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METHODOLOGICAL INSTRUCTIONS FOR STUDENTS

June 16, 2024

METHODOLOGICAL INSTRUCTIONS FOR STUDENTS

OF DENTISTRY FACULTY, 2-nd year

LESSON № 3 – 6 hrs.

THEME: I. Main principles and methods of pure cultures isolation. Isolation of pure cultures of bacteria from the caries tooth cavity.

II. Cultural properties of microorganisms. Bacteria enzymes and their significances for identification. Identification of the isolated pure cultures of aerobic bacteria.

III. Isolation and identification of pure cultures of anaerobic microorganisms.

Main principles and methods of pure cultures isolation. Isolation of pure cultures of bacteria from the caries tooth cavity. Cultural properties of microorganisms. Bacteria enzymes and their significances for identification. Identification of the isolated pure cultures of aerobic bacteria. Isolation and identification of pure cultures of anaerobic microorganisms

AIM: the student should familiarize with types of respiration, cultural properties, reproduction, and role in metabolism of enzymes of bacteria. To take possession creating technique of anaerobic conditions and stages of obtaining of pure culture of anaerobic bacteria.

To take possession of a bacteriological method of diagnostics of contagions, methods biochemical and seroljgical identification of bacteria.

PROFESSIONAL MOTIVATION: to take possession of bacteriological examination which is based on isolating a pure culture of causaive agents of infectious disease and their identification.

Practical activities (9⁰⁰-12⁰⁰)

I. Main principles and methods of pure cultures isolation. Isolation of pure cultures of bacteria from the caries tooth cavity.

1. To prepare stains from an mixture of bacteria, to stained by technique Gram’s method, to study by a microscope, to draw.

2. To inoculate a researched materials on Agar media by method of ‘strokes’.

3. To study and to sketch three Petri’s dishes with vegetation of an admixture. bacteria (by technique of Drygalskyy method).

4. Macro – and microscopic learning of colonies, which have grown on МPА with till sowings from the previous occupation. To describe and to sketch in protocols.

5. To prepare Gram-stained smears for microscopic examination from various types of colonies, to sketch in the protocol.

6. Sub-inoculation of colony of gramnegative bacteria onto slant agar to enrich for a pure culture.

II. Cultural properties of microorganisms. Bacteria enzymes and their significances for identification. Identification of the isolated pure cultures of aerobic bacteria.

The third stage of isolatione of pure culture of aerobic bacteria

1. To check of the purity of isolated culture, which has grown on slant agar, prepare Gram-stained smears for microscopic examination. To sketch in a protocol.

2. Inoculate the pure culture onto Hiss` media and MPB for examinate sugarlytic and peptolytic properties of bacteria.

3. To put a presumptive agglutination test on glass slides with pure culture and specific antiserums.

The first stage of isolation of pure culture of anaerobic bacteria

4. To sow a soil on milk and Кitt-Tarozzi medium.

III. Isolation and identification of pure cultures of anaerobic microorganisms.

The second stage of isolation of pure culture of anaerobic bacteria

1. To prepare smears from growth on Kitt-Tarozzi`s medium and milk, stain by Gram`s method, to sketch in the protocol.

2. Inoculate the studied material from Kitt-Tarozzi medium into Winyal-Weyon tubes for obtaining separate colonies.

3. To make conclusion about results of microbic growth in Hiss’ media.

4. To study colonies of anaerobes grown in Winyal-Weyon tube.

5. To make the conclusion about pure culture of aerobic

Break (12⁰⁰– 12³⁰)

Students’ independent study program

I. I. Main principles and methods of pure cultures isolation. Isolation of pure cultures of bacteria from the caries tooth cavity

1. To familiriaze cultural property of bacteria (R – and S-form).

2. What does it mean a colony and a pure culture?

3. To call methods and principles of isolation in pure culture.

4. To describe methods of obtaining in pure culture, are based on mechanical divorced of bacteria.

5. To describe methods of obtaining of pure culture of bacteria, taking into account their different biological .

6. Stages of pure culture isolation of aerobic microorganisms:

II. Cultural properties of microorganisms. Bacteria enzymes and their significances for identification. Identification of the isolated pure cultures of aerobic bacteria.

1. Enzymes of bacteria, their classification and practical value.

2. Identification of pure growths:morphological, tinktorial, cultural, biochemical, serological, biological.

3. With what purpose spend identification of pure growths?

4. To give the characteristic to диференциально-diagnostic mediums for definition of a fermentation of carbohydrates: Endo, Levin,Ploskirev, Hiss` media, Olkenitsky and others.

5. How study peptolytic, proteolytic and haemolytic properties of bacteria?

6. In what essence serological and biological identification of pure growths? How she is spent?

7. Stages of obtaining pure culture of anaerobes by Veinberg’s and Ceisler’s techniques.

III. Isolation and identification of pure cultures of anaerobic microorganisms.

1. Stages of obtaining pure culture of aerobes

2. Stages of obtaining pure culture of anaerobes

3. Stages of obtaining pure culture of anaerobes by Veinberg’s techniques.

4. Stages of obtaining pure culture of anaerobes by Ceisler’s techniques.

Discussion. Checking practical skills and theoretical material (12³⁰-14⁰⁰)

Break (14⁰⁰-14¹⁵)

Free of students’ work (14¹⁵-15⁰⁰)

Analysis of test exam objectives “Step”, evaluation of students who have not passed before the test control system «Moodel»; practicing and receiving practical skills

Tests and assignments for self–assessment

I. Main principles and methods of pure cultures isolation. Isolation of pure cultures of bacteria from the caries tooth cavity.

Choose the correct answers:

1. The replication of bacteria have been made by:

a – simple transversal division (segmentation);

b – mitosis;

c – sporulation;

d – budding;

e – sexual way.

2. Mechanical separation of bacteria performed by methods:

a – Drigalsky;

b – Pasteur;

c – Loeffler;

d – Gram;

e – streaks.

3. For isolation of pure culture of aerobic bacteria tested materials:

a – inoculate onto agar media;

b – inject into laboratory animal;

c – inject into chicken embryo;

d – infected cell cultures.

II. Cultural properties of microorganisms. Bacteria enzymes and their significances for identification. Identification of the isolated pure cultures of aerobic bacteria.

Choose the correct answers:

1. The S-forms of colony are:

a – convex;

b – with rough surface;

c – with fibrous edges;

d – with smooth surface;

e – with bumpy surface.

2. Till types of respiration the bacteria are divided on:

a – aerobes,

b – saprophytes,

c – lithotrophes,

d – microaerophiles,

e – capneic bacteria.

3. It is possible to make the anaerobic conditions using:

a – anaerostat,

b – thermostat,

c – handsets (tubes) Vinyale-Vinione,

d – Fortner method,

e – Drigalsky method.

4. For cultivation of anaerobes are used such media:

a – Ploskirev,

b – Kitt-Tarozzi,

c – blood agar,

d – Levenstein-Yensen,

e – Chystovych.

III. Isolation and identification of pure cultures of anaerobic microorganisms.

Select the correct statements:

a – the enzymes of bacteria are divided into exoenzymes, obligate, adaptable, anaerobic, design;

b – for the examining saccharolytic properties of bacteria apply such media: Endo, Kitt-Tarozzi, blood agar, Olkenitsky, Ploskirev;

c – for the examining proteolytic properties of bacteria apply such media: meat pepton gelatin, Levin, serum agar, Hiss, MPA;

d – the biological identification of bacteria are made onto express media, into cell cultures, by infected of laboratory animal, glass agglutination test.

1. Insert missed word in the next statement:

a –the serological identification of bacteria are made at them…………….. properties; for this purpose are used special.………. and………….. cultures; make .……….test.……..; b – onto medium Endo E.coli create ………. colonies, because it ferments…………., thus… medium becomes acid and coloures.…….…reduces.

Verification of knowledge level is checking by testing and answering the constrictive questions

Students must know:

1. Definition of constructive and power (fermentation, respiration, photosynthesis) metabolism of bacteria

2. The name of nutritional types of a bacteria.

3. The mechanism of a nutrition

4. Main growth factors of bacteria.

5. Enzymes of bacteria, their classification and function

6. Classification of media

7. Cultural properties of bacteria

8. Types and mechanism of bacterial respiration.

9. Main methods of creating of anaerobic conditions for cultivation of anaerobes.

10. Media which are used for anaerobes cultivation.

11. Stages of isolation and identification of anaerobes.

12. Classification bacterial enzymes, and their practical value.

13. Main principle morphological, tinctorial, cultural, biochemical, serological, biological Identification of pure culture.

12. Characteristic of differential diagnostic media

15. Serological and biological identification of pure culture

16. Cultural properties of bacteria

17. Types and mechanism of bacterial respiration.

18. Main methods of creating of anaerobic conditions for cultivation of anaerobes.

19. Media which are used for anaerobes cultivation.

20. Stages of isolation and identification of anaerobes.

Students should be able to:

1. To take possession of serial delutions procedure;

2. To take possession of determination procedure of common microbial number of water.

3. Prepare smear from different types of colonies;

4. Stain a smear by Gram’s technique;

5. Sub-inoculate the E. colі colony onto slant agar to enrich for a pure culture.

6. Inoculate a soil into milk and Kitt–Tarozzi medium.

7. Prepare smears from pure culture and tested materials;

8. Stain of smears by Gram method, microscopy;

9. Inoculate of pure culture into Hiss medium, MPB;

10. Production of an presumptive agglutination test on glass with pure culture;

11. To inoculate tested materials into Vinyale-Vinone tubes.

The answers to the self – assessment

I. Main principles and methods of pure cultures isolation. Isolation of pure cultures of bacteria from the caries tooth cavity.

1. A, d; 2. A, b, e. 3. A, b.

II. Cultural properties of microorganisms. Bacteria enzymes and their significances for identification. Identification of the isolated pure cultures of aerobic bacteria.

1. A, d; 2. A, d, e; 3. A, c, d; 4.B, c.

III. Isolation and identification of pure cultures of anaerobic microorganisms.

1. A – exoenzymes, adaptable, constructive; b – Endo, Olkenitsky, Ploskirev; c – MPG, serum agar; d – by infected of laboratory animals. 2. A – antigenic, immune, serum, pure, presumptive agglutination; red, lactose, рН, fuchsine.