Special bacteriology - СумДУlib.sumdu.edu.ua/library/docs/rio/2017/m4276.pdf · 2018-02-01 · Research and clinical experience are continually expanding our knowledge, the knowledge

1

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.

2

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 _______________

3

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.

4

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

5

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

6

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 ____________________

7

Date _____________________ Class № 2

MICROBIOLOGY AND LABORATORY DIAGNOSIS OF SHIGELLOSIS


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.


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


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


8

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:

9



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


preparations.


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.


10

Date _____________________ Class № 3

MICROBIOLOGY OF TYPHOID FEVER, А AND В PARATYPHOID


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.


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


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


11

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:

12

Task 6. Perfome the РНАT with erythrocyte Vi-diagnosticum.

Components of РНАT Result

1. ___________________________

2. ___________________________

3. ___________________________


1:10 1:20 1:40 1:80 1:160

Conclusion:


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

13

Typhoid fever erythrocyte О9

diagnosticum

Typhoid fever erythrocyte Vi

diagnosticum


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.


14

Date _____________________ Class № 4

MICROBIOLOGY AND LABORATORY DIAGNOSIS OF CHOLERA


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.


Task 1. Study the slide of V. cholerae; compare the results of microscopy, make conclusions,

and sketch the results of the microscopy.



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

15

Resistance to the

action of polymyxin B

Additional test

Haemolysis of sheep

erythrocytes

Hexsamine test

Voges-Proskauer test



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


preparations.


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



17

Date _____________________ Class № 5

MICROBIOLOGY AND LABORATORY DIAGNOSIS OF CAMPYLOBACTERIOSIS

AND HELICOBACTERIOSIS


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.


Task 1. Microscope and sketch the preparations of C. jejuni pure cultures microscopically. Make

a conclusion.



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


Task 3. Perfome the cito test for detection of H. pylory


campylobacteriosis laboratory diagnosis.


18

Date _____________________ Class № 6

MICROBIOLOGY AND LABORATORY DIAGNOSIS OF PSEUDOTUBERCULOSIS AND

INTESTINAL YERSINIOSIS


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.


Task 1. Microscope and sketch preparations of Yersinia pseudotuberculosis and Y. enterocolitica

pure cultures microscopically. Make a conclusion.



Yersinia

pseudotuberculosis,

Gram staining

Yersinia enterocolitica,

Gram staining

Task 2. Perfome the РНАT with erythrocyte O9 intestinal yersiniosis diagnosticum.


1. ___________________________

2. ___________________________

3. ___________________________


1:10 1:20 1:40 1:80 1:160

Conclusion:



method of obtaining

Purpose of using

(test, method)

Erythrocyte O9- intestinal

yersiniosis diagnosticum

Pseudotuberculin


pseudotuberculosis and intestinal yersiniosis laboratory diagnosis.


19

Date _____________________ Class № 7

LABORATORY DIAGNOSIS OF TOXIC INFECTIONS

AND ACUTE INTESTINAL INFECTIONS


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.


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.



E. coli O1:H27, Gram

staining

S. sonnei, Gram staining

S. enterica, Gram staining

P. vulgaris, Gram staining

Klebsiella pneumonia,

Burri-Gins staining

20

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.


1. ___________________________

2. ___________________________

3. ___________________________


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


21

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.


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.


23

Date _____________________ Class № 8

LABORATORY DIAGNOSIS OF FOOD POISONING


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.


Task 1. Microscope and sketch preparations of Clostridium botulinum and Staphylococcus

aureus pure cultures microscopically. Make a conclusion.



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


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.


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



Staphylococcus aureus food poisoning laboratory diagnosis.


25

Date _____________________ Class № 9

MICROBIOLOGY AND LABORATORY DIAGNOSIS OF STAPHYLOCOCCAL

INFECTIONS


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


4. Basic measures of prophylaxis and treatment of staphylococcal infections.


Task 1. Microscope and sketch the preparations of Staphylococcus aureus and S. epidermidis

pure cultures microscopically. Make a conclusion.



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.


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


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


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


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.


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.


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


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.


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.


29

Date _____________________ Class № 10

LABORATORY DIAGNOSTICS OF STREPTOCOCCAL INFECTIONS


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


4. Basic principles of streptococcal infections; prophylaxis and treatment.


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.



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


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.


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.


31

Date _____________________ Class № 11

LABORATORY DIAGNOSIS OF MENINGOCOCCAL

AND GONOCOCCAL INFECTIONS


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


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



Task 1. Microscope and sketch the demonstrations preparations. Make a conclusion.



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


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


Gonococcal

inactivated liquid

vaccine (gonovaccine)

33

Meningococcal

chemical vaccine

Gonococcal antigen


meningococcal infections and bacteria carriers laboratory diagnosis


gonococcal infection laboratory diagnosis.


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.


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.



C. tetani,

Gram staining

C. tetani,


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


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


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


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


Task 8. Fill in the card of particular microbiology test and sketch the scheme of the gas gangrene



37

Date _____________________ Class № 13

MICROBIOLOGY AND LABORATORY DIAGNOSIS OF DIPHTHERIA

AND WHOOPING COUGH (PERTUSSIS)


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.


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


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


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.


1. ___________________________

2. ___________________________

3. ___________________________


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


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


Task 11. Fill in the card of particular microbiology test and sketch the scheme of the whooping

cough (pertussis) laboratory diagnosis.


41

Date _____________________ Class № 14


OF TUBERCULOSIS AND LEPROSY


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.


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.



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


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.



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


Tuberculin

Lepromin

Erythrocytic

tuberculosis

diagnosticum

BCG

BCG-M


tuberculosis laboratory diagnosis.

Task 7. Fill in the card of particular microbiology test and sketch the scheme of leprosy



43

Date _____________________ Class № 15


OF PLAGUE AND TULAREMIA


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.


Task 1. Examine microscopically the smears of the patient's materials, fill in the protocol.

Name of the

preparation


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


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


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


Task 6. Fill in the card of particular microbiology test and sketch the scheme of the tularemia

laboratory diag


45

Date _____________________ Class № 16


OF ANTHRAX AND BRUCELLOSIS


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.


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,


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


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


Task 5. Fill in the card of particular microbiology test and sketch the scheme of the brucellosis



47

Date _____________________ Class № 17


OF SPIROCHAETOSIS (SYPHILIS, BORRELIOSIS, AND LEPTOSPIROSIS)


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.


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.



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.


leptospirosis laboratory diagnosis.


49

Date _____________________ Class № 18


OF RICKETTSIAL DISEASES


1. General description of bacteria of the family Rickettsia.

2. Microbiological diagnosis of rickettsial diseases.


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


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.


51

Date _____________________ Class № 19


OF CHLAMYDIAL DISEASES


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.


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


Dry chlamydia

diagnosticum for CFT

Task 3. Sketch the scheme of chlamydial diseases laboratory diagnosis.

Signature of teacher ____________________

52

Date _____________________ Class № 20


OF MYCOPLASMAL DISEASES


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.


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


Task 3. Sketch the scheme of the mycoplasmal diseases laboratory diagnosis.


53

Date _____________________ Class № 21


OF OPPORTUNISTIC MYCOSES AND PRIMARY MYCOSES


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.


Task 1. Study microscopically the material from the vagina.

Name of the

preparations


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


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.


Name of antifungal

preparates

Diameter of

inhibiting sone (mm)

Sensitiveness

Task 6. Sketch the scheme of the candidiasis laboratory diagnosis.


55

Date _____________________ Class № 22


OF SUBCUTANEOUS MYCOSES AND CUTANEOUS MYCOSES


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.


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.


56

Date _____________________ Class № 23

SANITARY AND MICROBIOLOGICAL INVESTIGATION OF WATER, AIR, SOIL,

FOODSTUFFS AND OBJECTS OF EXTERNAL ENVIRONMENT


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.


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


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

Documents

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