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METHODOLOGICAL INSTRUCTIONS FOR STUDENTS

June 1, 2024

METHODOLOGICAL INSTRUCTIONS FOR STUDENTS

OF DENTISTRY FACULTY, 2-nd year

LESSON № 4 – 6 hrs.

THEME: I. Morphology and biology of viruses. Basic methods of viruses cultivation. Methods of indication of the viruses.

II. Bacterial viruses (Bacteriophages). Structure, classification. Types of interaction of bacteriophages and bacterial cells. Virulent and moderate phages. Practical significance of bacteriophage phenomenon.

III. Sanitary-bacteriologic studying of water, soil, air. Sanitary-indicative microorganisms. Microbiologic control in stomatologic facilities.

AIM: To familiarise with modern classification of viruses, their morphology and chemical structure, cell cultures which are used in viruses examination, peculiarities of cytopathic effect, to learn the methods of chicken embryos inoculation

To familiarise with classification of bacteriophages, their morphology and chemical structure, methods of their cultivation, receipt and titration, practical importance of the phage in medicine

To study a microflora of soil, water and air. To take possession of methods of examination of water, soil, and air microflora. To learn to evaluate a sanitary and hygienic condition of water, soil, and air.

PROFESSIONAL MOTIVATION: Viruses are large, non-cellular, living, heterogeneous group of microorganisms and cause different diseases in man (influenza, encephalitis, AIDS et al.). It’s necessary to know about their biological properties, methods of cultivation, and main principle of laboratory diagnosis viral diseases.

A soil, water and air may be reservoir of different microorganisms which are causing agents of serious diseases such as dysentery, anthrax, cholerae, tuberculosis, diphtheria etc. It is necessary to control sanitary-epidemiological condition of water, air, soil to prevent diseases

Practical activities (9⁰⁰-12⁰⁰)

THEME: I. Morphology and biology of viruses. Basic methods of viruses cultivation. Methods of indication of the viruses

1. Inoculation of cell cultures by virus-containing material.

2. Inoculation of chicken embryos into the allantoic cavity by virus-containing material.

3. To familiarize with cell cultures, inoculated by the viruses. To estimate presence of cytopathic effect of viruses.

4. To study morphology of virions by using microphotographs and slides.

II. Bacterial viruses (Bacteriophages). Structure, classification. Sorts of interaction of bacteriophages and bacterial cells. Virulent and moderate phages. Practical significance of bacteriophage phenomenon

1. To carry out bacteriophages titration by Gracia’s technique.

2. To carry out the phagotyping of Staphylococcus aureus.

Dry plates with 1.5 per cent meat-peptone agar, draw squares on the bottom of the plate with their number being equal to the number of bacteriophages in the batch, and mark them. After that a 3-4-hour broth bacterial culture is streaked, dried, and a drop of the corresponding phage in the test dilution is transferred onto each square. Place the cultures into an incubator for 18-24 hrs and determine the range of the culture sensitivity to definite bacteriophages judging by the lytic effect.The phage type is reported as a specific genus and species followed by the types that can infect the bacterim

Determination of the sensitivity range makes it possible to refer a given culture to a definite phagovar or phagogroup.

3. Embryo is opened under sterile conditions, allantoic fluid is prepared

4. Virus indicate with haemagglutination test (HA)

The HA test is performed on special plexiglass plates. 0.5 ml allantoic fluid (virus-containing specimen) is added into one well and equal volume of virus-free isotonic sodium chloride solution is added into second well (to be used as control). Half a millilitre of 1°n erythrocyte suspension thrice washed in isotonic saline is added into two wells. The mixture may be incubated at 37°, 20° or 4 °C, depending on the properties of the tested virus.

Test results are assessed at 30-60 rain after complete erythrocyte sedimentation in the control, with the findings reading as follows:

(++++), intense and rapid erythrocyte agglutination with a star-like, marginally festooned sediment (“umbrella”}; (+++), residue of erythrocytes has clearings; (++), a less marked residue; (+), a floccular sediment surrounded with lumps of agglutinated erythrocytes, and (–), a markedly localized erythrocyte sediment (“rouleaus”), as in the control.

III. Sanitary-bacteriologic studying of water, soil, air. Sanitary-indicative microorganisms. Microbiologic control in stomatologic facilities

1. To make plating of 1 ml of drinking water in melted and cooled agar for determining of total microbic number of water.

2. To determine a coli-titer and coli-index of water using method of membrane filters.

3. To determine the number of bacteria in 1 m³ of the air by sedimentation method.

4. To determine the number of bacteria in 1 m³ of the air by aspiration method (with Krotov’s apparatus).

Break (12⁰⁰– 12³⁰)

Students’ independent study program

THEME: I. Morphology and biology of viruses. Basic methods of viruses cultivation. Methods of indication of the viruses

1. Classification, structure and chemical composition of viruses:

a – basis principles of classification of viruses; modern classification of viruses.

b – structure of virions, their dimensions; simple and complex viruses;

c – types of virus symmetry;

d – characteristics of the forms of viruses;

e – chemical composition of viruses;

f – methods of viruses examining.

2. Main methods of cultivation of viruses:

a – inoculation of laboratory animals;

b – inoculation of chicken embryos;

c – types of cell cultures, their classification, methods of inoculation of cell cultures, cytopathic effect, its types.

4. Types of interaction of viruses and sensitive cells.

5. Replication of viruses:

a – phase of interaction between viruses and sensitive cells;

b – features of reproduction of viruses with various types of nucleic acids (Baltimore’s classes of viruses reproduction).

6. Methods of indication of the viruses

8. The nature of viruses.

9. Prions and viroids as causative agents of different diseases. Their biological properties.

1. The structure of bacteriophages. The forms of bacteriophages.

2. Chemical structure and properties of bacteriophages.

3. The types of interaction of phage and bacterial cell. The notion about a prophage. Lysogenic bacteria. Phage conversion, its value.

4. The phases of interaction of bacteriophage and bacterial cell (reproduction of phages).

5. The methods of receipt, study and titration of bacteriophages.

6. Practical usage of phenomenon of bacteriophagia: indication of causative agents, bacterial phagovar determining, reaction of successive growth of the titer of specific phage, therapy, prophylaxis.

III. Sanitary-bacteriologic studying of water, soil, air. Sanitary-indicative microorganisms. Microbiologic control in stomatologic facilities

1. The main representatives of microflora of soil. The diseases transmitted by soil. Sanitary – exponential species. The significance OF soil microflora. Methods of studying of soil microflora.

2. The main representatives of microflora of the water. The diseases transmitted by water. Sanitary – exponential species.

3. The methods of studying of microbial number and coli-index of the water. The specifications of state standard.

4. The main representatives and sanitary – exponential species microflora of the air.

5. The diseases transmitted by air.

6. The methods of studying of air microflora (methods of sedimentation and aspiration).

7. Microbiologic control in stomatologic facilities

8. Microbiology of food products.

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

THEME: I. Morphology and biology of viruses. Basic methods of viruses cultivation. Methods of indication of the viruses

Choose the correct answers:

1. The absolutely new type of contagious agents of tobacco mosaic disease was described by:

a – Beyerink in 1898; b – D. Ivanovsky in 1892; c – L. Pasteur in 1885; d – F. Loeffler in 1887; e – F. d’Herell in 1917;

2. The classification of viruses is based on:

a – type of nucleic acid; b – contents of guanine/cytosine pairs; c – type of virus symmetry; d – presence or absence of envelope; e – peculiarities of virus replication.

3. The structural unit of virus capsid is:

a – neuraminidase; b – capsomer; c – hemagglutinin; d – proteins; e – lipids.

4. The chemical composition of viruses is represented by:

a – nucleic acids; b – proteins; c – lipids; d – carbohydrates. ???offsets

5. There are such hypothesis of viruses origin:

a – viruses became parasites of primitive cells, and the 2 evolved together; b – viruses evolved from parasitic bacteria; c – viruses may be components of host cells that become autonomous. They resemble genes that escape the regulatory control of the host cell.

Insert the necessary word or words:

6. A – the nucleic acid of virion is surrounded by …, which together with it form …. . Some viruses have a … ; b – there are three types of symmetry of virions – …, …, …; c – the dimensions of viruses can be determined by using …, …, …; d – in a nature the viruses can exist in two forms – … – virion and … – vegetative (reprodactive).

Choose the correct answers:

1. Bacteriophages are:

a – small microorganisms, which are filtrated through bacterial filters, b – mycoplasmas, c – viruses of bacteria, d – macro- and microphages.

2. By their morphology phages are divided on:

a – phages with icosahedral head and process, b – cubic, c – filamentous, d –- pleomorphic.

3. The phages perish quickly under the effect of boiling, acids, ultraviolet rays, chemical disinfectants.

4. There are some types of interaction of phages and sensitive cells:

a – productive (lytic) infection, b – abortive infection, c – lysogeny (virogeny).

5. The formation of “early” enzymes are taken under the control of the genetical apparatus of cells.

6. The lysogenic cultures are under certain conditions capable to product of temperate phages.

7. It is possible to titrate the phages with the help of:

a – Gracia method; b – Appelman’s method; c – Loeffler method.

8. For what purpose is used differential and diagnostic phages?

9. For what it is possible to utillize the staphylococcal bacteriophage (fluid, ointment, suppositories)?

Insert the necessary words:

10. The phages consist from …, surrounded by …. . For large phages is typical … structure, presence of …, … and …, which is ended by … .

11. The main phases of interaction of a bacteriophage and sensitive cell are:

a – adsorption (reversible and irreversible), b – penetration, c – …, d – … .

12. The change of properties of a cell under influence of the expression of the bacteriophage genes is named … .

Real-life situation to be solved:

13. On the Petri dish with 1,5 % of meat-peptone agar the 18 hour broth culture of microorganisms were streaked. In 10 minutes on the dried surface of the medium a few drops of filtrate which was examined on the presence of phages were transferred. After 24 hours of an incubation at 37 °C on the places of filtrate drops formed little sterile spots.

How to estimate the obtained results?

14. In the surgical unit of the hospital the cases of suppurations (pyosis) of postoperative wounds have appeared. Microbiologic investigation revealed that there were the cases of hospital infection. The causative agents of these complications were staphylococci. So, from the patient А., from the postoperative wound Staphylococcus aureus of phagovar 3A/3C/55/7, was isolated. From the scrub nurse S. from the stomatopharynx S. aureus of phagovar 79, from the surgeon P. – S. aureus of phagovar 80, from the junior sister Н. from postoperative ward S. aureus of phagovar 3A/3C/55/7 were isolated.

А. Who from the inspected persons is the carrier of conditional – pathogenic flora?

B. Who was a source of postoperative complications?

C. What measures are necessary to do immediately for prevention of circulation of hospital infection contamination?

III. Sanitary-bacteriologic studying of water, soil, air. Sanitary-indicative microorganisms. Microbiologic control in stomatologic facilities

Choose the correct answers:

1. Sanitary – exponential microorganisms of soil is:

a – S. faecalis, b – E. coli, c –C. perfrіngens, d – C. tetanі;

2. Sanitary – indicative microorganisms of water are:

A – M. candіcans, b – M. roseus, c – E. colі, d – S. faecalіs;

3. The coli-titer of drinking water should be: a – not less than 500 ml, b – not less than 100 ml, c– no less then 333 ml;

4. The coli-index of drinking water should be: a –not more then 1, b – not more then 3, c – not more then 5;

5. Sanitary–indicative microorganisms of air are: a – S. haemolyticus, b – Streptococcus viridans, c – S.aureus, d –S. epidermidis.

Real-life situation to be solved:

6. Using Krotov’s apparatus was made the plating of the air on meat-peptone agar during a planned inspection of the maternity hospital. In a day the number of microbe colonies was counted. The total number of colonies on two Petry dishes were 1600. High air pollution was concluded. The head physician received the orders from sanitary-epidemiological station to close the maternity hospital and to carry out urgent preventive measures.

A. Is it competent conclusion of sanitary-epidemiological station in virtue of the received data?

B. What supplementary methods of sanitary – bacteriological research of air can be utilized?

C. What parameters of microbiologic cleanness of the air is it necessary to take into consideration during such investigation?

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

Student must know:

1. Classification of viruses

2. Biological properties of viruses.

3. Replication of viruses.

4. The types of interaction of viruses and sensitive cells.

5. Methods of viruses cultivation.

6. Classification of phages.

7. Biological properties of phages.

8. Replication of phages.

9. The types of interaction of phages and bacteria.

10. The notion about a prophage, lysogenic bacteria, phage conversion and its value.

11. The methods of receipt, study and titration of bacteriophages.

12. Practical usage of phenomenon of bacteriophagia: indication of causative agents, bacterial phagovar determining, reaction of phage titer increasing, therapy

13. The microflora of soil, water and air.

14. The sanitary-indicative microbes of soil, water and air.

15. The diseases transmitted by of soil, water and air

16. The methods to determine of microflora of soil, water and air.

Students should be able to:

1. To inoculate the cell cultures with virus-containing material.

2. To inoculate of chicken embryos into the allantoic cavity with virus-containing material.

3. To reveale cytopathic effect of viruses on cultures of cells.

4. To titrate of bacteriophages by Appelman’s technique.

5. To determine of bacterial phagovar.

6. Carry out the plating of water for determining of total number of microbes;

7. Determine of a coli-titer and coli-index of water using method of membrane filters;

8. Carry out the plating of the air using sedimentation method;

9. Determine a number of bacteria in 1 m3 of the air by using aspiration and sedimentation method.

The answers to the self – assessment

THEME: I. Morphology and biology of viruses. Basic methods of viruses cultivation. Methods of indication of the viruses

1. B. 2. A, b, c, d, e. 3. B. 4. A, b, c, d. 5. A, b, c. 6. A – capsid, nucleocapsid, envelope (supercapsid membrane, lipid-containing membrane); b – helical, cubic (icosahedral), combined; c – electron microscopy, ultracentrifugation, filtration; d – extracellular, intracellular.

1. A, c. 2. A, b, c, d. 3. Correctly. 4. A, b, c. 5. Incorrectly. 6. Correctly. 7. A, b. 8. For express identification of vibriones species. 9. For treatment and prophylaxis of staphylococcal infection. 10. … the nucleic acids…, … capsid…, … spermatozoal…, head, hollow core, sheath, which is ended by hexagon base plate with tail fibers. 11. C – reproduction, e – liberation of new phages. 12. Phage conversions. 13. The formation of little sterile spots testifies about presence of bacteriophages in the tested material. 14. A – the carriers are the scrub nurse S., surgeon P., junior sister Н. B – junior sister of postoperative ward, as the cultures of staphylococci isolated from her and from the patient had the same phagovar. В – to make the sanitation of staphylococci carriers, junior nurse have to be transferred in another department of the hospital; to carry out disinfection in the surgical department, to keep mask regimen, to carry out bactericidal ultraviolet irradiating of the air by special lamps.

III. Sanitary-bacteriologic studying of water, soil, air. Sanitary-indicative microorganisms. Microbiologic control in stomatologic facilities

1. A – S. faecalіs, E. colі, C. perfrіngens; 2. C E colі; 3. C – no less then 333 ml; 4. B – not more then 3; 5. A – S. haemolyticus, b – S. vіrіdans, c – S. aureus. 6. A. No, because only aspiration method for determination of quantitative composition of the air was used. B. It is necessary to use sedimentation or aspiration methods to determine the qualitative composition of microflora of the air. C. Maternity hospital: the number of microbes in the birth ward – 1500 1 m³; operation room: before the operation the total microbes number – 500, after the operation – near 2000, Staphylococcus aureus, Staphylococcus haemolyticus and haemolytic streptococci should be absent.

References:

1. . Review of Medical Microbiology /E. Jawetz, J. Melnick, E. A. Adelberg/ Lange Medical Publication, Los Altos, California, 1982. – P. 97-115, 322-356.

2 Ronald M. Atlas. Microbiology in our World, 1995, P. 30-32, 257-285.

3. . Textbook of microbiology R. Ananthanarayan, C. K. J. Paniker – 2002.

4Materials to prepare for workshops №04