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 representations on cultivation, seeding techniques 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

Guidelines

for the implementation of practical work

for profession:

19.01.17 Cook. Confectioner

Developer:

Veretennikova O.M. teacher

Valuiki, 2016

Explanatory note

These guidelines for the implementation of practical work on the discipline "fundamentals of microbiology, sanitation and hygiene in food production » were developed on the basis of the Federal State educational standard(hereinafter - GEF) by profession:

01/19/17 Cook. Confectioner

Guidelines for the implementation of practical work are intended for first-year studentsThe implementation of practical and laboratory work is aimed at solving the followingtasks:

increase awareness and strength of knowledge acquisition;

develop the ability to analyze, compare the objects under study, conduct research, draw up tables, diagrams, clusters, draw conclusions;

develop in students logical thinking, cognitive abilities, independence;

learn how to use the acquired knowledge and skills in life.

When studying, fixing the material, the following types are used independent work:

Work with the text of the textbook.

Presentation work.

Work with the studied object.

Working with a table.

Work on drawing up a cluster, scheme.

Working with prepared micropreparations. Preparation of micropreparations.

Structure of guidelines:

1. theme
2. purpose of the work
3. equipment for work
4. work progress
5. control and updating of students' knowledge necessary to complete the work
6. working conditions

Each practical and laboratory work should be drawn up in a notebook for practical work in accordance with the recommendations. (Annex 1)

Control of the results of the work performed is carried out on the basis of a written report and the results of monitoring the student in the course of the work in accordance withevaluation criteria for the implementation of practical work.

List of practical works

Practical work No. 1

The device of the microscope and the rules for working with it.

Practical work No. 2 Microscopic examination of the morphology of yeast and mold

Practical work No. 3 Schemes of the structure of cells of bacteria, yeast, fungi.

Practical work No. 4-5

Practical work No. 6Schemes for the preparation of disinfectant solutions and their storage

Practical task aimed at checkingthe ability of students to apply theoretical knowledge in the discipline in practice

Practical work №1 MICROSCOPE DEVICE AND RULES OF WORKING WITH IT.

Goal of the work: To study the device of a light biological microscope and master the rules for working with it.

Equipment, materials: Microscope; finished micropreparations

Microscope (from Greek.micros- small andscopio- I look) is an optical device, consisting of three main parts: mechanical, optical and lighting.

The diagram of a light biological microscope is shown in fig. 1.

Mechanical or a tripod consists of a leg, a base, a tube holder, an object stage, a monocular attachment (tube), a revolving device, a coarse focusing handle (macrometric screw), a fine focusing handle (micrometric screw).

The tube is the telescope of a microscope. An eyepiece is freely inserted into the upper hole of the tube, at the lower end of the tube there is a revolving device (revolver) rotating around its axis, into which the lenses are screwed. By rotating the revolver, you can quickly change lenses while working with a microscope, bringing any lens under the tube. The lens must be centered, i.e. mounted on the optical axis of the microscope. To do this, the revolver is rotated around its axis until a click appears.

The object table is used to place the study drug on it. The drug is fixed on the table with clamps (terminals). In the center of the object stage there is a hole for the passage of light rays and illumination of the preparation. In some microscope designs, the stage can be moved using screws located around the periphery of the stage. This makes it possible to examine the drug in different fields of view.

1 – eyepiece

2 – monocular head

(tube)

3 - revolver

4 - lens

5 - subject table

6 - condenser

7 - Collector lens housing

8 - socket with a lamp

9 - hinge

10 – handle for moving the condenser bracket

11 – fine focus knob (micrometer screw)

12 – coarse focus knob (macrometer screw)

13 - tube holder

14 - screw for fastening the nozzle

Rice. 1Scheme of the device of a light biological microscope

Coarse and fine focus knobs (macro and micro screws) are used to move the tube up and down, which allows you to set it at the required distance from the preparation. Turning the screws clockwise lowers the tube, while turning it counterclockwise raises it. When turning the macrometric screw, the lens is approximately set to focus, i.e. at the distance from the preparation at which it becomes visible. The turn of the macro screw allows you to move the tube by 20 mm. The micrometer screw is used for precise focusing. A full turn of it moves the tube by 0.1 mm. The microscrew should be handled very carefully: it is permissible to rotate the microscrew no more than 180 0 C one way or the other.

Optical part is the most valuable part of the microscope. It consists of lenses and an eyepiece.

Eyepiece (from lat.oculus- eye) consists of two plano-convex lenses enclosed in a common metal frame. The upper lens is an eye lens (magnifying), the lower one is converging. The distance between the lenses is half the sum of their focal lengths. High magnification eyepieces have a shorter focus, so the length of the eyepiece is also shorter. Between the lenses there is a diaphragm that limits the field of view and delays the edge rays of light. Domestic microscopes are equipped with three interchangeable eyepieces, the magnification of which is indicated on the eyepiece body (x7; x10; x15).

The objectives are screwed into the sockets of the revolving device and consist of a system of lenses enclosed in a metal frame. The front (front) lens of the objective is the smallest and the only one that gives magnification. The remaining lenses in the lens only correct the imperfections of the resulting image (phenomena of spherical and chromatic aberration) and are called corrective.

Four lenses are screwed into the sockets of the revolving device, the magnification of which is indicated on the lens barrel (x8; x20; x40; x90 or 100). Each lens is characterized by its focal length (the distance between the glass slide and the front lens): the x8 lens has a focal length of about 9 mm, the x40 lens has a focal length of 0.65 mm, and the x90 lens has a focal length of 0.15 mm.

lighting part The microscope consists of a two-lens condenser, an iris diaphragm and a cartridge with a low-voltage incandescent bulb, powered through a step-down transformer from a voltage network of 120 ... 220 V.

The condenser serves for better illumination of the preparation. He collects the light rays into a beam and directs them through the opening of the stage to the preparation. The condenser arm can be moved up and down using the handle to move the condenser arm, which changes the angle of convergence of the beams and, consequently, the degree of illumination of the object. The higher the position of the condenser, the better the preparation is illuminated.

The iris diaphragm is located under the condenser and serves to regulate the flow of light entering the condenser. It consists of metal crescent-shaped plates. You can expand or narrow the aperture of the diaphragm using a special lever. When rotated clockwise, the aperture of the iris diaphragm increases and, consequently, the degree of illumination of the object increases.

When working with immersion lenses, the degree of illumination of the preparation should be maximum, so the shutter of the iris diaphragm is opened, and the condenser is raised to its highest position.

When working with dry lenses, as a rule, unpainted objects are considered. To achieve contrast, the condenser is lowered down, and the aperture of the iris diaphragm is reduced.

Rules for working with a microscope

On the desktop, the microscope is placed with the tube holder towards itself at a distance of 3 ... 5 cm from the edge of the table;

Turn on the microscope and set the correct lighting

The test preparation is placed on the object table and fixed with clamps;

The desired lens is placed under the tube and the focal length is set using the macro and micro screws. So, when working with immersion lenses, a drop of immersion oil is first applied to the preparation and the tube holder is carefully lowered with a macroscrew until it comes into contact with the glass. Then, carefully looking into the eyepiece, the tube holder is very slowly raised, rotating it counterclockwise, until the image is seen. Fine focusing of the lens is carried out with a micrometer screw. When working with dry objectives, the specimen is first examined with an x8 objective. Raising the tube holder with a macro screw and carefully looking into the eyepiece, set the focal length (about 9 mm) and achieve image clarity using a micrometer screw. Further, by moving the object table or glass slide, the area of ​​the preparation is set in the center of the field, in which the object under study is best seen. Then, rotating the revolving device around its axis, a lens of x20 or x40 is placed under the tube. In this case, the x90 lens should not fall under the tube. In the revolving device, the lenses are arranged in such a way that if an image with an x8 lens is found, then when examining a specimen with lenses of higher magnification, it is necessary to slightly adjust the clarity of the image using macro- and micrometer screws;

During microscopy, it is necessary to keep both eyes open and use them alternately;

After finishing work, remove the preparation from the object table, lower the condenser, place the x8 objective under the tube, remove the immersion oil from the front lens of the x90 objective with a soft cloth or gauze soaked in alcohol, put a gauze napkin under the objective, lower the tube holder.

What is the device of a biological microscope?

What parts and mechanisms does the mechanical part of the microscope consist of?

What is the optical system of a microscope?

What is included in the microscope lighting system?

How should the lighting system be set up when working with an immersion lens?

List the basic rules for working with a microscope.

Conditions for completing the task
1. Place (time) of the task
Biology class

Practical work №2 Microscopic examination of the morphology of yeasts and molds.

Goal of the work : Familiarize yourself with the morphological features of fungi and yeasts found in food production. To master the technique of microscopic examination of fungi and yeasts in “crushed drop” preparations.

Equipment, materials: Microscope; dissecting needles, glass slides and coverslips; filter paper; spirit lamp; cultures of mushroom generaMucor, Aspergillus, Penicillium, Alternaria; pure yeast cultureSaccharomycescerevisiae.

1. BRIEF THEORETICAL PROVISIONS

1. 1. Morphology and cultural features of microscopic fungi

The vegetative body of fungi is calledmycelium . Mycelium consists of many intertwining filaments called tubules.hyphae . The diameter of hyphae ranges from 5 to 50 microns. Depending on the structure of the mycelium, fungi are divided into higher and lower. In higher fungi, the hyphae are separated by partitions (septa) in the center of which there is a large pore. They grow and at the same time nuclear divisions occur, but cell divisions do not occur. Thus, the vegetative body of the fungus is one large multinucleated cell. All microscopic fungi can reproduce vegetatively with a piece of mycelium.

During asexual reproduction, phycomycetes producesporangiophores , and in Ascomycetesconidiophores .

Cultural signs of microscopic fungi

Colonies of microscopic fungi are many times larger than colonies of unicellular organisms (bacteria, fungi) and often grow over the entire surface. growth medium in Petri dishes. The consistency of mushroom colonies is different. Felt-like and leathery colonies are more often formed, less often crumbly. The surface of the colonies may be fluffy, like cotton wool, velvety, mealy, cobweb-like, filamentous, leathery or smooth. When growing on dense and liquid media, some of the hyphae grow into the nutrient medium, formingsubstrate mycelium, and the other part of the hyphae formsair mycelium in the form of a fluffy coating, visible to the naked eye. Mycelium can also be colorless (white, grayish) or colored (black, brown, green, yellow, etc.). Only the fruiting mycelium is pigmented.

Characteristics of microscopic fungi various classes

Morphological features of fungi of various classes are presented in fig. 5.

GenusMucor . They can reproduce asexually and sexually with the formation of sporangiophores (Fig. 5). Outside, the sporangium is covered with thin spikes of calcium oxalate crystals. At maturity, the sporangium ruptures, the sporangia spores are released and carried by air currents. On the sporangiophore, after the release of the sporangium from the spores, a column remains, and in its lower part - a collar. The color of the mycelium of mucor fungi is initially white, then grayish-olive, the appearance is felt-like.

A

b

V

G

Rice. 5Morphological features of fungi of various classes:

A - Mucor; b - Penicillium; V - Aspergillus; G - Alternaria

Mucor fungi grow on the surface of wet grain, malt, root crops, on food products, on the walls of damp rooms in the form of a grayish fluffy coating.Mucornigricansis the causative agent of the clamp rot of sugar beets. Many mucor fungi are used in industry for the production of various organic acids and alcohol (mushrooms of the speciesMucorjavanicus, Mucorracemosus), enzyme preparations, carotenoids, steroids.

Genus representativesAspergillus And Penicillium belong to the class Ascomycetes, which combines the highest microscopic perfect fungi. With asexual reproduction using spores, these fungi form conidiophores (Fig. 5). Aspergillus and penicillium are fungi. This means that during sexual reproduction, asci (bags) are formed on special fruiting bodies, in which there are 8 ascospores.

To the genusPenicillium accounts for about half of all mold fungi. They are widely distributed in the soil, in the air of poorly ventilated areas and cause deterioration of various products and materials. This mushroom has a branching septate mycelium (hyphae diameter - 2 ... 3 microns) and septate conidiophores (reminiscent of brushes), which branch out at the end in the form of processes - sterigmas. Conidia, consisting of chains of spores, depart from them. Depending on the type of conidia, they can be of different colors (white, green, etc.). Many penicilli are used in industry to obtain various valuable products. Among the isolated strains of this genus, 25% have antibiotic activity, and such species asPenicilliumnotatum, Penicilliumchrysogenumare used as producers of penicillin. Some types of penicillium are used as enzyme and lipid producers. Noble molds are used in the production of soft Roquefort and Camembert cheesesPenicilliumroquefortiAndPenicilliumcamamberti.

Mushrooms of the genusAspergillus there are more than 200 species. These mushrooms have a well-developed branching mycelium with numerous septa. Conidiophores are not septate, their upper ends are pear-shaped or spherically expanded in the form of a small head. On the head are pin-shaped sterigmata with chains of conidia, which resemble streams of water pouring out of a watering can. Hence the name "leak mold" (aspergerein Latin - to water, to spray). Aspergillus conidia acquire different colors when maturing, which, along with other features, determines their species affiliation.

As well as penicilli, representatives of the genusAspergillusare widely distributed in nature and play an important role in the mineralization of organic substances. They cause mold in many foods. These mushrooms are producers of many valuable substances and are widely used in industry. So,AspergillusNiger, used in industry for the production of citric acid;Aspergillusterreus- itaconic acidAspergillusflavusAndAspergillusterricolaform the most active complex of proteolytic enzymes;AspergillusoryzaeAndAspergillusawamoriare the best producers of amylolytic enzymes.

Mushrooms of the genusAlternaria belong to the class of imperfect fungi - deuteromycetes. These are the higher mushrooms. They have septate mycelium and short non-septate conidiophores, on which are pear-shaped or lemon-shaped multicellular conidia (Fig. 5). The fungus is the causative agent of black rot - a disease of root crops and fruits, as well as the causative agent of food spoilage.

Morphology of yeast and their characteristics

Yeast are higher unicellular fungi. Most yeasts belong to two classes of fungi - ascomycetes and deuteromycetes.

In relation to oxygen, yeasts are divided into facultative anaerobes (under aerobic conditions they respire and actively accumulate biomass, and under anaerobic conditions cause alcoholic fermentation) and aerobes.

Morphologically, yeasts are diverse. They differ from each other in the size and shape of the cells. The sizes of yeast cells, depending on the species, vary within the following limits; 2.5 to 10 µm across and 4 to 20 µm long. The morphological diversity of yeast forms is shown in Fig. 6.

A

b

V

G

d

e

and

h

Rice. 6Forms of yeast cells: a - oval ovoid;

b - cylindrical; c - apical; lemon-shaped; g - swept;

e - triangular; e - crescent; g - cone-shaped; h, i - myceloid

The shape and size of yeast cells depend on the type, age, nutrient medium, and cultivation method.

Depending on the species, yeast can reproduce vegetatively by budding (this is how oval-shaped yeast reproduces), binary fission (typical for cylindrical or rod-shaped yeast), or budding division. In addition to vegetative reproduction, yeast - ascomycetes can reproduce sexually with the formation of ascospores.

From yeast belonging to the class Ascomycetes, great importance have yeast-saccharomycetes of the genusSaccharomyces , which widely used in the food industry. The main biochemical feature of these yeasts is that they ferment sugars to form ethyl alcohol and carbon dioxide. Yeast used in industry is calledcultural yeast. So, in the baking industry and in the production of alcohol, riding yeast of the genusSaccharomycescerevisiae. Yeast speciesSaccharomycesminorfound application in the production of rye bread and kvass. Grassroots yeast is used in brewingSaccharomycescarlsbergensis. Saccharomycete yeasts are oval in shape, reproduce vegetatively by budding, and in unfavorable conditions reproduce sexually by ascospores.

Some sporogenic yeasts arewild yeast . These yeasts, like cultural ones, are capable of alcoholic fermentation, but in addition to alcohol they form many by-products (such as aldehydes, higher alcohols, esters, etc.) and therefore worsen the organoleptic characteristics of the product. These yeasts are pests in the production of various beverages (beer, wine, soft drinks), as well as spoilage agents in many foods.

Yeast - deuteromycetes can only reproduce vegetatively. Some of these yeasts (for example, yeasts of the genusCandida) are used in industry to obtain feed protein, organic acids, vitamins and other products of microbial synthesis. Yeast speciesTorulopsiskefirare part of the symbiotic sourdough - kefir fungus. Other imperfect (asporogenic) yeasts are wild yeasts and cause spoilage in many foods. Yeast pests of production include yeast generaPichia, Hansenula, Candida, Rhodotorula,Torula, Torulopsis, Mycoderma, Trichosporonetc. Among asporogenic yeasts there arefalse yeast , which form pseudomycelium and grow on liquid substrates in the form of films.

1. WORK PROCEDURE

A large drop of water is applied to a glass slide with a tube or pipette;

A small amount of mycelium is taken from a test tube or Petri dish, observing the rules of asepsis

The mycelium is carefully placed in a drop applied to a glass slide and, with the help of two needles, it is straightened in water;

The preparation is covered with a coverslip and lightly pressed down. Excess water is removed with filter paper.

The “crushed drop” preparation is microscoped first with an x8 lens, and then x40 in a darkened field of view (the condenser is lowered, the iris shutter is covered).

When selecting and microscopy of fungal preparations, the following recommendations are taken into account:

a) fungus Mucor . A blackish-gray fluffy aerial mycelium is selected. Under microscopy, attention is paid to hyphae with spore-filled sporangia and columns that form when the sporangium is released;

b) mushroom of the genus Aspergillus . A little fluffy mycelium with colored conidia is selected, slightly deepening with a needle into the nutrient medium. Pay attention to unsepted conidiophores;

c) mushroom of the genus Penicillium . When selecting, they try to take a young mycelium (on the border of the colored and white mycelium), deepening with a needle into the medium. Pay attention to septate hyphae with brushes.

d) mushroom of the genus Alternaria . They take the mycelium in black areas, deepening into it with needles. Pay attention to the septate mycelium, poorly developed conidiophores and large conidia, which look like rounded or pointed multicellular formations resembling "lemon pomegranates".

In the study of yeast a suspension of yeast is applied to a glass slide, covered with a coverslip, excess water is removed with filter paper. The specimen is microscoped with an x8 and x40 lens.

Registration and analysis of research results

Briefly outline the theoretical material. Microscopic pictures of the studied cultures of fungi and yeast are drawn, taking into account the morphological features of each microorganism. Under each figure, the Latin name and the increase in the preparation are signed. Describe the cultural properties of the studied fungi.

Answer Control questions

How are preparations of microscopic fungi and yeast prepared?

Describe the morphological and cultural properties of microscopic fungi.

What mushrooms are used in industry to obtain organic acids, enzymes, antibiotics and other valuable products?

Describe morphological properties yeast.

What is cultural yeast? In what areas of the food industry are they used?

Conditions for completing the task

Biology class

2. Maximum task completion time: 90 min

Practical work No. 3: Schemes of the structure of cells of bacteria, yeast, fungi.

Goal of the work: Study the structure of the cellbacteria, yeast, fungi

Material support: instruction cards for practical work, textbook, pencils

Exercise 1

Study the material in the textbook. According to the results of the study:

Draw in a notebook the structure of a cell of bacteria, yeast and fungi and indicate the distinguishing features

Answer the following questions in writing:

1. What shape do bacterial cells have?

2. What are the sizes of bacteria?

3. How does the reproduction of bacteria occur, the rate of reproduction?

4. How and under what conditions does spore formation occur in bacteria?

5. Are bacteria capable of independent movement?

Make a conclusion based on the results of your work.

Conditions for completing the task

1. Place (time) of the task

Biology class

2. Maximum task completion time: 90 min

Practical work on topic No. 4 Working with regulatory and technical documentation: SanPiN 2.3.6. 1079-01

Goal of the work: To study the sanitary requirements for the arrangement and maintenance of public catering establishments

Material support: instruction cards for practical work, SanPiN 2.3.6. 1079-01

Exercise 1

Study the material in the textbook. SanPiN 2.3.6. 1079-01. According to the results of the study:

1. Add the phrases: The site where the catering establishment is built must be

Manufacturing facilities include:

Warehouses are designed in ____________________ part of the building.

Drinking water must be of the same quality

Ventilation is used to purify the air

Type.

All production areas must be illuminated

Light.

Monthly housekeeping is called

2. Define the following terms:

Disinfection is...

Deratization is -

Disinsection is...

3. Using the educational material, fill in the table:

vegetable shop

Butcher shop

Fish shop

Hot shop

cold shop

Confectionary shop

Handout

Conditions for completing the task

1. Place (time) of the task

Biology class

2. Maximum task completion time: 90 min

Practical work No. 5 Working with regulatory and technical documentation: SanPiN 2.3.6. 1079-01

Goal of the work : To study the sanitary requirements for equipment, inventory, utensils, containers. Transportation and storage of food products.

material support : instructive cards for practical work, SanPiN 2.3.6. 1079-01

Exercise 1

Will study the material of the textbook, SanPiN 2.3.6. 1079-01. According to the results of the study:

1. Answer the following questions in writing:

What about kitchen utensils?

Why label dishes?

What about tableware?

What materials are allowed for the production of equipment and inventory

for catering establishments?

What is the fundamental difference between washing tableware and cutlery?

2. List the rules and requirements:

2.1. Sanitary rules for the transportation of semi-finished products:

2.2. Sanitary rules for food storage:

3. Add phrases:

Prior to distribution, the quality of the prepared meals must

When serving, first courses and hot drinks must be at a temperature

_______ °С, main dishes and side dishes temperature ______ °С, portioned dishes

temperature ______ °С, cold dishes and drinks ______ °С.

In medical and preventive and children's institutions in the winter-spring period, due to a lack of vegetable dishes ___________________, it is required to enrich some dishes with this.

________________ is responsible for the quality of the finished product and compliance with the rules for its release at catering establishments.

Conditions for completing the task

1. Place (time) of the task

Biology class

2. Maximum task completion time: 90 min

Practical work No. 6 Schemes for the preparation of disinfectant solutions and their storage

Target: to study the name of disinfectants, methods of preparing disinfectant solutions depending on the purpose. Prepare a solution of a given concentration.

material support : instructive cards for practical work, SanPiN 2.3.6. 1079-01, textbook

Exercise 1

Will study the material of educational literature, SanPiN 2.3.6. 1079-01. According to the results of the study:

1. Answer the questions:

What solutions are disinfectants?

What is the purpose of disinfectants?

What drugs are used as disinfectants?

How to recognize that the dishes were treated with disinfectants?

2. To study the preparation schemes and the purpose of disinfectants. Fill in the table.

3. Prepare 1 liter of 0.2% solution of chloramine B.

4. Make a conclusion based on the results of the work.

Conditions for completing the task

1. Place (time) of the task

Biology class

2. Maximum task completion time: 90 min

Evaluation criteria for the implementation of practical work:
A score of "5" is given if :
1. Correct independently determines the purpose of these works; performs the work in full in compliance with the necessary sequence of performance.
2. Independently, rationally selects and prepares the necessary equipment for the performance of work; carries out these works in conditions that provide the most accurate results.
3. Competently, logically describes the progress of work, correctly formulates conclusions; accurately and accurately performs all records, tables, drawings, drawings, graphs, calculations.
4. Shows organizational and labor skills: maintains a clean workplace, order on the table, uses materials economically; complies with safety regulations when performing work.
A score of "4" is given if :
1. Performs laboratory work in full accordance with the requirements when evaluating the results by "5", but allows two to three shortcomings or one minor error and one shortcoming in calculations, measurements.
2. When drawing up work, it allows inaccuracies in the description of the course of action; draws incomplete conclusions when generalizing.
A score of "3" is given if :
1.1 Correctly performs work by at least 50%, however, the volume of the completed part is such that it allows you to get the right results and draw conclusions on the main, fundamental important tasks work.
2. Selects equipment, material, starts work with the help of a teacher; or in the course of measurements, calculations, observations, makes mistakes, inaccurately formulates conclusions, generalizations.
3. Carries out work in irrational conditions, which leads to results with large errors; or in the report allows a total of no more than two errors (in recording numbers, measurement results, calculations, drawing up graphs, tables, diagrams, etc.), which are not of fundamental importance for this work, but affected the result of execution.
4. Makes a gross mistake in the course of the work: in the explanation, in the design, in observing the safety rules, which the student corrects at the request of the teacher.
A score of "2" is given if :
1. Does not independently determine the purpose of the work, cannot prepare the appropriate equipment without the help of a teacher; performs the work incompletely, and the volume of the completed part does not allow drawing correct conclusions.
2. Makes two or more gross mistakes in the course of work, which cannot be corrected at the request of the teacher; or makes measurements, calculations, observations incorrectly.

APPLICATION.

Annex 1

STUDENT REMINDER

When performing work, the student must:

Preliminarily familiarize yourself with the theoretical material in detail and have a good understanding of the microbiological patterns and processes that are to be studied in practice.

When performing an experiment, observe all precautions, the sequence of operations, making the necessary observations.

Write the results of the experiment in a notebook according to the scheme proposed in the work:

Tidy up after work is done workplace and hand it over to a laboratory assistant or teacher.

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.

Department of Education of the City of Moscow

Technological College No. 28

Zhukova L.A.

METHODOLOGICAL INSTRUCTIONS

To laboratory work №№1-4

For NGO students

Moscow

2012

"Fundamentals of Microbiology, Sanitation and Hygiene"

METHODOLOGICAL INSTRUCTIONS

to laboratory works №№1-4

for NGO students

34.17 Operator of sausage production processes.

_______________________________________________________________

The guide is intended for college students. Can be used for self-study to study sessions and extracurricular independent work.

Compiled by: Zhukova L.A. - teacher of special disciplines

Reviewer: Sukhanova N.V. - teacher of special disciplines

Editor: Malkova L.A. - Deputy Director for educational and methodological work

The manuscript was reviewed and discussed at a meeting of the Central Committee of Technological Disciplines, protocol No. __ dated __ _______ 2012.

EXPLANATORY NOTE

The guidelines were developed in accordance with the current program of the microbiology course adopted by the State Autonomous Educational Institution of Secondary Education of the city of Moscow Technological College No. 28 for students studying in the specialty 260301 "Technology of meat and meat products".

At the current level of development of the natural sciences, deep knowledge of the microbiological processes underlying many biotechnological industries and serving as a guarantee of protection is required. environment from anthropogenic impact.

In addition to acquiring theoretical knowledge in microbiology, future specialists need laboratory and practical classes, which are essentially small research projects.

The implementation of laboratory work ensures the consolidation of the theoretical knowledge of students, the development of skills in microbiological control and the ability to make independent conclusions and generalizations.

These guidelines for performing laboratory work suggest:

combine laboratory work on the same topic or having the same semantic content so that it is convenient to organize them in time, as required by the specifics of microbiological analyzes;

distribute classes so that the workload of the last lesson makes it possible to discuss the results of the analysis, to conduct a test on the work performed;

concentrate the most important educational material, allowing students to master the practical skills of working in microbiological laboratory;

give a methodology for conducting an analysis indicating the essence of the method, the technique of implementation, the rules for processing the results;

use those methods of microbiological analysis, which are provided by GOST.

Theme and purpose of the work.

Material support for laboratory work.

Recommendations for performing laboratory work, task, explanation for the work, methods of analysis, forms for compiling a report, control questions for laboratory questions and additional literature.

Due to the complexity of some methods and the duration of growing microorganisms, in some cases, part of the laboratory material is prepared in advance by the teacher, but the method of preparing it is given.

The skills acquired in laboratory classes are necessary for conducting microbiological control of production, the purpose of which is to timely identify violations of the sanitary condition of production and equipment, detect places and ways of microbial contamination, take the necessary measures to eliminate these dangerous foci and achieve high output. quality.

RULES OF WORK IN THE MICROBIOLOGICAL LABORATORY

Working in a microbiological laboratory requires strict adherence to special rules, which is determined by two main provisions.

First, in microbiological practice, mainly pure cultures of microorganisms are used, i.e., populations of microorganisms of the same species, often the offspring of one cell.

Since there is always a large amount of various microflora in the air, on the surface of the objects around us, on clothes, hands, hair, to ensure the sterility of studies and to avoid contamination of cultures, work must be carried out in compliance with asepsis rules.

Secondly, in studies with unidentified microorganisms, when they are detected from environmental objects and technogenic flows, pathogenic and conditionally pathogenic microorganisms can be isolated.

In addition, cells of both saprophytic and pathogenic microorganisms can be allergens for certain individuals. Thus, in order to obtain reliable research results, to ensure personal safety and the safety of others, certain rules must be observed.

When reseeding microorganisms, sterility is achieved due to the fact that all work is carried out near the flame burners. Bacteriological loops used for transferring microorganisms from solid media are ignited in a burner flame, and glassware and nutrient media are pre-sterilized in drying cabinets and autoclaves, respectively.

The surface of the desktop is disinfected both before and after work, wiping with a 3% solution of chloramine, lysol, or a 70% solution of isopropyl or ethyl alcohol. Solutions of these alcohols can also be used for hand disinfection.

Preparation of the premises includes wet cleaning and thorough ventilation, followed by irradiation with ultraviolet light from germicidal lamps. Depending on the degree of air pollution, irradiation from 30 minutes to several hours is required for its sterilization. Ultraviolet rays are dangerous to the eyes, therefore, when the bactericidal lamp is on, it is impossible to be in the room.

When working in a microbiology laboratory, students must observe the following rules:

1. Each student must work at a permanent place.
2. The workplace should be free of foreign objects (including briefcases and bags). While working with the burner, there should also be no notebooks on the table, which will be needed later for microscopy and sketching preparations.
3. All work is done in a clean dressing gown. Long hair must be tied up to prevent it from being caught in the flame of a heating pad.
4. Vessels used for cultivating microorganisms (test tubes, flasks, Petri dishes, mattresses) must be inscribed with the generic and specific name of the culture, the date of inoculation, the student's name and group number.
5. All items used when working with live cultures must be decontaminated either by burning in a burner flame or immersed in a disinfectant solution (subject and coverslips, pipettes, spatulas).
6. All inoculated test tubes, cups or flasks are placed in a thermostat or handed over to a laboratory assistant.
7. Waste material is placed in certain containers for their further disinfection.
8. Smoking and eating are strictly prohibited in the laboratory.
9. At the end of the class, each student must tidy up the workplace.

The obtained data should be recorded in the journal for laboratory work. Records should contain: the number and title of the work, the date of its start and end, the names of the objects of research, the conditions for conducting experiments, methods of analysis, as well as the results obtained and conclusions from them. When studying the morphology of cultures, their sketches are made at certain magnifications of the microscope. (Colored pencils, at least red and blue, should be available in class.) Numerical data is summarized in tables, graphs, or charts.

LAB #1

Organization and equipment of a microbiological laboratory. The device of an optical microscope and the rules for working with it. Mastering the technique of microscopy of microbes.

Purpose of the lesson.

To acquaint students with the organization and equipment of the microbiological laboratory. To study the device of an optical microscope. Familiarize yourself with the main forms of bacteria.

Material support.

Educational microbiological laboratory equipped with thermostats, sterilizers, refrigerators, water bath, distiller, bactericidal lamps, work tables with all necessary accessories, microscopes.

This lab contains a significant amount of educational material Therefore, it is desirable to familiarize students with the organization and equipment of the laboratory using a demonstration method.

When studying the microscope device, use a poster showing all its details.

Tasks.

1. Read the job description:

1. Equipment of the educational microbiological laboratory.
2. Equipment and organization of production

1. Rules of work in the microbiological laboratory.
2. The device of an optical microscope and its care.

Rules for working with the MBR-1 microscope.

1. Basic forms of bacteria.

1. Conduct microscopy of finished preparations with basic forms

bacteria.

Explanation for work

1.1 Equipment of the educational microbiological laboratory.

The premises of the educational microbiological laboratory should consist of educational, technical rooms and a teacher's room.

The training room should have the following inventory: laboratory tables, tables for microscopy, a table for the teacher, thermostats, stools, a shelf with titrated solutions, scales, a blackboard, a table for dishes, a sink.

For training sessions, an ordinary laboratory table can be used.

The microscopy table must be placed in front of a window; its height should be about 80 cm, width - about 40 cm. For greater stability, the table for microscopy is arranged on brackets.

Thermostats. Microbes must be grown under such conditions that the temperature of the surrounding air is subjected to perhaps the least fluctuations. For this, thermostats are used, which are cabinets in which a constant temperature is maintained.

There are air and water thermostats.

Air thermostats are heated by warm air, usually heated electric shock.

In water thermostats between double walls there is water heated by electric current, gas or other heat sources. Temperature fluctuations in these thermostats are less than in air ones.

There should be at least three thermostats in the laboratory: the temperature of one is 30°C, the second is 37°C and the third is 43°C. Microbes that grow well at 20°C can be grown at room temperature.

A constant temperature in thermostats is maintained by special devices - thermostats. In a thermostat with an accurate temperature controller, temperature fluctuations should not exceed 2 ° C.

Autoclave or sterilizer.The autoclave is a metal cylinder with double walls and a massive lid, which is closed with screw clamps.

A metal casing is put on the cylinder. The autoclave is equipped with a steam outlet tube, a manometer, a safety valve and a water gauge glass. It sterilizes nutrient media, various liquids and utensils.

Before sterilization, distilled or boiled water is poured into the autoclave through the funnel of the water gauge glass (when raw water is used, scale quickly forms) up to the line indicated on the autoclave casing. After that, the autoclave is loaded with the material to be sterilized (the latter must be covered with paper from above), the lid is screwed tightly, the valve of the steam pipe is opened and the heating begins.

Heating is carried out by electricity, steam (in this case, water is not poured into the autoclave) or several gas burners. As the autoclave heats up, first air begins to escape, and then steam, which enters the autoclave through holes in the upper part of the cylinder. When the steam begins to come out in a continuous stream, it is released for another 2-3 minutes to force air out of the autoclave, and then the valve is closed. As a result, the exit of steam stops and gradually the pressure in the autoclave begins to increase (lifting of the pressure gauge needle).

After establishing the required pressure, the degree of heating of the autoclave is reduced so that the pressure gauge needle remains at the same level for a certain time. The start of sterilization is considered from the moment the arrow reaches the proper pressure.

The pressure gauge readings correspond to a certain steam temperature.

Manometer reading in ati Steam temperature in ˚C

0,5 112,0

1,0 121,4

1,5 128,8

2,0 135,1

At the end of sterilization, the heating is stopped and the pressure gauge needle is allowed to fall to zero. Only after that, the tap of the steam outlet pipe is opened, steam is released and air is let in, then the lid is unscrewed, it is lifted and, after allowing the autoclave to cool in this position, the sterilized material is removed.

Crockery and inventory.

Petri dishes serve for sowing crops. Use cups with a diameter of 10 cm (height 1.5 cm). Glass should be thin, transparent, without air bubbles.

Pipettes. For bacteriological work, pipettes with a capacity of 1-5 and 10 ml are used. The most commonly used pipettes are 1 ml, about 28 cm long (without extension).

Test tubes. For pouring water into 10 ml, test tubes measuring 18 x 2.0 cm are used; 18 x 1.5 cm test tubes are also used. For culture media, 15 x 1.8 cm test tubes are used.

Needles and loops. Needles and loops are used for taking the test material and for inoculation.

For washing laboratory glasswaresink table.

When working with a microscope, usesubject and integumentary glass . Glasses should be colorless with a smooth surface, without air bubbles. For special studies,glass slides with wells.

1.2 Equipment and organization of production work

Microbiological (bacteriological) laboratory.

Bacteriological examination of food raw materials and products from it is carried out in a special laboratory that has a permit to work with microorganisms of III-IV pathogenicity groups. The microbiological (bacteriological) laboratory (or the bacteriological department as part of the production laboratory of a meat processing enterprise) is intended for sanitary and bacteriological control of raw materials, finished products, auxiliary materials, the sanitary and hygienic state of technological equipment, inventory, containers, clothing and personnel hands.

Laboratory premises.The general location of the laboratory and its infrastructure must comply with the requirements of GOST R 51446-99 (ISO 7218-96). The laboratory provides for the following separate rooms or isolated work areas:

for receiving, storing, preparing and processing samples;

for primary seeding, re-seeding, preparation of dilutions and other work in aseptic conditions;

for preparation, sterilization, bottling and storage of nutrient media, preparation of laboratory glassware and equipment;

for disinfection and cleaning of equipment, spent nutrient media and materials contaminated with microflora.

Thermostats, refrigerators, freezers can be located in separate rooms.

The premises must be located in such a way as to ensure their protection from the influence of external factors (elevated temperature, humidity, dustiness, noise, vibration), organize the flow of clean and contaminated materials.

Meat can be contaminated with pathogenic microflora, which poses a serious danger to human health, and the laboratory premises must be isolated from other departments. If it is not possible to organize a separate room, it is allowed to install a table in the common room for the initial inoculation of samples. It is advisable to equip a glazed box with a pre-box to ensure aseptic working conditions.

Laboratory rooms should be bright (table surface illumination within 500 lux) and spacious - for each analyst, the recommended working area is about 20 m2. Walls, ceiling and floor should be smooth, easy to clean, resistant to detergents and disinfectants. For ease of cleaning and disinfection, the surface of laboratory equipment and furniture is covered with a smooth impervious material, such as plastic, and the workplace is covered with a mirror table.

For bacteriological analysis of food products, in particular sausages and smoked meats, 1-2 boxes with pre-boxes are needed. Boxes are insulated, dust-protected glazed rooms; the maximum allowable number of particles larger than 0.5 microns in 1 m should not exceed 4000.

The laboratory premises are regularly cleaned and disinfected, the quality of which is periodically controlled by the microbiological method.

Each room should have cold and hot water supply, a bottle with a disinfectant solution for washing hands.

1.3 Rules for working in microbiology

laboratories

Direct contact with microorganisms and the need to comply with sterile conditions during all operations require knowledge and strict adherence to the following rules:

work in bathrobes;

maintain cleanliness and order in the workplace;

do not touch microbial deposits and condensation water in test tubes and culture dishes with your fingers;

do not wash microbial deposits from slides and coverslips and other objects with napkins, filter paper, disinfect them by placing them in alcohol or carbolic acid solution;

in the process of work and after sowing, destroy the remnants of microbial deposits on bacteriological needles and loops by calcining in the flame of an alcohol lamp or gas burner;

to neutralize used contaminated materials and spent cultures of microbes by sterilization in an autoclave (this work is performed by laboratory staff);

do not hold spirit lamps close to the face, light the spirit lamp only with a match;

clean and process the workplace at the end of work with a disinfectant solution under the supervision of a laboratory assistant; place cultures of microorganisms necessary for further work, in a refrigerator or safe, which is closed and sealed; put test tubes and Petri dishes inoculated with microflora into the thermostat;

wash hands thoroughly after work, do not eat in the laboratory.

1.4 The device of the optical microscope and its care.

A microscope is a complex optical instrument that magnifies an object by 1000-1500 times. A modern microscope consists of two main parts - mechanical and optical.

The mechanical part consists of a tripod, which distinguishes between a leg, a base, a tube holder, and an object table attached to the tripod base. The tube holder is raised and lowered using macrometric and micrometric screws designed for coarse and fine focusing of the object.

The optical part of the microscope consists of lighting and observation devices.

The lighting apparatus is located under the object table and consists of a mirror, a condenser and an iris diaphragm.

The mirror reflects the light rays and directs them towards the condenser.

The condenser is a system of lenses and serves to enhance the brightness of the illumination of the object in question.

Observation apparatus includes lenses and eyepieces.

Objectives are screwed into revolver sockets and consist of a system of lenses enclosed in a metal frame. The front or front lens of the objective is the smallest and the only one that gives magnification. The rest of the lenses in the lens are corrective.

Biological microscopes MBR-1 and MBI-1 usually have three or four lenses with digital designations x8, x20, x40, x90, showing the actual magnification of these objects.

The eyepiece is inserted into the upper end of the tube. The eyepiece is a system of two plano-convex lenses, convex facing the objective. The lens facing the eye is called the ophthalmic lens, and the lens facing the preparation is called the converging lens. The eyepieces of domestic microscopes are marked with numbers showing their own magnification: x5, x7, x10, x15.

The magnification of the microscope is equal to the product of the magnification of the eyepiece by the magnification of the objective.

Microscope care instructions:

The microscope is protected from dust, moisture and sunlight. After working with it, it is placed in a case or covered with a cap made of dense fabric.

Objectives and eyepieces are wiped with suede or flannel.

Do not leave the tube of the microscope open, i.e. without an eyepiece, as this leads to the accumulation of dust in the tube and contamination of the objectives.

Microscope consists of two main parts. The basis of the microscope is a tripod. A tube with an optical part enclosed in it and an object table are attached to it. The tripod consists of a base (or a horseshoe-shaped leg) and a tube holder. The tube holder is connected to the base with a hinge, which allows the top of the microscope to be tilted for ease of use. Objectives and eyepieces are placed in the tube. The lenses are screwed into the bottom of the tube, called the revolver. The eyepieces fit freely into the top of the tube. The magnification can be changed by using various lenses that are screwed into the rotating drum of the revolver.

The tube holder is raised and lowered using macrometric and micrometric screws designed for coarse and fine focusing of the object. In order for the preparation to be clearly visible, the tube must be moved in the direction of its optical axis using coarse or micrometric movement mechanisms. Rough movement should be carried out quite easily, but without spontaneous lowering of the tube. For a more accurate setting, a micrometer screw is used. The micrometer screw has complex structure, is one of the most easily damaged parts of a microscope. Do not turn the screw more than half a turn in one direction or another (after rough installation of the microscope). The object table is used to place the study drug.

The optical part consists of a mirror, a condenser with a diaphragm, objectives and eyepieces. The microscope mirror is movable. In artificial light, it is better to use a concave mirror, in diffused light - a flat one. The mirror reflects the light rays and directs them towards the condenser. The condenser is used to obtain a stronger illumination of the preparation. The diaphragm is used to regulate the illumination of the preparation. The diaphragm is located between the lower surface of the condenser and the mirror.

Objectives are the most important part of a microscope, determining its optical power. Objectives are screwed into revolver sockets and consist of a system of lenses enclosed in a metal frame. The front or front lens of the objective is the smallest and the only one that gives magnification. The remaining lenses in the lens are corrective. Eyepieces magnify the image given by the lens, but do not reveal any details of the study drug.

Lenses that need to be immersed in cedar oil during operation are called immersion, or submersible. A dry lens is a lens that has air between the preparation and the lens. Each microscope is equipped with dry and immersion objectives. Lenses with native magnification 8 and 40 are dry, objective with intrinsic magnification 90 is immersion.

Rules for working with the MBR-1 microscope

1. The microscope is stored in a cabinet to protect it from dust, moisture and light. The microscope is carried with the right hand, holding the tripod handle, with the left support from below.
2. Before working with the microscope, wipe the eyepiece, objective and mirror with a cloth.
3. Place the microscope with the tripod towards you, with the object stage away from you. In relation to the sitting microscope should be shifted closer to the left shoulder. To the right of the microscope are an album, pencils, colored pencils, and an eraser.
4. Put the low magnification lens in working position. To do this, turn the revolver until the desired objective is in the middle position (over the stage opening). When the lens is in the middle position, a special device is triggered in the revolver - a latch, while a slight click is heard and the revolver is fixed. Remember that the study of any object begins with a small increase.
5. Raise the lens above the stage by about 1 cm using the macrometric screw. Open the diaphragm, raise the condenser to the level of the stage.
6. Looking through the eyepiece with your left eye, use a mirror to direct the light so that the entire field of view is illuminated brightly and evenly.
7. Place the prepared specimen on the stage with the coverslip up so that the object is in the center of the opening of the stage. Lower the lens over the preparation by 0.2 cm using the macrometric screw.
8. Look into the eyepiece, at the same time slowly turn the rack towards you and gently raise the tube until (0.5 cm) until an image of the object appears in the field of view; carefully turning the micrometer screw no more than ½ or ¾ full turn, achieve a clear view.
9. Turning to the consideration of the object at high magnification, it is necessary to center the preparation, i.e. place the part of the object of interest in the very center of the field of view. To do this, move the preparation with the help of the preparations of the parents or with your hands until the object takes the desired position. If the object is not centered, then at high magnification it will remain out of view.
10. Switching from a smaller to a higher magnification, turn the revolver to place the lens of higher magnification against the lower opening of the tube. Look into the eyepiece and, by carefully turning the micrometer screw, achieve a clear image.
11. When sketching the preparation, look into the eyepiece with your left eye, and with your right eye into the album.
12. After working using a microscope with a revolver, replace the high magnification lens with a small lens, remove the preparation from the table and put it back in place.
13. Clean up your desk; drugs and guidelines on the teacher's desk.

1.5 Basic forms of bacteria.

According to their shape, bacteria are usually divided into spherical (cocci), rod-shaped (clostridia, bacilli) and convoluted (vibrio, spirilla, spirochetes).

Coccoid forms according to the location of cocci are subdivided into micrococci - cells located singly; diplococci - cocci connected by two; tetracocci - cocci connected by four; streptococci - cocci arranged in the form of a long or short chain; sarcins - cocci arranged in the form of packages; staphylococci - a random accumulation of cocci, often in the form of grapes.

Rod-shaped forms according to the location of the rods are divided into diplobacteria - rods connected in pairs; streptobacteria - sticks arranged in a chain. Among spore-forming bacteria, bacilli and clostridia are distinguished. In bacilli, the spore diameter does not exceed the width of the vegetative cell, and in Clostridium, the spore is larger than the width of the cell, so the cell takes the form of a spindle, tennis racket, drumstick.

The convoluted forms are subdivided into vibrios, having the form of a comma, spirilla, having several (up to 5) curls, and spirochetes with a large number of small curls.

Order of execution

2. Microscopy of finished preparations with the main forms of bacteria.

Microscopy technique.

The test preparation is placed on the object table and fixed with clamps.

When working with an immersion objective 90, a drop of immersion oil is applied to the preparation, and then, using a macrometric screw, the tube with a centered objective 90 is carefully lowered so that its front lens is immersed in the oil drop, and the focus is below the preparation. Then, looking into the eyepiece, the tube is very slowly raised with the same screw (by hundredths of a millimeter) until an image of microbes is seen. Precise installation of the drug in the focus of the lens is carried out using a micrometric screw.

Note.

The advantage of the immersion system is that when the lens is immersed in oil, the light rays passing through the glass-oil media are almost not refracted, since the light refractive index for these media is almost the same. As a result, the illumination during microscopy with oil is maximum.

At the end of the work, the immersion oil is removed from the front lens of the objective 90 with a soft tissue cloth, for a more complete removal of the oil, the front lens is wiped with a cloth moistened with a mixture of alcohol and ether in a 1: 1 ratio, then the lens is wiped dry. The microscope is placed in storage.

When microscopy, pay attention to the shape and size of the cells of the microorganism under study, to the structural features of the cells - the presence of spores and capsules in bacteria, buds - in yeast; in the mold preparation - for a variety of cell shapes: round, oval, elongated, cylindrical, branched.

Microscopy report.

The results of microscopy are recorded in the table (form 1).

Form 1

Control questions.

1. What equipment should be equipped with a microbiological laboratory, its purpose?
2. Basic rules for working in a microbiological laboratory.
3. What is the device optical system biological microscope?
4. What rules must be observed when using and caring for a microscope?
5. What are the main forms of bacteria?

LABORATORY WORK № 2.

Preparation of microbiological preparations for the study of living microorganisms ("crushed drop" and "hanging drop"). Preparation of smears from microbial cultures.

Any work on the preparation of preparations of microorganisms, as well as on transfers of microorganisms, is carried out in compliance with the rules of asepsis.

Laboratory table equipment.

To work with microorganisms, certain equipment is required. On the working laboratory table there should be an alcohol lamp or a gas burner, a bacteriological loop, slides and coverslips, a set of paints, a microscope, a napkin, cedar oil, tweezers, a cuvette with a bridge, a vessel with water, an hourglass, a pencil on glass, filter paper.

Purpose of the lesson.

Familiarize yourself with the methods of studying bacteria for motility.

Material support.

Young broth cultures of microorganisms in test tubes for the study of mobility, glass slides and coverslips, glass slides with a hole, bacteriological loops, Pasteur pipettes, microscopes, matches, alcohol or gas burners.

The teacher must demonstrate the technique of preparing unstained preparations of microorganisms, preparing the microscope for work, adjusting the illumination of the field of view, installing a magnifier, and microscopy of preparations.

During the work of students, the teacher should constantly check the illumination of the field of view in each microscope, if necessary, help to adjust it and look at the images of the objects found. Viewing an unstained preparation should be given Special attention, as students must see both the shape of the cells and their mobility here.

Tasks.

1. Prepare "crushed and hanging drop" preparations to determine the motility of bacteria.
2. Prepare a smear from microbial culture.
3. Learn the technique of microscopy and look at prepared preparations under a microscope.
4. Prepare a report on the results of microscopy.
5. Record the content of the lesson in a workbook.

Order of execution

1. Study of the mobility of microbes in preparations "crushed and hanging drop".

Crushed drop.

If the organisms are in a liquid medium, a drop of the suspension is applied to a defatted glass slide using a sterile pipette. (After use, the pipette is immediately placed in a porcelain glass with a disinfectant solution. Putting a dirty pipette on the desktop is unacceptable!). If the culture is grown on a solid nutrient medium, then a drop of water is first applied to the glass, and then cells of microorganisms or the test sample of the test product are placed in it with a bacteriological loop calcined in a burner flame, and thoroughly mixed.

A drop of liquid is covered with a coverslip. To prevent air bubbles from forming under it, the cover slip is first placed on the glass slide with one edge, and then gently lowered, pressing down to “crush” the drop. It is desirable to avoid excess liquid so that the suspension of microorganisms is not squeezed out from under the coverslip. If this happens, excess liquid is removed with filter paper. Used paper should immediately be placed in a disinfectant solution.

For microscopy of the “crushed drop” preparation, only objectives with a dry system are used, that is, without immersion. This type of drug allows you to determine the shape of the cells, their size, the method of sporulation, and if the cells are alive, then the presence or absence of mobility.

The advantage of the drug is that a thin layer of liquid is obtained, where you can see living organisms.

The disadvantage is that it dries quickly, and the movement stops, air often gets in, which violates the microscopic picture.

To prepare the drug"hanging drop" a small drop of a suspension of microorganisms is applied to the coverslip, turned upside down and placed on a special glass slide with a recess (hole) in the center. The drop should hang freely without touching the edges and bottom of the hole. If many days of observation are coming, then the edges of the hole are smeared with petroleum jelly and the drop is hermetically enclosed in a humid chamber. The "hanging drop" preparation is used to detect the mobility of microorganisms, to study the methods of reproduction, and to monitor the germination of spores.

For cooking swab preparation the studied culture of the microbe is carefully distributed in a uniform thin layer on a glass slide. When preparing cultures grown on solid media, proceed as follows. A drop of sterile tap water or saline is applied to a clean glass slide with a sterile pipette or bacteriological loop.

a) A culture tube is taken from which it is necessary to prepare a smear. The bacteriological loop is calcined in the flame of the burner, holding it in a vertical position.

b) Holding the cork between the little finger and the palm of the right hand, take it out of the test tube and hold it with the end down, avoiding contact with the hand of that part of the cork that was in the test tube.

c) We burn the open end of the test tube on a flame.

d) We introduce a loop into the test tube (once again passed through the flame), cool it by touching the wall of the test tube and collect a small amount of material, lightly touching the culture and not scratching the medium with the loop.

e) We burn the edges of the test tube and the end of the cotton plug over the flame of the burner.

f) Close the tube with a cotton stopper.

g) The taken material is emulsified into a drop present on the glass slide and evenly distributed over the surface (1.5 x 2 cm) with a thin layer of a loop.

h) The loop is calcined and put in place.

If the culture is grown in a liquid nutrient medium, then a sterile loop is lowered into the liquid, a drop is captured and rubbed on a glass slide.

3. Microscopy technique.

The microscope is placed on the desktop from the edge by 3-5 cm with the tube holder towards you.

A good uniform illumination of the field of view of the microscope is established, for which, looking into the eyepiece, a mirror directs a beam of light from the source into the lens. The condenser should be up and the diaphragm open. The field of view of the microscope should be well and uniformly illuminated at all points.

The test preparation is placed on the object table with a smear or drop upwards and fixed with clamps.

Unstained preparations are microscoped with x8 and x40 objectives, lowering the condenser by 1-0.5 cm to the preparation, achieving the best visibility of unstained cells.

When microscopy of "live" microbes (unstained preparation) note the mobility.

4.Report on the results of microscopy.

c) drawing of cells under a microscope (indicate the presence of spores, capsules, buds, mobility).

5. Write down the content of the lesson in a workbook.

Control questions

1. What is the technique for preparing preparations "crushed and hanging drop"?
2. What is the procedure for preparing a smear from a culture of bacteria grown on solid nutrient medium?
3. What allows you to install the drug "crushed drop"?
4. For what research is the drug "hanging drop" used?

LABORATORY WORK №3.

Preparation of stained preparations of bacteria.

The purpose of the lesson.

Learn how to prepare and stain bacterial preparations using the simple and Gram method.

Material support.

A set of ready-made paint solutions in bottles, a set of dry paints in bottles or in test tubes with rubber or cork stoppers: basic and acid fuchsin, gentian violet, methylene blue, safranin, malachite and brilliant green, a mixture of microbes: cultures of gram-positive bacteria (Bac. subtilis, Staph . saprophyticus), gram-negative microbes (E. coli) grown on slanted MPA, glass slides, aqueous fuchsin solution (Pfeiffer's magenta), bacteriological loops, filter paper, microscopes, alcohol or gas burners.

The teacher must demonstrate the technique of preparing stained preparations of microorganisms, preparing the microscope for work, adjusting the illumination of the field of view, installing a magnifier, and microscopy of preparations.

During the work of students, the teacher should constantly check the illumination of the field of view in each microscope, if necessary, help to adjust it and look at the images of the objects found. When viewing a colored preparation, the student should see the presence of spores and capsules in microorganisms.

Tasks.

1. Prepare a plaque smear and stain using a simple method.
2. Prepare a smear from a mixture of staphylococcus and Escherichia coli on one slide and stain by Gram.
3. View stained smears with an immersion lens.
4. Record the content of the lesson in a workbook.

Answer security questions.

Explanation for work

To study the shape of a microbial cell and some of its structures, a preparation (smear) from a material containing the microbe under study is stained.

Most microbes stain quickly and well with aniline dye solutions. By chemical properties they are usually divided into basic and acidic. For basic dyes, the chromophore (the ion that gives color) is a cation, for acidic dyes it is an anion.

The main dyes include red - neutral red, magenta basic, safranin, thionin, pyronin, hematoxylin; blue - methylene blue; violet - crystal violet, methylene violet; green - malachite green, methylene green. Basic dyes, as a rule, readily bind to the nuclear (acidic) components of cells.

Acid dyes include red and pink - acid fuchsin, eosin; yellow - picric acid, congo, fluorescene; black - nigrosin. Acidic dyes stain the (basic) components of cells more intensely.

Preparation of working solutions of paints.Before preparing working solutions of paints intended for staining microbes, often in advance from dry paints intended for staining microbes, saturated alcohol solutions are often prepared in advance from dry paints. To do this, the paint is poured with 96% alcohol in a ratio of 1:10. With this ratio, alcohol is saturated with paint (the undissolved part of the paints will precipitate). In order to save money, it is recommended to take methylene blue 7.0, gentian violet 4.8, basic fuchsin 8.1 per 100 cm³ of alcohol.

For better saturation, alcohol solutions are placed in a thermostat and kept until the paints are completely dissolved. Aqueous working solutions are prepared from saturated alcohol solutions as needed.

The most commonly used dye solutions are:

Tsil' fuchsin carbolic solution.To 100 ml of a 5% solution of crystalline carbolic acid, add 10 ml of a saturated alcoholic solution of basic fuchsin. A saturated solution of fuchsin is obtained by mixing 8 g of fuchsin crystals in 10 ml of alcohol. The paint is red.

Fuchsin Pfeifferis a carbolic solution of Ziel's fuchsin, diluted in distilled water 1:10. Prepared immediately before use. The paint has a rich pink color.

Carbolic solution of gentian violet.To 10 ml of a saturated alcohol solution of paint add 100 ml of a 2% aqueous solution of carbolic acid. A saturated solution is obtained by dissolving 1 g of crystalline gentian violet in 10 ml of 96% alcohol. The paint has a blue-violet color.

Safranin. Prepare immediately before use by adding 1 g of safranin to 100 ml of hot water. The paint is red-brown.

Methylene blue (Leffler's blue).Prepare by adding 30 ml of a saturated alcohol solution of paint and 1 ml of a 1% aqueous solution of KOH or NaOH to 100 ml of distilled water. The solution has a dark blue color.

Malachite green.1 g of paint is dissolved in 100 ml of distilled water, filtered. The solution has a blue-green color.

Lugol solution. Take 1 g of crystalline iodine and 2 g of potassium iodide per 300 ml of distilled water. First, potassium iodide is dissolved in a small amount of water, and then iodine crystals are added. After complete dissolution, the rest of distilled water is added.

Complex coloring methodsused for a detailed study of the structure of the cell, as well as for the differentiation of some microorganisms from others. Sophisticated staining methods are based on the features of the physicochemical structure of the cell and its parts. Gram stain has the greatest practical knowledge, which allows differentiating bacteria according to the chemical composition and structure of the cell wall.

The method was developed by the Danish scientist Christian Gram in 1885. When stained according to the Gram method, all bacteria are divided into two groups: gram-positive and gram-negative.

Order of execution

Gram stain technique.

1. Simple coloring method.At simple method coloring, one of some coloring solution is used, most often Pfeiffer fuchsine or methylene blue.

A few drops of a dye solution are placed on a fixed preparation with a pipette so as to cover the entire surface of the smear: Pfeiffer fuchsin for 1-2 minutes, methylene blue for 3-5 minutes. Then the paint is washed off with water, and the smear is dried between sheets of filter paper or in air.

2. Sophisticated painting methodsused for the purpose of diagnosis, identifying the distinctive structures of microbes in cases where the latter are not stained in a simple way.

To differentiate microbial cells that differ in the chemical composition and structure of the cell walls, a complex staining method is used - Gram stain.

Gram-staining bacteria are divided into two groups: gram-positive and gram-negative.

In gram-positive bacteria (G+), the cell wall thickness is 15-80 nm, it consists of peptidoglycan (from 60-90%) located in several layers, protein (about 1%) and lipids (about 1%). Polymers of a special type, teichoic acids, covalently bound to peptidoglycan, have been found. The high content of peptidoglycan ensures the formation of an alcohol-resistant complex with gentian violet and iodine when stained according to Gram, as a result of which they are colored blue-violet.

The thickness of the cell wall of gram-negative bacteria (G-) is 10-15 nm, but its structure is much more complicated than that of gram-positive bacteria. It is based on oriented internal lipids (10-20%), which form a lipopolysaccharide layer with superficially located polysaccharides. Proteins and polysaccharides are mosaically arranged on the surface of the cell wall. Peptidoglycan is represented by a single layer with a thickness of only 2-3 nm (about 5-10%), lies under the lipopolysaccharide layer and tightly covers the protoplast. Due to the high content of lipids, the cell wall of Gram-negative bacteria, when stained according to Gram, forms a complex with gentian violet and iodine, which is destroyed by alcohol treatment, as a result of which the cells become discolored.

The unequal attitude of microorganisms to Gram stain is associated with differences in the structure and chemical composition of the bacterial cell wall.

Gram-positive microorganisms include cocci, bacilli (spore-forming rods), actinomycetes, mycobacteria, clostridial bacteria.

Gram-negative microorganisms include: bacteria (non-spore-forming rods), spirilla, salmonella, E coli, Pseudomonas, Azotbacter, vibrios.

There are Gram-variable microorganisms whose attitude towards Gram stain changes at certain stages of their life cycle. Since the ability of cells to stain according to Gram depends on their age, cells of a young culture, often one-day old, are used for Gram staining. For comparison, simultaneously with the object being determined, it is advisable to stain the cells of microorganisms whose relationship to the Gram stain is known (control).

Gram stain is carried out as follows:

1. Three smears of culture of microorganisms are prepared on a glass slide: in the center - a smear of the studied culture, on the left and right - smears of control microorganisms.

The smears should be thin so that the cells are evenly distributed over the surface of the glass and do not form clusters, since the staining results may depend on the thickness of the smear. Dry the smears in air, fix over the flame of the burner.

1. Stain smears with carbolic gentian violet. A sufficient amount of dye is applied to the smears, held for 1-2 minutes, the dye is drained and, without washing off with water, the smears are treated with Lugol's solution until blackened (approximately 1-2 minutes).
2. The Lugol solution is drained and the preparation is treated with 0.5-1 min (strictly) 96% ethyl alcohol by immersion in a glass of alcohol or by applying alcohol to the smears (the result of the entire staining depends on the time the smear is treated with alcohol: with insufficient processing, all bacteria retain staining, with excessive - discolor).
3. The preparation is immediately washed with water and stained for 1-2 minutes with aqueous magenta. The dye is drained, the preparation is washed with water, dried with filter paper.
4. The preparation is microscoped with an immersion system. Gram-positive bacteria stain dark purple, Gram-negative bacteria stain red or magenta pink (the color of the additional dye).

Express method for determining the gram-type of microorganisms (according to Kregensen).

The method is based on the destruction of cells of gram-negative bacteria in alkaline environment and determination of free DNA.

A drop of a 3% KOH solution is applied to a glass slide, into which the studied bacteria (24-hour culture from slant agar) is added with a bacterial loop and mixed well with a loop.

After 5-10 s, when the loop moves along the glass, with a positive reaction, as a result of testing gram-negative bacteria, a slimy trace 1-2 cm long is formed. If mucus is not formed, then the reaction is negative, then a gram-positive culture is tested.

3. Report on the results of microscopy.

a) microscopy system;

b) microscopic culture;

c) drawing of cells under a microscope (indicate the presence of spores, capsules).

4. Write down the content of the lesson in a workbook.

Control questions

1. What dye solutions are used in the simple staining method?
2. Describe the procedure for Gram staining of bacteria.
3. Why do bacteria perceive Gram stains differently?
4. Which bacteria are Gram positive and which are Gram negative??

LAB #4

Nutrient media and techniques for their preparation.

The purpose of the lesson.

Master the technique of preparing nutrient media.

Material support.

Dry nutrient media, ready-to-use media, electric stove, cooking utensils, indicator paper, 20% sodium hydroxide, 20% hydrochloric acid, test tubes, pipettes, Petri dishes.

Before doing the work, students repeat the nutrition of microorganisms, nutrient media, the classification of nutrient media, the basic requirements for nutrient media.

When performing laboratory work, students prepare a nutrient medium.

Tasks.

1. Read the job description.
2. Prepare a nutrient medium (on the instructions of the teacher).
3. Record the content of the lesson in a workbook.

Answer security questions.

Explanation for work

Bacteriological laboratories cannot do without nutrient media. To determine the type of microorganism, to detect the causative agent of an infectious disease, i.e. to establish a diagnosis of a disease, to produce specific preparations (vaccines, serums), to treat and prevent infectious diseases, to obtain antibiotics, to conduct sanitary and microbiological studies of environmental objects, to bacteriologically study meat and meat products resort to the artificial cultivation of microorganisms on nutrient media.

In laboratories, microorganisms are cultivated in vitro, i.e. in glass test tubes, flasks or other vessels. In vitro cultivation requires substrates that microorganisms use as nutrients.

According to the need for nutrients, all microorganisms can be divided into three groups:

first group - unpretentious, growing on simple media (putrefactive, most coccal and other microorganisms);

second group - requiring additional nutrients for their growth: complete protein (chicken or whey), vitamins, a certain set of amino acids, trace elements (tuberculosis, tularemia bacillus, leptospira, etc.);

third group - not growing on artificial nutrient media, but capable of reproducing only inside living cells (viruses, rickettsiae).

The requirements for nutrient media are as follows:

1. They must contain sources of nitrogen and carbon, inorganic compounds, trace elements, as well as growth factors, vitamins, mainly group B.

Peptones are used as a universal source of nitrogen. Peptones are products of the hydrolysis breakdown of meat or casein. They contain polypeptides, amino acids and essential minerals.

As a universal source of carbon, carbohydrates (sugars) are added to nutrient media - glucose, lactose, sucrose, organic acids - lactic, citric, etc., polyhydric alcohols - mannitol, glycerin, sorbitol, etc.

2. Nutrient media must have a certain reaction of the medium. So, for the majority of coccal, putrefactive and pathogenic microorganisms, the optimum pH is 7.0 ... 7.4, and mold fungi, yeast, lactic acid microorganisms develop better at pH 6.0.

3. The nutrient medium must be sterile, i.e. do not contain microorganisms.

4. The nutrient medium must be moist, since the nutrition of microorganisms is carried out according to the laws of diffusion and osmosis. Many media must be transparent in order to be able to distinguish the growth of microorganisms on them and observe the physiological changes that occur as a result of their vital activity.

According to the consistency, nutrient media are liquid, semi-liquid and dense. To prepare dense media, agar-agar of 2 ... 3% concentration is added to liquid media, for semi-liquid - agar-agar of 0.2 ... 0.3% concentration.

By origin, natural and artificial environments are distinguished.

Natural growing mediaare products of animal origin (blood, blood serum, milk, bile ...) or vegetable origin (vegetables, fruits, cereals), which are used for the cultivation of microorganisms without special treatment with full preservation of their natural properties.

Artificial culture mediaprepared from products of plant processing, meat, milk, liver, etc., often with the addition of carbohydrates to them, table salt, paints, etc.

Among the artificial environments, synthetic environments are distinguished, i.e. environments with precisely known chemical composition. Artificial nutrient media are divided into simple, or conventional, and complex.

Simple nutrient media serve for the cultivation of most microorganisms. The protein base of all media is nutrient broth. Usually, two methods are used to prepare the main nutrient broth: on meat water with the addition of ready-made peptone - meat-peptone broth, on digestion of the products of hydrolysis of the feedstock using enzymes (trypsin - Hottinger broth, pepsin - Marten broth) or acids, such media are richer in amino acids.

The content of total and amino nitrogen in the medium depends on the nitrogen composition of meat-peptone media and on the degree of protein breakdown in meat and other hydrolysates.

For good growth of most microorganisms, it is necessary to contain at least 250 ... 300 mg of total nitrogen in 100 cm of broth in the presence of amino nitrogen (amino acids) in this amount, on average, about 25 ... 30%.

The most common simple nutrient media are meat-peptone broth (MPB), meat-peptone agar (MPA), meat-peptone gelatin (MPG).

Meat peptone agar (MPA) - used to determine the total number of microorganisms.

The basis for all three media is meat water, which is prepared from minced beef, filled with water and boiled for 1.5 ... 2 hours, filtered and sterilized for 20 ... 30 minutes at 120 ° C.

Meat-peptone broth.1% peptone and 0.5% common salt are added to 1 liter of meat water, alkalized with 10% NaOH solution to pH 7.4, filtered and sterilized for 15 ... 20 min at a pressure of 0.1 MPa.

Meat-peptone agar.Finely chopped agar-agar (2…3%) is added to the meat-peptone broth, the mixture is heated until the agar melts, the pH is set to 7.2…7.6, filtered through a cotton-gauze filter, poured into test tubes and sterilized in flasks for 20…30 min at 120˚С.

If necessary, MPA is heated in a water bath until complete melting. The test tubes are placed in special racks at an angle of 10˚, and after solidification, the so-called slant agar is obtained, which is used for microbial cultures. Melted meat-peptone agar is also poured into Petri dishes.

Meat-peptone gelatin is prepared similarly.

Order of execution

2. Preparation of nutrient medium from ready-made dry nutrient medium.

Dry nutrient media.Simple, elective and differential diagnostic dry nutrient media are produced. Dry nutrient media are hygroscopic powders, easily soluble in water. Dry culture media are available in glass jars with tight screw caps.

Such media are prepared according to the instructions by dissolving the powder in the indicated amount of distilled water, filtered and sterilized at a temperature of 120°C for 20 minutes.

Dry Nutrient Agarprepared as follows: to the dissolved dry agar (5 g of agar per 100 ml), a small amount of yeast extract, meat water is added as growth factors, filtered through a cotton-gauze filter, poured into test tubes or vials and sterilized at 0.1 MPa for 20 minutes.

3. Write down the content of the lesson in a workbook.

Control questions

1. What are the basic requirements for nutrient media?
2. What are the most commonly used nutrient media intended for the cultivation of microorganisms, their purpose?
3. What are the components of simple nutrient media?

Bibliography

1. Gradova N. B. Laboratory workshop on general microbiology - M .: DeLi print, 2010.
2. Marmuzova L.V. Fundamentals of microbiology, sanitation and hygiene in the food industry - M .: 2005.
3. Mudretsova-Viss K. A. Microbiology, sanitation and hygiene - M .: DL, 2010.

"Fundamentals of Microbiology, Sanitation and Hygiene".

METHODOLOGICAL INSTRUCTIONS

to laboratory works №№1-4

for NGO students

34.17 Operator of sausage production processes.

__________________________________________________________________

Zhukova Lyubov Anatolyevna, teacher of special disciplines of the State Autonomous Educational Institution of Secondary Education of the city of Moscow TK No. 28

State autonomous educational institution

middle vocational education Moscow city

College of Technology No. 28

Address: Moscow, st. Upper fields, 27

Submitted for printing 07.02.2012.

Paper size 60x90/16

Circulation 16 copies.

Printed in the printing house:

State autonomous educational institution

secondary vocational education in Moscow

ACTIVITY #1

PREPARATION OF NUTRIENT MEDIA AND METHODS OF THEIR STERILIZATION

FEATURES OF THE CULTIVATION OF MICROORGANISMS

Target: To study the rules of work in a microbiological laboratory, the features of cultivating microorganisms, methods of preparing nutrient media and methods of their sterilization.

Tasks

Familiarize yourself with the rules of conduct when performing a microbiological workshop.

To study the features of the cultivation of microorganisms.

Learn the methods of preparing and pouring culture media.

To learn methods of sterilization of glassware and culture media.

Prepare and pour the MPA nutrient medium.

Obtain an enrichment culture of hay and potato sticks.

Literature

Anikeev V.V., Lukomskaya K.A. Guide to practical exercises in microbiology. M.: Enlightenment, - 1983.

Gusev M.V. Microbiology: a textbook for universities / M.V. Gusev, L.A. Mineev. – Moscow, 2004

Emtsev V.T. Microbiology: a textbook for universities / V.T. Emtsev, E.N. Mishustin. – M.: Bustard, 2005.

Lukomskaya K.A. Microbiology with the basics of virology. M.: Enlightenment, - 1983.

Fundamentals of microbiology, virology and immunology. / Under. Edited by Vorobyov A.A. and Krivoshein Yu.S. - M.: Masterstvo, 2001.

Pimenova M.N. Guide to practical exercises in microbiology. Moscow, 1971.

Workshop on microbiology. Ed. Netrusova A.I. - M .: Academy, - 2005.

Tepper E.Z. Workshop on microbiology. – M.: Bustard, 2004.

Materials and equipment.Sterile Petri dishes, withsterile conical flasks 100-150 ml,nutrient agar, peptone, hay, potato tubers, chalk, tiles, alcohol lamps, flasks, cotton wool, gauze, pipettes, scales, weights, thermostat.

Basic concepts.Cultivation, culture, surface culture, deep culture, batch culture, continuous culture, pure culture, enrichment culture, inoculation, incubation, passaging, elective media, enrichment media, optimal media, natural media, synthetic media, semi-synthetic media, agar, sterilization, flaming, tyndalization, pasteurization, autoclaving, MPA.

Progress

Task number 1. Learn the rules of work when performing a microbiological workshop.

Task number 2. To study the classification of nutrient media, the features of their preparation and bottling, methods of sterilization of nutrient media and glassware.

Task number 3. Prepare and pour the MPA nutrient medium into sterile Petri dishes.

Task number 4. Obtain an enrichment culture of hay sticks(Bacillus subtilis).

The hay is finely chopped and placed in a 500 ml flask, filling it to one quarter of the volume, a pinch of chalk is added and boiled for 15-20 minutes until the medium acquires the color of strong tea infusion. Hay decoction is poured into sterile 100-150 ml conical flasks with a layer of 1.0-1.5 cm, closed with cotton plugs and placed in a thermostat at a temperature of 22-25 °C.

After two days, a whitish film develops on the surface of the medium. You. subtilis, which, during aging, on the 3-4th day, becomes grayish-greenish. Other microorganisms at the same time grow rarely and in small quantities.

Task number 5. Obtain an enrichment culture of the potato stick(Vas. subtilis var. mesentericus).

Washed potato tubers, without peeling, cut into slices. Their surface is rubbed with chalk to neutralize the medium and placed in sterile Petri dishes on a double layer of filter paper moistened with distilled water. Cups with potato medium are kept in an autoclave at 0.5 atm for 10 minutes and placed in a thermostat at a temperature of 27-30°C for 3-4 days.

A dense wrinkled film of potato stick culture is formed on the surface of potato slices. The color of the film can be different: whitish-gray, pinkish, yellow-brown, black, which depends on the varieties of culture that have received predominant development.

Control questions

Classification of nutrient media.

Methods for sterilization of laboratory glassware and culture media.

Methods for the preparation of individual nutrient media (MPB, MPA, MPZH, SA, KA, etc.) Pouring of nutrient media.

Features of the cultivation of microorganisms (surface and deep, periodic and continuous). enrichment and pure cultures.

Methods for the preparation of native and fixed preparations of microorganisms.

To study the main stages in the development of the science of microbiology, the contribution of Russian and foreign scientists to its development. Fill in table number 1.

Table number 1. History of development of microbiology

ACTIVITY #2

PREPARATION OF LIVE AND FIXED PREPARATIONS OF MICROORGANISMS AND ACQUAINTANCE WITH THEIR MORPHOLOGY

Target: To study the methods and techniques for preparing micropreparations in the study of microorganisms and get acquainted with their morphology.

Tasks:

To study the features of the morphology of bacterial cells.

Prepare intravital and fixed micropreparations of microorganisms.

Materials and equipment.Glass slides, bacteriological loop, spirit lamp, filter paper, crystallizer with a bridge for preparations, wash bottle with water, aqueous solutions of dyes - fuchsin, methylene blue, gentian violet (hereinafter referred to as equipment for the preparation of micropreparations); immersion oil, microscope; meat water, yeast, cultures of hay and potato sticks.

Progress

Task number 1. Conduct a lifetime study of bacterial cells by the following methods, make drawings:

crushed drop method.A drop of one of the liquids is applied to a glass slide with a microbiological loop. The cover glass is placed on the edge at the edge of the drop and gradually lowered onto it.

hanging drop method.In the center of the cover glass, a small drop of the test liquid is applied with a bacteriological loop and it is overturned over the recess of a special glass slide.

The drug "imprint".From the agar medium, on which the microorganisms grow in a continuous lawn or in the form of individual colonies, a small cube is cut out and transferred to a glass slide so that the surface with the microorganisms is facing upwards. Then a clean coverslip is applied to the lawn or colony, lightly pressed on it with a loop or tweezers and immediately removed, trying not to move. The resulting preparation is placed print down in a drop of water or methylene blue (1:40) on a glass slide.

Task number 2. Prepare a fixed preparationYou. subtilis, Vas. subtilis var mesentericus, St. lactis, S. cerevisiae, E. coli(Fig. No. 1, 2, 3, 4 applications),make drawings.

Application. The glass slide is dried on the flame of an alcohol lamp. A bacteriological loop sterilized on a burner flame is applied to the center of the glass slide with a smear of the test material. If the culture of the microorganism is grown on a dense nutrient medium, a drop of water is first applied to the glass, a small amount of material is introduced into it with a bacteriological loop and a smear is made.

Drying and fixing. The drug is dried in air, and then fixed. Conventional smears of microorganisms are fixed thermally by passing the glass 2-3 times through the burner flame with the smear up.Fixation of the smear leads to the death of microorganisms, their dense adhesion to the surface of the glass and the easier susceptibility of microbes to the dye.

Coloring. Pour the surface with a solution of any dye for 2-3 minutes (methylene blue, gentian violet, magenta). Distinguishsimple and differentialstaining of microorganisms. In the first case, the entire cell is stained, so that its shape and dimensions become clearly visible. Differential staining reveals only certain cell structures and storage substances.

Flushing. Then the dye from the smear is washed off with water from the wash bottle, the lower side of the preparation is wiped with a strip of filter paper, the upper side is carefully dried and microscoped.

General characteristics of microorganisms. Distinctive features of prokaryotes and eukaryotes.

The shape and size of bacterial cells. Morphological types of bacteria.

Fundamentals of systematics of microorganisms. The position of bacteria in the system of organisms and their variability. Characteristics of bacteria used in species identification.

a brief description of order Schizomycetales.

Brief description of the order Actinomycetales. Nocardia. Mycobacteria.

Brief description of the order Myxobacteriales.

Mushrooms. Brief description of zygo-, asco- and deuteromycetes.

Fill in the table number 2.

Table number 2. Differences between eukaryotes and prokaryotes

Linear chromosomes

Questions for self-study

Brief description of individual groups of bacteria (according to Bergey's determinant of bacteria).

Morphological characteristics and systematics of algae.

Morphological characteristics and systematics of protozoa.

ACTIVITY #3

COLORING OF INCLUSIONS, CAPSULES AND ENDOSPORES, GRAM Staining

Target: Learn how to stain spores, capsules and inclusions bacterial cell; to study its cytological properties using Gram stain as an example.

Tasks:

Detect lipid granules in yeast cells.

Detect glycogen-like polysaccharides in yeast cells.

Detect capsules of a bacterial cell by the method of negative contrast (for example, Azotobacter).

Perform differential staining of spores and cytoplasm according to the Ozheshka method.

Perform a Gram stain on bacteria.

Materials and equipment.Slides, bacteriological loop, alcohol lamp, filter paper, crystallizer with a bridge for preparations, wash bottle with water, microscope. Aqueous solutions of dyes - fuchsin, methylene blue, gentian violet, Tsilya carbolic fuchsin, Sudan III. Immersion oil, sulfuric acid 1%, ink, hydrochloric acid 0.5%, Lugol's solution. culturesVas.subtilis, Vas.mycoides, S.cerevisiae, E.coli.

Basic concepts.Murein, volutin, nucleoid, glycogen, capsules, cell wall, polysaccharides, polyphosphates, metachromatic grains, monotrichs, peritrichis, lofotrichs, bacillary form, clostridial form, plectridial form, exine, intine, metachromosia.

Progress

Task number 1. Detect inclusions of polyphosphates (volutin) in yeast cells.Volutin in fungal and yeast cells is localized in vacuoles, in bacteria and actinomycetes in the cytoplasm. It is a reserve phosphorus- and nitrogen-containing substance, a derivative nucleic acids. characteristic property- metachromosis, i.e., the ability to acquire a different color than the substances that color it.

Coloring of volutin according to the Omelyansky method.A thin smear from the culture of the microorganism is prepared on a glass slide, dried in air, fixed on a flame, stained with carbolic fuchsin for 30–40 s, and washed with water. Differentiate by immersing it in a flask with a 1% sulfuric acid solution for 20-30 seconds and immediately rinsing with water. Sulfuric acid discolors the cytoplasm, and the volutin grains remain stained with magenta. The preparation is counterstained with methylene blue (1:40) for 20-30 s, washed with water, dried in air and microscoped. On the preparation, the grains of the volutin are colored red, the cytoplasm of the cells - in blue (Fig. No. 1, 2 appendices).

Task number 2. Detect lipid granules in yeast cells. Reserve lipids in yeast and filamentous fungi are represented by neutral fats; in bacteria, this function is performed by hydroxybutyric acid.

Coloring of fatty inclusions.A drop of Sudan III solution is added to a drop of an aqueous suspension of microorganisms on a glass slide, covered with a coverslip and microscoped using a VI-40 objective. Sudan III dissolves in the fatty inclusions of the bacterial cell, staining them orange-red. The cytoplasm of the cell remains colorless.

Task number 3. Detect glycogen-like polysaccharides in yeast cells. Reserve substances of carbohydrate nature in bacterial cells accumulate in the form of granules. Granulose is a starch-like substance that, when interacting with Lugol's reagent, turns blue; glycogen is a polysaccharide that turns reddish-brown under the same conditions. Granulosis is found only in prokaryotic cells.

Glycogen and granulosa stain.A drop of a weak Lugol's solution is added to a drop of microorganism suspension on a glass slide, covered with a cover slip and microscoped using a VI-40 objective.

Task number 4. Detect bacterial cell capsules by negative contrast (in Azotobacter).

Identification of capsules on a negative intravital preparation.A large drop of black ink is applied to a well-defatted glass slide with a microbiological loop, and a drop of the microorganism culture under study is introduced into it, distributed using a glass slide and dried. Treat with an immersion system. On a dark background of the carcass, transparent zones of capsules around sharply defined cells are revealed (Fig. No. 8. Appendix).

Task number 5. Perform differential staining of spores and cytoplasm (in You. mycoides).

Ozheshka method. The dried preparation is poured with 0.5% hydrochloric acid and heated for 2 minutes until vapors appear. The smear is washed with water, covered with filter paper and filled with Ziel's carbolic fuchsin. Stain for 5 minutes with heating until vapors appear. Washed with water and differentiated in 1% sulfuric acid for 0.5-1 min (the time is determined empirically). Washed with water and stained for 30 with methylene blue. Rinse again, dry and microscope. The spores are stained pink, the cytoplasm is blue (Fig. No. 2 of the appendix).

Task number 6. Perform a Gram stain on bacteria. The difference in the chemical composition of the cell walls of bacteria affects their ability to stain according to Gram. On this basis, bacteria are divided into gram-positive and gram-negative (Table No. 3). Gram-positive shells contain more polysaccharides, murein and teichoic acids; shells of gram-negative have a multilayer structure with a high content of lipids (lipoproteins and liposaccharides). Gram-positive microorganisms, when stained, form an alcohol-insoluble iodine compound with the main dye; in Gram-negative microorganisms, this compound is soluble in alcohol.

Gram stain.Three smears are applied to a glass slide and then simultaneously processed at once: from a bacterial culture known to be gram-positive, from a bacterial culture known to be gram-negative, and between them a smear from the culture of the microorganism under study. Young one-day cultures are used.

The smears are dried in air, fixed on a burner flame and stained with a 1% aqueous solution of gentian violet for 1 min. The dye is washed off with Lugol's solution and smears are poured with the same solution for 1 minute. The preparation is washed with water and differentiated in a flask with 95% ethanol (0.5-1.0 min). After the smear is differentiated, the preparation is immediately thoroughly washed with water and stained with an additional dye - a 0.1% aqueous solution of fuchsin for 2-3 minutes. The preparation is finally washed with water, dried in air and microscoped with oil immersion. On the preparation, gram-positive bacteria are stained in lilac-violet color, gram-negative - in pink-raspberry (Fig. No. 2 of the Appendix).

Table number 3. The ratio of bacteria to Gram stain

Staphyloccocus aureus

Escherichia coli

Streptococcus

Proteus vulgaris

Bacillus subtilis

Pseudomonas aeruginosa

Sacharomyces

Shigella sp.

Control questions and tasks

The structure of the bacterial cell wall. Capsule formation.

The ratio of bacteria to Gram stain. Distinctive features of gram-positive and gram-negative microorganisms.

Cell membrane and intracellular membrane structures.

Nuclear apparatus, composition, organization and replication.

Ribosomes, gas vacuoles and other bacterial organelles; their meaning.

Bacterial cell inclusions (polysaccharides, polyphosphates, lipids, sulfur inclusions).

Ways of reproduction of bacteria. Sporulation in bacteria.

Flagella. movement of bacteria. Taxis.

Questions for self-study

Hereditary factors of microorganisms.

Mechanisms causing change genetic information microorganisms.

ACTIVITY #4

QUANTITATIVE ACCOUNTING OF AIR AND WATER MICROFLORA

MICROBIAL NUMBER DETERMINATION

Target: To carry out a quantitative account of microorganisms of air and water.

Tasks

To study methods of quantitative accounting of air and water microflora.

Carry out a microbiological analysis of air and a sanitary-bacteriological study of water.

Materials and equipment.Petri dishes with sterile MPA, sterile 1 ml pipettes, water bath, electric stove, thermometer, tap water, water from a pond, spirit lamp, matches.

Basic concepts.total microbial count of water,total microbial number of air, if-index, if-titer, sanitary-indicative microorganisms, autochthonous microflora, allochthonous microflora, saprobity zones.

Progress

Task number 1. Lay the experience to determine the TMC (total microbial number) of the air.

For infection, Petri dishes are opened in the test room or outdoors for 5 minutes. The lid of the Petri dish is removed and placed side by side without turning over. On the lid of the Petri dish indicate the variant of the experiment, the date of sowing. Infected Petri dishes are placed in a thermostat at a temperature of 25-28°C.

After 2-3 days, the number of colonies of microorganisms that have developed on an agar plate of a Petri dish is counted.In this case, the following are taken into account: according to rough estimates (Omelyansky) on an area of ​​100 cm 2 within 5 minutes, as many microorganisms and spores settle as there are in 10 liters of air. We assume that each colony originated from a single cell or spore. EThis method gives only approximate data, but it allows you to detect the relative number of microorganisms in the air of different rooms quite accurately.Fill in table number 4, draw conclusions.

For example: 5 colonies were found in a Petri dish, the radius of the dish is 2 cm. The area is S=πr 2 =3.14*4=12.5. Then 10 liters contains

Table number 4. Total microbial count (TMC) of indoor air.

Colonies of microorganisms isolated from the air on MPA plates can be used for species identification. Most often, colonies of micrococci, sarcines, some bacilli and bacteria are isolated from the air.

Task number 2. Lay the experience to determine the TMF of water.

For research, tap water is taken into sterile flasks. 1 ml of water is taken from the flask with a sterile pipette and added to a Petri dish with a straightened medium (MPA) (its temperature should not be higher than 45°C). The cup is closed and, gently tilting, the nutrient medium is mixed, the plate is allowed to harden, and placed in a thermostat. After 3-5 days, colonies developed in Petri dishes are counted and the number of bacteria in 1 ml of water is determined.

After counting the colonies, the microbial number of water is determined - the number of microorganisms per 1 ml of the test water, taking into account the dilution, if any.

The data is drawn up in the form of table No. 5, conclusions are drawn.

Table number 5. Total Microbial Count (TMC) drinking water

tap water

Control questions

Features of air microflora. Spread of microorganisms in the air.

Standards for the sanitary condition of indoor air.

Sanitary and microbiological study of air.

Microflora of drinking water.

Microflora of natural waters. Distribution of microorganisms in water.

Sanitary indicators of drinking water. Total microbial count of water. If-index, if-titer of water.

Pollution and self-purification of water bodies. The role of microorganisms in the processes of self-purification of water bodies.

Biological treatment of drinking and waste waters.

Sanitary-bacteriological study of water. Determination of TMC, if-index, if-titer.

Sanitary-indicative microorganisms of air, water, soil.

Requirements for sanitary-indicative microorganisms.

Microflora of food products (sour-milk, fish, meat, sausages, canned food).

Characteristics of the groups of microorganisms used in the assessment of the safety of food raw materials and food products.

Sanitary and bacteriological standards for various food products.

Methods of sanitary-bacteriological research of food products.

Soil as a habitat for microorganisms.

Ecological factors of the spread of soil microorganisms.

Relationships between soil microorganisms.

Participation of soil microflora in the processes of mineralization of organic substances and the formation of humus.

Sanitary and bacteriological study of the soil.

ACTIVITY #5

OBTAINING A PURE CULTURE OF MICROORGANISMS

NEPHELOMETRIC METHOD OF QUANTITATIVE MICROORGANISMS

Target: To carry out the isolation of pure cultures of microorganisms, to master the nephelometric method of accounting for the number of microorganisms.

Tasks

Carry out the isolation of pure culture by the method of "depleting" stroke.

Quantify microorganisms by nephelometric method

Materials and equipment.Petri dishes with sterile MPA, FEK, Goryaev's camera, microscopes, microbiological loops, spirit lamp, matches.

Basic concepts. Pure culture, growth, reproduction, binary fission, budding, cell cycle (monomorphic, dimorphic, polymorphic), generation time, specific growth rate, biomass yield, economic factor, stationary growth phase, lag phase, exponential reproduction phase, stationary phase, dying phase, stagnant culture, flow culture, synchronous culture.

Progress

Task number 1. Obtaining a pure culture of microorganisms

The isolation of pure cultures of aerobes is carried out by sieving the enrichment culture on the surface of a solid nutrient medium. Sowing is carried out by the exhaustive stroke method. After the colonies have grown, the purity of the isolated colonies is checked visually and by microscopy. After 3-6 days, an isolated colony of microorganisms is selected and described (using the appendix drawings).The colony is described as follows:

The size of the colony: dotted (D - less than 1 mm), small (D - 1-2 mm), medium (D - 2-4 mm) and large (D - 4-6 mm or more).

The shape of the colony is round, amoeboid, rhizoid.

Optical properties - transparent, matte, fluorescent, translucent (translucent), opaque, shiny.

Color - note the color of the colony and the release of pigment into the medium.

The surface is smooth, rough, folded, bumpy.

Profile - flat, convex, crater-like, growing into agar, etc.

The edge of the colony is smooth, wavy, lobed, rhizoid, etc.

The structure of the colony is homogeneous, fine or coarse-grained.

Consistency - oily, pasty, viscous, filmy.

Task number 2. Carry out a quantitative assessment of microorganisms by the nephelometric method.

a) Nephelometric method.cell density S.cerevisiae in a liquid medium is determined nephelometrically (FEC) using a cuvette with an optical path length of 0.5 cm and a green light filter (A = X 540 nm). To obtain reliable results, the cell density should be in the range of 0.1-0.6. At cell concentrations above 0.6, secondary light scattering occurs, resulting in underestimated results. Therefore, suspensions of high densities should be diluted with water 2, 4, 6, 8 and 10 times before measuring light transmission. The results of measurements of the optical density of each suspension (water serves as a control) are recorded in table No. 6.

b) Cell counting in the Goryaev-Tom chamber.To switch from photoelectrocolorimeter (FEC) readings to the number of microorganism cells in the medium, their number is counted using a Goryaev-Toma counting chamber and yeast suspension dilutions, which were used to determine their density by the nephelometric method. Cells are counted in large grid squares using an 8* objective. Total number counted cells should be at least 600. The number of cells in 1 ml of the corresponding suspension is calculated by the formula M = 1000*a*n/h*S, where M is the number of cells in 1 ml of suspension; a is the average number of cells in a large grid square; h is the chamber depth (1/10 mm); S is the area of ​​the large grid square (1/25 mm 2 ); n is the degree of dilution of the suspension; 1000 mm 3 - 1 ml. The results obtained, million cells in 1 ml are recorded in table. No. 6.

Table number 6. The number of yeast cells in suspensions, established using the Goryaev camera, and the corresponding FEC readings

FEK indications

Build a calibration curve based on the data in Table. No. 6, plotting the number of yeast cells on the abscissa axis, and the optical density of the corresponding suspension on the ordinate axis. A calibration curve is built to quickly determine the number of cells of a certain type of microorganisms according to FEC readings.

Control questions and tasks

Accumulative cultures and the principle of electivity. Pure cultures, methods of preparation and significance.

Reproduction of microorganisms.

Cell cycles of bacteria. Growth of individual microorganisms and populations. Balanced and unbalanced growth.

Crop growth parameters: generation time, specific growth rate, biomass yield, economic factor.

Growth curves, features of individual phases. Growth of microorganisms during continuous cultivation. Synchronous cultures.

ACTIVITY #6

SANITARY-BACTERIOLOGICAL STUDY OF FOOD PRODUCTS

Target: Conduct a sanitary-bacteriological assessment of the state of food.

Tasks

1. Determine the total microbial count of certain foods.

2. Determine the presence of BGKP (bacteria of the Escherichia coli group).

Materials and equipment.Petri dishes with MPA, Petri dishes with Endo medium, 1 ml pipettes, mortars with pestles, scalpels, test tubes with 9 ml of sterile water, test tubes with Kessler medium, sterile flasks with 100 ml of water, scales.

Basic concepts.Specific microflora of foodstuffs, nonspecific microflora of foodstuffs, BGKP, sanitary indicative microorganisms.

Progress

Task number 1. Determine the presence of BGKP and the total contamination of food products by performing the following steps in sequence:

1. Preparation of food products for research.

Grind in a sterile porcelain mortar 10 g of a sample (taken sterile from different places), gradually adding 90 ml of isotonic sodium chloride solution, and leave at room temperature for several minutes. Then, a suspension for inoculation and dilution (1 ml) is beaten off with a sterile pipette with a wide nose. It is assumed that 1 ml of the prepared suspension contains 0.1 g of the initial product (dilution 10-1 ). Products of a liquid consistency are sown and bred for crops without prior preparation.

2. Preparation of food dilutions for sowing.

For products of a liquid consistency, dilutions are prepared as follows: 1 ml of the product is taken with a sterile pipette and added to a test tube containing 9 ml of a sterile isotonic sodium chloride solution, mixed. The resulting dilution (10-2 ) is subjected in the same way to further dilution in the required number of times (a multiple of 10).

Drawing No. 1. Preparation of dilutions and sowing of soil suspension on solid nutrient media

3. Determination of the total contamination.

The dilutions chosen for inoculation are added 1 ml into sterile Petri dishes with melted at 45-50 0 MPA, after which they are mixed. The medium is allowed to solidify, the crops are grown during the day at 37°C, after which the number of grown colonies (CFU) is counted. Determine the total number of bacteria in 1 ml or 1 g of the product.

4. Determining the presence of BGKP.

For sowing, the amount of the product is used, in which, in accordance with the established norms, the absence of CGB is provided. From the selected dilutions of the studied product samples, 1 ml is taken and inoculated into test tubes with Kessler medium. The inoculated tubes are incubated at 37°C for 24 hours. In the absence of signs of growth (gas formation or changes in the color of the medium), a conclusion is made about the compliance of the test product with the standard. If there are signs of growth, they are sown on Endo medium. Crops are incubated for 18-20 hours at 37°C. When viewing crops, colonies are noted that are suspicious or typical for CGB (form red, pink, pale pink colonies with or without a metallic sheen). They are used to make preparations of living and fixed cells, Gram-stained, and microscoped. The detection of Gram-negative, non-spore-forming rods with rounded ends indicates the possible presence of CGB.

Data on the total contamination and the presence of Escherichia coli are entered in the table. Draw conclusions about the microflora of various food products using sanitary and microbiological standards (SanPiN 2.3.2.560-96) (Table No. 7).

Table number 7. Some sanitary and bacteriological standards (SanPiN 2.3.2.560-96)

Cheese Russian

0,001

Ice cream

1*10 5

0,01

Control questions, see lesson number 4.

ACTIVITY #7

SOIL MICROBOCENOSES

Target: To study the species composition and distribution of soil microorganisms.

Tasks

1. To study the nature of the soil microflora and the dominant species.

2. Make a quantitative and qualitative account of soil microflora.

Materials and equipment.Slides, sterile Petri dishes with MPA,scales, weights, scalpel, mortar, rubber glove, flask with sterile distilled water 90 ml, sterile flask 250 ml, test tubes with 9 ml sterile water, pipettes 10 ml and 1 ml, micropipettes 0.1 ml, glass spatulas.

Progress

Task number 1. To study the nature of soil and rhizosphere microbiocenoses by the method of glass fouling (according to Kholodny).

On a flat soil surface, a cut is made with a knife, the depth of which depends on the horizon under study. Washed and degreased glasses (several glasses are taken at the same time) are pressed tightly against the vertical wall of the cut and covered with soil. Glasses are kept in the soil from a week to several months.

After the expiration of the exposure time, the soil is removed from the back side of the glasses, the experimental surface of the glasses is dried in air and fixed three times, passing the back side over the burner flame. After fixing, the glass is immersed in water with the experimental surface down, without bringing it to the bottom. After washing, the preparation is immersed in a solution of carbolic erythrosin for a period of 30 minutes to 24 hours. The stained preparations are examined under a microscope with an immersion system. When microscopy, the nature of the microflora, the density of fouling of glasses and the dominant forms are noted.

Task number 2. To carry out the determination of TMC of the soil.

Preparation of soil suspension by dilution method.Observing sterility, weigh 10 g of soil and transfer it to a sterile mortar. Moisten a sample of soil in a mortar to a paste-like state by adding 2-3 ml of water from the first flask containing 90 ml of sterile water, and rub for 5 minutes with a finger in a rubber glove. After grinding, the soil from the mortar is transferred with water to the second dry flask, the first dilution is obtained - 1/10. The soil suspension in the flask is shaken for 5 min, allowed to stand for 30 s, and then 1 ml of the soil suspension is transferred from the flask to test tube No. 1 with 9 ml of sterile distilled water with a sterile pipette, the second dilution is obtained - 1/100. Similarly, a series of subsequent dilutions of the soil suspension is prepared -1/1000, 1/10000, 1/100000 and more, depending on the expected number of microorganisms (Fig. No. 1, lesson No. 6).

To isolate and quantify bacteria, the soil suspension is sown on one of the nutrient media (MPA, KAA, soil agar, Ashby medium; KAA or Chapek medium is used to account for actinomycetes). Colonies of bacteria on Petri dishes are counted after 3-5 days, fungi and yeast - after 5-7, actinomycetes - after 7-15. The content of microorganisms in 1 g of soil is determined by the formula: a \u003d b * c, where a - the number of microorganisms in 1 g of soil, b is the average number of colonies, V - breeding. Compare the species composition and diversity of soil, water and air microflora and draw a conclusion.

Control questions and tasks, see lesson number 4.

ACTIVITY #8

CONVERSION OF NITROGEN COMPOUNDS BY MICROORGANISMS

Target: To study the features of the processes of transformation by microorganisms organic compounds nitrogen.

Tasks

To study the microorganisms involved in the processes of protein ammonification.

To study the microorganisms involved in the processes of ammonification of urea.

Materials and equipment:Environment for reproduction of the ammonification process of protein and urea, microscopes, equipment for the preparation of micropreparations.

Basic concepts.Ammonification, proteases, peptidases, urease, deamination, decarboxylation.

Progress

Task number 1. To study the microorganisms that carry out protein ammonification, draw a picture. To study the ammonification of protein substances, meat broth with the addition of 3% peptone can serve as a nutrient medium.30 ml of the medium is poured into a 100 ml flask and 1/3 teaspoon of soil is added. The flasks are closed with cotton plugs. Two pieces of paper are suspended above the medium - red litmus, or universal indicator paper, moistened with distilled water, to detect evolving ammonia and filter paper, moistened with an alkaline solution of lead acetate, to detect hydrogen sulfide and mercaptan. Fix them between the cork and the walls of the neck of the flask. The papers must not touch the medium. On days 3–5 of incubation at 28–30°C, the contents of the flask are analyzed. The causative agents of the protein ammonification process and their metabolic products are determined.

Microscopy.To detect pathogens of putrefactive decomposition of protein substances, a preparation of live bacteria is prepared in a crushed drop, as well as a fixed and stained preparation. More often than others, mobile cells are found on the preparation. Proteus vulgaris (Fig. No. 4 of the application)prevailing in the first stages of protein breakdown. These are non-spore-forming, sticks of unequal length. In addition, there are many spore-forming cells on the preparation. Bacillus mycoides and Clostridium sp. (Fig. No. 1, 4 attachments). In the latter, spores are located terminally and their diameter exceeds the width of the cell. You. mycoides causes ammonification of proteins under aerobic conditions, and C. putrificus - in anaerobic, but can also develop under aerobic conditions, if the environment contains aerobic microorganisms that absorb oxygen.

NH released into the atmosphere 3, stains a suspended strip of red litmus paper blue. Lead paper blackens under the influence of hydrogen sulfide if it is covered with a silvery coating, which means that along with H 2 S mercaptans are also released (for example, methyl mercaptan CH 3SH).

Task number 2. To study the microorganisms involved in the processes of ammonification of urea, make a drawing. To monitor the process of ammonification of urea, you can use a nutrient medium of the following composition (g/l of distilled water): potassium or sodium tartrate (tartaric acid, you can salt malic acid) - 5.0; urea - 5.0; TO 2 NRO 4 - 0.5; MgSO 4 7H 2 O - 0.2.

The medium is poured into 100 ml flasks, 30 ml each, inoculated with soil, and placed in a thermostat at 25–30°C. To detect ammonia, a strip of red litmus paper is hung under a cotton plug. After 3-5 days, the culture is analyzed. Establish the release of ammonia on the blue of red litmus paper.

Microscopy.To study the causative agents of ammonification of urea, a preparation is prepared from a barely noticeable film on the surface of the medium and stained with fuchsin. Cells are most commonly seen under a microscope. Urobacillus pasteurii (you. probatus), less often - Planosarcina ureae (Fig. No. 5 of the application).

Control questions and tasks

mineralization of nitrogen. Bacteria involved in protein ammonification.

Degradation of nucleic acids.

Decomposition of urea, uric and hippuric acids.

Characterization of nitrification processes. Microorganisms that produce nitrates in the soil.

Recovery of nitrates and nitrites in nature.

Denitrification (dissimilation nitrate reduction).

Assimilation nitrate reduction.

Nitrogen fixation by free-living microorganisms.

Associative nitrogen fixation.

Symbiotic nitrogen fixation. Characteristics of nodule bacteria.

Interaction of nodule bacteria with host plant. The formation of bacteroids.

Symbiotic nitrogen fixation of non-legume plants.

Chemistry of nitrogen fixation processes.

14. Fill in the table number 8.

Table number 8. Characteristics of the nitrogen cycle processes and microorganisms involved in the conversion of nitrogen compounds

Ammonification of urea

I phase of nitrification

Symbiotic nitrogen fixation

ACTIVITY #9

CONVERSION OF NITROGEN COMPOUNDS BY MICROGANISMS

Target: To study the features of the processes of transformation of mineral compounds of nitrogen by microorganisms.

Tasks

1. To study the features of the course of nitrification processes (I, II phase) and the microorganisms involved in them.

2. To study the features of the denitrification processes and the microorganisms involved in them.

3. To study the features of the processes of nitrogen fixation and the microorganisms involved in them.

Materials and equipment.Flasks with liquid Ashby medium for cultivating nitrogen fixers, Winogradsky medium for cultivating nitrifiers, Giltai medium for cultivating denitrifiers, equipment for staining micropreparations, microscopes.

Basic concepts.Nitrification (I and II phases), nitrogen immobilization, assimilation nitrate reduction, dissimilation nitrate reduction (denitrification), nitrogen fixation by free-living microorganisms, associative nitrogen fixation, symbiotic nitrogen fixation, leghemoglobin, nitrogenase, nitrate reductase.

Progress

Task number 1. To study the microorganisms involved in the processes of nitrification, to draw a picture. For the accumulation of nitrifying bacteria, the nutrient medium of S. N. Vinogradsky is used (g / l of distilled water):

The media are sterilized and poured into flasks with a layer of 1.0-1.5 cm and infect with a lump of greenhouse soil. The flasks are closed with cotton plugs and placed in a thermostat at a temperature of 25–28 °C on the 14–21st day.

Microscopy.To study nitrifying bacteria, micropreparations are prepared from the liquid in flasks. In the field of view of the microscope, clusters of oval or coccoid cells are visible Nitrosomonas (first phase) and short rod cells Nitrobacter (second phase) . Representatives of the genus Nitrosomonas (Fig. No. 6 of the application)have an oval shape, in some cases approaching a ball, have mobility and are equipped with one long flagellum. Different types Nitrosomonas are very widespread in the soil and differ from each other in the size or shape of the cells, as well as in their relation to the active reaction of the environment. Most studied Nitrosomonas europea. The cells are coccoid or oval, 0.5-1.5 µm in size, motile, with a long polar flagellum. Nitrobacter - small rounded, ovoid or pear-shaped sticks, immobile, single or connected in small groups, surrounded by a mucous capsule(Fig. No. 7 of the application). Among the bacteria of this genus, it is customary to distinguish two types: Nitrobacter winogradskii and Nitrobacter agilis.

Oxidation of the ammonium form to the nitrite form is also carried out Nitrosocystis and Nitrosospira , nitrite to nitrate Nitrospina.

Task number 2. To study the microorganisms that carry out the processes of denitrification, draw a picture. For the accumulation of denitrifying bacteria, the following medium is used (in g/l): Rochelle salt - 20; KNO 3 - 2.0; K 2 NRO 4 - 0.5; MgSO 4 7H 2 O - 0.2; FeSO 4 7H 2 Oh, traces. The medium is poured in a high layer into large test tubes to the brim and sterilized. Thoroughly mix with soil to remove air bubbles and cover with a rubber tube, into which is inserted a glass tube open from 2 sides, expanded in the middle part. The cork displaces some of the liquid into the glass tube. There should be no air bubbles under the cork. Vaseline oil is poured into a tube above the medium in a small layer to create anaerobic conditions, placed in a thermostat at a temperature of 30-35 ° C for 5-6 days.

Microscopy.A drop is taken from the middle of the substrate with a clean pipette and a fixed and stained preparation is prepared, which is then examined under a microscope with an immersion system. The preparation is dominated by non-spore-forming spherical cells or short rods Paracoccus denitrificans (Bacterium denitrificans ) (Fig. No. 7 of the appendix). On the medium with Rochelle salt often develops P. stutzeri (Achromobacter stutzeri - rod-shaped cells with a size of 2.0 - 4.0 microns, mobile) (Fig. No. 7 of the appendix). In this case, the culture forms a greenish-yellow fluorescent pigment. Also denitrifiers include: Pseudomonas aeruginosa - small rods (1.0 - 1.5 microns in size), single or paired, mobile, carry 1 - 2 polar flagella. Greening of the medium is often observed, especially when using citric acid carbon, which indicates the development Pseudomonas fluorescens - small rods (1.0 - 2.0 microns in size), mobile, have 3 - 4 polar flagella.

Task number 3. To study the microorganisms involved in the fixation of atmospheric nitrogen, to draw a picture. Ashby medium is used to accumulate free-living nitrogen fixers. The composition of the nutrient medium (g/l of distilled water):mannitol, glucose, or sucrose - 20.0; TO 2 NRO 4 - 0.2; MgSO 4 - 0.2; NaCl - 0.2; K 2 SO 4 - 0.1; CaCO3 - 5.0.

The nutrient medium, without sterilizing, is poured into flasks with a volume of 100 - 150 ml, with a layer of 1 - 1.5 cm and infected with greenhouse soil (1/3 teaspoon). The flasks are closed with cotton plugs and placed in a thermostat at a temperature of 28–30 °C.

After 5-7 days, a brown-brown film of Azotobacter is formed on the surface of the medium. The liquid in the flask foams and emits the smell of butyric acid, which indicates the development of bacteria in the environment. Cl. pasteurianum.

Microscopy.A fixed micropreparation is prepared from the surface film. The two most common types of Azotobacter are: Azotobacter chroococcum - in a young culture has rod-shaped cells, mobile, 3 - 7 microns in size(Fig. No. 8 of the application). In the old culture, the cells are coccoid, paired and sarcine-like packets, usually surrounded by a mucous capsule. There are many shiny grains of volutin in the cells. Colonies on dense nutrient media are slimy, spreading or convex, colorless or dark brown to black; Azotobacter vinelandii - in a young culture, the cells are rod-shaped, 2-3 microns in size. In the old culture, the shape of the cells is spherical. A drug is prepared from a drop of liquid Clostridium pasteurianum - motile rod-shaped cells, 3-7 microns in size, single or arranged in pairs and short chains(Fig. No. 1.10 of the application). Spores are oval, form excentrally, with spore-bearing cells taking the shape of a lemon. A lot of granulosa accumulates in the cells. Colonies whitish, smooth, shiny.

Control questions, see lesson number 8.

ACTIVITY #10

Target:

Tasks:

To study the mechanism of alcoholic fermentation and the morphology of its pathogens.

To study the mechanism of the course and types of lactic acid fermentation, the morphology of its pathogens.

To study the features of the conversion of alcohol into acetic acid and the morphology of acetic acid bacteria.

To study the mechanism of butyric fermentation and the morphology of its pathogens.

Materials and equipment. Brine, kefir, baker's yeast, beer, butyric acid bacteria culture medium, preparation staining equipment, microscopes.

Basic concepts.Alcoholic fermentation, Pasteur effect, homofermentative lactic fermentation, heterofermentative lactic fermentation.

Progress

Task number 1. To study the microorganisms involved in alcoholic fermentation, draw a picture. 50 ml of 20% sucrose solution and about 1 g of yeast diluted in 10 ml of sucrose solution (20%) are poured into a 250 ml flask. Close and maintain the temperature around 35-40°C for several days.

Microscopy.Most of the cultivated yeasts used in practice belong to the genus Saccharomyces (Saccharomyces cerevisiae, rice. No. 1, 2 applications) and Schizosaccharomyces. These yeasts differ from wild ones in that they are able to withstand high concentrations of sugar (up to 70%) and alcohol (up to 14%), develop at pH 4-6, and form fewer fermentation by-products.

Task number 2. To study the microorganisms involved in lactic acid fermentation, draw a picture.To get acquainted with the bacteria of lactic acid fermentation (homofermentative fermentation), you can use ready-made lactic acid products (curdled milk, acidophilus, kefir) and brine (heterofermentative fermentation). These products are looped onto a glass slide and a thin smear is made. Stain for 3-5 minutes with an aqueous solution of methylene blue.

Microscopy.In lactic acid products, the following pathogens of homofermentative fermentation are most often found - Streptococcus lactis (Fig. No. 9 of the appendix), having the form of oval cocci with a diameter of 0.5-1.0 microns, are located in culture in pairs (diplococci) or in short chains (streptococci), less often in single cells; Lactobacillus bulgaricus (Fig. No. 9 of the appendix) - a large rod (4-5 microns long), immobile, gram-positive, located in the form of individual cells and short chains, the optimum development temperature is 40-45 ° C; Lactobacillus acidophilus - in morphology it is close to Bulgarian, but has a different temperature optimum of development - 37°C.

Among the causative agents of heterofermentative fermentation are usuallyLactobacillus brevis, Lactobacillus brassicae, Leuconostoc mesenteroidesand others (brine microflora). A white or creamy velvety wrinkled film on the surface of the brine or kefir indicates the presence Geotrichum candidum ( Oidium lactis) (Fig. No. 9 of the appendix) - milk mold, which always accompanies lactic acid fermentation.

Task number 3.To study the microorganisms involved in the conversion of alcohol to acetic acid, draw a picture.A thin layer of beer (0.5-1.0 cm3) is poured into conical flasks. The flasks are closed with cotton plugs and placed in a thermostat at a temperature of 30°C. After 5-7 days, the nature of the formed films is described, stained smears are microscopically examined.

Microscopy.Acetic acid bacteria are united in the genusAcetobacter, type viewAcetobacter aceti(Fig. No. 9 of the appendix) is represented by weakly mobile rod-shaped elliptical cells 0.6-0.8 wide, 1.0-3.0 µm long, single, in pairs or chains. Often forms involutional forms in the form of swollen, branched or filiform formations. Acetic acid bacteria are easily isolated from beer.

Task number 4.To study the microorganisms involved in butyric fermentation. Cut the unpeeled potatoes into slices, fill the test tube with them by 1/3 of the volume, add a pinch of chalk and fill with water to the brim. The test tubes are placed in a water bath at a temperature of 80°C for 10-15 minutes. Then the test tubes are stoppered and transferred to a thermostat with a temperature of 250 C. After 2-3 days, bacteria of butyric fermentation are found in the liquid.

Microscopy.On fixed preparations are foundCl. pasteurianum, Cl. butiricum(Fig. No. 1, 10 of the appendix) - movable sticks with rounded ends, single and paired. In old cultures, a spore is found at one end of the cell.

Control questions

Alcoholic fermentation.

Yeast. Form, structure, reproduction and classification.

Oxidation of ethyl alcohol to acetic acid.

Chemistry and causative agents of lactic acid fermentation (homofermentative type).

Chemistry and causative agents of lactic acid fermentation (heterofermentative type).

Butyricand acetobutyl fermentation.

Butyric fermentation of pectin substances.

Anaerobic degradation of fiber.

Oxidation of fiber by microorganisms.

Fill in table No. 9 "Characteristics of fermentation processes."

Table number 9.Characterization of carbon conversion processes (fermentation and oxidation processes of compounds that do not contain nitrogen)

Questions for self-study

Methods of nutrition and intake of nutrients into the cell.

Nutritional needs of microorganisms.

Types of nutrition of microorganisms.

Metabolism of microorganisms. Fermentation, respiration, photosynthesis.

ACTIVITY #11

CONVERSION OF CARBON COMPOUNDS BY MICROORGANISMS

Target:Explore Features various kinds fermentation and the morphology of their pathogens.

Tasks:

To study the mechanism of pectin fermentation and the morphology of its pathogens.

To study the features of the transformation of cellulose under aerobic conditions and the morphology of its pathogens.

To study the features of cellulose fermentation under anaerobic conditions and the morphology of its pathogens.

Materials and equipment. Accumulative cultures of microorganisms that carry out the fermentation of pectin substances, decomposition of fiber, microscopes, equipment for staining preparations.

Basic concepts.Butyric fermentation, pectin, pectinase, cellulose, cellulase.

Progress

Task number 1.To study the microorganisms involved in the fermentation of pectin substances, to draw a picture. To isolate pectin-destroying bacteria, flaxseed or nettle nutrient medium is used. Sheaves 5-7 cm long are prepared from the stems, placed in test tubes, poured with tap water and boiled for 10-15 minutes. Boiling removes extractives, which can serve as a carbon source for accompanying butyric acid bacteria. The water is drained, the sheaves are filled with a new portion of water, the test tubes are tightly closed with cotton plugs and sterilized in an autoclave at 1 atm for 20 minutes. When the tubes are cool, the medium is infected with a piece of fresh flax straw. Infected test tubes are placed in a thermostat at a temperature of 35°C. After 3-5 days, the process of pectin fermentation begins, the liquid becomes cloudy and foams.

Microscopy.To study the morphology of pectin fermentation bacteria, life-long preparations are prepared. The snopik is removed from the test tube, a drop of liquid is squeezed onto a glass slide, fixed and lifetime preparations are prepared (in Lugol's solution). Cells are visible on the preparationClostridium pectinovorumAndClostridium felsineum, vegetative and sporulating, stained with iodine for granulosa in dark blue (Fig. No. 9 of the appendix).

Task number 2.To study the microorganisms involved in the decomposition of fiber (anaerobic conditions), draw a picture.To obtain an accumulation culture of anaerobic formscellulose-destroying bacteriause the Imshenetsky environment. INabout 1-2 g of filter paper or cotton wool are added to a round flat-bottomed flask, and filled to the top with a medium of the following composition (in g / l): KNH4 HPO4 – 1.0; KN2 RO4 – 0.5; TO2 NRA4 – 0.5; CaCl2 – 0.03; peptone - 5; MgSO4 – 0.4; CaCO3 – 2.0; NaCl - 0.5; MnSO4 and FeSO4 footprints;medium pH 7.0-7.4. The environment is infected with a small amount of soil, the flask is closed with a cork stopper with a hole for the release of gases and placed in a thermostat at 30-35°C. Elective conditions in this case are determined by the presence of cellulose, a source of carbon that can be consumed only by specific cellulose-decomposing bacteria that have the cellulase enzyme, as well as anaerobic conditions. Peptone, introduced into the medium in a small amount, strongly stimulates the fermentation process. After 7-10 days, cellulose fermentation begins, which lasts 2-3 weeks. The filter paper, as it ferments, becomes slightly slimy, turns yellow and gradually collapses.

Microscopy.Microscope a piece of paper. Under anaerobic conditions, the process of decomposition of cellulose is carried out by bacteria of the genusClostridium. Cl. omelianskii(Fig. No. 9 of the appendix) has the appearance of long rod-shaped cells up to 8 microns, in old cultures the length is about 10-15 microns, located singly or connected in threads. Spores are spherical, form at the end of the cell, the cell takes the form of a drumstick. It is isolated from soil and water. The optimum growth temperature is 30-35°C.

Task number 3.To study the microorganisms involved in the decomposition of fiber (aerobic conditions), draw a picture.For the isolation of aerobic fiber-destroying bacteria, Hutchinson's nutrient medium is used. Medium composition (g/l): K2 NRA4 – 1.0; NaCl - 0.1; CaCl2 – 0.1; FeCl3 – 0.01; MgSO4 – 0.3; NaNO3 - 2.5; medium pH 7.2-7.3. A sterile nutrient medium is poured into conical flasks with a layer of 1.5-2.0 cm, the medium is infected with a lump of soil, and a pleated filter is lowered onto the surface of the medium with the cone up. The flasks are closed with cotton plugs and placed in a thermostat at a temperature of 25-30°C for 10-14 days. At the border of the nutrient medium and air, optimal conditions for nutrition and aerobic respiration of fiber-destroying bacteria are created. In this zone, the most intensive process of destruction of the filter paper. After 8-10 days, yellow-orange spots of colonies of fiber-destroying bacteria appear on the filter. The paper decomposes and the filter gradually settles to the bottom.

Microscopy.A smear in a drop of liquid is prepared from the destroyed masses of fiber in a flask with a microbiological loop. Most often, representatives of the orders are found on the preparation.Fly bacterialesAndCytophagalesgroups of gliding bacteria (Fig. No. 10 of the appendix).GenusCytophagarepresented by long rod-shaped cells with pointed ends, somewhat curved (2-10 microns). Colonies are yellow, orange, brown-brown, smooth, slimy.

GenusСellvibrlorepresented by small, slightly curved movable rods (2-4 microns). Colonies are ocher-yellow mucous. RollCellfalciculahas the form of short thick curved sticks with pointed ends (2.0 * 0.7 microns).

Genussorangiumin a young culture, it looks like thick, slightly curved rod-shaped cells with rounded ends (2-5 microns). With the aging of the culture, fruiting bodies are formed, consisting of microcysts. Rod-shaped cells are shortened, covered with a thick membrane, acquiring irregular outlines. Colonies bright orange, purple.

GenusPolyangiumrepresented by almost straight rod-shaped cells with rounded ends (3.5-8 microns). With aging, oval microcysts are formed, which are connected and pear-shaped fruiting bodies. Fruiting bodies are yellow, orange, sit directly on the substrate.

In addition to bacteria, mold fungi are involved in the aerobic destruction of fiber.Aspergillus, Penicillium, Fusarium, Cladosporium, Trichodermaand some actinomycetes.

Control questions, see lesson number 10.

ACTIVITY #12

ECOLOGY OF MICROORGANISMS

Target:To study the influence of environmental factors on the growth and development of microorganisms.

Basic concepts.Obligate psychrophiles, psychrotrophic microorganisms (facultative psychrophiles), thermophiles, osmotolerant microorganisms, gallophiles, lyophilization, obligate aerobes and anaerobes, microaerophiles, aerotolerant anaerobes, antiseptics.

Control questions

The effect of temperature on the growth and development of microorganisms. Ecological groups of bacteria in relation to temperature.

Influence of humidity on the growth and development of microorganisms.

Influence of light and various radiations on the growth and development of microorganisms. Influence of the magnetic field.

Influence of oxygen concentration on the growth and development of microorganisms.

Influence of the environment reactionon the growth and development of microorganisms. Compounds and ions toxic to bacteria.

Adaptive reactions of microorganisms.

ACTIVITY #13

DETERMINATION OF SENSITIVITY OF BACTERIA TO ANTIBIOTICS

Target:To determine the sensitivity of microorganisms to antibiotics.

Tasks:

1. Determine the sensitivity of microorganisms to antibiotics by diffusion into agar using paper discs

Materials and equipment.Petri dishes with MPA, sterile discs moistened with antibiotic solutions, pure cultures of saprophytic bacteria and mold fungi, pipettes, spirit lamps, spatulas.

Basic concepts.Antibiotics, bacteriostatic and bactericidal effects, R-factors, resistance.

Progress

Task number 1.Determine the sensitivity of microorganisms to antibiotics by diffusion into agar using paper discs.

The disk diffusion method for determining the sensitivity of microorganisms to antibiotics is based on recording the diameter of the growth inhibition zone around a paper disk with an antibiotic. On the surface of nutrient agar in Petri dishes, inoculated with test microbes, paper disks impregnated with antibiotics are applied, andcups are incubated at 37°C. The presence of a zone of inhibition of microbial growth arounddiscs indicates the sensitivity of the pathogen to the drug, the absence of a growth inhibition zone indicates resistance.

Definition progress.A culture of microorganisms is applied to sterile Petri dishes with MPA. A daily broth culture is used as an inoculation material - 0.2 ml of a bacterial suspension is applied to the surface of the MPA and evenly distributed by shaking the cup, followed by sucking off the excess liquid with a pipette.

The cups are dried for 30-40 minutes at room temperature. Then, disks impregnated with various antibiotics are placed on the surface of the seeded medium with tweezers. The discs are applied tightly to the surface for close contact with the medium. The discs are placed at an equal distance from one another and about 2 cm from the edge of the cup. The cups are placed in a thermostat upside down or put a circle of filter paper under the lid of the cup to avoid erosion of the lawn with condensation water, and incubated at 37°C for 18 hours.

Evaluation of results.Using a ruler, determine the diameter of the microbial growth inhibition zones around the disks, including the diameter of the disk itself. Single colonies or a thin film of microorganism growth within the zone of growth inhibition is not taken into account. The absence of zones of microbial growth inhibition around the discs indicates the absence of sensitivity of the microbe to this antibiotic. With a zone of microbial growth inhibition with a diameter of up to 10 mm, the strain is regarded as a little sensitive. A microbial growth inhibition zone of more than 10 mm indicates the sensitivity of the strain. How more zone growth retardation, the higher the sensitivity of microorganisms to the antibiotic. Record the results in table No. 10.

Table number 10.Susceptibility of microorganisms to antibiotics.

Antibiotic properties of streptomycin. Antibiotic properties of penicillin. ACTIVITY #14 BASICS OF MEDICAL MICROBIOLOGY Target:To study microorganisms - pathogens of animals and humans. Literature Lebedeva M.N. Microbiology. - M .: "Medicine", 1969. Fundamentals of microbiology, virology and immunology / Ed. Edited by Vorobyov A.A., Krivoshein Yu.S. – M.: Masterstvo, 2001. Issues for discussion Characterization of pathogenic cocci on the example of staphylococci. Inflammatory-purulent diseases of the skin. Septicemia. Characterization of pathogenic cocci on the example of streptococci. Localized pyoinflammatory infections, tonsillitis, scarlet fever. Characterization of pathogenic cocci on the example of pneumococcus. Pneumonia. Characterization of pathogenic cocci on the example of meningococcus. Meningitis. Characterization of pathogenic cocci on the example of gonococcus. Gonorrhea, blenorrhea. Characterization of gram-negative non-spore-forming bacteria on the example of the plague bacillus. Plague. Characterization of gram-negative non-spore-forming bacteria on the example of tularemia bacillus. Tularemia. Characterization of gram-negative non-spore-forming bacteria using the example of Brucella. Brucellosis. The family of intestinal bacteria on the example of Escherichia coli. Characteristics of the group of typhoid and paratyphoid bacteria. Typhoid and paratyphoid. Causative agents of food poisoning. Salmonellosis. Characteristics of causative agents of dysentery. Types of dysentery. Characteristics of cholera vibrio. The structure of capsular bacteria on the example of Klebsiella. Characterization of anthrax bacilli. Forms of anthrax. Characteristics of hemophilic bacteria on the example of whooping cough. Pathogenic anaerobes on the example of causative agents of gas gangrene. Pathogenic anaerobes on the example of tetanus bacillus. Pathogenic anaerobes on the example of causative agents of botulism. Characteristics of the diphtheria bacillus. Characterization of acid-fast bacteria on the example of tubercle bacillus and leprosy bacillus. Characterization of pathogenic fungi on the example of actinomycetes. Dermatomycosis. Characterization of pathogenic spirochetes on the example of pale treponema. Syphilis. Characteristics of pathogenic spirochetes on the example of relapsing fever spirochete. This guide briefly provides theoretical basis sanitary microbiology, as well as some experiments related to the determination of various indicators of the microflora of air, water and soil. Miscellaneous Consists of two sections - General Applied Medical Microbiology and Clinical Microbiology. The first section outlines the main methods of microbiological research, methods of specific therapy and prevention of infectious diseases, the second - the methods of their laboratory... 1.32 MB added 06/11/2011 Tutorial. KemTIPP. - Kemerovo, 114 p. Subject and tasks of microbiology. Basic properties of microorganisms Historical sketch of the development of microbiology. Prospects for the development and achievements of modern microbiology in national economy, Food Industry. Principles of systematics. Structural organization microorganism...