METHODOLOGICAL INSTRUCTION

 

METHODOLOGICAL INSTRUCTION

FOR 2nd COURSE-STUDENTS

of MEDICAL FACULTY

 

Lesson 4 (6 hours)

Themes:

I. Types of bacterial respiration. The methods of creation of anaerobic conditions. Isolation of pure culture of anaerobic bacteria. Identification of microorganisms.

II. Chemotherapeutic preparations. Antibiotics. Methods of examination of bacterial antibiotic susceptibility. The main principles of rational antibiotic therapy of diseases.

 

Aim: To learn methods of bacteria cultivation and their respiration.. To learn methods of bacteria cultivation and isolation of anaerobic bacterial pure cultures. To learn methods of examination of bacterial antibiotic susceptibility.

Professional motivation of students: to take possession of bacteriological examination which is based on isolating a pure culture of causaive agents of infectious disease and their identification, to understand the main principles of a rational chemotherapy.

 

 

Methodology of Practical Class. 9.00-12.00

 

Theme I. Types of bacterial respiration. The methods of creation of anaerobic conditions. Isolation of pure culture of anaerobic bacteria. Identification of microorganisms.

Work 1. To familiarize with methods of making of anaerobic conditions using anaerostat, tubes Vinyale-Vinione, Fortner biological method.

Work  2. To inoculate a soil into milk and  Kitt-Tarozzi medium.

Work  3. Checking of the purity of the isolated culture, which has grown on slant agar, making smear, staining by Gram method. Sketching in album.

Work  4. Sub-inoculating culture on Hiss medium, Olkenitsky medium and MPB for determination of saccharolytic and peptolytic properties of bacteria.

Work  5. Perfoming presumptive agglutination test on glass with pure culture and specific serums.

Work  6. Preparing smears from cultures on Kitt-Tarozzi medium and milk, staining by Gram method.

Work  7. Sub-inoculating tested material from Kitt-Tarozzi medium into Vinyale-Vinone tubes for obtaining separate colonies.

Work  8. To familiarize with growth character of E.coli and Salmonella onto Endo, Levin media

 

Theme II. Chemotherapeutic preparations. Antibiotics. Methods of examination of bacterial antibiotic susceptibility. The main principles of rational antibiotic therapy of diseases.

Work 1. To study the phenomenon of microbial antagonism (special Petri dish).

Work  2. Work . To familiarize with the collection of antibiotics and others chemotherapeutical  drugs.

Work  3. To examine the sensitivity of staphylococci to benzylpenicillin by serial dilutions method in a liquid media.

 

Method of serial dilutions in a liquid medium. Hottinger’s broth (or another medium suitable for the growth of the given micro-organism) is poured by 2-ml portions into test tubes mounted in a tube rack by ten in each row. Prepare antibiotic solution containing 100 U per ml and add 2 ml of this solution into the first test tube. Following thorough mixing, transfer with a new sterile measuring pipette 2 ml of the culture from this tube into the next one, and so on until the ninth tube is reached, from which 2 ml is poured off. The tenth tube containing no antibiotic serves as a control of culture growth.

Wash the 24-hour agar culture of the studied microorganism with isotonic sodium chloride solution, determine the density of the sus­pension by the turbidity standard, and dilute to a concentration of 10000 microorganisms per ml. A sample of  0,2 ml of the obtained suspension is inoculated into all tubes of the row beginning from the control one. Thus, all tubes contain 1000 microorganisms per 1 ml. The results of the experiment are read following incubation of the tube at 37 °C for 18-20 hrs. The minimal concentration of the anti­biotic suppressing the growth of the given microorganism is deter­mined by the last test tube with a transparent broth in the presence of an intensive growth in the control one.

One may also prepare antibiotic solution in molteutrient agar to subsequently streak the tested culture onto the surface of this medium.

Another approach to antimicrobial susceptibility testing is the determination of the minimum inhibitory concentration (MIC) that will prevent microbial growth (fig. 1). The MIC is the lowest concentration of antimicrobic that prevents the growth of a microorganism in vitro.

The minimum inhibitory concentration indicates the minimal concentration of the antibiotic that must be achieved at the site of infection to inhibit the growth of the microorganism being tested. By knowing the MIC and the theoretical levels of the antibiotic that may be achieved in body fluids, such as blood and urine, the physician can select the appropriate antibiotic, the dosage schedule, and the route of administration. Generally, a margin of safety of 10 times the MIC is desirable to ensure successful treatment of the disease.

MIC is not designed to determine whether the antibiotic is microbicidal. It is, however, also possible to determine the minimal bactericidal concentration (MBC). The MBC is also known as the minimal lethal concentration (MLC) (fig. 1). The minimal bactericidal concentration is the lowest concentration of an antibiotic that will kill a defined proportion of viable organisms in a bacterial suspension during a specified period of exposure. Generally, a 99.9% kill of bacteria at an initial concentration of 10⁵-10⁶ cells/mL during a 17- to 24-hour exposure period is used to define the MBC.

To determine the minimal bactericidal concentration, it is necessary to plate the tube suspensions showing no growth in tube dilution (MIC) tests onto an agar growth medium. This is done to determine whether the bacteria are indeed killed or whether they survive exposure to the antibiotic at the concentration being tested.

Determination of the MIC is adequate for establishing the appropriate concentration of an antibiotic that should be administered for controlling the infection in patients with normal immune response levels. Determination of the MBC is essential for patients with endocarditis (inflammation of the endocardium or lining to the heart) because the patient’s immune response cannot be relied on to remove the infecting microorganisms. It is particularly useful in determining the appropriate concentration of an antibiotic for use in treating patients with low erred immune defense responses, such as may occur in patients receiving chemotherapy treatment in cancer.

Figure 1. The minimum bactericidal concentration (MBC) of an antibiotic requires the demonstration that microorganisms have lost the ability to reproduce. In this example, although cell growth is inhibited at concentrations of 100 and 6,25 (MIC – 6,25 mg/vL), viable cells remain, which is shown by the formation of colonies (growth) on an agar plate lacking the antimicrobic No vable cells are detected (no growth on agar plates) at 25 mg/mL, which therefore is the MBC.

 

4. To examine sensitivity of staphylococci to antibiotics by disks diffusion method.

Disk method. Into sterile Petri dishes placed on a horizontal surface, pour 15 ml of solid nutrient medium (most often 2 per cent agar on Hottinger’s broth containing 0.11-0.13 per cent of amine nitrogen). On the surface of solidified and slightly dried agar, pour 1 ml of suspension of 24-hour culture of the causative agent or, if no pure culture has been isolated, of the pathological material (pus, exudate) obtained for the study and diluted with isotonic saline. Spread uniformly over the agar surface the bacterial suspension, removing its remainder with a Pasteur pipette. Disks with antibio­tics (5-6 disks per plate) are placed onto the surface of the inoculated plate at a distance of 25 mm from its centre. The plates are incubated at 37 °C for 16-18 hrs, after which the results of the test are read by measuring the zones of growth retardation of microorganisms around the disks, including the diameter of the disk itself (fig.2). The size of the zones depends on the degree of sensitivity of the causative agent to a given anti­biotic (tabl. 1). Yet, this method cannot be considered strictly quantitative.

 

Figure 2. Testing sensitivity of bacteria to antibiotics by the “disk method”

 

 

Table 1. Zone Size Interpretive

 

R = Resistant, I = Intermediate, MS = Moderately Susceptible, S = Susceptible

 

 

Break 12.00-12.30

 

Individual Students Program.

  You should be  prepared for the practical class using the existing textbooks and lectures. Special attention should be paid to the following questions:

 

Theme I. Types of bacterial respiration. The methods of creation of anaerobic conditions. Isolation of pure culture of anaerobic bacteria. Identification of microorganisms.

1.                Main methods of creating anaerobic conditions for cultivation of bacteria (mechanical, chemical,  biological and others).

2.                Media which are used for cultivation of anaerobic bacteria.

3.                Stages of isolation of pure culture of anaerobic bacteria by Veinberg’s and Zeissler’s techniques.

4.                Identification of pure culture (morphological, tinctorial, cultural, biochemical, serological, biological).

5.                What purpose has identification of pure culture?

6.                Characteristic of differential diagnostic media for the determination of fermentation of the saccharolytic action of bacteria (Endo, Levin, Ploskirev, Hiss, Olkenitsky, Ressel and others).

7.                How we study the peptilytic, proteolytic and hemolytic properties of bacteria?

8.                What are the methods of serological and biological identification of pure culture?

9.                Cultural properties of bacteria (R – and S-forms).

10.           Types and mechanism of bacterial respiration. Toxical influence of oxygen on bacteria and mechanisms of its warning.

11.           Main methods of creating anaerobic conditions for cultivation of bacteria (mechanical, chemical, biological and others).

12.           Media which are used for cultivation of anaerobic bacteria.

 

Theme II. Chemotherapeutic preparations. Antibiotics. Methods of examination of bacterial antibiotic susceptibility. The main principles of rational antibiotic therapy of diseases.

1. Phenomenon of microbial antagonism:

a – to explain, what is microbial antagonism, provide  the examples;

b – methods of microbial antagonism studying;

c – practical meaning of the phenomenon of microbial antagonism.

2. Concept of chemotherapeutic drugs:

a – main groups of chemotherapeutic drugs, demands to them;

b – chemotherapeutic index, its value;

c – mechanisms of action of the main chemotherapeutic preparations;

3. Antibiotics and mechanisms of their action:

a – what does term “antibiotics” mean;

b – classification of antibiotics according their origin, action spectrum, mechanism of action, chemical structure;

c – units of determination of antibiotics activity;

d – antimicrobial susceptibility testing (serial dilutions, standard disks, accelerated methods, automated liquid diffusional method);

e – determination of concentration of antibiotics in biological fluids and tissues;

f – main principles of a rational chemotherapy;

g – side effects of antibiotics, complications of chemotherapy.

4. Resistance of microbes to antibiotics:

a – mechanisms, which cause drug resistance;

b – factor of a multiple resistance to drugs (R –, r–factors), transmission of them in bacteria;

c – methods of warning of derivation of resistant causative agents.

 

Seminar discussion of theoretical issues (12.30 – 14.00 hour).

 

Break 14.00-14.15

 

 

Individial student’s work. 14.15-15.00

Checking practical skills. Checking with quizes  and  constructive questions to verify knowledge level of student who did not do it using “Moodle” system.

 

Test evaluation and situational tasks.

 

Theme I. Types of bacterial respiration. The methods of creation of anaerobic conditions. Isolation of pure culture of anaerobic bacteria. Identification of microorganisms.

1.     Select the correct statements:

a – the enzymes of bacteria are divided into exoenzymes, obligate, adaptable, anaerobic, design;

b – for the examining saccharolytic properties of bacteria apply such media: Endo, Kitt-Tarozzi, blood agar, Olkenitsky, Ploskirev;

c – for the examining proteolytic properties of bacteria apply such media: meat pepton gelatin, Levin, serum agar, Hiss, MPA;

d – the biological identification of bacteria are made onto express media, into cell cultures, by infected of  laboratory animal, glass agglutination test.

2.     Insert missed word in the next statement:

a –the serological identification of bacteria are made at them…………….. properties; for this purpose are used special.………. and………….. cultures; make .……….test.……..; b – onto medium Endo E.coli create ………. colonies, because it ferments…………., thus… medium becomes acid and coloures.…….…reduces.

 

Theme II. Chemotherapeutic preparations. Antibiotics. Methods of examination of bacterial antibiotic susceptibility. The main principles of rational antibiotic therapy of diseases.

Choose the correct answers:

1. Chemotherapeutic index is the ratio of maximum tolerated doses of a drug to minimal curative one. It should be: a – not less than 1.5; b – not less than 3;  c – not less than 2.5; d – is more than 5.

2. Antibiotics of animal origin are: a – gramicidins; b – coli–bacterin; c – lysozyme; d – spermine; e – erythryn.

3. Main demands to antibiotics: a – high selective antimicrobic effect in doses, nontoxic for an organism; b – absence or low level of a toxicity of a drug and products of its metabolism; c – slow development of resistance during its application; d – preservation of antimicrobial effect in fluids and tissues of an organism, e – absence or low level of antibiotic inactivation by serum proteins and tissues enzymes; f – convenient form for using for different age–grades and localization of the process, which provides maximal effect; g – stability in usual conditions of storage.

4. To antibiotics, which are inhibitors of ribosome protein synthesis belong: a – Chloramfenicol; b – Erythromycin; c – Rifampicin; d – Penicillins and Cephalosporins; e – Kanamycin, Gentamicinu; е – Tetracyclins.

5. Choose antibiotics which are cell–wall inhibitors:

a – Polymyxins; b – Oleandomycin; c – Penicillins; d – Linkomycin; e – Cephalosporins; f – Vancomycin.

6. There are such types of fastness (resistance) of microbes to antibiotics: a – natural, which is determined by properties of species or family of microorganisms; b – acquired; c – chromosomal; d – extrachromosomal (R-palsmids carry genes for resistance to one – and often several – antimicrobial agents and heavy metal).

Real- life situational to be solved:

7. The woman after labors complains of a fever, pain in the left mamma, bad state. The doctors made the diagnosis “Purulent mastitis”.  Staphylococcus aureus was isolated from the pus. S. aureus antibiotic resistance was determined by agar diffusion antimicrobial susceptibility testing using AGV medium. There were such diameters of the zones of inhibition: Penicillin – 8 mm, Streptomycin – 8 mm, Oxacillin – 15 mm, Lincomycin – 29 mm, Gentamycin – 22 mm, Tetracyclin – 10 mm. This answer received from bacteriological laboratory on 4^th day. During this term the patient has been treated by Penicillinum.

A. Is the correction of an antibiotic therapy necessary?

B. What antibiotic or their combination is it necessary to use for treatment and why?

8. To the patient with a fever,  tissuis, pain in his side, grave general state the diagnosis  of low lobar pneumonia has been made. Penicillin injections has been made. After the first injection the patient was found to have reducing of arterial pressure, rapid puls, loss of consciousness, cool and clammy sweat (the clinical symptoms of an anaphylactic shock). After of noradrenalin, prednisolon, dimedrol, calcium chloride injections the patient’s state was improved.

А. What mistake was made during first injection of Penicillin?

B. How is it possible to explain origin of the anaphylactic shock?

 

Student should know:

1.                Types of bacterial respiration

2.                Enzymes of bacteria, their classification and function

3. Principles and methods of isolation of pure culture of aerobic and anaerobic microorganisms.

4. Main groups of chemotherapeutic drugs and demands to them.

5. Antibiotics, classification of antibiotics according their origin, action spectrum, mechanism of action, chemical structure, and mechanisms of their action

6. Antimicrobial susceptibility testing (serial dilutions, standard disks, accelerated);

7. Principles of determination of concentration of antibiotics in biological fluids and tissues;

8. Main principles of a rational chemotherapy;

9.Side effects of antibiotics, complications of chemotherapy.

10.Mechanisms, which cause drug resistance.

 

Student should be able to:

1. To inoculate a soil into milk and Kitt-Tarozzi medium.

2. To carry out a presumptive agglutination test with pure culture.

3. To carry out identification of bacteria

4. Determine antibiotic sensitivity of bacteria by agar diffusion susceptibility testing;

5. Determine antibiotic sensitivity of bacteria by serial dilution method in liquid fluid.

 

Answers to the Self-Assessment:

Theme I. 1. A – exoenzymes, adaptable, constructive; b – Endo, Olkenitsky, Ploskirev; c – MPG, serum agar; d – by infected of laboratory animals. 2. A – antigenic, immune, serum, pure, presumptive agglutination; red, lactose, рН, fuchsine.

Theme II. І. A. 2. C, d, e. 3. A, b, c, d, е, f, g. 4. A, b, e, f. 5. C, e, f. 6. A, b, c. 7. A. Yes, it is necessary, as isolated from the pus Staphylococcus aureus strain is resistant to Penicillin. B. Ampicillin and Gentamycin, because zones of growth inhibition around these disks testify about sensitivity of the microbes to these antibiotics; it is possible to use also combination Lincomycin / Gentamycin as they do synergistic effect.        8. A. it was necessary to make a sensitivity test before injection of antibiotics; B. The patient had high sensitivity to Penicillin.

 

 

Reference:

Basic:

Materials for practical class 04

1. Medical microbiology /Patrick R. Murray, 2009, 948 p.

2. Kone man’s color atlas and textbook of diagnostic microbiology /Washington C. Winn. Jr. 2006, 1536 p.

3. W. Levinson, E. Jawetz/ Medical Microbiology and Immunology/ International edition, 2001.– 582 p.

4. http://intranet.tdmu.edu.ua/data/kafedra/theacher/micbio/pyatkovskiy/English/Lectures/Microbiology,%20virology%20and%20immunology/MEDICAL/2%20year/The%20physiology%20of%20microorganisms.ppt

5 http://intranet.tdmu.edu.ua/data/kafedra/theacher/micbio/klymjuk/English/Lectures/Microbiology,%20virology%20and%20immunology/medical/2%20year/Doctrine%20about%20Antibiotics.ppt

Additional:

1. E. Jawetz, J.Melnnick, E.Adelberg  Review of Medical Microbiology, 2000,  553 p.

2. W. Levinson, E. Jawetz. Medical Microbiology and Immunology. Examination and Board Review/ Lange medical book. Sixth edition, 2000, 536 p.

 

 

 

The methodical instruction has been done

by prof. Sergey I. Klymnyuk, ass. prof. Olena V. Pokryshko

 

Methodical instruction was discussed at the Department sitting

24.09.2013. Minute № 3