SIGNIFICANCE OF MICROBIOLOGY IN NURSES’ PRACTICE. THE HISTORY OF

MICROBIOLOGY. CLASSIFICATION AND

STRUCTURE OF MICROORGANISMS.

Chair of Medical Biology, Microbiology, Virology, and Immunology

Lecturer Prof. S.I. Klymnyuk

Lecture schedule

1. History of Microbiology.

2. Classification of bacteria.

3. Structure of bacterial cell

Microbiology is a science, which study most shallow living creatures - microorganisms. They were frequently named by microbes. Before inventing of microscope humanity was in dark about their existence. But during the centuries people could make use of processes vital activity of microbes for its needs. They could prepare a koumiss, alcohol, wine, vinegar, bread, and other products. During many centuries the nature of fermentations remained incomprehensible.

Microbiology learns morphology, physiology, genetics and microorganisms systematization, their ecology and the other life forms. The taxonomy of microbes is very variable. They include prions, viruses, bacteria, water-plants, fungi, protozoon and even some multicellular organisms.

The Microorganisms are extraordinarily widely spread in nature. They literally ubiquitous forward us from birth to our death. Daily, hourly we eat up thousands and thousands of microbes together with air, water, food. On our skin, in mouth and nasal cavities, on mucous membranes and in bowels enormous amount of microorganisms live and act. Many of them are found in earth cortex and in the air, and in the ocean’s, sea’s, river’s water, on all of latitudes, mainlands and continents.

Some microbes bring enormous use to humanity. They are our good friends.

But among enormous amount of microorganisms, which populate our planet, there are such, which bring death to people, animals and plants. For them, our enemies, does not exist geographic and state boundaries. Disease which they cause, spread with striking speed. Epidemics of flue and plague, cholera and smallpox, measles and diphtheria affected sometimes whole mainlands of our planet.

Only during one plague epidemic in 14 century in Europe was killed 25 mln. people and 35 mln. in Аsia. The countries Europe needed 100 years, to bring population level down to outgoing level.

For the first time term “microbe" was offered by French scientist Sh.Sedillot in 1878. It derives from Greek “microbe", that means briefly living, or most shallow living creature. Science, which learns the microorganisms, was named by E.Duclaux microbiology. For short development period this science accumulated great factual material. The separate microbiological branches such as bacteriology, mycology, protistology, virology quickly appeared.

Periods of microbiology development

• Morphologic

• Physiologic

• Prophylactic

Development of microbiological science was interlinked with art of glass and diamonds grinding. This brought to creation of the first microscope by Hans and Zacharian Jansen in Holland in 1590.

The discovery of microorganisms is associated with the name of Antony van Leeuwenhoek (1632-1723).

In 1683 Leeuwenhoek described the basic bacterium forms. His scientific supervisions Leeuwenhoek described in special letters and sent off them to the London Royal Scientific Society. He sent away about 300 letters. The Leeuwenhoek’s letters brought on enormous surprise among English scientists. They opened a fantastic world of invisible creatures. He named them “living animals" (animalcula viva) and in one of letter wrote: “In my mouth there are more, than peoples in all English kingdom".

These wonderful discovery of Dutch naturalist were the embryo, with which science of bacteria developed. Namely from these times starts the so-called morphological period in microbiology history (XVII middle of age). It is also called micrographycal period, as the study of microorganism came only to description of their dimensions and forms. Biological properties and their significances for man still a long time remained incomprehensible.

However, using the primitive microscopes of that time it was difficult to determine the difference between separate bacteria species. Even celebrated founder of scientific systematization of all of living organisms Karl Linney renounced to classify the bacteria. He gave them general name “chaos".

The important attempt to make microbes systematization belongs to Danish naturalist O. Muller. In 1786 year he described 379 infusorians and some species of bacteria. Ph. Ehrenberg. Was the first who offered the terms “bacterium ", “spirillum", “spirochaeta ", “vibrio". The great contribution in microorganisms systematization was made by one of founders of Ukrainian microbiology L. Zemkovsky (1822-1887). He described 43 new species of microbes, got original vaccine against anthrax.

In the second half of XIX century microbiology strongly affirms as independent science. Namely these sciences were fruitful soil, onwhich Pasteur's talent evinced.

He studied wine "illness“, fermentation, made Pasteurization method, offered to grow microbes on artificial nutrient media, he proved, that on definite cultivation conditions the pathogenic bacteria lose its virulence, made vaccine against anthrax, rabies.

Not less important are scientific works of celebrated German scientist R. Koch.

He performed classic researches on etiology of anthrax, opened tuberculosis bacilli, cholera vibrio, proposed to isolate pure bacterial cultures on solid nutrient media (gelatin, potatoes), developed the preparations staining methods by aniline dye-stuffs, method of hanging drop for examination of bacteria motility, offered apparatus for sterilization

The Patriarch of world and Ukrainian microbiology - I. Metchnikov

He studied inflammation pathology, phagocytosis, bases about antagonism of bacteria.

From all microbes-antagonists I.Metchnikov preferred the lactic bacteria. On their base he offered three medical preparations -sour clotted milk, yogurt and lactobacillin.

Now they are called by eubiotics

Classic Metchnikov's researches defined a prophylactic period in microbiology history.

In 1892 D. Ivanovskiy described an virus of mosaic tobacco illness – new class of infectious agents

Microorganisms constitute a very antique group of living organisms which appeared on the Earth's surface almost 3000 million years ago.

There are natural and artificial classifications system.

Bergey's Manual of Determinative Bacteriology - the "bible" of bacterial taxonomy.

There are such levels of microorganisms’ organization: Species – Genus – Family – Class – Division – Kingdom

Procaryotae Kingdom has 4 Divisions according to the structure of cell wall and Gram staining:

Gracilicutes (gracilis - thin, cutis - skin) – Gram-negative bacteria,

Firmicutes (firmus - firm) – Gram-positive bacteria,

Tenericutes (tener – soft, tender) – microbes without cell wall,

Mendosicutes (mendosus - mistaket) – microbes wuth atipical peptidoglican

35 of the major groups of bacteria are distinguished primarily on morphological characteristics, namely: cell shapes (rods, cocci, curved, or filament forming); spore production; staining reactions; motility

Other groups are defined based on their metabolism, on combinations of morphological and physiological characteristics.

Some of the Major Groups of Bacteria in Bergey's Manual

Spirochetes

Very slender rods that are helically coiled around a central axial filament; includes the bacteria that cause syphilis and Lyme disease

Gram-positive cocci

Bacteria that have a cell wall structure that results in their staining blue-purple by the Gram stain procedure and that are spherical; include the

streptococci and staphylo cocci Endospore-forming rods

and cocci Bacteria that form heat-resistant bodies called endospores within their cells; include the bacteria that cause gas gangrene, botulism, tetanus, and

anthrax

There are such levels of microorganisms’ organization: Species – Genus – Family – Class – Division – Kingdom

Species is population of microbes, which have the only source of origin, common genotype, and during the present stage of evolution are characterized by similar morphological, biochemical, physiological and other signs

If deviations from the typical species properties are found on examination of the isolated bacteria, then culture is considered a subspecies.

Infrasubspecies subdivisions

serovar (antigenic properties)

morphovar (morphological properties)

chemovar (chemical properties)

biovar (biochemical or physiological properties)

pathovar (pathogenic properties)

phagovar (relation to phages)

The term clone was applied to designate a group of individuals arising from one cell

Population is an elementary evolutional unit (structure) of a definite species

The term strain designates a microbial culture obtained from the different sources or from one source but in different time

Morphological Classification of Bacteria

Bacteria (Gk. bakterion - small staff) are unicellular organisms lacking chlorophyll.

Morphologically, bacteria possess four main forms:

spherical (cocci)

rod-shaped (bacteria, bacilli, and clostridia)

spiral-shaped (vibrios, spirilla and spirochaetes)

thread-shaped (non-pathogenic)

Cocci (Gk. kokkos berry). These forms of bacteria are spherical, ellipsoidal, bean-shaped, and lanceolate. Cocci are subdivided into six groups according to cell arrangement, cell division and biological properties

Micrococci (Micrococcus). The cells are arranged singly or irregularly. They are saprophytes, and live in water and in air ( M. roseus, M. luteus, etc.).

Diplococci (Gk. diplos double) divide in one plane and remain attached in pairs. These include: Meningococcus (causative agent of epidemic cerebrospinal meningitis, and gonococcus, causative agent of gonorrhoea and blennorrhoea) Pneumococcus (causative agents of pneumonia)

Streptococci (Gk. streptos curved, kokkos berry) divide in one plane and are arranged in chains of different length. Some streptococci are pathogenic for humans and are responsible for various diseases.

Tetracocci (Gk. tetra four) divide in two planes at right angles to one another and form groups of fours. They very rarely produce diseases in humans.

Staphylococci (Gk. staphyle cluster of grapes) divide in several planes resulting in irregular bunches of cells, sometimes resembling clusters of grapes. Some species of Staphylococci cause diseases in man and animals

Sarcinae (L. sarcio to tie) divide in three planes at right angles to one another and resemble packets of 8, 16 or more cells. They are frequently found in the air. Virulent species have not been encountered

Rods. Rod-shaped or cylindrical forms are subdivided into bacteria, bacilli, and clostridia. Bacteria include those microorganisms which, as a rule, do not produce spores (colibacillus, and organisms responsible for enteric fever, paratyphoids, dysentery, diphtheria, tubercu losis, etc.). Bacilli and clostridia include organisms the majority of which produce spores (hay bacillus, bacilli responsible for anthrax, tetanus, anaerobic infections, etc.)

According to their arrangement, cylindrical forms can be subdivided into three groups: monobacteria monobacilli

E. coli Y. pestis

C. tetani

C. botulinum

diplobacteria diplobacilli

K. pneumoniae

streptobacteria streptobacilli

Haemophilus ducreyi

(chancroid)

Bacillus anthracis

(anthrax)

Spiral-shaped bacteria

Vibriones (L. vibrio to vibrate) are cells which resemble a comma in appearance. Typical representatives of this group are Vibrio cholerae, the causative agent of cholera, and aquatic vibriones which are widely distributed in fresh water reservoirs.

Spirilla (L. spira coil) are coiled forms of bacteria exhibiting twists with one or more turns. Only one pathogenic species is known {Spirillum minus} which is responsible for a disease in humans transmitted through the bite of rats and other rodents (rat-bite fever, sodoku)

Spirochaetes (L. spira curve, Gk. chaite cock, mane) differ from bacteria in structure with a corkscrew spiral shape

Borrelia. Their cells have large, obtuse-angled, irregular spirals, the number of which varies from 3 to 10. Pathogenic for man are the causative agents of relapsing fever transmitted by lice (Borrelia hispanica), and by ticks (Borrelia persica, etc.). These stain blue-violet with the Romanowsky-Giemsa stain

Leptospira (Gk. leplos thin, speira coil) are characterized by very thin cell structure. The leptospirae form 12 to 18 coils wound close to each other, shaping small primary spirals. The organisms have two paired axial filaments attached at opposite ends (basal bodies) of the cell and directed toward each other.

Leptospira interrogans which is pathogenic for animals and man cause leptospirosis

Treponema (Gk. trepein turn, nema thread) exhibits thin, flexible cells with 6-14 twists. The micro-organisms do not appear to have a visible axial filament or an axial crest when viewed under the microscope

A typical representative is the causative agent of syphilis Treponema pallidum

Properties of prokaryotes and eukaryotes Prokaryotes Eukaryotes

The nucleoid has no membrane separating it from the cytoplasm

Karyoplasm is separated from the cytoplasm by membrane

Chromosome is a one ball of double twisted DNA threads. Mitosis is absent

Chromosome is more than one, There is a mitosis

DNA of cytoplasm are represented in plasmids

DNA of cytoplasm are represented in organelles

There aren’t cytoplasmic organelle which is surrounded by membrane

There are cytoplasmic organelle which is surrounded by membrane

The respiratory system is localized in cytoplasmic membrane

The respiratory system is localized mitochondrion

There are ribosome 70S in cytoplasm

There are ribosome 80S in cytoplasm

Peptidoglycan are included in cell’s wall (Murein)

Peptidoglycan aren’t included in cell’s wall

The structure of procaryotes

Nucleus. The prokaryotic nucleus can be seen with the light microscope in stained material. It is Feulgen-positive, indicating the presence of DNA. Histonelike proteins have recently been discovered in bacteria and presumably play a role similar to that of histones in eukaryotic chromatin

The DNA is seen to be a single, continuous, "giant" circular molecule with a molecular weight of approximately 3 X 109. The unfolded nuclear DNA would be about 1-3 mm long (compared with an average length of 1 to 2 µm for bacterial cells)

Plasmids are small circular DNA molecules that can be thought of as carrying extra genes that can be used for special situations. There may be several different plasmids in one cell and the numbers of each may vary from only one to 100s in a cell.

Plasmids: R, Col, Hly, Ent, Sal

The cytoplasmic constituents of prokaryotic cells invariably include the prokaryotic chromosome and ribosomes.

The ribosomes of prokaryotes are smaller than cytoplasmic ribosomes of eukaryotes. Prokaryotic ribosomes are 70S in size, being composed of 30S and 50S subunits. The 80S ribosomes of eukaryotes are made up of 40S and 60S subunits. Ribosomes are involved in the process of translation (protein synthesis), but some details of their activities differ in eukaryotes, Bacteria and Archaea. Protein synthesis using 70S ribosomes occurs in eukaryotic mitochondria and chloroplasts, and this is taken as a major line of evidence that these organelles are descended from prokaryotes

Some inclusions in bacterial cellsCytoplasmic inclusions

Where found Composition

Glycogen Many bacteria e.g. E. coli

Polyglucose

Polybetahydroxyutyric acid (PHB)

Many bacteria e.g. Pseudomonas

Ppolymerized hydroxy butyrate

Polyphosphate (volutin granules)

Many bacteria e.g. Corynebacterium diphtherieae

Linear or cyclical polymers of PO4

Sulfur globules Phototrophic purple and green sulfur bacteria and lithotrophic colorless sulfur bacteria

Elemental sulfur

Volutin granules

Loeffler's technique Neisser's staining

The cell envelope is a descriptive term for the three layers of material that envelope or enclose the protoplasm of the cell. The cell protoplasm (cytoplasm) is surrounded by the plasma membrane, a cell wall and a capsule

Bacterial plasma membrane are composed of 40 percent phospholipid and 60 percent protein. The phospholipids are amphoteric molecules with a polar hydrophilic glycerol "head" attached via an ester bond to two nonpolar hydrophobic fatty acid tails, which naturally form a bilayer in aqueous environments. Dispersed within the bilayer are various structural and enzymatic proteins which carry out most membrane functions.

Mesosome

The predominant functions of bacterial membranes are:

1. Osmotic or permeability barrier;

2. Location of transport systems for specific solutes (nutrients and ions);

3. Energy generating functions, involving respiratory and photosynthetic electron transport systems, establishment of proton motive force, and transmembranous, ATP-synthesizing ATPase;

4. Synthesis of membrane lipids (including lipopolysaccharide in Gram-negative cells);

5. Synthesis of murein (cell wall peptidoglycan);

6. Assembly and secretion of extracytoplasmic proteins;

7. Coordination of DNA replication and segregation with septum formation and cell division;

8. Chemotaxis (both motility per se and sensing functions);

9. Location of specialized enzyme system.

Cell wallMost prokaryotes have a rigid cell wall. The cell wall is an essential structure that protects the cell protoplast from mechanical damage and from osmotic rupture or lysis.

The osmotic pressure against the inside of the plasma membrane may be the equivalent of 10-25 atm.

The cell walls of all Bacteria contain a unique type of peptidoglycan called murein. Peptidoglycan is a polymer of disaccharides (a glycan) cross-linked by short chains of amino acids (peptides), and many types of peptidoglycan exist. All Bacterial peptidoglycans contain N-acetylmuramic acid and N-acetylglucosamine, connected by a beta1,4-glycoside bond. Then there is connections with peptide side chain that contains L-alanine, (L-ala), D-glutamate (D-glu), Diaminopimelic acid (DAP), and D-alanine (D-ala).

Structure of peptidoglycan

Differences between Gram-positive and Gram negative cells

Capsules Most prokaryotes contain some sort of a polysaccharide layer outside of the cell wall polymer

Only capsule of B. anthracis consist of polypeptide (polyglutamic acid)

Functions of capsules

Like fimbriae, capsules, slime layers, and glycocalyx often mediate adherence of cells to surfaces. Capsules also protect bacterial cells from engulfment by predatory protozoa or white blood cells (phagocytes), or from attack by antimicrobial agents of plant or animal origin. Capsules in certain soil bacteria protect them from perennial effects of drying or desiccation.

They provide virulent properties of bacteria (S. pneumoniae, B. anthracis)

Flagella

Flagella are filamentous protein structures attached to the cell surface that provide the swimming movement for most motile prokaryotes. Prokaryotic flagella are much thinner than eukaryotic flagella, and they lack the typical 9 + 2 arrangement of microtubules. Their diameter is about 20 nanometers, well-below the resolving power of the light microscope. The flagellar filament is rotated by a motor apparatus in the plasma membrane allowing the cell to swim in fluid environments. Bacterial flagella are powered by proton motive force (chemiosmotic potential) established on the bacterial membrane, rather than ATP hydrolysis which powers eukaryotic flagella

The ultrastructure of a bacterial flagellum

Outer membrane

Peptidoglуcan layer

Periplasmic space

Plasma membrane

According to a pattern in the attachment of flagella motile microbes can be divided into 4 groups: (1) monotrichates, bacteria having a single flagellum at one pole of the cell (cholera vibrio, blue pus bacillus), (2) amphitrichates, bacteria with two polar flagella or with a tuft of flagella at both poles (Spirillum volutans), (3) lophotrichates, bacteria with a tuft of flagella at one pole (blue-green milk bacillus,Alcaligenes faecalis), (4) peritrichates, bacteria having flagella distributed over the whole surface of their bodies (colibacillum, salmonellae of enteric fever and paratyphoids A and B)

Flagella

The flagella of Proteus vulgaris demonstrated by

electron microscopy

Fimbriae and Pili are interchangeable terms used to designate short, hair-like structures on the surfaces of prokaryotic cells

Like flagella, they are composed of protein. Fimbriae are shorter and stiffer than flagella, and slightly smaller in diameter. Generally, fimbriae have nothing to do with bacterial movement

Fimbriae are most often involved in adherence of bacteria to surfaces, substrates and other cells in nature

Common pili (almost always called fimbriae) are usually involved in specific adherence (attachment) of prokaryotes to surfaces in nature. In medical situations, they are major determinants of bacterial virulence because they allow pathogens to attach to (colonize) tissues and/or to resist attack by phagocytic white blood cells.

Pili of the second class (sex-pili) provide conjugation process

Endospores. Endospores are highly heat-resistant, dehydrated resting cells formed intracellularly in members of the genera Bacillus and Clostridium. Endospores are small spherical or oval bodies formed within the cell. A spore is formed at a certain stage in the development of some microorganisms and this property was inherited in the process of evolution in the struggle for keeping the species intact.

Spore

The sporulation process begins when nutritional conditions become unfavorable, depletion of the nitrogen or carbon source (or both) being the most significant factor. Sporulation involves the production of many new structures, enzymes, and metabolites along with the disappearance of many vegetative cell components.

Spore formation

Properties of Endospores

1. Core. The core is the spore protoplast. It contains a complete nucleoid, all of the components of the protein-synthesizing apparatus, and an energy-generating system based on glycolysis. A number of vegetative cell enzymes are increased in amount (eg, alanine racemase), and a number of unique enzymes are formed (eg, dipicolinic acid synthetase). The energy for germination is stored as 3-phosphoglycerate rather than as ATP.

2. Spore wall. The innermost layer surrounding the inner spore membrane is called the spore wall. It contains normal peptidoglycan and becomes the cell wall of the germinating vegetative cell.

3. Cortex. The cortex is the thickest layer of the spore envelope. It contains an unusual type of peptidoglycan, with many fewer cross-links. Cortex peptidoglycan is extremely sensitive to lysozyme, and its autolysis plays a key role in spore germination.

4. Coat. The coat is composed of a keratinlike protein containing many intramolecular disulfide bonds. The impermeability of this layer confers on spores their relative resistance to antibacterial chemi cal agents.

5. Exosporium. The exosporium is a lipoprotein membrane containing some carbohydrate.

Germination

The germination process occurs in 3 stages:

1. Activation. Even when placed in an environment that favors germination (eg, a nutritionally rich medium), bacterial spores will not germinate unless first activated by one or another agent that damages the spore coat. Among the agents that can overcome spore dormancy are heat, abrasion, acidity, and compounds containing free sulfhydryl groups.

Germination

2. Initiation. Once activated, a spore will initiate germination if the environmental conditions are favorable. Different species have evolved receptors that recognize different effectors as signalling a rich medium: thus, initiation is triggered by L-alanine in one species and by adenosine in another. Binding of the effector activates an autolysin that rapidly degrades the cortex peptidoglycan. Water is taken up, calcium dipicolinate is released, and a variety of spore con stituents are degraded by hydrolytic enzymes.

Germination

3. Outgrowth. Degradation of the cortex and outer layers results in the emergence of a new vegetative cell consisting of the spore protoplast with its surrounding wall. A period of active biosynthesis follows; this period, which terminates in cell division, is called outgrowth. Outgrowth requires a supply of all nutrients essential for cell growth.

In bacilli and clostridia, spores are located (1) centrally, in the centre of the cell (causative agent of anthrax); (2) terminally, at the ends of the rod (causative agent of tetanus); (3) subterminally, towards the ends (causative agents of botulism, anaerobic infections, etc.)

The spores of certain bacilli are capable of withstanding boiling and high concentrations of disinfectants. They are killed in an autoclave exposed to saturated steam, at a temperature of 115-125 C, and also at a temperature of 150-170 C in a Pasteur hot-air oven.