KURSK STATE MEDICAL UNIVERSITY

DERPATMENT OF MICROBIOLOGY

Guide-lines for practical classes

on medical microbiology

Methodical directions for independent students work

during the preparations for practical
classes on medical microbiology

KURSK - 2004
 Guide-lines for practical classes on medical microbiology. Methodical
directions for independent students work during the preparations for practical
classes on medical microbiology / P.V. Kalutsky, O.A. Medvedeva, S.N. Sergeeva.
- Kursk: KSMU, 2004. - 44 p.

Reviewers:
Professor A.A. Dolzzhikov,
Associated professor M.J. Smarhtin.

Methodical directions for independent students work during the preparations

for practical classes on medical microbiology. These directions will be used like
additional materials for teaching English-speaking students medical faculty on
microbiology department. It contains plan of study, task for self-training, test
questions and plan for practical classes.

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 КУРСКИЙ ГОСУДАРСТВЕННЫЙ МЕДИЦИНСКИЙ
УНИВЕРСИТЕТ

Кафедра микробиологии

Руководство к практическим занятиям

по медицинской микробиологии

Методические рекомендации для самостоятельной

работы студентов и самоподготовки
по медицинской микробиологии

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 Курск - 2004

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УДК 57961=111(072) Печатается по решению
ББК 28.44581.2 Англ я 7 редакционно-издательского
совета КГМУ

Руководство к практическим занятиям по медицинской микробиологии

Методические рекомендации для самостоятельной работы студентов и
самоподготовки по медицинской микробиологии. П.В. Калуцкий, О.А.
Медведева, С.Н. Сергеева. - Курск КГМУ, 2004. – 44 с.

Рецензенты
Профессор А.А. Должиков
Доцент М.Ю. Смахтин

Учебно-методическое пособие по медицинской микробиологии

предназначено в качестве дополнительного материала при обучении
англоговорящих студентов лечебного факультета на кафедре микробиологии.
Подразделено в соответствии с планом практических и содержит
дополнительный материал, вопросы для самоконтроля и план работы студента
на занятии.

ISBN 5-7487-0776-4 ББК 28.44581.2 Англ я 7

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© Коллектив авторов, КГМУ, 2004

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 Time-table of practical classes in medical microbiology

1. Microbiology diagnosis of enteric fever.

2. Microbiological diagnosis of enteric fever, bacillary dysentery, E.coli-
associated diarrheal diseases.
3. Microbiology diagnosis of enteric fever, bacillary dysentery, E.coli-
infection. Microbiology diagnosis of salmonellosis.
4. Microbiological diagnosis of enteric fever, bacillary dysentery, coli-
infection (completion). Microbiological diagnosis of cholera.
5. Microbiology diagnosis of cholera (completion). Quiz on ”Pathogens of
intestinal infections.”
6. Microbiology diagnosis of infections caused by pathogenic cocci.
7. Microbiology diagnosis of wound and pus-inflammatory infections caused
by pathogenic cocci (contamination) and anaerobic microorganisms.
8. Microbiology diagnosis of wound and pus inflammatory infections caused
by sporing and nonsporing anaerobes (continuation). Microbiology diagnosis of
diphtheria.
9. Microbiology diagnosis of anaerobic infections, diphtheria (completion).
Microbiology diagnosis of whooping cough, legionellosis, tuberculosis,
leprosy, actinomycosis.
10. Quiz on “Microbiology diagnosis of suppurative, wounds and air
transmitted infections.”
11. Microbiology diagnosis of zoonotic infections.
Microbiology diagnosis of plaque, tularemia, brucellosis, anthrax.
12. Microbiology diagnosis of zoonotic diseases (completion).
Microbiology diagnosis of infections caused by pathogenic spirochetes and
fungi.
13. Microbiology diagnosis of diseases caused by rickettsia, chlamydia,
mycoplasma.
14. Morphology and physiology of viruses. Methods of viral cultivation and
detection.
15. Causative agents of acute viral respiratory infections.
16. Causative agents of enteroviral (polyomielitis, Coxacie, ECHO), neuroviral
(rabies, viral encephalitis) infections and viral hepatitis.
17. Causative agents of AIDS. Oncogenic viruses.
18. Quiz on "Viral infection".

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 INTRODUCTION
You have studied a course of general microbiology with the part, describing
infection and immunity. It is necessary for the mastering of the clinical microbiology
course. At practical classes on general microbiology and infectious immunology you
already acquired knowledge of basic microscopical, microbiological and
immunological methods of investigation. The theoretical knowledge and practical
skills formed the essential basis for successful work at practical classes in medical
microbiology. During the classes you must independently make microbiological
diagnosis of infectious diseases by the usage of previous knowledge and experience.
Detection of etiologic and pathogenic role of isolating microorganisms is a
peculiarity of the microbiological and immunological laboratory diagnosis of the
infectious diseases.
Immunological changes and allergic conditions of the patient can be detected
by laboratory methods. It gives the possibility to choose effective immunological
drugs in certain clinical conditions. Examination of the isolated pathogen
characteristics and especially determination of its antibiotic sensitivity will help the
physician in the antibiotic strategy of successful treatment.
The main aim of clinical microbiology is detection of the ethiologic role of
microbes as causative agents of infection diseases and (or) immunological disorders
in patient's organism.
The main tasks of clinical microbiology are:
1) Detection of the ethiologic and pathogenic role of isolated pathogens;
2) Detection of the character and level of immunological and allergic disorders
in patient's organism;
3) Provision for the possibility of choice effective chemotherapeutic and
immunologic medicine for patient's treatment.
The solution of these tasks will help:
a) the physician: make the final diagnosis of an infection disease and
administrate the most effective antibacterial and immunological therapy;
b) the epidemiologist: make an epidemiological analysis for detection of
source, routes of infection transmission; detect carriers for prevention of the infection
spreading and make the decision about the specific prophylaxis in specific
communities.
Successful laboratory diagnosis of infectious diseases may take place only
during a good interaction and understanding between the physician and
microbiologist. Correct collection and transportation of patient's samples have a great
meaning for investigation. The choice of the investigated material are provided by
preliminary clinical diagnosis, by the stage of a disease , routes of transportation and
by the peculiarities of a pathogen.
During the collections of a specimens from a patients or a carriers for
laboratory investigation it is necessary to follow special rules:
1. Samples material must be taken before the usage of antibiotics or other
chemotherapeutic drugs.

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 2. Collection of the material must be done in the correlation with the stage of
the disease, pathogenesis of infectious process and must state probable pathogen
localization.
3. Materials must be taken in sufficient amount , according to the aseptic rules
for protection of its contamination by microbes from the environment and from
human normal flora.
4. Materials must be delivered to the laboratory as soon as it possible in sterile
glass or in special containers under the primary temperature or in a frozen condition
(depending on the microbial characters).
5. Any laboratory material have be covering by letter, containing: full patient’s
name, kind of the material, date of its collection, preliminary clinical diagnose.
At practical classes in special (clinical) microbiology you must independently
make all the investigation for detection (depending on the material and direction of
the physician) of pathogenic species, determination of specific antibodies presence in
the organism or presence of allergy.
For this work you will use different methods studied before:
1) microscopical;
2) microbiological (bacteriological, mycological, virological);
3) biological;
4) immunological (serological, skin allergic tests, tests of the immune status
evaluation ).
During the preparation for practical classes in medical microbiology you must
remember the characteristics of the pathogens. We recommend you to describe
microorganisms according to the following scheme:
1. Morphology: form, cells arrangement in the slide, existence of spores,
flagella, capsule, inclusions and other morphological peculiarities.
2. Staining characteristics: ability to stain by aniline dyes, Gram's stain, special
method of staining.
3. Cultural characteristics: necessity of special culture media, culture media
used more often than others, temperature of the growth, type of respiration, character
of the growth on solid and liquid media, colonies characters etc.
4. Resistance to environment; resistance to temperature, drying; duration of
their presence in water, soil, air, other peculiarities.
5. Enzymes characteristics: possibility to ferment sugars, proteins, presence of
biological variants.
6. Antigenic characteristics: somatic (O), flagella (H), capsule (K) antigens and
their characteristics; division into serological variants.
7. Characteristics of pathogenicity: enzymes of pathogenicity, capsule,
adhesion, factors of toxicity etc.
Later during the study of different microorganisms groups you will have to
find and mark common characteristics for each group. After characterizing of
separated pathogens you will add in this common scheme peculiarities of each
pathogen. For example: all Enterobacteriaceae (pathogens of intestinal diseases) are
rods of middle sizes with irregular arrangement, peritrichous (except shigellae), gram
negative, but they differ one from another according to the enzymatic and antigenic

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characteristics. All pathogens of clostridia anaerobic infections are large gram
positive rods, spore-forming, peritrichous (except Clostridium perfringens),
anaerobes. They differ in the spore position, morphology of deep colonies, antigenic
structure of toxins and their effect in organism.
Preparation of these schemes will help you to study the characteristics of
microbial properties and shorten the time of your preparation. It is necessary to note
that these recommendations may be used only for the characteristic of bacteria. Other
groups of microorganisms (fungi, rickettsiae, viruses) have their peculiarities and will
be described in other chapters.
Studing the characteristic of the pathogen we start to prepare the scheme of the
investigation for microbiology diagnosis. At practical classes you will receive
patients samples and directions from the hospital with all information about the
patient and hypothetical diagnosis. Basing on the obtained specimen and patients
data you will prepare the scheme of the investigation for concrete material and make
the examination. You will write down all data, the order of the investigation, the
results and recommendations in a special copy-book.
In addition to the pathogens characteristic, scheme of microbiological and
immunological investigation of patient's samples you will discuss epidemiology,
treatment and specific prophylaxis of the infectious diseases.
At the lessons of special microbiology you will acquire the material necessary
for the doctor if you will do practical work with preliminary home preparations.
Good knowledge may be received only during the systematic study of the material
and when the theoretical knowledge is supported by practical work in the
laboratories.

COLLECTION OF SPECIMENS
Sterile Swabs. - Many specimens are collected by means of sterile swabs.
These are made by attaching a small piece of cotton to the end of a 6-inch wooden
applicator. The swabs are put into test tubes which are plugged with cotton stoppers
and autoclaved to ensure sterility. The applicator should be about an inch longer than
the tube, so that the end of the swab can be grasped without contaminating the part
that is inside the tube. Using a swab, be careful, doesn’t let the cotton or any other
part of the swab touch to anything, except the infected area from which the culture
is being taken.
For usage in the operating room, it is convenient to put the tube containing the
swab into a larger tube which in turn is stopped with a cotton plug and autoclaved.
Then the inner tube can be delivered to the operator, and can be handled without
danger of contamination, as the outer part of this tube is sterile.
Do not allow a swab to dry out before taking it to the laboratory. Many
organisms are extremely delicate, and bacteriological research for them will be
entirely futile if the specimen is permitted to dry.
Throat Cultures. - Material from the tonsils or pharynx may be collected with
sterile swabs. If anyone suspects a streptococcus infection of the tonsils, the swab
should be held against the tonsil for an appreciable number of seconds and gently but
firmly rotated over the surface of the tonsil.

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 Blood for Culture. - Clean the skin in the region of the bend of the elbow as
for surgical operation, using the iodine and allowing the iodine to dry and act for
three minutes before inserting the needle. Place a tourniquet (a length of soft rubber
tubing is satisfactory for the purpose) around the arm above the elbow and draw it
tightly enough to close off the veins without interfering with the entrance of the blood
through the arterial system; in other words, be sure that the radial pulse is palpable.
The operator then enters the vein by means of sterile needle and syringe, usually
withdrawing about 15 cc. of blood. This is transferred to a small flask containing a
sterile sodium citrate solution to prevent clotting of the blood.
Blood for Wassermann, Widal, etc. - Blood for these purposes is collected in
the same way as outlined above, except that it is not necessary to keep the blood
sterile after collection. It is usually placed in a small test tube (without containing of
sodium citrate) and allow to clot. Microscopic Widal reactions can be done with I or
2 drops of blood obtained by pricking the finger with a sterile needle; so collected
blood can be dried either on a slide or on a small piece of filter paper. The latter
method is recommended only in isolated districts where the blood must be sent to a
distant laboratory for examination. Even then, it is advisable to collect about 5 cc. of
blood by venipuncture, so that the laboratory will have sufficient blood to perform
the necessary tests.
Urine. - Urine for bacteriological study must be collected under sterile
precautions. Female patients are usually catheterized. Male patients are usually
cleaned by vigorous scrubbing with green soap and make final sterilization of the
external parts are made with bichloride solution. The first urine that is voided is
discarded, the last few cubic centimeters are collected in a sterile test tube.
Feces. - A small specimen should be placed in a container and sent to the
laboratory while still fresh. Particles of blood or pus that may be present there should
be included.
Sputum. - The patient should rinse out his mouth well by water or by mild
antiseptic solution. Sputum for culture is result of a deep cough or clearing of the
throat, and should be collected in a sterile container, such as a Petri plate. Do not
allow the patient to contaminate the exterior part of the container, this infection
may be spread to those who are to handle the material subsequently. Specimens of
sputum from babies or from unconscious patients can be obtained by holding a sterile
swab at the back of the throat until the patient coughs.
Sputum for microscopical examination of tubercle bacilli need not be
collected in a sterile container.
Cerebrospinal Fluid. – Collect it in a sterile culture tube by lumbar puncture.
The opened end of the tube should be flamed before and after the fluids adding.
Labeling Specimens. - It is essential that all specimens for bacteriological or
serological study be labeled properly. The seriousness of mixing tubes of blood for
the Wassermann reaction, for example, is too apparent to require emphasis. The label
should include the patient's name, his history number, the date, the source of the
material (throat, vein, bladder, etc.), the diagnosis, and the examination desired.
Many hospitals have blanks for this purpose, and the nurse should be acquainted with
the forms used in the hospital where she is working.

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 LESSON 1.
Microbiological diagnosis of enteric fever.

Plan of study.
1. Aim and tasks of clinical microbiology.
2. Directions and methods used in clinical microbiology diagnosis.
3. General characteristic of Enterobacteria.
4. Characteristics of the enteric fever causative agent.
5. Pathogenesis of enteric fever.
6. Methods of early diagnosis of enteric fever.
7. Serological diagnosis.
8. Diagnosis of the enteric fever pathogens carrier stage .

Task for self-training.

Write in Latin the genus names of microorganisms belong to
Enterobacteriaceae family and enteric fever causative agents .
Enterobacteriaceae Pathogens of enteric fever
_____________________ ___________________________

Test questions.
Subject and tasks of medical microbiology. Characteristics of methods of
investigations used in clinical microbiology: microscopical, microbiological,
biological, immunological. Rules of samples collection from patients and carriers.
Rules of the samples transportation to the laboratory.
Classification of the intestinal bacteria. General characteristic of the family
Enterobacteriaceae. Evolutionary relations between the enteric gram-negative rods.
Importance of nutrient media. Differential media used for diagnosis of digestive tract
infection : composition, character of different microorganisms growth . What
intestinal bacteria are motile and nonmotile ?
Salmonella genus: it’s characteristic, classification, Latin name of enteric fever
pathogens. Their morphology,cultural properties, factors of pathogenicity.How we
may differentiate the growth of enteric fever patogens from the growth of E.coli
using differential media.
Pathogenesis of enteric fever. Methods of the enteric fever diagnosis according
to the duration of the disease. Methods of enteric fever early diagnosis. Method of the
blood culture isolation: specimen (place of taking and volume), media and conditions
of cultivation.
Serological diagnosis of enteric fever: material for diagnosis. Enumerate
methods for the enteric fever pathogens carrier-stage diagnosis. When is it necessary
to make this investigations?

Practical classes.
1. Study the morphology of the enteric fever pathogens .
2. Early diagnosis of enteric fever. Blood inoculation for obtaining the
bloodculture .

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 3. Detection of the specific antibodies presence and determination their titre
using passive hemagglutination test.
4. Diagnosis of enteric fever pathogens carrier-stage .

LESSON 2.
Microbiological diagnosis of enteric fever, bacillary dysentery, E.coli-associated
diarrheal diseases.

Plan of study.
1. Diagnosis of enteric fever: blood culture (2-nd day), stool culture (2-nd day).
2. Antigenic structure and classification of Salmonella.
3. Characteristic and classification of Shigella.
4. Pathogenesis of bacillary dysentery, material for investigation, beginning of
the investigation.

Task for self-training

Latin name of Shigella species, classification of Shigella and causative agents
of escherichiosis.
Classification of Shigella.
Group Species Presence of serological variants.
A
B
C
D

Classification and characteristics of pathogenic E.coli.

Group Character of the pathological process Who may be infected
name (according to the localization) (according to the age)

Test questions.
Antigenic structure of Salmonella spp, localization of antigens in bacterial
cell. Classification of Salmonella by the antigenic structure (Kauffman-Wait scheme).
What antigens are genus specific? What antigens are species specific? Composition
and usage of the bile broth and Rappoport , s medium? How growth of S.typhi and
S.paratyphi A (S.paratyphi B) on the Rappoport’s medium can be differentiated? Giss
sugar media, selenite media: their composition and usage, detection of the results.
Methods of enteric fever pathogens carrier-stage diagnosis. Scheme of
investigation(day by day). Principles and method of the Vi-hemagglutination.
Treatment and prophylaxis of enteric fever.
Characteristic of the Shigella: name of species, classification. Antigenic
structure and toxin production . Culture media for the Shigella pure culture isolation.
Characteristic of immunity against bacillary dysentery. Why is the material from the
patient put into the conservant? What is the composition of the conservant?

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 Task. 1. 3 months later after the recovery from enteric fever a man was
examined on the enteric fever pathogens carrier-stage. What material must be taken
and how must be used?
2. What results of Vi-hemagglutination test will be after 3 months (positive or
negative):
a) after the recovery the absence of the enteric fever pathogens is tested;
b) after the vaccination against enteric fever;
c) carrier of the enteric fever pathogens.

Practical classes.
1. Continuation of haemoculture obtaining. Indicate changing on Rappoport’s
medium: presence of the growth, production of acid and gas. Prepare slide from
Rappoport’s medium. Make transinoculation on MacConkey agar.
2. Continuation of the stool culture obtaining. Study the pathogens growth on
EMB medium (Gram’s staining). Inoculate material from the isolated colony on
triple-sugar iron containing medium.
3. Examination of Vi-hemagglutination test result (diagnosis of enteric fever
pathogens carrier-stage).
4. Morphology and biochemical characteristic of Shigella.
5. Start the laboratory diagnosis of bacillary dysentery. Inoculate the
specimen from the conservant on the bile- containing medium.
6. Start the laboratory diagnosis of coli-infection. Inoculate the specimen on
the MacConkey agar.

LESSON 3.
Microbiology diagnosis of enteric fever, bacillary dysentery, E.coli-infection.
Microbiology diagnosis of salmonellosis.

Plan of study.
1. Identification of previously isolated pathogens of enteric fever, bacillary
dysentery, intestinal coli-infections.
2. Characteristic of causative agents of food poisoning and food-intoxications.
3. Mechanism of the food poisoning and food-intoxications beginning.

Additional material.
Identification of Salmonella cultures by the usage of monoreceptor
agglutination sera.
After the study of morphology and biochemical activity of the culture, isolated
from the patient it is necessary to detect it’s antigenic structure. It is done by the
usage of agglutination test with monoreceptor Salmonella sera.
Monoreceptor Salmonella serum is known as one type antibodies (receptors)
containing serum. For example, O-serum, receptor "4", contains antibody against
somatic salmonella antigen "4": H-serum "b" contains antibodies against flagella
antigen "b". For culture identification firstly make a slide agglutination test with the
mixture of some O-sera and after the positive result identify Salmonella. For this

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reason agglutination tests with different types of O-sera are done (for detection of the
pathogen’s group) and an agglutination test with specific H-sera is done. Species’
recognition of isolated salmonella culture are made in a similar way.

Test questions.
How is an agglutination test used for the detection of the microbial species?
Monoreceptor serum: what is this, method of it’s preparation. How is Salmonella
antigenic structure identification made by the usage of monoreceptor sera?
Phagotyping of enteric fever pathogens. What is the aim of phagotyping test?
Explain the terms "food poisoning" and "food intoxications". Enumerate
Salmonella species most frequently causing food poisoning. What another bacteria
may cause food poisoning? Conditions limited ability of the food poisoning. Scheme
of microbiological diagnosis of Salmonella food poisoning, specimens for diagnosis,
nutrient media, classification of pathogens, serological investigation.
What bacteria may cause food-intoxications? Scheme of microbiological
diagnosis of Staphylococcal food-intoxications. Scheme of botulism’s
microbiological investigation.
Scheme of the bacillary dysentery laboratory diagnosis. Principles of antigenic
classification of E.coli, antigens and their position in cell. Categories of pathogenic
E.coli. Characteristics of different representatives. Serological types of E.coli most
frequently cause coli-infection. Describe microbiological diagnosis of coli-infection
(day by day).

Practical classes.
1. Continue investigation of blood culture. On MacConkey medium choose the
isolated lactose negative colony and inoculate it on triple-sugar iron containing
medium.
2. Continue investigation of coproculture. Examine the result of pathogen’s
growth on triple-sugar iron containing medium: fermentation of lactose, glucose,
production of gas, H2S. Make the slide agglutination tests with specific monoreceptor
sera. Formulate an answer.
3. Continue investigation on bacillary dysentery. Study the growth of lactose
negative colony and make the slide agglutination test with S.flexneri and S.sonnei
sera.
4. Continue investigation of coli-infection. Study the growth of the colonies on
the MacConkey agar. Choose isolated lactose positive colonies, make a slide
agglutination tests using complex serum and microbes from different colonies till
getting positive results (up to 10 colonies). Colony given positive result inoculate on
nutrient medium for obtaining of pure culture.

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 LESSON 4.
Microbiological diagnosis of enteric fever, bacillary dysentery, E.coli-infection
(completion). Microbiological diagnosis of cholera.

Plan of study.
1. Completion of microbiological diagnosis of enteric fever, bacillary
dysentery, E.coli-infection. Conclusions of the investigations, formulation of
answers.
2. Study of biological preparations for diagnosis, specific prophylaxis and
treatment of digestive tract’s infection.
3. Characteristic of pathogenic vibrios.
4. Rules of speciemen’s collection and transportation for the diagnosis of
cholera. The first day of the cholera diagnosis.
5. Characteristic of parahaemolytic vibrio and campylobacters. Scheme of the
microbiological diagnosis of infections caused by halophilic vibrios and
campylobacters.

Task for self-training

Write in Latin name of causative agents of cholera, name of halophilic vibrios
and campylobacters (basic species).

Additional material.
Conditions for the collection and transportation of samples for cholera
diagnosis.
Collection of samples for the diagnostic investigations must be taken before
the beginning of the antimicrobial chemotherapy. It is better to collect native
samples, but in case of absence of this ability conservant media can be used.
1. Native samples (stool mass, vomiting mass) are collected from the
individual (washed by disinfectants) bedpan on the bottom of which a smaller vessel
(that may be disinfect by boiling) is put. Samples in volume 10-20 ml are transferred
into a sterile glass by the sterile metal spoons or spoons of another material. The
flack must be closed by the stopper or cork with the parchment.
2. In some cases (in convalescents) bile is taken from the duodenum probe and
examined.
3. In diseases similar to cholera the specimen is taken from the cadavers part of
upper, middle and low part of intestine in length of 10 sm. Parts must be separated by
ligatures which are put on the both ends of the intestine part. Gall bladder must be
taken off after the ligation of the duct.
4. During the convalescents investigation healthy persons preliminary are
given laxative (15-20 ml of MgSO4) for the collection of liquid stool from the upper
part of intestine. Samples must be put into the 1% peptone water. In case of massive
investigation of V.cholerae samples from 5-10 persons may be collected in 1 tube. If
we’ll get the positive result a sample from each person must be done. Conservant: 1%
alkaline peptone water (pH 9,1), peptone water or the potassium tellurite containing
medium etc. Food, soil, water, swabs from different subjects, flies, contaminating by

16
the patients excrements, clothing, inhabitants of rivers (fish etc.) must be examined.
Material must be put into the metal box and then into the container. It must be closed,
sealed up and sent with the special person.

Test questions.
Classification of pathogenic vibrios, diseases causes by them. Characteristic of
cholera vibrios: morphology, cultural characteristics, staining, enzyme activity,
resistance in environment. Antigenic structure, serological types. Toxin production.
Biochemical groups according to Haiberg’s classification. Non-O1/non-O139 vibrios.
Samples for cholera diagnosis. Sanitary regime of work with the samples from the
cholera patients and cholera carriers. Complete cholera diagnosis (step by step).
Rapid diagnosis of cholera. Examination of the water for V.cholerae detection.
Differentiation between V.cholerae classical type and V. cholerae eltor. Drugs for the
diagnosis, treatment and specific prophylaxis of cholera.
Campylobacter: classification (main species), morphology, Gram’s staining,
specificity of respiration and cultivation. Sources of infection. Diseases caused by
campylobacter. Specificity of patients’s samples collection and transportation.
Scheme of examination for campylobacter diagnosis Role of Helicobacter pylori in
ethiology of gastritis and ulcer of stomach and duodenum. Drugs for treatment.
Characteristic of parahaemolitic vibrio: morphology, culture, place of habitate
(of life), conditions for infection beginning, sensitivity to high salt’s concentration.
Diseases caused by parahaemolitic vibrio.

Practical classes.
1. Finish investigation of blood culture (4th day, demonstration). Describe the
character of microbes growth on triple-sugar iron containing medium. Make a slide
agglutination test with monoreseptor sera. Formulate an answer.
2. Finish investigation of E.coli-infection (3 day). Using pure culture of E.coli
make a slide agglutination test with type specific OB-E.coli serum. Describe the
results of tube agglutination test with alive and heated cultures. Formulate the result
of investigation.
3. Study morphology and biochemical characteristics of cholera vibrio.
4. Start investigation of the patient’s specimen infected by cholera vibrio. Read
the recommendations. Prepare a slide from stool mass, stain it by the fucshin and
examine under the microscope. Inoculate stool mass in tube with 1% peptone water
and on the alkaline agar.
5. Start water investigation for detection of cholera vibrio. Make inoculation of
water (450 ml) in sterile alkaline peptone solution (50 ml), (see p. suppl.).

LESSON 5.
Microbiology diagnosis of cholera (completion). Quiz on ”Pathogens of intestinal
infections, food poisoning and intoxications.”

Practical classes.
1. Finish an investigation of cholera. Study the growth of bacteria in alkaline
peptone water and on alkaline agar: pay attention to character of the growth, prepare

17
a slide, stain it by the fucshsin. Examine under the light microscope. Prepare "weat
smear" and examine motility of the microorganisms. Using isolated colonies and sera
(cholera "O1", "Inaba", "Ogawa") make the slide agglutination tests. Register the
results of polymyxin and phage sensitivity tasts. Formulate an answer.
2. Complete an investigation of the water. Count up a result. Clean the working
place.
3. Answer the questions of the test and take part in discussion: “Pathogens of
intestinal infections, food poisoning and food intoxications”.

LESSON 6.
Microbiology diagnosis of infections caused by pathogenic cocci.

Plan of study.
1. Characteristic of staphylococci, streptococci and their role in human
pathology.
2. Characteristic of gonococci, meningococci.

Task for self-training

Write Latin name of staphylococci, streptococci, gonococci and meningococci.

Test questions.
Staphylococci. Species name in Latin, what species frequently cause diseases?
Morphology, media for cultivation, selective media. Basic criteria for staphylococci
differentiation from micrococci and for differentiation of different staphylococci
species. Factors of staphylococci virulence. Preparations for treatment of acute and
chronic staphylococcal infections, for diagnosis and prophylaxis.
Streptococci. Form, arrangement of cells, production of spore, flagella,
capsule, Gram's staining. Culture media for cultivation. Toxins and enzymes of
virulence. Classification of Streptococci according to the blood hemolysis (write in
Latin). Other streptococci species which can cause diseases in a human. What species
frequently cause disease? Group specific antigen: chemical structure, serological
groups and their designation. What group includes the pathogenic streptococci? Type
specific antigens: chemical structure, which of them is factor of virulence, its action
on the organism, how many serological types are known, how are they designated,
what reaction is used for this detection? Role of streptococci in ethiology of scarlet
fever, erysepelas, rheumatic fever.
Streptococci of pneumonia: Latin name, morphology (form, arrangement,
spores, capsule), Gram's staining, type of respiration, media for cultivation. Antigenic
structure: localization and chemical nature of main antigens, serological types.
Diseases frequently caused by pneumococci. Microbiology diagnosis: material for
investigation, methods of cultural and animals’ inoculation. Identification of
pneumococci by inulin fermentation, sensitivity and bile, phenomenon of capsule
swelling. Character and intensity of immunity. Preparations for treatment, diagnosis
and prophylaxis.

18
 Meningococci. Name in Latin. Morphology and physiology: form,
arrangement, spores, flagella, capsule, Gram's staining. Media for cultivation, type of
respiration, optimum temperature for the growth, attitude to low temperature, factors
of pathogenicity. Antigenic structure: what pathogens of different serological groups
cause diseases frequently. Enumerate forms of meningococcal infection. Where are
they located? Enumerate specimens for investigation of different forms of
meningococcal infection, peculiarities of their collection and transportation to
laboratory. Make different schemes of microbiology investigation depended on form
of infection. Drugs for treatment and specific prophylaxis.
Gonococci. Name in Latin. Morphology and physiology: form, arrangement,
spores, flagella, capsule, Gram's staining. Media for cultivation, type of respiration,
optimum temperature for the growth, attitude to low temperature, factors of
pathogenicity. Antigenic structure. Microbiology diagnosis of acute and chronic
gonorrhea. Microbiology diagnosis of acute gonorrhea: material for investigation.
Bacterioscopical method of examination: methods of staining, peculiarities of
pathogen. Methods for diagnosis of chronic gonorrhea. Drugs for the diagnosis and
treatment of gonorrhea.

Practical classes.
1. Examine under the light microscope and draw slides: staphylococci,
streptococci in pure culture and staphylococci, streptococci in pus.
2. Study peculiarities of staphylococci and streptococci growth:
a) on blood agar
b) in meat-peptone broth.
3. Bacterioscopical and bacteriological examination of patient’s specimen
infected by staphylococci. Prepare a slide from pus, stain it by Gram’s method,
examine under the microscope and draw. Inoculate the pus by the strikes loop method
on blood agar for obtaining of isolated colonies. After that make antibiotic sensitivity
test.
4. Continue the culture isolation form the blood (streptococcal septicemia, 2-nd
day). 1-st: 10 ml of patient's blood has been inoculated into 100 ml of glucose
containing broth. 2-nd day. Describe the character of the growth. Make a slide, stain
it by Gram’s method, examine under the microscope and draw. After slide
preparation transinoculate microorganism on blood agar.
5. Detect staphylococcal carriers. Take mucous from the nose by a sterile
tampon and inoculate it on the milk salt agar.
6. Make laboratory diagnosis of chronic gonorrhea. Describe the result of
ready-prepeared complement fixation test and write down the result.
7. Bacterioscopical diagnosis of epidemic cerebrospinal meningitis. Examine
under the light microscope a slide of cerebrospinal fluid stained by Gram's method
and draw it.

19
 LESSON 7.
Microbiology diagnosis of wound and pus-inflammatory infections caused by
pathogenic cocci (contamination) and anaerobic microorganisms.

Plan of study.
1. Determination of the pathogenic criteria and species differentiation of
staphylococci.
2. Staphylococcus carrier stage, its diagnosis and meaning.
3. Streptococcal diseases: purulent, septicemia, scarlet fever, rheumatic fever
and it's diagnosis.
4. Characteristic of the gram negative aerobic-pathogens of wound and
purulent infections.
5. Characteristic of nonsporing and sporing anaerobic pathogens of wound and
pus-inflammatory infections.

Additional material.
Microbiological methods of
Staphylococcus identification.
The aim of the identification is determination of the genus and species of
isolated pure culture. Genus Staphylococcus is included by the Micrococcaceae
family. This family also includes Micrococcus and Planococcus genuses.
General features of Micrococcaceae family representatives are:
1) morphology, arrangement (grape-like), positive Gram's stain;
2) presence of catalase;
Genus Staphylococcus consists of 3 main species of clinical importance:
S.aureus, S.epidermidis and S.saprophyticus. Representatives of the two last species
are coagulase-negative and for a long time they were considered to nonpathogenic
microorganisms. Today it is proved that Staphylococcus epidermidis may cause such
diseases as miocarditis, sepsis, conjunctivitis, infection of wound and urino-genital
tract. Staphylococcus saprophyticus may cause acute urethritis and cystitis.
On the 1-st step detect membership of cocci to Micrococcaceae family.
For the detection of the culture membership to the genus Staphylococcus use
cultural and biochemical methods.
Cultural method. The basic difference of staphylococci from micrococci is the
colonies color. Gold or white colonies are significant for staphylococci. Micrococci
colonies are yellow or pink.
Biochemical method. It is based on the knowledge that Staphylococci are
facultative anaerobes and Micrococci are obligate aerobes. According to this
Staphylococci may grow and ferment glucose in anaerobic conditions but Micrococci
do not have this ability.
For the detection of these characteristic inoculate microorganisms into deep
layer of the glucose indicator containing medium. On the surface of the medium put
sterile vaseline oil and place the tube into incubator (37 0C) for 5 days. The culture
growth and media color changing indicates the availability of the genus
Staphylococcus.

20
 Specie identification of Staphylococci.
Staphylococcus aureus differ from other species by the presence of golden or
straw-colored pigment, enzymes (coagulase, lecithinase and DNA-se). Coagulase is
detected by the inoculation of culture into citrate rabbit’s plasma, incubation at 37°C
and registration of results after 1, 2, 4 and 18 hours. Appearance of gel on the bottom
of the tube is the positive result of the reaction.
Lecithinase is detected by the inoculation of the culture into egg salt agar. In
the composition of egg yolk there is lecithin. Inoculations are incubated at 37 0C
during 18-24 hours. Formation of inducible corolla may testify the lecithinase
production.

DNA-se detection.
Principles of the method. High polymeric DNA is added into the medium, it
splits into low polymeric fragments by action of microbial enzyme DNA-se. Muddy
medium is transformed into a transparent one.
Protocol of investigation: Using strikes-method inoculate the culture of
Staphylococci on medium containing 50 - 200 mg of DNA and 0,5 ml 10% solution
of CaCl and incubate it at 370C for 18-24 hours. After that put 5-7 ml solution of HCl
on the surface of the agar. If in 2-3 minutes there transparent zones around the
inoculations appear the reaction will be positive.
If culture of staphylococci produce golden (strawcolored) pigment, coagulate
plasma and have one of two other enzymes (lecithinase, DNA-se) this is
Staphylococcus aureus culture.
In case of absence of the full set of these criteria (the main one is presence of
coagulase) you have to continue identification of Staphylococcus epidermidis and
Staphylococcus saprophyticus by three criteria.

Staphylococcus Novobiocyne Phosphatase Oxidation of

specie resistance mannitol
S. epidermidis - + -
S. saprophiticus + - +

Novobiocyne resistance is detected by the culture inoculation on the

novobiocyne containig medium (2 mg/ml of novobiocyne).
Oxidation of mannitol is done by the m/o inoculation on the mannitol and
indicator containing medium. Chaging of medium color testifies the ability of the
m/o to ferment mannitol.
For strains of Staphylococcus epidermidis the most significant signs are:
- sensitivity to novobiocyne;
- presence of phosphatase and impossibility to fermentate mannitol.
For strains of Staphylococcus saprophyticus contrary properties are significant.
Isolated cultures of Staphylococcus aureus are usually subjected to phage typing with
the international phage set (22 phages).

21
 Rapid methods of anaerobic gas gangrene diagnosis.
1. Investigated material is put into 10 tubes with semisolid medium, 5 tubes are
heated for 20 minutes at 800C. After that different monovalent gangrene sera are
added to each tube. After 10-18 hours the appearance of microbes in streptobacilli
form and their growth as the isolating colonies shows the conformity of pathogen to
the specific serotype.
Growth as uniform turbidity and irregular position of clostridia shows the
absence of pathogen’s conformity to the specific serotype.
2. Skin test on guinea pigs. Material from the patient (tissue fluid) in mixture
with monovalent gangrene sera are injected intracuteneously to some guinea pigs.
One animal (control) is injected only by specimen from the patient.
Result of the skin test will be observed after 24 hours. If the neutralization of
the exotoxin by the specific serum takes place skin reactions there will be no. If
serum and exotoxin are nonspecific – one to another the guinea pigs skin in the place
of injection will become blue or violet in color.

Test questions.
Describe day by day the order of investigation and the results of
Staphylococcus aureus isolation from the pus. Describe day by day the order of
investigation and the results of septicemia:what volume of blood will be taken, in
what culture medium it must be inoculated, what volume of culture medium must be
used for inoculation, under what condition the collection and the inoculation of blood
will be done. Describe the investigations for the detection of Staphylococcus and
Streptococcus separately. What material will be choosen for diagnosis of the
Staphylococcal food poisoning, what will be detected in specimen, what animals are
used? Identification of staphylococcus pure culture: what criteria of genes and species
detection do you know?
Methods of the detection of Staphylococcus pathogenic factors: coagulase,
lecithinase, DNA-se. Staphylococcal phage typing: what is the aim of this test; by
what phage is it set. Describe day by day the motion of investigation and the results
of the Streptococci isolation from the pus.
Characteristic of nonsporing anaerobes. Classification: families, genuses, main
species (name in Latin). Main characteristics: morphology, Gram's staining,
conditions for cultivation.
Characteristic of the main species of gram-negative microorganisms. Rod of
blue-green pus: name in Latin, morphology, cultivation, pigment production, main
factors of virulence. Patient groups frequently suffered this infection. Proteus: name
in Latin, morphology, physiology, peculiarities of cultural characteristics.
Associations of microbes in pus and wound: frequency of association, peculiarities of
microbiology diagnosis of pathogens in association.
Klebsiella: Latin name of species. Morphology (spores, flagella, capsule),
necessity in special culture media, Gram's staining, staining methods for capsule
detection. Antigenic structure: O- and K-antigens. Diseases caused by Klebsiella.
Microbiology diagnosis: material for investigation; bacteriological and

22
bacterioscopical investigations, detection of serological variants, serological
diagnosis. Drugs for treatment.
Characteristic of anaerobic gas gangrene pathogens (3 main species): name in
Latin, morphology (spores, flagella, capsule), Gram's stain, type of respiration, media
for cultivation, character of the colonies growth in the deep layer of agar, changing
on the Wilson-Blaer medium, changing in milk; characteristics of toxin; it’s effect in
organism, serological types of toxin. Factors of invasiveness.
For the help: write Latin names of nonsporing anaerobes, gram negative
aerobes-pathogens of wound and pus inflammatory infections, write Latin names of
gas gangrene infection pathogens.

Practical classes.
1. Continuation of pus investigation (2nd day). Examine and describe colonies
on the Perti plate with blood agar. Measure diameter of growth inhibition zones
around the disks with antibiotics and make the conclusion about resistance to
antibiotics. Identify pure culture of staphylococci:
a) check up the results of anaerobic mannitol fermentation;
b) make the reaction of plasma coagulation: in the tube with 5 ml citrated
rabbit plasma in dilution 1:4 (experiment) and in tube with 0,5 ml of NaCl solution
(control) inoculate by the loop Staphylococci culture. Put the tubes into the
incubator. At the end of class check the result, make the conclusion and write it down
in the journal;
c) check up the results of lecythinase and DNA-ase tests.
2. Continuation of blood investigation (3rd day). Examine colonies on the blood
agar, pay attention on the sizes, hemolysis zones. Detect Streptococci specie (by
hemolysis). Prepare a slide from the colony, stain it by the Gram's method, examine
under the microscope and draw it. Write down the results into the notebook, make the
conclusion and in part "notes" write down what else is necessary to do for the
detection of Staphylococci specie and its antigenic structure.
3. Continue to detect the staphylococci carrier. Examine and describe the
character of microorganisms growth on the milk-salt agar. Make a slide and after
Gram's staining, examine it under the microscope. Describe the results in the journal
and make the conclusion. In the part "notes" describe what other investigations must
be done for the conclusion about absence or presence of the staphylococci carrier
state.
4. Investigate the wound samples of anaerobic gas gangrene pathogens.
Prepare a slide from the wound’s specimen, stain it by the Gram's method, examine it
under the microscope and draw it. Inoculate it on the Robertson-cooked medium,
Wilson-Blaer medium and in the sterile milk. Work’s description must be put down
in the notebook.

23
 LESSON 8.
Microbiology diagnosis of wound and pus inflammatory infections caused by
sporing and nonsporing anaerobs (continuation). Microbiology diagnosis of
diphtheria.

Plan of study.
1. Methods of pure culture isolation and methods of anaerobes cultivation.
2. Characteristic of pathogens and microbiology diagnosis of tetanus.
3. Schemes of microbiology investigation for isolation of nonsporing
anaerobes and gram negative rods - causative agents of pus inflammatory and wound
infections.
4. Specific prophylaxis and treatment of tetanus and anaerobic gas gangrene.

Test question.
Enumerate methods of anaerobic cultivation. How are the anaerobic conditions
organized by these methods? Methods of anaerobes pure culture isolation (name and
describe). Rules of patient's sample collection with preliminary diagnosis of
anaerobic gas gangrene. Describe the results of Clostridium perfringens inoculations
on Robertson-cooked medium, Wilson-Blaer medium, milk? When can you count up
results? What drugs are used for active immunization against anaerobic gas
gangrene? When passive prophylaxis be done and by what preparations? Rules of
usage.
Rules of samples collection and transportation from patient suspected of
anaerobic infection, (infection is caused by nonsporing anaerobes). Cultivation of
nonsporing anaerobes: nutrient media, gas mixture, duration of cultivation. Methods
of nonsporing anaerobes differentiation.
Methods of isolation and identification of Pseudomonas aeruginosa, Proteus
spp.
Characteristic of tetanus pathogen: Latin name, morphology (spores, flagella,
capsule), Gram's staining, type of respiration spores resistance. Character of toxin:
effect on organism, serological toxin’s types. Tetanus in human: mechanism of
contamination, main factor of virulence. Biological method of toxin detection.
Preparations for specific prophylaxis and treatment. Plan prophylaxis of tetanus and
tetanus prophylaxis by the evidences.
Task. Two patients come with traumas (abrasion of skin) in hospital. After
discussion it became known that the first (a constructor worker M., 22 years old)
during the service in the army was injected by tetanus toxoid. The second patient
(woman farmer worker, 50 years old) during last 30 years did not have any
prophylaxis against tetanus. The physician administrated to the first patient the
injection of tetanus toxoid. The injection of tetanus toxoid and tetanus antitoxic
serum have been administrated to the second patient. What is your opinion: are the
administrations correct? Why did the physician decide to administrate the toxoid
injection only in the first case? How can you explain the administration to another
patient?

24
 Characteristic of botulism pathogen: Latin name, morphology (spores,
flagella, capsule), Gram's staining, type of respiration, spores resistance. Resistance
to digestive enzymes, temperature, high concentration of NaCl. Character of toxin:
effect on an organism, serological toxin types. Botulism in a man: mechanism of
beginning, main factor of virulence. Scheme of microbiological investigation of
botulism. Method of toxin detection in a food, blood. Detection of toxin types.
Preparations for specific prophylaxis and treatment.
Characteristic of diphtheria pathogen: Latin name, morphology (form,
arrangement, spores, flagella, capsule, inclusion), Gram's staining, other methods of
staining, physiology (type of respiration, media for cultivation, character of the
growth on clotted serum and tellurite medium, biological types). Diphtheria toxin:
action on organism, unit of toxin power measuring, toxoid preparation. Describe
method of diphtheria culture toxigenicity detection (gel precipitation test); frequent
localization of local process in diphtheria. What samples must be taken from a
diphteria patient? Describe day by day the investigation and the result of toxigenic
diphtheria pathogen isolation? Methods of diphtheria pathogens from diphtheroids
differentiation: morphology, chemical activity, urease and cystinase tests, antigenic
structure and virulence. Immunity in diphtheria: character of immunity, duration of
immunity after the plan vaccination. Specific diphtheria prophylaxis: what
preparations are used, in what way the vaccination can be started? Drugs for specific
diphtheria therapy: preparation, dosage, usage.

Task for self-training

Latin name of tetanus pathogen and scheme of heterologic serum
administation.

Practical classes.
1. Examine under the microscope and draw morphology of pathogenic
anaerobes and gram negative rods (Gram's staining).
2. Study the cultural characteristic of Pseudomonas aeruginosa and Proteus
spp. Pay your attention on color of Pseudomonas aeruginosa pigment and character of
Proteus spp. growth.
3. Continue to investigate a wound’s sample (2-nd day). Study the character of
anaerobes growth on of Robertson-cooked medium, Wilson-Blaer medium, milk.
Prepare a slide from Robertson-cooked medium growth, stain it by the Gram's
method, examine it under the microscope and draw it.
For isolation of the pure culture inoculate material by the Veinberg’s method.
4. Make inoculations of student’s specimen for diphtheria carrier state
detection.

25
 LESSON 9.
Microbiology diagnosis of anaerobic infections, diphtheria (completion).
Microbiology diagnosis of whooping cough, legionellosis, tuberculosis, leprosy,
actinomycosis.

Plan of study.
1. Microbiology diagnosis, treatment & prophylaxis of whooping cough,
diphtheria.
2. Peculiarities of epidemiology, diagnosis & treatment of legionellosis..
3. Microbiology diagnosis, treatment & prophylaxis of tuberculosis, leprosy,
actinomycosis.

Test questions.
Whooping cough pathogen. Names in Latin. Morphology: form, arrangement,
spores, flagella, capsule, Gram's staining. Physiology: necessity for special culture
media, growth factors, basic culture media, colonies and their characteristic,
enzymatic activity. Antigenic structure, production of toxins, characteristic of toxins.
Source of infection, routes of transmission, pathogenesis. Microbiology diagnosis of
infection on the early stage: methods of samples collection, culture media, isolation
of pure culture, criteria for differentiation of whooping cough pathogens from another
Bordetella. Preparations for specific prophylaxis and treatment.
Legionellae. Name of pathogen in Latin. Morphology, Gram's staining.
Peculiarities of culture: optimum temperature, necessity for special culture media,
type of respiration, enzymes activity. Antigenic structure, serological groups.
Ecology of legionellae:natural reservuar, conditions for the multiplication and
accumulation. Main forms of legionellosis, routes of transmission. Microbiology
diagnosis. Drugs for treatment.
Tuberculosis pathogen. Name of the discoverer. Explanation of the name
"mycobacterium"? Latin name. Morphology: form, morphological variants, granules,
spores, flagella, capsule. Peculiarities of the tubercular bacteria chemical structure.
What method of staining is used and what pathogen's characteristics is it based on?
Type of respiration, media for cultivation, speed of growth. Types of tubercular
bacilli (name in Latin), differences between them (morphology, sensitivity to
glycerol, pathogenicity for humans and laboratory animals). Toxic substances of
tubercular bacilli and their effect on human’s organism. Tuberculosis in humans:
sources of infection, routes of transmittion: what organs are frequently contaminated?
Peculiarities of tuberculosis immunity, mechanism of immunity, allergy?
Microbiology diagnosis of tuberculosis: specimens, method of sputum enrichment,
schemes of diagnosis, rapid methods of diagnosis, differentiation of tubercular bacilli
types and detection of drug resistance. Skin allergy test: medicine and result. What
state of organism indicates by positive result? What is the aim of allergy test?
Specific prophylaxis: name of vaccine, what does it consist of, how is it prepared?
Forms of immunity. Enumerate drugs for tuberculosis treatment. Characteristic of
opportunistic mycobacteria: classification, name of the main species in Latin. Sources

26
of infection and conditions for the disease start. Differentiation of them from
tuberculosis pathogens. Drugs for treatment.
Characteristic of leprosy pathogen: Latin name, morphology and culture, type
of parasitism. Spreading of leprosy, clinical forms, microbiology diagnosis. Drugs for
treatment.
Causative agents of actinomycosis: Latin name, morphology and physiology.
Actinomycosis in humans: place of localisation. Specimens and methods of
diagnosis. Preparations for the diagnosis, prophylaxis and treatment.

Practical classes.
1. Complete investigation of wound sample. Describe colonies in Pasteur
tubes, mark colonies character for anaerobic gas gangrene pathogens. After tube
cutting prepare a slide from colonies, stain it by the Gram's method. Basis on
microscopy examination detect presence of microorganisms morphologically similar
to anaerobic gas gangrene pathogens. Write down the preliminary diagnose, write
what is necessary for the final conclusion.
2. Study inoculations for diphtheria carrier detection. Examine colonies
microscopically and after slide preparation, stain it by the acidic gentian-violet,
compare your slide with the slide from pure culture of diphtheria pathogens. Make
the conclusion about the presence of pathogenic microorganisms. Study the method
for detection of diphtheria pathogen toxigenicity.
3. Examine morphology and study cultural characteristic of tubercular bacilli.
Make bacterioscopical diagnosis of lungs tuberculosis. Study and describe character
of the tubercular bacilli growth on culture medium. Make a conclusion.
4. Study microbiology diagnosis of leprosy. Examine under the microscope
and draw slide from leprosy patient's nasal mucous.
5. Make bacterioscopical diagnosis of actinomycosis. Examine and draw a
slide actinomycetes in patient’s tissue.

LESSON 10
Quiz on “Microbiology diagnosis of supporative, wound infection and air
transmitted infections.”

LESSON 11.
Microbiology diagnosis of zoonotic infections.
Microbiology diagnosis of plaque, tularemia, brucellosis, anthrax.

Plan of study.
1. Characteristic of plaque, tularemia, brucellosis, anthrax pathogens.
2. Rules of collection, transportation and work with the dangerous infections
samples.
3. Schemes of microbiology diagnosis of plaque, tularemia, brucellosis and
anthrax.
4. Experimental method of investigation: animal’s infection for diagnosis.

27
 Test questions.
Enumerate in Latin the pathogens of zoonotic infections. What diseases may be
caused by them? Why these infections are named as zoonotic, why are they
dangerous infections?
Plaque pathogen. Name, morphology (spore, flagella, capsule production),
peculiarities of staining, Gram's staining. Type of respiration, optimal temperature of
the growth. May it grow on simple media? Character of plaque pathogen colonies’
growth on solid medium. What types of colonies are formed by non-virulent forms?
Describe the character of the growth in liquid medium. Explain antigenic structure
and virulence factors of the plaque pathogen. Plaque in a humans. Enumerate sources
of infection. Ways of pathogen’s penetration into organism. Enumerate clinical forms
of diseases. Is there bacteremia in plaque? Microbiology diagnosis of plaque: in what
laboratory microbiology diagnosis may be done? What must be done when the plaque
pathogen is discovered? What samples may be taken from the patient according to the
from of disease? Other materials for examination. Enumerate methods of
investigation. Results of the slide microscope investigation. What is the significance
of microscopic method? What medium is used for bacteriological method? What is
the temperature for culturation of plaque pathogen culture? What are the
characteristics of the culture identification? How to differentiate pathogen of plaque
from the pathogen of pseudotuberculosis? What animals may be infected and by what
method?
Causative agent of tularemia: name in Latin, morphology (spore, flagella,
capsule production), Gram's staining. Describe the type of respiration and culture
media for cultivation. May tularemia pathogen produce toxins? What antigens does it
consist of and what microbe does it have a common antigen? Tularemia in humans.
Name source of infection, places of entry. What biological changes take place in the
human's organism? How are they developing? May tularemia be transmitted from
sick person to healthy person? Microbiology diagnosis of tularemia. Enumerate
methods of microbiology diagnosis. Explain what samples may be taken and what
may be detected by each method? What is the meaning of primary microscopical
examination of the samples? By what method is pure culture of the pathogen
isolated? In what laboratories an investigation and isolation of tularemia pathogen
may be done?
Pathogens of brucellosis: name in Latin. Morphology of Brucella: spore,
flagella, capsule production, Gram's staining. Media for Brucella cultivation and time
of the growth. Cultural peculiarities of Brucella abortus. Toxin production.
Brucellosis in a humans: sources of infection, place of entry, pathways of spreading
and place of localization. Character of immunity: may there be cross-immunity
between different Brucella species? Enumerate methods of brucella diagnosis. What
may be detected by each of these methods? What methods can't be used in an
ordinary laboratory? What is the aim of Rait test? What can it show? What is the
specimen and diagnostic preparation for Rait test. Describe the technique of reaction.
Describe the Haedllson test and Burne test.
Pathogen of anthrax: name in Latin, morphology, arrangement, presence of
spore, flagella, capsule, Gram's staining. How may material contaminated by anthrax

28
pathogen be sterilized? How may this microorganism be cultivated? Describe
character of the pathogen growth on the solid and liquid media. From what antigens
does the anthrax pathogen consist of? Enumerate factors of virulence. Anthrax in a
humans: name sources of infection and pathways of spreading. Enumerate clinical
forms of anthrax. Microbiology diagnosis of anthrax. Enumerate methods of
investigations and samples for investigations. What is the meaning of microscopical
method of examination? Scheme of pure culture isolation and it’s results.
Differentiation of anthrax bacilli from nonpathogenic bacilli. What diagnostic test can
be done on patient? Askoli test: what can be detected by this test? What is the result
of the reaction? Drugs for specific prophylaxis and treatment of plaque, brucellosis,
tularaemia, anthrax.

Task for self-training

Write in Latin the names of causative agents of plaque, tularemia, brucellosis
(3 species), anthrax.

Practical classes.
1. Make serological diagnosis of tularemia. Make agglutination test using
serum of tularemia patient. Write scheme of reaction and results.
2. Make serological diagnosis of brucellosis (Haedllson reaction). Write
scheme of reaction and results.
3. Examine slides of plaque, tularemia, brucellosis and anthrax pathogens.
4. Start microbiology diagnosis of anthrax. Prepare a slide from carbuncule,
stain it by the fucshin (draw this picture). Inoculate carbuncule’s samples on meat-
peptone agar. Infect white mouse by carbuncule’s material.

LESSON 12.
Microbiology diagnosis of zoonotic diseases (completion).
Microbiology diagnosis of infections caused by pathogenic spirochetes and fungi.

Plan of study.
1. Characteristic of causative agents of leptospirosis and schemes of the
diagnosis of this infection.
2. Characteristic of pathogenic Borrelia.
3. Scheme of microbiology diagnosis of relapsing fever, Lyme disease.
4. Characteristic of the syphilis causative agent, pathogenesis and stages of
infection. Microbiology diagnosis of syphilis according to the stage of disease.

Test questions.
Causative agents of leptospirosis: name in Latin. Describe Leptospira
morphology, character of their movement, staining, media for cultivation, optimal
temperature for growth. Enumerate sources of infection and pathways of human’s
contamination. Microbiology diagnosis of leptospirosis: enumerate material and
methods of investigation (according to the different stages of disease). Peculiarities of

29
microscopical, bacteriological methods of diagnosis. Serological diagnosis of
leptospirosis. Preparations for diagnosis, active and passive prophylaxis, treatment.
Pathogenic Borrelia.
What kinds of relapsing fever do you know (according to the vector and region
of spreading)? Name sources and mechanism of contamination. Causative agent of
epidemic relapsing fever (louse-borne): name in Latin, morphology and physiology,
staining. Pathogenesis of fever attacks. Where is Borrelia localised during the attack?
Where is Borrelia localized between the attacks? What are the reasons for the next
attack? Causative agents of endemic relapsing fever (tick-borne): Latin name,
morphology and culture, methods of staining. Microbiology diagnosis of relapsing
fever: material for investigation. When it must be taken? What may be detected?
What methods are used? Drugs for treatment.
Causative agent of Lyme disease: name in Latin, morphology and culture,
methods of staining. Sources and pathways of infection. Clinical symptoms of
disease. Microbiology diagnosis: material for investigation on different stages of
disease, detection of pathogen, serological diagnosis. Drugs for treatment.
Syphilis pathogen: Latin name, morphology, character of movement, staining.
Type of respiration, media for cultivation. “Cultural” and “tissue” Treponema:
difference between them. Susceptibility to the temperature, drying, chemical agents.
Human’s infection: source, places of entry, pathways of contamination.
Immunity to syphilis: peculiarities and appearance. Microscopical method of
syphilis diagnosis: specimens, methods of living Treponema examination.
Serological diagnosis of syphilis: in what period of disease may it be done?
Wasserman’s test: principle of reaction.What may be detected by this test (antigens or
antibodies)? What antigen is used for Wasserman’s test. Reaction of immobilization
of Treponema pallidum movement (principles and accounting of reaction).
Fluorescent treponemal antibody test (FTA-ABS). Medicines for diagnosis and
treatment of syphilis.
Causative agents of fungal infections: classification of pathogenic fungi,
characteristic of the main pathogens and diseases caused by them. Candida fungi:
morphology, similarity and difference from Saccharomyces. Culture media for
Candida cultivation. Microbiology diagnosis: meaning of bacteriological and
bacterioscopical methods of investigations, serological methods of diagnosis.
Dermatomycosis: Latin name of the pathogens and diseases caused by them.
Microscopical method of diagnosis: samples for investigation, preparation of slides,
method of microscopical examination, criteria for the differentiation of the pathogens

Practical classes.
1. Make microbiology diagnosis of anthrax. Examine colonies in Petri plate,
prepare slides, stain them, examine under the microscope. Write down the results of
colony staining and microscopical examination in suppl.
2. Examine under the dark-field microscope Leptospira culture.
3. Examine under microscope and draw thick slide from the patient’s blood
(patient had been infected by pathogen of relapsing fever).
4. Study morphology of syphilis pathogen (slide from patient’s chanker).

30
 5. Make serological diagnosis of syphilis. Make Wasserman’s test. Detect the
result and write down it in suppl.
6. Prepare the slide using Candida albicans culture, stain it by methylen blue.
Examine under microscope.

LESSON 13.
Microbiology diagnosis of diseases caused by rickettsiae, chlamydia,
mycoplasma.

Plan of study.
1. Characteristic of pathogenic Rickettsia.
2. Scheme of microbiology diagnosis of spotted fever and Brill’s disease.
3. Scheme of microbiology diagnosis of Q – fever.
4. Characteristic of pathogenic Mycoplasma and scheme of laboratory
diagnosis of infections caused by them.
5. Characteristic of Chlamydia and scheme of laboratory diagnosis of
infections caused by pathogenic Chlamydia.

Test questions.
Systematic position of rickettsia. Enumerate pathogenic Rickettsia and diseases
caused by them. Causative agent of spotted fever: name in Latin, morphology and
culture, methods of staining. Antigenic structure, toxin production. Sources of
infection, localization of the pathogen (in sources). Who is a vector of spotted fever
and how is he infected? Localization of pathogen in vector. Mechanism of human’s
contamination. Serological diagnosis of spotted fever: what tests and what antigens
are used? What is Brill's disease? How can it be differentiated from primary spotted
fever? Drugs for prevention and treatment.
Causative agent of Q-fever. Name in Latin. Morphology and culture, resistance
in environment, difference from another rickettsia by antigens. Sources of infection.
Pathways of Q – fever spreading. Microbiology diagnosis of Q – fever. Drugs for
treatment.
Chlamydia: name of the pathogen in Latin, morphology and culture. Life cycle
of chlamydia. Antigenic structure. Enumerate infections caused by Chlamydia spp.
Microbiology diagnosis: bacterioscopical method, serological investigations. Drugs
for treatment.
Mycoplasma: name in Latin, morphological structure and culture properties.
Factors of mycoplasma’s pathogenesity. Infections caused by mycoplasma. Methods
of microbiology diagnosis of mycoplasmosis. Medicines for treatment.

Practical classes.
1.Make tube agglutination test (RAR) and detect antibodies titre of patient’s
serum (patient has been infected by louse – borne typhus). Write down the result of
the test.
2. Examine under light microscope a slide of rickettsia (pure culture of
microorganisms stained by Zdrodovsky’s method).

31
 3. Examine under microscope a slide from the patient with Chlamydia
infection. Find and detect the intracellular and extracellular forms of chlamydia.
4. Study the morphology of mycoplasma using ready prepared slide.
Mycoplasma pneumonia stained by silver impregnation method.

LESSON 14.
Morphology and physiology of viruses. Methods of viral cultivation and
detection.

Plan of study.
1. Morphology, physiology and classification of viruses.
2. Methods of viral cultivation.

Additional material.
Methods of viral cultivation.
For viral cultivation the following methods are used:
1. Animals contamination (intraperitoneally, intravienn, intramuscular,
intranasal, brain inoculation etc.).
2. Chicken embryo contamination (chorionallantoic envelope, in allantoic
cavity, in amniotic cavity, in yolk sack).
3. Contamination of tissue’s cells culture. Cell’s cultures are tissue cells
growing outside of the organism in special culture medium. In the artificial
conditions tissue cells save their metabolism and susceptibility to certain viruses.
Cells with rapid growth and high level of metabolism are the most suitable for viral
cultivation. For this reason embryonatic tissues are widely used(chicken's embryos,
fibroblasts, human amnion cells, tumor's cells).
Cell culture grows in special flacks and tubes. Cell culture need a support for
the growth (for example glass).
Flack’s walls or glass plate are coated by the grown up tissue cell culture is
infected by virus. Work in sterile conditions. For the inhibition of other microbial
flora growth (except viruses) virus containing specimen must be treated by the
antibiotic (frequently penicillin and streptomycin).
Viral multiplication are detected by the cytopathic effect (CPE): under the
microscope degenerative changing and finally cells destruction are detected. This is
the result of viral multiplication.
In virology practice fresh cell's culture (primary or primary-trypsinised) and
transinoculated cell’s cultures are frequently used.
Primary-trypsinised cell's cultures are prepared from organs of adult animals
(frequently from monkey’s kidney and other animals) human's embryo, chicken’s
fibroblasts. After the trypsinisation tissue cells are cultivated in the culture medium.
Tissue pieces are reduced to fragments, washed by Haenk’s buffer solution for blood
removing and treated by 0,25-0,3% trypsin solution. Trypsin destroy intracellular
brigdes and make cells free. Then using Gorjaev’s chamber the cell’s number are
counted and diluted till the certain concentration (400000 cells in ml). Prepared cell’s
suspension (being in tubes) close tightly by sterile plugs and put into incubator

32
(37°C) almost in horizontal position – (under corner 5°) on special support. After 3-4
days tubes wall surface will be covered by all-round layer of multiplying cells. Tubes
with the good growth of tissue cells will be taken for viral inoculations.
Examples of transinoculated tissue cell culture.
1) line Hela - cells of carcynoma of the uterus neck;
2) line Hep-2- malignant human tumors pharynx cells;
3) line Detroit-6- cells isolated from bone narrow of a person with lung cancer;
4) line A-0 and A-1 - human amnion cells;
5)line CMC - cells of the core of monkey cynomolgus specie and others.
Transinoculated or diploid cell cultures – are cells of human’s tissue preserving
in process of going out (till year) diploid chromosome set. Diploid humans’ cells do
not expose to malignisation and by this differ from tumor cells.

Methods of viral detection in tissue culture.

1) Cytopathic effect (CPE);
2) Hemadsorbtion test;
3) Color probes method;
4) Immunofluorescent assay;
5) Complement fixation test;
6) Hemagglutination reaction;
7) Gell precipitation test;
8) Animal’s infection.

Method of viral specie and type detection.

The identification of viruses is made by the test of viral neutralization by
specific antisera. The result of neutralisation can be detected by the following sings:
1. neutralization of CPE.
2. neutralization of hemadsorbtion.
3. inhibition of hemagglutination.
4. color test.
5. fluorescence of the virus containing cells after their treating by some
specific fluorescent antisera.
6. neutralization of viruses in experiment ( some experimental animals are
resistant to viral infection).

Test questions.
Position of viruses among living organisms: what kingdom are they belong to?
What are the main peculiarities of viruses? Viral sizes (small, middle, big), methods
of viral sizes detection. What is the name for viral particle? Structure of simple and
complex viruses: types of symmetry, structural components of viral particle, external
coat. Examples of simple and complex viruses. Chemical structure of viruses.
Principles of viral classification. Enumerate the main families, genus and species of
RNA- and DNA-containing viruses. Methods of viral cultivation. Kinds of tissue
cultures, their characteristics and methods of their preparations. Culture media for the

33
tissues cultures cultivation. Chicken’s embryo: structure, methods and conditions for
infection of chicken’s embryo.
Main steps of viral replication. What methods of viral detection do you know?
What are intracellular inclusions? What is cytopathic effect of viruses? Methods of
CPE detection. Methods of virus specie and type detection.

Practical classes.
1. Using tables, slides study morphology of viruses. Study different viruses’
structure, peculiarities of structure and biological role of different structural elements.
2. Morphology study of virions (Pashen’s bodies). Why they may be observed
under the light microscope. Examine virions’ particles under light microscope and
draw it.
3. Using light microscope examine ready-prepared slide of haemadsorbtion.
4. Using light microscope examine normal and infected by viruses tissue
culture cells. Draw it.

LESSON 15.
Causative agents of acute viral respiratory infections.

Plan of study.
1. Characteristics of causative agents of influenza and acute viral respiratory
infections.
2. Schemes of laboratory diagnosis of influenza and acute viral respiratory
infections.

Additional material.
Causative agents of acute respiratory viral infection (ARVI) are the following:
RNA- containing and DNA- containing viruses.
RNA – containing viruses:
1. Orthomyxoviruses (influenza virus A, B, C)
2. Paramyxoviruses (parainfluenza viruses, mumps virus, Newcastle’s virus,
measles virus, respiratory- cyncitical virus).
3. Picornaviruses: more than 110 serotypes of rhinoviruses, Coxsackie virus
(A and B) some serotypes and ECHO virus.
4. Reoviruses (3rd serological type)
5. Coronaviruses.
DNA – containing viruses.
6. Adenoviruses (8 types from 41 types are pathogens of ARVI).
To ARVI diagnosis the following methods are used:
1. Virological – infection of tissue culture cells and chicken’s embryos.
2. Immunofluorescense (method of rapid diagnosis)
3. Selogical tests – inhibition of passive haemagglutination, complement
fixation test, ELISA.
4. Usage of RNA.

34
 Test questions.
Enumerate main pathogens of acute respiratory viral infections (ARVI).
Influenza virus: what viral family it is included, form, type of symmetry, scheme of
structure, peculiarities of genome, antigens. What antigens are used for detection of
type and subtype of influenza virus. Antigenic changing of influenza virus. Methods
of diagnosis that used on the first days of the infection and for retrospective
diagnosis. Reaction of hemagglutination. Reaction of hemagglutination inhibition.
What can be detected by this reaction (two variants)? What diagnostic preparations
are necessary for this? Write a scheme of hemagglutination inhibition for the
detection of influenza virus type and subtype. Specific prophylaxis and treatment of
influenza; drugs and their characteristics.
Parainfluenza virus: classification, characteristic, diseases caused by this virus.
Mumps virus: classification, characteristics, diagnosis and specific prophylaxis
of mump.
Measles virus: it characteristic. Immunity. Diagnosis of measles, preparations
for prophylaxis.
Herpes virus: classification, characteristics, diseases caused by it. Diagnosis of
herpesvirus infections. Prophylaxis and treatment.

Practical classes
1. Make reaction of haemoagglutination using washing water from patient’s
nasopharyngs.
2. Count up the results of serological diagnosis of influenza (demonstration):
reaction of inhibition haemagglutination for the detection of the influenza virus type
and subtype.
3. Rapid method of influenza diagnosis – immunofluorescent test
(demonstration).

LESSON 16.
Viral cultivation (completion). Causative agents of enteroviral (polyomielitis,
Coxacie, ECHO), neuroviral (rabies, viral encephalitis) infections and viral
hepatitis.

Plan of study.
1. Characteristics of causative agents of poliomyelitis, viral encephalitis, viral
hepatitis.
2. Schemes of laboratory diagnosis of poliomyelitis, rabies, viral hepatitis.
3. Specific prophylaxis of poliomyelitis, rabies, viral encephalitis, viral
hepatitis.

Additional material.
Main pathogens of the intestinal viral infections.
1. Polyoviruses (3 serological types).
2. ECHO viruses (34 serological types).
3. Coxacie A viruses (24 serological types).

35
 4. Coxacie B viruses (6 serological types).
5. Enteroviruses (serological types 68-71).
6. Hepatitis A virus (72-nd serological type).
7. Rotaviruses (4 serological types).

Nosological forms and clinical syndromes caused by enteroviruses.

N Nosological forms and syndromes Type of viruses

1 Polyomyelitis Polyovirus I, II, III
2 Polyomyelitis like diseases Coxacie A 6, 7, 9, 10, 14
Coxacie B 1 - 5
ECHO 2, 4, 6, 9, 11, 16.
3 Gastroenteritis Coxacie A 2, 9
Coxacie B 1 - 5
ECHO 2, 5, 6 - 12, 14 18, 22 - 24
Rotavirus 1 - 4
Enterovirus 68 - 69
4 Acute respiratory viral infections Coxacie A 21
Coxacie B 3, 4
ECHO 7, 11, 20
Enterovirus 68, 69
5 Acute serum encephalitis Coxacie A 2, 4, 7, 9, 15,
()Meningitis, (encephalitis, Coxacie B 1 - 6
meningoencephalitis) ECHO 1 - 7, 9, 11, 13, 14, 16, 18, 20, 21
Enterovirus 71
6 Myocarditis, pericarditis Coxacie 1 – 6
7 Vesicular stomatitis Coxacie A 5
8 Viral hepatitis Enterovirus 72

Laboratory diagnosis methods of enteroviral infections.

Specimens for investigation: feces, washing water from nasopharyng,
cerebrospinal fluid, serum.
Virological method.
Coxacie and ECHO viruses can cause polyomyelitis like disease. Investigation
must be done for isolation of anyone from 3 viral species. That’s why for inoculation
of patient’s specimen tissue cells cultures and newborn mice are used.
Polioviruses and ECHO viruses cause CPE in tissue culture, Coxacie viruses A
and B cause paralysis and death of a newborn mice.
Type detection of isolated viruses is made by neutralization test. Type specific
antisera against polioviruses, Coxacie viruses A and B, ECHO virus are used.
Serological method.
For the detection of antibodies titre the patient’s blood serum is used.
Haemagglutination inhibition test is used for diagnosis of ECHO virus infection;
neutralization; color and complement fixation tests are useful for diagnosis of
Coxacie and polioviruses infections. For the detection of rotaviruses stool mass are
examined, virus is detected by the immunoelectronic microscopy method and by the
way of tissue cell culture infection. Presence of viral antigen is detected by

36
serological tests: complement fixation test, immunofluorescent assay,
radioimmunoassay, ELISA.

Classification of viral hepatitis.

Disease name Pathogen’s name Routes of Taxonomic position

s name transmission
Viral hepatitis A Hepatitis A virus Feаcal – oral Family Picarnoviridae Genus
Enterovirus №72
Viral hepatitis B Hepatitis B virus Parenteral Family Hepadnaviridae
Viral hepatitis D Delta – virus Parenteral ?
(delta infections)
Viral hepatitis C Hepatitis C virus Parenteral Flaviviridae
Viral hepatitis E Hepatitis E virus Feacal-oral Caliciviridae
Viral hepatitis G Hepatitis G virus Parenteral Flaviviridae
Viral hepatitis T Transfusion Parenteral ?
transmitted virus Fecal – oral

Test question.
Enumerate the main nosological forms and clinical syndromes caused by
enteroviruses.
Characteristic of family Picornaviridae: classification, sizes of virions, virion's
structure, methods of cultivation, serological types, diseases. Methods of eneroviral
infections laboratory diagnosis.
Poliomyelitis viruses: what family and genus are included, type of nucleic acid,
virion's sizes, antigenic properties (serological types), resistance to environment,
cultivation. Morphological manifestations of viruses and host-cell interaction; source
of a human’s infection, main places of viruses localization in human’s organism,
routes of viruses excretion. Laboratory diagnosis: what specimens are taken from the
patient. Methods of laboratory diagnosis.
Color test: mechanism, ingredients of reaction, registration of results. Usage of
color test for viral specie and type detection (technique of color test for antiviral
antibodies titer detection). Specific prophylaxis of poliomielitis: medicines for the
active immunity formation. What groups of people must be vaccinate? What
medicine for the passive immunization do you know?
General characteristic of Coxacie and ECHO-virus: family they are included
in. Antigenic classification (serological types), diseases, methods of laboratory
diagnosis.
Pathogens of hepatitis. Modern classification of viral hepatitis and their
pathogens. Hepatitis A virus: family and genus it is included in, type of nucleic acid?
Source of human infection, resistance to environment, routes of transmission,
laboratory diagnosis. Materials for investigations, methods of serological diagnosis,
general and specific prophylaxis. Hepatitis B virus: what is the type of this virus?
Virion’s structure, antigenic structure (name of antigens): what is the "australian
antigen" and Dein's particles? Source of infection, routes of transmission, resistance
to environment, Carrier stage. Methods of laboratory diagnosis, drugs for specific

37
prophylaxis. Delta-virus: peculiarities of structure, interaction between delta-virus
and hepatitis B virus, source of infection, routes of transmission. Hepatitis C, E, G
and T viruses: sources and routes of transmission, laboratory diagnosis.
Rabies virus: viral name (synonyms), what family is it included in?
Peculiarities of structure and cultivation. What is formed in the host cells during viral
multiplication? Sources of infection for humans, natural reservoirs. Laboratory
diagnosis: material for investigation, methods of investigation. What is "fixed" rabies
virus, how can receive it? What is the difference of the "street" rabies virus from
“fixed rabies virus”, what can it be used for? Specific prophylaxis of rabies. Cultural
antirabies vaccine: what does it consist of and when can it be used? Antirabies
gamma-globulin: preparation, ingridients, usage.
Pathogens of tick-born encephalities: general characteristic, classification.
Sources and routes of transmission. Laboratory diagnosis. Specific prophylaxis and
therapy.

Practical classes.
1. Examine under the light microscope and draw slides of tissue culture
infected by the poliovirus (EPE) and noninfected (control) tissue culture. Register the
results of color test, make the poliovirus detection and antibodies titer detection
(demonstration). Draw the results and make the conclusion.
2. Examine under the light microscope slides of brain tissue with Negri, bodies
in nerve cells and draw it.
3. Study the schemes of viral hepatitis diagnosis.
4. Study the diagnostic medicines for ELISA.

LESSON 17.
Causative agents of AIDS. Oncogenic viruses.

Plan of study.
1. Characteristic of human immunodeficiency viruses (HIV) and oncogenic
viruses.
2. Laboratory diagnosis of AIDS.
3. Elaboration of the AIDS treatment and specific prophylaxis methods.

Test questions.
Human immunodeficiency virus: family type, peculiarities of this family
representatives, type of nucleic acid, role of enzyme revertase. Virion's structure,
functions of different structural elements, resistance to environment. In what
biological liquids of organism can HIV be detected? Mechanism of transmission and
main routes of contamination. Can people be infected by the insects, through the
blood-sacking insects, by direct contact? Main "groups of risk". Pathogenesis of
AIDS - duration of incubation period; what cells of the organism are affected; what
receptor do they have? What is the result of this affect? How and why is T-helper/T-
suppressor ratio changed? Reasons for the immunodeficiency development. AIDS
clinic: early clinical manifestations, clinical symptoms during the peak of disease.

38
What complications (pus-inflammatory processes and tumors) are frequently
observed? Laboratory diagnosis of AIDS: what is necessary to detect? Serological
method: material for investigation, main principles of investigation (on the first and
second stage)? Principal scheme of EZISA and Western blott. Prerequisites for
development of therapeutic drugs, usage of the therapeutic drugs. How is the problem
of AIDS specific prophylaxis solving? Role of information in AIDS prophylaxis.
Oncogenic viruses. History of oncogenic viruses discovery. Essence of Zilber's
virogenetic theory. Characteristic of Retroviridae family viruses: virion's structure,
role of revertase, integration into the host genome. Evidences of oncogenic viruses
association with some human's tumors. Evidences of infections viruses role in the
human’s tumors development.

LESSON 18.
Quiz on "Viral infection".

QUESTIONS FOR FINAL EXAM ON MICROBIOLOGY.

1. Meaning of the medical microbiology for the physician. The achievements

of microbiology, virology & immunology in the development of medicine & their
tasks in modern conditions.
2. The invention of a microscope & the discovery of microbes
(A.Leeuwenhoek). Basic stages of the development of microbiology & their
characteristics.
3. Luis Pasteur. His discoveries in the field of microbiology.
4. Works of R.Coch & their significance for microbiology & infectious
pathology.
5. The role of Russian scientists in the development of microbiology.
6. The discovery of viruses by D.Ivanovsky & their significance for the
beginning & development of virology. Etiological role of viruses in human
pathology.
7. Subject, aims & parts of medical microbiology. Methods using in
microbiology.
8. Methods of microscopy: microscopy with immersion system; dark-field
microscopy; phase-contrast microscopy; fluorescent microscopy. Electronic
microscope.
9. The main principles of bacterial systematic. Taxonomic category.
Principles of classification. Specie like basic taxonomic unit. Subspecies
(biovariant, serovariant, phagovariant), culture, population.
10. Forms of bacteria. Morphology, ultrastructure, chemical composition of
bacterial cell. Main differences between prokaryotes & eukaryotes. Protoplast,
spheroplast. L-forms of bacteria.
11. Examination of coloring microorganisms. Simple & complicated methods
of bacterial coloring. Gram method. Gram positive & gram-negative groups of
microorganisms.

39
 12. Bacterial spores, capsules, flagella, inclusions. Biological role & methods
of detection.
13. Morphology & ultrastructure of actinomycetes. Pathogenic actinomycetes.
Actinomycetes like antibiotic productors.
14. Spirochetes: classification, morphology & physiology. Pathogens from
different genus (named in Latin).
15. Microscopic fungi. Classification, structure of different groups.
Pathogenic fungi.
16. Morphology & physiology of mycoplasma. Human pathogenic species.
17. Rickettsia: morphology & physiology. Chlamydia: morphology &
physiology. Pathogens for human.
18. Morphology & chemical composition of viruses. Difference of viruses
from other organisms. Methods of viral classification. Tissue cultures & their
characteristics.
19. Principles of viral classification. Reproduction of viruses (phases of viral -
host cell interaction).
20. Phages (viruses of microorganisms): morphology & ultrastructure. Phases
of interaction with cell of virulent & moderate phages. Titer of phage. Prophage.
Methods of phage-typing. Practical using of phages.
21. Bacterial nutrition. Types of nutrition. Mechanisms of the transport of
substances inside the cell. Nutritional requirements for growth of microorganisms.
22. Culture media. Classification by the assignment, origin, composition. Main
requirements for culture media.
23. Growth & division of microbes. Phases of division. Cultural conditions.
24. Classification of microorganisms by the type of respiration. Scheme of the
biological oxidation of glucose in aerobes & anaerobes.
25. Methods for the cultivation of obligate anaerobes: principles, apparatuses.
26. Significance of identification of morphological & cultural properties of
microorganisms in microbiological diagnostics.
27. Microbial enzymes. Classification by the biological role & substrate
specificity; using for identification of microbes.
28. Methods of detection proteolytic & shugarolytic enzymes: culture media,
products of metabolism & methods for their detection.
29. Differentiation media: enumerate main kinds, principles of composition,
assignment & practical using.
30. Isolation of pure cultures of aerobic & anaerobic microorganisms..
Enumerate methods & principles of preparing of isolating colonies of aerobes.
Methods of isolation of pure culture of aerobes.
31. Influence of environmental factors on microorganisms. Influence of
physical factors: temperature, sun energy, drying. Method of lyophylic drying.
32. Sterilization, disinfection, aseptic, antiseptic. Methods of sterilization.
Their characteristics: apparatuses, factors of sterilization, regime of sterilization,
objects of sterilization.

40
 33. Influence of disinfectants on the microorganisms. Enumerate groups of
disinfectants by the mechanism of interaction; enumerate the main substances of each
group.
34. Microbial flora of water, air, soil. Sanitation microorganisms. Living time
of the pathogens in environment.
35. Bacteriological examination of water.
36. Interactions between microorganisms in association: symbiosis,
metabiosis, synergism, antagonism. Microbes-antagonists, their using in production
of antibiotics & other drugs.
37. Chemotherapy. Main groups of chemotherapeutic preparations.
38. Antibiotics. Classification by the chemical composition, mechanism of
action, antimicrobial specter. Units of the measuring of antibiotic activity.
39. Complications after the using of antibiotics. Prophylaxis & treatment.
40. Drug resistance of microorganisms. Types & mechanisms of formation,
role of plasmids. Ways of overcoming. Methods of examination of antibiotic
sensitivity.
41. Bacterial gene apparatus & their peculiarities in viruses. Genotype &
phenotype of microorganisms.
42. Kinds of changing. Mutations, their types. Mutagens: physical, chemical,
biological.
43. Genetic changing in microorganisms (recombination’s). Kinds of
recombination’s & their characteristics. Plasmids: main types & their characteristics.
44. Role of mutations in evolution of microorganisms, recombination’s &
selection.
45. Bacterial changeability & its role in diagnostics, therapy & prophylaxis of
infectious diseases.
46. Human microbial flora & its role under normal & pathogenic conditions.
Characteristic of the skin flora, mucous surfaces, respiratory tract, oral cavity.
Changing of the flora according the age of human.
47. Microbial flora of colon. Main aerobic & anaerobic microorganisms.
Disbacteriosis: causative factors, methods of prophylaxis; drugs rebuilding of
microbial flora of intestine.
48. Infection & infectious disease. Main factors necessary for the beginning
of infection. Differentiation of infectious diseases from other human diseases.
49. Pathogenicity & virulence of microorganisms. Basic factors of virulence.
Methods for measuring of virulence.
50. Microbial toxins: types, units of the toxin strength, characteristics. Gene
determinants of toxigenicity. Preparing & practical using of toxins & anatoxins. Main
toxigenic bacteria.
51. Ways of invasion of microbes in human. Dynamic of development of
infectious process; its periods. Antroponosis, zoonosis, antropozoonosis.
52. Forms of infectious process. Persistence of pathogens in organism.
Reinfection, superinfection, secondary infection; exogenic & endogenic infection;
localized & generalized infection. Bacteremia, septicemia, toxemia. Carriage of
pathogenic microbes.

41
 53. Role of microorganisms, human organism, environment & social
conditions in the beginning & development of infectious diseases. Mixt infection,
types of interactions between microorganisms.
54. Hospital infections, conditions for the beginning. Hospital strains of
opportunistic microorganisms; conditions for their formation, main characteristics.
Methods of prophylaxis of hospital infections.
55. Specialty of viral infections. Infectiveness of viruses. Kinds of viral
infections: productive, persistence (characteristic).
56. Immunity. Basic parts of modern immunology. Kinds of immunity. Sterile
& nonsterile immunity.
57. Nonspecific factors of human defense: superficial surfaces, humoral &
cell factors, role of normal human flora.
58. Phagosytic theory of immunity. Phagosytosis: phagosytic cells, stages
of phagocytosis & their characteristics. Indicators for the characteristic of
phagosytosis.
59. Complement: chemical structure, routes of activation, role in the
antiinfectious defense, sources of preparations, practical using.
60. Antigens. Main characteristic of antigens. Complete & incomplete
antigens. Specificity of antigens. Group antigens, species antigens, type antigens.
Autoantigens.
61. Antigenic structure of bacterial cell: indication, position of antigens,
characteristics, preparation, practical using. Group & species antigens of microbes.
Antigenic structure of viruses.
62. Immune system of a men. Immunocompetent organs, cells; their main
functions; subpopulations of T-lymphocytes & their function. Cell & humoral
immunity.
63. Mechanism of immune answer. Interaction between T- & B-lymphocytes
& macrophages. Their role in cell & humoral immunity.
64. Antibody, immunoglobulins, their main characteristics. Structure. Basic
classes of immunoglobulins, their physical, chemical & biological properties.
Specificity of antibodies.
65. Production of antibodies: cells & their interactions; phases; primary &
secondary immune answers; practical using of these knowledge.
66. Complete & incomplete antibodies. Methods of detection. Autoantibody,
their role in pathology. Theory of antibody formation process. Regulation of these
process on the cell level, gene
level, mediators level, molecular level.
67. Localized immunity: main mechanisms, Specialty of the structure of
secretory immunoglobulins; phases of their formation & functions.
68. Transplantational immunity: kinds of grafts, human HLA complex.
Principles of donor & recipient selection, overcoming of the transplantational barrier
(immunosupressors).
69. Antiviral immunity: nonspecific factors of defense, role of phagocytosis
& antibodies. Interferon: conditions for production, types, mechanism of antiviral
action. Inductors of interferon. Practical using.

42
 70. Mechanisms of connection between antibody & antigen & reactions of
immunity. Types of antibody. Monoclonal antibody: scheme of preparation,
practical using.
71. Reaction of neutralizing of toxin: mechanisms & ingredients. Usage for
the measuring of the level of antitoxic immunity (the name of reaction, description of
the method), usage for diagnostic aim.
72. Precipitins & reaction of precipitation: mechanisms & ingredients;
preparing of antibody & antigens; practical usage.
73. Agglutinins & reaction of agglutination: mechanism of reaction &
ingredients; preparation of polyvalent & monovalent serums; reaction of passive
agglutination, practical use.
74. Reaction of immunofluorescent (direct & indirect methods), ELISA,
radioimmunoassay. Practical using.
75. Reaction of immune lysis: reaction of hemolysis (mechanism,
ingredients), reaction of complement fixation (ingredients, mechanism, indications
of results).
76. Application of reactions of immune answer for diagnostics of viral
infections. ELISA. Western-blott.
77. Pathology of immune answer. Classification by the mechanism of breach
of immune system. Immunodeficiency conditions; primary immunodeficiencies,
their genetic mechanisms; secondary immunodeficiencies: causative agents, clinical
symptoms & principles of their treatment.
78. The valuation of immune status. Tests for the valuation of the factors of
nonspecific defense, humoral immunity, cell immunity, hypersensitivity.
79. Role of medical microbiology in prophylaxis infectious diseases. Drugs for
prophylaxis, diagnostics. Main directions of applied immunology.
80. Prophylaxis by vaccines. Kinds of vaccines, their preparations. Main
directions of improvement of vaccines. Vaccines of the 3-rd generation. Adjutants.
Vaccine therapy.
81. A live vaccines. Methods of preparation of vaccine strains. Conservation
of alive vaccine, specificity of its using. Dead vaccines, their preparation,
conservation, using.
82. Anatoxins, their preparation. Native & adsorbed anatoxins.
83. Antitoxic serums, their preparation, purification, using. Homologic &
heterologic gamma-globulin’s. Preparation & using. Methods of injections of
heterologic preparates for the prophylaxis of allergic complications.
84. Methods of prophylaxis & therapy of toxemic infections. Drugs,
directions for use.
85. Allergy, its types. Main peculiarities & mechanisms of formation of the
reactions of immediate type of hypersensitivity: anaphylaxis, cytotoxic, diseases of
immune complexes.
86. Hypersensitivity of delayed type: main specialties, mechanism of
formation, role in antimicrobial immunity. Infectional allergy.

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 87. Staphylococci: morphology, structure, classification (species); factors of
virulence. Staphylococcal diseases. Laboratory diagnosis. The problem of hospital-
acquired staphylococcal infections. Drugs for prophylaxis & treatment.
88. Streptococci: morphology, physiology, classification on hemolysis,
antigenic structure, factors of virulence. Streptococcal diseases. Laboratory diagnosis.
Pathogenic role in rheumatic fever, scarlet fever & other diseases. Immunity. Drugs
for prophylaxis & treatment.
89. Streptococcus pneumoniae: morphology & physiology, antigenic
structure (serological groups), role in human pathology. Laboratory diagnosis,
prophylaxis & treatment.
90. Meningococci: morphology & physiology; antigenic structure
(serogroups), factors of virulence, diseases. Laboratory diagnosis in sick people &
carriers. Drugs for specific prophylaxis & treatment.
91. Gonococci: morphology & physiology; conditions of cultivating; factors
of virulence, diseases. Laboratory diagnosis of acute & chronic gonorrhea. Drugs for
prophylaxis & treatment.
92. Escherichia: morphology & physiology. Antigenic structure &
classification. Physiological role in organism & sanitary-significant meaning. of
E.coli-associated diarrheal diseases: classification of microorganisms & diseases.
Laboratory diagnosis. Principles of prophylaxis & treatment.
93. Salmonella: morphology & physiology. Antigenic structure &
classification. Pathogens of enteric fever: morphology & physiology, factors of
virulence. Pathogenesis of enteric fever. Methods of early diagnosis.
94. Pathogens of enteric fever: morphology & physiology, factors of
virulence. Laboratory diagnosis of the early stages of disease; methods of discovery
of blood culture; serological diagnosis of carriage. Drugs for prophylaxis.
95. Salmonella - pathogens of food toxikoinfections: morphology and
physiology. Conditions of the beginning of food poisoning. Laboratory diagnosis.
Peculiarities of the epidemiology of hospital-acquired diseases in modern conditions.
Changes of epidemiology and in bacterial qualities.
96. Shigella: morphology & physiology; classification; factors of virulence &
pathogenesis of diseases. Laboratory diagnosis. Drugs for treatment & prophylaxis.
97. Pathogenic vibrios: morphology, physiology, classification. Cholera
vibrios, qualities. Biotypes. Antigenic structure, classification. Pathogenesis of
cholera. Laboratory diagnosis. Drugs for treatment & prophylaxis.
98. Campylobacters: morphology, physiology. Peculiarities of cultivation;
antigenic structure; species, clinical forms. Laboratory diagnosis. Drugs for
treatment.
99. Pathogens of plague. Morphological and cultural peculiarities. Sources of
infection. Clinical forms of plague. Laboratory diagnosis. Peculiarities of work with
the plague materials. Specific prophylaxis and therapy. Drugs for treatment.
100. Clostridium tetani: morphology, physiology. Production of toxin. Tetanus
in man: conditions of it beginning, pathogenesis. Specific prophylaxis and therapy.

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 101. Anaerobic gangrene; characteristic of pathogens. Production of bacterial
toxins. Conditions of diseases beginning. Laboratory diagnosis. Specific prophylaxis
and therapy.
102. Clostridium botulinum: it characteristic. The toxin-production. Types of
the bacterial toxins. Conditions of the onset of the diseases. Laboratory diagnosis.
Specific prophylaxis and therapy.
103. Non-spore-forming anaerobic bacteria: classification, morphology,
physiology. Basic peculiarities of clinic. Laboratory diagnosis. Drugs for treatment.
104. Pathogens of diphtheria: peculiarities of morphology & cultural qualities.
The toxin-production. Diphtheria in man. Immunity, methods of it discovering.
Bacteria-carriage. Specific prophylaxis & therapy. Epidemiology.
105. Mycobacteria: morphology, physiology, peculiarities of staining &
cultivation, classification. The pathogens of leprosy, it characteristic. Laboratory
diagnosis, prophylaxis & treatment.
106. Mycobacterium tuberculosis: morphology, physiology, peculiarities of
staining & cultivation, classification. Laboratory diagnosis, prophylaxis and
treatment. Peculiarities of immunity. Allergic probes. Drugs for treatment &
prophylaxis.
107. Pathogenic fungi: classification, morphology & physiology. Mycosis.
Superficial mycoses. Candidiasis. Laboratory diagnosis; antifungal drugs for
treatment.
108. Candida fungi: morphology, physiology, it difference from the yeast’s.
Conditions, which assists to beginning of candidiasis. Role of fungi in human
pathology. Laboratory diagnosis, therapy.
109. Pathogenic spirochete: classification; characteristic of unvenereal
treponematosis. Pathogenesis of syphilis; periods of disease; methods of laboratory
diagnosis. Immunity. Drugs for treatment; prophylaxis.
110. Spirochete of relapsing fever. Epidemiology of epidemic & endemic
relapsing fever. Pathogenesis of attacks. Laboratory diagnosis. The role of Minch,
Mechnicoff, Gabrichevsky in science.
111. Pathogens of Lyme disease: morphology, physiology, epidemiology,
spreading; sources & ways of transmission. Clinic, stages of disease. Laboratory
diagnosis. Drugs for treatment.
112. Leptospira: morphology, physiology. Sources of infection & ways of
transmission. Laboratory diagnosis. Specific prophylaxis & therapy.
113. Mycoplasma: characteristic, classification; peculiarities of morphology
and cultivation. Pathogenic species. Mycoplasma pneumonia. Laboratory diagnosis,
therapy.
114. Rickettsia: characteristic, classification. The louse-borne typhus:
mechanism of infectioning, pathogenesis. Brill's disease. Laboratory diagnosis.
Specific prophylaxis & therapy.
115. Pathogens of Q-fever: morphology, physiology. Sources & ways of
transmission. Stability in the nature. Laboratory diagnosis. Treatment & prophylaxis.
116. Chlamydia: morphology & physiology. Pathogenic species; diseases.
Pathogens of psittacosis. Laboratory diagnosis, treatment & prophylaxis.

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 117. Pathogens of trachoma & unveneral urethritis: morphology and
physiology. Clinical forms. Ways of transmission.
118. Rabies virus, it characteristic. Sources of infection & mechanism of the
infecting. Pathogenesis of hydrophobia. Laboratory diagnosis. Antirabic
immunoglobulin; antirabic vaccine: types & tactic of employment, mechanism of
acting.
119. Orthomyxoviruses: structure of influenza virus; classification:
changeability of viruses & it mechanisms. Immunity. Laboratory diagnosis. Drugs for
treatment & prophylaxis.
120. Paramyxoviruses: morphology & physiology, classification.
Characteristic of the parainfluenza virus. Mumps virus. Laboratory diagnosis, drugs
for treatment & specific prophylaxis.
121. Measles virus, it characteristic. Immunity; specific prophylaxis;
peculiarities of vaccine. Herpesviruses, cytomegalovirus, Epstein-Bar virus.
Characteristic of these diseases. Laboratory diagnosis, treatment and prophylaxis.
122. Picornaviruses: classification, structure of virions, chemical structure.
Poliovirus: characteristic, types & pathogenesis of poliomyelitis. Laboratory
diagnosis, specific prophylaxis. Echoviruses, Coxacieviruses; characteristic of
diseases in man.
123. Togaviruses: characteristic & classification. Japanese B encephalitis virus,
tick-borne encephalitis virus. Sources & ways of transmission. Laboratory diagnosis.
Specific therapy & prophylaxis.
124. The modern classification of pathogens of the infectious hepatitis.
Infectious hepatitis A virus, it characteristic, ways of transmission & pathogenesis of
infectious hepatitis A. Laboratory diagnosis, prophylaxis. Infectious hepatitis C, E &
G viruses.
125. Infectious hepatitis B virus, it characteristic, classification, antigenic
structure. Sources & mechanism of transmission; pathogenesis. Infectious hepatitis D
virus, it characteristic; peculiarities of clinic; laboratory diagnosis & specific
prophylaxis. Hepatitis T virus.
126. Oncogenic viruses: characteristic; classification; oncogenic viruses of
animals. Peculiarities of cooperation of oncogenic viruses with body cells. Zilber's
virus-genetic theory of the beginning of malignant tumors.
127. Human Immunodeficiency Virus (HIV), it characteristic, ways of
transmission, groups of risk. Peculiarities of its interaction with the human body
cells; mechanism of immunodeficiency formation. Laboratory diagnosis of HIV.
Social, legal & ethic aspects of AIDS. Prophylaxis & treatment of AIDS.
128. Characteristic of the causative agents of slow & unconventional virus
diseases. Enumerate pathogens & diseases. Prion’s characteristic.

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