General bacteriology and immunologylib.sumdu.edu.ua/library/docs/rio/2018/m4275.pdf ·...

1

Ministry of Education and Science of Ukraine

Sumy State University

Medical Institute

Department of Public Health

Ivakhnyuk T.V., Ivakhnyuk U.P., Holubnycha V.N., Kornienko V.V.

Microbiology, Virology

and Immunology

Part 1 4275

General bacteriology and immunology

Practical Workbook

Sumy

Sumy State University

2018

2

Practical Workbook of «Microbiology, Virology and Immunology» Part 1 «General

bacteriology and immunology» / T. V. Ivakhnyuk, U. P. Ivakhnyuk, V. M. Holubnycha,

V. V. Korniienko – Sumy : Sumy State University, 2018. – 60 p.

Department of Public Health

General bacteriology and immunology

Practical Workbook

Student's name ____________________________________________

Group number _______________

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INTRODUCTION

Medical microbiology is the study of the background material on various pathogenic

microorganisms and describes the epidemiology, transmission, clinical manifestations, diagnosis, and

treatment of diseases caused by those pathogens.

The purpose of this workbook is to provide students with basic knowledge of the etiology of the

microrganisms causing the disease and the pathogenetic mechanisms leading to a clinically manifestation

of the infection. This knowledge is necessary for diagnosis, therapy, and appropriate actions to prevent

infection. This workbook designed for novice medical students. Besides academic purpose this notebook

can be usefull to health professionals of various specialisation especially to the physicians.

This workbook vast and complex field of medical microbiology easier to understand thanks to

numerous illustrations with detailed explanatory legends. The many tables present knowledge in a cogent

and clear form.

Part 1 introduces basic techniques of microbiology. It includes general requirements for work in the

laboratory, precautions for handling microorganisms, the use of a microscope, microscopic morphology

of microorganisms in wet and stained preparations, pure culture techniques, and an exercise in

environmental microbiology. It provides instructions to gain some experience in methods of the killing

microorganisms in order to allow students understand the principles of disinfection and sterilization

before they continue to study pathogenic microorganisms. There is an exercise on antimicrobial agents

that includes antimicrobial susceptibility testing. Part 1 reflects the essential role of antigen detection

techniques in the daily work of the laboratory, and the more rare use of methods for detecting serum

antibodies.

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GENERAL INFORMATION Every practical lesson on the special microbiology is based on knowledge from sections

«Morphology and Physiology of Bacteria» and «Infectious Process. Immunity».

Turning to the study of each topic, a student shoud:

KNOW basic principles of microbiological diagnisis of bacterial infections, сonformities to the

law of functioning of the human immune system, аntigenic structure of the bacterial cell, methods

of creating of artificial immunity, the basic concepts of epidemiology, and general compliance

with the laws of development of infectious diseases;

be ABLE to prepare a preparation from material, taken from a patient or from a colony on nutrient

media, to stain smear by simple staining techniques or to perform a Gram stain to use microscopic

examination of preparation and to estimate morphological and staining properties of bacteria; to

make inoculation in liquid and on solid media; to perform serological testing to find out antibodies

in the blood serum of a patient and to study antigenic structure of bacteria.

A course map of practical lesson

Step

Time,

minutes

Teaching methods Location

Relevance of the topic,

practical application,

purpose and objectives

10 Multimedia slide show A classroom for

practice

Assessment and

correction of initial

level of knowledge

10 Computer tests

Presentation, practical

work, and feeling

protocols

50 Preparing smears by different staining

techniques of bacteria. Growing of

bacteria in the culture medium. Reading

charts of microbiological diagnosis in

an infectious diseases.

Immunobiological preparations.

Multimedia slideshow

Concluding the results

of practical lesson

10 Charts, multimedia presentations

In every practical lesson a student writes lesson protocol of lesson, which which should be

signed by the teacher, if it is designed correctly.

Rules for practical lesson protocol registration:

1. Write a theme of the lesson, a scheme of the microbiological diagnosis (should be prepared at

home before the lesson).

2. Demonstration microslides, growth of the studied bacteria, must be sketched on nutrient media.

3. If the biochemical properties of bacteria are studied in practical lesson, a student writes a

conclusion in the protocol.

4. In every practical lesson a student describes immunobiological preparations according to the

following chart:

4.1. If the preparation is used for treatmentor for prevention:

Preparation Сomposition and method

of preparing

Purpose of use

(characteristics of immunity)

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Appendix A

Immunity description:

Postinfectious

Postvaccine

Active

Passive

Humoral

Cellular

Antibacterial

Antiviruses

Antitoxins

Antifungal

Specific

Nonspecific

Group-specific

Species-specific

Type-specific

4.2 If this preparation is for diagnosis, it should be described according to the following chart:


of preparing

Purpose of use

(test using this preparation)

At the end of the section a student takes a module control.

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Date _____________________Class №1

ORGANIZATION OF THE MICROBIOLOGICAL LABORATORY ACTIVITY.

MORPHOLOGY OF THE MICROORGANISMS.

SIMPLE METHODS OF STAINING SPECIMENS

Relevance of the topic: Every experienced physician should be able to prepare a smear from any

material of a patient or bacterial culture on the glass slide, stain and examine it under a microscope in

order to determine the morphology of the causative agent for approximate diagnosis of infectious diseases

or complications.

Primary objective: To be able to carry out microscopic examination of the stained specimens and

to distinguish cell morphology and staining properties.

QUESTIONS FOR DISCUSSION

1. The rules of practice in microbiology department and in practical bacteriological laboratory.

2 Morphology and classification of microorganisms.

3. Rules of microscopy using an oil-immersion objective.

4. The rules for the preparation of a smear from broth and solid media cultures.

5. Simple techniques of the microorganisms staining.

PROTOCOL OF THE PRACTICAL SESSION

Practical tasks to be done:

Task 1. Learn the safety rules in microbiology laboratory and write them down.

GENERAL LABORATORY SAFETY RULES FOR MICROBIOLOGY

1. No smoking, eating, drinking, chewing gum, or applying cosmetics in laboratory area,

including laboratory office. No foodstuffs are to be stored in laboratories (including, refrigerators, and

freezers).

2. Disposable laboratory gloves are not to be worn in communal areas. Door handles,

telephones, computer keyboards, and mice (except in clearly defined circumstances), lift buttons, etc. are

not to be touched with gloves. If needed, wear one glove and use the ungloved hand to open doors,

operate lifts, etc.

3. Rubber or disposable gloves should be worn when handling/working with human blood or

other body fluids, dangerous chemicals, infectious, or potentially infectious materials. Select an

appropriate glove type.

4. Laboratory gowns or labcoats must always be worn in laboratories, but they should be taken

off before entering “clean areas”, e.g., tea room, stores, media, toilets, library, and office rooms.

5. Clothing and footwear must be suitable for laboratory conditions. Flip-flops, sandals or high-

heeled shoes are not to be worn in the laboratory. Bare feet are prohibited in the building. Sandals with an

enclosed toes and heels are acceptable, but for your own protection, an enclosed shoes are preferable.

Long hair must be tied back to avoid contact with microorganisms and equipment.

6. Protective glasses must be worn for all kinds of work involving corrosive or toxic chemicals,

radioactivity, and ultraviolet light.

7. The best protection from microorganisms ingestion is avoidance of their penetration in your

mouth. Labels and envelopes must not be licked. Pencils and pens must not be placed in the mouth. Biting

of fingernails, playing with hair, applying lipstick, eating, drinking, etc., are not allowed. Wash your

hands when leaving the laboratory for lunch, etc.

8. Do not pipette by mouth. The use of pipettes with cotton plugs to reduce contamination is

preferable. Place pipettes in disinfectant solution to minimize aerosol production. Submerge them for 18–

24 hours. Residual volumes from pipettes create aerosols; use mechanical devices that are calibrated to

deliver. Fit pipettes to a soft bulb by holding it at the plugged end to avoid the risk of cuts in case the

pipette is broken.

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9. Minimize the use of “sharps”. Do not bend needles, or try to replace the caps after use. Use

syringes fitted with blunt cannulas where possible. Avoid using syringes to mix infectious liquids (if

essential, hold the tip of the needle under the surface of the fluid and avoid excessive force). Discard used

syringes and needles into an approved sharps container.

10. Hazardous chemical and biological spills and blood spills on the floor, benches or equipment

should be cleaned up immediately. Special treatment is required for spills of a biohazardous nature.

11. Hands should be washed after completing each task and always before leaving the laboratory.

12. No running or “horse play”. Report all potential hazards and problems immediately. Try to

anticipate potential problems.

13. Any faulty equipment should be removed from service for repair or disposal.

14. When flaming wire loops, draw the loop gradually from the cooler to the hotter part of the

flame to minimize spattering, or use electric heaters. Make sure the loop circle is completely closed and

the loop wire is not longer than 6 cm.

15. Disposable plastic loops must be placed loop-end down in disinfectant for 18–24 hours.

16. Petri dish cultures of fungi should be imprinted and incubated with the lid up permost to

prevent the dispersal of fungal spores. Recognize fungi as potential pathogens and be aware of the ability

of some species to produce mycotoxins.

17. Take care when handling Petri dishes that contain condensate. This may contain viable

microorganisms that can be spread via droplets or aerosols when the plates are opened or dropped.

18. Open and operate tissue grinders in a biological safety cabinet. Hold glass grinders in a wad

of absorbent material and wear gloves. Wait 10 minutes before opening a blender bowl to allow aerosols

to settle. Refrigerate to condense aerosols. Use models designed to prevent leakage from rotor bearings

and O-ring gaskets or use a “stomacher”.

Task 2. Use the microscope to identify bacteria and draw the samples of the main cocci and rod-shaped

bacterial forms. Make a conclusion.

Preparation

Picture Cell morphology and staining

Staphylococcus aureus,

gentian-violet stain

Streptococcus pyogenes in pure

culture, gentian-violet stain

Sarcina lutea in pure culture,

carbol fuchsin staining

Neisseria meningitidis

(diplococci) in spinal fluid,

fuchsin staining

Escherichia coli,

fuchsin staining

Corynebacterium diphtheriae

in pure culture,

gentian-violet staining

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Preparation


Clostridium tetani in pure

culture,

fuchsin staining

Bacillus cereus in pure culture,

gentian-violet staining

Task 4. Study the course of rays in the dry and oil immersion system.

Work with a microscope consists of two main stages:

1) Proper installation of visual field illuminance of the specimen.

2) Microscopy of specimens with different lenses.

Stained bacteria specimens are microscopically examined only by means of immersion lens (x90).

Rules of work with immersion system:

1. Lift condenser up to the level of the objective table, open the diaphragm of the microscope.

2. By means of eyepiece 15×, objective 8×, and flat mirror, achieve the maximal visual field

illuminance.

3. Put a preparation on the objective table, fix its plugs, by means of the macroscrew find the

contours of the image, and by means of the microscrew – obtain precise image.

4. Turning a nosepiece of the microscope, place an immersion objective 90× above the slide; dip it

in the oil immersion.

5. Turning the microscrew of the microscope, obtain precise image of the preparation, study

microorganism morphology, determine the increase, at which the preparation was investigated.

6. After the termination of microscopy lift a tube, take the preparation away, wipe an objective

from oil immersion, carefully turn it aside and lower a tube.

Possible errors when using a microscope:

1. Wrong choice of mirror.

2. Incorrect installation of condenser and iris diaphragm, which leads to inadequate illumination of

the object.

3. Wrong choice of the lens for investigation.

4. Incorrect installation of the eyepiece, etc.

Morphological and staining features of the microorganisms cells can be studied using various

methods of microscopy and some methods of staining. The choice of methods and means of microscopic

investigation of stain is determined by specific study purpose. So, if we want to detect motion, we should

prepare wet-mount preparation or hanging drop; capsules are detected by means of Burri-Gins staining,

etc.

Microscope maintenance

The microscope should be kept clean and in prevention from mechanical damage. It should be taken

by the column and supported by the other hand at the bottom of the rack. Before work it should be

checked. Particular attention should be paid to optical part. Do not touch the lens surface – fingerprints

are always produced on it. Eyepiece lens pollution can be detected by moving it under direct vision. Do

not disassemble lenses and adjust mechanical parts.

After completion of work the microscope should be brought in order. Oil should be wiped carefully

from the immersion lens with the help of special cloth. Nosepiece is transferred to a small dry lens 8x.

Aperture is fully opened, condenser is slightly lower. The cloth is put on the objective table. The

microscope tube is lowered down as much as possible. Microscope is covered with polyethylene cover.

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The immersion oil has the same refractive

index as the glass lens and the glass slide. This

prevents distortion because the light waves follow a

homogeneous path.

In microscopic examination with the help of

immersion objective the latter is immersed in oil

(cedar, peachy, “immersion”, etc.) whose refractive

index is close to that of glass. When such a medium

is used, a beam of light emerging from the slide is

not diffused and the rays arrive at the objective

without changing their direction. The resolving

power of the immersion objective is about 0,2 μm.

Resolution is the capacity of optical system to

distinguish or separate two adjacent object or point

from one other. The maximum magnification of modern light microscopes is as high as 2000-3000. Total

power of magnification formed by the combined lenses (objective and ocular) is product of the separate

powers of each lens: Power of objective x Power of ocular = Total magnification.

Task 4. Prepare a smear of staphylococcal culture on nutrient agar, stain the smear with gentian

violet, perform the microscopy, and draw it.

Technique of cultures smear preparation grown on the solid nutrient medium

Manipulation 1. Smear preparation of the culture grown on the solid nutrient medium For smear preparation take a clean slide. By means of glass pencil draw a circle 1.5–2.0 cm in

diameter–a place of the material drawing. On the opposite part of the glass prepare smear test. In the left

hand take a test tube with a physiological solution (NaCl), and in the right–a bacteriological loop. The

loop is calcinated in a flame of a spirit-lamp (sterilized) for

destruction of extraneous bacteria. By rotary movements take a

wadded fuse out of the test tube, pressing it by the 5th and 4th

fingers of the right hand to a palm, and burn the edge of the test

tube. A loop is cautiously entered into a test tube, cooled about

an internal surface of the glass. Then put 1–2 drops of the sodium

chloride on the glass slide, burn the edge of the test tube again

and close its fuse. Then take a test tube with the grown up culture

in the left hand, similarly open it, sterilize the edge of the test

tube, enter the loop into the test tube cautiously, grasp a material

with a sliding movement, take out a loop from the test tube, burn

the edge of the test tube again and close its fuse. The material on

the bacteriological loop is brought in the drop of the sodium

chloride and prepared for a suspension, not falling outside the

limits of the circle (at correct distribution of the material in smear

test at microscopy isolated bacterial cells are visible). After

preparation of smear test, bacteriological loop is carefully burned in a flame of the spirit-lamp. Manipulation 2. Smear test drying

Smear test is dried up on air or in warm air stream above a flame of the spirit lamp, not allowing a

drop to begin to boil.

Manipulation 3. Smear test fixation Dried up smear tests are physically or chemically fixed for speciments obtaining. During fixation

bacteria die, they are better stained and are densely attached to the surface of the glass. Fixe the smear by

passing it through the flame of the spirit lamp three times (in 3–5 seconds), carrying out physical fixation

(smear test upwards). Smear blood tests and smear-prints from organs are fixed, by immersing them for

5–20 minutes in methyl or ethyl spirit (chemical fixation).

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Manipulation 4. Smear staining by simple method

The prepared specimen is stained by the certain dye, for

example, methylene blue (for 3–5 minutes), solution of Pfeiffer's

fuchsine (for 1–2 minutes), and gentian violet (for 1–2 minutes). The

simple method of staining allows revealing microorganisms in a

material, to define their quantity, shapes, and arrangement.

Examine under the microscope and sketch preparations of Staphylococcus spp., stained with gentian

violet. Draw a conclusion.

Define morphological and tinctorial properties of the

microorganisms: _______________________________________________________________

_______________________________________________________________

Task 5. Prepare a smear of coli bacterial culture on nutrient broth, stain the smear with using

water solution of fuchsin, perform the microscopy, and draw it.

Technique of cultures smear preparation grown on the liquid nutrient medium

Manipulation 1. Prepare the smear of the culture grown on the liquid nutrient medium

On the prepared glass slide a drop of the liquid nutrient medium with microorganisms is put with a

bacteriological loop and in regular intervals it is distributed on the slide.

Manipulation 2. Dry the smear.

Manipulation 3. Fix the smear.

Manipulation 4. Stain the smear by simple method – this stage of smear preparation is conducted

similar to the task 4.

Examine under the microscope and sketch preparations of E. coli culture that is stained gentian violet.

Draw a conclusion.

Define morphological and tinctorial properties of the

microorganisms:

_______________________________________________________________

_______________________________________________________________

Teacher's signature ____________________

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Date _____________________Class № 2

STRUCTURE OF THE BACTERIAL CELL. COMPLEX METHOD OF SPECIMEN STAINING.

MICROSCOPIC METHOD OF DIAGNOSIS

Relevance of the topic: Each microorganism has a specific morphological structure and tinctorial

properties; knowledge of these properties helps to identify the causative agent. Methods of complex

staining allow to differentiate bacteria and to study the structural characteristics of bacterial cells.

Therefore, for causative agent identification, it is important to be able to stain specimens and to

distinguish different forms of the major groups of microorganisms.

Primary objective: To be able to detect and identify different structures of the microorganisms.


1. Cell walls of gram-positive and gram-negative bacteria: structure, chemical composition, and

thickness. Cell walls of acid-fast bacteria.

2. Complex methods of the microscopic specimens staining – Gram staining: principle and staining

procedure. Purpose of each step in the Gram staining procedure. Examples of gram-positive and gram-

negative bacteria (in Latin).

3. Features of the morphological organization of protoplasts, spheroplasts, and L-forms of bacteria.

4. Acid-fast bacteria. The method of acid-fast bacteria staining: Ziehl-Neelsen staining technique.

5. Cytoplasmic membrane, mesosome, cytoplasm, nucleoid, ribosome, inclusion: structure and

functions.

6. Bacterial capsule: structure, chemical composition, and functions. Complex methods of the

specimens staining. Burri-Gins staining technique, principles and stain procedure.

7. Spores: structure and functions. Spore formation and germination.

8. Spore staining: Anjesky`s method.

9. Flagella: the structure, chemical composition and functions. Methods of motility testing: the

hanging drop and wet mount techniques.

10. Microscopic method for diagnosis of infectious diseases.



Task 1. Prepare the smears from mix-cultures (staphylococcal and colibacillar) and stain them

using Gram method; perform the microscopy and draw it.

The Gram stain – a differential staining technique - is the most useful and cost-effective method

applied in the clinical microbiology laboratory. The differential stain is the most commonly used

technique for direct microscopy of specimens and bacterial colonies because it has a broad staining

spectrum. First devised by Hans Christian Joachim Gram late in the 19th century, it has remained the

same and serves to divide bacteria into 2 groups: gram-positive organisms, which retain the primary

gentian violet dye and appear deep blue or purple, and gram-negative organisms, which can be

decolorized, thereby losing the primary stain and the subsequent fuchsin counterstaining and appearing

red or pink.

Gram staining procedure

1. Smear the material to be stained on the slide. Allow it to dry in the air. Fix it by gentle heating to

kill bacteria and allow the material to be attached to the slide.

2. Apply an appropriate solution of gentian violet to the smear and let stand for one minute. Wash the

slide carefully.

3. Add iodine solution for one minute (a mordant that makes the stain more prominent and fixes

gentian violet dye to the cell wall). Rinse the slide.

4. Apply 95% ethyl alcohol until all but the thickest parts of the smear are decolorized, but no more

than 10 to 15 seconds. Wash the slide.

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5. Apply carbol fuchsin countersta into the slide and incubate for 1 minute. Wash and dry the slide.

6. Examine the smear using the oil-immersion lens of the light microscope.

7. Gram-positive organisms are blue-purple and gram-negative bacteria are pink-red. If the spesimen

is stained correctly, gram-positive (violet) and gram-negative (red) bacteria will be viewed under the

microscope. If the staining process vas performed incorrectly, all bacteria are the same colour (violet or

red)

Perform the microscopy of the smears and sketch the preparations of mixed culture. Make a conclusion.

Describe the morphological and tinctorial properties of the

microorganisms: _______________________________________________________________

_______________________________________________________________

Task 2. Study the preparations of the specimen under a microscope and draw them in the protocol.

Preparation Picture Cell morphology and staining

Escherichia coli,

Gram staining

Salmonella typhi,

Gram staining


in pure culture, Gram staining

Mycobacterium tuberculosis

from patient's sputum, Ziehl-

Neelsen staining


in pure culture, Neisser stain

Klebsiella pneumoniae,

Burri staining

Task 3. Prepare smears of spore-forming culture, stain them by Ziehl-Neelsen method, perform the

microscopy and draw the results.

Causative agents of tuberculosis and leprosy of Mycobacterium genus have a distinctive character

called «acid fastness» due to the presence of lipids (waterline mycolic acid) in the cell wall. These

organisms quickly absorb carbolic fuchsin a red dye, in the presence of a detergent or when decolored and

retain the dye after washing with an acidified alcohol solution. All non-acid-fast bacteria, pus, cells, etc.

lose carbolic fuchsin when treated with addition of alcohol and take a contrasting counter stain (e.g.,

methylene blue, brilliant green). Tubercle bacilli are more strongly acid-fast than other members of the

acid-fast group and have a characteristic beaded appearance. Both Gram staining and acid-fast staining

depend on the integrity of the cell wall. Broken or disintegrated bacilli or their parts are neither gram-

positive nor acid-fast.

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Ziehl-Neelsen staining procedure

1. Fix the smear.

2. Flood the slide with carbolic fuchsin, steam gently for 5 minutes over low flame, do not allow to

dry and add more stain if necessary. Cool. Alternatively, carbolic fuchsin-containing phenol and alcohol

(cool) may be used without heating.

3. Apply 90 % alcohol containing 3 % to 5 % HC1 until all but the thickest parts of the smear cease to

give off colour (approximately 1 to 3 minutes). Wash the slide.

4. Counterstain with methylene blue for 1 minute. Wash the slide.

5. Examine the smear using the oil-immersion lens of the light microscope.

Sources of error:

1. Overheating (burning) during fixation can be avoided by just touching the back of the slide to the

back of the hand each time the slide has been passed though the flame.

2. Do not stain smears which have only been air-dried. Smears must also be “fixed”.

3. Smears should not be too thick. After air drying, examine them under the microscope. If there are

no areas of bacteria separation, more water should be added to dilute the smear.

4. After staining it is essential that the back surface of the slide is wiped clean.

5. If washing with distilled water is not done adequately, crystallization of the stain may appear on the

slide.

Microscope and sketch preparations of spore-forming culture, stained Ziehl-Neelsen. Make a conclusion.

Describe the morphological and tinctorial properties of the

microorganisms: _______________________________________________________________

_______________________________________________________________

Task 4. Study motile microorganisms by wet-mount and hanging drop techniques.

1. Wet-mount technique 1. A drop of the test material, usually a 24-hour broth culture of microorganisms, is placed into the

centre of the glass slide.

2. The drop is covered with a cover slip in such a way that the fluid fills the entire space without

overflowing.

2. Hanging drop technique. To prepare this kind of specimen, special glass slides with an

impression (well) in the centre are used.

1. A small drop of the test material is put in the middle of the cover slip.

2. The edges of the well are ringed with petrolatum.

3. The slide is placed onto the cover slip so that the drop appears in the centre of the well.

4. The slide is carefully inverted and the drop hangs in the centre of the cavity of the cavity slide,

which prevents it from drying.

The prepared specimens are examined microscopically, slightly darkening the microscopic field by

lowering the condenser and regulating the entrance of light with a concave mirror. At first low power

magnification is used (objective 8x) to detect the edge of the drop, after which a 40x or an oil-immersion

objective is mounted. Occasionally, molecular (Brownian) motility is mistaken for the motility of

microrganisms. To avoid this error, it should be borne in mind that microorganisms propelled by flagella

may traverse the entire microscopic field and make circular and rotatory movements.

After the examination, the wet-mount and hanging-drop preparations should be immersed in the

separate bath with disinfectant solution to kill the investigated microorganisms.

Toothpick

Coverslip

14

Task 5. Study the chart of microscopic method of diagnostics of gonorrhea and sketch it in the

protocol.

Chart of microscopic method of diagnostics of gonorrhea

A microscopic method of research can not be used as general method of laboratory diagnostic.

Because it does not let to identify the etiology of infectious diseases. In some cases a microscopic method

can be utilized. For example at gonorrhea.

Teacher's signature____________________

Mat

her

ial

for

test

ing

-

pat

ien

t's-

sp

inal

flu

id

Preparation of the

fixed preparation

Gram stain or other staining

technique

Microscopic

research of

preparation

Extracellular

normal flora

Leucocytes Gonococcus

RESPONSE

15

Date _____________________Class № 4

FEATURES OF CHLAMYDIAE, SPIROCHAETES, RICKETTSIA,

AND MYCOPLASMAS ULTRASTRUCTURE.

MODERN METHODS OF MICROSCOPIC EXAMINATION

Relevance of the topic: The microscopic method is commonly used in the laboratory diagnosis

for various infectious diseases. Such bacteria as spirochaetes, rickettsia, chlamydia, and mycoplasma have

special features in the cellular structure. These features must be considered during identification.

Therefore, every experienced physician must know the structure peculiarities of different bacteria and be

familiar with modern methods of microscopic investigation.

Primary objectives: To know structures of spirochaetes, rickettsia, chlamydia and mycoplasma;

to be able to conduct microscopic examination of native and fixed specimens using different methods of

microscopy.


1. Morphology of spirochaetes. Features of spirochaetes ultrastructure. Differences between structures

of Treponema spp., Borrelia spp., and Leptospira spp.

2. Morphology of Ricketsia spp., Chlamidia spp., Mycoplasma spp. Ultrastructural features of these

bacteria. Examples.

3. Life cycle of chlamydia.

4. Modern methods of microscopic examinations: purpose of use and principles.



Task 1. Study demonstration preparates.

Preparation Picture Cell morphology and staining

Treponema pallidum,

direct immunofluorescense

method

Treponema pallidum,

dark field microscopy

Rickettsia spp.,

Zdrodovsky stain (rickettsiae

are found in the cytoplasm of

infected cell)

Borrelia burgdorfery,

Romanowsky-Giemsa stain of

the blood smear

Leptospira interrogans,

dark field microscopy


16

Date _____________________Class № 5

STERILIZATION AND DISINFECTION

Relevance of the topic: In practice, it is often necessary to control undesirable growth of

microorganisms, to reduce the growth rate and their manifestation, either partially or completely

neutralizing bacteria in the external environment or in tissues. There are many ways to do this at thus

stage of medical development. Knowledge of basic methods of sterilization, disinfection, and antisepsis is

a contribution to the success of the doctor's work.

Primary objective: To be able to use basic knowledge of microbiological disinfection, sterilization,

and antiseptics in medical practice.


1. Sensitivity of microorganisms to physical and chemical factors.

2. Physical factors affecting microorganisms: filtration, drying, radiation, ultrasound, and temperature.

3. Chemical factors affecting microorganisms: phenols, halogens, alcohols, acids, alkalis, oxidizers,

aldehydes.

4. Disinfection: definition, purpose, types (physical and chemical methods). Monitoring the

effectiveness of disinfection.

5. Basic microbiology asepsis and antisepsis.

6. Sterilization: definition, purpose, types (thermal methods and monitoring of the ionizing radiation

impact, filtering and chemical methods). Medical equipment sterility control.



Task 1. Study demonstration.

1.1 Seitz and Berkefelda filters

1.2 Autoclave

1.3. Koch's steam sterilizer

1.4 Paster's hot air oven

Task 2. Determination of antibacterial action of disinfectants substances.

For this experiment, on a nutrient medium was made by grow a pure culture of bacteria (by making

a bacterial lawn). Then at planting were superimposed paper disks impregnated with specific disinfecting

solution. This Petri dish was incubated at 370C for 18-24 hours. After this procedure study zone of growth

inhibition around the disks with a ruler. Explore this area and make a conclusion about the effectiveness

of the disinfecting solution with respect to this pure bacterial culture.

Picture Interpretation of the result

Task 3. Study bacterial contamination of a nonsterile bandage.

One thread from a nonsterile bandage was put into sterile peptone water. A test tube was cultivated

in a thermostat at 370С one day.


Gram stain


17

Date _____________________Class № 6

BACTERIAL PHYSIOLOGY. BACTERIAL NUTRITION AND RESPIRATION.

CULTURE MEDIA. ISOLATION OF PURE CULTURE OF AEROBIC BACTERIA

(STAGE 1)

Relevance of the topic: Bacteriological method in diagnosis is the main method of microbiological

diagnostics of infectious diseases. For each experienced physician it is important to know how to

diagnose it, starting with the selection of material for inoculation, and ending with the evaluation of the

obtained results.

Primary objective: To be able to cultivate bacteria in culture media, to isolate a pure culture and to

evaluate the results of the tests.


1. Tasks and methods of culturing bacteria.

2. Rules for working with bacterial cultures.

3. Nutrition of bacteria. Classification of the bacteria accoding to the types of nutrition.

4. Respiration of bacteria. Aerobes, anaerobes, microaerophiles.

5. Mechanisms of nutrient transmission in the bacterial cell.

6. Culture media; classification according to the purpose and requirements.

7. Pure culture isolation techniques based on biological principles.

8. Culture medium for anaerobic bacteria cultivation.

9. Stages for isolation of aerobic bacteria in pure cultures – stage 1.



Task 1. Take the first stage of isolation pure culture of E. coli from the mixed culture of bacteria.

Scheme for isolating Escherichia coli bacteria grown

in a pure culture from the mixed culture

Stage1 (1 day) data ________________

Mixed bacteria’s (emulsion of

faeces of sick person)

Microscopy:

Study morphology and

staining properties of the

Gram’s stained bacteria

Inoculate material on the

selective medium and

differential diagnostic media -

Endo agar

Endo agar contains lactose and

pH indicator, so lactose-positive

microorganisms form red

colonies. Lactose negative

microorganisms form pink or

colorless colonies . Incubate in the thermostat at 37° C for 18 - 24 hours (1 day).

18

Task 2. Take the first stage of isolation pure culture of staphylococcus from nose.

Scheme for isolating Staphylococcus aureus bacteria grown in pure aerobic culture

Stage1 (1 day) data ________________


Using a cotton swab, take the

rheum (mucus) from a nose).

Inoculate the selective medium - Egg yolk agar – with the material.

Selective media contain 8 to 10% NaCl. Staphylococci are tolerant to sodium

chloride in concentrations of 5-10%. Salt-containing media are used for isolating

of staphylococci from samples containing large numbers of other bacteria

Incubate in the thermostat at 37° C for 18-24 hours (1 day)

19

Date _____________________Class № 7

GROWTH AND REPRODUCTION OF BACTERIA. ISOLATION OF PURE CULTURES OF

AEROBIC BACTERIA (2-4 stages). BACTERIAL CULTURE METHODS.

PRINCIPLES OF ANTIBIOTIC THERAPY


1. Principles of cultivation of microorganisms in culture media.

2. Growth and reproduction of microorganisms. Phases of growth in bacterial population.

3. Cultural characteristics of microorganisms (growth in liquid and solid culture media).

4. Bacteriological method in the diagnosis of infectious diseases.

5. Enzymes of bacteria. Classification of enzymes: exoenzymes, endoenzymes, constitutive enzymes

and adaptive enzymes.

6. Three groups of enzymes classified according to their chemical properties. Classification of

enzymes on the basis of the type of reactions.

7. The third and fourth stages of isolation of bacteria in pure culture (identification of bacteria):

morphological characteristics, physiological characteristics and chemical characteristics.

8. Antibiotics: definition and classification.

9. Spectrum of activity and efficacy of antibiotics (bacteriostasis, bactericidal activity, postantibiotic

effect (L-forms)).

10. Mechanisms of action of antibiotics.

11. Side effects of antibiotics: toxic effects, allergic reactions, side effects of biological therapies.

12. The problem of antibiotic resistance: definition, species, clinical and biochemical drug resistance.

13. Tests for antimicrobial drug susceptibility: sensitivity tests, disk-diffusion antimicrobial test.

Interpret the results.



Task 1. Study macroscopic characteristics of a microorganisms isolated on liquid media.

Growth of

Streptococcus in

sugar peptone broth

Growth of

Vibrio cholerae

in nutrient broth

Growth of

S. aureus

in nutrient broth

Growth of Leptospira

interrogans

in liquid media

Picture

Cultural characteristics

Task 2. Study the isolated colonies in a differential diagnostic media (Endo agar, Levin agar and

Ploskirev’s media).

20

The growth of E. coli

in the Endo's

medium

The growth of E. coli

in the EMB medium

Growth S. typhi in the

Ploskirev's medium

Growth S. typhi in the

Endo's medium

Picture


Task 3. Study the biochemical properties of bacteria in pure cultures isolated from patient (Hiss'

media and nutrient broth). Fill in the table below with your results.

Bacterium Glucose Lactose Mannitol Maltose Sucrose Meat infusion

broth

H2S indole

Escherichia

coli

Salmonella

typhi

Salmonella

paratyphi A

Salmonella

paratyphi B

Symbols: A – acid; G – gas

Task 4. Study the biochemical properties of bacterial pure cultures isolated from patient in Ressel

medium. Fill in the table below with your results.


21

Task 5. Study the isolated colonies of Staphylococcus aureus and Staphylococcus epidermidis in egg-

yolk salt agar and blood agar.

Staphylococcus aureus and Staphylococcus

epidermidis on yolk-salt agar Staphylococcus aureus and Staphylococcus

epidermidis on blood agar Picture


Task 6. Define the positive and negative reactions of plasma coagulase test.

This test is used to determinate coagulase (it is an enzyme of pathogenic bacteria causes blood

plasma coagulation, allowing bacteria to penetrate deeper into host tissues).


Task 7. Examine the Petri dish using Kirby-Bauer disk-diffusion method, indicating antibiotic

ingibiting the growth of isolated strain. Draw a conclusion.


Antibiotic Diameter of zone of

growth inhibition

(mm)

Antibiotic sensitivity

Task 8. Define sensitivity of staphylococci to phytoncide. Draw a conclusion.


22

Task 9. Examine sensitivity of staphylococci to penicillin by using serial dilutions method in liquid

media.

Ingredient Test tube

1 2 3 4 5 6 7 8 9 10 Control

of

bacteria

Control

of

antibiotic

Nutrient broth, ml 2.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

Preparation of serial

dilutions

1.0

→

1.0

→

1.0

→

1.0

→

1.0

→

1.0

→

1.0

→

1.0

→

1.0

→

1.0

↓

Antibiotic

concentration

(mg/ml)

50 25 12.5 6.2 3.1 1.6 0.8 0.4 0.2 0.1 50

Bacterial suspension 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1

Incubation in the thermostat at 37 °C during 18–24 hours

Result

Twelve test-tubes are needed for the experiment Two ml of nutrient broth are added into first tube.

Put 1 ml of the media in other tubes ntaining 32 units of penicillin are poured in the 1st and 11th test-

tubes. Then the numbers of consecutive dilutions are added by transferring 2 ml from the first tube in the

second and so on up to the 10th tube. There will be 0.008 units of penicillin in 10 test tubes with 2 ml of

nutrient broth.

In each tube add 0.4 ml of staphylococcus suspension. Control of bacteria tube contains 2 ml of nutrient

broth with microbe culture without antibiotics. Control of antibiotic tube contains 2 ml of nutrient broth

with antibiotics without culture. After inoculation in the thermostat for 24 hours record the results of

investigation.

Sign “–” marks the absence of growth, sign “+” denotes the growth of investigated microorganism.

After the 1st day, the minimum bacteriostatic (that suppresses) of antibiotic concentration is determined.

It is considered to be the lowest antibiotic concentration at which bacteria do not propagate and the tube

content remains transparent.

Conclusion: minimum inhibitory concentration (MIC) _________________________________

minimum bacteriocide concentration (MBC) _______________________________

Task 10. Describe the stages of isolation of aerobic bacteria in pure culture (draw a general

algorithm for isolation of aerobic bacteria in pure culture).

Teacher's signature ___________________

23

Date _____________________Class № 8

OBLIGATE ANAEROBIC BACTERIA. METHODS FOR ISOLATION OF ANAEROBIC

CULTURE. METHOD FOR BIOLOGICAL RESEARCH.

Relevance of the topic: Bacteriological and biological methods of diagnosis are the main methods

of microbiological diagnosis of anaerobic infectious diseases such as gas gangrene and tetanus. Every

experienced physician must be able to know how to conduct it, beginning with the selection of the

material for staining, and ending with the evaluating of the obtained results.

Primary objective: To be able to cultivate anaerobic bacteria in culture media, isolate pure

culture, and evaluate the results of identification tests.


1. Classification of obligate anaerobes: spore-forming obligate anaerobes (Clostridium), nonspore-

forming microbes (Bacteroides, Fusobacterium, Propionobacterium, Eubacterium, Peptococcus and

Peptostreptococcus).

2. Physiology of obligate anaerobic bacteria.

3. Methods for isolation of anaerobic bacteria in pure culture.

4. Anaerobic jar.

5. Biological research method: its application in the study of aetiology, pathogenesis, immunogenesis,

diagnosis, therapy, and prevention of infectious diseases. Use of laboratory animals, animal lines.



Task 1. Perform the microscopy and draw anaerobic species of pathogenic bacteria.

Preparates


C. tetani,

Gram staining

C. perfringens,

Ziehl-Neelsen staining

Peptostreptococcus spp.,

Gram staining

B. bifidum,

Gram staining

24

Task 2. Draw the growth of the obligate anaerobic bacteria (C. perfringens) in special culture

medium.

Growth of

C. perfringens

in the blood-sugar

Zeissler agar

Growth of

C. perfringens

in Litmus milk

Growth of

C. perfringens in

Kitt-Tarozzi medium

Growth of

C. perfringens in

Wilson-Blair medium

Picture


Task 3. Study the Fortner's principle, the method of Brewer’s anaerobic Petri dish, and the method

of Veyon-Vinyal.

Fortner's principle method Veyon-Vinyal

method

Brewer’s anaerobic Petri dish

Picture

Principle of operation

Task 4. Perform the 2nd stage for isolation of pure culture from colonies of anaerobic bacteria

taken from the soil.

Kitt-Tarozzi medium with isolated

1. Growth in the Kitt-Tarozzi medium; soil bacteria

2. Incubation in the thermostat at 37 ○C for 18 h

Soil solution


25

Date _____________________Class № 9

BACTERIAL GENETICS. BACTERIOPHAGES

Relevance of the topic: Genetic mechanisms have opened new perspectives in the diagnosis of

infectious diseases. In addition, it is the reason of the microorganisms resistance to antibiotics.

Polymerase chain reaction, which is now widely used for diagnostics is also based on genetic principles.

Primary objectives: To be able to conduct and evaluate the experiments in the genetic

recombination; to learn the principles of phagotyping and the use of bacteriophage in medicine.


1. Genetics of microorganisms.

2. Genotypic and phenotypic variations.

3. Transmission of genetic material.

4. Plasmids: definition, classification and function.

5. Bacteriophages: structure of bacteriophages and the life cycles of bacteriophages.

6. Practical application of bacteriophages.

7. Polymerase chain reaction (PCR).



Task 1. Perform and read a conjugation reaction.

Too cultures are used for a conjugation reaction:

1. E. coli F+ Pro+, Ura+, His+, StrS. This culture has fertility-factor, F-plasmid, and is able to produce

proline, uracil and histidin, but it is streptomycin-sensitive.

2. E.coli F-, Pro-, Ura-, His-, StrR. It is streptomycin-resistant. Base medium does not contain amino acid

and contains streptomycin is used for this experiment.

Conclusion:__________________________________________________________________________

____________________________________________________________________________

26

Task 2. Perform and read a transformation reaction.

In this experiment, one should perform a transformation using:

1. S. aureus as a recipient culture, it is streptomycin-sensitive.

2. Foreign DNA contains streptomycin-resistant genes.

3. Selective medium that contains streptomycin.

Bacterial transformation principle

Conclusion:__________________________________________________________________________

_____________________________________________________________________________________

_______________________________________________________________________

Task 3. Perform and read the manifestation of multiple antibiotic resistance of bacterial culture of

Staphylococcus aureus.

MICROBIOLOGY LABORATORY REPORT

Patient’s Name:___________________________________________________________________

Sex: __________________ Age: __________________________ _Date:_____________________

Antimicrobial Susceptibility Report

Name of Organism: _______________________________________________________________

Source: _________________________________________________________________________

Antimicrobial Agent S I R

27

Task 4. Study the

bacteriophages for

treatment, prophylaxis

and for diagnostic

purposes.Preparation

Purpose of use

Polyvalent Shigella

bacteriophage (liquid)

Polyvalent Shigella

bacteriophage (in tablets)

Polyvalent bacteriophage of

salmonella group

А,В,С,D,Е (in tablets)

Vibrio cholera O1 biotype

El Tor bacteriophage

(liquid)

Task 5. Identify the bacterial cultures by means of species phages by method of phage typing.

Phage typing (phagotyping method):

_____________________________________________________________________________________

_____________________________________________________________________________________

_____________________________________________________________________________________

_____________________________________________________________________________________

_____________________________________________________________________________________

_____________________________________________________________________________________

_____________________________________________________________________________________

_____________________________________________


28

Task 6. PCR applications (master the principle of PCR testing to diagnose a wide variety of

infections and noninfectious diseases to fullfill all the tasks required)

Application areas of PCR

__________________________________________________________________________

__________________________________________________________________________

__________________________________________________________________________

__________________________________________________________________________

__________________________________________________________________________

__________________________________________________________________________

__________________________________________________________________________

PCR-based techniques

PCR-based techniques are methods that rely on the polymerase chain reaction (PCR) to amplify stretches

of DNA by creating many identical or near-identical copies

PCR components:

___________________________________________

___________________________________________

___________________________________________

___________________________________________

___________________________________________

___________________________________________

PCR steps Denaturation: __________________________________________________________________

Annealing: ____________________________________________________________________

Extension: ____________________________________________________________________


29

Date _____________________Class № 10

NORMAL MICROFLORA. BACTERIAL OVERGROWTH SYNDROME

Relevance of the topic: The state of normal microflora of human body is an important component

of the innate immunity. The breach of the quantitative and qualitative composition of the normal flora

(overgrowth syndrome) has causes and consequences that complicate the course and treatment of the

different diseases. The study of human normal microflora is held for suspected overgrowth syndrome,

diagnosis of endogenous infections and in patients working in harmful conditions or living in countries

with a disturbed ecosystem.

Primary objectives: to be able to diagnose of the intestine and other overgrowth syndrome biotopes

according to the results of the bacteriological investigation of the infectious diseases.


1. Conception of normal microflora of the human body. The stages of normal microflora development.

2. The normal microflora of the different biotopes of the human body.

3. The physiologic meaning of the normal microflora.

4. Overgrowth syndrome. Origin, development stages, diagnosis, treatment and prevention.



Task 1. Take dental plaque by use of sterile cotton swab. Make a smear, stain it by Gram’s method.

Microscopically examine the smear.

Preparation


Task 2. Study the growth of microorganisms from the skin of the hand on the nutrient agar.


Microscope picture Cell morphology and staining

30

Task 3. Study under the microscope and draw a smear preparation, write down a conclusion.

Preparation

Figure in color Cell morphology and staining

Smear of a newborn faeces,

Gram staining

Bifidobacterium bifidum,

Gram staining

Task 4. Biological preparation (eubiotic products)

Name of preparation Purpose of the use

Colibacterin

Bifidumbacterin

Bificol

Lactobacterin

Simbiter


31

Date _____________________Class № 10

DOCTRINE OF INFECTION

Relevance of the topic: Knowledge of the infection and infection components is important for the

formation of a physician’s knowledge about nature (etiology), pathogenesis, diagnosis, and treatment of

infectious diseases and complications.

Primary objectives: To be able to identify the main components of infectious process

(macroorganism, microorganism, and environment) and to choose the right treatment and prevention.


1. Pathogenicity and virulence of the microorganisms. Virulence factors, methods of determination.

2. Meaning of macroorganism, microorganisms. Natural and social conditions and heredity in the

emergence and development of infectious diseases.

3. Sources, routs, and mechanisms of infections transmission.

4. Pathogenesis of infectious diseases. Periods of infection.

5. Types of infection depending on the cause, pathogenesis, methods of infection transmission, clinical

manifestations.



Task 1. Study the pathogenic activity of Staphylococcus spp. (plasmocoagulase, DNAse,

lecitovitelase, haemolysin). Draw a conclusion about investigated patogenecity of the culture.

Demonstration Picture Interpretation of the result

Haemolytic properties of

bacteria

Coagulase test

DNAse test

Lecitinase activity

32

Task 2. Study the demonstration, draw it in the protocol. Draw a the conclusion

Preparation

Figure in color Cell morphology and staining

Task 3. List the bacteria that produce exotoxins or have endotoxins

Exotoxin producing bacteria Endotoxin having bacteria


33

Date _____________________Class № 11

IMMUNITY. FACTORS AND MECHANISMS OF THE INNATE IMMUNITY

Relevance of the topic: Immune system is the most important for sustainability of internal

environment (homeostasis) of the human body. There are no any inflammatory pathological processes,

which are not linked with the state of immunity. The termination of the pathological process depends

primarily on the state of immunity. Different specialists (infectionist, therapists, surgeons, oncologist,

etc.) need knowledge of immunology for understanding the diseases essence of illness, timely diagnosis,

treatment, and prevention of diseases complications. This theme contains information about congenital

factors and mechanisms of the immunity.

Primary objectives: to be able to essess the state of cellular and humoral factors of innate

immunity.

QESTIONS FOR DISCUSSION

1. Definition of immunity. Role of immunity factors and reactions in the infectious and noninfectious

acquired human pathology.

2. Innate and acquired factors of the immunity. The first line of the protection.

3. Types of the anti-infectious immunity.

4. Humoral factors and mechanisms of innate resistance (IR). Their definition and function.

5. Cellular factors and mechanisms of IR. Their definition and function. Importance of Mechnikov’s

scientific works.

6. The role of immunology in the development of medicine. Immune-based diagnosis, therapy, and

immunization.



Task 1. Study macrophages phagocytosis for latex beads. Stain the smears with methylene blue.

Figure in color Conclusion

Task 2. Evaluate phagocytic activity in the preparation (by counting phagocytic cells and

calculating the phagocytic index).

Figure in color Conclusion

Task 3. Determine lysozyme activity in human saliva.

Lysozyme is a proteolytic muramidase enzyme (lat. murus – wall). Hydrolysis of acetylamino-

polysaccharides of bacterial cells causes a violation of cell wall synthesis. Lysozyme is mucosal

protective factor and it is present in tears, saliva, blood, and mother's milk.

In order to determine of the lysozyme titre in saliva, do a series of consecutive 10-fold dilutions of

saliva (1:10, 1:100, 1:1000, 1:10000). The last tube is a control one, and it does not contain lysozyme.

34

Add 1 ml of Micrococcus lysodeikticus suspension that contains 1 billion microbial cells in all five

tubes. After incubation in a thermostat for 3 hours, the titre of lysozyme is definite. It is the last dilution in

which lysis of bacteria occurs.

The purpose of the reactions: ___________________________________________________________

_____________________________________________________________________________________

Dilution of saliva Control

1:10 1:100 1:1000 1:10000

Сonclusion:

Task 4. Phagocytosis in neonates (a) and adults (b).

Figure in color

a)

b)

Conclusion

a) b)

Task 5. Determination of blood serum bactericidal activity.

The purpose of the test: ________________________________________________________________

_____________________________________________________________________________________

Experiment 1. Prepare a series of serum dilutions. Then add 0,1 ml of the bacterial suspension in

every tube.

Blood serum dilution Control

1:10 1:20 1:40 1:80

Сonclusion:

35

Experiment 2. After incubation in a thermostat, inoculate each dilution of a serum on the medium.

The highest dilution of serum with no visible growth of bacteria is considered as a bactericidal titer of

serum .

Figure in color Interpretation of result

Task 6. Evaluate the bactericidal action of neutrophils with the help of nitroblue tetrazolium

reduction test (NBT test).

The purpose of the reaction productions: ____________________________________________

________________________________________________________________________________

Picture Conclusion


36

Date _____________________ Class № 12

ANTIGENS

Relevance of the topic: Antigens have a critical role in the development of the immune responses

and in infectious process. Various antigens of the microorganisms are used in medical practice for

diagnostics, prevention, and sometimes treatment of the infectious diseases.

Primary objectives: to be able to distinguish different antigens and to use them in medical practice.


1. Antigens: Identification, structure (epitopes, carriers).

2. Classification of antigens by origin, chemical nature, immunogenicity.

3. The main properties of the antigens: antigenicity, immunogenicity, specificity.

4. Antigens of the human body: blood group antigens, self-antigen. Major histocompatibility complex

(MHC) antigens: identification, localization, HLA system, nomenclature, functions, role in the immune

response.

5. CD antigens of the cells of the immune system.

6. Bacteriak and viral antigens. Microbial mechanism of pathogenicity.

7. Antigen processing in the body. Superantigens.

8. Practical use of the microbial antigens.



Task 1. Learn demonstration antigenic preparations and fill out the table below:

Diagnostic preparations:

Antigen suspensions) and related antigens


of preparing

Purpose of preparation use

Brucelosis antigen suspension

Salmonella О and H antigen

suspension

Polysaccharide antigen of

Candida albicans

Salmonella antigen suspension

Shigella Sonnei and Flexneri

antigen suspension

37

Typhoid fever erythrocytic

antigen suspension

Whooping cough erythrocytic

antigen suspension

Gonococcal antigen

Whooping cough (pertissis)

antigen

Salmonellosis erythrocytic O

antigen suspension

Diagnostic allergens for skin allergy tests

Preparation Сomposition and method of

antigen preparing

Purose of preparation use

Tuberculin

Brucellin

Antraxin

Tularin

Candida allergens

Trichophytin

38

Task 2. Learn demonstration preparations for treatment and prevention and fill out the table

below:

Live (attenuated) vaccines

Vaccine preparation Сomposition and method of

vaccine preparing

Purpose of use

(characteristics of immunity)

BCG tuberculosis (TB) vaccine

Polio vaccine

Measles vaccine

Mumps vaccine

Rubella vaccine

Brucellosis vaccine

Influenza vaccine

Anthrax

vaccine

Plague vaccine

Tularemia vaccine

Yellow fever vaccine

Q-fever vaccine

Killed (inactivated) vaccines

Pertussis vaccine

Gonorrhea vaccine

39

Influenza vaccine

Rabies vaccine

Autovaccine

Leptospirosis vaccine

Brucellosis vaccine

Chemical vaccine

Typhoid fever vaccine

Meningococcal infection

vaccine

Influenza vaccine

Haemophilus influenza type B

(Hib)

vaccine

Recombinant (genetically engineering) vaccines

Hepatitis B

vaccine

Toxoid vaccines

Diphtheria toxoid

Tetanus toxoid

Staphylococcus aureus toxoid

Associated vaccines

DTaP (adsorbed diphtheria,

tetanus and pertussis) vaccine

Sexta toxoid (C. botulinum

types A, B and E, C. tetani,

C. perfringens, C. novyi

toxoids)


40

Date _____________________Class № 13

ADAPTIVE HUMORAL IMMUNE RESPONCE.IMMUNOGLOBULINS. FLOCULATION

AND NEUTRALISATION TESTS.

Relevance of the topic: The humoral immune response is one of the main reactions to the

introduction of the antigens in the human or animal body. Humoral immune response determines the

evaluation of the immunogenicity antigens. The result of the humoral immune response is mediated by

antibodies. One of the basic properties of the antibodies is their specificity. It determines their use for

diagnostic purpose: to detect different antigens. Precipitation reaction and its variations are used in

medical practice to identify pathogens and toxins (anthrax, meningococcal infection, diphtheria, etc.). The

antitoxic serum is used in treatment and prevention of diphtheria, tetanus, botulism, and other diseases.

Primary objectives: To be able to use precipitation and agglutination tests in medical practice and

to evaluate their results.


1. Adaptive humoral immune response: Definition, scheme, and phases. The role of cellular and humoral

factors in the immune response. Antigens stimulated humoral immune response.

2. Definition, structure, functions, and properties of antibodies. The obtaining and practical use of the

antibodies.

3. Immunoglobulins (Іg): Types, properties, and functions.

4. Exotoxin, toxoid, and antitoxin: definition, properties, flocculation test, and practical use.

5. Neutralisation test: components, purpose of their use, and procedure.



Task 1. Draw a scheme of the adaptive humoral immune response.

41

Task 2. Study and make a record in the protocol of the demonstrated immune serum (antitoxins)

and antibodies.


of preparing

Purpose of use

Antidiphtheria serum

(antitoxin)

Antibotulinum serum

(antitoxin)

Antipertussis (whooping

cough) immune globulin

Task 3. Titrate the antitoxic serum by means of the flocculation method and evaluate the result.

Scheme of the antitoxic serum titration

Tube

number

Component Result after 20 minutes

incubation in the thermostat Antitoxic serum Diphtheria toxin (50 lf/ml)

1

2

3

4

5

0.1

0.15

0.2

0.25

0.3

2.0

2.0

2.0

2.0

2.0

Initial

flocculation

Result:

Conclusion:

_____________________________________________________________________________________

_____________________________________________________________________________________

_____________________________________________________________________________________

_________________________________________________________________

Task 4. Study the viability of the mice in neutralisation test (NT) to confirm the presence of

botulinum toxin in food under examination: components, purpose of use, and procedure.

Components:

_____________________________________________________________________________________

_____________________________________________________________________________________

_____________________________________________________________________________________

_____________________________________________________________________________________

_____________________________________________________________________________________

_______________________________________________________ Purpose of use: _____________________________________________________________________________________

_____________________________________________________________________________________

_____________________________________________________________________________________

_________________________________________________________________

42

Procedure:

_____________________________________________________________________________________

_____________________________________________________________________________________

_____________________________________________________________________________________

_____________________________________________________________________________________

_____________________________________________________________________________________

_______________________________________________________ _____________________________________________________________________________________

_____________________________________________________________________________________

_____________________________________________________________________________________

_____________________________________________________________________________________

_____________________________________________________________________________________

_______________________________________________________

Conclusion:

_____________________________________________________________________________________

_____________________________________________________________________________________

_____________________________________________________________________________________

_____________________________________________________________________________________

____________________________________________________________


43

Date _____________________Class № 14

SEROLOGICAL TESTS: AGGLUTINATION, PRECIPITATION, COMPLEMENT

FIXATION TEST (CFT), AND TESTS USING LABELED ANTIBODIES

AND ANTIGENS (ELISA, IFT, RIA)

Relevance of the topic: Serological reactions are used in modern medical practice to diagnose

infectious and other diseases, as well as to determine their effective treatment and prevention.

Primary objectives: To know the purpose of conducting serological reactions in medical practice

and to be able to evaluate their results correctly.


1. The main goals and principles of conducting serological tests in medical practice.

2. Agglutination test and indirect (passive) haemagglutination test (PHAT): definition, mechanism,

and practical use.

3. Precipitation reaction: identification, mechanism, types, and practical use.

4. Agglutination and precipitation sera: preparation, titration, and practical use.

5. Complement fixation test (CFT): the purpose of the test, components, and mechanisms.

6. Immunofluorescent test (IFT): the variety, purpose of the test, components, and mechanisms.

7. Enzyme linked immunosorbent assay (ELISA): the purpose of the assay, components, and

mechanisms.

8. Radioimmunoassay (RIA): the purpose of of the assay, components, and mechanisms.



Task 1. Carry out a slide agglutination test. Prepare a smear on a glass slide, stain it with useof the

fuchsin, and examine it microscopically for agglutination.

Components:

_____________________________________________________________________________________

_____________________________________________________________________________________

_____________________________________________________________________________________

Result of the slide agglutination test:

Basic testing principle:

44

Stain it using fuchsin, and examine microscopically:

Conclusion:

_____________________________________________________________________________________

_____________________________________________________________________________________

_____________________________________________________________________________________

Task 2. Carry out a tube agglutination test. Record the results in the protocol and draw a

conclusion.

Components:

_____________________________________________________________________________________

_____________________________________________________________________________________

_____________________________________________________________________________________

Agglutination test

1 2 3 4 5 6 7

Dilution

Conclusions:

Task 3. Carry out a ring precipitation test to detect species that belong to the blood spot. Write

down the response to the test in the protocol, estimate the results, and draw the conclusion.

Components:

_____________________________________________________________________________________

_____________________________________________________________________________________

_____________________________________________________________________________________

_____________________________________________________________________________________

Basic testing principle:

45

Ingredients Number of the tube

1 2 3

Precipitating serum to the human blood protein

(ml).

0,2 - -

Precipitating serum to the sheep blood protein (ml). - 0,2 -

Saline (ml) - - 0,2

Extracting bloodstain «X» (ml) 0,2 0,2 0,2

The result (precipitate formation)

Drawing

Conclusion:

_____________________________________________________________________________________

_____________________________________________________________________________________

_____________________________________________________________________________________

Task 4. Evaluate the result of the gel precipitation test. Record the result in the protocol and draw a

conclusion.

Components:

_____________________________________________________________________________________

_____________________________________________________________________________________

_____________________________________________________________________________________ Purpose of use _____________________________________________________________________________________

_____________________________________________________________________________________

Testing principle and result:

Conclusion:

_________________________________________

_________________________________________

_________________________________________

_________________________________________

_________________________________________

_________________________________________

_________________________________________

_________________________________________

_________________________________________

Task 5. Evaluate the result of the complement fixation test (CFT). Write it in the protocol and draw

a conclusion.

Components:

_____________________________________________________________________________________

_____________________________________________________________________________________

_____________________________________________________________________________________

46

Purpose of use: _____________________________________________________________________________________

_____________________________________________________________________________________

Test-tubes Components

Pre

sence

of

hem

oly

sis

Exam

ine

a pat

ient's

seru

m i

n t

he

dil

uti

on 1

: 5

Anti

gen

(w

ork

ing

dose

)

Com

ple

men

t

(work

ing d

ose

)

Sal

ine

Ther

most

at:

tem

per

ature

: 37º

C,

tim

e: 40 m

inute

s

Hem

oly

tic

syst

em

Ther

most

at:

tem

per

ature

: 37º

C,

tim

e: 40 m

inute

s

1 step (research)

2 step (serum

control)

3 step (antigen

control)

0.5

0.5

-

0.5

-

0.5

0.5

0.5

0.5

-

0.5

0.5

1.0

1.0

1.0

Records of the

results

1st (research)

2nd (serum control)

3d (antigen control)

Drawing

Conclusions

Task 6. Evaluate the result of the passive (indirect) hemagglutination test (PHAT). Write it in the

protocol and draw a conclusion. Components:

_____________________________________________________________________________________

_____________________________________________________________________________________

_____________________________________________________________________________________

Purpose of use: _____________________________________________________________________________________

_____________________________________________________________________________________

Result:

47

Conclusion:

_____________________________________________________________________________________

_____________________________________________________________________________________

_____________________________________________________________________________________

Task 7. Evaluate the results of the direct (A) and indirect (B) immunofluorescence tests. Write the

results it in the protocol and draw a conclusion. Figure in color (result)

A) IFT

Components:

B) iIFT

Components:

Picture (Result)

Conclusion

Task 8. Conduct an ELISA and evaluate the result. Draw a conclusion.

Components:

_____________________________________________________________________________________

_____________________________________________________________________________________

_____________________________________________________________________________________

Purpose of use: _____________________________________________________________________________________

_____________________________________________________________________________________

Testing principle:

Positive ELISA Negative ELISA

Principle

Result (figure in color)

Conclusion


48

Date _____________________Class № 15

CELLULAR IMMUNE RESPONSE. IMMUNOTOLERANCE.

REGULATION OF THE IMMUNE RESPONSE.Relevance of the topic: The adaptive immune

responses lead to infectious diseases caused by intracellular parasites, cancer, autoimmune diseases,

delayed hypersensitivity, and in transplantation immunity. Knowledge of the mechanisms and assessment

of the cell type immune response are necessary for a doctor to understand the pathogenesis, diagnosis,

and treatment of these states. Establishment of artificial immunological tolerance is used in organ

transplantation. There are many situations in medical practice when a doctor has to interfere with the

regulation of patients’ immune responses (stimulation or depression and knowledge of the mechanisms of

these processes).

Primary objective: To be able to estimate the state of cellular immunity to detect

immunodeficiency.


1. Adaptive cellular immune response: Definition and varieties of the adaptive cellular immune

response. Antigens are caused the cellular immune response.

2. Cytotoxic type of the cellular immune response: pattern (scheme, cytokines, effector cells,

mechanisms of action, apoptosis.

3. Diseases in which the leading role belongs to the cytotoxic type of the immune response.

4. Delayed type of the cellular immune response: skim, cytokines, effector cells, mechanism of action.

5. Diseases in which the leading role belongs to the delayed type immune response.

6. Primary and secondary immune responses. Memory cells, practical value.

7. Immune tolerance: definition, types, mechanisms of action, practical use.

8. Regulation of immune responses: exhaustive factors and mechanisms.



Task 1. Examine antigenic preparations and record the results in the protocol, and indicate their

composition and purpose of use.


of preparing

Purpose of preparation use

Live vaccine

BCG vaccine

Brucelosis vaccine

Anthrax vaccine


Tuberculin

Brucellin

49

Antraxin

Tularin

Task 2. Study the microscopic preparation of lymphocyte apoptosis viewed under light microscopy.

Picture Conclusion

Task 3. Draw the scheme of cytotoxic cellular immune response.

Task 4. Draw the scheme of the delayed-type hypersensitivity.


50

Date _____________________Class № 16

ANTI-INFECTIOUS IMMUNITY. IMMUNE SYSTEM PATHOLOGY

Relevance of the topic: The state of the immune system affects the onset of an infection disease.

The disease in turn, affects the humoral and cellular indices. The disease that cause the immune system

disorders are rather common among the population and tends to increase incidence of their occurence.

These are immunodeficiency, allerges, autoimmune and lymphoproliferative diseases.

Primary objective: Knowledge of the state of the immune system and the nature of the immune

response various infectious diseases. To be able to treat, prevent and diagnose these diseases relying on.

To know the pathogenesis of immune diseases and principles of the immunodiagnosis and

immunotherapy.

QESTIONS FOR DISCUSSION 1. Anti-infectious immunity: definition, classification. Variety of types of anti-infection immunity:

humoral, cellular (cytotoxic and inflammatory), antitoxic, antibacterial, antiviral, local, general,

antifungal, antiprotozoal, protective, nonprotective, sterile and nonsterile.

2. Types of the immune system reactions, which are formed in response to pathogen entry into the

body and the role of immunity in the pathogenesis of infectious diseases.

3. Participation factors of innate immunity in the pathogenesis of infectious diseases (phagocytosis,

natural killer, complement system, acute phase proteins: pentraxin, biogenic amines, histamine and

seratonin, eicosanoids, prostaglandins, leukotrienes and cytokines).

4. Participation factors of humoral immunity in the pathogenesis of infectious diseases.

5. Participation factors of cellular cytotoxic immunity in the pathogenesis of infectious diseases.

6. Participation factors of cellular immunity in the pathogenesis of the inflammatory-type infectious

diseases.

7. Interaction between innate and adaptive factors in anti-infectious immunity.

8. Mechanism that microorganisms use against immune system.

9. Components of the agents that modify the immune response (locus of the pathogen, exo- and

endotoxins, enzymes, peptidoglicans, capsule antigens, immunoglobulin-binding proteins, etc.).

10. Use of humoral and cellular immunity in the diagnosis, treatment and prevention of infectious

diseases.

11. The types of the immune system disorders (immunopathology).

12. Immunodeficiency: definition, classification and clinical manifestations.

13. Principles of the diagnosis and treatment of the immunodeficiency.

14. Allergy (hypersensitivity): definition, types and immunological mechanisms.

15. The principles of the diagnosis and treatment of allergic diseases.

16. Autoimmune (autoaggressive) diseases: definition and mechanisms of development.

17. The principles of treatment and prevention of autoimmune diseases.



Task 1. Examine and enter into the protocol immunological preparations providing the purpose of

their appointment: diagnosis, treatment, and prevention.

Preparation Сomposition and method of

preparing

Purpose of use

Diphtheria antitoxic (DAT)

serum

51

Tetanus antitoxic serum

Botulism antitoxic serum

Gas gangrene antitoxic serum

Vaccine against tuberculosis

(BCG)

Vaccine against brucellosis

Vaccines against anthrax and

tularemia

Vaccine against plague

Tuberculin

Brucellin

Antraxin

Tularin

52

Task 2. Evaluate the results of PHAT conducted with the serum of patient with whooping cough at

the 2d and 12th days from the onset of the disease. Draw a conclusion.

Serum dilution

1:2 1:4 1:8 1:16 1:32 1:64 1:128 DC SC

І

ІІ

Conclusion:

Task 3. Evaluate the results of CFT. It was conducted with the paired serum of the patient with

syphilis, evaluate results and make a conclusion.

Serial dilution of serum

1:10 1:20 1:40 1:80 1:160 1:320 1:640 DC SC

І

ІІ

Conclusion:


53

Date _____________________Class № 17

EVALUATION OF THE IMMUNE STATUS.

PRINCIPLES OF THE IMMUNE SYSTEM FUNCTIONING

Relevance of the topic: The state of the immune status of both patients and healthy people should

be determined under condition that they have immunodeficiency, and also that they will live and work

under extreme conditions. The immune system as well as other systems of the body has its own

peculiarities of functioning. A doctor should know and take into account these peculiarities in his

professional activity.

Primary objectives: To be able to evaluate the state of the immune system and to recognize human

immunodeficiency. To learn the principles of functioning of the immune system and features of the anti-

infectious immunity.

QESTIONS FOR DISCUSSION 1. Immune status of the human body: definition, principles and indications for examination.

2. Purposes and methods of determining cellular immunity.

3. A two-stage principle in the immune status assessment.

First-stage tests (tentative): - clinical analysis of peripheral blood: relative and absolute numbers of lymphocytes in the blood;

- determination of the number of T-and B-lymphocytes in the blood;

- evaluation of the major immunoglobulin classes (M, G, A) in serum;

- identification of the phagocytic activity of the leukocytes.

Second-stage tests (analytical): - identification of subpopulations of the regulatory T-lymphocytes (T-helper and T-suppressor cells);

- identification of spontaneous migration of e leukocytes and leukocyte migration inhibition testing with

the use of PHA;

- tuberculin skin allergic test and other allergy skin tests subject to identify which of the allergens the

most the population is sensitive;

- study of the proliferative activity of T-lymphocytes in response to mitogens and antigens in blast-

transformation test;

- determination of the dynamics of major cytokines that regulate cellular and humoral immune

responses (ІNF-γ, TNF-α, IL-2 and IL-4, 5, 6, 10) and modulate inflammation.

4. Principles of the immune system functioning.

5. The aims and methods of determining the state of humoral and cellular immune responses to

various infections.

6. Components of the pathogens (bacteria) that modify immune response (localization of the causative

agents, exotoxins and endotoxins, enzymes, peptidoglicans, capsules, immunoglobulin-binding proteins,

antigens, etc.).

7. Mechanisms that bacterial and viral pathogens use to evade host immune defenses.



Task 1. Study the technique of determining the total number of T-lymphocytes (CD3) in stained

smears of peripheral blood by a spontaneous rosette test

Picture Principle and interpretation of the result

54

Task 2. Study the blast transformation of lymphocytes, stimulated by non-specific mitogen

(phytohaemagglutinin -PHA), in the stained smear of the blood.


Task 3. Study the technique of the leukocyte migration inhibition (LMI) test in capillary tubes.


Task 4. Study the technique of Radial immunodiffusion ( Mancini technique).


Task 5. Study the immunograms.

Example 1. Define the type of the immunodeficiency in 55-year old the man, who has been

subjected to a three-time examinations for the presence of the major immunoglobulins in the peripheral

blood over the past two years. Results of the study:

Man (55 years

of age)

Ig (g/l)

A M G

a 0 2.7 30.3

b 0 1.9 39.0

c 0 2.1 28.5

Conclusion:

55

Example 2. A 60-year old man was operated due to rectal polyposis. The results of the

immunological investigation are given in the table. Determine the presence of secondary

immunodeficiency and the possibility of inflammatory complications after surgery.

A 60-year

old man

with rectal

polyposis

Leu

cocy

tes

Eosi

nophil

s

Monocy

tes

Sta

b n

eutr

ophil

s

Seg

men

ted

neu

trophil

s

Lym

phocy

tes

T-

lym

phocy

tes

B-

lym

phocy

tes

0-

lym

phocy

tes

T-h

elper

cel

ls

T-c

yto

toxic

cell

s

Phag

ocy

tic

index

Ery

thro

cyte

sedim

enta

tion

rate

Before

surgery

4.

3

3 1 2 71 23 71 13 16 61 10 21 11

2nd day

after

surgery

4.

8

4 2 3 63 28 68 6 26 64 4 36 12

6th day

after

surgery

4.

0

2 1 1 68 27 54 6 40 50 4 30 11

Conclusion:


56

Date _____________________Class № 18

SPECIFIC PREVENTION AND TREATMENT OF INFECTIOUS DISEASES. THERAPEUTIC,

PREVENTIVE, AND DIAGNOSTIC IMMUNOLOGICAL PREPARATIONS

Relevance of the topic: The use of specific immunological preparations in practical infectology is

one of the most effective means of diagnosis, treatment and prevention of many infectious diseases and

complications. Every doctor, regardless of specialty, should know the principles of production of

immunological preparations, rules for their use, mechanisms of action, and how to prevent disease

complications.

Primary objectives: to be able to use diagnostic, preventive, and curative immunologic

preparations in medical practice.


1. The principles of use of the immunological preparations for diagnosis, therapy, and prevention.

2. Principles of vaccination. Vaccination according to schedule of immunization and epidemiological

indications.

3. Types of vaccines to prevent infectious disease. The principles of preparation and action of the live,

killed, chemical, toxoid, subunit, recombinant and DNA vaccines.

4. Principles of vaccine therapy.

5. The use of allergens.

6. Immune serum (antitoxins) and antibodies for therapy: preparation, titration, and application.

Prevention of complications.

7. Diagnostic immune serum (agglutinated, haemolytic, precipitated, antiviral, and luminiscent),

preparation, titration, and application.

8. Monoclonal antibodies: preparation and application.

9. Antigen suspensions and antigens: preparation and application in serodiagnosis.



Task 1. Study and draw into the protocol the main antimicrobial drugs used in the treatment,

prevention, and diagnosis of infectious diseases.

Diagnostic preparations:

Antigen suspensions and antigens

Name of preparation Сomposition, method of

producing

Purpose of use

(name of the test)

Brucelosis antigen suspension

Salmonella О and H antigen

suspensions

Polysaccharide antigen of

Candida albicans

Salmonella antigen suspension

57

Shigella sonnei and flexneri

antigen suspension

Typhoid fever erythrocytic

antigen suspension

Whooping cough erythrocytic

antigen suspensions

Gonococcal antigen

Pertusis (whooping cough)

antigen

Salmonella erythrocytic

O-antigen suspensions


Tuberculin

(PPD skin test reagents)

Brucellin

Antraxin

Tularin

Allergens for Candida fungal

infections testing

Trichophytin

Preparations for treatment and prevention:

Live (attenuated) vaccines

Preparation Сomposition Purpose of use (characteristics

of immunity)

BCG tuberculosis (TB) vaccine

Polio vaccine

Measles vaccine

58

Mumps vaccine

Rubella vaccine

Brucellosis

Influenza vaccine

Anthrax vaccine

Plague vaccine

Tularemia vaccine

Yellow fever vaccine

Q fever vaccine

Killed (inactivated) vaccine

Pertussis vaccine

Gonorrhea vaccine

Influenza vaccine

Rabies vaccine

Tick-borne encephalitis (TBE)

vaccine

Autovaccine

Cholera vaccine

Leprosy vaccine

59

Leptospirosis vaccine

Brucellosis vaccine

Toxoids

Diphtheria toxoid

Tetanus toxoid

Staphylococcus aureus toxoid

Cholera toxoid

Polyvalent botulinum

toxoid(A-E)

Gas gangrene toxoid.

Chemical vaccine

Typhoid fever vaccine

Meningococcal infection

vaccine

Influenza vaccine

Haemophilus influenza type B

(Hib) vaccine

Recombinant (genetically engineering) vaccine

Hepatitis B vaccine

Associated vaccines

DTaP (adsorbed diphtheria,

tetanus and pertussis) vaccine

Sexta toxoid (C. botulinum

types A, B and E, C. tetani,

C. perfringens, C. novyi

toxoids)

60

Antitoxic sera

Antidiphtheria serum

(antitoxin)

Antitetanus serum (antitoxin)

Antibotulism serum (antitoxin)

Antigas gangrene

serum (antitoxin)


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