Microbiology, virology: guidelines for practical classes

The Ministry of education and science of Russian Federation

Ulyanovsk State University

Institute of Medicine, Ecology and physical culture

Department of General and Clinical Pharmacology with Microbiology course

M. N. Artamonova, N.I. Potaturkina- Nesterova, I. S. Nemova

Microbiology, virology:

guidelines for practical classes for foreign students

Part 1

Ulyanovsk 2016

2

УДК 535.317.68 (07.07)

ББК 52.64 я 73

G 36

It was recommended by the scientific-methodological council of Institute of

Medicine, Ecology and physical culture of Ulyanovsk State University

Reviewer:

A. S. Nesterov, PhD of Medicine, professor

M. N. Artamonova, N.I. Potaturkina- Nesterova, I. S. Nemova

Microbiology, virology: guidelines for practical classes for foreign students.

Part 1/ M. N. Artamonova, N.I. Potaturkina- Nesterova. - Ulyanovsk: ULSU,

2016.-85 p.

Guidelines for practical training on "Microbiology, Virology" is

recommended for students of English department and build in accordance with the

existing curriculum, the federal state educational standards in the direction of

preparation 31.05.01.- "General Medicine".

The handout is structured on topics that focused on the basic concepts of the

discipline. Each practice lesson includes a description of the learning objectives,

job occupation, questions for self-control, tasks for students' independent work,

which describe the types of performed work; basic terms of the subject; examples

of typical case studies; list of recommended literature, additional material,

comprising - instructions for students on labor protection.

© M. N. Artamonova, N.I. Potaturkina- Nesterova,

I. S. Nemova 2016

© Ulyanovsk State University

3

CONTENTS

Practical lesson 1. Subject and aims of medical microbiology. The

morphology and taxonomy microorganisms. Microscopic research method.

Sterilization and disinfection……………………………………….…………

4

Practical lesson 2. Physiology of microorganisms. Metabolism. Nutrition

and respiration of bacteria. Culturing methods of aerobic and anaerobic

bacteria ………………………………………………………………………

19

Practical lesson 3. Viruses. The discovery of viruses, its classification.

Bacteriophages. The practical importance of phages in biology and medicine.

The genetics of microorganisms. Biotechnology in microbiology. Molecular-

biological methods of diagnosis……………………………………………….

30

Practical lesson 4. Infection: the role of microorganisms in infectious

process. Biological diagnostic method. The role of host in the infectious

process. Microbiological basics of antimicrobial therapy. Methods for

determination sensitivity of bacteria to antibiotics………………………..

38

Practical lesson 5. Ecology of microorganisms. The human microflora and

its function. Dysbiosis, methods of correction. Biofilms. Sanitary

demonstration water microorganisms, soil, air, and methods of

research………………………………………………………………………..

43

Practical lesson 6. Immunity. Serological methods – agglutination test,

precipitation test, complement fixation test ………………………………..

47

Practical lesson 7. Clinical Microbiology. Etiology and pathogenesis of

nosocomial infections. The causative agents, properties and microbiological

diagnostics of nosocomial infections. Pathogenic and opportunistic gram-

positive and gram-negative cocci…………………………………………….

51

Practical lesson 8. Pathogens of escherichiosis and dysentery. The causative

agents of typhoid, salmonella poisoning and cholera………………………

64

Practical lesson 9. Pathogens of diphtheria, whooping cough. Pathogens of

legionellosis. Pathogenic mycobacteria. Concluding lesson 2. Colloquium.

4

The final lesson ………………………………………………………………. 75

The list of literature………………………………………………………….. 85

5

Practical lesson 1

Subject and aims of medical microbiology. The morphology and

taxonomy microorganisms. Microscopic research method.

Objective: formation of knowledge about the structure, aims and methods of

microbiology as a science, the history of its formation and position among other

sciences.

Tasks:

1. Learn to work with bacterial cultures.

2. Prepare fixed preparations

Questions:

1. Subject of Medical Microbiology and its importance for practical health care.

The history of microbiology.

2. The system and nomenclature of microorganisms.

3. Research microbiology methods.

4. Simple and complex staining techniques. The mechanism of staining smears.

Tinctorial properties of microorganisms.

5. The light microscope, its main characteristics. Types of light microscopy (dark-

field, phase - contrast, fluorescent). Immersion microscopy principles. Electron

microscopy, atomic force microscopy.

6. The forms of the bacteria.

7. The structure of the bacterial cell: genome, cytoplasm, ribosome. Its structure,

functions and methods of detection.

8. The shell of bacteria: cytoplasmic membrane, cell wall, capsule. The structure,

functions and methods of detection.

9. Fimbria and pili. Its structure, functions and methods of detection.

10.Bacterial spores. Its role and structural features. Spore formation and methods

of detection.

6

Morphology of microorganisms. Simple methods of staining preparations

Notion Definition/ explanation

Microbiology

Microbiology is the study (logy) of very small (micro)

living (bio) organisms. Often, people are scared by the

topic's name of the theme and by science itself

Bacteria Bacteria are relatively simple in structure. They are

procaryotic simple unicellular organism with no nuclear

membrane, mitochondria,

Golgi bodies, or endoplasmic reticulum that

reproduces by asexual division. Although the cell wall

encircling bacteria is itself complex, there are two basic

forms: gram-positive cell wall with a thick peptidoglycan

layer and gram-negative cell wall with a thin

peptidoglycan layer and an overlaying outer membrane.

Principle of the

microscopy with oil

immersion

Placing a drop of oil with the same refractive index as

glass between the cover slip and objective lens eliminates

two refractive surfaces, so that magnifications of 1000x or

greater can be achieved while still preserving good

resolution

Types of bacteria Bacteria have three basic shapes: spherical (round), rod-

shaped, and spiraled. A round bacterium is called a coccus

(plural, cocci). A rod-shaped organism is called a bacillus

(plural, bacilli) or simply a rod. A spiraled bacterium with

at least two or three curves in its body is called a spirillum

(plural, spirilla). Long sinuous organisms with many loose

or tight coils are called spirochaetes

Cocci The patterns formed by bacterial cells grouping

together as they multiply are often characteristic for

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individual bacterial genera or species. Cocci may occur in

pairs (diplococci), chains (streptococci), clusters

(staphylococci), or packets of four (tetrads), and are

seldom found singly

Rod-shaped

bacteria (bacilli)

Rod-shaped bacteria (bacilli) generally occur as

individual cells, but they may appear as end-to-end pairs

(diplobacilli) or line up in chains (streptobacilli). Some

species tend to palisade, that is, line up in bundles of

parallel bacilli, others may form V, X, or Y figures as they

divide and split. Some may show great variation in their

size and length (pleomorphism)

Spiral forms of

bacteria

Vibrio, spirochaete, spirilla

Structure of the bacterial cells. Complex method of preparations staining.

Notion Definition/explanation

Cell wall Outer covering of most cells that protects the bacterial cell

and gives shape to it

Gram staining The Danish bacteriologist J. M. C. Gram (1853–1938)

devised a method of bacteria staining using a dye called

crystal (gentian) violet. Gram's method helps distinguish

different types of bacteria.

The characteristics of bacteria Gram staining are denoted

as positive or negative, depending upon whether the bacteria

take up and retain the crystal violet stain or not.

Gram-positive bacteria retain the colour of the crystal

violet stain in the Gram stain. This is characteristic of

bacteria that have a cell wall composed of a thick layer of a

8

particular substance (specifically, peptidologlycan containing

teichoic and lipoteichoic acid complexed to the

peptidoglycan).

Gram-positive bacteria include staphylococci (“staph”),

streptococci (“strep”), pneumococci, and the bacterium

responsible for diphtheria (Corynebacterium diphtheriae) and

anthrax (Bacillus anthracis).

Gram-negative bacteria lose the crystal violet stain (and

take the colour of the red counterstain) in Gram's method.

This is characteristic of bacteria that have a cell wall

composed of a thin layer of a particular substance

(specifically, peptidoglycan is covered by an outer membrane

of lipoprotein and lipopolysaccharide - containing

endotoxin).

Gram-negative bacteria include most of the bacteria

normally found in the gastrointestinal tract that can be

responsible for disease as well as gonococci (venereal

disease) and meningococci (bacterial meningitis). The

organisms responsible for cholera and bubonic plague are

gram-negative

Gram-positive

bacteria

Gram-positive cells, peptidoglycan makes up as much as

90% of the thick, compact cell wall, and is the outermost

layer of the cell. The location and thickness of peptidoglycan

is the important factor that results in gram-positive cells

staining differently than gram-negative cells

http://www.medterms.com/script/main/art.asp?articlekey=2061

http://www.medterms.com/script/main/art.asp?articlekey=13044

http://www.associatedcontent.com/article/1971712/gram_positive_bacterial_stain.html?cat=4

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Gram-negative

bacteria

The cell walls of gram-negative bacteria are more

chemically complex, thinner and less compact, with

peptidoglycan comprising only 5 to 20% of the cell wall. In

gram-negative cells, peptidoglycan is not the outermost layer,

but is located between the plasma membrane and an outer

lipopolysaccharide (LPS) membrane

Cytoplasm A gel-like substance composed mainly of water that also

contains enzymes, salts, cell components, and various

organic molecules

Nucleoid

Prokaryotic nucleoid, the equivalent of the eukaryotic

nucleus, can be seen with the light microscope in stained

material. It is Feulgen-positive, indicating the presence of

DNA

Pili Hair-like structures on the surface of the cell that attach to

other bacterial cells. Shorter pili called fimbriae help bacteria

attach to surfaces

Flagella

Flagella are composed entirely of a single protein subunit

called flagellin, which differs in primary structure among

different bacterial speciese, and are responsible for bacterial

motility that may inhance bacterial invasion. The surface of

flagella is made up of protein antigens with diverse epitopes

useful in the identification and classification of organisms.

They can be single (monotrichous) or multiple (peritrichous).

Spirochaetes contain similar motility apparatus, protein in

nature, which leis in the periplasmic space between the

cytoplasmic membrane and the outer membrane

Number and

arrangement of

1. Monotrichate: one flagellum at one end of the

organism, e.g., vibrio, pseudomonas, spirillum, etc.

http://www.associatedcontent.com/article/1971854/gram_negative_bacterial_stain.html?cat=4

10

flagella on bacterial

body

2. Amphitrichate: one flageilum at both poles, e.g.,

Alcaligenes fecalis.

3. Lophotrichate: a tuft of flagella at the end, e.g.,

pseudomonas.

4. Peritrichous: several flagella are present all over the

surface of bacterium, e.g. Escherichia coli, Salmonella spp.

Capsules, slime

layers

Some bacteria have a layer of material lying outside the

cell wall. When the layer is well organized and not easily

washed off, it is called a capsule. A slime is a zone of diffuse,

unorganized material that is removed easily. A glycocalyx

network of polysaccharides extending from the surface of

bacteria and other cells (in this sense it could encompass both

capsules and slime layers). Capsules and slime layers usually

are composed of polysaccharides, but they may be

constructed of other materials. For example, Bacillus

anthracis has a capsule of polyD-glutamic acid. Capsules

contain a great deal of water and can protect bacteria against

desiccation

Function of the

capsule in bacteria

Attachment to surfaces; protection against phagocytic

engulfment, occasionally killing or digestion; reserve of

nutrients or protection against desiccation

Spores

Bacteria in genera such as Bacillus and Clostridium

produce quite a resistant structure capable of surviving for

long periods in an unfavourable environment and then giving

rise to a new bacterial cell. This structure is called an

endospore since it develops within the bacterial cell.

Endospores are spherical to elliptical in shape and may be

either smaller or larger than the parent bacterial cell.

11

Endospore position within the cell is characteristic and may

be central, subterminal, or terminal

Cycle of spore

formation and

germination

At the beginning of spore formation, a septum forms,

separating the nascent spore from the rest of the cell and all

of the genetic material of the cell is copied into the newly-

forming cell. The spore contents are dehydrated

Function of the

bacterial spore

Bacterial spores are the highly resistant spores. In fact the

spores indicate the resting phase of some types of

bacteria,which help to tide over the unfavourable conditions.

These spores can also be called as endospores. These

endospores contain calcium and dipicolenic acid and hence

they are very resistant. They are resistant to temperature, UV

light, disinfectants, etc. and thus they protect the bacterium.

In the favourable conditions these develop into a new

bacterium.

PROCEDURE OF PRACTICAL WORK

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

them down.

GENERAL LABORATORY SAFETY RULES FOR MICROBIOLOGY

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

laboratory area, including laboratory office. No foodstuffs are to be stored in

laboratories (including cold rooms, refrigerators, and freezers).

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

handles, telephones, computer keyboards, and mice (except in clearly defined cir-

cumstances), lift buttons, etc. are not to be touched with gloves. If needed, wear

one glove and use the ungloved hand to open doors, operate lifts, etc.

12

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

human blood or other body fluids, dangerous chemicals, infectious, or potentially

infectious materials, ultraviolet light boxes. Select an appropriate glove type.

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

they should be taken off before entering “clean areas”, e.g., tea room, stores,

media, toilets, library, and office rooms.

5. Clothing and footwear must be suitable for laboratory conditions. Flip-

flops, sandals or high- heeled shoes are not to be worn in the laboratory. Bare feet

are prohibited in the building. Sandals with an enclosed toes and heels are

acceptable, but for your own protection, an enclosed shoes are preferable. Long

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

6. Protective glasses must be worn for all kinds of work involving corrosive

or toxic chemicals, radioactivity, and ultraviolet light.

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

penetration in your mouth. Labels and envelopes must not be licked. Pencils and

pens must not be laced in the mouth. Biting of fingernails, playing with hair,

applying lipstick, eating, drinking, etc., are not allowed. Wash your hands when

leaving the laboratory for lunch, etc.

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

contamination is preferable. Place pipettes in disinfectant solution to minimize

aerosol production. Submerge them for 18–24 hours. Residual volumes from

pipettes create aerosols; use mechanical devices that are calibrated to deliver. Fit

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

case the pipette is broken.

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

caps after use. Use syringes fitted with blunt cannulas where possible. Avoid using

syringes to mix infectious liquids (if essential, hold the tip of the needle under the

surface of the fluid and avoid excessive force). Discard used syringes and needles

into an approved sharps container.

13

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

benches or equipment should be cleaned up immediately. Special treatment is re-

quired for spills of a biohazardous nature.

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

leaving the laboratory.

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

immediately. Try to anticipate potential problems.

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

disposal.

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

the hotter part of the flame to minimize spattering, or use electric heaters. Make

sure the loop is completely closed and the loop wire is not longer than 6 cm.

15. Disposable plastic loops must be placed loop-end down in disinfectant

for 18–24 hours.

16. Petri dish cultures of fungi should be imprinted and incubated with the

lid up permost to prevent the dispersal of fungal spores. Recognize fungi as

potential pathogens and be aware of the ability of some species to produce

mycotoxins.

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

contain viable microorganisms that can be spread via droplets or aerosols when the

plates are opened or dropped.

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

glass grinders in a wad of absorbent material and wear gloves. Wait 10 minutes

before opening a blender bowl to allow aerosols to settle. Refrigerate to condense

aerosols. Use models designed to prevent leakage from rotor bearings and O-ring

gaskets or use a “stomacher”.

Task 2. Prepare the smears of mixed (staphylococcal and colibacillar)

cultures, stain them by Gram’s method, perform microscopy and draw it.

Technique of cultures smear preparation grown on the solid nutrient

14

medium

Manipulation 1. Smear preparation of the culture grown on the solid

nutrient medium

For smear preparation take a clean slide. By means of glass pencil draw a

circle 1.5–2.0 cm in diameter – a place of the material drawing. On the opposite

part of the glass prepare smear test. In the left hand take a test tube with a

physiological solution (NaCl), and in the right – a bacteriological loop. The loop is

calcinated in a flame of a spirit-lamp (sterilized) for destruction of extraneous

bacteria. By rotary movements take a wadded fuse out of the test tube, pressing it

by the 5th and 4th fingers of the right hand to a palm, and burn the edge of the test

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

of the glass. Then put 1–2 drops of the sodum chloride on the glass slide, burn the

edge of the test tube again and close its fuse. Then take a test tube with the grown

up culture in the left hand, similarly open it, sterilize the edge of the test tube, enter

the loop into the test tube cautiously, grasp a material with a sliding movement,

take out a loop from the test tube, burn the edge of the test tube again and close its

fuse. The material on the bacteriological loop is brought in the drop of the sodum

chloride and prepared for a suspension, not falling outside the limits of the circle

(at correct distribution of the material in smear test at microscopy isolated bacterial

cells are visible). After preparation of smear test, bacteriological loop is carefully

burned in a flame of the spirit-lamp.

Manipulation 2. Smear test fixation

Fixe the smear by spent it three times (in 3–5 seconds) through the flame of

the spirit-lamp (smear test upwards), carrying out physical fixation.

Manipulation 3. Smear staining by Gram’s method.

Gram staining procedure

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

gentle heating, which kills

bacteria and allows the material to be attached to the slide.

15

2. Apply an appropriate solution of gentian violet. Stain it for about 1 minute.

Wash carefully.

3. Apply iodine solution (a mordant, which strengthens the bond between dye

and substrate) for 1 minute. Wash carefully.

4. Apply 95% ethyl alcohol until all but the thickest parts of the smear are

decolourized, or for not more than 10 to 15 seconds. Wash.

5. Counterstain with fuchsin for 1 minute. Wash. Dry.

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

7. Gram-positive organisms are blue-purple and gram-negative bacteria are

pink-red. If the spesimen is stained correctly, gram-positive (violet) and gram-

negative (red) bacteria will be visible on microscopy. At the wrong staining all

bacteria are the same colour (violet or red).

Manipulation 4. Performing of microscopy with immersion system .

Rules of work with immersion system:

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

of the microscope.

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

maximal visual field illuminance.

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

macroscrew find the contours of the image, and by means of the microscrew –

obtain precise image.

4. Turning a nosepiece of the microscope, place an immersion objective 90×

above the slide; dip it in the oil immersion.

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

preparation, study microorganism morphology, determine the increase, at which

the preparation was investigated.

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

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

Manipulation 5. Drawing it.

16

Tasks for self-guided work:

1. Draw microscope and fill in the gapes of structure of the microscope

2. Study the principles of microbiological methods and draw the scheme.

3. Draw the scheme of Gram’s staining

17

4. Draw the pictures and write down forms of bacteria:

18

5. Fill the table.

Structure of bacterial cell Its functions Methods of detection

Cell wall

Cytoplasmic membrane

Bacterial capsule

Cytoplasm

Nucleoid

Ribosomes

Spores

Flagella

Conclusion.

19

Practical lesson 2

Physiology of microorganisms. Metabolism. Nutrition and respiration of

bacteria. Culturing methods of aerobic and anaerobic bacteria

Objective: To systematize the knowledge of the metabolism of

microorganisms.

Tasks:

1. To study the mechanisms of microbial nutrition.

2. To study kinds of culture media.

3. To consider microbial seeding technique on nutrient media

Questions:

1. Anabolism and catabolism.

2. Mechanisms of nutrients transmission in the bacterial cell.

3. Autotrophs and heterotrophs, auxotrophs and prototrophs.

4.Classification of culture media.

5. Simple and complex nutrient medium.

6. Method for sowing nutritive medium.

7. Growth phases on medium.

8. Mechanism of bacterial respiration. Aerobes and anaerobes.

9. Methods of cultivation of anaerobic bacteria: culture media, equipment.

10. Isolation of pure culture of anaerobes.

11. Identification of the selectioned pure cultures of bacteria.

12. The main groups of bacteria enzymes.

13. Determination saccharolytic enzymes of bacteria.

14. Determination of proteolytic enzymes.

15. Determination peptolytic enzymes.

16. Aggression enzymes: coagulase, hyaluronidase, neuraminidase, DNA -

ase, hemolysin.

20

Physiology of bacteria. Bacteria nutrition and respiration. Nutrient

media. Isolation of pure cultures of aerobic bacteria (stage 1)


Metabolism Metabolism is the total of all chemical reactions occurred

in the cell. Metabolism may be divided into major parts.

In catabolism larger and more complex molecules are

broken down into smaller, simpler molecules with the release

of energy. The bacterial cell obtains the energy for

biochemical reaction due catabolism (energy-generating or

energy-yielding process).

Anabolism is the synthesis of complex molecules from

simpler ones with the input of energy (energy-requiring

process)

Pure culture Pure culture is a population of cells arising from a single

cell.

Can be accomplished from mixtures by a variety of

procedures, including streak plates and pour plates

Culture media

Much of microbiology depends on the ability to grow and

maintain microorganisms in the laboratory.

This is possible only if suitable culture media is available.

Media can be defined (synthetic) or complex, and liquid

or solid.

Classification of

culture media

Based on the consistency:

- liquid – peptone water, nutrient broth;

- semisolid – nutrient agar stabs;

- solid – blood agar, serum agar.

Based on oxygen requirement:

- aerobic medium;

21

- anaerobic medium

Synthetic

(defined) media

Synthetic (defined) media are media in which all

components and their concentrations are known.

1. Peptones: protein hydrolysates prepared by partial

proteolytic digestion of various protein sources.

2. Extracts: aqueous extracts, usually of beef or yeast

Liquid media Liquid media: the easiest to prepare and use. Good for

growing quantities of microbes needed for analysis or

experiments. Unless inoculated with pure culture, cannot

separate different organisms

Solid media Solid media is needed for surface cultivation of

microorganisms

Selective media Inhibits the growth of some bacteria while selecting for

the growth of others. Example: brilliant green agar – dyes

inhibit the growth of gram-positive bacteria; selects for gram-

negative bacteria

Differential media Differential media distinguish between different groups of

bacteria on the basis of their biological characteristics. They

cause observable change in medium when biochemical

reaction occurs. For example: MacConkey agar has color

indicator that distinguishes presence of acid. Bacteria that

ferment a particular sugar (e.g., glucose in culture media) will

produce acid wastes on plates, turn pH indicator red. Bacteria

that cannot ferment the same sugar

will grow but not affect pH, so colonies remain white

Complex media Enriched media: blood agar (nutrient agar + 5 to 10%

sheep blood).

Melt the sterile nutrient agar by steaming, cool to 45 °C.

22

Add the blood aseptically with constant shaking.

Mix the blood with molten nutrient agar thoroughly but

gently avoiding froth formation.

Immediately pour in to the Petri dishes or tubes and allow

setting.

Use: cultivate all the fastidious organisms

Growth and reproduction of bacteria. Enzymes


Isolation of microbes

from clinical

material

1. Microscopic examination: Gram staining, acid fast

stain, dark field microscopy, phases contrast.

2. Culture: use streak plate to culture sample on

appropriate media to get pure culture. Use in addition

selective media for isolation of the suspected organism.

3. Identification: use staining, differential media,

serology and or gene probe for identification of the isolated

microbe.

4. In vitro antibiotic sensitivity test.

5. Epidemiology in case of outbreak to determine the

source of infection

Clone A race of cells derived from a single ancestral cell and

sharing a single function

Colony A number of microorganisms living or multiplying

together on solid culture media in the result of

multiplication of a single cell

Pure culture Pure culture is a population of microorganism of the

same species isolated on a nutrient medium

23

Characteristic of

stroke culture

1. The degree of growth – scanty, moderate or profuse.

2. Their nature – discrete or confluent, filiform,

spreading or rhizoid

3. Their elevation, surface, edges, colour, structure,

odour, emuisifiability, consistency and cha the medium are

noted

Classification of

enzymes

1. Exoenzymes are transported extracellularly, where they

break down large food molecules or harmful chemicals;

cellulase, amylase, penicillinase.

2. Endoenzymes are retained intracellularly and function

there.

3. Constitutive enzymes are always present, they are

produced in equal amounts or at equal rates, regardless of

amount of substrate; enzymes are involved in glucose

metabolism.

4. Induced enzymes are not constantly present, they are

produced only when substrate is present; prevent cells from

resources loss

Facultative

anaerobes

Microorganisms that can live and grow with or without

molecular oxygen

Obligate anaerobes Microorganisms that can grow only in the complete

absence of molecular oxygen; some are killed by oxygen

Examples of obligate

anaerobic bacteria

Clostridium perfringens, C. tetani, C. histolyticum, C.

novyi, C. septicum, C. botulinum, Bacteroides fragilis, B.

gracilis

Facultative anaerobic

organism

A facultative anaerobic organism is an organism, usually a

bacterium, that makes ATP by aerobic respiration if oxygen

is present but is also capable of switching to fermentation.

http://www.answers.com/topic/organism

http://www.answers.com/topic/bacteria

http://www.answers.com/topic/adenosine-triphosphate

http://www.answers.com/topic/cellular-respiration

http://www.answers.com/topic/oxygen

http://www.answers.com/topic/fermentation-biochemistry

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In contrast, obligate anaerobes die in presence of oxygen

Examples of

facultative anaerobic

bacteria

The examples of facultative anaerobic bacteria are

Staphylococcus (gram-positive), Escherichia coli (gram-

negative), Corynebacterium (gram-positive), and Listeria

(gram-positive)

Methods of

anaerobic culture

isolation

Physical method → cultivated the anaerobic bacteria’s

in agar deep or in fluid media, but on surface presence oil,

Vinyal-Veyon’s method.

Chemical method → used in media substance for

absorption O2.

Biological method → Fortner method (subcultured to

aerobic and anaerobic)


Task 1. Obtaining of isolated colonies of bacterial culture by seeding using the

streak-plate technique

1. Sterilize the inoculating loop by flaming.

2. Remove the cap and sterilize the mouse of the test tube in the flame.

3. Using the sterile loop take a small amount of material from the tube.

http://www.answers.com/topic/obligate-anaerobe-2

http://www.answers.com/topic/staphylococcus

http://www.answers.com/topic/gram-positive

http://www.answers.com/topic/escherichia-coli

http://www.answers.com/topic/gram-negative

http://www.answers.com/topic/gram-negative

http://www.answers.com/topic/corynebacterium-1


http://www.answers.com/topic/listeria


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4. Sterilize the mouse of the test tube after taking the sample, recap it and place it

back. The inoculating loop is not flamed.

5. Carefully lift the top of the dish containing sterile agar just enough to insert

your inoculating loop easily and make a streak over the agar plate spreading out

the bacteria.

6. Remove the inoculating loop, close the Petri dish and rotate it counter

clockwise about 90 degrees.

7. Sterilise the inoculating loop in order to kill any remaining bacteria by flaming

them.

8. Open the agar Petri dishes again, insert the loop under the lid and cool it at the

edge of the agar. Keeping in mind where the initial streaks ended make a streak

over the agar plate spreading out bacteria starting from the previous streak.

9. Close the Petri dish and turn the Petri dish counter clockwise about 90 degrees

again.

10. It is necessary to repeat the sequence of the streaking procedures described

26

above twice to make a set of four cross-streaks.

12. After you will finish your work finally sterilise the inoculating loop and put it

out.

Task 2. Describe cultural properties of colonies which grew on Endo medium.

Use picture from Task 2 for self-guided work. Write down these properties in

the protocol.


Task 1. Write down in your copybook.

The chart of bacteriological method of diagnostics of infectious diseases

27

Task 2. Draw the scheme of the streak plate technique

Task 3. Fill the table.

Types of medium Compounds Purpose

Endo medium

28

Ploskirev’s medium

Blood agar

Salt agar

MacConkey agar

Task 4. Study and draw types of bacterial colonies.

bottom or parietal growth (example: streptococcus on sugar broth)

tender film (ex.:Vibrio cholerae on nutrient broth)

diffusion growth (ex.: staphylococcus on nutrient broth)

Task 5. Fill the scheme.

Sheme of pure culture isolation of aerobic bacteria’s

Stage 1 of pure culture isolation of aerobic bacteria – 1st day

29

Stage 3 of pure culture isolation of aerobic bacteria – 3rd day

30

Conclusion.

Practical lesson 3.

Viruses. The discovery of viruses, its classification. Bacteriophages. The

practical importance of phages in biology and medicine. The genetics of

microorganisms. Biotechnology in microbiology. Molecular-biological

methods of diagnosis

Objective: formation of knowledge about the structure of the features of viruses

and bacteriophages.

Tasks:

1. To review the structure, allocation methods, identification of viruses.

2.To study the principles of the using of bacteriophages preparations for treatment

and prevention of infectious diseases.

Questions:

1. Classification of viruses. Virus and virion.

2. Morphology of viruses. The functions of RNA and DNA.

31

3. Chemical composition nukleoprotein. Enzymes.

4. Methods of viruses cultivation.

5. Interaction of viruses with cell virus. The mechanism of transcription and

replication viral genome.

6. The mechanism of DNA and RNA viral integration into the cellular genome.

7. Ways transmission of viral infections.

8. The morphology of phages.

9. The mechanism of interaction between phages and the bacterial cell.

10.Virulent and temperate phages. Lysogenesis.

11.Titr phage. Methods for its determination.

12. Obtaining of phage culture. The using of phages in medicine.

13. Genotype and phenotype of bacteria.

14. Extrachromosomal factors in bacteria – plasmids, transposons, Is – sequence;

its role.

15. Forms of variability in microorganisms.

16. Mutations, kinds of mutations in bacteria.

17. Genetic recombination in bacteria (transformation, transduction,conjugation).

18. The concept of the modifications.

19. The theoretical and practical importance of genetics: molecular-biological

methods of diagnosis The practical using of genetic engineering.

Table 4. Viruses. Bacteriophages


Two types of

phages

There are two types of phages: virulent phages and

temperate phages. Virulent phages infect the bacteria,

reproduce, and then lyse and kill the bacteria. On the other

hand, temperate phages have a good temperament and do not

immediately lyse the bacteria they infect.

Types of phage Productive type: bacteriophages lyse their host bacterial

32

reproduction in

bacterial cell

cells after penetration and reproductive cycle.

Abortive type: after penetration the new phage particles

will not forming and bacteria live.

Integrative type: phage genome is integrated in bacterial

chromosome and bacterial host cell is live

Classification of

bacteriophages

Polyvalent – destroy kindred bacteria, for example,

polyvalent salmonella bacteriophages destroy all Salmonella

spp. (S. enteritidis, S. typhimurium).

Monovalent – destroy one species of bacteria, for

example, typhoid fever Vi- bacteriophages – destroy only

typhoid fever agent.

Typospecific – destroy one type of bacteria species

Prophage The integrated temperate phage genome is called a

prophage. Bacteria that have a prophage integrated into their

chromosome are called lysogenic because at some time the

repressed prophage can become activated.

Bacterial genetics.


Genotype and

phenotype

The genotype of an organism is its genetic makeup, the

information that codes for all the particular characteristics

of the organism. The genotype represents potential

properties, but not the properties themselves. Phenotype

refers to actual, expressed properties, such as the

organism's ability to perform a particular chemical

reaction. Phenotype, then, is the manifestation of genotype

Transcription

Transcription is the syn thesis of a complementary

strand of RNA from a DNA template

33

Transformation Naked DNA fragments from one bacterium, released

during cell lysis,

bind to the cell wall of another bacterium. The

recipient bacterium must be competent, which means that

it has structures on its cell wall that can bind the DNA and

take it up intracellularly. Recipient competent bacteria are

usually of the same species as the donor. The DNA that

has been brought in can then incorporate itself into the

recipient's genome if there is enough homology between

strands (another reason why this transfer can only occur

between closely related bacteria)

Transduction Transduction occurs when a virus that infects bacteria,

called a bacteriophage, carries a piece of bacterial DNA

from one bacterium to another.

Types of

phage

reproduction

in bacterial

cell

Productive type: bacteriophages lyse their host

bacterial cells after penetration and reproductive cycle.

Abortive type: after penetration the new phage particles

will not forming and bacteria live.

Integrative type: phage genome is integrated in

bacterial chromosome and bacterial host cell is live

Conjugation Conjugation is bacterial sex at its best: hot and heavy.

In conjugation DNA is transferred directly by cell-to-cell

contact, resulting in an extremely efficient exchange of

genetic information. The exchange can occur between

unrelated bacteria and is the major mechanism for transfer

of antibiotic resistance

Mutation Any alteration made to the DNA sequence of an

organism is called a mutation. This may or may not have

34

an effect on the phenotype (physically manifested

properties) of the organism.

1. Morphological mutations change microorganisms

appearance, colony or cellular morphology.

2. Lethal mutations-result in death of microorganism.

3. Conditional mutations are expressed under certain

environmental conditions, e.g., not expressed at a low

temperature (permissive temp), but expressed at high

temperature (non-permissive temp).

4. Biochemical mutations alter a biosynthetic

pathway and the organisms ability to grow on minimal

media.

5. Resistant mutations – mutant that is now resistant

to an antibiotic or virus

Plasmids

Plasmids are small ds DNA molecules, usually circular

that can exisit independently of the host chromosome.

They have their own replication origin so can replicate

automonously (replicon) and

Conjugative

plasmids

Conjugative plasmids have genes for pili and can

transfer copies of themselves to other bacteria during

conjugation.

Fertility factor or

F factor

Fertility factor or F factor – these plasmids can also

intergrate into the host chromosome or be maintained as

an episome (independent of chromosome)

R factor Conjugative plasmids which have genes that code for

antibiotic resistance for the bacteria harbouring them.

These do not integrate into the host chromosome

Col Plasmids Harbour bacteriocins which are proteins that destroy

35

other bacteria (e.g., cloacins kill Enterobacter species)

Virulent plasmids Virulent plasmids – have genes which make bacteria

more pathogenic because the bacteria is better able to

resist host defences or produce toxins/invasions



Bacteriophages for treatment, prophylaxis, and diagnosis purposes.

Notion Content Purposes of its using

Polyvalent

shigellosis

bacteriophage

(liquid)

It is used for phagotyping the

selected pure culture of

shigellae (to confirm isolated

pure culture of shigellae to

Shigella genus)

Polyvalent

shigellosis

bacteriophage (in

tablet)

It is used for the emergency

prophylaxis of shigellosis and

treatment of acute shigellosis

Salmonellosis

polyvalent

А,В,С,D,Е

bacteriophage

(tabletted)

It is used for salmonella

infection urgent prophylaxis

and treatment

Cholera

bacteriophage El

Tor (liquid)

It is used for phagotyping the

selected pure culture of

cholera (for confirm isolated

36

pure culture

Task 2. Write down definition for terms.

Virulent phages – ____________________________________________________

Temperate phages – ________________________________________________

Polyvalent phages – __________________________________________________

Monovalent phages – _______________________________________________

Task 3. Fill the gaps in picture “Bacteriophage”. Draw it.

Task 4. Draw the picture in your copybook. Explain and write down stages of

phage titration.

37

Stages of phage titration

1._________________________________________________________________

2.________________________________________________________________

3.________________________________________________________________


Extrachromosomal factors in bacteria and its functions

Factors Functions

Plasmids

-F- plasmids

-R- plasmids

- col Plasmids

- virulent plasmids (vir-)

Transposons

Is – sequence

Task 6. Write down, draw the picture and answer on the question.

For conjugation reaction 2 cultures are used:

1. E. coli F+ Pro+, Ura+, His+, StrS. This culture has fertility-factor, plasmid

and is able to produse proline, uracile, histidin, but it is sensitive to streptomycin.

38

2. E.coli F-, Pro-, Ura-, His-, StrR, it is resistant to streptomycin.

Basal medium is used for this experiment. In this medium amino acid is

absent, but streptomycin is present.

Question: why do F+

and F- E.coli (which isn’t able to grow on the medium with

streptomycin) grow on this medium after mixing?

Conclusion.

Practical lesson 4

Infection: the role of microorganisms in infectious process. Biological

diagnostic method. The role of host in the infectious process. Microbiological

basics of antimicrobial therapy. Methods for determination sensitivity of

bacteria to antibiotics

The objective: the formation of knowledge on mechanisms of variation bacteria,

antimicrobial therapy.

39

Tasks:

1. To study with features of variability of species microorganisms.

2. To consider the modern methods of genetic research microorganisms and their

using in medical practice.

3. Determine the sensitivity of bacteria to antibiotics by Indicator disc.

Questions:

1. The notion of infection and infectious disease. Forms of infection.

2. Exogenous and endogenous infections. The term "entrance gate and infective

dose."

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

4. The local and generalized infectious process. The spread ways of infections in

the body. Notions: bacteremia, viremia, toksinemiya, sepsis, pyosepticemia.

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

clinical manifestations.

6. Notions: monoinfection and mixed infection, primary and secondary infection,

reinfection, superinfection, relapse.

7. Pathogenesis of infectious diseases. Periods of infection.

8. The pathogenicity and virulence of microorganisms, measuring of virulence.

9. Virulence factors: adhesion, colonization, penetration, invasion.

10. Toxicity. Exotoxins. Classification of action mechanism.

11.Endotoxins. The chemical compound, action on the macroorganism.

12.Role microorganism in the occurrence of infection.

13. The notion of antibiotics, its discovery. Classification of antibiotics: on origin,

method of preparation, action on a microorganism, antimicrobial spectrum.

14. The mechanism of action of antibiotics on microbial cells.

15. The principle of obtaining antibiotics. Activity of antibiotics.

16. The mechanism of bacterial resistance to antibiotics and how to deal with it.

17. Methods of determining the bacterial sensitivity to antibiotics.

18. Side effects of antibiotics.

40

Infection doctrine. Antibiotics


Infectious process Dynamic interaction between sensitive macroorganisms and

pathogenic or potential pathogenic microorganism in specific

environmental conditions

Pathogenicity Property (capacity) of the definite species of bacteria to cause

(excite) infectious process in sensitive people and animals

Virulence Degree of the microorganism pathogenicity

Invasiveness Ability of the microorganisms to penetrate and spread in

the body due to the presence of specific enzymes that change

the permeability of cells, and tissues. For example, presence

of the staphylococcus hyaluronidase

Toxigenicity Ability (property) of the microorganisms to produce

toxin that has large molecular mass, antigenic properties, and

affects the basic cell and body tissue

Susceptibility Ability of the macroorganism to interact with pathogens

and create conditions for its existence. Susceptibility is a

specific concept

Persistence Long causative agent keeping in the body without the

occurrence of the infection

Antibiotics Antibiotics are large group of drugs, which can inhibit

selectively growth of bacteria, fungi or inhibit growth of

tumor (cancer), without causing serious damage to the host.


Determination of antibacterial action of antibiotics with disk diffusion

technique.

41

For this experiment, on a nutrient medium there was grown a pure bacterial

culture. Small filter paper discs containing various antibiotics to be tested are then

placed on the surface of the medium.

After getting the growth of bacteria as a lawn you have to evaluate the

inhibition zones which will appear (or not) around the disks and to make the

conclusions about susceptibility of test bacteria to antibiotic using the next criteria:

Diameter of inhibition zone The level of susceptible of bacteria to

the antibiotic

1 Absent or diameter of zone is

less 10 mm

Resistant

2 The zone of inhibition is from 10

to 14 mm

Low susceptible

3 The zone of inhibition is from 15

to 25 mm

Susceptible

4 More than 25 mm High susceptible

Antimicrobial susceptibility disk diffusion test: 1 – resistant, 2 – low

susceptible, 3 – susceptible, 4–high susceptible

Task 1. Determine antibacterial action of antibiotics and fill the table.

42

The level of antimicrobial activity of antibiotics.

Antibiotic Diameter of inhibition zone The level of susceptible


Task 1. Write down notions and its explanations.

Infectious process – __________________________________________________

Pathogenicity – ____________________________________________________

Virulence – ________________________________________________________

Invasiveness – ______________________________________________________

Toxigenicity – ______________________________________________________

Persistence – _______________________________________________________


Characteristic of the toxins

Properties Exotoxins Endotoxins

Chemical composition

Secreted by

The level of toxigenicity

Toxic action

Thermolabile or thermally

stable

Task 3. Examples of bacterial cultures synthesizing exotoxin and endotoxin

43

Examples of bacteria producing

exotoxin

Examples of bacteria having endotoxin

Conclusion.

Practical lesson 5

Ecology of microorganisms. The human microflora and its function.

Dysbiosis, methods of correction. Biofilms. Sanitary demonstration water

microorganisms, soil, air, and methods of research.

Objective: To systematize the knowledge of microbial ecology.

Tasks:

1. Learn how to count the microbial count of air, water.

2. Learn the composition of the normal skin microflora

Questions:

1. Ecology of microorganisms. Forms of interspecies relationships.

2. Role, value, and tasks of sanitary microbiology. 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.

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.

5. Microflora of air and methods of bacteriological examination: sedimentation and

aspiration methods. Sanitary indicators of air microorganisms.

44

6. General principles of sanitary and microbiological investigation of foodstuffs.

7. The normal microflora of the human body and its significance.

8. Factors that change the normal microflora of the organism. Dysbiosis, ways of

its elimination.

Sanitary and microbiological investigation of water, air, soil, foodstuffs,

and objects of external environment


Sanitary indicatory

microorganisms

Permanent inhabitants of surfaces and digestive

tract of the humans and animals that are excreted from

an organism like pathogenic bacteria. The sanitary

indicatory microorganisms are indicator of pathogenic

microorganisms presence in the external environment

objects

Coli titre of water

(determination)

The least of water (ml), in which coliform bacteria

(bacteria group of Escherichia coli-BGEC) is revealed

Coli index of water

(determination)

An amount of bacteria of Escherichia coli bacteria

group, which are contained in 1 liter of the investigated

water


Task 1. Define the microbal number of the air by the sedimentation method

(by Koch)

Sedimentation method is an inoculation of microbes on the surface of solid

medium under the action of gravity. For investigation of air in apartments take

Petri dish with media, keep it opened for 60 minutes (for determination of general

semination air). Incubate Petri dish in the thermostat at 37 °C for 24 hours and then

at a room temperature (one days). Determine the amount of microbes in 1 m3 of air

45

(microbial number) and evaluate the degree of air pollution according to amount of

colonies.

After incubation count up the amount of growing colonies, calculate the

amount of microbes in 1 m3 air by using a formula:

“a” is an amount of colonies; “b is area of Petri dish (63 cm2); “t” is time of exposition.

Task 2. Inoculate the microorganisms from the skin of the hand on the

nutrient agar.

The predominant resident microorganisms of the skin are aerobic and

anaerobic diphtheroid bacilli (e.g., Corynebacterium, Propionibacterium);

nonhaemolytic aerobic and anaerobic staphylococci (Staphylococcus epidermidis,

occasionally S aureus, and peptostreptococcus species); gram-positive, aerobic,

spore-forming bacilli that are ubiquitous in air, water, and soil; alpha-

haemolytic streptococci (viridans streptococci), and enterococci (enterococcus

species); and gram-negative coliform bacilli and acinetobacter. Fungi and yeasts

are often present in skin folds; acid-fast, nonpathogenic mycobacteria occur in

areas rich in sebaceous secretions. Touch by fingers a surface of the nutrient agar

in Petri dish for inoculation of the microorganisms that inhabit the skin or got there

from environment. Incubate Petri dish in the thermostat at 37 °C for 24 hours and

then at a room temperature (one day).

Draw colonies in the protocol, make the conclusion.


1. Draw and fill the scheme.

Normal microflora. Disbacteriosis

46

Task 2. Write down notions and its explanations.

Sanitary indicatory microorganisms – ___________________________________

Coli titre – _________________________________________________________

Coli index – _______________________________________________________

Normal microflora – ________________________________________________

Dysbiosis – ________________________________________________________


Characteristic of habitats for microorganisms

Propertis Soil Water Air

Favourable or

unfavourable habitat

for microorganisms

The source of

microbial

contamination

47

Species of sanitary

indicatory

microorganisms

Normal indexes of its

parameters (amount)

Methods for detection

of microorganisms

Task 4. Fill the scheme and draw it.

Conclusion.

Practical lesson 6

Immunity. Serological methods – agglutination test, precipitation

test, complement fixation test

Objective: To acquire knowledge on immunity, antigens and antibodies, its

methods of detection.

48

Tasks:

1. To consider the concept of an antigen and an antibody.

2. To study the types of immunity.

3. To learn some basic serological reactions

Questions:

1. The notion of immunity. Classification of immunity.

2. The notion of an antigen, a chemical composition.

3. Bacterial antigens.

4. Antigens of viruses.

5. Notion of antibody, its structure and properties.

7. Serological tests – notion and its using in microbiological diagnostics.

8. Agglutination test - definition, components, using.

9. Precipitation test - definition, components, using.

10. Complement fixation test – components, phases of reaction and using.

Immunity. Factors and mechanisms of the innate immunity


Immunity Dynamic condition of the organism that consists of

the set of specific

Antigens Foreign macromolecules that when introducted into the

body cause the formation of the immune response, on

condition of their recognition by specific receptors of

lymphocytes

Antibodies Specific proteins that are synthesized in the body in

response to input (falling) of an antigen


Practical use of the

agglutination test

1. In serological test for detection of the specific

antibodies formed in the serum or other body fluids by the

49

infectious diseases and other pathological conditions.

2. For identification of the bacterial antigens in the

bacteriological method

Types of the

agglutination test

1. Slide agglutination test is made on the glass or

porcelain plates with a smooth surface.

2. Tube agglutination test is made in the test tube or in

the polisterol hole in plates with consecutive dilution of

the serum. In every tube (hole) the same amount of

antigen is contributed. The titre of the antibody is detected

Precipitation test

mechanism

Interaction of a soluble antigen (precipitinogen) and

antibodies (precipitin) in the presence of electrolyte

(0.85% solution of NaCl). An aggregation of the antigen

makes opacity of transparent liquid (precipitate).

Deposition from solution complexes of the antigen-

antibody become in equivalent ratio in the range of the

interacting molecules concentrations

Practical use of

the PT

PT is used for the diagnosis of a number of infectious

diseases - anthrax, plague, tularemia, epidemic cerebrospinal

meningitis and others. Extracts from affected organs, and

other liquor are used as the antigens, and diagnostic

precipitating serum is used as antibodies (precipitins).

PT is also used to study the antigenic structure of the

bacteria. In addition, it is used to determine the species

proteins, including proteins of blood


Task 1. Fill the scheme.

50

Task 2. Write down an antigens of bacteria (left picture) and viruses (right

picture).Draw.


Serological tests.

Test Components Positive result of

test

Application

Agglutination test

Precipitation test

51

Complement

fixation test

Task 4. Fill the scheme and draw it.

Structure of immunoglobulin.

Conclusion.

Practical lesson 7

Clinical Microbiology. Etiology and pathogenesis of

nosocomial infections. The causative agents, properties and microbiological

diagnostics of nosocomial infections. Pathogenic and opportunistic gram-

positive and gram-negative cocci

Objective: formation of knowledge about nosocomial infections and its pathogens.

Tasks:

1. To study with the pathogens of nosocomial infections.

2. To study the epidemiology and pathogenesis of nosocomial infections.

3. To study methods of laboratory diagnostics of nosocomial infections.

Questions:

52

1. Clinical microbiology, its tasks.

2. The causes of hospital infections.

3. Classification of nosocomial infections.

4. The main causative agents of nosocomial infections.

5. The sources and pathways of hospital infections.

6. Characteristics of opportunistic microorganisms that causes nosocomial

infections.

7. The features of nosocomial infections.

8. Microbiological diagnostics and prevention of nosocomial infections.

9.Biological properties of bacteria Klebsiella: morphology, tinctorial properties,

cultivation, biochemical properties, antigens, toxins, other pathogenicity factors.

10. Epidemiology and pathogenesis. Diseases.

11. Microbiological diagnostics, treatment and prevention of klebsiellosis.

12. Classification and biological properties of Proteus.

13. Diseases. Epidemiology and pathogenesis.

14. Microbiological diagnosis, treatment and prevention of Proteus-infections.

15. Biological properties of Pseudomonas aeruginosa.

16. Epidemiology and pathogenesis. Caused diseases.

17. Microbiological diagnostics, treatment and special prevention of Pseudomonas

-infection.

18. The biological properties of Helicobacter pylori.

19. Epidemiology. Caused disease and pathogenesis.

20. Microbiological diagnostics, treatment, prevention of caused diseases.

21. General characteristics of pyogenic cocci group.

22. Taxonomy and biological properties of staphylococci.

23. Epidemiology and pathogenesis of diseases caused by staphylococci.

24. Microbiological diagnosis, treatment and specific prevention of staphylococcal

infections.

25. Taxonomy and biological properties of the streptococci.

53

26. Epidemiology and pathogenesis of streptococcal infections.

27. Streptococci is causative agents of scarlatina and rheumatism.

28. The microbiological diagnosis, treatment and specific prevention of

streptococcal infections.

29. Taxonomy and biological properties of meningococcus.

30. Epidemiology and pathogenesis of meningococcal infections.

31. Microbiological diagnosis, treatment and specific prevention of meningococcal

infections.

32. Taxonomy and biological properties of gonococci.

33. Epidemiology and pathogenesis of gonococcal infections.

34. Microbiological diagnosis, treatment and specific prevention of gonococcal

infections.

Clinical microbiology. Laboratory diagnosis of opportunistic and hospital

infections

Clinical

microbiology

Part of microbiology that studies the interaction between

macroorganism and microorganisms in norm and pathology, in

the dynamics of the inflammatory processes taking into account

conduct therapy to the convalescence

Nonpathogenic

microorganisms

Microorganisms that have small degree of pathogenicity for

human and may cause the diseases in definite conditions

Opportunistic

infections

Diseases appear in immunocompromised patients and

caused by non-pathogenic microorganisms

Mixed infection Infection that is caused by a few species of microbes

Hospital infection The onset of infection is in the hospitals. It complicates the

clinical course of basic disease

Laboratory diagnosis of infections that caused by cocci


54

Staphylococcus:

classification and

biological

properties

The term Staphylococcus is derived from the Greek term

staphyl, meaning “a bunch of grapes”. This name refers to the

fact that the cells of these gram-positive cocci grow in a

pattern resembling a cluster of grapes; however, organisms in

clinical material may also appear as single cells, pairs, or short

chains. Most staphylococci are 0.5 to 1 µm in diameter and are

nonmotile, aerobic or facultative anaerobic, and catalase-

positive and grow in a medium containing 10% sodium

chloride and at temperature ranging from 18 ºC to 40 ºC.

The organisms are present on the skin and mucous

membranes of humans and other mammals, and birds.

Staphylococcus is an important pathogen in humans, causing a

wide spectrum of life-threatening systemic diseases; infections

of the skin, soft tissues, bones, and urinary tract; and

opportunistic infections. The species most commonly

associated with human diseases are S. aureus (the most

virulent and best-known member of the genus), S. epidermidis,

S. saprophyticus, and S. haemolyticus.

Diseases of

S. aureus

1. Localized skin infections include impetigo, folliculitis,

furuncles, and carbuncles. Impetigo, a superficial infection

affecting mostly young children, occurs primarily on the face

and limbs. Initially, a small macule (flattened red spot) is seen,

and then a pus-filled vesicle (pustule) on an erythematous base

develops.

2. Toxic shock syndrome is a disease that was initially

evident in children, although it is now recognized as primarily

a disease in menstruating women.

3. Food poisoning is one of the most common food

55

poisoning in the world. The organisms are usually introduced

into food, such as processed meat, pastries, potato, salad, and

ice cream. Staphylococcal food poisoning is characterized by

severe vomiting, xudates, and abdominal pain.

4. Pneumonia is a disease that occurs among

immunosuppressed patients, the aged, children under one year

of age, and frequently in children with measles and influenza.

5. Other diseases like osteomyelitis, septicemia, and

septic arthritis

Principle of the

laboratory

diagnostics

Specimens obtained depend on the disease process and

include lesion material, pus, sputum, blood, spinal fluid, and

faeces.

İsolation and identification of S. aureus requires initial

cultivation on blood agar and/or specific medium and

overnight incubation under aerobic conditions at 37 ºC. The

organism may be identified as a gram-positive, catalase-

positive coccus exhibiting coagulase

Treatment The antibiotics of choice are oxacillin (or other

penicillinase-resistant penicillin) or vancomycin for oxacillin-

resistant strains.

The focus of infection (e.g., abscess) must be identified

and drained. Treatment is symptomatic for patients with food

poisoning

Streptococcus The genus Streptococcus is a diverse collection of gram-

positive cocci typically arranged in pairs or chains. Most

species are facultative anaerobes. Their nutritional

requirements are complex, necessitating the use of blood or

serum-enriched media for isolation. Carbohydrates are

56

fermented, resulting in the production of lactic acid, and unlike

Staphylococcus species, streptococci are catalase-negative

Group A, B

haemolytic

streptococci

Streptococcus pyogenes is the most virulent member of

this group of gram-positive cocci. This bacterium is an

important cause of a variety of suppurative and nonsuppurative

diseases

Classification of

streptococci

1. Haemolitic properties on blood agar (alfa-, betta-, and

gamma-haemolitic streptococci).

2. Serological grouping (A and B).

3. Biochemical properties

Diseases caused

by streptococci

Streptococcus pyogenes.

Pharyngitis. Group A streptococcus is the major cause of

bacterial pharyngitis, occasionally involved with group C

and G.

Scarlet fever is a complication of streptococcal pharyngitis

seen when the infecting strain is lysogenized by a temperate

bacteriophage that stimulates production of erythrogenic

toxin.

Erysipelas. This disease can affect all age groups.

Puerperal sepsis. This infection is initiated during or

following soon after the delivery of a newborn.

The major clinical manifestations are carditis, polyarthritis,

and subcutaneous nodules.

Acute haemorrhagic glomerulonephritis occurs most

commonly in children.

S. xudates is the most common cause of lobar and

lobular (broncho) pneumonia.

Treatment Adequate drainage, debridement, and antibiotic therapy

57

are essential for the treatment of localized, suppurative skin

lesions. Penicillin is the drug of choice for acute diseases.

Penicillin has no effect upon established rheumatic heart

disease and acute haemorrhagic glomerulonephritis.

Penicillin resistant strains have not been reported.

Erythromycin is the drug of choice for penicillin allergic

patients

Neisseria

gonorrhoeae

N. gonorrhoeae is a fastidious organism, requiring

complex media for growth and adversely affected by drying

and fatty acids. Soluble starch is added to the media to

neutralize the toxic effect of the fatty acids. It is highly

susceptible to environmental, physical, and chemical agents.

The optimum growth temperature is 35 ºC to 37 ºC, with poor

survival of the organism at lower temperature. A humid

atmosphere supplemented with CO2 is required or enhances

growth of N. gonorrhoeae

Diseases caused

by N.gonorrhoeae

Gonorrhoea is one of the most commonly reported

sexually transmitted diseases. A higher proportion of females

than males are generally asymptomatic; these individuals act

as the reservoir for maintaining and transmitting gonococcal

infections.

Laboratory

diagnosis


include urethral, cervical, rectal, pharyngeal, and/or

conjunctival exudates.

The direct demonstration of gram-negative intracellular

xudates ic within PMNs is diagnostic only when observed

in the urethral exudates of males with characteristic clinical

manifestations.

58

Gram stains of smears from female urethral and cervical

exudates, from rectal, pharyngeal, and conjunctival exudates

of male and female, are unreliable due to the potential

presence of nonpathogens resembling gonococcal

morphology, and presence of meningococci. All such

specimens must be cultured and the isolated organism

xudates i.

Treatment

Ceftriaxone, cefixime, ciprofloxacin, or ofloxacin can be

administered in uncomplicated cases. In vitro susceptibility

should be determined in cases unresponsive to therapy,

because antibiotic resistance is increasing.

N. meningitidis N. xudates ic causes endemic or epidemic disease

of worldwide prevalence. The most commonly recognized

form of this disease is meningitis.

N. xudates ic is an obligate parasite of humans,

harboured in the nasopharynx, and transmitted by droplet

nuclei or direct intimate contact with a sick people or bacteria

carrier.

This organism is a gram-negative, nonmotile,

encapsulated, piliated diplococcus flattened on one site to

give the appearance of a “kidney bean” or “coffee bean”.

Pathogenesis and

clinical

manifestations

The basic pathogenesis process of primary

meningococcal disease is initiated in the nasopharynx from a

sick people or carrier. Disseminated meningococcal disease

manifests most often as septicemia and meningitis.

Meningitis is the most common complication of

meningococcal septicemia. Clinical manifestations are fever,

stiff neck, vomiting, severe headache, convulsions, bulging

59

of the fontanels, and progression to coma within a few hours.

Laboratory

diagnosis


mainly include nasopharynx swabs, blood culture, and

cerebrospinal fluid. Gram stains of specimens may show

gram-negative, intracellular and extracellular xudates ic in

association with PMNs.

Primary isolations require initial blood cultures and

culture on chocolate agar plates or, if a mixed flora is

anticipated, Thyer-Martin medium, which is an enriched

chocolate agar medium, containing antibiotics to inhibit

gram-positive organisms and gram-negative rods. Incubation

for 48 hours at 35–37 ºC under aerobic conditions in the

presence of 3–10% carbon dioxide is optimal for isolation of

the organisms, which may be identified as gram-negative,

oxidase-positive, diplococcus that ferments glucose and

maltose, and agglutinates in the presence of serogroup-

specific anticapsular antibody

Treatment Early treatment with penicillin reduces the case fatality

rate in disseminated disease from 40–90% to 10–15%, but the

antibiotic is ineffective in eradicating the carrier state.

Chloramphenicol and ceftriaxone are effective in penicillin-

allergic individuals. Polysaccharide vaccines conjugated with

60

protein carriers offer protection for infants younger than 2

years


Task 1. Study the preparation of the pure cultures of Klebsiella

pneumoniae microscopically; draw it in the protocol.

Klebsiella pneumonia

The total magnification – 16x100

Task 2. Study the growth of Proteus vulgaris on nutrient agar. Draw the picture.

The feature of proteus is its ability to creeping

growth after innoculation by Shukhevych (in

liquid condensation on the nutrient agar slant).

Growth of bacteria is observed as veiled

incrustation on the surface of media. This

feature is typical for growth H-form proteus

inoculation.

Task 3. Study the preparation of the pure cultures of Staphylococcus

aureus microscopically; draw it in the protocol.

Staphylococcus aureus


61

Task 4. Study the growth of S. aureus in nutrient agar (left picture) and

blood agar (right picture). Fill in the protocol with their cultural properties.

Task 5. Study the preparation of pure cultures of Streptococcus pyogenes

microscopically and draw it in the copy book.

Streptococcus pyogenes


Task 6. Study the preparation of incomplete phagocytosis of Neisseria

gonorrhoeae microscopically and draw it in the copy book.

Neisseria gonorrhoeae



62

Task 1. Study the biochemical activity of Proteus mirabilis in the Hiss

medium. Draw colour of tubes in accordance its biochemical properties.

glucose lactose maltose mannitol sucrose


Characteristics of nosocomial pathogens

Pathogens Morphology

(picture)

Nutrient

medium

A source

of disease

Transmissi

on paths

Diseases

K. pneumoniae

P. vulgaris,

P. mirabilis,

P. aeruginosa

H. pylori


Characteristics of pyogenic cocci.

Pathogens Morphology

(picture)

Nutrient

mtdium

Transmission

paths

Diseases

63

Staphylococci

Streptococci

Meningococcus

Gonococci

Task 4. Solve the case. Answer on the questions and write down it.

At the Mount Union hospital, a 5-year old white male child in good general

health and physical condition was presented at the Saturday walk-in clinic by his

mother. He was brought in because he had a fever, was cranky and had complained of

a sore throat for about 24 hours. On physical examination by the attending resident, the

patient had a fever of 39.30 C, and he had considerable swelling and drainage of the

pharynx and in the conjunctivae. His tonsils were enlarged and coated with a white

patchy xudates. He had a red throat and swollen anterior cervical lymph nodes. His

ears were clear. His chest sounded clear and he had no additional remarkable findings

on routine examination.

1. What would be your presumptive diagnosis for this child? Why?

2. What diagnostic testing would be indicated to follow this exam?

3. What is the most likely treatment for this illness? Why is it important?

4. What factors of this case allowed you to make a presumptive diagnosis?

Task 5. Fill the scheme. Draw it.

Scheme meningococcal infections and bacteria carriers

laboratory diagnosis

64

Conclusion.

Practical lesson 8

Pathogens of escherichiosis and dysentery. The causative agents of typhoid,

salmonella poisoning and cholera

Objective: formation of knowledge about the biological properties of pathogenic

enterobacteria

Tasks:

1. To study the biological properties of pathogenic enterobacteria.

2. To study the epidemiology and pathogenesis of diseases caused by its.

3. To learn the methods of microbiological diagnosis, treatment and specific

prevention of colienterit and dysentery

Questions:

1. The general characteristics of the family Enterobacteriaceae.

2. Opportunistic Escherichia: its role in the life of the human organism, caused

diseases.

3. The biological properties of Escherichia.

65

4. Epidemiology and pathogenesis of diarrhoea-causing E. coli.

5. Microbiological diagnostics, treatment and prevention escherichiosis.

6. The taxonomy and biological properties of Shigella.

7. Epidemiology and pathogenesis of dysentery.

8. The microbiological diagnosis, treatment and specific prevention of dysentery.

9. Taxonomy and biological properties of pathogens Salmonella

10. Epidemiology and pathogenesis of typhoid - paratyphoid diseases, immunity.

11. Microbiological diagnosis of typhoid fever. Specific prevention and treatment of

typhoid – paratyphoid diseases.

12. Epidemiology and pathogenesis of salmonellosis. Immunity.

13. Microbiological diagnosis, treatment and prevention of salmonellosis.

14. Classification of vibrio. The morphological, tinctorial, cultural property of agents

cholera. Biochemical properties and antigens of V. cholerae.

15. Epidemiologiya and pathogenesis of cholera, immunity.

16. Bacteriological method of cholera diagnostic. Rapid methods and serological

diagnostic of cholera. Prevention of cholera.

Laboratory diagnosis of the infection caused by Enterobacteriaceae


Morphological,

physiological, and

tinctorial features of

Enterobacterium

Rod-shaped asporogenic capsular or acapsular

peritrichous or immobile gram-negative monobacteria of

Enterobacteriaceae family: chemoorganothrophes, aerobes

or facultative anaerobes

Bacteria from

Enterobacteriaceae

family, that are of

high importance for

human

Escherichia, Shigella, Salmonella, Citrobacter,

Enterobacter, Klebsiella, Yersinia, Proteus

Diseases caused by 1. Intestinal infections (colienteritis, shigellosis,

66

Enterobacteriaceae typhoid fever, food toxic infections, and yersiniosis) which

are caused by the representatives of genus of Escherichia,

Shigella, Salmonella, Yersinia. They are characterized by

the faecal-oral or oral mechanism of transmission via water,

food or contact and house routes of the infection

transmission.

2. Opportunistic (extraintestinal) infections

(bacteriemia, meningitis of newborn, traumatic, genito-

urinary, and respiratory infections) which are caused by the

opportunistic pathogenic bacteria of Escherichia,

Klebsiella, Citrobacter, Enterobacter, Proteus genus on the

background of immunodeficiency

Systematic position

of colibacillosis

causative agents

(family, genus,

Family: Enterobacteriaceae.

Genus: Escherichia.

Species: Escherichia coli

Morphological

and tinctorial features

of Escherichia

Straight asporogenic gram-negative rods with rounded

ends, which have capsule or microcapsule, peritrichous

Types of E. coli,

which cause

gastrointestinal

disease

EPEC – enteropathogenic E. coli

EIEC – enteroinvasive E. coli

ETEC – enterotoxigenic E. coli

EHEC – enterohaemorrhagic E. coli

Enterotoxigenic

E. coli and

mechanism of

pathogenic action of

ETEC on epithelium

ETEC causes cholera-like gastrointestinal disease in

children

67

Enteropathogeni

c E. coli and

mechanism of


EPEC on intestinal

cells

EPEC are agents of colienteritis (salmonella-like

infection) mainly in children of the first year of life with the

house, contact, and food routes of infection transmission.

EPEC propagates on the surface of epithelium of small

intestines, stipulating thinning, fragmentation, and tearing

away of microvilli, damage of apical surface of epithelium,

resulting in moderate inflammation and appearance of

shallow erosions

Enteroinvasive

E. coli and

mechanism of


EIEC on intestinal

cells

EIEC causes a disease closely resembling that caused

by Shigella spp. in children of senior age and adults with

the water and food routes of infection transmission. EIEC is

capable of immersion and reproduction in the epithelium of

mucous membrane of the colon, cytoplasmic damage and

death of intestinal cells, invasion to nearby epithelium,

causing focal ulcerous inflammation with surplus

infiltration and accumulation of exudation in the cavity of

colon, by erosions and acute inflammation

Enterohaemorrh

agic E. coli,

mechanism of


EHEC on epithelium

and endothelium

EHEC are agents of haemorrhagic diarrhoea

(haemorrhagic colitis) in adults and in new-born can

predetermine necrotic enterocolitis with the high

percentage of lethal consequencies with the damage of

sigmoideus, ascending and transversal large intestines;

alimentary route of infection transmission. EHEC

propagates oneself on the surface of epithelium of colon

with destruction of microvilli, damage of apical surface of

epithelium. Cytotoxins cause the death of intestinal

epithelium by interfering with protein synthesis. Some of

the toxins are identical to the Shiga toxins of Shigella

68

dysenteriae. Verocytotoxin destroys endothelium of the

blood capillaries. Formation of fibrin results in violation of

Bangui motion. About 4% of infected individuals develop

haemolytic uremic syndrome, marked by anaemia due to

lysis of red blood cells, kidney failure, and central nervous

system damage

Properties are

necessary for E. coli

identification

Antigenic structure (in slide agglutination test and

tube agglutination test with groupspecific and monovalent

escherichiosis sera – basic) and morphological, tinctorial,

cultural, and biochemical properties

Taxonomic position

of the shigellosis

causative agents

Enterobacteriaceae family.

Shigella genus

Species: S. dysenteriae, S. flexneri, S. boydii, S. sonnei

Features of

shigellosis

The heaviest course of shigellosis is caused by S.

dysenteriae, which produces Shiga toxin (exotoxin), less

difficult course of shigellosis is caused by S. sonnei

Morphological and


the shigellosis

causative agents

Short lines with rounded ends gram-negative

immobile monobacteria. Spores and capsules are not

formed

Transmission of

the infection

Through contaminated water (prevails at shigellosis

Flexneri), contaminated food: especially through milk and

milk products (S. sonnei is kept and propagated for a long

time), contact route of infection (especially for S.

dysenteriae species). Insect vectors (flies, cockroaches)

transfer causative agents on foodstuffs

Mechanism of

local immunity

Prevention of shigella adhesion on epithelium,

penetration of shigellae in a cell, prevention of

69

immunological damage of the intestine

Material

investigated at

shigellosis

Bacteriological method: faeces (basic), foodstuffs

(especially milk, cheese, sour cream) at outbreaks of

shigellosis, autopsy material (fragment of large colon,

pieces of parenchymatous organs, mesenterial lymphatic

nodes), blood and urine (at suspicion on bacteriemia).

Serological method: blood analysis

Methods of

shigellosis

microbiological

diagnosis

The basic method is bacteriological, additional –

serological (tube agglutination test, PHAT), express-

methods of antigens diagnosis: IFT (investigation of faeces

and urine), ELISA (investigation of blood serum),

molecular and biological: PCR

Media for

primary cultivation of

causative agents

Differential media: MacConkey, EMB or DCA

(desoxycholate citrate agar), selective media: Hektoen

enteric agar; enrichment medium – selenite medium

Cultural

properties of

shigellosis causative

agents

It forms colourless (lactose-negative), shallow (1–1.5

mm), convex, circular transparent smooth (S-colonies)

colonies with intact edges. S. sonnei forms also R-colonies

– flat dull with a rough surface and unequal edges

Laboratory diagnostics of typhoid fever, A and B paratyphoid and

cholera


Typhoid fever

It is acute anthroponosis infection systemic disease with

faecal-oral transmission mechanism, that is caused by

Salmonella typhi, characterized by small bowel lymphatic

apparatus lesion, bacteraemia, fever, typhoid maculopapular

rash, organism intoxication and cyclic course

Taxonomic Enterobacteriaceae family.

70

classification of

typhoid fever, A and

B paratyphoid

causative agents

Salmonella genus.

Species:

1. Salmonella enterica subspecies choleraesuis serotype

typhi – causative agent of typhoid fever.


paratyphi A – causative agent of A paratyphoid.


paratyphi B (S. schottmuelleri) – causative agent of B

paratyphoid

Morphological and


the causative agent

Short straight with rounded ends gram-negative mostly

movable (peritrichaetes) monobacteria. They do not generate

spores or capsules. Various types of salmonellas do not

differ from one another morphologically

Source of typhoid

fever, A and B

paratyphoid

infection

At typhoid fever and A paratyphoid – patient and bacteria

carrier (employees of catering facilities, water supply objects

– anthroponosis infection). В paratyphoid – patients, bacteria

carriers, and animals (cattle stock, poultry) –

zooanthroponosis infection

Methods of


B paratyphoid

microbiological

diagnosis

1. Bacteriological (basic): diseases diagnosis, contact

persons examination, bacteria carriers identification and

infection source detection.

2. Serological method.

3. Express method

Material for


B paratyphoid

bacterial diagnostics

Basic material is blood (haemoculture), faeces (stool

culture), urine (urine culture), bile (bile culture), bone

marrow aspirates (myeloculture). Additional material is

roseolas content (roseola culture), autopsy material, and

drinking water

71

Typhoid fever

specific prophylaxis

Chemical adsorbed Vi-typhoid fever monovaccine

administration according to epidemiological indications

Cholera Acute extremely dangerous anthroponosis quarantine disease

caused by Vibrio cholerae O1 and O139 serogroups, which

is characterized as the spread of epidemics, profuse watery

diarrhoea, vomiting, dehydration, severe intoxication, faecal-

oral mechanism of transmission and high death-rate

Taxonomic position

of cholera germs

Vibrionaceae family.

Vibrio genus.

V. cholerae type.

Serogroup O1, serotype Ogáva (AB), Inába (AU),

Hikodzhúma (FAA), biovar: classic – V. cholerae cholerae

and El Tor – V. cholerae eltor serogroup, V. cholerae O139

(Bengál)

Morphological and

staining properties

of the cholera

causative agents

Short slightly curved or straight gram-negative rods are of

medium size (1.5–4 × 0.2–0.4 mm), they form clusters in the

form of “fish shoats”, mobile (monotrixous). Spore and

capsules are not forming

Source of

infection of the

cholera causative

agents

The source of infection is patient and carrier. The patient

excretes from 100 mln to 1 billion vibrios in 1 ml of excreta.

The reservoir of infection is also water

Routes of

V. cholerae

transmission

The principal routes are through water, milk, vegetables,

fruits, often – food or water. All epidemics and pandemics of

cholera have water in nature. Some role is played by flies

Material taken from

the contact persons

with cholera

Faeces after the use of the laxatives (25–30 g of

magnesium sulphate). The upper part of faeces is taken

72

Methods of

cholera diagnosis

Bacteriological.

Method of rapid diagnosis (IFT, reaction of

immobilization of vibrios, PCR).

Microscopic.

Serological (for retrospective diagnosis) – ELISA, IFT,

PHAT (definition of anti-cholera Ig titres and antitoxin)


Task 1. Study the preparation of the pure cultures of E. coli

microscopically; draw it in the protocol.

E. coli


Task 2. Study the growth of E. coli on Endo agar. Fill in the protocol with

their cultural properties.

Bacteria E. coli which ferment lactose will

produce sufficient acid (pH change) to cause a

colour shift in the indicator and to change as a

result the colour of their colonies. E. coli is a main

fermenter of lactose, while Shigella and

Salmonella are non-fermenters.

Task 3. Study the preparation of the pure cultures of V. cholerae


V. cholera


73



Types of E. coli, which cause gastrointestinal disease

Types of E. coli Strains

(O..H..)

Toxins Mechanism of

pathogenic action

Enterotoxigenic

E. coli (ETEC)

Enteropathogenic

E. coli (EPEC)

Enteroinvasive

E. coli (EIEC)

Enterohaemorrhagic

E. coli (EHEC)


A 4-year old girl presents at the emergency room with bloody diarrhea, fever and

vomiting. The child’s mother reports that the child has had these symptoms for about

24 hours and she has not passed any urine for about 12 hours.

The child is enrolled in a day care center and the group had recently made a field

trip to a fast food place to learn about different jobs. The children had a lunch of

ground beef, fries and cola after meeting with different workers. This field trip was 4

days earlier on Friday. The child had a temperature of 39C and showed physical signs

of dehydration. Blood samples drawn showed evidence of greatly reduced kidney

function and lysed red blood cells.

1. What is your diagnosis here? What is organism is responsible?

2. What were the critical features to your diagnosis?

74


S. typhi S. paratyphi A S. paratyphi B S. enteritidis

Source of

infection

Transmission

paths

Caused disease

Task 4. Fill the scheme. Draw it.

3.

I – _____________________________________________________________

II – _______________________________________________________________

III – ______________________________________________________________

IV – ______________________________________________________________

V – _______________________________________________________________

VI – _____________________________________________________________


75

The laboratory received blood from a patient suspected cholera, but with

unclear clinical symptoms. What kind serological research tests can you use for

diagnostic? What instructional techniques should be applied to solve this problem?

Conclusion.

Practical lesson 9

Pathogens of diphtheria, whooping cough. Pathogens of legionellosis. Pathogenic

mycobacteria.

Purpose: formation of knowledge about the biological properties of pathogens

diphtheria, pertussis, legionellosis and methods of microbiological diagnostics.

Tasks:

1. To study the biological properties of the diphtheria, pertussis, legionellosis.

2. To study the epidemiology and pathogenesis of diphtheria, pertussis, legionellosis.

3. To consider the methods of microbiological diagnostics.

Questions:

1. The taxonomy and biological properties of the diphtheria bacteria.

2. Epidemiology and pathogenesis of diphtheria. The immunity.

3. Methods of laboratory diagnosis, treatment and specific prevention of diphtheria.

4. The taxonomy and biological properties of Bordetella.

5. Epidemiology and pathogenesis of whooping cough.

6. Laboratory diagnosis and specific prevention of pertussis.

7. The taxonomy and biological properties of Legionella.

8. Epidemiology of legionellosis.

9. Methods for the laboratory diagnosis of legionellosis.

10. Taxonomy and biological properties of the tuberculosis pathogen.

11. Epidemiology and pathogenesis of tuberculosis.

12. Methods of microbiological diagnostics and specific prevention of tuberculosis.

13. Biological features of leprosy pathogen, its methods of culturing.

14. Epidemiology, pathogenesis and clinical forms of leprosy.

76

15. Microbiological diagnostics of leprosy. Treatment and prevention of leprosy.

Laboratory diagnosis of the diphtheria and pertussis


Diphtheria

Corynebacterium diphtheriae causes diphtheria. Usually the

bacteria multiply on the surface of the mucous membranes of

the throat where they


cause inflammation. The inflammation may spread to

the voice box (larynx) and may cause throat swelling, and

airway narrowing. Disease-causing strains of C. diphtheriae

release a damaging substance (toxin), which can also involve

the heart, brain and nerves.

The bacteria may cause a thick, gray covering in the

nose, throat or airway a marker of diphtheria that

distinguishes it among other respiratory diseases. This

covering is usually fuzzy gray or black and causes respiratory

difficulties and painful swallowing

Morphology,

staining, and

culturing of

Corynebacterium

diphtheriae

Diphtheria bacteria are gram-positive, pleomorphic,

often club-shaped rods. The individual cells tend to group in

V, Y, or palisade arrangements.

Loeffler’s nutrient medium, which consists of

coagulated serum and nutrient broth, is used for the primary

cultures.

Transmition of

diphtheria

The disease is usually transmitted by droplet infection,

or less frequently indirectly via contaminated objects

Laboratory

diagnosis of

diphtheria


include a nose, throat, nasopharyngeal, and wound swab.

Specimens are cultivated on cysteine-tellurite agar and

77

Loeffler’s coagulated serum. In addition, differential

diagnosis necessitates primary cultivation on blood and

chocolate agar.

Treatment of

diphtheria

Specific antitoxic serum must be administered

immediately. Inasmuch as the antitoxin is generated in

horses, a skin test to ensure the absence of hypersensitivity to

horse protein is essential prior to its use. Although antibiotics

have no effect upon the toxemic disease process, penicillin or

erythromycin is effective in killing organism and thus

preventing further toxin production

Prevention and

control of

diphtheria

1. Active immunization with formalin-inactivated

toxoid. Vaccination is highly effective. It is first

administered early in infancy along with tetanus toxoid and

killed Bordetella pertusis (DTaP) and must be followed by

periodic toxoid boosters throughout childhood and

adulthood.

2. The schick test

Bordetella

pertussis

B. pertussis organisms do not grow on universal laboratory

media. Bordet-Gengou medium contains potatoes, glycerol,

and sheep blood. It is traditionally the medium of choice.

These organisms are nonmotile and oxidize amino acids but

do not ferment carbohydrates. The other bordetella species

are less fastidious and can grow on blood and MacConkey

agars

Laboratory

diagnosis of

pertussis

Secretions collected on nasopharyngeal smears

consisting of calcium alginate or dacron on wire handles are

the best specimen. The smears must be inoculated

immediately onto media at the patient’s bedside or

78

transported in a moist, protective medium to the laboratory

for cultivation.

Direct fluorescent antibody staining of the organism is

the fastest diagnostic tool. Although method is only 60%

sensitive, it is very specific.

Treatment of

pertussis

Treatment with macrolide (erythromycin, azithromycin)

is effective in eradicating organisms and reduces the duration

of infectious stage. Treatment does not alleviate symptoms

Laboratory diagnostics of the tuberculosis and leprosy


Etiopathogenesis

of tuberculosis

Tuberculosis is an infectious disease and its causative

agent is Mycobacterium tuberculosis (MBT) of the genus

Mycobacterium of Mycobacteriaceae family

Morphology of

Mycobacterium

tuberculosis

MBT looks like a bacillus. MBT can exist in various

forms: typical rods, chips, L-forms and filtrating forms

Groups of

mycobacteria

3 groups of mycobacteria are distinguished: true

(pathogenic for a human being), atypical and acidic-stable

saprophytes. There are three types (species) of pathogenic

MBT: human (M. tuberculosis), bovine (M. bovis) and African

(M. africanum)

Sourse of

tuberculosis

infection

Sick people and animals, secreting MBT, are the source of

human tuberculosis infestation. A pathogene, depending on the

affected organ, is secreted into the environment with sputum,

excrements, urine, milk, sperm, etc. Infectation occurs most

often by aero-genic (90%), contact (5–6%), rarely alimentary

(1–2 %) and extremely rarely by intrauterine route

Matherial for The materials for MBT revealing are sputum, bronchial

79

microbiological

investigation of

tuberculosis

lavage waters, faeces, urine, fistula pus (matter), pleural cavity

exudate, spinal fluid, punctates and biopsy matherial of various

organs and tissues

Methods of

mycobacteria

investigation

Bacterioscopy (flotation method, luminescent microscopy).

Bacteriological method.

Biological method

Tuberculin skin test

Disposition

prophylaxis of

tuberculosis

An active vaccination reduces the risk of contracting the

disease by about one-half. It contains the live vaccine BCG

(lyophilized bovine TB of the Calmette-Guerin type).

Morphology and

culture properties

of

Mycobacterium

leprae

In morphological terms, these acid-fast rods are identical

to tuberculosis bacteria. They differ, however, in that they

cannot be grown on nutrient media or in cell cultures

Epidemiology of

leprosy

Person-to-person spread by direct contact or inhalation of

infectious aerosoles. People in close contact with patients who

have lepromatous disease are at greatest risk


Task 1. Study the preparation of the pure cultures of C. diphtheria


C. diphtheria



80

Task 1. Study the differentiative properties of corynebacteria. Write

down.

Type of diphtheria bacilli differentiation

Properties Gravis Intermedius Mitis

Morphology

Colony on

tellurite

blood agar

Consistency

of colonies

Haemolysis

Growth in

broth

Glycogen

fermentation


Differentiating features of bordetella

Feature B. pertussis B. parapertussis

Motiliti

Appearance on

MacConkey agar

Appearance on Bordet-

Gengou medium (days)

Urease

Nitrate to nitrite

Citrate use

Oxidase

Antitoxic

antidiphtheria serum

Colonies of

toxigenic

Corynebacteria

diphtheriae

81

Heat labile toxin and

tracheal cytotoxin

Adenylate cyclase toxin

Pertussis toxin

Task 3. Study the antimicrobial agents for treatment of tuberculosis.

Write the main data to the protocol.

1.____________________________________________________________

2._____________________________________________________________

3.____________________________________________________________

Conclusion.

82

Questions for the final lesson.

1. Value of medical microbiology in practical activities of the doctor.

2. Structure and a chemical compound of a bacterial cell. Peculiarities of a

structure of Gram-positive and Gram- negative bacteria.

3. Tinctorial properties of bacteria. Methods of coloring.

4. Structure and a chemical compound of a bacterial cell. Capsules, flagella,

sporulation.

5. Peculiarities of biology of viruses.

6. Structure and a chemical compound of viruses and bacteriophages.

7. Types of virus and cell interaction. Stages of multiplication of viruses.

8. Bacteriophagia. Interaction of a phage with a bacterial cell. Temperate and

virulence bacteriophages. Lysogeny.

9. Application of phages in medicine and biotechnologies.

10. Artificial nutrient mediums, their classification. Requirements. Showed to

nutrient mediums.

11. Microbial growth and multiplication of bacteria.

12. Methods for cultivation of anaerobes.

13. Principles and methods of isolation of pure cultures of bacteria.

14. Enzymes of bacteria, their value in identification of bacteria.

15. Methods of cultivation of viruses.

16. Normal microbial flora of human body. Dysbioses. Probiotics.

17. Microflora of air and methods of its research.

18. Methods of sanitary - bacteriological research of water: definition of

microbic number.

19. Concept of disinfection. Methods. Disinfectants.

20. Concept of sterilizations, methods, the equipment.

21. Concept of chemotherapy and antibiotics. The mechanism of action of

antibiotics.

83

22. Antibiotics, classification bayed on a source of reception, a way of

reception.

23. Antibiotics. Sources and ways of reception. Classes of antibiotics.

24. Classification of antibiotics on chemical structure, the mechanism and a

spectrum of activity.

25. Methods of antibiotic sensitivity test.

26. A structure of bacterial genome. Concept of a genotype and a phenotype.

Kinds of variability.

27. Plasmids, their functions and properties. Use plasmids in genetic

engineering.

28. The mechanism of transfer of a genetic information.

29. Concept of an infection. Pathogenicity and virulence of bacteria. Factors of

pathogenicity.

30. Toxins of bacteria, their nature, properties, reception.

31. Concept of infectious disease. Stages of development and characteristic

properties.

32. Concept of clinical microbiology. A role of opportunictic microorganisms in

a pathology of human.

33. Methods of microbiological diagnostics of infectious diseases.

34. Concept of immunity. Classification of immunity.

35. Antibodies, their characteristics.

36. Meningococci. Taxonomy. The characteristic of biological properties.

Pathogenesis. Forms of an infection. Microbiological diagnostics. Principles

of specific prophylaxis and treatment.

37. Causative agents of a gonorrhoeae. Taxonomy. Microbiological diagnostics.

Specific treatment.

38. Staphylococci. Taxonomy. The characteristic of biological properties.

Microbiological diagnostics of the diseases caused by staphylococci.

Principles of specific prophylaxis and treatment.

84

39. Streptococci. Taxonomy. The characteristic. Microbiological diagnostics of

streptococcal infections. Principles of specific prophylaxis and treatment. A

role of streptococci at a scarlet fever.

40. Causative agents of tuberculosis. Taxonomy. The characteristic.

Microbiological diagnostics of a tuberculosis. Principles of specific

prophylaxis at children.

41. Causative agents of tularemia. The characteristic. Microbiological

diagnostics. Principles of specific prophylaxis and treatment.

42. Causative agents of anthrax. The characteristic. Microbiological diagnostics.

Principles of specific prophylaxis and treatment.

43. Causative agents of brucellosis. Taxonomy. The characteristic.

Microbiological diagnostics. Principles of specific prophylaxis and

treatment.

44. Causative agents of a plague. Taxonomy. The characteristic.


treatment.

45. Causative agents of wooping cough. Taxonomy. The characteristic.


treatment.

46. A problem of the hospital infections.

85

List of literature

1. Brooks G.F., Carroll K.C. & others. Jawetz, Melnick, Adelberg's Medical

microbiology-McGraw-Hill Education Medical .- 2015. - 864 р.

2. Chamberlain N. The Big Picture. Medical Microbiology -The McGraw-Hill

Companies.- 2009. - 456 p.

3. Hart C. Anthony, Shears Paul. Color Atlas of Medical Microbiology- Elsevier-

2004. - 698 p.

4. Murray P.R., Rosenthal K.S., Pfaller M.A. Medical Microbiology-

Saunders/Elsevier. - 2013. -1023 p.

5. Ryan, Kenneth, Sherris. Medical Microbiology - McGraw-Hill Medical.- 2010.-

1040 p.

6. Tortora G.J., Funke B.R., Case C.L. Microbiology. An Introduction - Pearson

Education.- 2012. - 960 p.

Documents

Microbiology, virology: guidelines for practical classes