programme of medical studies module basis of preclinical ... studies-2009-08... · programme of...

Programme of Medical studies

MODULE Basis of Preclinical Studies

Faculty of Medicine Kaunas University of medicine

Second year

Autumn semester

TABLE OF CONTENTS

1. General information..................................................................................................................... 4 2. General Content........................................................................................................................... 5 3. Aim of the Module ...................................................................................................................... 6 4. Departments, aims and objectives of the course.............................................................................. 7 5. Lectures ......................................................................................................................................... 14

5.1. Protein structure and functions. (2 hours) ............................................................................. 14 5.2. Enzyme catalysis and its mechanisms. Kinetics. (2 hours) ................................................... 14 5.3. Regulation of enzyme activity. Isoenzymes (2 hours). ............................................................ 14 5.4. Vitamins: biochemical functions and deficiency diseases. (2 hours). .................................... 14 5.5.Principles of metabolism in the human body. Degradation of carbohydrates in a cell under anaerobic and aerobic conditions. (2 hours) ................................................................................ 15 5.6. Metabolism of glycogen (2 hours). ......................................................................................... 15 5.7. Structure and functios of mitochondria. Mechanism of oxidative phosphorylation. (2 hours)....................................................................................................................................................... 15 5.8. Carbohydrate synthesis. Pentose phosphate pathway. Regulation of carbohydrate metabolism. (2 hours). ................................................................................................................... 16 5.9. Fatty acid catabolism and biosynthesis. Formation of ketone bodies. Regulation of fatty acid metabolism (2 hours). .................................................................................................................... 16 5.10. Metabolism of nucleotides and its disorders. Gout. Orotic aciduria. (2 hours) .................. 16 5.11. Principles of regulation of metabolism, molecular mechanisms of hormonal signal transduction(2 hours) .................................................................................................................... 17 5.12. Passive and active transport across the cell membranes. Epithelial transport and its mechanisms. (2 hours). .................................................................................................................. 17 5.13. General principles of pathology in preclinical studies. (2 hours)........................................ 17 5.14. Structural basis of homeostasis. Objects and methods of pathological anatomy study. (2 hours.)............................................................................................................................................ 18 5.15. Pharmacology, drugs, drug development and regulation, pharmacovigilance……………..18

5.16.Pharmacokonetics (2h)……………………………………………………………………………18

5.17. Pharmodynamics (2h) .......................................................................................................... 18 5.18. Classification and taxonomy of microorganisms. Structure of prokaryotes and viruses.(2 hours)............................................................................................................................................. 19 5.19. Basics of microorganisms’ heredity (2 hours). .................................................................... 19 5.20. Pathogenicity and virulence of microorganisms (2 hours). ................................................. 19

6. Practicals........................................................................................................................................ 21 6.1. Introduction to biochemical studies. Determination of protein amount in blood serum by biuret method. (3 hours) ................................................................................................................ 21 6.2. Precipitation of proteins (by heating, by organic and inorganic acids, by ethanol, by salts of heavy metals, by reagents of alkaloids. (3hours ) ......................................................................... 21 6.3. Specifity and thermolability of enzymes (3 hours.)................................................................. 21 6.4. Effect of pH on enzyme activity (3 hours)............................................................................... 22 6.5. Digestion of carbohydrates. Determination of activity of salivary α-amylase (3 hours)....... 22 6.6. Determination of the amount of ascorbic acid. (3 hours) ...................................................... 22 6.7. Determination of glucose concentration in blood serum. (3 hours)....................................... 23 6.8. Study of respiration of mitochondria (3 hours) ...................................................................... 23 6.9. Determination of calcium amount in blood serum. (3 hours.) ............................................... 23

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6.10. Epithelial ion transport (Computerized laboratory work) (3 hours)........................................ 24 6.11. Basis of structural homeostasis. Objects and methods of the study of pathological anatomy. (3 hours) ........................................................................................................................................ 24 6.12 Practicals in pharmacology. ................................................................................................. 25 6.13. Practicals in pharmacology. ................................................................................................ 25 6.14. Practicals in pharmacology. ................................................................................................ 25 6.15. Microscopic method of study in the diagnosis of infectious diseases. Morphology and structure of prokaryotic and eukaryotic microorganism. (2 hours) .............................................. 25 6.16. Bacteriological method. Cultivation of microorganisms. Obtaining of pure cultures. (2 hours)............................................................................................................................................. 34 6.17. Methods of identification of microorganisms, determination of resistance to antibiotics. (2 val.)................................................................................................................................................. 40 6.18. Immunological diagnosis. Immunological techiques, characteristics and forms. Agglutination and precipitation (2 hours)..................................................................................... 44 6.19. Immunological techniques in diagnosis of infectious diseases. Complement fixation test (CFT), enzyme immunoassay (EIA), immunofluorescence assay, and radioimmunoassay (RIA) and neutralization tests (2 hours)............................................................................................................ 47

7. Seminars ........................................................................................................................................ 52 7.1. Pharmacology seminar........................................................................................................... 52 7.2. Pharmacology seminar........................................................................................................... 52 7.3. Protein structure and properties. Kinetics of enzymatic catalysis and inhibition of enzymes. Solution of problems (2 hours.) ..................................................................................................... 53 7.4. Aerobic and anaerobic metabolism of carbohydrates. (2 hours) ........................................... 53 7.5. Analysis of metabolism of fats and fatty acid using the maps of metabolic pathways. Biochemical aspects of obesity. (2 hours). .................................................................................... 54 7.6. Signal transduction: second messengers, their formation, inactivation and significance in regulation of metabolism (2 hours.) .............................................................................................. 54 7.7. Humoral and cellular antimicrobial immune response. (2 hours)........................................ 55 7.8. Principles and methods of laboratory diagnosis of fungal and viral infections. Cultivation of viruses, determination of identity, immunologic and genetic methods (2 hours).......................... 56

8. Module exam questions ................................................................................................................. 58 8.1 Biochemistry ............................................................................................................................ 58 8.2 Physiology and pathological physiology................................................................................. 58 8.3. Pathological anatomy............................................................................................................. 58 8.4.Teoretical and Clinical Pharmacology. .................................................................................. 58 8.5.Microbiology ........................................................................................................................... 59

Appendix 1 ........................................................................................................................................ 60 Tables of computerized exercise results........................................................................................ 60

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1. General information

Supervisors of the module: professor L.Ivanovienė ([email protected]) and

associate professor A.Milašius ([email protected])

Departments

Physiology

Pathological anatomy

Theoretical and Clinical Pharmacology

Biochemistry

Microbiology

Subject matters and teachers in-charge

Physiology (doc. G.Civinskienė 366053 )

Pathological physiology (prof. A.Kondrotas; 327258)

Pathological anatomy (prof. R.Gailys; 327013)

Theoretical and Clinical Pharmacology (ass. prof. Milašius; 327242)

Biochemistry (prof. L.Ivanovienė; 327323)

Microbiology (ass.prof. D.Janulaityte-Guntter/ass.prof.R.Plančiūnienė;

327365)

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mailto:[email protected]

mailto:[email protected]

2. General Content 1. Protein structure and functions.

2. Enzymes and enzymatic reaction kinetics.

3. Regulation of enzyme activity.

4. Vitamins and their biological function.

5. Organization of human metabolism

6. Release of energy from nutrients and mechanisms of energy transformation.

7. Principles of metabolism regulation.

8. Mechanisms of transport of molecules and ions across membranes.

9. Common principles of pathological processes.

10. Principles of tissue homeostasis maintaining.

11. Pharmacology, drugs, drug development and regulation, pharmacovigilance

12. Pharmacokinetics and pharmacodynamics.

13. Classification, structure and pathogenicity of microorganisms.

14. Techniques used in microbiology.

15. Principles of immunology.

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3. Aim of the Module

The aim of this module is to help students to obtain theoretical knowledge and practical skills from

biochemistry, physiology, pathological physiology, pharmacology, pathological anatomy,

microbiology that are essential for study of other modules.

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4. Departments, aims and objectives of the course.

1. Department of Biochemistry The aim of the course

To obtain knowledge about relationships between protein structure and function (structural, motor,

transport and catalytic proteins), importance of enzymes to human tissue functioning, disorders of

enzyme functions and diagnostic significance. To understand how metabolism within a human body

is organized and controlled, and how mechanisms of signals transduction (at the human body, tissue

and cellular levels) work. To learn consequences of their disorders.

The objectives of the course

• Protein structure, classification, and functions

Subject– Biochemistry

Unit –Department of Biochemistry

References:

1. P. C, Champe, R.A. Harvey, D.R. Ferrier Biochemistry. Lippincott’s Illustrated

Reviews. Lippincott's Williams &Wilkins, 2005, p.13-43.

2. R.K. Murray, D.K.Granner, P.A. Mayes, V.W. Rodwell. Harper’s Biochemistry. 24th

edition. A Lange medical book. Prentice-Hall International, 1996, p.41-63.

Supplementary readings:

Devlin MD. Textbook of Biochemistry with Clinical Correlations. Wiley-Liss; 6th ed., 2006.

Useful Internet sites: http://www.indstate.edu/thcme/mwking/

• Peculiarities of enzyme structure, their properties, the mechanisms of enzymatic catalysis,

kinetics of enzymatic reactions, principles of enzyme activity control.



References:


Reviews. Lippincott’s Williams &Wilkins, 2005, p.59-68.




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http://www.indstate.edu/thcme/mwking/

Devlin MD, Textbook of Biochemistry with Clinical Correlations. Wiley-Liss; 6th ed., 2006


• Vitamins and their biochemical functions.


Unit – Department of Biochemistry

References:








• Organization of metabolism, peculiarities of the structure of high-energy compounds and

their significance for the processes of metabolism.



References:








http://www.biocarta.com/genes/Metabolism.asp

http://www.iubmb-nicholson.org/chart.html

• Metabolism of principal nutrients and nucleotides in human organism.



References:


Reviews. Lippincott’s Williams &Wilkins, 2005, p. 83-154; 171-198; 289-304.

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edition. A Lange medical book. Prentice-Hall International, 1996, p.135-235; .p.370-

385.






• Principles of metabolism regulation. Mechanisms of hormonal signal transduction.



References:


Reviews. Lippincott’s Williams &Wilkins, 2005, p. 92-94; 305-334.






http://www.biocarta.com/genes/CellSignaling.asp

2. Department of Physiology

The aim of the course

To obtain knowledge on passive and active transport across the membranes, epithelial transport and

its mechanisms, establishment of transepithelial potential, resting membrane potential and

equilibrium potential. To understand the common principles of pathological process and general

nosology, etiology and pathogenesis of diseases.

The objectives of the studies

• Passive and active transport. Epithelial transport. Transepithelial potential. Resting

membrane potential. Equilibrium potential.

Subject – Physiology

Unit – Department of Physiology

References:

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http://www.biocarta.com/genes/CellSignaling.asp

1. Ganong WF. Review of Medical Physiology. 22nd edition. Lange Medical Books, McGraw-

Hill, 2005, p. 7-8, 28-35.

2. Guyton AC, Hall JE. Textbook of Medical Physiology. 11th edition. Elsevier, Saunders,

2006, p.45-61.

• General principles of pathology in preclinical studies

Subject – Pathological physiology

References:

Copstead LE, Banaski JL. Pathophysiology. Elsevier, 2005, p. 2-23.

3. Clinic of Pathological Anatomy


To obtain knowledge relevant in understanding of principles of tissue homeostasis maintaining

The objectives of the studies

• The structural basis of homeostasis

Subject– Pathological anatomy

Unit – Clinic of Pathological anatomy

References:

Pathologic Basis of Disease / Eds I. L Robbins, R.S. Cotran. 7 th edition, 2005.

4. Department of Theoretical and Clinical Pharmacology


To obtain knowledge of principles of general pharbamology

The objectives of the course

• Drug research methods. Basis of pharmacology.

Subject– Theoretical and clinical pharmacology

Unit – Department of Theoretical and clinical pharmacology

• Major definitions in Pharmacology. Drug development and regulation, benefit-risk ratio.

• Routes of drug administration. Absorption, bioavailability, distribution, elimination. Linear and non-linear kinetics. Drug binding to plasma proteins.

• Targets of drug action. Major receptor families. Dose-response relationships. Agonists and antagonists.

References:

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1. Mysek MJ, Harvey RA, Champe PC. Lippincott‘s Illustrated Reviews:

Pharmacology. 4th ed. Philadelphia: Lippincott Williams & Wilkins; 2009. p. 1-34

2. Katzung BG. Basic and Clinical Pharmacology. 10th ed. Boston: McGraw-Hill;

2007. p. 1-73.

3. Trevor AG, Katzung BG, Masters CB. Katzung & Trevor’s Pharmacology:

Examination & Board Review. New York: Lange Medical Books / McGraw-Hill;

2005. p. 1-4

4. Rang HP, Dale MM, Ritter JM. Pharmacology. 5th ed. Edinburgh: Churchil

Livingstone; 2003. p. 3-127

5. Brunton LL. Goodman & Gilman’s Manual of Pharmacology and Therapeutics. New

York: McGraw-Hill Companies, Inc; 2008. p. 1-84.

6. Waller DG. Medical Pharmacology and Therapeutics. Edinburg: Harcourt Publishers

Limited; 2001. p. 3-78.

7. Mutschler E, Geisslinger G, Kroemer HK, Schafer-Korting M.

Arzneimittelwirkungen. Lehrbuch der Pharmakologie und Toxicology. 8 Aufl.

8. Stuttgart; 2001. S. 5-134.

5. Department of Microbiology


• Master knowledge about morphology and structure of microorganisms and their

relationships with pathogenicity.

• Master the diagnostic methods of microbiology and their significance in determining the

causative agents of infection.

• Master the techniques of aerobic and anaerobic bacteria cultivation and isolation of pure

bacterial culture.

Objectives of the course

• Learn to prepare the smears of bacterial cultures and stain them by simple and Gram

staining methods.

• Be able to evaluate the morphology of bacteria and their staining by Gram method

properties by light microscopy using immersion objective lens.

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• Be able to recognize acid-fast bacteria in the smears stained by Ziehl-Neelsen (acid-fast

stain) method.

• Be able to stain encapsulated microorganisms by Burri-Giemsa method and determine a

capsule by microscope.

• Be able to recognize the spores of bacteria by simple and complex staining methods in the

stained microorganisms.

• Learn to prepare the micropreparations from fungal colonies and be able to recognize the

structures of fungal morphology.

• Be able to inoculate test material into solid and liquid nutrition media, to determine the types

of single bacteria colonies.

• Acquaint with the growth of anaerobic bacterial cultures using Gas-Pack method.

• Master the methods of microorganism identity determination.

• Master the concepts of microorganisms: species, subspecies, serogroup, serotype, biotype,

phagotype, ecotype, and the methods of their determination.

• Master the determination of bacteria identity for epidemiologic purposes.

• Master the purposes and determination tests for microbial susceptibility to chemotherapeutic

agents.

• Be able to evaluate the antibiogram (disk-diffusion test).

• Master the mechanisms of the resistance of microorganisms to antibiotics and chemical

preparations.

• Master the criteria of antigenicity, the concepts of microorganism species, strains,

serogroups, serotypes, O, K, H antigens.

• Master the possibility of identification of microorganisms based on determination of specific

antigens.

• Master the methods of diagnostic immunology, their purposes, the phases of immunologic

reactions, variety of techniques, the essence of agglutination and precipitation tests.

• Master the classes of immunoglobulins, their structure and functions in the antimicrobic

immunity.

• Master the dynamics of antibody synthesis.

• Master antigens determination of microorganisms performing agglutination test.

• Immunologic diagnostic techniques, their purposes and essence.

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• Diagnosis by serology of infectious diseases, its purposes and evaluation of results.

• Complement fixation technique, its stages and practical use.

• Immunofluorescence technique: direct and indirect, its essence and practical use.

• Enzyme-linked immunosorbent assay (ELISA): types of competition and „sandwich”; their

essence and practical use.

• Immunoblot in serodiagnostics of infectious diseases; its essence.

• Radioimmunoassay (RIA): its essence and practical use.

References:

1. E. Jawetz, J.L. Melnick, E.A. Adelberg. Medical Microbiology. Twenty-Third Edition. Lange

medical Books. 2004. p. 8-70, 96-195, 367-381, 623-627.

2. P.R.Murray, K.S.Rosenthal, G.S.Kobayashi, M.A.Pfaller. Medical Microbiology. Fourth

edition. Morsby. 2002. p. 5-201, 429-457, 629-638.

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5. Lectures 5.1. Protein structure and functions. (2 hours)

Unit– Department of Biochemistry

In charge – professor L.Ivanovienė, professor V.Borutaitė

Description

Protein structure: primary, secondary, tertiary and quaternary. Relationship between protein

structure and function. Fibrous and globular proteins. Simple and conjugated proteins, their

prosthetic groups. Blood plasma proteins, methods of their identification, functions and diagnostic

significance. Catalytic proteins, peculiarities of their molecular structure. Active site, catalytic

amino acids.

5.2. Enzyme catalysis and its mechanisms. Kinetics. (2 hours)



Description

Evaluation of enzyme activity , enzyme units, velocity of enzymatic reaction and its dependence on

concentration of substrate, pH value, temperature and concentration of enzyme. Michaelis-Menten

equation, affinity of enzyme for substrate. Mechanism of enzymatic catalysis.

5.3. Regulation of enzyme activity. Isoenzymes (2 hours).



Description

Activators and inhibitors of enzymes. Types of inhibition. Reversible and irreversible enzyme

inhibition. Determination of type of inhibition. Suicide inactivators. Drugs as enzyme inhibitors.

Regulatory enzymes, mechanisms of activity control, significance of regulatory enzymes in control

of cell metabolism. Effect of hormones on activity of regulatory enzymes.

5.4. Vitamins: biochemical functions and deficiency diseases. (2 hours).



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Description

Classification and functions of vitamins. Vitamins as coenzymes: functions of coenzymes and

classification. Coenzymes of oxidoreductases, coenzymes of transferases, coenzymes of isomerases,

coenzymes of ligases and their respective vitamins. Symptoms of vitamin deficiency. .Biochemical

mechanism of action of water-soluble vitamins. Mechanisms of toxicity of water-insoluble

vitamins.

5.5. Principles of metabolism in the human body. Degradation of carbohydrates in a cell under anaerobic and aerobic conditions. (2 hours)



Description

The major carbohydrates of human organism. Stages of metabolism, ATP homeostasis. Pathways of

glucose 6-phosphate metabolism. Glycolysis – production of ATP under breackdown of glucose

molecule. Anaerobic glycolysis: energy value, physiological significance and mechanisms of

regulation. Activity of anaerobic glycolysis during muscle contraction and relaxation. Metabolism

of lactic acid. Cori cycle.

5.6. Metabolism of glycogen (2 hours).



Description

Relationship between structure of glycogen molecule and its metabolism. Mechanism of

degradation of glycogen and physiological significance. Regulation of glycogen breakdown –

regulatory cascades. Glycogen synthesis: localisation, substrates, enzymes, and mechanisms of

glycogen synthesis regulation. Glycogen storage diseases: causes and manifestations.

5.7. Structure and functios of mitochondria. Mechanism of oxidative phosphorylation. (2 hours)


In charge – associate professor R. Morkūnienė, professor V. Borutaitė

Description

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Processes taking place in mitochondria. Mitochondrial respiratory chain substrates and their origin.

Mechanisms of generation of electrochemical proton gradient in mitochondria. Mechanisms and

reguliation of oxidative phosphorylation. . Efficiency of oxidative phosphorylation. Mechanisms of

the respiratory chain inhibition and uncoupling.

5.8. Carbohydrate synthesis. Pentose phosphate pathway. Regulation of carbohydrate

metabolism. (2 hours).


In charge – associate professor R. Morkūnienė, professor V.Borutaitė

Description

Glucose biosynthesis (gluconeogenesis): substrates, differences and similiarities of gluconeogenesis

and glycolysis; enzyme localization, specific reactions of gluconeogenesis; mechanisms of

gluconeogenesis regulation – control of amounts of specific enzymes and their activity. Pentose

phosphate pathway, enzymes, regulation, biological significance. Fructose and galactose

metabolism and disorders.

5.9. Fatty acid catabolism and biosynthesis. Formation of ketone bodies. Regulation of fatty acid metabolism (2 hours).



Description

Enzymatic systems required for fatty acid oxidation: their localization and sequence of action.

Enzymatic system of beta-oxidation, its link to tricarboxylic acid cycle. Discrepancy between

activities beta-oxidation system and tricarboxylic acid cycle – synthesis of ketone bodies. Effect of

hormones on the synthesis of keton bodies, diagnostic significance of ketone bodies. Essential and

nonessential fatty acids. Principles of fatty acid biosynthesis, enzyme systems implicated.

Reciprocal regulation of fatty acid synthesis and fatty acid oxidation.

5.10. Metabolism of nucleotides and its disorders. Gout. Orotic aciduria. (2 hours)



Description

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Sources of substrates for purine nucleotide biosynthesis. IMP synthesis. AMP and GMP synthesis

from IMP. Regulation of purine nucleotide synthesis. Purine nucleotide synthesis from purine basis

(,,Salvage pathway“). Degradation of purine nucleotide. Gout, pathogenesis, drugs used for

treatment. Substrates of pyrimidine nucleotide synthesis. UMP synthesis. UTP and CTP synthesis.

Orotic aciduria. Degradation of pyrimidine nucleotide. Importance of nucleotide analogs for

medicine.

5.11. Principles of regulation of metabolism, molecular mechanisms of hormonal signal transduction(2 hours)


In charge – professor L. Ivanovienė, professor V.Borutaitė

Description

Coordination pathways of metabolic activity of cells and tissues in human organism. Nervous,

endocrine, paracrine and autocrine signal transduction. Hierarchy of regulation of hormone

secretion. Mechanisms of transduction of hormonal signal. Second messengers: their synthesis and

inactivation. Impact of second messengers to cell metabolism. Regulation of gene expression by

hormones and its molecular mechanisms.

5.12. Passive and active transport across the cell membranes. Epithelial transport and its mechanisms. (2 hours).


In charge – professor E.Kėvelaitis, associate professor R. Miliauskas

Description

Passive transport: transcellular and paracellular. Active transport: primary and secondary. Epithelial

transport: channels, carriers, and pumps. Functions of the epithelium. Transepithelial potential.

Resting membrane potential and equilibrium potential.

5.13. General principles of pathology in preclinical studies. (2 hours).


In charge – professor A. Kondrotas

Description

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Tasks of pathological and clinical physiology, general nosology, etiology and pathogenesis of

diseases. General etiology: risk factors of diseases, their causes and conditions, role in prevention.

General pathogenesis: mechanisms of the onset of diseases, course and outcome. Typical pathologic

process, basis of disease model.

5.14. Structural basis of homeostasis. Objects and methods of pathological anatomy study. (2 hours.) Unit – Clinic of Pathological Anatomy

In charge– professor R. Gailys

Description

Principles of morphological study on biopsy from living and deceased human body. Basic

morphological expressions of adaptive and compensating reactions: hypertrophy and hyperplasia,

atrophy, regeneration and organisation. Peculiarities of some tissue regeneration and wound

healing.

5.15. Pharmacology, drugs, drug development and regulation, pharmacovigilance (2h) Definitions of pharmacology and drugs (medicinal products). Drug development, drug regulation and its history. Drug benefit-risk ratio, adverse reactions, pharmacovigilance. Drug abuse.

Unit– Department of Theoretical and Clinical Pharmacology

In charge– associate professor A. Milašius

5.16. Pharmacokinetics (2h) Mechanisms of drug absorption and physical factors influencing absorption. Bioavailability, bioequivalence, and therapeutic equivalence. Drug distribution, steady-state, and drug binding to plasma proteins. Drug metabolism and excretion. Linear and non-linear kinetics. Unit– Department of Theoretical and Clinical Pharmacology


5.17. Pharmodynamics (2h) Targets of drug action. Ligands and receptors. Major receptor families. Dose-response relationships. Agonists, antagonists and allosteric modulators. Functional antagonism. Dose-kinetics-response relationships

Unit– Department of Theoretical and Clinical Pharmacology


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5.18. Classification and taxonomy of microorganisms. Structure of prokaryotes and viruses.(2 hours)

Unit– Department of Microbiology

In charge– associate professor D. Janulaitytė-Gunther, associate professor R. Plančūnienė

Description

Medical microbiology. Clinical microbiology: its objectives and abilities. The significance of

microorganisms in the living world, systematics and nomenclature. Principles of classification.

Conception of species as the key taxon. Differences between prokaryotes and eukaryotes. Structure

of prokaryotes: nucleus, cell wall, plasma membrane, capsule, flagella, and pili. Relationships of the

structure of prokaryotes with their pathogenicity. Sporogenesis of prokaryotes. Peculiarities of

structure of spirochaetes, rickettsiae, chlamydiae, mycoplasmas, actinomycetes.

Peculiarities of DNA and RNA viral reproduction. Classification of viruses. Origin and

nature of viruses. Virion structure: genome, capsid, envelope, their composition, and functions.

Differences between enveloped and naked viruses. Forms of virus-cell interaction. Autonomic and

integrated viral infections. Productive, abortive, persistent infections. Stadiums of reproduction.

Viral cytopathologic effects. Types of persistent viral infections.

5.19. Basics of microorganisms’ heredity (2 hours).


In charge – associate professor D. Janulaitytė-Gunther, associate professor R. Plančūnienė

Description

Variability forms of microorganisms. Mechanisms of genetic transfer and recombination:

transformation, transduction, and conjugation. Plasmids. Determinated transfer of plasmids.

Molecular diagnosis in identification, detection and epidemiology of microorganisms. Molecular

hybridization. Genetic probes. Polymerase and ligase chain reactions.

Mechanisms of genetic resistance to antimicrobic preparations: chromosome and plasmid

determinated resistance. Beta-lactamases. Methods of determination of resistance genes.

5.20. Pathogenicity and virulence of microorganisms (2 hours).



Description

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Significance of microorganisms for the infectious process. Infectious process and infectious disease.

The most important peculiarities of infectious diseases. Concept of epidemiology. The most

important mechanisms of exposure to infectious diseases. Concept of infection source and infection

reservoir. Factors of virulence of microorganisms: components of structure (adherence,

colonization), enzymes, and toxins. Endotoxins and exotoxins, the mechanisms of their action.

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6. Practicals 6.1. Introduction to biochemical studies. Determination of protein amount in blood serum by biuret method. (3 hours)


Aim: To determine the amount of protein by biuret method and be able to evaluate the significance

of protein amount in blood serum.

Objectives:

1. Introduce the objects of the course of Biochemistry.

2. Determine the amount of proteins in blood serum by biuret method.

3. Learn to calculate protein amount and express it in systemic and not systemic units.

4. Be able to discuss the significance of alterations of protein amount in blood serum.

References:

L.Ivanovienė, R.Morkūnienė, R.Banienė, L.Ivanovas, V.Borutaitė. Laboratory manual on

Biochemistry. Part I. KMU leidykla, Kaunas, 2005, p. 20-23.

6.2. Precipitation of proteins (by heating, by organic and inorganic acids, by ethanol, by salts of heavy metals, by reagents of alkaloids. (3hours )

Unit– Department of Biochemistry Aim: To understand protein physical-chemical properties and mechanisms of protein precipitation

and denaturation.

Objectives

1. Ascertain the mechanisms of reversible protein precipitation.

2. Understand the mechanisms of protein denaturation under exposure to different physical

and chemical factors.

3. Understand the significance of protein precipitation in medicine.

References:



6.3. Specifity and thermolability of enzymes (3 hours.)

Unit – Department of Biochemistry Aim: To evaluate specifity and thermolability of enzymes.

Objectives:

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1. Determine the specifity of salivary α-amylase and yeast sucrase.

2. Evaluate the effect of temperature on activity of salivary α-amylase and sucrase.

References: L.Ivanovienė, R.Morkūnienė, R.Banienė, L.Ivanovas, V.Borutaitė. Laboratory manual

on Biochemistry. Part I. KMU leidykla, Kaunas, 2005, p. 40-.

6.4. Effect of pH on enzyme activity (3 hours)

Unit – Department of Biochemistry Aim: To evaluate the dependance of enzyme activity on pH.

Objectives:

1. Determine the effect of pH on activity of salivary α-amylase.

2. Ascertain why enzyme activity depends on pH.


on Biochemistry. Part I. KMU leidykla, Kaunas, 2005, p. 38-40.

6.5. Digestion of carbohydrates. Determination of activity of salivary α-amylase (3 hours) Unit – Department of Biochemistry Aim: To evaluate effect of salivary α-amylase on dietary carbohydrates.

Tasks:

1. Determine the activity of salivary α-amylase.

2. Evaluate effect of activators and inhibitors on activiy of salivary α-amylase.

References:



6.6. Determination of the amount of ascorbic acid. (3 hours)

Unit– Department of Biochemistry Aims: To determine the amount of ascorbic acid in the specimen of one of biological fluids.

Objectives:

1. Determine the amount of ascorbic acid by 2,6-dichlorphenolindophenol.

2. Learn to calculate the amount of ascorbic acid in the specimen.

3. Discuss the biological meaning of results.

4. Know the role of ascorbic acid in enzymatic catalysis.


on Biochemistry. Part I. KMU leidykla, Kaunas, 2005, p. 59-61.

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6.7. Determination of glucose concentration in blood serum. (3 hours)

Unit – Department of Biochemistry Aim: To learn to determine and calculate glucose concentration in blood serum and interpret results

of the study.

Objectives:

1. Determine glucose concentration in blood serum by o-toluidine method and by using

CARDIO-Check analyser.

2. Calculate glucose concentration in blood and express it in systemic and nonsystemic units.

3. Evaluate alterations of glucose concentration and be able to intepret them.

4. Get acquainted with glucose tolerance test and its significance for identification of glucose

consumption disorders in organism.

References:



6.8. Study of respiration of mitochondria (3 hours)


Aim: To get acquainted with both the mechanism of oxidative phosphorylation and methods used in

its study.

Objectives:

1. Understand the principles of methods used in examination of mitochondria respiration.

2. Understand the mechanism of oxidative phosphorylation.

3. Discuss the principles of methods used to study the intensity of oxidative

phosphorylation.

References:

L.Ivanoviene, V.Borutaite Advanced practical course in biochemistry, Kaunas, 1993, p.3-8.

6.9. Determination of calcium amount in blood serum. (3 hours.)

Unit– Department of Biochemistry Aim: To determine calcium amount in blood serum.

Objectives:

1. To determine calcium amount in blood serum.

2. Learn to calculate calcium amount in specimen of blood serum.

3. Understand the role of calcium in the human body.

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4. Be able to evaluate the significance of changes of calcium amount in the body.


on Biochemistry. Part II. KMU leidykla, Kaunas, 2005, p. 56-59.

6.10. Epithelial ion transport (Computerized laboratory work) (3 hours)


Aim: To evaluate epithelial ion transport and its influencing factors.

Objectives:

1. To determine dependence of transepithelial potential (TEP) and short circuit current (SCC)

on Na+ concentration.

2. To determine dependence of TEP and SCC on temperature of solution.

3. To determine dependence of TEP and SCC on permeability of apical membrane to Na+ ions.

4. To determine dependence of TEP and SCC on activity of Na+-K+ pump.

Written report of this laboratory work is included in Appendix 1

6.11. Basis of structural homeostasis. Objects and methods of the study of pathological anatomy. (3 hours)

Unit – Clinic of pathological anatomy

Objectives: 1. Acquaint with the main categories of biopsies and their methods of study by means of

concrete study objects.

2. Observe the signs of biological death in the body of diseased and patholocical changes of

organs, found during autopsy.

3. Study the mechanisms of organisation development, morphological expressions and their

funcional significance of adaptive and compensation reactions – hypertrophy and

hyperplasia, atrophy, regeneration by means of presented macro- and micro preparations and

electronogramms.

Hypertrophia et hyperplasia mitochondriarum myocyti. Electronogramm (x21 000). Rat cardiac

muscle-adaptation to a long lasting exertion. Pay attention to the increased in size with exsessive

cristae mitochondrias (hypertrophy) and increase in their number (hyperplasia).

Regeneration epithelii bronchi (metaplasia).Histological slide (H+E). The normal columnar ciliated

epithelial cells are replaced by stratified squamous ones.

24

Cicatrix myocardii et hypertrophia myocytorum. Histological slide (H+E). Find the myocardium

postinfarction scar composed of the connective tissue. Pay attention to the myocytes in the vicinity

of the scar (increased nuclei an increase in cell size); evaluate the cind and type of regeneration.

Granulationes. Histological slide (H+E). Find plenty of thin wall capillaries and cells of young

connective tissue: fibroblasts, which are matturing to fibrocytes; also ther can be found monocytes,

plasma cells, lymphocytes, granulocytes and seldom giant cells.

References:

1. Pathologic Basis of Disease /Eds I.L. Robbins, R.S. Cotron. 7 th edition, 2005, p. 4-10.

2. Guide to laboratory practice in general pathological anatomy. R. Butkus, J. Čeponis, R.

Gailys, V. Lesauskaitė, Kaunas, 1991, p. 1-8.

6.12 Practicals in pharmacology.

Unit – Department of Theoretical and Clinical Pharmacology Prescription: structure and types. Rules of writing of prescriptions. Abbreviations in prescriptions. Solid drug forms. Propriatory names of medicines. Task- based writing of prescriptions. (3 hours)

6.13. Practicals in pharmacology.

Unit – Department of Theoretical and Clinical Pharmacology Soft drug forms. Ointments, pastes, suppositories. Specificity of their use, the peculiarities of writing of the prescriptions for these drug forms. Task- based prescription writing. (3val)

6.14. Practicals in pharmacology.

Unit – Department of Theoretical and Clinical Pharmacology Liquid drug forms: solutions, mixtures, suspensions, emulsions. Peculiarities of their use and prescription. Task- based prescription writing. (3 hours)

6.15. Microscopic method of study in the diagnosis of infectious diseases. Morphology and

structure of prokaryotic and eukaryotic microorganism. (2 hours)


Aims

Know the morphology, structure of different prokaryotic microorganisms, and functions of cell

structures.

Know the different causes of staining by Gram, Ziehl-Neelsen methods, the significance of these

staining methods for determination of the identity of microorganisms.

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Know the peculiarities of morphology and structure of eukaryotic microorganisms (fungi, protozoa)

and their methods of examination.

Know the differences between prokaryotes and eukaryotes.

Know the significance of microscopic examination for diagnosis of infectious diseases.

Objectives

Learn to prepare the smear from bacterial cultures and stain them by simple and Gram staining

methods.

Be able to evaluate the morphology of bacteria and their staining by Gram method using immerse

objective microscope.

Be able to recognize acid-fast bacteria in the smears stained by Ziehl-Neelsen method.

Be able to stain encapsulated microorganisms by Burri-Giemsa method and to determine a capsule

on microscopic examination.

Be able to recognize the spores of bacteria by simple and complex staining methods in the stained

smear of microorganisms.

Learn to prepare the smear from fungal colonies and be able to recognize the structures of fungal

morphology.

The following questions should be studied before laboratory work:

1. The equipment and structure of the bacteriological laboratory.

2. The instructions of work in the bacteriological laboratory.

3. Microscopic examination and its purposes.

4. Preparation of test smears and fixation. The aim and methods of fixation.

5. Simple staining methods of microorganisms.

6. The phase contrast, luminescent, dark field microscopy. Principles of taxonomy of

microorganisms: classification, nomenclature and identification.

7. The structure of prokaryotes: plasma membrane, genome, spores, capsule, flagella, pili,

plasmids, inclusions and other stuctural elements.

8. Bacterial cell wall: structure and functions.

9. Complex staining methods: the essence, indication of use.

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10. Gram staining method, its essence and significance for diagnosis of infectious diseases.

11. The peculiarities and differences of structure and chemical composition of gram-positive

and gram-negative bacteria. Inclusions of bacterial cytoplasm (volutin granules, glycogen,

lipids, pigment storages and the others), their biological significance and methods of

examination (staining by Neisser, Loeffler technique).

12. The bacterial plasma membrane: structure, functions. Differences between eukaryotic and

prokaryotic membrane of cytoplasm.

13. A capsule of prokaryotes: chemical composition, biological significance. Burri-Giemsa

method of staining.

14. Flagella and fimbriae (pili) of prokaryotes: structure and functions, methods of study.

15. Spores of bacteria: structure and chemical composition. Rate of sporogenesis. The

mechanism of the resistance of spores to environmental factors. Bacilli and clostridia. The

techniques of examinations.

16. The structure and morphology of spirochaetes, mycoplasmas, actinomycetes, chlamydiae

and rickettsiae.

17. Acid-fast bacteria: morphology, structure, and chemical composition.

18. Ziehl-Neelsen staining method and its essence. Groups of microorganisms-eukaryotes: the

peculiarities of their morphology and structure.

19. Essential differences of morphology and structure of fungi and protozoa.

20. The differences of prokaryotic and eukaryotic cell structure.

During laboratory the following is carried out:

1. A smear is prepared from mixture of gram-positive and gram-negative bacteria and stained

by Gram method. Morphology of the bacteria and the staining peculiarities are determined.

2. Acid-fast bacteria in the Ziehl-Neelsen stained smear of sputum are determined on

microscopic examination.

3. A smear of capsulated bacteria is prepared and stained by Burri-Giemsa method.

Morphology of bacteria and a capsule are determined.

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4. The smears of the cultures of sporulated bacteria are stained by simple and Gram methods

and examined under microscope. Morphology of bacteria and localization of spores are

evaluated.

5. A smear of Candida fungi is prepared and stained by simple staining methods, the properties

of morphology are determined.

6. Pseudomycellium and yeast forms of Candida fungi are demonstrated in the smear of

clinical specimen.

7. Micropreparation of the colonial prints of mould fungi grown in Sabouraud’s medium is

prepared. Type of hyphae and distribution of spores are determined.

8. Proteus mirabilis motility in „wet mount” micropreparation is demonstrated.

Questions for self-control: 1. What are the tasks of the microbiological laboratory?

2. Species of microorganisms. Subspecies. Variant (type). Populiation. Clon. Strain

3. Which microorganisms are called prokaryotes?

4. Forms of morphology of bacteria and their differences.

5. Polymorphism of bacteria.

6. Methods of examination of bacterial morphology. Microscopy methods.

7. Methods of bacterial smear preparation.

8. Purposes and methods of fixation of smears.

9. When are simple staining methods of bacteria applied?

10. Why is microscopy using immersion objective lens applied for examination of micropreparations?

11. How is immerse objective system used?

12. Phase contrast, dark field and luminiscent microscope.

13. What are classifications of microorganisms known and which is used presently?

14. How are the species names of microorganisms made up?

15. Spores of prokaryotes, their formation. What are the causes of resistance to environmental factors of spores?

16. How are sporogenic bacteria called?

17. Describe the process of sporogenesis.

18. How are sporogenic bacteria stained?

19. Which bacteria have a capsule? What is its function?

20. How is bacterial capsule determined?

21. How is bacteria grouped in relation to the number of flagella and their distribution?

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22. What is the mechanism of motility of bacteria?

23. What are bacterial fimbriae (pili)? What kind of pili do you know and what are their functions?

24. Do spirochetes belong to prokaryotes?

25. What are the peculiarities of structure of spirochetes, actinomycetes, mycoplasmas, chlamydiae and rickettsiae?

26. How do eukaryotes and prokaryotes differ in their structure?

27. What is the difference between genome of prokaryotes and eukaryotes?

28. Where is genetic information of bacteria?

29. Which structures of bacteria perform respiratory function?

30. Are the bacterial and human ribosomes of a cell the same?

31. Structure, chemical composition and functions of gram-positive and gram-negative bacterial cell wall?

32. What are L-forms of bacteria, filtered-form bacteria, protoplasts, and spheroplasts?

33. What is the essence of Gram staining and practical value?

34. How are acid-fast bacteria stained by Ziehl-Neelsen method and why?

35. Which microorganisms are called eukaryotes?

36. What is the difference between eukaryotes and prokaryotes?

37. What is the difference between fungi and protozoa?

38. How are fungi classified?

39. What function do fungal spores perform?

40. How are spores classified?

41. What are hyphae and myceiuml of fungi?

42. Which fungi are called moulds?

43. What is fungal dimorphism?

44. Is it possible to determine the class and species of fungi on microscopic examination?

45. Are there any fungi with multicellular organism?

46. How are micropreparations from clinical substance done and stained to determine protozoa?

47. Is it possible to determine the species of protozoa on microscopic examination?

INSTRUCTIONS OF WORK IN THE LABORATORY Students working in the laboratory have to wear a medical gown and a cap.

Consuming of food and drinks, smoking are prohibited.

All employees must work only in his/her work place, which is cleaned after work. Hands must be

washed with soup and treated by disinfectants if necessary.

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Used pipettes, cover slip and glass slide, gauze swab or /and cotton wool must be put in the utensil

with disinfectant.

Pincettes, bacteriological loops and needles used to prepare micropreparations or cultures before

and after preparation must be strerilized by the burner flame.

The used culture of microorganisms, the corpses of laboratory animals must be put into special

utensil.

If bacteria are accidentally spilt on the work place, hands or coats they must be treated by 3 per cent

solution of chloroamine.

METHODS OF FIXATION OF MICROPREPARATIONS (SMEARS) Physical – prepared and dried smear is exposed to the burner’s flame. The objective glass should

not be too hot because if the bacterial cell is exposed to high temperature (60–70°C) irreversible

changes of morphology occur.

Chemical – prepared and dried smear is immersed in the solution of fixation or some of it is poured

on the smear.

Prepared smears are treated:

1) Ethanol: 10—15 min. (ethyl alcohol);

2) Acetone: 5 min.;

3) Nikiforow’s mixture (equal volumes of ether and ethyl alcohol): for 10—15 min;

4) Methyl alcohol: 3 min.;

5) Fumes of osmic acid – for several seconds

6) Formalin solution for several seconds.

THE TECHNIQUE OF MICROSCOPY USING UN IMMERSION OBJECTIVE SYSTEM The diagram is opened, condenser lifted and the light is corrected to achieve a good field of vision

(OBJECTIVE LENS 8x). The light field of vision should have a disc form and be well illuminated.

In the beginning a smear is examined under microscope with objective lens 8x (10x), then by mean

of the oil immersion (90x, 100x) objective lens. Examining under the microscope with objective

lens 8x (10x), the distance between objective and preparation is aproximally 9 mm, with objective

lens 40x – 0,6mm and oil immersion 90x objective – 0,15 mm. On the preparation one drop of oil

(cedar oil) is dripped as its refractive index is close to that of glass (n ≈1,51). Objective 90x is

immersed in the oil.

30

By rotating of coarse focusing knob a good field of vision is achieved, by fine focusing knob – its

optimal focus. Several fields of vision are viewed until the testing objects are seen the best.

After work body tube of microscope is lifted, preparation is removed , light is switched off,

condenser is lowered , the objective lens 8x (10x) is put into working position. With cotton wool

immerse objective lens is cleaned and microscope is put back in its place.

METHODS OF STAINING Gram stain (proposed by H.C.Gram in 1884)

1. A fixed smear is covered with a slip of filter paper and exposed to aqueous solution of

gentianviolet. If the slip is impregnated with dyes some water is poured to wet it. In 1–2 min the

slip is taken and the remains of dyes are spilt.

2. Lugol’s solution is poured on the preparation. (for 1–2 min )

3. Lugol’s solution is spilt and the preparation is decolourised with ethanol 96° (30–60 sec) .

4. Then preparation is washed with water.

5. Preparation is stained with fuchsine or safranin solution (1–2 min.), then washed with water,

dried and examined by the light microscope using an oil immersion objective lens.

According to this staining test all microorganisms are classified into gram-positive or gram-

negative: gram-positive stain violet, gram-negative – red.

Ziehl-Neelsen stain. This test is used to stain acid-fast bacteria (infection agents of tuberculosis,

leprosy) as well as bacterial spores.

1. A fixed smear is covered with a slip of filter paper and carbol-fuchsine is poured on it. The

preparation is left for 3–5 min and heated over the flame until steam appears.

2. The filter paper is removed and the preparation washed with water.

3. The preparation is decolourised in 5% solution of sulfuric acid (immersed for 20–30 sec in the

acid or it is poured on the preparation) until the red colour is washed.

4. The preparation is washed with water and stained with aqueous-alcoholic solution of methylene

blue (3–5 min.)

5. The preparation is washed with water, dried and examined by the light microscope using an oil

immersion objective lens.

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Non acid-fast bacteria exposed to 5% solution of sulfuric acid are discolourised and after staining

with methylene blue stains blue. Acid-fast bacteria are stained red after exposure to carbolfuchsine

and remain resistant to decolourization. The other elements (leucocytes, fibrin, mucus) stain blue.

Romanovsky-Giemsa (azure II-eosin) stain. This method of staining is used to examine the cell

structure as well as morphology of spirochetes, protozoa, and rickettsiae.

1. A smear fixed chemically is immersed into glass with dye (2 drops of dye are mixed with 1 ml

distilled water) and left for 20 min.

2. The preparation is washed with water, dried and examined under microscope.

Burri technique

1. A drop of Indian-ink is dripped on the edge of glass slide then, next to the drop another drop of

distilled water is added. In the drop of water examining bacteria are placed by means of

bacteriological loop. Shortly both drops are mixed.

2. The second glass slide is pressed (angle 45°) to the first in the way that the drop spreads

uniformly. Then the second glass is slid uniformly spreading ink in a thin layer on the surface of

objective glass.

3. The preparation is dried and examined under microscope using an oil immersion objective lens.

Bacteria are seen unstained in a dark background.

Aujeskie stain. Used for staining of bacterial spores. As the envelope of spores is resistant to stain

before dyeing it is exposed to HCl solution.

1. Non- fixed dried smear is exposed to 0,5% HCl solution and heated until steam appears for 2–3

min.

2. Acid is spilt, the preparation washed with water, dried and fixed above the flame.

3. Stained with Ziehl-Neelsen technique.

Spores stain red, vegetative bacterial forms appear blue. Under microscope vision of field is blue.

Burri-Giemsa stain. Used for demonstration of bacterial capsule.

1. The smear is prepared by Burri technique, then dried and fixed (by alcohol and flame).

2. Stain with fuchsine (1–2 min).

3. Washed with water, dried and examined under microscope using an oil immersion objective lens.

Capsule is seen unstained around red bacterium in dark pink background.

32

Neisser stain. Used to stain microbial cell volutin inclusions.

1. Fixed smear is dyed with acetic-acid methylene blue for 2–3 min.

2. The smear is exposed to Lugol solution and left for 10–30 sec.

3. Then the smear is washed with water and dyed with vesuvin or chrisoidin for 30–60 sec.

4. The smear is washed, dried and examined under microscope.

Bacteris stain yellow, volutin granules appear dark blue (black).

„Wet mount”and „hanging” drop preparation.

„Wet mount” drop preparation. If culture is grown in solid nutrition media a drop of water or

sodium chloride isotonic solution is placed on the glass slide. Then test material is placed in

solution by means of bacteriological loop and homogenic suspension is prepared. The drop is

covered with cover slip in a way to prevent air bubbles. Fluid should not flow over the edges of a

cover slip. Bacterial cultures grown in liquid nutrition media are taken by pipette or loop and

dripped on the glass slide.

„Hanging ”drop preparation. Preparation is prepared similarly as “Wet mount” but in this case the

drop of culture is dripped on the cover slip, which is inverted and placed on the glass slide with a

well. The edges of the well are ringed with petrolatum. Then the cover slip is pressed to the glass

slide firmly that leads to the creation of hermetic chamber, which prevents the drop from drying. If

done properly, the drop “hangs” without touching the edges and the bottom of the well.

Both preparations are examined under microscope with objective lenses 8x, 10x and 40x

without oil. Condenser is lowered and diaphragm is partially closed. Immersion objectives system

can be used in examination too. In most cases phase-contrast or dark-field microscopy are used.

“Thin” and “Thick” blood smear preparations:

“Thin” blood smear. On the edge of glass slide a drop of blood is dripped. The drop is fixed with

another glass slide by 45° angle. The second glass slide is slid towards the first one to spread blood

uniformly. Dried smear is fixed with chemical solutions.

“Thick” blood smear. On the glass slide 2–3 drops of blood are dripped and mixed. The smear is

dried and distilled water (2-3 drops) are dripped to produce haemolysis of erythrocytes and left for

10–15 min. The preparation is dried and stained by Romanosky-Giemsa method.

Attention. The smears are dried at room temperature.

33

1 topic. Microscopic method for diagnostic of infectious diseases. Morphology and structure of

prokaryotes and eukaryotes.

References:

1. E. Jawetz, J.L. Melnick, E.A. Adelberg. Medical Microbiology. Twenty-Third Edition. Lange medical Books. 2004. p. 8-42, 623-626.

2. Patrick R. Murray, Ken S. Rosenthal, George S. Kobayashi, Michael A. Pfaller. Medical

Microbiology, Fourth Edition, 2002, p. 7-24, 67-70, 157-161.

6.16. Bacteriological method. Cultivation of microorganisms. Obtaining of pure cultures. (2 hours).


Aims 1. Obtain knowledge of essence and scheme of bacteriological examination.

2. Obtain knowledge of methods of aerobic and anaerobic bacteria cultivation and isolation of pure

bacterial cultures.

Objectives 1. Be able to inoculate test material into solid and liquid nutrient media, to determine the types of

single bacterial colonies.

2. Acquaint with the growth of anaerobic bacteria using “Gas-Pack” system.


1. Mechanisms of microorganism’s nutrition.

2. Types and character of bacterial multiplication in solid and liquid nutrition media.

3. Phases of bacterial population growth in liquid nutrient media.

4. Purpose of nutrition media, types and principles of creation.

5. Principles of cultivation of microorganisms.

6. Types of bacterial respiration, the essence of respiration.

7. Mechanisms of respiration of aerobic and anaerobic organisms.

8. Bacteriological examination: objectives and stages.

9. Pure culture of microorganisms, the purposes and methods of obtaining.

10. Principles and methods of isolations of aerobic microorganisms in pure culture.

34

11. Colonies of microorganisms, their morphology and diversity.

12. Pigments of bacteria, types and functions.

During laboratory the following is carried out:

1 task. Isolation of pure cultures by the method of single (isolated) colonies.

The first day of examination:

The test material smear is prepared and stained by Gram method; diversity of bacterial forms,

distribution and staining is determined.

In order to get isolated colonies the test material is inoculated onto meat-peptone agar (MPA) in the

Petri dish. The dish is placed in the incubator for 24 hours at 37oC .

The second day of examination: the records of an earlier prepared test material are studied.

Two different colonies are chosen (No.1 and No. 2).

The smears are prepared from the microorganisms grown in these colonies.

The smear is stained by Gram method.

The smears are examined under light microscope, bacterial morphology and staining properties are

determined.

The properties of colonies are studied and evaluated. The properties are transferred into Table 1.

To get pure culture colonies No.1 and No. 2 are inoculated into the slant agar.

The third day. The cultures on the slant agar (Nr.1 ir Nr.2) are examined under microscope. The

growth (pigment, the multiply along the loop direction, etc.) is evaluated. To evaluate biochemical

activity cultures are inoculated into “colour row”.

35

Table 1. Properties of bacterial colonies.

Colonies Properties

No.1 No.2

Macroscopy(examination by naked eye):

Form of colony: regular or irregularly rounded;

Dome, convex, flat form;

Surface: brilliance or mat;

Size: in mm;

Solour: colony is grey or having pigment;

Transparency: transparent, semitransparent, no transparent.

Microscopy (colony is examined under microscope with objective lens 8x):

Nature of the edge of colony: smooth, curve;

Structure: homogenous, granulated, filamentous

During preparation of smear the following is determined:

Consistency of colony: soft, mucous, solid;

Emulgation of bacteria with saline

(homogenous suspension or flakes)

The following is evaluated by microscopy of the smear:

Bacterial form, distribution;

staining properties (Gram stain).

2 task. The scheme of isolation of pure cultures of anaerobes and microaerophils is studied

and described. To find anaerobes or microaerophiles a test inoculums is taken with special swabs

or syringes, put into transport media and in the laboratory are inoculated into solid (or liquid)

nutrition medium. The cultures are cultivated in jars (with candle or special “Gas-Pack” chemical

substances) or carbon dioxide incubators. The colonies grown from these microorganisms are

examined under microscope; cultures are also prepared to reproduce microorganisms and later their

biochemical activity is determined.

36

If microorganisms catabolize fatty acids and release some volatile substances, these

substances can be identified by gas chromatography.

3 task. Determination of types of different bacterial species growth (types of colonies, haemolysis,

and biochemical activity) in the various nutrition media: blood agar, differential and selective

media.

The following questions should be studied before laboratory work: 1. What are the mechanisms of bacteria nutrition?

2. What are the types of nutrition of microorganisms?

3. Which type of nutrition do pathogenic microorganisms belong to?

4. Who determined anaerobiosis? When?

5. What are the differences between aerobic and anaerobic respiration?

6. What are facultative and obligate anaerobes? What are microaerophiles?

7. Why does molecylar oxygen block the existence of anaerobes?

8. In which nutrition media are anaerobs cultivated? Under what conditions?

9. What is the purpose of liquid and solid nutrition media?

10. What are selective, differential, selective and preserve nutrition media?

11. What are the growth factors of microorganisms? Do these factors are necessary for all bacteria reproduction?

12. How do bacteria multiply in nutrient media?

13. What are the phases of bacteria population growth in the liquid nutrition media?What is the significance of these phases?

14. What are clone, colony, culture, biotype, population?

15. Why is pure culture of microorganisms isolated?

16. How is pure cultures of aerobs isolated?

17. What purposes and stages of bacteriological examination do you know?

18. How long does the bacteriological examination last? Is it possible to shorten this examination? If yes, how?

19. Who was the first to use solid nutrition media? Why?

20. Describe morphology of bacterial colony. What are the main forms of bacteria? What is the character of these forms?

21. What pigments do bacteria synthesize? What are their functions?

ISOLATION OF PURE CULTURE OF MICROORGANISMS

Pure culture of microorganisms is the population of single species cells grown in the sterile

nutrient media.

37

Colony is circumscribed, visible by naked eye aggregation of microorganisms grown from single

cell in the solid media.

Isolation of pure culture of microorganisms from test material often results in a single

colony. 1. Methods of isolated colonies. Inoculation takes place slowly by dilution of

microorganisms.

Agar nutrition media in the Petri dish (Fig. 1) is divided into 4 segments (the lines are marked on

the bottom of the outside dish by special pencil). The test substance is inoculated by bacteriological

loop producing streaks: the first inoculation is streaked in the 1st segment, then without burning

loop – 2nd, later – 3rd and then – 4th segment (the distance between lines - 5 mm). In most cases

single bacteria colonies are grown in the 3rd or 4th segment.

Test substance is inoculated by a loop from the edge of Petri dish: 3–5 streaks are produced

(Fig. 2). Microorganisms are inoculated on the traces of streaks by the burn and cooled loop: 3–5

perpendicular lines of cultures to the streaks of previous cultures are made. The action is repeated

for the third and then for the fourth time.

1 2

4 3

Figure 1. Result of single colonies by method of segments

Figure 2. Result of single colonies by method of streaks

2. Filtration. This method may be used when there are not many microorganisms in test substance.

A test substance is filtrated through special with small pores membrane filters, which trap bacteria.

Then the filter is placed on differential or selective media; microorganisms grow on the surface of

38

the filter. In other cases a test inoculum is placed on the filter, which is permeable to small motile

bacteria (campylobacter) but the others are trapped. After some time the filter is removed.

3. Bacteriostatic (inhibition) method. Some chemical substances and antibiotics inhibit the

multiply of microorganisms. For instance, penicillin and streptomycin are used to inhibit bacteria by

cultivation with solution (5%) of sulfuric acid for non acid-fast bacteria in test material.

4. Method of enrichment. Test material is inoculated into selective media, where only single

species bacteria grow, whereas the other species are inhibited.

5. Biological method. Laboratory animals that are the most susceptible to a certain microorganism

are infected by test substance. With the manifestation of disease the specimen from damaged organs

and tissues are inoculated into nutrition media. This results in growth of single species of bacteria.

The pure culture of bacteria is identified.

METHODS OF ANAEROBES AND MICROAEROPHILES CULTIVATION

Anaerobs are cultivated only under anaerobic conditions, that is, by decreasing oxidation-reduction

potential in media.

Microaerophiles as well as oxygen-tolerant anaerobes are cultivated when the concentration of

oxygen is decreased. Such conditions are acheved by physical, chemical and biological methods.

Physical methods.

1. Specimens are inoculated into oxidation-reduction substance-rich media (with blood, glucose,

lactose, thioglycollate ).

2. Specimens are inoculated by injection into the depth of solid media.

3. The air is pumped or pushed by inert gas from the utensils (special jars, anaerobic incubators),

where microorganisms are cultivated.

4. Media with cultures are placed into anaerobic chamber, where air is removed using vacuum

pump. Air is often replaced by indifferent gas (nitrogen, hydrogen, carbon dioxide or special “Gas-

Pack” mixtures).

Chemical methods. Packets with special chemical substances (e.g. „Gas-Pack” mixtures) are

housed in a hermetically sealed utensils. Sets of packets to cultivate microaerophiles, obligate

anaerobs and aerotolerant anaerobes are made. Chemical reactions taking place in the sets remove

39

atmospheric oxygen and produce gas, which has a required concentration (e.g. CO2) In other cases,

pyrogallol or sodium hydrosulfite are placed in a jar or a candle is lit.

Biological methods. Agar in Petri dish is divided into two parts: in the first part aerobes (e.g.

Staphylococcus aureus or Serratia marcescens) are inoculated, in the second – anaerobes. The

content of Petri dish is hermetically closed: the edges are sealed with plasticine (adhesive tape) or

parafin ; the cultures are housed in a thermostat. Firstly, due to oxygen aerobes multiply, then in 3–

4 days –after consumption of oxygen - anaerobes. The layer of agar should be enough thick then

less air will be in the dish.

Mixed methods. Two or three above mentioned methods are used.

References:

1. E. Jawetz, J.L. Melnick, E.A. Adelberg. Medical Microbiology. Twenty-Third Edition. Lange medical Books. 2004. p. 62-70. 2. Patrick R. Murray, Ken S. Rosenthal, George S. Kobayashi, Michael A. Pfaller. Medical Microbiology, Fourth Edition, 2002, p. 25-34, 195-201.

6.17. Methods of identification of microorganisms, determination of resistance to antibiotics. (2 val.) Unit – Department of Microbiology

Objectives:

1. Master the methods of determination of microorganism identity.

2. Master conceptions of microorganisms: species, subspecies, strains, serogroup, serotype,

biotype, phagetype, ecotype and methods of determination of their identity.

3. Master determination of bacterial identity for epidemiological purposes.

4. Master the purposes and methods of determination of antibiogram (disk-diffusion test for

determining the activity of antimicrobials).

5. Be able evaluate antibiogram.

6. Master the mechanisms of resistance of microorganisms to antibiotics and chemical

preparations.

The following questions are sudied before the laboratory work:

1. Biochemical activity of microorganisms – the property of genus, species, biotype.

2. Methods of determination of biochemical activity: differential - diagnostic media, “color

row”, half-automatic and automatic systems.

3. Identification of microorganisms based on antigen structure.

40

4. Identification of microorganisms based on determination of nucleic acid individual

sequence.

5. Epidemiological markers to determine hospital strains of bacteria.

6. Mechanisms of antagonism of microorganisms and their biological significance.

7. Antimicrobial chemotherapeutical preparations.

8. Antibiotics. Classifikacion based on pharmacodynamics, targets in microorganisms.

9. Antibiogram, methods of determination, significance. Determination of minimal inhibition

concentrations (MIC).

10. Resistance of microorganisms to antibiotics. Mechanisms of resistance. Chromosomal and

plasmid acquired resistance. Determination of immunogens and enzymes (beta-lactamases).

During the laboratory the following is carried out: 1 task. Isolation of pure cultures using the method of isolated colonies (continued).

Determination of identity of microorganisms based on their biochemical activity.

Grown bacterial cultures No.1 and No.2 are inoculated into media, which possess certain substances

and indicators.

Biochemical activity is evaluated by determining changes in colour of media. Data on bacteria are

filled in the Table 1.

Table 1. Evaluation of morphological properties and biochemical activity of bacteria

Bacterial cultures Bacterial properties No. 1 No. 2

Morphology Endospores: + or – Gram stain Pigmentation The form of colonies (S, R)

Glucose Sucrose Mannitol Indol Urease Inosid Phenylalanine Lysine H2S (hydrogen sulfide)

B

ioch

emic

al a

ctiv

ity o

f en

zym

es

Lactose Note: Glucose, sucrose, mannitol, and lactose: yellow colour – “+”, blue colour – “– ”; Indol, urease, and lysine: red colour – “+”, yellow colour – “– ”; Phenylalanine: green colour – “+”, yellow colour – “– ”;

41

H2S: black colour – “+”, colourless – “– ”; Inosid: yellow colour – “+”, green colour – “– ”. 2 task. Determination of microorganism sensitivity to antibiotics by disk-diffusion method

(Kirby-Bauer test).

The diameter of clear zone surrounding the disk, where the growth of microorganisms was

inhibited, is measured and using special tables degree of sensitivity of bacteria to antribiotics is

determined.

Data are filled in table 2.

Table 2 . Resistance of microorganisms to antibiotics.

Resistance Test bacterial culture

Antibiotics

Diameter of zone of growth

inhibition, mm

Sensitive Moderate resistant

Resistant

1. 2. 3. 4. 5. 6.

Conclusion 3 task. Determination of minimal inhibitory concentration (MIC) of antibiotics.

An antibiotic is diluted and poured into liquid media afterwards test bacterial cultures (standartized

suspensions) are inoculated into every tube. The results are evaluated after 24 hours of incubation in

a thermostat. Bacteria are sensitive to the concentration of antibiotic if the media in tube remains

transparent. In this way the minimal inhibitory concentration (µg/ml), which inhibits the growth of

bacteria, is determined.

The scheme of the study is presented in Table 3.

Table 3. Determination of the minimal inhibitory concentration (MIC) of antibiotics In vitro Components

1 2 3 4 5 6 7 8 Nutrition media, ml 2 2 2 2 2 2 2 2 Concentration of antibiotic in media, µg/ml

50,0

25,0

12,5

6,25

3,12

1,56

0,78

–

Test culture, ml 0,1 0,1 0,1 0,1 0,1 0,1 0,1 0,1 Results Conclusion

4 task. Determination of concentration of antibiotics in blood serum. The test is caried out in

vitro arranged in the two rows. In the first row of in vitro antibiotic undergoes a series of dilutions

42

in liquid media. In the second row - patient’s blood serum. Then in every vitro the test culture,

which is the standard to determine resistance of antibiotics (S. aureus – for beta-lactamic

antibiotics, E. coli – aminoglycosides), is inoculated. The cultures are incubated for 24 hours at

37°C .

The growth of bacteria is indicated by turbidity in the media. The concentration of antibiotic

is calculated by multiplying the MIC (from the first row) by the highest dilution in blood serum, in

which the growth of test culture is still being blocked. For example, the MIC (from the data of the

first row) is 6,25 µg/ml, the highest dilution of blood serum (test culture is being inhibited) - 1:640

– the concentration of antibiotic in the patient’s blood serum is 640 x 6,25 = 4000 µg/ml.

Calculate the concentration of antibiotic in a patient’s blood serum on the basis of the data of

demonstrated test.

5 task. Determination of β-lactamase of microorganisms.

Synthesis of β-lactamase of bacteria is controlled by R-plasmids. These lactamase are determined

by several methods.

Evaluate the data of demonstrated test.

Self-learning 1. What are the purposes of the study of bacterial enzyme activity?

2. Which properties of enzyme of microorganisms are more frequently determined?

3. How is the carbohydrate-degrading property of bacteria studied? What end products of this process are known?

4. How are proteins, amino acids and urea degradation determined?

5. How is catalase and cytochrome oxidase activity of bacterial determined?

6. Which microorganisms are auxotrophs and prototrophs?

7. How are volatile and aromatic substances synthesized by microorganisms determined?

8. The automatic systems to determine enzyme activity of microorganisms.

9. What are probiotics and prebiotics?

10. Who was a pioneer of chemotherapy?

11. What is the chemotherapeutical index?

12. What are antibiotics?

13. Who discovered antibiotics? When?

14. How are antibiotics classified?

15. The activity of antibiotics against microorganisms.

43

16. What is the mechanism of antibiotic exposure to a microbic cell?

17. Which factors promote the development of antibiotic-resistant microorganisms?

18. What methods are used to determine sensitivity of microorganisms to antibiotics? What is antibiogram?

19. Which method to determine sensitivity of microorganisms to antibiotics is the most accurate?

20. How quantitatively is the sensitivity of microorganisms determined by the disk-diffusion method?

21. How are the minimum inhibitory concentrations are determined (MIC), MIC50, MIC90?

22. How do you understand the natural resistance of microorganisms to antibiotic?

23. What are the genetic mechanisms of acquired resistance of microorganisms to antibiotics?

24. What causes disbacteriosis? Prophylaxis.

25. What do know about R-plasmids controlling products?

26. The key role of antibiotics in spreading of resistant microorganisms populations.

27. Why is the prevalence of R-plasmid controlling resistance to antibiotics higher if compared to that of chromosomal?

References:

1. E. Jawetz, J.L. Melnick, E.A. Adelberg. Medical Microbiology. Twenty-Third Edition. Lange medical Books. 2004. p. 42-51, 161-195. 2. Patrick R. Murray, Ken S. Rosenthal, George S. Kobayashi, Michael A. Pfaller. Medical Microbiology, Fourth Edition, 2002, p. 25-34, 185-194.

6.18. Immunological diagnosis. Immunological techiques, characteristics and forms. Agglutination and precipitation (2 hours).


Objectives: 1. Master the criteria of antigen; the concept of antigen of genus, species, serogroups,

serotypes, and O, K, H antigens of microorganism.

2. Master the possibilities of microorganism identification based on detection of specific antigens.

3. Master immunological techniques, their purposes, the phases of immune reactions, variety of tests, the essence of agglutination and precipitation.

4. Master immunoglobulin classes, structures and functions in antimicrobial immunity.

5. Master the dynamics of antibodies synthesis.

6. Master to detect antigens of microorganisms by the agglutination test.

The following questions should be studied before laboratory work: 1. Acquired humoral immunity.

2. Antigens: criteria of antigen, complete antigens and haptens.

44

3. The antigenic determinants or epitopes.

4. The antigens of microorganisms: their location and functions.

5. The heterologous antigens of microorganisms.

6. Superantigens. Protective antigens.

7. The izotype, allotype and idiotype of immunoglobulins.

8. The structure and functions of immunoglobulin in antimicrobial immunity.

9. The dynamics of the synthesis of immunoglobulins during primary and secondary response.

10. The immuno-diagnostics of infectious diseases: serology, allergodiagnosis and detection of

antigens.

11. The mechanisms of agglutination and precipitation tests, modifications of the tests, practical

use.

During the laboratory the following is carried out:

1 task. Determination of microorganism antigens by agglutination test. Agglutination test using X culture, which was isolated from a patient and grown in slant agar, is

carried out on slide glass. Drops of „A” and „B” serum (or imuunoglobulins solution) are dripped

by means of sterile loop (or pipette) on the glass. Then by means of loop X culture is added and

mixed with each serum („A” ir „B”). During agglutination granules are made up (appear).

After evaluation of the test results answer the following questions:

1. Why was agglutinations test carried out and how should its results be evaluated?

2. Which method of diagnostic was applied?

2 tasks. Evaluation of direct agglutination test.

Agglutination test with a patient’s blood serum and Salmonella enterica Typhi serotype O

antigen diagnosticum based on the following scheme (Tab.1) is carried out.

Table1. Scheme of direct agglutination test.

In vitro Controls

Components, ml

1

2

3

4

5

6

Seru

m

Ant

igen

Isotonic NaCl solution 1,0 1,0 1,0 1,0 1,0 1,0 – 1,0Patient’s blood serum, (1:25)

1,0 → → → → → 1,0 –

Dilutions 1:50 1:100 1:200 1:400 1:800 1:1600

1:25 –

O diagnosticum 0,1 0,1 0,1 0,1 0,1 0,1 – 0,1Test data

Data are evaluated after 24 hours of incubation and exposure at 37°C temperatures.

45

The following questions should be answered:

1. Which diagnostic method was used?

2. What was in patient’s blood serum looked for?

3. Was the titer of antibodies determined?

4. Was the class of immunoglobulins determined?

3 task. Indirect latex agglutination test using ‘’pair blood serum’’.

Blood serum of a patient suspected having pseudotuberculosis, is examined on the 5th and

10th day after revealing of symptoms. Sepecomens of patients blood serum are diluted with (from

1:5 to 1:160) isotonic NaCl solution in tubes then in every tube Yersinia pseudotuberculosis

antigens, which are adsorbed on latex particles, are dripped. In antigenic control tube - latex

particles in isotonic solution.

Evaluate test data and answer the following questions:

1. Which diagnostic method was applied?

2. What was in blood serum looked for?

3. Why are Y. pseudotuberculosis antigens adsorbed on latex particles?

4. Why is patient’s blood serum examined twice?

5. Can the diagnosis be confirmed by test results?

4 task. Coagglutination test using antibody diagnosticum of staphylococcus.

The drop of antibody diagnosticum is mixed with secreted pure culture on the slide glass. The

test is positive if granules, flakes, appear. Perform the test. ( Tab.2):

Table 2. Coaggliutination test scheme. Test substance Diagnosticum

Pure culture of gram-negative bacteria secreted from an organism (Haemophilus influenzae ?)

Staphylococcus with adsorbed immunoglobulins against H. influenzae.

Test data Evaluate test data and answer the following questions:


2. Is this test immunological?

3. Your comments on data.

5 task. Precipitation immunodiffusion test in gel to determine toxigenicity of Corynebacterium

diphtheriae.

46

A strip of filter paper soaked with antitoxic serum (antidiphtherial serum) is placed on the

surface of nutrition media on Petri dish. Then cultures of test Corynebacterium diphtheriae and

control toxygenic strain are perpendicularly streaked into the strip. The dish is incubated for 24—28

hours at 37°C. Precipitin lines are formed when bacterial toxins are excreted.

Evaluate the test data and answer the following questions:


2. Is this test immunological?

3. Which type of toxin is determined by the test (exotoxin, endotoxin)?

Self-learning

1. What is acquired immunity?

2. What are the mechanisms of acquired immunity?

3. Classes of immunoglobulins, their structure and functions in antimicrobial immunity.

4. What are immunoglobulin allotype, idiotype, isotype?

5. What are Fc and Fab immunoglobulin fragments? What are their functions?

6. Which antibodies are called monoclonal?

7. The dynamics of antibody synthesis, its significance in serological diagnosis, vaccination?

8. What are the methods of immunological diagnosis?

9. What is serology?

10. What reactions are called immuno-? Immunological? Serological?

11. Phases of serological reactions.

12. Peculiarities of agglutination test. Agglutinins.

13. Cases of agglutination tests. Direct and indirect agglutination.

14. What is coagglutination test?

15. Agglutinating serums; monoreceptors (adsorbed )serums?

16. Precipitation test, its modifications. Practical value of precipitation.

References:

1. E. Jawetz, J.L. Melnick, E.A. Adelberg. Medical Microbiology. Twenty-Third Edition. Lange medical Books. 2004. p.119-146.

2. Patrick R. Murray, Ken S. Rosenthal, George S. Kobayashi, Michael A. Pfaller. Medical Microbiology, 4 Edition, 2002, p. 91-146.

6.19. Immunological techniques in diagnosis of infectious diseases. Complement fixation test

(CFT), enzyme immunoassay (EIA), immunofluorescence assay, and radioimmunoassay (RIA) and

neutralization tests (2 hours).

Unit – Department of Microbiology

47

Aims:

1. Master the practical use of immunological techniques, which are performed using labeled

components.

2. Be able to explain the essence of immunological techniques, which are carried out using

antibodies or antigens labelled by fluorochrome, enzyme and radioisotope.

Objectives of the laboratory work:

1. Be able evaluate immunological techniques, using labeled antibodies and immunoglobulins in

order to detect antigens of microorganisms in test material.

2. Be able to evaluate immunological techniques using labeled antibodies and antigens in order to

detect immunoglobulin isotypes in blood serum.

3. Master theoretical aspects and practical value of immunoblotting.


1. Immunological diagnostic techniques, their purposes and essence.

2. Serology of infectious processes, purposes and evaluation of results.

3. Complement fixation test: stadiums and practical use.

4. Immunofluorescence: direct, indirect, the essence and practical use.

5. Enzyme-linked immunosorbent assay (ELISA): competition and „sandwich” types, the essence,

practical use.

6. Immunoblot in serology of infectious diseases, the essence.

7. Radioimmunoassay (RIA): the essence and practical use.

During the laboratory works the following is carried out:

1 task. Complement fixation test.

Patients (A and K) suspected having mycoplasmic infection were subjected to examination.

Examination of their blood serum was carried out using complement fixation test based on the

following scheme. (Table1)

48

Table1. Complement fixation scheme. Tube number Components

1 2 3 Patient’s serum diluted 1:5, ml 0,5 0,5 – Mycoplasma pneumoniae antigen, ml 0,5 – 0,5 Isotonic NaCl solution, ml – 0,5 0,5 Complement in work concentration, ml 0,5 0,5 0,5

Incubation for 30 min at 37°C Haemolytic system, ml 1 1 1

Incubation for 30 min at 37°C Test results

Evaluate test results ( „+” - haemolysis; „–” – haemolysis absent) and answer the following

questions:

1. Which metod of diagnosis was applied?

2. What did we look for in blood serum?

3. Was the test positive?

4. What is the function of complement in the study?

5. What is haemolytic system and its significance in CFT?

2 tasks. Enzyme immunoassay.

2.1. Suspecting that a patient is ill with hepatitis A (HAV) his/her serum was examined by ELISA

(Enzyme-liked immunosorbent assay) based on the schema (Table2 )

Table 2.. ELISA scheme Wells Components

1 2 3 1. Hepatitis A virus Ag fixed in well + + + 2. Patient’s blood serum (1:1000) + - - 3. Blood serum of healthy person (1:1000) - + - 4. IgM to HAV antigens - - + 5.Peroxydase-labelled rabbit antibodies to human IgM + + + 6. Substrate for peroxydase + + + 7. Test results (colour reaction)

Evaluate the results, draw the schema of the test and answer the following questions.

1. Which method of diagnosis was used?

2. What did we look for in patient’s serum?

3. What is the role of rabbit antibodies to human IgM in this test?


5. Was the test performed by ELISA competition or “sandwich” principle?

49

2.2. Blood serum of 10 donors were examined to detect virus of B hepatitis (HBV) infection.

ELISA based on Table 3 was carried out.

Evaluate the results of the test, explain the schema and answer the following questions:

1. Which diagnostic method was used?

2. What did we look for in blood serum?

3. Were there any donors infected with HBV detected?

4. Was this technique performed by competition or „sandwich” principle?

Table 3. Enzyme immunoassay scheme.

Wells Components 1 – 10 11 12

Immunoglobulin to HBV antigens, fixed in well + + + Donor’s blood serum + – – Ill person‘s with hepatitis B blood serum – + – Healthy person’s blood serum – – + Ig against HBV antigens labeled by peroxidase + + + Substrate for peroxidase + + + Results (colour reaction)

2.3. A patient suspected having cytomegalovirus (CMV) infection; his/her urea was examined using enzyme immunoassay based on the schema (Table 4): Table 4. Enzyme immunoassay scheme.

Number of well Components 1 2 3

Immunoglobulins against CMV + + + Patient’s urea + – – Healthy person’s urea – + – CMV antigen – – + Immunoglobulin labelled by peroxydase against CMV + + + Substrate for peroxydase + + +

Results (colour reaction)

Evaluate the results of the test, draw the schema and answer the following questions:

1. Which technique was used?

2. What did we look for in the patient’s urine?


4. Was this technique performed by competition or „sandwich” principle?

Notice. During all enzyme immunoassays wells are washed with buffer solution after every filling with reagent and incubation.

50

Self-learning 1. What is immunodiagnosis of infectious diseases?

2. What does serological diagnosis of infectious process mean?

3. Do we need to know (and why?) dynamics of antibody synthesis to perform serology?

4. Which components do take part in immunolysis?

5. Significance of antibodies and complement in the immunolysis test.

6. Are all antigens lysed in immunolysis test?

7. Can immunolysis take part in the body?

8. How are haemolytic sera produced?

9. Which components do take part in CFT?

10. How are positive and negative CFT evaluated?

11. What is the essence of immunoflourescence technique?

12. What are direct and indirect immunoflourescence test?

13. What is the essence of enzyme immunoassay? What are its modifications?

14. How are positive and negative results of enzyme immunoassay evaluated?

15. What is the essence of radioimmune technique?

16. Which toxins are neutralized by antibodies?

17. How is toxin neutralization examination performed?

18. What is immunoblot?

19. Why is it important during serology to determine class of immunoglobulins or to use

immunoblot analysis?

References:

1. E. Jawetz, J.L. Melnick, E.A. Adelberg. Medical Microbiology. Twenty-Third Edition. Lange medical Books. 2004. p.119-146. 2.Patrick R. Murray, Ken S. Rosenthal, George S. Kobayashi, Michael A. Pfaller. Medical Microbiology, 4 Edition, 2002, p. 91-146.

51

7. Seminars 7.1. Pharmacology seminar.

Unit – Department of Theoretical and Clinical Pharmacology

In charge – associated professor A.Milašius Pharmacodynamics. Categories and types of receptors. Second messengers. Interdependence

between dose and response. Agonists and antagonists (full, functional, partial). Therapeutic index.

Effective dose. Its proportionality to dose-response reaction in population. (2 hours).

References:

1. Katzung BG. Basic & Clinical Pharmacology. Eighth edition.2001: 753-823, 845-853.

2. Howland RD, Mycek MJ, Harvey RA, Champe PC. Pharmacology. 3 rd edition. Lippincott‘s

Illustrated Reviews. 2006:341-452.

3. Rang HP, Dale MM, Ritter JM, Moore PK. Pharmacology. Fifth Edition. 2003: 620-692.

4. Mutschler E, Geisslinger G, Kroemer HK, Schafer-Korting M. Arzneimittelwirkungen. Lehrbuch

der Pharmakologie und Toxicology. 8 Auflage. Stuttgart, 2001: 757-871.

Supplementary reading:

1. Goodman and Gilman’s. The Pharmacological Basis of Therapeutics. Elevent edition. 2006:

1095-1314.

7.2. Pharmacology seminar.

Unit – Department of Theoretical and Clinical Pharmacology

In charge – associated professor A.Milašius Routes of drug administration. Absorption of active substances from digestive system: passive

diffusion and active transport. Influence of physical factors to drug absorption. The surface of

absorption and duration of this contact. Biological drug availability and biological and therapeutical

equivalence of medicaments. Volume of drug distribution , cummulation. Pharmacokinetic

synergism and antagonism. Pharmacokinetics of drugs used on daily basis. Equilibrium

concentration. (2 hours)

52

7.3. Protein structure and properties. Kinetics of enzymatic catalysis and inhibition of enzymes.

Solution of problems (2 hours.)



Aim:

To learn the principles of protein structure formation and properties; to be able to determine kinetic

constants of enzymes and types of enzymatic reaction inhibition.

Objectives:

1. Master to evaluate electric charge of amino acids and proteins dependaning on pH of

solutions.

2. Understand the principles of formation of three-dimentional structure protein molecule.

3. Learn to calculate the kinetic constants of enzymes and understand their meaning.

4. Learn to determine the types of enzyme inhibition.

References:

1. P.C.Champe, R.A.Harvey, D.R.Ferrier. Biochemistry. Lippincott’s illustrated reviews.

Lippincott Williams&Wilkins, 2005, p. 1-12; 53-67.

2. R.K.Murray, D.K.Granner, P.A.Mayes, V.W.Rodwell. Harper’s Biochemistry.Prentice-Hall

International Inc. 1996, p. 23-52; 75-90.

7.4. Aerobic and anaerobic metabolism of carbohydrates. (2 hours)



Aim

Using the map of metabolic pathways, to find out the metabolic reactions of carbohydrates and

product of their degradation during aerobic and anaerobic conditions.

Objectives:

1. Master to use the scheme of metabolic pathways.

2. Find out the sequences of enzymatic reactions of carbohydrate metabolism taking place in

aerobic and anaerobic conditions.

3. Find out the principle mechanisms of aerobic and anaerobic metabolism of glucose.

4. Master to calculate the energy value of aerobic and anaerobic breakdown of carbohydrates.

References:

53


Lippincott Williams&Wilkins, 2005, p. 69-154.


International Inc. 1996, p. 135-145; 158-184.

7.5. Analysis of metabolism of fats and fatty acid using the maps of metabolic pathways.

Biochemical aspects of obesity. (2 hours).



Aim

Uing the map of metabolic pathways to follow up metabolic reactions of fatty acid breakdown and

synthesis as well as degradation and synthesis of triacylglycerols.

Objectives:

1. Master to follow up triacylglycerol degradation and synthesis reactions in the map of

metabolic pathways.

2. Master to follow up fatty acids degradation reactions in the map of metabolic pathways.

3. Master to follow up reactions of fatty acids synthesis in the map of metabolic pathways.

4. Master to calculate energy yield under decomposition of triacylglycerold, fatty acids, and

indermediary products of their breakdown.

References:


Lippincott Williams&Wilkins, 2005, p. 171-216.


International Inc. 1996, p. 216-253.

7.6. Signal transduction: second messengers, their formation, inactivation and significance in

regulation of metabolism (2 hours.)



Aim

To get aquainted with mechanisms of cell-to-cell communication.

Objectives:

1. Aquaint with mechanisms of intracellular signal transduction.

2. Analyze the significance of enzymes implicated in intracellular signal transduction.

54

3. Understand the mechanisms of intracellular signal amplification.

4. Know Multiple functions of cAMP.

5. Aquaint with types phosphatidylinositol metabolites as second messengers.

6. Understant significance of protein kinase in regulation of metabolism, cell division and

differentiation.

References:


Lippincott Williams&Wilkins, 2005, p. 92-94;


International Inc. 1996, p. 509-521.

7.7. Humoral and cellular antimicrobial immune response. (2 hours)

Unit– Department of Microbiology In charge– associate professor D. Janulaitytė-Gunther, associate professor R. Plančūnienė

Objectives: 1. Master the mechanisms of the humoral response in antimicrobial immunity.

2. Master the dynamics of antibody synthesis in the course of infectious diseases.

3. Master types of immune cells and their co-operation, production of cytokines, their function.

Tasks:

1. Antimicrobial antibodies and their function.

2. Isotypes of antibodies.

3. Dynamics of antibody synthesis during primary and secondary immune response.

4. Antigen-presenting cells, TH1 and TH2 the helper cells of lymphocytes, B lymphocytes, plasma

cells. Immune memory in response to microbe’s antigens.

5. Antigens, chemical and functional description. Antigens of microorganisms.

6. Superantigens of microorganisms, the mechanisms of their action.

7. Haptens, their inability to cause IgG synthesis and immune memory – the problem of vaccine

production.

8. Antimicrobial antibodies – the cause of immunopathologic reactions. Heterogenic antigens of

microorganisms.

9. T lymphocytes (CD4, CD80, antigen-presenting cells). Endogenic and exogenic presentation of

antigens.

10. CD4 and CD8 lymphocytes, macrophages interaction.

11. Cytokines - producing CD4, CD8 and macrophages, their variety, functions.

12. Interferons, lymphokines, monokines - nonspecific to antigens of microorganisms, cytokines.

55

References:

1.Patrick R. Murray, Ken S. Rosenthal, George S. Kobayashi, Michael A. Pfaller. Medical Microbiology, 4 Edition, 2002, p. 91-146

7.8. Principles and methods of laboratory diagnosis of fungal and viral infections. Cultivation of

viruses, determination of identity, immunologic and genetic methods (2 hours)



Objectives:

1. Master the peculiarities of biology of fungi and viruses.

2. Master the methods of viral cultivation and identification.

3. Master the methods of virology, immunology, and molecular diagnosis of viral infections.

4. Master the principles of microscopic, mycologic, immunologic and molecular diagnosis.

Tasks: 1. Viral intracellular parasitism-the cause of viral growth in the living cells.

2. Cultivation of viruses in cell cultures, embrionated eggs, infected laboratory animals.

3. Determination of viral cytopathic effect in cell cultures.

4. Viral detection by haemagglutination and haemadsorbtion tests.

5. Viral detection of viruses by immunologic and genetic tests.

6. Immunological techniques of viral infections: serodiagnosis, detection of antigens in test

material.

7. Criteria of serology of viral infections; the growth of antibodies titer, determination of IgM,

immunoblot.

8. Detection of viral antigens: enzyme immunoassay (EIA), immunofluorescence,

radioimmuneassay (RIA), their specificity and sensitivity.

9. Molecular diagnosis in viral infections: in situ hybridization, the polymerase chain reaction

(PCR), ligase chain reaction (LCR).

10. Fungi – the causatives of infectious diseases, mycotoxicosis.

11. Peculiarities of morphology, structure and biology of fungi as eukaryotic microorganisms.

12. Fungal polymorphism. Yeast, hyphae, dimorphic forms.

13. Fungal spores, their variety and significance in diagnostic of mycosis.

14. Factors of fungal virulence.

15. Fungi produced toxins, mycotoxicoses, and methods of their detection.

16. Fungal cultivation in nutrition media and determination of identity principles.

56

17. Determination of fungal antigens in a test substance.

18. Opportunistic mycoses, principles of diagnosis

Reference:

1. Patrick R. Murray, Ken S. Rosenthal, George S. Kobayashi, Michael A. Pfaller. Medical Microbiology, 4 Edition, 2002, p. 67-70, 627-38, 449-457.

57

8. Module exam questions 8.1 Biochemistry

1. Protein structure, characteristics, classification and functions.

2. Characteristics of enzymes as biologic catalyzators, classification, nomenclature.

3. Structure of enzyme and the mechanisms of enzyme catalysis.

4. Vitamins and coenzymes: their structure, role in enzyme catalysis.

5. Kinetics of enzyme catalysis, kinetic constants and their significance

6. Enzyme inhibitors, types, application to medicine.

7. Regulation of enzyme action.

8. Metabolism. Catabolism and anabolism.

9. Basis of metabolism regulation.

10. Classes of hormones and common mechanisms of their action.

8.2 Physiology and pathological physiology 1. Passive and active transport, its mecahanisms.

2. Epithelial transport, its significance, mechanisms. Functions of epithelium.

3. Transepithelial potential and it influencing factors.

4. Resting membrane potential, its origin, equilibrium of ions potential.

5. General nosology. Norm, health, disease. Principles of disease classification.

6. General etiology. Causes of diseases and conditions.

7. General pathogenesis. Stages of diseases, peculiarities of course of disease, outcome of

diseases.

8.3. Pathological anatomy

1. Structural basis of adaptive and compensation reactions.

2. Hyperthropy and ir hyperplasia, the essence, morphology and functional significance.

3. Causes of atrophy, morphology and functional significance.

4. Reparative regeneration, its pathogenesis and morphology. Metaplasia.

5. Regeneration of the epithelial and concective tissue. Wound healing and its organization.

8.4.Teoretical and Clinical Pharmacology. 1. Routes of administration and their choice. Absorption, bioavailability, and AUC

58

2. Relationships between pharmakokinetics and therapeutics. Therapeutic window.

3. Factors that may influence drug kinetics and effects. Clearance and elimination

4. Possible changes in drug effect following repeated or long-term use. Tolerance and

dependence.

5. Adverse effects. Principles of overdose treatment

6. Drug interactions and incompatibilities 7. Multiple-dose kinetics. Distribution. Steady-state.

8.5. Microbiology 1. Sistematics and nomenclature of microorganisms. Principles and criteria of classification. Conception of species as the cornerstone taxon. Subspecies. Biotype. Chemotype. Serotype. Strain . Culture . 2. Structure of prokaryotes: nucleus, cell wall, plasma membrane, capsule, flagella, and pili. Sporogenesis of prokaryotes. Links of the structure of prokaryotes with their pathogenicity. 3. Principles of cultivation of microorganisms. The rate of reproduction and phases of the growth of microorganisms in liquid nutrition media. 4. Bacteriological method of investigation. Its purposes and stadiums. The concept of pure culture of microorganisms. The peculiarities of isolation of pure aerobic and anaerobic cultures. Methods of determination of species of microorganisms. 5. Basics of microorganisms’ heredity. The genotype and phenotype of microbes. Mutation of microorganisms. Genotypic and phenotypic inheritance. The recombinations of bacteria: transformation, transduction, and conjugation. Plasmids, the most important their genotype and phenotype functions. The role of mutations, recombinations and selection in the evolution of microorganisms. 6. Immunoglobulins, their structure and function. Classes of immunoglobulins and their functions. Theories based on immunoglobulin synthesis mechanisms. Complete, incomplete and normal antibodies. Monoclonal antibodies. Autoantibodies.

59

Appendix 1

Tables of computerized exercise results

60

Name ....................................................................... Faculty ...............Group.......... Date ..................

TRANSPORT ACROSS MEMBRANES computerized exercise STEP

Directions: Take measurements from the computer screen and fill in the tables. Exercise F1. Dependence of transepithelial potential (TEP) and short circuit current (SCC) on the apical Na+ concentration

Na+ concentration 110 mmol / l 10 mmol / l 0 mmol / l 110 mmol / l

Time TEP SCC TEP SCC TEP SCC TEP SCC

0'15”

0'45”

1'15”

1'45”

Mean:

Exercise F2. Dependence of TEP and SCC on the temperature of the bathing solution

Exercise F3. Dependence of TEP and SCC on the Na+ permeability of the apical membrane

Time TEP SCC Time TEP SCC

Control -0'15” Control -0'15”

Effect of temperature 0'15” Effect of

amilorid 0'05”

0'30” 0'10”

0'45” 0'15”

1'00” 0'20”

1'15” 0'25”

1'30” 0'30”

1'45” 0'35”

Mean: 0'40”

0'45”

0'50”

0'55”

1'00”

61

Exercise F4. Changes of TEP and SCC after activation of the Na+/K+ pump

Exercise F5. Changes of TEP and SCC after inhibition of the Na+/K+ pump

Time TEP SCC Time TEP SCC

Control -0'15” Control -0'15”

Effect of isoproterenol 0'15” Effect of

ouabain 0'15”

0'30” 0'30”

0'45” 0'45”

1'00” 1'00”

1'15” 1'15”

1'45” 1'45”

2'15” 2'15”

2'45” 2'45”

3'15” 3'15”

3'45” 3'45”

4'15” 4'15”

4'45” 4'45”

5'15” 5'15”

5'45” 5'45”

6'15” 6'15”

6'45” 6'45”

7'15” 7'15”

7'45” 7'45”

8'15” 8'15”

8'45” 8'45”

9'15” 9'15”

9'45” 9'45”

Conclusions:

62

programme of medical studies module basis of preclinical ... studies-2009-08... · programme of...

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