contaminated to less contaminated areas in the following order: rear of the hand, palm side, interdigital spaces and nail beds. Firstly, leaf hand should be cleaned, followed by the right one.

• In case of contamination of hands with potentially pathogenic microorganism or infectious clinical specimen, the area should be covered with cotton wad wetted in disinfected solution, followed by washing with soap.

All microorganisms are divided into 4 groups based on their pathogenic potential:

1. group (the most dangerous) — e.g. Yersinia pestis

2. group — Vibrio cholerae, Bacillus anthracis, Coxiella burnetii, Blastomyces dermatitidis, HIV, hepatitis B, C viruses and others

3. group — Bordetella pertussis, Clostridium botulinum, Corynebacterium diphtheriae, poliovirus, etc.

4. group — Streptococcus pneumoniae, Clostridium perfringens, Candida spp.,

etc.

Any work with microorganisms belonging to the I and II groups is allowed only in designated laboratories built and equipped in connection with special instructions of Ministry of Health of Russian Federation (e.g. SanPiN). Activities with microorganisms belonging to III and IV groups are permitted in laboratories built according with corresponding requirements of Ministry of Health of Russian Federation.

1. Bacterial morphology and modern methods

of microscopic examinations

Bacteria are unicellular prokaryotic microorganisms. There are three common shapes of bacteria: the coccus, the bacillus, and the spiral.

1. Coccus. A coccus-shaped bacterium is usually spherical, although some appear oval, elongated, or flattened on one side. Cocci are approximately 0. 5 micrometer (pm) in diameter and may be seen, based on their planes of division and tendency to remain attached after replication, in one of the following arrangements.

• Division in one plane produces either a diplococcus and or streptococcus arrangement.

• Division in two planes produces a tetrad arrangement a tetrad: square of 4 cocci

• Division in three planes produces a sarcina arrangement

• Division in random planes produces a staphylococcus arrangement

As you observe different bacteria, keep in mind that the procedures used

in slide preparation may cause some arrangements to break apart or clump together. The correct form, however, should predominate. Also remember that each coccus in an arrangement represents a complete, single, one-celled organism.

2. Bacillus (rod). A bacillus or rod is a hotdog-shaped bacterium having one of the following arrangements:

• bacillus: a single bacillus;

• streptobacillus: bacilli in chains;

• coccobacillus: oval and similar to a coccus.

A single bacillus is typically 0. 5-1. 0 μm wide and from 1- 4 μm long. Small bacilli or bacilli that have just divided by binary fission may at first glance be confused for cocci so they must be observed carefully. You may, however, be able to see bacilli that have not divided and are definitely rod-shaped as well as bacilli in the process of dividing.

2. Spiral. Spiral-shaped bacteria occur in one of three forms.

• vibrio: incomplete spiral or comma-shaped;

• spirillum: a thick, rigid spiral;

• spirochete: a thin, flexible spiral.

In addition to the above-mentioned types, there are bacteria that do not have cell wall — Mycoplasma spp. Thus you can observe different forms: spherical, ellipse-, pear-, disk-shaped, etc.

The main purposes of microscopy are as follows:

1. Detection of microorganisms in different materials.

2. Presumptive identification of microorganisms.

3. Study of some morphological features and structures of microorganisms (e.g. capsules).

4. Study of smears from pure cultures.

During the studies of microorganisms, different models of microscopes are used. Microscope is characterized by two main properties: magnification and resolution.

In general, magnification of microscope is equal to multiplication of magnification of objective to magnification of eyepiece.

Resolution is a property that allows to distinguish details of preparation and/ or to distinguish two dots lying close to each other. It depends only on resolution of objective and condenser and does not depend on magnification of eyepiece.

The highest resolution of light microscope is achieved when working with immersion objective. It is called immersion because requires usage of immersion oil which is placed between object glass and objective.

Aphase-contrast microscope illuminates a specimen wit ordinary light. Light is diffracted differently by a microbe than by its surrounding liquid, and the phase-contrast microscope collects only certain diffracted wavelengths. The result is that the microscope not only magnifies the microbes, but also makes their edges and features clearer.

A fluorescent microscope is used to view organisms containing molecules that can be activated by one wavelength of light to emit light of a different wavelength. This type of microscope displays those microbes that contain fluorescent compounds as bright forms against a dark background. In the case

9

of samples that contain a lot of debris, microbes can be visualized more clearly and distinguished from the background by staining them with dyes that intercalate with DNA and fluoresce only under this condition.

Tindale’ effect provides a basis for dark-field microscopy. Rays which enlighten the object does not go to objective of microscope, so field remains dark, but object on its background (because of reflection of rays) seems to be luminescent. Dark-field effect can be created using a special condenser or regular condenser with central part closed by dark paper.

For electron microscopy it is required to prepare smear on colloidal film (because electrons does not go via glass). Then preparations are stained with salts of heavy metals or covered by molecules of heavy metals. In addition, high vacuum needed to be created to allow passage of bunch of electrons. In addition, instead of glass lenses, special electromagnetic ones are used. For the time being, modern electron microscopes have resolution 5-3 angstrom and magnification of 100, 000 and more.

There are other different applications of electron microscopy using, for example, freezing-thawing method at -196°C and others.

Apart from above-mentioned, there are some other visualization methods available as follows:

1. Computer interference microscopy which allows to obtain highly contrasted view for detection of subcell structures;

2. Laser confocal microscopy allowing to detect different objects in focus in all areas;

3. Roentgen microscopy;

4. Positron emission tomography.

In microbiology for study of bacterial morphology stained fixed smears or non-stained, native smears prepared either from clinical specimens or from grown microorganisms.

Preparation of smear

The fust consideration is the correct preparation of the smear.

• On degreased microscope slide, put 1 drop of water.

• Flame the bacteriological loop until it becomes red, open the tube with a little finger of right hand and harvest bacteria from the surface of the media with a cooled-down loop and then smear the bacteria in a drop of water on a slide.

• For preparation of smear by imprint method, cut a piece of agar with grown bacterial colony, place on cover glass (bacteria down!), firmly pressing towards slide and immediately heat fix the slide.

• Allow the smear to air dry and then briefly heat fix the slide by passing it 2-4 times through a flame (the slide should not become too hot to touch). Failure to follow these directions may cause staining artifacts and disrupt the normal morphology of bacteria and cells!

10

Fixation is needed due to the following reasons:

1. To inactivate microorganisms;

2. To fix them on the surface of slide and avoid washing away;

3. To improve susceptibility of bacteria to staining.

Staining of smears is performed using dyes, which could be divided into positive (e.g. methylene blue, fuchsine), and negative (e.g. nigrosine). Positive dyes are those staining microorganisms and other objects on the slide; negative - filling space, surrounding microorganisms that results them to become visible.

Also they can be divided into acid (e.g. eosin, Congo red), which stain alkaline structures (e.g. cytoplasmic proteins) and alkaline (e.g. — with acid (basophilic) structures (e.g. nucleic acids, ribosomes).

Capability of cells to accept different dyes reflects their tinctorial features.

There are simple and complex staining techniques. Simple staining technique is the staining of smears using one dye. In majority of cases, methylene blue, crystal violet or fuchsine is used.

To make a simple stained smear, fixed smear is placed onto two glass sticks placed in parallel over the tray. Smear is covered with 1% aqueous solution of methylene blue or fuchsine for 1-2 min. (do not allow dye to be dried on the slide). Upon the completion of staining, smear is rinsed with water until the water runs clear. Then, using filter paper, dry off the slide, followed by placing immersion oil and microscopy with immersion system.

Demonstrations

1. Acquaintance with rules of work in the microbiological laboratory.

2. Modem methods of work with immersion objectives.

3. Organization of phase-contrast, electron and fluorescent microscopes.

4. Dark-field microscopy.

Students' activities

1. Every student performs microscopy with dry and immersion objectives ready-to-use smear of yeasts, anthracoids, staphylococci and staphylococci in the blood.

2. Every student prepares 3 smears using sarcinas, staphylococci, anthracoids, E. coli, Vibrio spp., stains them with methylene blue and Pfeiffer fuchsine, performs microscopy with immersion objective and makes a drawing in the workbook.

3. To design a scheme of bacteriological laboratory and substantiate connection between different rooms.

4. To learn and write down to the workbook methods of disinfection of contaminated materials and environmental objects and hands of laboratory personnel contaminated by potentially infectious materials.

11

Control questions

1. Describe main rules of work in the microbiological laboratory.

2. How to determine magnification of microscope?

3. What is the resolution of microscope and on which factors does it depend?

4. What are the advantages of immersion objective?

5. What are the main rules of work with immersion objective?

6. What are the two main systems of electron microscope?

7. What are the main differences between electron and light microscope?

8. How specimens are prepared for electron microscopy?

9. Describe the main principles of work of phase-contrast microscope.

10. Describe the main principles of work of fluorescent microscope.

11. Describe the main principles of dark-field microscopy.

12. Name the main forms of cocci, bacilli and spiral bacteria,

13. What stages does the preparation of smear include?

14. What purpose the fixation is used for and how is it performed?

15. What are the tinctorial features of bacteria?

16. Name the main dyes used for staining smears.

17. What is the purpose of studying of tinctorial features of bacteria?

18. What is the simple staining method?

12

PRACTICAL SESSION No. 2

Structure of bacterial cell. Complex staining techniques.

Plan of the session

1. Composition of bacterial cell.

2. Complex staining techniques. Gram stain.

3. Motility of bacteria. Study of live microorganisms.

Foreword notes

The life arose on Earth approximately 4 billion years ago. The simplest of cells, and the first types of cells to evolve are prokaryotic cells, organisms that lack a nuclear membrane. For approximately 2 billion years, prokaryotic-type cells were the only form of life on Earth. The oldest known sedimentary rocks, from Greenland, are about 3. 8 billion years old. The oldest known fossils are prokaryotic cells, 3. 5 billion years in age, found in Western Australia and South Africa. The nature of these fossils, and the chemical composition of the rocks in which they are found, indicate that lithotrophic and fermentative modes of metabolism were the first to evolve in early prokaiyotes, and that photosynthesis developed in prokaryotes at least 3 billion years ago. Anoxygenic (non O₂-producing) photosynthesis preceded oxygenic (O₂ -producing) photosynthesis. The larger, more complicated eukaryotic cells appeared much later, between 1. 5 and 2 billion years ago.

Figure 1. Schematic drawing of a typical prokaryotic cell

13

Procaryotic structure and function

Prokaryotes are distinguished from eukaryotes on the basis of nuclear organization, specifically their lack of a nuclear membrane.

Prokaryotes also lack any of the intracellular organelles and structures, which are characteristic of eukaryotic cells. Most of the functions of organelles such as mitochondria, chloroplasts and the Golgi apparatus are taken over by the prokaryotic plasma membrane. Prokaryotic cells have three architectural regions (Fig. 1): appendages (proteins attached to the cell surface) in the form of flagella and pili; a cell envelope consisting of a capsule, cell wall and plasma membrane; and a cytoplasmic region that contains the cell genome (DNA) and ribosomes and various sorts of inclusions.

Flagella are filamentous protein structures attached to the cell surface that provide swimming movement for most motile prokaryotic cells. 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. Prokaryotes are known to exhibit a variety of types of tactic behavior, i.e., the ability to move (swim) in response to environmental stimuli. For example, during chemotaxis a bacterium can sense the quality and quantity of certain chemicals in their environment and swim towards them (if they are useful nutrients) or away from them (if they are harmful substances).

Fimbriae and Pili are interchangeable terms used to designate short, hair- like structures on the surfaces of prokaryotic cells. Fimbriae are shorter and stiffer than flagella, and slightly smaller in diameter. Like flagella, they are composed of protein. A specialized type of pilus the F or sex pilus, mediates the transfer of DNA between mating bacteria, but the function of the smaller, more numerous common pili is quite different. Common pili (almost always called fimbriae) are usually involved in 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 to resist attack by phagocytic white blood cells.

Most prokaryotes have a rigid cell wall. The cell wall is an essential structure that protects the delicate cell protoplast from osmotic lysis. The cell wall of Bacteria consists of a polymer of disaccharides cross-linked by short chains of amino acids (peptides). This molecule is a type of peptidoglycan which is called murein. In the Gram-positive bacteria (those that retain the purple crystal violet dye when subjected to the Gram-staining procedure) the cell wall is a thick layer of murein. In the Gram-negative bacteria (which do not retain the crystal violet) the cell wall is relatively thin and is composed of a thin layer of murein surrounded by a membranous structure called the outer membrane. Murein is a

14

substance unique in nature to bacterial cell walls. Also, the outer membrane of Gram-negative bacteria invariably contains a unique component, lipopolysaccharide (LPS or endotoxin), which is toxic to animals. The cell walls of Archaea may be composed of protein, polysaccharides, or peptidoglycan- like molecules, but never do they contain murein. This feature distinguishes the Bacteria from the Archaea.

Although prokaryotes lack any intracellular organelles for respiration or photosynthesis, many species possess the physiologic ability to conduct these processes, usually as a function of the plasma membrane. For example, the electron transport system that couples aerobic respiration and ATP synthesis is found in the plasma membrane. The photosynthetic chromophores that harvest light energy for conversion into chemical energy are located in the membrane. Hence, the plasma membrane is the site of oxidative phosphorylation or photophosphorylation in prokaryotes, analogous to the functions of mitochondria and chloroplasts in eukaryotic cells. The prokaryotic plasma membrane is also a permeability barrier, and it contains a variety of different transport systems that selectively mediate the passage of substances into and out of the cell.

The membranes of Bacteria are structurally similar to the cell membranes of eukaryotes, except that bacterial membranes consist of saturated or monounsaturated fatty acids (never polyunsaturated fatty acids) and do not normally contain sterols. The membranes of Archaea form phospholipid bilayers functionally equivalent to bacterial membranes, but archaeal lipids are saturated, branched, repeating isoprenoid subunits that attach to glycerol via an ether linkage, as opposed to the ester linkage found in glycerides of eukaryotic and bacterial membrane lipids. The structure of archaeal membranes is thought to be an adaptation to their survival in extreme environments.

Most prokaryotes contain some sort of a polysaccharide layer outside of the cell wall or outer membrane. In a general sense, this layer is called a capsule or glycocalyx. Capsules, slime layers, and glycocalyx are known to mediate adherence of prokaryotes to particular surfaces. Capsules also protect bacteria from engulfment by predatory protozoa or white blood cells (phagocytes) and from attack by antimicrobial agents of plant or animal origin. Capsules in certain soil bacteria protect them from perennial effects of drying or desiccation.

All of the various surface components of a prokaryotic cell are important in its ecology since they mediate the contact of the cell with its environment. The only "sense" that a prokaryote has results from its immediate contact with its environment. It must use its surface components to assess the environment and respond in a way that supports its own existence and survival in that environment. The surface properties of a prokaryote are determined by the exact molecular composition of its plasma membrane and cell wall, including LPS, and the function of surface structures such as flagella, fimbriae and capsules.

15

Some important ways that prokaryotes use their surface components are (1) as permeability barriers that allow selective passage of nutrients and exclusion of harmful substances; (2) as "adhesins" used to attach or adhere to specific surfaces or tissues; (3) as enzymes to mediate specific reactions on the cell surface important in the survival of the prokaryote; (4) as "sensing proteins" that can respond to temperature, osmolarity, salinity, light, oxygen, nutrients, etc., resulting in a signal to the genome of the cell that will cause a beneficial response to the new environment.

The cytoplasmic constituents of prokaryotic cells invariably include the prokaryotic chromosome and ribosomes. The chromosome is typically one large circular molecule of DNA, more or less free in the cytoplasm. Prokaryotes sometimes possess smaller extrachromosomal pieces of DNA called plasmids. The total DNA content of a prokaryote is referred to as the cell genome. During cell growth and division, the prokaryotic chromosome is replicated in the usual semi-conservative fashion before for distribution to progeny cells. However, the eukaryotic processes of meiosis and mitosis are absent in prokaryotes. Replication and segregation of prokaryotic DNA is coordinated by the membrane, possibly by mesosomes.

The distinct granular appearance of prokaryotic cytoplasm is due to the presence and distribution of 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.

Often contained in the cytoplasm of prokaryotic cells is one or another of some type of inclusion granule. Inclusions are distinct granules that may occupy a substantial part of the cytoplasm. Inclusion granules are usually reserve materials of some sort. For example, carbon and energy reserves may be stored as glycogen (a polymer of glucose) or as polybetahydroxybutyric acid (a type of fat) granules. Polyphosphate inclusions are reserves of PO₄ and possibly energy; elemental sulfur (sulfur globules) are stored by some phototrophic and some lithotrophic prokaryotes as reserves of energy or electrons. Some inclusion bodies are actually membranous vesicles or intrusions into the cytoplasm, which contain photosynthetic pigments or enzymes.

16

Figure 2. The universal tree of life (N. Pace)

Taxonomy and classification of procaryotes

In the 1980's, Woese began phylogenetic analysis of all forms of cellular life based on comparative sequencing of the small subunit ribosomal RNA (ssrRNA) that is contained in all organisms. A new dichotomy was revealed, this tune among the prokaryotes: their existed two types of prokaryotes, as fundamentally unrelated to one another as they are to eukaryotes. Thus, Woese defined the three cellular domains of life as they are displayed in Fig. 2: Eucarya, Bacteria and Archaea. Phenotypic differences between them are indicated in the Table 1.

Main objective of the session

To become familiar with structural (differential) features of prokaryotes and learn some methods of their studies (Gram stain and determination of motility).

17

Table 1. PHENOTYPIC PROPERTIES OF BACTERIA AND ARCHAEA COMPARED WITH EUKARYA

Property Cell configuration	Eukarya Eukaryotic	Cellular Domain Bacteria Prokaryotic	Archaea Prokaryotic
Nuclear membrane	Present	Absent	Absent
Number of chromosomes	>1	1	1
Chromosome topology	Linear	Circular	Circular
Murein in cell wall	—	+	—
Cell membrane lipids	Ester-linked	Ester-linked glycerides;	Ether-linked
	glycerides;	unbranched; saturated or	branched;
	unbranched; polyunsaturated	monounsaturated	saturated
Cell membrane sterols	Present	Absent	Absent
Organelles (mitochondria and chloroplasts)	Present	Absent	Absent
Ribosome size	80S (cytoplasmic)	70S	70S
Cytoplasmic streaming	+	-	-
Meiosis and mitosis	Present	Absent	Absent
Transcription and translation coupled	—	+	+

Educational tasks

To know: 1. Permanent and non-permanent structures of bacteria cell wall

and their functions.

2. Differences in the structure of gram-positive and gram-negative bacteria.

3. Methods of studies of microorganisms in native and stained conditions:

applied techniques and peculiarities of microscopic features of native and stained preparations.

To be 1. To make Gram stain.

capable: 2. To make and microscopy preparations from live microor-

ganisms.

Methodical guidelines

1. Complex staining techniques (Gram stain).

During the complex staining techniques, more than one dye is applied to smear. In addition to dye, different decolorizing agents are used: alcohols, acids, acetone, etc. Using complex staining techniques, it is possible to identify cytological peculiarities of microorganisms (cell structures, inclusions, etc. )

18

Gram stain is the most widely used staining procedure in bacteriology. It is called a differential stain since it differentiates between gram-positive and gram- negative bacteria. Bacteria, which stain violet or purple with the gram staining procedure, are termed gram-positive; those, which stain pink, are said to be gram-negative. The terms positive and negative designate two distinct morphological groups of bacteria based on the chemistry and structure of their cell wall. Examples of gram-positive bacteria are as follows: Staphylococcus spp., Streptococcus spp., Bacillus spp., Clostridium spp. and others. Escherichia spp., Pseudomonas spp., Neisseria spp., Rickettsia spp. belong to gram-negatives.

Gram-positive and gram-negative bacteria stain differently because of fundamental differences in the structure of their cell walls. The bacterial cell wall serves to give the organism its size and shape and prevents osmotic lysis. The material in the bacterial cell wall which confers rigidity is peptidoglycan.

1. Chemically, 60 to 90% of the gram-positive cell wall is peptidoglycan. Interwoven in the cell wall of gram-positive are teichoic acids. Teichoic acids, which extend through and beyond the rest of the cell wall, are composed of polymers of glycerol, phosphates, and the sugar alcohol ribitol. Some have a lipid attached (lipoteichoic acid). The outer surface of the peptidoglycan is studded with proteins that differ with the strain and species of the bacterium.

2. The gram-negative cell wall, on the other hand, contains only 2-3 layers of peptidoglycan and is surrounded by an outer membrane composed of phospholipids, lipopolysaccharide, lipoprotein, and proteins. Only 10-20% of the gram-negative cell wall is peptidoglycan. The phospholipids are located mainly in the inner layer of the outer membrane, as are the lipoproteins that connect the outer membrane to the peptidoglycan.

With the current theory behind Gram staining, it is thought that in gram- positive bacteria, the crystal violet and iodine combine to form a larger molecule that precipitates out within the cell. The alcohol/acetone mixture then causes dehydration of the multilayered peptidoglycan, thus decreasing the space between the molecules and causing the cell wall to trap the crystal violet-iodine complex within the cell. In the case of gram-negative bacteria, the decolorizer, being a lipid solvent, dissolves the outer membrane of the cell wall and may also damage the cytoplasmic membrane to which the peptidoglycan is attached. The single thin layer of peptidoglycan is unable to retain the crystal violet-iodine complex and the cell is decolorized.

Gram stain technique (Sinyov modification)

1. Place strip of filter paper, wetted in carbolic gentian violet, put on the surface couple drops of water and leave for 1.5-2 min.

2. Take off the filter paper and place iodine (Lugol) solution for 1 min.

3. Pour out Lugol solution (without flooding with water) and place alcohol decolorizer for couple seconds until the purple color stops flowing.

19