Special bacteriology - СумДУlib.sumdu.edu.ua/library/docs/rio/2017/m4276.pdf · 2018-02-01 ·...
Transcript of Special bacteriology - СумДУlib.sumdu.edu.ua/library/docs/rio/2017/m4276.pdf · 2018-02-01 ·...
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Ministry of Education and Science of Ukraine
Sumy State University
Medical Institute
Department of Public Health
Microbiology, Virology
and Immunology
Part 2 4276
Special bacteriology
Practical Workbook
Sumy
Sumy State University
2017
Ivakhnyuk T. V., Ivakhnyuk U. P., Holubnycha V. M., Kornienko V. V.
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Practical Workbook of «Microbiology, Virology and Immunology» Part 2
«Special bacteriology » / T. V. Ivakhnyuk, U. P. Ivakhnyuk, V. M. Holubnycha,
V. V. Kornienko – Sumy : Sumy State University, 2017. – 60 p.
Department of Public Health
Special bacteriology
Practical Workbook
Student's name ____________________________________________
Group number _______________
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INTRODUCTION
Medicine is an ever-changing science undergoing continual development. Research and clinical
experience are continually expanding our knowledge, the knowledge of proper treatment and drug
therapy in particular. Medical microbiology is the study of the background material on various pathogenic
organisms. It delineates the epidemiology, transmission, clinical manifestations, diagnosis, and treatment
of diseases caused by these organisms.
The objective of this study aid is to instil a broad-based knowledge of the aetiologic organisms
causing disease and pathogenetic mechanisms leading to clinically manifested infections. This knowledge
is a necessary prerequisite for the diagnosis, therapy, and prevention of infectious diseases. Beyond this
academic purpose, its usefulness extends to all medical professions and most particularly to physicians
working in both clinical and private practice settings.
The workbook makes the vast and complex field of medical microbiology more accessible by the
use of numerous illustrations with detailed explanatory legends. Many tables present knowledge in a
cogent and useful form. The material is organized into four sections of increasing complexity designed to
give students first a sense of familiarity with the nature of microorganisms, then practice in aseptic
cultural methods in clinical settings. Instructors may select among the exercises or parts of exercises they
wish to perform, according to the focus of their courses and time available.
PROPER SAFETY PROCEDURES
To insure safety of those working in the lab, as well as the integrity of each experiment, each of the
following rules must be met.
1. Clothing should be protected by a lab coat or an apron. No shorts are allowed – you will be asked
to return home and change if worn to the lab class.
2. Hair that is long should be tied back to avoid contamination as well as safety when working near
the Bunsen burners.
3. Lab stations must be wiped down at the beginning of a lab to lower contamination rates of
cultures by organisms being already on the stations as well as safety for the students. Stations must also
be wiped down at the end of every lab session. Station clearing is best accomplished with fresh 10%
bleach. If there is any visible contamination on the bench, wash it with soap and water before the bleach.
4. Avoid direct contact with any microbes being tested by keeping all cultures well below mouth,
nose and eye area. Microbial agents normally move with gravity, so downward is the basic direction.
Thus, to insure integrity of cultures, avoid coughing, excessive talking, laughing, etc. while working with
cultures being open. Keep cultures at a minimum of exposure to the air for the best results.
5. Lab stations should be kept clear of any extra materials (non-lab books, book bags, purses, keys,
etc.) to avoid contamination as well as accidents.
6. All lab materials must be stored in the appropriate locations at the end of each session.
7. Tubes and racks should be placed in the appropriate location for autoclaving.
8. Bunsen burners should be lit from the beginning of each session to the end as this decreases the
risk of contamination of cultures and helps the safety of the lab worker.
9. All spills should be reported immediately to the lab instructor for proper cleanup. Unreported
spills can result in biohazardous conditions. To keep spills, burns, contamination and other accidents to a
minimum, it is wise to stay alert and pay attention to your surroundings all the times.
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Date _____________________ Class № 1
MICROBIOLOGY AND LABORATORY DIAGNOSIS OF INFECTIONS CAUSED
BY ESCHERICHIA COLI
QUESTIONS FOR DISCUSSION
1. General description of the Enterobacteriaceae family bacteria.
2. Biological properties of E. coli, antigenic structure and classification of pathogenic E. coli.
3. Microbiological diagnosis of E. coli associated with diarrhoeal diseases and colibacillosis.
4. Epidemiology and pathogenesis of diarrhoea-causing E. coli. Specific features of the immunity in
diarrhoea caused by E. coli.
5. Principles of prophylaxis and medical treatment of such type of diarrhoea.
PROTOCOLE OF PRACTICAL SESSION
Task 1. Microscope and sketch preparations of “E. coli O1:K1”and “E. coli O55:K59:H1” pure
cultures microscopically. Make a conclusion.
Name of the preparation
Picture Morphological and staining properties
E. coli O1:K1, Gram staining
E. coli O55:K59:H1 , Gram
staining
Task 2. Study the cultural properties of E. coli on Endo and EMB agar plate: sketch
colonies, make conclusions, and mark the plan of the further investigation.
The growth of E. coli O1:K1 and
potentional pathogenic E. coli on Endo
medium
The growth of E. coli O1:K1 and potentional
pathogenic E. coli on EMB medium
Picture
Cultural characteristic
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Task 3. Study the antigenic structure of Escherichia and estimate the result of slide
agglutination test with Escherichia polyvalent agglutination OK- and monovalent group sera. Make
a conclusion.
Components of
the reaction
Serum
the
Escherichia
polivalent
agglutinating
OKA-serum
the
Escherichia
polivalent
agglutinating
OKB-serum
the
Escherichia
polivalent
agglutinating
OKC-serum
the
Escherichia
polivalent
agglutinating
OKD-serum
the Escherichia
polivalent
agglutinating
OKE-serum
Pure culture
from patient
Result
Conclusion:
Task 4. Study the growth of E. coli on the triple sugar iron (TSI) agar. Make a conclusion.
Conclusion:
Task 5. Examine biochemical activity of E. coli on Hiss’ media.
Microbe
Fermentation of
Production of
Glucose Mannitol Lactose Sucrose H2S Indole
E. coli
Task 6. Examine antibiotic susceptibility test of E. coli. Make a conclusion.
Picture Interpretation of result
Name of antibiotic Diameter of
inhibiting sone
(mm)
Sensitiveness
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Task 7. Study and record in the protocol the demonstration of diagnostic preparations.
Name of the preparation Сomposition,
method of obtaining
Purpose of using
(test, method)
Escherichia agglutinating
polyvalent OKA-serum
Escherichia agglutinating
adsorbed monovalent serum
O55
Task 8. Study and record in the protocol the demonstration of therapeutic and prophylactic
preparations.
Name of the
preparation
Сomposition, method of obtaining Purpose of using
Colibacterin
Bifidumbacterin
Bificol
Lactobacterin
Bacteriophagum
intestinalis
Bacteriophagum coli
fluidum
Task 9. Fill in the card of particular microbiology test and sketch the scheme of the
diarrhoea-causing E. coli laboratory diagnosis.
Teacher's signature ____________________
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Date _____________________ Class № 2
MICROBIOLOGY AND LABORATORY DIAGNOSIS OF SHIGELLOSIS
QUESTIONS FOR DISCUSSION
1. Biological features of Shigella spp.
2. Microbiological diagnostics of shigellosis.
3. Epidemiology and pathogenesis of shigellosis. Features of shigellosis immunity.
4. Basic measures for prophylaxis and treatment of shigellosis.
PROTOCOLE OF PRACTICAL SESSION
Task 1. Study the slide of S. flexneri 2a, S. sonnei and E. coli О55:К59:Н1; compare results of
microscopy, make conclusions, and sketch the results of the microscopy.
Name of the
preparations
Picture Morphological and staining properties
S. flexneri 2a,
Gram staining
S. sonnei , Gram
staining
E. coli О55:К59:Н1,
Gram staining
Task 2. Study the cultural properties of Shigella ssp. on EMB agar and Hektoen enteric
agar, sketch the colonies, make conclusions, and mark the plan of the further investigation.
The growth of Shigella spp.
on EMB agar
The growth of Shigella spp.
on Hektoen enteric agar
Picture
Cultural characteristic
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Study the antigenic structure of Shigella spp. in slide agglutination test with shigellosis
agglutination polyvalent serum for identification of the causative agent.
Components of slide agglutination test:
Result
1. ______________________________________
2. ______________________________________
Conclusion:
Task 3. Study the growth of Shigella spp on the triple sugar iron (TSI) agar. Make a
conclusion.
Conclusion:
Task 4. Examine biochemical activity of Shigella spp. on Hiss’ media.
Microbe Fermentation of Production of
Glucose Mannitol Lactose Sucrose H2S Indole
Task 5. Make the consideration of РНАT with erythrocyte Flexneri diagnosticum for
serological diagnostics of shigellosis.
Components of РНАT: Result
1. ___________________________
2. ___________________________
3. ___________________________
Dilution of serum DC
1:2 1:4 1:8 1:16 1:32
Conclusion:
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Task 6. Study and record in the protocol the demonstration of diagnostic preparations.
Name of the preparation Сomposition,
method of obtaining
Purpose of using
(test, method)
Shigellosis adsorbed
agglutinating polyvalent
serum
Adsorbed agglutinating
typical (serospecific) serum
for Shigella flexneri (type II)
Shigellosis luminescent
serum
Shigellosis Sonnei
diagnosticum
Shigellosis erythrocyte
Sonnei diagnosticum
Polyvalent shigellosis (liquid)
bacteriophage
Task 7. Study and record in the protocol the demonstration of therapeutic and prophylactic
preparations.
Name of the preparation Сomposition,
method of obtaining
Purpose of using
Polyvalent shigellosis
(tableted) bacteriophage
Lactoglobulin
Eubiotics: colibacterin,
bifidumbacterin, bificol,
lactobacterin
Task 8. Fill in the card of particular microbiology test and sketch the scheme of the shigellosis
laboratory diagnosis.
Teacher's signature ____________________
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Date _____________________ Class № 3
MICROBIOLOGY OF TYPHOID FEVER, А AND В PARATYPHOID
QUESTIONS FOR DISCUSSION
1. General characteristics of the bacteria genus Salmonella. Classification of Salmonella according to
the biochemical properties and antigenic structure. Kauffmann-White classification. Pathogenesis of
salmonellosis in human and animals.
2. Biological properties of typhoid fever, A and B paratyphoid.
3. Pathogenesis and features of immunity of the typhoid fever, A and B paratyphoid.
4. Microbiological diagnostics of typhoid fever, A and B paratyphoid.
5. Basic measures of specific prophylaxis and medical treatment of typhoid fever, A and B
paratyphoid.
PROTOCOLE OF PRACTICAL SESSION
Task 1. Study the slide of S. typhi, E. coli; compare the results of microscopy, make
conclusions, and sketch the results of the microscopy.
Name of the preparations
Picture Morphological and staining properties
S. typhi, Gram staining
E. coli, Gram staining
Task 2. Study the cultural properties of causative agents on bismuth-sulphite agar and on
nutrient agar with bile (Ploskirev’s) medium, sketch colonies, make conclusions, and mark the plan
of the further investigations.
The growth of S.typhi
on bismuth-sulphite agar
The growth of S.typhi on nutrient agar
with bile (Ploskirev’s) medium
Picture
Cultural characteristic
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Task 3. Identify the haemoculture isolated from the patient with enteric fever (by the slide
agglutination test).
haemoculture
О-serum Н-serum
IX subspecies I subspecies, d phase
Conclusion:
_____________________________________________________________________________________
_____________________________________________________________________________________
Task 4. Study the antigenic structure of salmonella and estimate the slide agglutination test
with salmonellosis polyvalent O serum.
Components of slide agglutination test
Result
1. ______________________________________
2. ______________________________________
Conclusion:
Task 5. Perfome the Widal test.
Table 1 - Schematic Description of the Widal test
Ingredient Number of the test tubes
1 2 3 4 5 SC DC
Sodium
chloride solution 0.9 %, ml – 1,0 1,0 1,0 1,0 – 1,0
Patient's serum
in 1:100 dilution, ml 1,0 1,0 1,0 –
Diagnosticum, drops 1,0 1,0 1,0 1,0 1,0 – 1,0
Serum dilution obtained 1:50 1:100 1:200 1:400 1:800 1:50 –
Results Diagnosticum Dilution of patient’s serum DC SC
1:50 1:100 1:200 1:400 1:800
S. typhi
S. paratyphi A
S. schottmuelleri
Conclusion:
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Task 6. Perfome the РНАT with erythrocyte Vi-diagnosticum.
Components of РНАT Result
1. ___________________________
2. ___________________________
3. ___________________________
Dilution of serum DC
1:10 1:20 1:40 1:80 1:160
Conclusion:
Task 7. Study and record in the protocol the demonstration of diagnostic preparations.
Name of preparation Сomposition,
method of obtaining
Purpose of using
(test, method)
Adsorbed agglutinating
polyvalent O serum
Adsorbed salmonellosis
agglutinating diagnostic sera:
polyvalent group О (О1, О4, О5,
О12) agglutinating diagnostic
serum and monovalent О4
agglutinating diagnostic serum
Adsorbed typhoid fever
agglutinating monovalent Нd-
serum
Luminescent typhoid fever serum
Monovalent paratyphoid О2
diagnosticums
Monovalent typhoid fever Vi
diagnosticum
Salmonellosis complex
erythrocyte А, В, С, D, Е
diagnosticum
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Typhoid fever erythrocyte О9
diagnosticum
Typhoid fever erythrocyte Vi
diagnosticum
Task 8. Study and record in the protocol the demonstration of therapeutic and prophylactic
preparations.
Name of preparation Сomposition,
method of obtaining
Purpose of using
(characteristic of immunity)
Polyvalent
salmonellosis А, В, С,
D, Е bacteriophage
(tableted)
Typhoid fever
bacteriophage
(tableted)
Alcohol typhoid fever
vaccine enriched with
Vi antigen
Chemical absorption
of typhoid-
paratyphoid-tetanus
vaccine
Typhoid fever vaccine
with sixtoxoid
Task 9. Fill in the card of particular microbiology test and sketch the scheme of typhoid fever, A
and B paratyphoid laboratory diagnosis.
Teacher's signature ____________________
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Date _____________________ Class № 4
MICROBIOLOGY AND LABORATORY DIAGNOSIS OF CHOLERA
QUESTIONS FOR DISCUSSION
1. Biological properties of Cholerae spp.
2. Epidemiology and pathogenesis of the diseases caused by Cholerae spp. Specific features of
immunity in such cases.
3. Microbiological diagnostics of cholera.
4. Basic measures of prophylaxis and treatment of cholera.
PROTOCOLE OF PRACTICAL SESSION
Task 1. Study the slide of V. cholerae; compare the results of microscopy, make conclusions,
and sketch the results of the microscopy.
Name of the preparation
Picture Morphological and staining properties
V. cholerae, Gram staining
Task 2. Study biochemical properties of the causative agents of Cholera on Hiss media, make
conclusions and mark the plan of the further investigations.
Heiberg differentiated vibrios into biochemical types according to their ability to ferment mannose,
arabinose, and saccharose. Eight groups of vibrios are known to date; the cholera vibrios of the
cholerae and El Tor biovar belong to biochemical variant 1.
mannose arabinose sucrose
Task 3. Determine the biovar of the V. cholerae according to the table. Make the conclusion.
Test V. cholerae
serogroup O1
V. cholerae
O1 El Tor
V. cholerae
O139
Non-01 /non-O139
V. cholerae
The main tests
Agglutination by
cholera О1-serum
Agglutination by
Ogáva and Inába
serum
Lysis by El-Tor
bacteriophage
Lysis by Cholera
monophagous C IV
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Resistance to the
action of polymyxin B
Additional test
Haemolysis of sheep
erythrocytes
Hexsamine test
Voges-Proskauer test
Task 4. Study and record in the protocol the demonstration of diagnostic preparations.
Name of the preparation Сomposition,
method of obtaining
Purpose of using
(test, method)
Cholera O1 agglutinating
serum
Cholera H-agglutinating
serum
Agglutinating Ogáva serum
Cholera bacteriophage El
Tor (liquid)
Cholera bacteriophage C
(liquid)
Сholera fluorescent serum
16
Task 5. Study and record in the protocol the demonstration of therapeutic and prophylactic
preparations.
Name of the preparation Сomposition,
method of obtaining
Purpose of using
(characteristic of immunity)
Vibrio polyvalent
bacteriophage (tablets)
Corpuscular inactivated dry
cholera vaccine
Cholera vaccine
(choleragen-anatoxin + O
antigen)
Bivalent cholera chemical
vaccine (pelletized)
Choleragen-toxoid
Task 6. Fill in the card of particular microbiology test and sketch the scheme of the cholera
laboratory diagnosis.
Teacher's signature ____________________
17
Date _____________________ Class № 5
MICROBIOLOGY AND LABORATORY DIAGNOSIS OF CAMPYLOBACTERIOSIS
AND HELICOBACTERIOSIS
QUESTIONS FOR DISCUSSION
1. Biological features of campylobacteriosis and helicobacteriosis causative agents.
2. Microbiological diagnostics of campylobacteriosis and helicobacteriosis
3. Epidemiology and pathogens of campylobacteriosis and helicobacteriosis. Features of the
immunity.
4. Principles of campylobacteriosis and helicobacteriosis prophylaxis and treatment.
PROTOCOLE OF PRACTICAL SESSION
Task 1. Microscope and sketch the preparations of C. jejuni pure cultures microscopically. Make
a conclusion.
Name of the preparations
Picture Morphological and staining properties
C. jejuni, Gram staining
Task 2. Study the cultural properties of Campylobacter spp. on special medium: sketch
colonies, make conclusions, and mark the plan of the further researches.
The growth of Campylobacter spp.
on Skirrow agar
The growth of Campylobacter spp.
on chocolate agar
Picture
Cultural characteristic
Task 3. Perfome the cito test for detection of H. pylory
Task 4. Fill in the card of particular microbiology test and sketch the scheme of the
campylobacteriosis laboratory diagnosis.
Teacher's signature ____________________
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Date _____________________ Class № 6
MICROBIOLOGY AND LABORATORY DIAGNOSIS OF PSEUDOTUBERCULOSIS AND
INTESTINAL YERSINIOSIS
QUESTIONS FOR DISCUSSION
1. Pseudotuberulosis and intestinal yersiniosis: epidemiology, pathogenesis, immunity.
2. Microbiological diagnosis of pseudotuberulosis and intestinal yersiniosis.
3. Preparations for the diagnosis, prevention and treatment of pseudotuberulosis and intestinal
yersiniosis.
PROTOCOLE OF PRACTICAL SESSION
Task 1. Microscope and sketch preparations of Yersinia pseudotuberculosis and Y. enterocolitica
pure cultures microscopically. Make a conclusion.
Name of the preparation
Picture Morphological and staining properties
Yersinia
pseudotuberculosis,
Gram staining
Yersinia enterocolitica,
Gram staining
Task 2. Perfome the РНАT with erythrocyte O9 intestinal yersiniosis diagnosticum.
Components of РНАT: Result
1. ___________________________
2. ___________________________
3. ___________________________
Dilution of serum DC
1:10 1:20 1:40 1:80 1:160
Conclusion:
Task 3. Study and record in the protocol the demonstration of diagnostic preparations.
Name of the preparation Сomposition,
method of obtaining
Purpose of using
(test, method)
Erythrocyte O9- intestinal
yersiniosis diagnosticum
Pseudotuberculin
Task 4. Fill in the card of particular microbiology test and sketch the scheme of the
pseudotuberculosis and intestinal yersiniosis laboratory diagnosis.
Teacher's signature ____________________
19
Date _____________________ Class № 7
LABORATORY DIAGNOSIS OF TOXIC INFECTIONS
AND ACUTE INTESTINAL INFECTIONS
QUESTIONS FOR DISCUSSION
1. Biological properties of salmonella, their antigenic structure and classification.
2. Biological properties of acute intestinal infections and toxic infection agents: ETEC, S. sonnei,
Salmonella typhimurium, Salmonella enterica, Klebsiella pneumoniae, Proteus vulgaris, Proteus
mirabilis, Citrobacter freundii, Citrobacter diversus.
3. Epidemiology and pathogenesis of salmonellosis, acute intestinal infection caused by
opportunistic pathogenic bacteria and toxic infections, features of the immunity.
4. Microbiological diagnosis of salmonellosis.
5. Features of microbiological diagnosis of acute intestinal infection and toxic infection, caused by
opportunistic pathogenic bacteria.
6. The principles of prevention and treatment of salmonellosis, acute intestinal infection caused by
opportunistic pathogenic bacteria and toxic infections bacterial aetiology.
PROTOCOLE OF PRACTICAL SESSION
Task 1. Microscope and sketch the preparations of E. coli O1:H27, S. sonnei, S. enterica, and
P. vulgaris pure cultures microscopically. Make a conclusion.
Name of the preparations
Picture Morphological and staining properties
E. coli O1:H27, Gram
staining
S. sonnei, Gram staining
S. enterica, Gram staining
P. vulgaris, Gram staining
Klebsiella pneumonia,
Burri-Gins staining
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Task 2. Study the biochemical activity of salmonella in Hiss medium, draw it in the protocol.
Bacterium Glucose Lactose Mannitol Maltose Sucrose Nutrient broth
H2S Indole
E.coli
S. sonnei
S. enterica
P. vulgaris
Symbols: A – acid; G – gas
Task 3. Study the results of PHAT with salmonellosis polyvalent (A, B, C, D, E) erythrocyte
diagnosticum. Fill the results in the protocol.
Components of РНАT: Result
1. ___________________________
2. ___________________________
3. ___________________________
Dilution of serum DC
1:10 1 20 1:40 1:80 1:160
Conclusion:
Task 4. Study the cultural properties of Klebsiella pneumoniae on Ploskirev’s medium and
Proteus vulgaris on nutrient agar.
The growth of Klebsiella pneumoniae
on Ploskirev’s medium
The growth of Proteus vulgaris
on nutrient agar
Picture
Cultural characteristic
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Task 5. Make quantitative accounting of the opportunistic pathogenic microorganisms in the
medium (inoculation of the faeces of healthy and sick person) and analyze the results.
At bacteriological investigation of the material a bacteriologist uses the quantitative method of
sectoral inoculation, which allows to determine the number of bacteria in 1 g of the investigated
material – CFU/g. The bacteriologist prepares faeces dilution 1:10. The nutrient medium surface is
divided into 4 sectors: A, 1, 2, 3. Platinum loop (0.1 ml of the investigated material) inoculates the
material in section A, making 40 streaks. After the loop is sterilized in a flame of spirit lamp and four
innoculations are made from sector A, in sector 1, from sector 1 in sector 2, and from sector 2 in sector 3
(each time the loop is sterilized). The culture is incubated in the thermostat at t = 37 ºC for 18-24 hours,
then the number of colonies that have grown up is counted up, and sets the number of bacteria in 1 g of
the investigated material is determined (according to the table).
Table 1 – Bacteria count according to the number of colonies in the sectors
Bacteria count in 1 ml
of the investigated
material
Number of colonies in the sector
А 1 2 3
Less than 1 thousand 1–6 – – –
1 thousand 8–20 – – –
5 thousand 20–30 – – –
10 thousand 30–60 – – –
50 thousand 70–80 – – –
100 thousand 100–150 5–10 – –
500 thousand Innumerable 20–30 – –
1 million Innumerable 40–60 – –
5 million Innumerable 100–14 – –
10 million Innumerable Innumerable 10–20 –
50 million Innumerable Innumerable 60–80 Single growth
The results show the evidence that in
the patient’s material bacteria grew in all
sectors. In sector 3 there grew a very large
number of colonies of the bacteria that is
> 108 CFU/g of the investigated material.
In the material of healthy people,
bacteria grew only in the sector A and the
number of bacteria was 5, that according to
this table 2 is 1×103 CFU/g of the
investigated material. The number of
opportunistic pathogenic microorganisms in
microbiocenosis is in the normal range.
The results and conclusion: _______________________________________________________
____________________________________________________________________________________
____________________________________________________________________________________
____________________________________________________________________________________
____________________________________________________________________________________
Figure 1 – Inoculation of the investigated material for
determining the number of bacteria in 1 g
22
Task 6. Study the preparations for the diagnostic, therapeutic, and preventive purposes of
the acute intestinal infections, food poisoning and toxic infection.
Name of the preparation Сomposition,
method of obtaining
Purpose of using
Coliproteus lactoglobulin
Coliproteus bacteriophage
Colibactein, lactobacterin,
and simbiter
Agglutinating adsorbed
salmonella O- and H-sera
(dry)
Intesti bacteriophage
(liquid)
Salmonellosis coliproteus
lactoglobulin
Task 7. Fill in the card of particular microbiology test and sketch the scheme of acute
intestinal infections caused by nonpathogenic bacteria laboratory diagnosis.
Task 8. Fill in the card of particular microbiology test and sketch the scheme of Salmonellosis.
Teacher's signature ____________________
23
Date _____________________ Class № 8
LABORATORY DIAGNOSIS OF FOOD POISONING
QUESTIONS FOR DISCUSSION
1. Biological properties of Clostridium botulinum.
2. Biological properties of agent staphylococcal food poisoning.
3. Epidemiology and pathogenesis of food poisoning, features of the immunity.
4. Microbiological diagnosis of botulism and staphylococcal food poisoning.
5. Features of microbiological diagnosis of botulism and staphylococcal food poisoning.
6. The principles of prevention and treatment of botulism and staphylococcal food poisoning.
PROTOCOLE OF PRACTICAL SESSION
Task 1. Microscope and sketch preparations of Clostridium botulinum and Staphylococcus
aureus pure cultures microscopically. Make a conclusion.
Name of the preparations
Picture Morphological and staining properties
Clostridium botulinum,
Gram staining
Clostridium botulinum,
Ziehl-Neelsen staining
Staphylococcus aureus,
Gram staining
Task 4. Study the cultural properties of Staphylococcus aureus on EYA and blood agar.
The growth of Staphylococcus aureus
on EYA
The growth of Staphylococcus aureus
on blood agar
Picture
Cultural characteristic
24
Task 5. Study the neutralization test (NT) to botulotoxin in foodstuff examination:
components, purpose of their use, procedure.
Components:
Purpose of their use:
Procedure:
Result:
Conclusion:
Task 6. Study the preparations for the diagnostic, therapeutic, and preventive purposes of
the acute intestinal infections, food poisoning and toxic infection.
Name of the preparation Сomposition,
method of obtaining
Purpose of using
Monovalent antitoxic
antibotulinum serum (A, B,
C, E, F
Dry diagnostic botulinic
sera of A, B, C, E, F type
Staphylococcal
bacteriophage (liquid)
Task 7. Fill in the card of particular microbiology test and sketch the scheme of the botulism
laboratory diagnosis.
Task 8. Fill in the card of particular microbiology test and sketch the scheme of the
Staphylococcus aureus food poisoning laboratory diagnosis.
Teacher's signature ____________________
25
Date _____________________ Class № 9
MICROBIOLOGY AND LABORATORY DIAGNOSIS OF STAPHYLOCOCCAL
INFECTIONS
QUESTIONS FOR DISCUSSION
1. Biological properties of Staphylococcus spp.
2. Staphylococcal infections (furuncles, carbuncles, sinusitis, ostitis, postinfluenza pneumonia,
sepsis, etc.), microbiological diagnosis.
3. Epidemiology and pathogenesis of the diseases caused by Staphylococcus spp. Specific features of
immunity in such cases.
4. Basic measures of prophylaxis and treatment of staphylococcal infections.
PROTOCOLE OF PRACTICAL SESSION
Task 1. Microscope and sketch the preparations of Staphylococcus aureus and S. epidermidis
pure cultures microscopically. Make a conclusion.
Name of the preparation
Picture Morphological and staining properties
Staphylococcus aureus,
Gram staining
Staphylococcus
epidermidis, Gram staining
Task 2. Prepare and stain by Gram’s method the preparation taken from the blood of the
patient suspected of staphylococcal sepsis, draw it in the protocol.
Picture Morphological and staining properties
Task 3. Study the cultural properties of S. aureus and S. epidermidis in milk nutrient agar
The growth of S. aureus
on milk nutrient agar
The growth of S. epidermidis
on milk nutrient agar
Picture
Cultural characteristic
26
Task 4. Study the cultural properties of S. aureus and S. epidermidis in blood agar
The growth of S. aureus on blood agar The growth of S. epidermidis on blood agar
Picture
Cultural characteristic
Task 5. Study the cultural properties of S. aureus and S. epidermidis in egg yolk salt agar
The growth of S. aureus
on egg yolk salt agar
The growth of S. epidermidis
on egg yolk salt agar
Picture
Cultural characteristic
Task 6. Study the growth of other types of Staphylococcus spp. in citrate plasma. Draw the
plasma in the test tubes before streaking of Staphylococcus spp. and after 24-hour growth.
Picture Interpretation of result
Task 7. Study the sensitivity of the isolated staphylococcal culture to antibiotics; make a
conclusion. Write the results in the protocol.
Picture Interpretation of the result
Name of antibiotic Diameter of inhibiting
sone (mm)
Sensitiveness
27
Task 8. Study the sensitivity of the isolated staphylococcal culture to phintonside garlic and
make a conclusion. Write the results in the protocol.
Picture Interpretation of result
Task 9. Examine the sensitivity of staphylococci to penicillin by serial dilutions method in a
liquid media.
Place 14 sterile tubes in a rack and add 2 ml of sterile nutrient broth to each tubes. Then add 2 ml of
penicillin to te first tube. Add 2 ml of the penicillin broth to the first tube, the concentration of peniicillin
in this tube is 50 unit per ml. Take a fresh pipette, introduce it into the first tube, mix the contents
thoroughly, and transfer 2 ml from this tube into the second tube (25 unit per ml). Discard the pipette.
With a fresh pipette, mix the contents of the second tube and transfer 2 ml to the third tube (12.5 unit per
ml). Continue the dilution process through tube number 10.
After the contents of the each tube are mixed, discard 2 ml of broth so that the final volume in all
tubes is 2 ml. From the plate culture of E. coli prepare a suspension of the organism in 5 ml of saline
equivalent to a McFarland 0.5 standard. With a sterile pipette, transfer 0.1 ml of the staphylococci
suspension to the antibiotic containing broth tubes 1 through 10 and to the growth control tube.
Shake the rack gently to mix the tube contents and place the tubes in the incubator for 18 to 24
hours. Examine the absence and presence of growth. In 1st day determine the minimum inhibition
concentration of antibiotic (MIC). It is the lowest antibiotic concentration at which bacteria does not
multiplies and the content of tube remains transparent.
Ingredient Test tube 1 2 3 4 5 6 7 8 9 10 control of
bacteria
control of
antibiotic
Nutrient broth, ml 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
Antibiotic
(containing 100
units per 1 ml), ml
1.0 – – – – – – – – – – 1.0
Preparation of
serial dilutions
1.0
→
1.0
→
1.0
→
1.0
→
1.0
→
1.0
→
1.0
→
1.0
→
1.0
→
1.0
↓
Antibiotic
concentration
(units/ml)
50 25 12.5 6.2 3.1 1.6 0.8 0.4 0.2 0.1 50
Bacterial
suspension
0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1
Incubation in thermostat at 37 °C during 18–24 hours
Result
Conclusion: minimum inhibitory concentration (MIC) _________________________________
minimum bacteriocide concentration (MBC) _______________________________
28
Task 10. Study and record in the protocol the demonstration of diagnostic preparations.
Name of the preparation Сomposition, method of
obtaining
Purpose of using (test, method)
Staphylococcal bacteriophage
(liquid)
Task 11. Study and record in the protocol demonstration a therapeutic and prophylactic
preparations.
Name of the preparation Сomposition, method of
obtaining
Purpose of using
Staphylococcal toxoid
Antistaphylococcal gamma-
globulin
Task 12. Fill in the card of particular microbiology test and sketch the scheme of staphylococcal
infections (furuncles, carbuncles, sinusitis, ostitis, postinfluenza pneumonia, sepsis) laboratory
diagnosis.
Teacher's signature ____________________
29
Date _____________________ Class № 10
LABORATORY DIAGNOSTICS OF STREPTOCOCCAL INFECTIONS
QUESTIONS FOR DISCUSSION
1. Classification of streptococci. Biological properties of Streptococcus spp.
2. Streptococcal infections (streptococcal pharyngitis or tonsillitis, scarlet fever, rheumatic fever,
glomerulonephritis, sepsis and pneumoniae), microbiological diagnosis.
3. Epidemiology and pathogenesis of the diseases caused by streptococcus. Specific features of
immunity in such cases.
4. Basic principles of streptococcal infections; prophylaxis and treatment.
PROTOCOLE OF PRACTICAL SESSION
Task 1. Microscope and sketch preparations of pure cultures of Streptococcus pyogenes,
Streptococcus spp. microscopically in the material taken from the patient and in pure culture. Make
a conclusion.
Name of the preparation
Picture Morphological and staining properties
Streptococcus pyogenes in
pleural puncture biopsy
material, Gram staining
Streptococcus pyogenes in
pure culture, Gram staining
Streptococcus pneumonia,
Burri-Gins method
Task 2. Study the cultural properties of S. pyogenes and S. pneumoniae in blood agar.
The growth of S. pyogenes in blood agar The growth of S. pneumoniae in blood agar
Picture
Cultural characteristic
30
Task 3. Study the main antimicrobial drugs used for treatment, prevention and diagnostics
of suppurative diseases and write them down in the copy book.
Name of the preparation Сomposition, method of
obtaining
Purpose of using
O-streptolysin
Pneumococcal vaccine
Task 4. Fill in the card of particular microbiology test and sketch the scheme of the streptococcal
pneumonia laboratory diagnosis.
Teacher's signature ____________________
31
Date _____________________ Class № 11
LABORATORY DIAGNOSIS OF MENINGOCOCCAL
AND GONOCOCCAL INFECTIONS
QUESTIONS FOR DISCUSSION
1. Biological properties of Neisseria spp.
2. Gonococcal infections (gonorrhoea, conjunctivitis of the newborn, pelvic inflammatory disease
(PID); microbiological diagnosis. Principles of Bordet-Gengou test (CFT) for diagnosis of gonorrhoea.
3. Epidemiology and pathogenesis of the diseases caused by gonococci. Specific features of
immunity in such cases.
4. Principles of gonococcal infections; prophylaxis and treatment (features of chronic gonorrhoea
treatment).
5. Principles of meningitis microbiological diagnosis.
6. Epidemiology and pathogenesis of the diseases caused by meningococci. Specific features of
immunity in such cases.
PROTOCOLE OF PRACTICAL SESSION
Task 1. Microscope and sketch the demonstrations preparations. Make a conclusion.
Name of the preparations
Picture Morphological and staining properties
Smear from urethra of
women with gonorrhea,
Gram staining
The spinal fluid of the child
suspected of meningitis,
Gram staining
Task 2. Study the cultural properties of Neisseria meningitides on blood agar
Picture Cultural characteristic
32
Task 3. Study CFT with a gonococcal antigen. Fill in the result of reaction in the protocol,
explaining the mechanism of its formation.
Components of РНАT: Principle
1. ___________________________
2. ___________________________
3. ___________________________
4. ___________________________
5. ___________________________
Result
Dilution of serum CS CA
1:2 1:4 1:8 1:16 1:32 1:64 1:128
Conclusion:
Task 5. Examine the sensitivity of N. gonorrhoeae to antibiotics, make the conclusion. Write
down the results of the experiment in the protocol.
Picture Interpretation of the result
Name of antibiotic Diameter of
inhibiting sone
(mm)
Sensitiveness
Task 6. Study the main antimicrobial drugs used for diagnosis, treatment, and prevention of
meningococcal and gonococcal diseases, write them down in the protocol.
Name of the
preparation
Сomposition, method of obtaining Purpose of using
Gonococcal
inactivated liquid
vaccine (gonovaccine)
33
Meningococcal
chemical vaccine
Gonococcal antigen
Task 7. Fill in the card of particular microbiology test and sketch the scheme of the
meningococcal infections and bacteria carriers laboratory diagnosis
Task 8. Fill in the card of particular microbiology test and sketch the scheme of the
gonococcal infection laboratory diagnosis.
Teacher's signature ____________________
34
Date _____________________ Class № 12
MICROBIOLOGY AND LABORATORY DIAGNOSIS
OF ANAEROBIC INFECTIONS
QESTIONS FOR DISCUSSION
1. General characteristics of the pathogenic spore-forming anaerobes (clostridia) – C. tetani,
C. perfringens, C. septicum, C. histoliticum, C. novyi.
2. Epidemiology, pathogenesis, microbiological diagnostics, treatment and prevention of tetanus and
gas gangrene.
3. General characteristics of non-clostridia anaerobes – bacteroides, fusobacteria, propionibacterium,
veillonella, eubacterium, peptococcus, peptostreptococcus, bifidobacterium.
PROTOCOLE OF PRACTICAL SESSION
Task 1. Microscope and sketch the preparations of pure cultures of obligate anaerobic bacteria
microscopically in the material taken from the patient and in pure culture. Make a conclusion.
Name of the preparation
Picture Morphological and staining properties
C. tetani,
Gram staining
C. tetani,
Ziehl-Neelsen staining
B. bifidum,
Gram staining
Peptostreptococcus spp.,
Gram staining
Task 2. Study the cultural properties of anaerobes on the media.
The growth of C. perfringens
on the blood-sugar Zeissler’s agar
The growth of C. perfringens
on the lactose egg-yolk medium
Picture
Cultural characteristic
35
Task 3. Study the Fortner dish with Zeissler’s agar and study the cultural properties of
C. perfringens on Kitt-Tarozzi medium, Litmus milk.
Fortner dish C. perfringens on
Kitt-Tarozzi medium
C. perfringens on
Litmus milk
C. perfringens on
Wilson-Blerr
Picture
Cultural characteristic
Task 4. Examine the biochemical properties of bacteria and make the conclusion. Write
down the results of the experiment.
Causative agent Fermentation of
carbohydrates
Curdle of milk Dilution of gelatin
C. perfringens
C. tetani
Task 5. Study the main antimicrobial drugs used for diagnosis, treatment and prevention of
meningococcal and gonococcal diseases, write them down in the protocol.
Name of the
preparation
Сomposition, method of obtaining Purpose of using
Tetanus toxoid (TT)
Sextatoxoid
DTaP
DT
Tetanus antitoxic
serum
36
Task 6. Prepare the smear from soil and stain it by Zeihl-Neelson method.
Ziehl-Neelsen staining procedure
1. Fix the smear.
2. Flood the slide with carbolic fuchsin, steam gently for 5 minutes over low flame, do not allow to
dry and add more stain if necessary. Cool. Alternatively, carbolic fuchsin-containing phenol and alcohol
(cool) may be used without heating.
3. Apply 90% alcohol containing 3% to 5% HC1 until all but the thickest parts of the smear cease to
give off colour (approximately 1 to 3 minutes). Wash.
4. Counterstain of 1 minute with methylene blue. Wash.
5. Examine smear using the oil-immersion lens of the light microscope.
Sources of error:
1. Overheating (burning) during fixation can be avoided by just touching the back of the slide to the
back of the hand each time the slide has been passed though the flame.
2. Do not stain smears which have only been air dried. Smears must also be “fixed”.
3. Smears should not be too thick. After air drying, examine them under the microscope. If there are
no areas of bacteria separation, more water should be added to dilute the smear.
4. After staining it is essential that the back surface of the slide is wiped clean.
5. If washing with distilled water is not done adequately, crystallization of the stain may appear on
the slide.
Microscope and sketch preparations of spore-forming culture, stained by Ziehl-Neelsen technique.
Make a conclusion.
Describe the morphological and tinctorial properties of the microorganisms: _______________________________________________________________
_______________________________________________________________
Task 7. Fill in the card of particular microbiology test and sketch the scheme of the tetanus
laboratory diagnosis.
Task 8. Fill in the card of particular microbiology test and sketch the scheme of the gas gangrene
laboratory diagnosis.
Teacher's signature ____________________
37
Date _____________________ Class № 13
MICROBIOLOGY AND LABORATORY DIAGNOSIS OF DIPHTHERIA
AND WHOOPING COUGH (PERTUSSIS)
QESTIONS FOR DISCUSSION
1. Biological properties of diphteria and pertussis causative agent.
2. Pathogenesis of diphtheria and pertussis in humans.
3. Features of antidiphtherial and antipertussis immunity.
4. Laboratory diagnostics of diphtheria and pertussis.
5. Specific prophylaxis and treatment of the disease.
PROTOCOLE OF PRACTICAL SESSION
Task 1. Microscope and sketch preparations of pure cultures of C. diphtheria by Gram’s
method, Neisser’s method, and Loeffler’s method; Bordetella pertussis by Gram’s method. Make a
conclusion.
Name of the
preparation
Picture Morphological and staining properties
C. diphtheria,
Gram staining
C. diphtheria,
Neisser’s method
C. diphtheria,
Loeffler’s method
Bordetella pertussis,
Gram staining
Task 2. Study the cultural properties of Corynebacterium diphtheriae cultures on Loeffler
nutrient medium and Buchin's medium.
The growth of Corynebacterium diphtheriae
cultures on Loeffler nutrient medium
The growth of Corynebacterium diphtheriae
cultures on Buchin's medium
Picture
Cultural characteristic
38
Task 3. Study the diphtheria bacillus cultures toxigenicity in gel-precipitation assay
(Elek test).
Componets:
Result
Conclusion:
Task 4. Study the cistinase and urease test. Make a conclusion.
Result of the cistinase test Result of the urease test
Conclusion
Task 5. Read the indirect haemagglutination test, make the conclusion about antidiphtherical
immunity.
Components of РНАT: Result
1. ___________________________
2. ___________________________
3. ___________________________
Dilution of serum DC
1:20 1:40 1:80 1:160 1:320
Conclusion:
39
Task 6. Study the differentiating features of bordetella and fill in the table.
Table 1 – Differentiating features of bordetella
Feature B. pertussis B. parapertussis B. bronchiseptica B. avium
Motiliti
Appearance on
MacConkey agar
Appearance on Bordet-
Gengou medium (days)
Urease
Nitrate to nitrite
Citrate use
Oxidase
Heat labile toxin and
tracheal cytotoxin
Adenylate cyclase toxin
Pertussis toxin
Task 7. Examine the agglutination test with the patient's paired sera and whooping cough
diagnosticum. Make the conclusion.
Components of agglutination test: Principle
1. ___________________________
2. ___________________________
3. ___________________________
4. ___________________________
5. ___________________________
Result
Dilution of serum CD
1:40 1:80 1:160 1:320 1:640
Conclusion:
40
Task 8. Describe the immunological preparations for treatment and prophylaxis of diphtheria
and pertussis.
Name of the
preparation
Сomposition, method of obtaining Purpose of using
DTaP
DT
ADT-M
Diphtheria toxoid
Antidiphtheria
antitoxic serum
Antidiphtherial
gamma globulin
Task 9. Describe the principle of Schick test.
Task 10. Fill in the card of particular microbiology test and sketch the scheme of the diphtheria
laboratory diagnosis.
Task 11. Fill in the card of particular microbiology test and sketch the scheme of the whooping
cough (pertussis) laboratory diagnosis.
Teacher's signature ____________________
41
Date _____________________ Class № 14
MICROBIOLOGY AND LABORATORY DIAGNOSIS
OF TUBERCULOSIS AND LEPROSY
QUESTIONS FOR DISCUSSION
1. Biological properties of Mycobacterium tuberculosis, Mycobacterium bovis,
Mycobacterium leprae.
2. Epidemiology and pathogenesis of tuberculosis and leprosy.
3. Features of antituberculosis immunity.
4. Laboratory diagnosis of tuberculosis and leprosy.
5. The tuberculin skin test, the interpretation of the results.
6. Specific prophylaxis and treatment of the disease.
PROTOCOLE OF PRACTICAL SESSION
Task 1. Prepare the smear from the vaccine BCG strain, stain it by Ziehl-Neelsen method,
perform the microscopy and draw it, make the conclusion.
Name of the preparation Picture Morphological and staining properties
The smear from vaccine BCG strain
(stained by Ziehl-Neelsen method)
Ziehl-Neelsen staining procedure
1. Fix the smear.
2. Put carbolfuchsin on the slide and steam it gently for 5 minutes over low flame, and do not
allow drying, add more stain if necessary. Cool. Alternatively, carbolfuchsin containing phenol and
alcohol (cool) may be used without heating.
3. Apply 90% alcohol containing 3% to 5% HC1 until all but the thickest parts of the smear
cease to give off color (approximately 1 to 3 minutes). Wash.
4. Stain for 1 minute with methylene blue. Wash.
5. Examine smear using the oil-immersion lens of the light microscope.
Task 2. Perform the microscopy and draw the ready preparation from the patient's sputum
(stained by Ziehl-Neelsen method), make the conclusion.
Name of the preparation
Picture Morphological and staining properties
Preparation from patient's sputum
(stained by Ziehl-Neelsen method)
Task 3. Learn the chemical structure of media for cultivation of tubercle bacilli. Examine
the growth of mycobacteria (M. tuberculosis) on Löwenstein-Jensen medium, make the conclusion.
M. tuberculosis on Löwenstein-Jensen medium
Picture Cultural characteristic
42
Task 4. Read the results of sensitivity tests to antibiotics of M. tuberculosis, make the
conclusion. Draw the slant and the growth of mycobacteria in the test tubes. Make the conclusion.
Picture Interpretation of result
Name of antibiotic Diameter of
inhibiting sone
(mm)
Sensitiveness
Task 5. Study and write into the protocol the information about the main diagnostic and
prophylactic preparation.
Name of the
preparation
Сomposition, method of obtaining Purpose of using
Tuberculin
Lepromin
Erythrocytic
tuberculosis
diagnosticum
BCG
BCG-M
Task 6. Fill in the card of particular microbiology test and sketch the scheme of the
tuberculosis laboratory diagnosis.
Task 7. Fill in the card of particular microbiology test and sketch the scheme of leprosy
laboratory diagnosis.
Teacher's signature ____________________
43
Date _____________________ Class № 15
MICROBIOLOGY AND LABORATORY DIAGNOSIS
OF PLAGUE AND TULAREMIA
QESTIONS FOR DISCUSSION
1. Special danger infections.
2. Biological properties of F. tularensis causative agent of tularemia.
3. Microbiological diagnosis of tularemia.
4. Biological properties of Y. pestis causative agent of plague.
5. Microbiological diagnosis of plague.
6. Specific prevention of special danger infections.
PROTOCOLE OF PRACTICAL SESSION
Task 1. Examine microscopically the smears of the patient's materials, fill in the protocol.
Name of the
preparation
Picture Morphological and staining properties
Yersinia pestis,
Gram staining
Francisella tularensis,
Gram staining
Task 2. Study the growth of the Francisella tularensis on the solid media.
The Francisella tularensis on the solid media
Picture Cultural characteristic
Task 3. Study and write in the protocol information about immunobiological preparations
for diagnostics, treatment and prophylaxis of the plague and tularemia.
Name of the
preparation
Сomposition, method of obtaining Purpose of using
Tularemia
diagnosticum
Pestilential
erythrocytic antibody
diagnosticum
44
Pestilential
bacteriophage
Tularin
Plague live EV
vaccine
Tularemia live vaccine
Immunoglobulin
antipestilential
Plague serum labeled
FITS
Tularemia serum
labeled FITS
Task 4. Read the PHAT with the patient's paired sera and make the conclusion.
Conclusion:
_______________________________________
_______________________________________
_______________________________________
_______________________________________
_______________________________________
_______________________________________
_______________________________________
Task 5. Fill in the card of particular microbiology test and sketch the scheme of the plague
laboratory diagnosis.
Task 6. Fill in the card of particular microbiology test and sketch the scheme of the tularemia
laboratory diag
Teacher's signature ____________________
45
Date _____________________ Class № 16
MICROBIOLOGY AND LABORATORY DIAGNOSIS
OF ANTHRAX AND BRUCELLOSIS
QUESTIONS FOR DISCUSSION
1. Biological properties of the B. anthracis and brucellae, antigenic structure.
2. Microbiological diagnosis of athrax and brucellosis.
3. Epidemiology and pathogenesis of athrax and brucellosis. Features of immunity at such diseases.
4. Principles of prophylaxis and medical treatment of athrax and brucellosis.
PROTOCOLE OF PRACTICAL SESSION
Task 1. Carry out the precipitation test in tube according to the table.
Table 1 - Precipitation test with investigated material
Ingredient
0.9% NaCl solution, ml - 0.5 0.5
Diagnostic serum, ml 0.5 - 0.5
Diagnostic material extract, ml 0.5 0.5 -
Result:
Conclusion:
Task 2. Study the smears of the patient’s materials.
Name of the
preparation
Picture Morphological and staining
properties
B. anthracis,
Ziehl-Neelsen staining
B. anthracis,
Gram staining
B. melitensis,
Gram staining
46
Task 3. Study and write in the protocol the information about immunobiological preparations
for diagnostics, treatment and prophylaxis of athrax and brucellosis.
Name of the
preparation
Сomposition, method of obtaining Purpose of using
Tularemia
diagnosticum
Anthraxin
Brucelin
Tularin
Live brucellosis
vaccine
Killed brucellosis
vaccine
Antianthrax gamma
globulin
Antianthrax
precipitating serum
Anthrax luminescent
serum
Task 4. Fill in the card of particular microbiology test and sketch the scheme of the anthrax
laboratory diagnosis.
Task 5. Fill in the card of particular microbiology test and sketch the scheme of the brucellosis
laboratory diagnosis.
Teacher's signature ____________________
47
Date _____________________ Class № 17
MICROBIOLOGY AND LABORATORY DIAGNOSIS
OF SPIROCHAETOSIS (SYPHILIS, BORRELIOSIS, AND LEPTOSPIROSIS)
QUESTIONS FOR DISCUSSION
1. General description of bacteria of the family Spirochaetes.
2. Microbiological diagnosis of leptospirosis.
3. Microbiological diagnosis of boreliosis.
4. Biological properties of T. pallidum.
5. Microbiological diagnosis of T. pallidum.
6. Principles of prophylaxis and medical treatment of syphilis.
PROTOCOLE OF PRACTICAL SESSION
Task 1. Carry out venereal disease research laboratory (VDRL) or rapid plasma reagin
(RPR).
When you carry out venereal disease research laboratory (VDRL) you must put 0.1-0.2 ml of the
patient’s serum in the tubes, then add carefully the dilution of the antigen. If you do everything correct,
the measure between serum and antigen will be clear. The tubes are incubated at 37 °С for 30 minutes.
While working you must be careful because the patient’s serum may contain spirochaetes. If the result is
positive, white ring will be at the measure between serum and antigen.
Task 2. Carry out the Wassermann’s test according to the table.
Table 1 - The Wassermann’s test with patient’s sera
Tube number
Content
1 (test) 2 serum control) 3 (antigen control)
Patient’s serum 0.5 0.5 –
Аntigen 0.5 – 0.5
Complement 0.5 0.5 0.5
0.9 % NaCl – 0.5 0.5
37 °С, 30 minutes
Haemolytic mixed 1.0 1.0 1.0
37 °С, 60 minutes
Result
Conclusion:
48
Task 3. Study the smears of the patient’s materials and draw them in the protocol.
Name of the preparations
Picture Morphological and staining properties
T. pallidum
in IFT
T. pallidum,
in the dark field examination
T. pallidum.
silver impregnation method
B. persica,
Giemsa’s staining
B.persica
in the dark field
L.interrogans
in the dark field examination
Task 4. Describe the immunobiological preparations for diagnostics, treatment and
prophylaxis of spirochetosis.
Name of the
preparation
Сomposition,
method of obtaining
Purpose of using
Tularemia
diagnosticum
Cardiolipin antigen
for Wassermann’s test
Specific antigen
for Wassermann’s test
Cardiolipin antigen
for venereal disease
research laboratory
Killed leptospirosis
vaccine
Task 5. Fill in the card of particular microbiology test and sketch the algorithm of action
during conduction of syphilis microbiological diagnosis.
Task 6. Fill in the card of particular microbiology test and sketch the scheme of the epidemic
relapsing fever microbiological diagnosis.
Task 7. Fill in the card of particular microbiology test and sketch the scheme of the
leptospirosis laboratory diagnosis.
Teacher's signature ____________________
49
Date _____________________ Class № 18
MICROBIOLOGY AND LABORATORY DIAGNOSIS
OF RICKETTSIAL DISEASES
QUESTIONS FOR DISCUSSION
1. General description of bacteria of the family Rickettsia.
2. Microbiological diagnosis of rickettsial diseases.
PROTOCOLE OF PRACTICAL SESSION
Task 1. Carry out and estimate the haemagglutination test.
Table 1- Haemagglutination test with patient's paired sera
Ingredient,
ml
Number of the test wells Control
1 2 3 4 5 6 7 8 9 10
0.9% solution
of NaCl
0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 –
Assayed
serum 1:62.5
0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 – –
Diagnosticum 0.1 0.1 0.1 0.1 0.1 0.1 0.1 – 0.1 0.1
Serum
dilution
1:125 1:250 1:500 1:1000 1:2000 – – 1:125 – –
Control
erythrocytes
– – – – – – – 0.1 – –
Standard
immune
serum, diluted
to the titre
– – – – – – – – – 0.4
Result:
Conclusion:
Task 2. Study the smears of the patient’s materials and draw them in the protocol.
Name of the preparations Picture Morphological and staining properties
R. prowazekii, IFT
R. prowazekii,
Zdrodovsky staining
R. prowazekii,
dark field examination
50
Task 3. Describe the immunobiological preparations for diagnosis, treatment, and
prophylaxis of spirochaetosis.
Name of the
preparation
Сomposition, method of obtaining Purpose of using
Type-specific
Muzer’s serum for
CFT
Ricketsia epidemic
typhus prowazekii
erythrocyte
diagnosticum for
PHAT
Coxiella burnetti dry
antigen for CFT
Task 4. Fill in the card of particular microbiology test and sketch the scheme of the rickettsial
diseases laboratory diagnosis.
Teacher's signature ____________________
51
Date _____________________ Class № 19
MICROBIOLOGY AND LABORATORY DIAGNOSIS
OF CHLAMYDIAL DISEASES
QUESTIONS FOR DISCUSSION
1. General description of bacteria of the family Chlamydia.
2. Microbiological diagnosis of the diseases caused by Chlamydia.
3. Epidemiology and pathogenesis of the diseases caused by Chlamydia. Features of immunity in
such diseases.
4. Principles of prophylaxis and treatment of the diseases caused by Chlamydia.
PROTOCOLE OF PRACTICAL SESSION
Task 1. Inoculate the patients’ sputum with candidiasis pneumonia on the medium.
Inoculate 0.1 ml of the diluted (1:100) sputum on Petri dish with potatoes medium. Spread the
material on the surface of the media in order to obtain growth in the form of lawn. After that, put Petri
dish for incubation.
Task 2. Study and write in the protocol the information about immunobiological
preparations for diagnosis of chlamydial infection.
Name of the
preparation
Сomposition, method of obtaining Purpose of using
Dry chlamydia
diagnosticum for CFT
Task 3. Sketch the scheme of chlamydial diseases laboratory diagnosis.
Signature of teacher ____________________
52
Date _____________________ Class № 20
MICROBIOLOGY AND LABORATORY DIAGNOSIS
OF MYCOPLASMAL DISEASES
QUESTIONS FOR DISCUSSION
1. 1.General description of bacteria of the family Mycoplasma.
2. Microbiological diagnosis of the diseases caused by Mycoplasma.
3. Epidemiology and pathogenesis of the diseases caused by Mycoplasma. Features of immunity in
such diseases.
4. Principles of prophylaxis and treatment of the diseases caused by Mycoplasma.
PROTOCOLE OF PRACTICAL SESSION
Task 1. Inoculate the patients’ sputum with candidiasis pneumonia on the medium.
Inoculate 0.1 ml of the diluted (1:100) sputum on Petri dish with potatoes medium. Spread the
material on the surface of the media in order to obtain growth in the form of lawn. After that, put Petri
dish for incubation.
Task 2. Study the growth of the Mycoplasma hominis on the solid medium.
The Mycoplasma hominis on the solid medium
Picture Cultural characteristic
Task 3. Sketch the scheme of the mycoplasmal diseases laboratory diagnosis.
Teacher's signature ____________________
53
Date _____________________ Class № 21
MICROBIOLOGY AND LABORATORY DIAGNOSIS
OF OPPORTUNISTIC MYCOSES AND PRIMARY MYCOSES
QUESTIONS FOR DISCUSSION
1. General characteristics of fungi (definition and taxonomy, morphology).
2. General aspects of fungal disease (primary mycoses, opportunistic mycoses, subcutaneous
mycoses, cutaneous mycoses, fungal allergies, and fungal toxicoses).
3. Primary mycoses (coccidioidomycosis, histoplasmosis, blastomycoses): characteristics of
pathogen, pathogenesis, principle diagnosis, treatment and prophylaxis.
4. Opportunistic mycoses (surface and deep yeast mycoses, aspergillosis, mucormycoses,
phaeohyphomycoses, hyalohyphomycoses, cryptococcoses; penicilliosis, pneumocystosis): characteristics
of pathogen, pathogenesis, principle diagnosis, treatment and prophylaxis. Features of candidiasis
diagnosis.
5. Principle of antifungal immune response.
PROTOCOLE OF PRACTICAL SESSION
Task 1. Study microscopically the material from the vagina.
Name of the
preparations
Picture Morphological and staining properties
Material from the vagina,
Gram staining
Task 2. Study the growth of Candida from the patient’s sputum (phase 2).
1. Count colonies of Candida spp., determining the amount of fungus cells in 1 ml of the diluted
sputum (1:100).
2. Prepare a smear, stain the preparation by Gram’s method, microscopic examination, draw it in
protocol.
3. Make a conclusion.
At determining the amount of fungi in 1 ml of sputum it is necessary to take into account, that a
sputum was preliminary diluted 100 times, and 0.1 ml of the diluted sputum was inoculated on Petri dish.
It is needed to count up the amount of colonies of fungus which grow on Petri dish and to increase the
obtained number by 1000 (taking into account aforesaid). If amount of fungus ≥ 100 000/ml material, it
gives ground to diagnose candidiasis. The amount of fungi from 50 000 to 100 000/ml material is an
index higher than norm and for clarification of diagnosis, in this case, it is needed to perform the
investigation in a few days. The isolation of the fungi colonies from the patient without clinical
manifestation are estimated as carrier.
The growth of Candida on the Sabouraud medium
Picture Cultural characteristic, conclusion
54
Task 3. Study and write in the protocol the main diagnosis of suppurative diseases.
Name of the
preparation
Сomposition, method of obtaining Purpose of using
Polysaccharide
antigen of Candida
spp. for CFT
Erythrocytes Candida
diagnosticum
Task 4. Study PHAT (demonstration) for serological diagnosis of candidiasis. Write the
conclusion in the protocol.
Conclusion:
______________________________________
_______________________________________
_______________________________________
_______________________________________
_______________________________________
_______________________________________
Task 5. Read the results of sensitivity tests to antifungal preparates of Candida albicans,
make the conclusion. Draw the slant and the growth of mycobacteria in the test tubes. Make the
conclusion.
Picture Interpretation of result
Name of antifungal
preparates
Diameter of
inhibiting sone (mm)
Sensitiveness
Task 6. Sketch the scheme of the candidiasis laboratory diagnosis.
Teacher's signature ____________________
55
Date _____________________ Class № 22
MICROBIOLOGY AND LABORATORY DIAGNOSIS
OF SUBCUTANEOUS MYCOSES AND CUTANEOUS MYCOSES
QUESTIONS FOR DISCUSSION
1. General characteristics of fungi (definition and taxonomy, morphology).
2. General aspects of fungal disease (primary mycoses, opportunistic mycoses, subcutaneous
mycoses, cutaneous mycoses, fungal allergies, and fungal toxicoses).
3. Subcutaneous mycoses (sporotrichosis, chromoblastomycosis, Madura foot (mycetoma):
characteristics of pathogen, pathogenesis, principle diagnosis, treatment and prophylaxis.
4. Cutaneous mycoses (pityriasis versicolor, dermatomycoses): characteristics of pathogen,
pathogenesis, principle diagnosis, treatment and prophylaxis.
5. Principle of antifungal immune response.
PROTOCOLE OF PRACTICAL SESSION
Task 1. Examine microscopically the smears from the patient's materials, fill in the protocol.
Name of preparation Сomposition, method of obtaining Purpose of using
Microsporum spp.
Task 2. Study the growth of the fungi on the solid media.
The growth of Trichophyton rubrum
on the Sabouraud medium
The growth of Trichophyton violaceum
on the Sabouraud medium
Picture
Task 3. Sketch the scheme of the dermatomycosis laboratory diagnosis.
Teacher's signature ____________________
56
Date _____________________ Class № 23
SANITARY AND MICROBIOLOGICAL INVESTIGATION OF WATER, AIR, SOIL,
FOODSTUFFS AND OBJECTS OF EXTERNAL ENVIRONMENT
QUESTIONS FOR DISCUSSION
1. Role, value, and tasks of sanitary microbiology.
2. Sanitary indicators of microorganisms.
3. Microflora of water and methods of its bacteriological examination (fermenting method, method
of membrane filters). Sanitary indicators of water microorganisms. Methods of the pathogenic
microorganisms’ indication.
4. Microflora of the soil and methods of bacteriological examination. Sanitary indicators of the soil
microorganisms. Principles of the determination of microbial number, coli titre, perfringens titre and titre
of soil termophylic bacteria. Methods of the pathogenic microorganisms indication.
5. Microflora of air and methods of bacteriological examination: sedimentation and aspiration
methods. Sanitary indicators of air microorganisms. Methods of the pathogenic microorganisms’
indication.
6. General principles of sanitary and microbiological investigation of foodstuffs.
7. Bacterial flora of milk and dairy products. Methods of sanitary and microbiological investigation
of milk and dairy products. Change of microflora of milk at storage: bactericidal phase; phase of the
mixed microflora; phase of lactobacillus; phase of yeasts and moulds development.
PROTOCOLE OF PRACTICAL SESSION
Task 1. Define the microbal number of the open reservoir water.
Table 1 - Determination of microbal number of water
Ingredient Dilutions of the investigated water
1:10 1:100 1:1000
Investigated water, ml 1.0 1.0 1.0
Nutrient agar, ml
10.0 10.0 10.0
Amount of growing colonies
Result
Task 2. Define the microbal number of the air by the sedimentation method (by Koch) and
by the aspiration method (by Krotov).
Table 2 - Result of the sedimentation method
Picture
Conclusion:
57
Task 3. Define coli index (plumbing) of drinking water by the method of membrane filters.
Picture Conclusion:
Task 4. Define coli index and coli titre (plumbing) of drinking water by the fermentation
method.
Table 3 – Determination of coli titre and coli index of water
Investigation stage Volume of the inoculated water, ml
100 100 100 10 10 10 1 1 1
1. Inoculation in test tubes with
the glucose-peptone medium, ml
10 10 10 1 1 1 10 10 10
Result: ( + or – )
2. Inoculation of glucose-
peptone medium on the Endo
agar
Result: colour of colonies
microscopy
test on oxidase
Conclusion:
Figure 1 – Method of membrane filters
58
Table 4 – Determination of coliforms index in the investigated water
Amount of positive results of water analysis from:
Coli index
Coli titre Three small
bottles for 100 ml
Three small bottles
for 10 ml
Three small
bottles for 1 ml
0 0 0 3 >333
0 0 1 3 333
0 1 0 3 333
1 0 0 4 250
1 0 1 7 143
1 1 0 7 143
1 1 1 11 91
1 2 0 11 91
2 0 0 9 111
2 0 1 14 72
2 1 0 15 67
2 1 0 20 50
2 2 1 21 48
2 2 0 28 86
3 0 0 23 43
3 0 1 39 26
3 0 2 64 16
3 1 0 43 23
3 1 1 75 13
3 1 2 120 8
3 2 0 93 11
3 2 1 150 7
3 2 2 210 5
3 3 0 240 4
3 3 1 460 2
3 3 2 1100 0,9
Note: Water is suitable for the use if coli titre ≥ 333 ml, and coli index ≤ 3.
Task 5. Define microbial number of soil (demonstration is used for investigation): count up
the amount of colonies growing on Petry dish with a nutrient agar, define the number of microbes
in 1 g of the investigated soil.
Table 5 – Indexes of soil pollution
Soil Microbial number,
million in 1 g
Coli titre
Perfringes titre
Strongly muddy ≥ 3-5 ≤ 0.001 ≤ 0.0001
Moderately muddy 2.5-3 0.0-0.001 0.01-0.0001
Poorly muddy 2 0.1-0.01 0.1-0.01
Clean 1-1.5 ≥ 1.0 ≥ 0.1
Picture Conclusion:
59
Task 6. Carry out microscopic investigation of kefir: prepare staining, stain methylene blue,
examinatione microscopically.
Name of the
preparation
Picture Morphological and staining properties
Material – kefir,
methylene blue
staining
Signature of teacher ____________________
60
Appendix A
Description of immunity
Postinfection
Postvaccine
Active
Passive
Humoral
Cellular
Antibacterial
Antiviruses
Antitoxins
Antifungal
Specific
Nonspecific
Group specific, species specific,
Type-specific