and sodium are called macronutrients, or ash elements. They account for up to 5-10% of the dry mass of cells. Iron, manganese, molybdenum, cobalt, copper, vanadium, zinc, nickel and some other heavy metal cations are called trace elements and make up fractions of a percent of the dry mass of cells. Carbon is of the greatest importance for any living organism. It is included in all organic molecules in the cell and accounts for about 50% of the dry biomass. In relation to carbon, all organisms are divided into autotrophic and heterotrophic. Autotrophs use carbon dioxide as a source of carbon. Heterotrophs need ready-made organic compounds. Various organic substances can serve as a source of carbon for most microorganisms: proteins and their decay products, carbohydrates, fats, hydrocarbons. Nitrogen nutrition in its value is in second place after carbon. Nitrogen is part of the amino acids and other cellular components that ensure the viability of organisms. Nitrogen makes up 14% of the dry matter of the cells. The source of nitrogen is nitrogen-containing organic or mineral compounds. The most common sources of mineral nitrogen are ammonium salts and nitrates. Proteins, amino acids, nucleotides are used as organic sources of nitrogen. Some prokaryotes can use atmospheric nitrogen. 35
36 Phosphorus and sulfur are part of important cell biopolymers. Phosphorus (3% of cell dry matter) is part of nucleotides and ATP, and sulfur (less than 1%) is part of some amino acids. As a source of phosphorus, phosphates are usually used, and sulfurs are sulfates. Phosphorus and sulfur can also be used in the form of organic compounds. For the growth of microorganisms, macronutrients are required in small quantities: ions alkali metals(Na+, K+) and alkaline earth metals(Mg 2+, Ca 2+) that play important role in the activity of microorganisms. Macronutrients in microbial cells are necessary to regulate permeability, osmotic pressure, and pH. In addition to these metals, a number of trace elements (trace elements) are required for microbial growth. Mineral composition nutrient medium forms the distribution of electrical charges on the cell surface. Changing the electrical potential of cells can change their physiological activity. One of the main functions of trace elements is participation in enzymatic catalysis. Currently, the action of the fourth part of all enzymes in the cell is associated with metals. In addition to the main components of the nutrient medium, for the normal development of some microbes, additional substances are also needed, which are called "growth factors". Growth factors is the combined name of various chemical nature connections. These are mainly organic substances, the addition of which in very small quantities stimulates the growth and reproduction of microorganisms. They are to microbes what vitamins are to higher organisms. Growth factors are mainly B vitamins, which play the role of regulators and stimulants of microbial metabolism, or amino acids. Yeast autolysate, yeast extract, native proteins (blood, serum), etc. are used as growth factors. Microorganisms' nutritional requirements are very diverse. For this reason, there is no universal 36
37 medium, equally suitable for the cultivation of any microorganism. Depending on the goals of cultivation and the needs of a given microorganism, nutrient media differ in three ways: in composition, physical condition and appointment. In terms of composition, nutrient media are divided into two groups: 1) natural (natural); 2) artificial (synthetic). Natural media are those that have an indefinite chemical composition, since they include products of plant or animal origin, waste from various industries containing organic compounds. Natural nutrient media provide intensive growth of a wide variety of microorganisms. They contain a rich set of organic and inorganic compounds, including all the necessary elements and additional substances. For example, the following culture media are most commonly used in the laboratory. 1. Meat-peptone broth (MPB) extract from meat, contains products of incomplete protein breakdown peptones, which contain organic carbon, organic nitrogen, phosphorus-containing, sulfur-containing organic substances. The BCH also contains all the minerals necessary for microorganisms. MPB is used in the cultivation of many types of bacteria. 2. Beer wort extract from germinated barley grains, contains sugar (mainly maltose) as a carbon source, nitrogenous substances, ash elements, various growth factors and B vitamins. Beer wort is a good environment for the development of many microorganisms, in particular yeast, mold fungi. Good natural media are milk, potatoes, decoctions of fruits and vegetables. In industry, semi-synthetic or natural media are usually used. Very often, microbiological or food industry waste is used as a carbon source: molasses (waste from sugar production), 37
Ministry of Agriculture of the Russian Federation FEDERAL FISHING AGENCY KAMCHATKA STATE TECHNICAL UNIVERSITY DEPARTMENT OF BIOLOGY AND CHEMISTRY CHEMISTRY AND WATER MICROBIOLOGY PREPARATION
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Laboratory work "The structure of the bodies of cap mushrooms" Purpose: to study the structure of the bodies of cap mushrooms Equipment: cap mushrooms (porcini mushroom, russula, champignon, boletus, butterdish, honey agaric, etc.), ready
FEDERAL STATE EDUCATIONAL INSTITUTION OF HIGHER PROFESSIONAL EDUCATION "OMSK STATE AGRARIAN UNIVERSITY NAMED AFTER P.A. STOLYPIN" INSTITUTE OF VETERINARY MEDICINE AND BIOTECHNOLOGY
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Research Yeast: the exciting life of small mushrooms Author of the work: student of 5 "B" class GBOU Gymnasium 1748 "Vertical" Kutaytsev Georgy Valerievich Head: Nosova Elena Vladimirovna,
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GENERAL MICROBIOLOGY
Chapter I. Microscope and microscopy technique
1. Light-optical microscopy
2. Dark field microscopy
3. Phase contrast microscopy
4. Luminescent (fluorescent) microscopy
Chapter II. General understanding of cultivation, seeding technique and the necessary equipment for working with microorganisms
Chapter III. Methods for the preparation of preparations of microorganisms
Chapter IV. microbial cell research
1. Forms of cells of microorganisms
2. The structure of microorganism cells (cytochemical research methods)
3. Gram staining of microorganism cells
4. Coloring of spores in bacteria
5. Capsule coloring
6. Coloring of flagella
7. Coloring of the nuclear substance of bacteria
8. Staining of microorganism cell inclusions
Chapter V. Nutrition of microorganisms
1. Importance of individual nutrients
2. Preparation of culture media
3. Sterilization methods
Chapter VI. Accounting for the number of bacteria and isolation of pure culture
1. Accounting for the number of bacteria in the soil
2. Determination of the qualitative composition of bacteria
3. Accounting for the number of microorganisms in water and other liquids
4. Accounting for the number of bacteria in the air
5. Isolation of pure bacterial cultures
Chapter VII. Determination of the type of bacteria
Chapter VIII. The transformation by microorganisms of nitrogen-free organic matter
Fermentation processes
1. Alcoholic fermentation
2. Lactic acid fermentation
3. Butyric fermentation
4. Fermentation of pectin substances
5. Cellulose fermentation
6. Fiber oxidation
7. Fat oxidation
8. Hydrocarbon oxidation
Chapter IX. Transformation of organic and mineral nitrogen compounds by microorganisms
1. Ammonification
2. Nitrification
3. Denitrification (nitrate respiration)
4. Biological fixation of atmospheric nitrogen
Chapter X
1. Conversion of sulfur compounds by microorganisms
2. Participation of microorganisms in the transformation of iron
3. Transformation of phosphorus compounds by microorganisms
AGRICULTURAL MICROBIOLOGY
Chapter XI. General microbiological analysis of the soil
1. Research methods
2. Groups of microorganisms, composition and preparation of culture media
3. Taking an average soil sample and preparing the sample for microbiological analysis
4. Preparation of soil suspension
5. Accounting for different groups of microorganisms
6. Determination of the total number of microorganisms in the soil by direct counting under a microscope
Chapter XII. The study of cenoses of microorganisms
1. Glass fouling method according to N. G. Kholodny
2. The study of microbial cenoses in the soil according to the method of Perfiliev and Gabe
3. Perfiliev capillary method modified by Aristovskaya
4. Identification of microorganisms of the autochthonous group involved in the decomposition of humic substances, according to the method of Winogradsky in the modification of Tepper
5. Identification of microorganisms involved in the decomposition of humic substances, according to the Tepper method
Chapter XIII. Determination of soil biological activity
1. Determination of the biological activity of the soil by the intensity of decomposition of the canvas (method of Mishustin, Vostrov and Petrova)
2. Determination of the total microbiological activity of the soil by excretion carbon dioxide
3. Determination of soil ammonifying activity
4. Determination of the ammonifying activity of microorganisms
5. Determination of the nitrifying activity of the soil
6. Determination of soil denitrifying activity
7. Determination of the nitrogen-fixing activity of microorganisms
Chapter XIV. Study of bacteria in the root zone of plants and on roots
1. Accounting for bacteria in the rhizosphere by the Krasilnikov method
2. Accounting for rhizosphere and root microflora by the method of successive washing of roots according to E. 3. Tepper
3. Isolation of pure cultures of nodule bacteria, quantitative accounting in the soil, determination of their activity and virulence
Chapter XV. Analysis of bacterial preparations
Chapter XVI. Feed microbiology
1. Epiphytic microflora of grain and its change during feed storage
2. Silo analysis
3. Feed yeasting
Chapter XVII. Microflora of milk and dairy products
1. Bacteriological analysis of milk
2. Methods for isolating lactic acid bacteria into pure culture
3. Acquaintance with the microflora of butter
Literature index
