HOST RANGE

• Viruses infect all major groups of organisms: vertebrates, invertebrates, plants, fungi, and bacteria.

• Some viruses have a broader host range than others, but none can cross the eukaryotic/prokaryotic boundary.

Factors which affect host range include:

• whether the virus can get into the host cell

• if the virus can enter the cell, is the appropriate cellular machinery available for the vims to replicate?

• if the vims can replicate, can infectious vims get out of the cell and spread the infection?

Virus structure

Viruses range in size from less than 100 nanometers in diameter to several hundred nanometers in length in the case of the Filoviridae.

Viral components — general

Viruses contain:

• a nucleic acid genome (RNA or DNA)

• a protective protein coat (called the capsid)

The nucleic acid genome plus the protective protein coat is called the nucleocapsid

The nucleocapsid may have icosahedral or helical symmetry Viruses may or may not have an envelope made of lipid derived from the host cell

Viral envelope

Enveloped viruses obtain their envelope by budding through a host cell membrane. In some cases, the vims buds through the plasma membrane but in other cases the envelope may be derived from other membranes such as those of the Golgi body or the nucleus.

• The envelope consists of a lipid bilayer and proteins and always includes at least one virally coded protein involved in attachment.

• Enveloped viruses do not necessarily have to kill cell in order to be released, since they can bud out of the cell - a process which is not

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necessarily lethal to the cell — hence some budding viruses can set up persistent infections.

• Enveloped viruses are readily infectious only if the envelope is intact (since the viral attachment proteins which recognize the host cell receptors are in the viral envelope if it is an enveloped virus). So agents which damage the envelope reduce infectivity.

Bacteriophages are obligate intracellular parasites that multiply inside bacteria by making use of some or all of the host biosynthetic machinery (i.e., viruses that infect bacteria.). At one time it was thought that the use of bacteriophage might be an effective way to treat bacterial infections, but it soon became apparent that phage are quickly removed from the body and thus, were of little clinical value. However, bacteriophages are used in the diagnostic laboratory for the identification of pathogenic bacteria (phage typing). Although phage typing is not used in the routine clinical laboratory, it is used in reference laboratories for epidemiological purposes. Recently, new interest has developed in the possible use of bacteriophages for treatment of bacterial infections and in prophylaxis. Whether bacteriophages will be used in clinical medicine remains to be determined.

Although different bacteriophages may contain different materials they all contain nucleic acid and protein.

Depending upon the phage, the nucleic acid can be either DNA or RNA but not both and it can exist in various forms. The nucleic acids of phages often contain unusual or modified bases. These modified bases protect phage nucleic acid from nucleases that break down host nucleic acids during phage infection. The size of the nucleic acid varies depending upon the phage. The simplest phages only have enough nucleic acid to code for 3-5 average size gene products while the more complex phages may code for over 100 gene products.

Bacteriophages come in many different sizes and shapes. The basic structural features of bacteriophages are listed below.

1. Size — T4 is among the largest phages; it is approximately 200 nm long and 80-100 nm wide. Other phages are smaller. Most phages range in size from 24-200 nm in length.

2. Head or Capsid — All phages contain a head structure which can vary in size and shape. Some are icosahedral (20 sides) others are filamentous. The head or capsid is composed of many copies of one or more different proteins. Inside the head is found the nucleic acid. The head acts as the protective covering for the nucleic acid.

3. Tail — Many but not all phages have tails attached to the phage head. The tail is a hollow tube through which the nucleic acid passes during infection. The size of the tail can vary and some phages do not even have a tail structure. In the more complex phages like T4 the tail is surrounded by a contractile sheath which contracts during infection of the bacterium. At the end of the tail the more complex phages like T4 have a base plate

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Educational tasks

Methodical guidelines

1. For study of morphology of fungi place 1 drop of water onto the microscopic slide. Then place and spread mycelium by preparation needles, followed by coverage of preparation with cover glass and microscopy under dry system. Under microscopy with weak and strong dry systems pay attention on composition of mycelium and fruiting body. Mucor spp. has a single-celled, non-septate mycelium; Aspergillus spp. and Penicillium spp. - multi-cell. Fruiting bodies: large sporangia with endospores in Mucor spp.; non-septate conidiophores Aspergillus spp. and septate — in Penicillium spp.

2. Slide prepared with pure culture of Candida spp., stained with methylene blue, pay attention to form of cells, presence of budding protrusions and composition of cells.

Demonstrations

1. Preparations of slide from filamentous fungus (mold).

2. Preparation smear from Candida albicans.

3. Schemes of morphology and composition of rickettsia and chlamydia.

4. Toxoplasma. Plasmodium in ready-to-use preparations.

5. Schemes of composition of virions.

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6. Babesh-Negri bodies in brain tissues of animal died from rabies.

7. Scheme of composition of bacteriophages.

8. Titration of bacteriophage by Appelman method.

9. Biopreparations of bacteriophages.

Students’ activities

1. To make one preparation of filamentous fungus, examine under microscope and draw the observations in the workbook.

2. To make one preparation of yeast culture, stain by Neisser' method, examine under microscope and draw the observations in the workbook.

3. Preparation of smear from rickettsia diagnosticum.

4. To make one preparation of Candida albicans, make Gram stain (or methylene blue), examine under microscope and draw the observations in the workbook.

5. Study of preparations from bacteriophages.

Control questions

1. What are morphological differences between ascomycetes and phicomycetes?

2. What are the morphological differences between fungi and bacteria?

3. What is the place of spirochetes in the systematics of microorganisms?

4. What species of spirochetes have a medical significance?

5. What features are in common between rickettsia and bacteria?

6. What is the main biological feature in common with rickettsia and viruses?

7. What are the forms of rickettsia?

8. What mechanism is favoring intracellular survival of rickettsia?

9. Which bacteria belong to obligate intracellular parasites?

10. What are the peculiarities of live cycle of chlamydia?

11. What is the specific feature of functional organization of protozoa on comparison with bacteria?

12. Name the types of protozoa which have the species pathogenic for humans.

13. What lies in the basis for diagnostics of protozoal infections?

14. What are the structural elements of virions?

15. What biological properties of viruses differ them from other microorganisms?

16. What structural elements does bacteriophage have?

17. What are the differences between virulent and moderate bacteriophage?

18. What stages of interaction between virulent bacteriophage and bacterial cell you know?

19. What is lysogeny?

20. What purposes bacteriophages are used in medicine?

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Physiology of bacteria. Nutrition and growth of bacteria and viruses. Methods of isolation of pure cultures of aerobic bacteria.

Plan of the session

1. Nutrition and growth of bacteria. Classification of nutrient media.

2. Cultivation of rickettsia, chlamydia and viruses.

3. Method of culture and re-culture of bacteria. Methods of isolation of pure cultures: Drigalski, Gold, Koch and biobacteriological methods.

Foreword notes (source:http://textbookofbacteriology.net/nutgro.html with additions).

Every organism must find in its environment all of the substances required for energy generation and cellular biosynthesis. The chemicals and elements of this environment that are utilized for bacterial growth are referred to as nutrients or nutritional requirements. In the laboratory, bacteria are grown in culture media which are designed to provide all the essential nutrients in solution for bacterial growth.

Major elements

At an elementary level, the nutritional requirements of a bacterium such as E coli are revealed by the cell's elemental composition, which consists of C,

H, O, N, S. P, K, Mg, Fe, Ca, Mn, and traces of Zn, Co, Cu, and Mo. These elements are found in the form of water, inorganic ions, small molecules, and macromolecules which serve either a structural or functional role in the cells.

The general physiological functions of the elements are outlined in the Table 4.

Table 4 ignores the occurrence of trace elements in bacterial nutrition. Trace elements are metal ions required by certain cells in such small amounts that it is difficult to detect (measure) them, and it is not necessary to add them to culture media as nutrients. Trace elements are required in such small amounts that they are present as "contaminants" of the water or other media components. As metal ions, the trace elements usually act as cofactors for essential enzymatic reactions in the cell. One organism's trace element may be another's required

element and vice-versa, but the usual cations that qualify as trace elements in bacterial nutrition are Mn, Co, Zn, Cu, and Mo.

Carbon and energy sources for bacterial growth

In order to grow in nature or in the laboratory, a bacterium must have an

energy source, a source of carbon and other required nutrients, and a permissive rage of physical conditions such as O₂ concentration, temperature, and pH.

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Sometimes bacteria are referred to as individuals or groups based on their patterns of growth under various chemical (nutritional) or physical conditions. For example, phototrophs are organisms that use light as an energy source; anaerobes are organisms that grow without oxygen; thermophiles are organisms that grow at high temperatures.

All living organisms require a source of energy. Organisms that use radiant energy (light) are called phototrophs. Organisms that use (oxidize) an organic form of carbon are called heterotrophs or chemo(hetero)trophs. Organisms that oxidize inorganic compounds are called lithotrophs.

The carbon requirements of organisms must be met by organic carbon (a chemical compound with a carbon-hydrogen bond) or by CO₂ Organisms that use organic carbon are heterotrophs and organisms that use CO₂ as a sole source of carbon for growth are called autotrophs.

Thus, on the basis of carbon and energy sources for growth four major nutritional types of prokaryotes may be defined (Table 5).

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Table 5. MAJOR NUTRITIONAL TYPES OF PROKARYOTES

Almost all eukaryotes are either photoautotrophic (e.g. plants and algae) or heterotrophic (e.g. animals, protozoa, fungi). Lithotrophy is unique to prokaryotes and photoheterotrophy, common in the Purple and Green Bacteria, occurs only in a very few eukaryotic algae. Phototrophy has not been found in the Archaea, except for nonphotosynthetic light-driven ATP synthesis in the extreme halophiles.

Growth factors

This simplified scheme for use of carbon, either organic carbon or CO₂, ignores the possibility that an organism, whether it is an autotroph or a heterotroph, may require small amounts of certain organic compounds for growth because they are essential substances that the organism is unable to synthesize from available nutrients. Such compounds are called growth factors.

Growth factors are required in small amounts by cells because they fulfill specific roles in biosynthesis. The need for a growth factor results from either a blocked or missing metabolic pathway in the cells. Growth factors are organized into three categories.

1. Purines and pyrimidines: required for synthesis of nucleic acids (DNA and RNA)

2. Amino acids: required for the synthesis of proteins

3. Vitamins: needed as coenzymes and functional groups of certain enzymes. Some bacteria (e.g. E. coli) do not require any growth factors: they can synthesize all essential purines, pyrimidines, amino acids and vitamins, starting with their carbon source, as part of their own intermediary metabolism. Certain other bacteria (e.g. Lactobacillus spp. ) require purines, pyrimidines, vitamins and several amino acids in order to grow. These compounds must be added in advance to culture media that are used to grow these bacteria. The growth factors are not metabolized directly as sources of carbon or energy, rather they are assimilated by cells to fulfill their specific role in metabolism. Mutant strains of bacteria that require some growth factor not needed by the wild type (parent)

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strain are referred to as auxotrophs. Thus, a strain of E. coli that requires the amino acid tryptophan in order to grow would be called a tryptophan auxotroph and would be designated E. coli trp-.

Some vitamins that are frequently required by certain bacteria as growth factors are listed in Table 6. The function(s) of these vitamins in essential enzymatic reactions gives a clue why, if the cell cannot make the vitamin, it must be provided exogenously in order for growth to occur.

Table 6. COMMON VITAMINS REQUIRED IN THE NUTRITION OF CERTAIN BACTERIA

Culture media for the growth of bacteria

For any bacterium to be propagated for any purpose it is necessary to provide the appropriate biochemical and biophysical environment. The biochemical (nutritional) environment is made available as a culture medium, and depending upon the special needs of particular bacteria (as well as particular investigators) a large variety and types of culture media have been developed with different

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purposes and uses. Culture media are employed in the isolation and maintenance of pure cultures of bacteria and are also used for identification of bacteria according to their biochemical and physiological properties.

The manner in which bacteria are cultivated, and the purpose of culture media, vary widely. Liquid media are used for growth of pure batch cultures while solidified media are used widely for the isolation of pure cultures, for estimating viable bacterial populations, and a variety of other purposes. The usual gelling agent for solid or semi-solid medium is agar, a hydrocolloid derived from red algae. Agar is used because of its unique physical properties (it melts at 100 degrees and remains liquid until cooled to 40 degrees, the temperature at which it gels) and because it cannot be metabolized by most bacteria. Hence as a medium component it is relatively inert; it simply holds (gels) nutrients that are in aqueous solution.

Types of culture media

Culture media may be classified into several categories depending on their composition or use. A chemically-defined (synthetic) medium is one in which the exact chemical composition is known. A complex (undefined) medium (Tables 7 and 8) is one in which the exact chemical constitution of the medium is not known.

Table 7. COMPLEX MEDIUM FOR THE GROWTH OF FASTIDIOUS BACTERIA

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Defined media are usually composed of pure biochemicals off the shelf; complex media usually contain complex materials of biological origin such as blood or milk or yeast extract or beef extract, the exact chemical composition of which is obviously undetermined. A defined medium is a minimal medium if it provides only the exact nutrients (including any growth factors) needed by the organism for growth. The use of defined minimal media requires the investigator to know the exact nutritional requirements of the organisms in question.

Chemically-defined media are of value in studying the minimal nutritional requirements of microorganisms, for enrichment cultures, and for a wide variety of physiological studies. Complex media usually provide the full range of growth factors that may be required by an organism so they may be more handily used to cultivate unknown bacteria or bacteria whose nutritional requirement are complex (i.e., organisms that require a lot of growth factors).

Most pathogenic bacteria of animals, which have adapted themselves to growth in animal tissues, require complex media for their growth. Blood, serum and tissue extracts are frequently added to culture media for the cultivation of pathogens. Even so, for a few fastidious pathogens such as Treponema pallidum, the agent of syphilis, and Mycobacterium leprae, the cause of leprosy, artificial culture media and conditions have not been established. This fact thwarts the ability to do basic research on these pathogens and the diseases that they cause.

Other concepts employed in the construction of culture media are the principles of selection and enrichment. A selective medium is one which has a component(s) added to it which will inhibit or prevent the growth of certain types or species of bacteria and/or promote the growth of desired species. One can also adjust the physical conditions of a culture medium, such as pH and temperature, to render it selective for organisms that are able to grow under these certain conditions. For example, Corynebacterium diphtheriae grow much better on serum or blood media with added tellurite sodium or potassium which delays or prevents growth of other bacteria (Tinsdale and Klauberg media). Vibrio cholerae can grow on 'hunger' medium which contains only 1% of peptone but pH of medium should be > 8.0.

A culture medium may also be a differential medium if allows the investigator to distinguish between different types of bacteria based on some observable trait in their pattern of growth on the medium. Thus a selective, differentia] medium for the isolation of Staphylococcus aureus, the most common bacterial pathogen of humans, contains a very high concentration of salt (which the staphylococci will tolerate) that inhibits most other bacteria, mannitol as a source of fermentable sugar, and a pH indicator dye. From clinical specimens, only staphylococci will grow. S. aureus is differentiated from S. epidermidis (a nonpathogenic component of the normal flora) on the basis of its ability to ferment mannitol. Mannitol-fermenting colonies (S. aureus) produce acid

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Figure 5. Picking a single colony off of a Petri plate in order to obtain a pure culture

(Source: http: //www. cat. cc. md. us/courses/biol41/labmanua/lab3/atppc. html)

2. Then, an isolated colony can be aseptically "picked off’ the isolation plate (Fig. 5) and transferred to new sterile medium (Fig. 6). After incubation, all organisms in the new culture will be descendants of the same organism, that is, a pure culture.

Figure 6. Obtaining pure cultures from an isolation plate

(Source: http://www.cat.cc.md.us/courses/biol41/labmanua/lab3/pickofT12.html)

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Methodical guidelines

Pure cultures of bacteria are needed for diagnostic investigations — identification, which is performed by determination of morphological, cultural, biochemical and other properties of microorganisms. Morphological and tinctorial properties of bacteria are studied under microscopic examination of smears, unstained or stained by different techniques. Cultural properties of characterized by nutritive requirements, conditions and type of growth of bacteria of solid media and broth. They are established based on colony morphology and peculiarities of growth. Biochemical properties of bacteria are established by set of constitutive and inducible enzymes, which are characteristics of a particular genus, species and variants. In bacteriological practice sugar fermentation and proteolytic activities of bacteria, which is detected on differentia! media, have taxonomic significance. During the identification of bacteria up to genus and species, special attention is drawn to pigments which color colonies and media in different colors. For example. Micrococcus luteus produce a produces a yellow, water insoluble pigment, while E. coli is nonchromogenic (Fig. 7).

Figure 7. Single isolated colonies of Micrococcus luteus and Escherichia coli on TSA

(Source: http://www.cat.cc.md.us/courses/ bio l41/labmanua/lab3/ecmlisol.html)

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Methods of isolation of pure cultures of aerobic bacteria (continuation). Main types of biological oxidation of substrates (aerobic and anaerobic). Action of physical and chemical factors on microorganisms. Asepsis and antiseptics.

Plan of the session

1. Isolation of pure cultures of aerobic bacteria (2^nd day). Identification: cultural, morphological and tinctorial properties.

2. Methods of cultivation of anaerobes.

3. Methods of isolation and identification of pure cultures of anaerobic bacteria.

4. Methods of sterilization. Disinfection.

Foreword notes

During the isolation of pure cultures of bacteria, there is a need to pay attention to the characteristics of microbial growth, e.g. its cultural characteristics.

When bacteria are cultivated in broth, growth of bacteria can be observed as:

• Diffuse turbidity;

• Near-bottom or parietal (near-walls) growth in transparent medium;

• Growth as ‘cotton nubbins’.

In semi-solid media the following can be detected:

• Diffuse growth around inoculation stab (motile bacteria);

• Growth by inoculation stab (non-motile bacteria);

• Media breaches or formation of gas.

On solid media growth can be characterized as:

• Isolated colonies;

• Bacterial lawn.

Determination of purity of isolated cultures

Isolated colonies grew on solid media are re-inoculated by loop onto the surface of agar slant in the tube. Taking into consideration that isolated colonies sometimes might be formed not only from separate cells, determination of their homogeneity is a compulsory step of isolation of pure culture. It could be achieved by the different methods: visual. Microscopic and re-culture on corresponding media. When visual control is used growth of bacteria is studied on streak on the surface of agar slant. In case of presence of non-homogeneity of growth, it is considered that culture is contaminated and additional steps for isolation of pure cultures are required.

For differentiating of colonies of bacteria the following characteristics are very helpful:

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