Morphology and biology of viruses. Basic methods of viruses cultivation.

Methods of indication of the viruses

Bacterial viruses (Bacteriophages). Structure, classification. Sorts of interaction of bacteriophages and bacterial cells. Virulent and moderate phages. Practical significance of bacteriophage phenomenon

Sanitary-bacteriologic studying of water, soil, air. Sanitary-indicative microorganisms. Microbiologic control in stomatologic facilities

 

 

Viruses are the smallest infectious agents (20-300 nm in diameter), containing one kind of nucleic acid (RNA or DNA) as their genome, usually as single molecule.

The nucleic acid is encased in a protein shell,  and the entire infectious unit is termed a virion. Viruses replicate only in living cells. The viral nucleic acid contains informatioecessary for programming the infected host cell to synthesize a number of virus-specific macromolecules required for the production of virus progeny. During the replicative cycle, numerous copies of viral nucleic acid and coat proteins are produced. The coat protein assemble together to form the capsid, which encases and stabilizes the viral nucleic acid against the extracellular environment and facilitates the attachment and perhaps penetration of the virus upon contact with new susceptible cells.

The nucleic acid once isolated from the virion, can be hydrolyzed by either ribo- or deoxyribonuclease, whereas the nucleic acid within the intact virus is not affected by such treatment. In contrast, viral antiserum will neutralize the virion because it reacts with the antigens of protein coat. However, the same antiserum has no effect on the free infectious nucleic acid isolated from the virion.

The host range for a given virus may be extremely limited, but viruses are known to infect unicellular organisms such as mycoplasmas, bacteria, and algae and all higher plants and animals.

Much information on virus-host relationships has been obtained from studies on bacteriophages, the viruses that attack bacteria.

 

Some Useful Definitions in Virology

Capsid: The symmetric protein shell that en closes the nucleic acid genome. Often, empty capsids are by-products of the viral replicative cycle.

Nucleocapsid: The capsid together with the enclosed nucleic acid.

Structural units: The basic protein building blocks of the capsid.           

Capsomeres: Morphologic units seen in the elec­tron microscope on the surface of virus parti­cles. Capsomeres represent clusters of polypeptides, which when completely assembled form the capsid.

Virion: The complete infective virus particle, which in some instances  (adenoviruses, papovaviruses, picornaviruses) may be identical with the nucleocapsid. In more complex virions (herpesviruses, myxoviruses), this in­cludes the nucleocapsid plus a surrounding envelope.

Detective virus: A virus particle that is function­ally deficient in some aspect of replication. Defective virus may interfere with the replication of normal virus.

Pseudovirus: During viral replication the capsid sometimes encloses host nucleic acid rather than viral nucleic acid. Such particles look like ordinary virus, particles when observed by elec­tron microscopy, but they do not replicate. Pseudovirions contain the “wrong”  nucleic acid.

Primary,  secondary, and tertiary nucleic acid structure: Primary structure refers to the se­quence of bases in the nucleic acid chain. Sec­ondary structure refers to the spatial arrange­ment of the complete nucleic acid chain, i.e., whether it is single- or double-stranded, circu­lar or linear in conformation. Tertiary structure refers to other elements of fine spatial detail in the: helix, eg, presence of supercoiling. break­age points, regions of strand separation.

Transcription: The mechanism by which spe­cific information encoded in a nucleic acid chain is transferred to messenger RNA.

Translation: The mechanism by which a particu­lar base sequence in messenger RNA results in production of a specific amino acid sequence in a protein.

Evolutionary origin of viruses. The origin of viruses is not known. Three hypotheses have been proposed:

  (1) Viruses became parasites of primitive cells, and the 2 evolved together. Many viruses today cause no host cell damage and remain latent in the host.

  (2) Viruses evolved from parasitic bacteria. While this possibility exists for other obligatory intracellular organisms, eg, chlamydiae, there is no evidence that viruses evolved from bacteria.

  (3) Viruses may be components of host cells that        become autonomous. They resemble genes that escape the regulatory control of the host cell. There is evidence that some tumor viruses exist in host cells as unexpress genes. The likehood is great that some small viruses evolved in this fashion. On the other hand, large viruses of the pox or herpes groups show very limited resemblance to host cell DNA.       

 

CLASSIFICATION OF VIRUSES.  Basis of Classification.  The following properties, listed in the order of preference or importance, have been used as a basis for the classification of viruses. The amount of information available in each category is not uniform for all viruses. For some agents, knowledge is at hand about only a few of the properties listed.

 

(1)      Nucleic acid type: RNA or DNA; single-stranded or double-stranded; strategy of replication.

(2)      Size and morphology, including type of symmetry, number of capsomeres, and presence of membranes. 

(3)      Presence of specific enzymes, particularly RNA and DNA polymerases concerned with genome, and neuraminidase necessary for release of certain virus particles (influenza) from the cells in which they were formed.

(4)      Susceptibility to physical and chemical         agents, especially ether.

(5)      Immunologic properties.

(6)      Natural methods of transmission.

(7)      Host, tissue, and cell tropisms.  

(8)      Pathology; inclusion body formation.

(9)      Symptomatology.

 

Classification by Symptomatology. The oldest classification of viruses is based on the diseases they produce, and this system offers certain conveniences for the clinician. However, it is not satis­factory for the biologist because the same virus may appear in several groups, since it causes more than one disease depending upon the organ attacked.

A. Generalized Diseases: Diseases in which virus is spread throughout the body via the blood­stream and in which multiple organs are affected. Skin rashes may occur. These include smallpox, vaccinia, measles, rubella, chickenpox, yellow fever, dengue, enteroviruses, and many others.

B. Diseases Primarily Affecting Specific Or­gans: The virus may spread to the organ through the bloodstream, along the peripheral nerves, or by other routes.

1. Diseases of the nervous system – Poliomy­elitis, aseptic meningitis (polio-, coxsackie-, and echoviruses), rabies, arthropod-borne encephalitides, lymphocytic choriomeningitis, herpes simplex, meningoencephalitis of mumps, measles, vaccinia, and “slow” virus infections.

2. Diseases of the respiratory tract – Influenza, parainfluenza, respiratory syncytial virus pneumonia and bronchiolitis, adenovirus pharyngitis, common cold (caused by many viruses).

3. Localized diseases of the skin or mucous membranes – Herpes simplex type 1 (usually oral) and type 2 (usually genital), molluscum contagiosum, warts, herpangina, herpes zoster, and others.

4. Diseases of the eye – Adenovirus con­junctivitis, Newcastle virus conjunctivitis, herpes keratoconjunctivitis, and epidemic hemorrhagic con­junctivitis (enterovirus-70).

5. Diseases of the liver-Hepatitis type A (infec­tious hepatitis) and type B (serum hepatitis), yellow fever, and, in the neonate, enteroviruses, herpesviruses, and rubella virus.

6. Diseases of the salivary glands – Mumps and cytomegalovirus.

7. Diseases of the gastrointestinal tract – Rotavirus, Norwalk type virus.

8. Sexually transmitted diseases – Until re­cently, only bacteria (Neisseria gonorrhoeae, Treponema pallidum, and Chlamydia trachomatis) were in­cluded in this category of disease. It is now recognized that herpes simplex virus, hepatitis B virus, papilloma virus, molluscum contagiosum virus, and probably cytomegalovirus are all venereal pathogens.

 

Classification by Biologic, Chemical, and Physical Properties.

Viruses can be clearly separated into families on the basis of the nucleic acid genome and the size, shape, substructure, and mode of replication of the virus particle. Table 1 shows one scheme used for classification. However, there is not complete agree­ment among virologists on the relative importance of the criteria used to classify viruses.

Within each family, genera are usually based on antigenicity. Properties of the major families of animal viruses are summarized in Table 1, are discussed briefly below.

 

DNA-Containing Viruses

A. Parvoviruses: Very small viruses with a par­ticle size of about 20 nm. They contain single-stranded DNA and have cubic symmetry, with 32 capsomeres 2-4 nm in diameter. They have no envelope. Replica­tion and capsid assembly take place in the nucleus of the infected cell. Parvoviruses of rodents and swine replicate autonomously. The adenoassociated satellite viruses are defective, i.e., they require the presence of an adenovirus or a herpesvirus as a ”helper”. Some satellite viruses occur in humans.

B. Papovaviruses: Small (45-55 nm), ether-resistant viruses containing double-stranded circular DNA and exhibiting cubic symmetry, with 72 capso­meres. Known human papovaviruses are the papilloma (wart) virus and agents isolated from brain tissue of patients with progressive multifocal leukoencephalopathy (JC virus) or from the urine of immunosuppressed renal transplant recipients (BK vi­rus). In animals, there are papilloma, polyoma, and vacuolating viruses. These agents have a slow growth cycle and replicate within the nucleus. Papovaviruses produce latent and chronic infections in their natural hosts, and all can induce tumors in some animal species.

C. Adenoviruses: Medium-sized (70-90 nm) vi­ruses containing double-stranded DNA and exhibiting cubic symmetry, with 252 capsomeres. They have no envelope. At least 37 types infect humans, especially in mucous membranes, and they can persist in lymphoid tissue. Some adenoviruses cause acute respi­ratory diseases, pharyngitis, and conjunctivitis. Some human adenoviruses can induce tumors iewborn hamsters. There are many serotypes that infect ani­mals.

D. Herpesviruses: Medium-sized viruses con­taining double-stranded DNA. The nucleocapsid is 100 nm in diameter, with cubic symmetry and 162 capsomeres. It is surrounded by a lipid-containing envelope (150-200 nm in diameter). Latent infections may last for the life span of the host.

Human herpesviruses include herpes simplex types 1 and 2 (oral and genital lesions); varicella-zoster virus (shingles and chickenpox); cytomegalovirus; and EB virus (infectious mononucleosis and association with humaeoplasms). Other herpesviruses occur in many animals.

E. Poxviruses: Large brick-shaped or ovoid (230 x 400 nm) viruses containing double-stranded DNA, with a lipid-containing envelope. All poxviruses share a commoucleoprotein antigen and contain several enzymes in the virion, including a DNA-dependent RNA polymerase. Poxviruses replicate entirely within cell cytoplasm. All poxviruses tend to produce skin lesions. Some are pathogenic for humans (smallpox, vaccinia, molluscum contagiosum), others for animals. (Some of the latter can infect humans, eg, cow-pox, monkeypox).

RNA-Containing Viruses

A. Picornaviruses: Small (20-30 nm), ether-resistant viruses containing single-stranded RNA and exhibiting cubic symmetry. The groups infecting hu­mans are rhinoviruses (more than 100 serotypes caus­ing common colds) and enteroviruses (polio-, coxsackie-, and echoviruses). Rhinoviruses are acid-labile and have a high density; enteroviruses are acid-stable and have a lower density. Picornaviruses infect­ing animals include foot-and-mouth disease of cattle and encephalomyocarditis of rodents.

 

Table 1. Classification of viruses into families based on chemical and physical properties

 

 

“Diameter, or diameter X length.

**The naked virus, i.e., the nucleocapsid, is 100 nm in diameter; however, the enveloped virion varies up to 200 nm.

***The genus Orthopoxvirus, which includes the better studied poxviruses, eg, vaccinia, variola, cowpox, ectromelia, rabbitpox,

monkeypox, is ether-resistant. Some of the poxviruses belonging to other genera are ether-sensitive.

****Reoviruses contain an outer and an inner capsid. The inner capsid appears to contain 32 capsomeres, but the number on the outer capsid has not been definitely established. A total of 92 capsomeres has been suggested.

 

B. Reoviruses: Medium-sized (60-80 nm), ether-resistant viruses containing a segmented double-stranded RNA and having cubic symmetry. Reoviruses of humans include rotaviruses, which cause infantile gastroenteritis and have a distinctive wheel-shaped appearance. Antigenically similar reoviruses infect many animals. Orbiviruses constitute a distinct subgroup that includes Colorado tick fever virus of humans and other agents that infect plants, insects, and animals (blue tongue of cattle and sheep).

C. Arboviruses: An ecologic grouping of vi­ruses with diverse physical and chemical properties. All of these viruses (more than 350) have a complex cycle involving vertebrate hosts and arthropods as vec­tors transmitting the viruses by their bite. Arboviruses infect humans, mammals, birds, and snakes, and mos­quitoes and ticks as vectors. Human pathogens include dengue, yellow fever, encephalitis viruses, and others. Arboviruses belong to several groups, including toga-, bunya-, rhabdo-, arena-, and reoviruses, described here.

D. Togaviruses: Most arboviruses of antigenic groups A and B and rubella virus belong here. They have a lipid-containing envelope, are ether-sensitive, and their genome is single-stranded RNA. The en­veloped virion measures 40—70 nm. The virus parti­cles mature by budding from the host cell membrane. Some togaviruses, eg, Sindbis virus, possess a 35-nm nucleocapsid and within it a spherical core 12-16 nm in diameter. Sindbis virus may have 32 capsomeres in an icosahedral surface lattice.

E. Arenaviruses: RNA-containing, enveloped viruses ranging in size from 50 to 300 nm. They share morphologic, biologic, and antigenic properties of ar­boviruses of the Tacaribe complex, Lassa fever, and lymphocyticchoriomeningitis. Some produce “slow” virus infections.

 F. Coronaviruses: Enveloped, 80- to 130-nm particles containing an unsegmented genome of single-stranded RNA; the nucleocapsid is probably helical, 7-9 nm in diameter. They resemble orthomyxoviruses, but coronaviruses have petal-shaped surface projections arranged in a fringe like a solar corona. Coronavirus nucleocapsids develop in the cytoplasm and mature by budding into cytoplasmic vesicles. Human coronaviruses have been isolated from acute upper respiratory tract illnesses— “colds”.  Coronaviruses of animals include avian in­fectious bronchitis virus among many others.

G. Retroviruses: Enveloped viruses whose genome contains duplicate copies of high-molecular-weight single-stranded RNA of the same polarity as viral messenger RNA. The virion contains various enzymes including reverse transcriptase (RNA – DNA). Leukemia and sarcoma viruses of animals, foamy viruses of primates, and some “slow” viruses (visna, maedi of sheep) are included.

H. Bunyaviruses: Spherical, 90- to 100-nm par­ticles that replicate in the cytoplasm and acquire an envelope by budding through the cell membrane. The genome is made up of a triple-segmented, single-stranded RNA. About 70 are antigenically related to Bunyamwera virus; 50 others are not but are mor-phologically similar.

Orthomyxoviruses: Medium-sized, 80- to 120-nm enveloped viruses containing a segmented single-stranded RNA genome and exhibiting helical symmetry. Particles are either round or filamentous. Most Orthomyxoviruses have surface projections as part of their outer wall (hemagglutinin, neuraminidase). The internal nucleoprotein helix measures 6-9 nm, and the RNA is made up of 8 segments. During replication, the nucleocapsid is formed in the nucleus, whereas the hemagglutinin and neuraminidase are formed in the cytoplasm. The virus matures by bud­ding at the cell membrane. Orthomyxoviruses are sen­sitive to dactinomycin. All Orthomyxoviruses are in­fluenza viruses that infect humans or animals.

J. Paramyxoviruses: Similar to but larger (150-300 nm) than Orthomyxoviruses. The internal nucleocapsid measures 18 nm, and the molecular weight of the single-stranded nonsegmented RNA is 4 times greater than that of Orthomyxoviruses. Both the nucleocapsid and the hemagglutinin are formed in the cytoplasm. Paramyxoviruses are resistant to dac­tinomycin. Those infecting humans include mumps, measles, parainfluenza virus, and respiratory syncytial virus. Others infect animals.

K. Rhabdoviruses: Enveloped virions resem­bling a bullet, flat at one end and round at the other (Fig 27-35), measuring about 70 x 175 nm. The envelope has 10-nm spikes. The genome is single-stranded RNA. Particles are formed by budding from the cell membrane. Rabies virus is a member of this group along with many other viruses of animals and plants.

L. Other Viruses: Insufficient information to permit classification. This applies to hepatitis viruses, to agents responsible for some im­mune complex diseases and for sortie “slow” virus diseases, and to some viruses of gastroenteritis

M. Viroids: Small infectious agents causing diseases of plants and possibly animals and humans. They are nucleic acid molecules (MW 70,000-120,000) without a protein coat. Plant viroids are single-stranded, covalently closed circular RNA molecules consisting of about 360 nucleotides and comprising a highly base-paired rodlike structure with unique properties. They are arranged in 26 double-stranded segments separated by 25 regions of unpaired bases embodied in single-stranded internal loops; there is a loop at each end of the rodlike molecule. These features provide the viroid RNA molecule with struc­tural, thermodynamic, and kinetic properties very similar to those of a double-stranded DNA molecule of the same molecular weight and G + C content. Viroids replicate by an entirely novel mechanism in which infecting viroid RNA molecules are copied by the host enzyme normally responsible for synthesis of nuclear precursors to messenger RNA. Thus, DNA-dependent RNA polymerase purified from healthy plant tissue is capable of synthesizing linear (—) viroid RNA copies of full length from (+) viroid RNA templates in vitro.

The infectious agents of degenerative neurologic disorders such as kuru or Creutzfeldt-Jakob disease, or scrapie of sheep, may fit into this category. (The agent of the latter may be a DNA molecule similar in size to plant viroid RNA).

 

CULTIVATION; QUANTIFICATION; INCLUSION BODIES; CHROMOSOME DAMAGE

 

Cultivation of Viruses

At present, many viruses can be grown in cell cultures or in fertile eggs under strictly controlled conditions. Growth of virus in animals is still used for the primary isolation of certain viruses and for the study of pathogenesis of viruses and of viral on-cogenesis.

A.                      Chick Embryos: Virus growth in an embryonated chick egg may result in the death of the embryo (eg, encephalitis virus), the production of pocks or plaques on the chorioallantoic membrane (eg, herpes, smallpox, vaccinia), the development of hemagglutinins in the embryonic fluids or tissues (eg, influenza), or the development of infective virus (eg, polio virus type 2).

 

 

B. Tissue Cultures: The availability of cells grown in vitro has facilitated the identification and cultivation of newly isolated and previously known viruses. There are 3 basic types of cell culture. Primary cultures are made by dispersing cells (usually with trypsin) from host tissues. In general, they are unable to grow for more than a few passages in culture, as secondary cultures. Diploid cell strains are secondary cultures which have undergone a change that allows their limited culture (up to 50 passages) but which retain their normal chromosome pattern. Continuous cell lines are cultures capable of more prolonged (perhaps indefinite) culture which have been derived from cell strains or from malignant tissues They invariably have altered and irregular numbers of chromosomes.

The type of cell culture used for virus cultivation depends on the sensitivity of the cells to that particular virus In the clinical laboratory, multiplication of the virus can be followed by determining the following.

1 The cytopathic effect, or necrosis of cells in the tissue culture (polio-, herpes-, measles-, adenovirus, cytomegalovirus, etc).

2 The inhibition of cellular metabolism, or failure of virus-infected cells to produce acid (eg, enteroviruses).

3 The appearance of a hemagglutinin (eg, mumps, influenza) or complement-fixing antigen (eg, poliomyelitis, varicella, measles).

4 The adsorption of erythrocytes to infected cells, called hemadsorption (parainfluenza, influenza). This reaction becomes positive before cytopathic changes are visible, and in some cases it is the only means of detecting the presence of the virus 5 Interference by a noncytopathogenic virus (eg, rubella) with replication and cytopathic effect of a second, indicator virus (eg, echovirus).

6 Morphologic transformation by an oncogenic virus (eg, SV40, Rous sarcoma virus), usually accompanied by the loss of contact inhibition and the piling up of cells into discrete foci Such alterations are a heritable property of the transformed cells.

 

Quantification of Virus

A. Physical Methods: Virus particles can be counted directly m the electron microscope by comparison with a standard suspension of latex particles of similar small size However, a relatively concentrated preparation of virus is necessary for this procedure, and infectious virus particles cannot be distinguished from noninfectious ones.

Hemagglutination. The red blood cells of humans and some animals can be agglutinated by different viruses Both infective and noninfective particles give this reaction, thus, hemagglutination measures the total quantity of virus present The orthomyxoviruses contain a hemagglutinin that is an integral part of the viral envelope Once these viruses have agglutinated with the cells, spontaneous dissociation of the virus from the cells can occur The dissociated cells cao longer be agglutinated by the same virus species, but the recovered virus is able to agglutinate fresh cells. This is due to the destruction of specific mucopolysaccharide receptor sites on the surface of the erythrocyte by the enzyme neuraminidase of the virus particles.

 

 

Paramyxoviruses growing in cell culture can be detected by hemadsorption Erythrocytes adsorb to each infected cell. 

Poxviruses have an agglutinin for red cells (a phosphohpid-protein complex) that can be separated from the infective virus particle.

Arboviruses and others have hemagglutinins that appear to be identical with the virus particle The union between hernagglutinin and red blood cells is irreversible.

B. Biologic Methods: Quintal assays depend on the measurement of animal death, animal infection or cytopathic effects in tissue culture upon end point dilution of the virus being tested The titter is exprcssei.1 as the 50% infectious dose (ID5o), which is the reciprocal of the dilution of virus that produces the effect 111 50% of the cells or animals inoculated Precise assays require the use of a large number of test subjects.

The most widely used assay for infectious virus is the plaque assay. Monolayers of host cells are inoculated with suitable dilutions of virus and after adsorption are overlaid with medium containing agar or carboxymethylcellulose to prevent virus spreading Alter several days, the cells initially infected have produced virus that spreads only to surrounding cells, producing a small area of infection, or plaque Under controlled conditions a single plaque can arise from a single infectious virus particle, termed a plaque-forming unit (PFU) The cytopathic effect of infected cells within the plaque can be distinguished from uninfected cells of the monolayer, with or without suitable staining. and plaques can usually be counted macroscopically. The ratio of infectious to physical particles vanes widely, from near unity to less than 1 per 1000.

Certain viruses such as herpes or vaccinia form pocks when inoculated onto the chorionallantoic membrane of the embryonated egg. Such viruses can be quantitated by relating the number of pocks counted to the virus dilution.

Inclusion Body Formation. In the course of virus multiplication within cells, virus-specific structures called inclusion bodies maybe produced. They become far larger than the individual virus particle and often have an affinity for acid dyes (eg, eosin) They may be situated in the nucleus (herpesvirus), in the cytoplasm (pox virus), or in both (measles virus) In many viral infections, the inclusion bodies are the site of development of the virions (the virus factories) In some infections (molluscum contagiosum), the inclusion body consists of masses of virus particles that can be seen in the electron microscope to ripen to maturity within the inclusion body. In others (as in the intranuclear inclusion body of herpes), the virus appears to have multiplied within the nucleus early in the infection, and the inclusion body appears to be a remnant of virus multiplication. Variations in the appearance of inclusion material depend largely upon the fixative used

The presence of inclusion bodies may be of considerable diagnostic aid The intracytoplasmic inclu-
sion ierve cells, the Negri body, is pathognomonic for rabies.

Negri body

 

Chromosome Damage. One of the consequences of infection of cells by viruses is derangement of the karyotype. Most of the changes observed are random. Frequently, breakage,  fragmentation, rearrangement of the chromosomes, abnormal chromosomes, and changes in chromosome number occur Herpes zoster virus interrupts the mitotic cycle of human cells in culture, resulting in formation of micronuclei and fragmentation of some chromosomes. Chromosome breaks have also been observed in leukocytes from cases of chickenpox or measles These viruses, as well as rubella virus, cause similar aberrations when inoculated into cultured cells. Cells infected with or transformed to malignancy by SV40, polyoma, or adenovirus type 12 also exhibit random chromosomal abnormalities.

The Chinese hamster cell has a stable karyotype composed of 22 chromosomes Inoculation of these hamster cells with herpes simplex virus results in chromosome aberrations that arc not random in distribution Most of the breaks occur in region 7 of chromosome No. 1 and in region 3 of the X chromosome The Y chromosome is unaffected Replication of the virus is necessary for induction of the chromosome aberrations. To date, no pathognomonic chromosome alterations have been identified in virusinfected cells in humans.

 

STRUCTURE AND SIZE OF VIRUSES

Virus Particles

Advances in x-ray diffraction techniques and electron microscopy have made it possible to resolve fine differences in the basic morphology of viruses. The study of virus symmetry in the electron microscope requires the use of heavy metal stains (eg, potassium phosphotungstate) to emphasize surface structure The heavy metal permeates the virus panicle as a cloud and brings out the surface structure of viruses by virtue of “negative staining “

Virus architecture can be grouped into 3 types based on the arrangement of morphologic subunits. (1) those with helical symmetry, eg, paramyxo- and orthomyxovimses, (2) those with cubic symmetry, eg, adenoviruses, and (3) those with complex structures, eg, poxviruses All cubic symmetry observed with animal viruses to date is of the icosahedral pattern. The icosahedron has 20 faces (each an equilateral triangle), 12 vertices, and 5-fold, 3-fold, and 2-fold axes of rotational symmetry. Capsomeres can be arranged to comply with icosahedral symmetry in a limited number of ways, expressed by the formula N = 10(n-I)² + 2, where N is the total number of capsomeres and n the number of capsomeres on one side of each equilateral triangle shows the number of capsomeres where n varies from 2 to 6, in several virus groups.

Icosahedral structures can be built from one simple, asymmetric building unit, arranged as 12 pentamer units and x number of hexamer units. The smallest and most basic capsid is that of the phage fX-174, which simply consists of 12 pentamer units Viruses exhibiting icosahedral symmetry can also be grouped according to their tnangulatioumber, T, which is the number of small triangles formed on the single face of the icosahedron when all its adjacent morphologic subunits are connected by lines. One class has T values of 1,4,9,16, and 25; a second class, values of 3 and 12, and a third class, values of 7, 13, 19, and 21 The number of morphologic units (capsomeres) is expressed by the formula M = 10T + 2. Table 2 shows the tnangulatioumber for several virus groups This formula for tnangulatioumber originated in the idea that those viruses would be  formed from small subunits so as to give a surface lattice representing the minimum-energy design for closed shells arranged from identical units.

An example of icosahedral symmetry is seen in Fig 1. The adenovirus (n = 6) model illustrated shows the 6 capsomeres along one edge (Fig 1[a]). Degradation of this virus with sodium lauryl sulfate releases the capsomeres in groups of 9 (Fig 1 [b], [c]) and possibly groups of 6 The groups of 9 lie on the faces and include one capsomere from each of the 3 edges of the face, and the groups of 6 would be from the vertices The groups of 9 form the faces of the 20 triangular facets, making the adenovirus icosahedron account for 180 subunits, and the groups of 6 which form the 12 vertices account for 72 capsomeres, thus totaling 252.

Figure 1. (a) Representation of the capsomere arrangement of an adenovirus particle, as viewed through the 2 fold axis of symmetry. (b) Arrangement of capsomere group of 9, obtained by treatment of an adenovirus with sodium lauryl sulfate. (c) Orientation of the capsomere group of 9 on the adenovirus particle If the model were marked to show the maximum number of small triangles formed on one face of the icosahedron by drawing a line between each adjacent morphologic subunit, it would yield the tnangulatioumber for the adenovirus particle, which in this case turns out to be 25.

Measuring the Size of Viruses. Small size and ability to pass through filters that hold back bacteria are classic attributes of viruses. However, because some bacteria may be smaller than the largest viruses, filtrability is no longer regarded as a unique feature of viruses.

The following methods are used for determining the sizes of viruses and their components.

A. Filtration Through Collodion Membranes of Graded Porosity: These membranes are available with pores of different sizes. If the virus preparation is passed through a series of membranes of known pore size, the approximate size of any virus can be mea­sured by determining which membranes allow the in­fective unit to pass and which hold it back. The size of the limiting APD (average pore diameter) multiplied by 0.64 yields the diameter of the virus particle. The passage of a virus through a filter will also depend on the physical structure of the virus; thus, only a very approximate estimate of size is obtained.

B. Sedimentation in the Ultracentrifuge: If particles are suspended in a liquid, they will settle to the bottom at a rate that is proportionate to their size. In an ultracentrifuge, forces of more than 100,000 times gravity may be used to drive the particles to the bottom of the tube. The relationship between the size and shape of a particle and its rate of sedimentation permits determination of particle size. Once again, the physical structure of the virus will affect the size esti­mate obtained.

C. Direct Observation in the Electron Mi­croscope: As compared with the light microscope, the electron microscope uses electrons rather than light waves and electromagnetic lenses rather than glass lenses. The electron beam obtained has a much shorter wavelength than that of light, so that objects much smaller than the wavelength of visible or ultraviolet light can be visualized. Viruses can be visualized in preparations from tissue extracts and in ultrathin sections of infected cells. Electron microscopy is the most widely used method for estimating particle size.

D. Ionizing Radiation: When a beam of charged particles such as high-energy electrons, alpha parti­cles, or deuterons passes through a virus, it causes an energy loss in the form of primary ionization. The release of ionization within the virus particle pro­portionately inactivates certain biologic properties of the virus particle such as infectivity, antigenicity, and hemagglutination. Thus, the size of the biologic unit responsible for a given function in a virus particle can be estimated.

E. Comparative Measurements: (See Table 1.) For purposes of reference, it should be recalled that: (1) Staphylococcus has a diameter of about 1000 nm. (2) Bacterial viruses (bacteriophages) vary in size (10-100 nm). Some are spherical or hexagonal and have short or long tails. (3) Representative protein molecules range in diameter from serum albumin (5 nm) and globulin (7 nm) to certain hemocyanins (23 nm).

The relative size and morphology of various virus families see Lecture 4. Particles with a 2-fold difference in diameter have an 8-fold difference in volume. Thus, the mass of a pox virus is about 1000 times greater than that of the poliovirus particle, and the mass of a small bacterium is 50,000 times greater.

 

CHEMICAL COMPOSITION OF VIRUSES

Viral Protein. The structural proteins of viruses have several important functions. They serve to protect the viral genome against inactivation by nucleases, participate in the attachment of the virus particle to a susceptible cell, and are responsible for the structural symmetry of the virus particle. Also, the proteins determine the antigenic characteristics of the virus.

Virus structural proteins may be very specialized molecules designed to perform a specific task: (1) vaccinia virus carries many enzymes within its particle to perform certain functions early in the infectious cycle; (2) some viruses have specific proteins for at­tachment to cells, eg, influenza virus hemagglutinin;

and (3) RNA tumor viruses contain an enzyme, reverse transcriptase, that makes a DNA copy of the virus RNA, which is an important step in transformation by these viruses.

Viral Nucleic Acid. Viruses contain a single kind of nucleic acid, either DNA or RNA, that encodes the genetic informa­tioecessary for the replication of the virus. The RNA or DNA genome may be single-stranded or double-stranded, and the strandedness, the type of nucleic acid, and the molecular weight are major characteristics used for classifying viruses into families (Table 1).

The molecular weight of the viral DNA genome ranges from 1.5 X 10⁶ (parvoviruses) to 160 X 10⁶ (poxviruses). The molecular weight of the viral RNA genome ranges from 1 x 10⁶ (for bromegrass mosaic virus) to 15 x 10⁶ (for reoviruses).

The sequence and composition of nucleotides of each viral nucleic acid are distinctive. One of the properties useful for characterising a viral nucleic acid is its guanine + cytosine (G + C) content.

Most viral genomes are quite fragile once they are removed from their protective protein capsid, but some nucleic acid molecules have been examined in the electron microscope without disruption, and their lengths have been measured. If linear densities of approximately 2 X 10⁶ per mcm for double-stranded nucleic acid and 1 x 10⁶ per mcm for single-stranded forms are used, molecular weights of viral genomes can be calculated from direct measurements (Table 1).

All major DNA virus groups in Table 1 have genomes that are single molecules of DNA and have a linear or a circular configuration. This circle is often supercoiled  in the virion.

Viral RNAs exist in several forms. The RNA may be a single linear molecule (eg, picomavirus). For other viruses (eg, orthomyxovirus), the genome con­sists of several segments of RNA that may be loosely linked together within the virion. The isolated RNA of picomaviruses and toga viruses is infectious, and the entire molecule functions as a messenger RNA within the infected cell. The isolated RNA of other RNA viruses is not infectious. For these virus families, the virions carry an RNA polymerase which in the cell transcribes the genome RNA molecules into several complementary RNA molecules, each of which may serve as a messenger RNA.

Molecular hybridization techniques (DNA to DNA, DNA to RNA, or RNA to RNA) permit the study of transcription of the viral genome within the infected cell as well as the relatedness of different viruses.

The number of genes in a virus can be approxi­mated if one makes certain assumptions about (1) triplet code, (2) the molecular weight of the genome, and (3) the average size of a protein (Table 1).

Viral Lipids. A number of different viruses contain lipids as part of their structure (eg, Sindbis virus [Fig 2]). Such lipid-containing viruses are sensitive to treatment with ether and other organic solvents (Table 1), indicating that disruption or loss of lipid results in loss of infectivity. Non-lipid-containing viruses are gener­ally resistant to ether.

Figure 2. Proposed structure of Sindbis virus

 

 

The specific phospholipid composition of a virion envelope may be determined by the ‘ ‘budding ” of the virus through specific types of cell membranes in the course of maturation. For example, herpes viruses bud through the nuclear membrane of the host cell, and the phospholipid composition of the purified virus reflects the lipids of the nuclear membrane. The different ways in which various animal viruses acquire an envelope are suggested in Fig 3. Budding of virions occurs only at sites where virus-specific pro­teins have been inserted into the host cell membrane.

Figure 3. Diagrammatic relationship between lipid-containing viruses and host cell membranes

Glycosphingolipids occur in the surface mem­brane of animal cells. When cultured cells are trans­formed by some oncogenic viruses, there are altera­tions in the various sphingolipids. These may be re­lated to the loss of contact inhibition and to changes in surface antigens that result from viral transformation.

Viral Carbohydrates. Virus envelopes contain glycoproteins. The sugars added to vims glycoproteins often reflect the host cell in which the vims is grown. The glycopro­teins are important vims antigens. As a result of their position at the outer surface of the virion, they are frequently involved in the interaction of the vims with neutralizing antibody.

 

PURIFICATION AND IDENTIFICATION OF VIRUSES

Purification of Virus Particles. For purification studies, the starting material is usually large volumes of tissue culture medium, body fluids, or infected cells. The first step involves concen­tration of the virus particles by precipitation with ammonium sulfate, ethanol, or polyethylene glycol or by ultrafiltration. Hemagglutination and elution can be used to concentrate myxoviruses. Once concentrated, virus can be separated from host materials by differential centrifugation, density gra­dient centrifugation, column chromatography, and Rhabdovirus electrophoresis.

The minimal criteria for purity are a homogene­ous appearance in electron micrographs and the failure of additional purification procedures to remove  “con­taminants” without reducing infectivity.

Rate-Zonal Centrifugation. A sample of concentrated virus is layered onto a preformed linear density gradient of sucrose or glycerol, and during centrifugation the virus sediments as a band at a rate determined primarily by the size and weight of the virus particle. Samples are collected by piercing a hole in the bottom of the centrifuge tube. The band of purified virus may be detected by optical methods, by radiolabeling the virus, or by assaying for infectivity.

Equilibrium Density Gradient Centrifugation. Viruses can also be purified by high-speed cen­trifugation in density gradients of cesium chloride (CsCI), potassium tartrate, potassium citrate, or su­crose. The gradient material of choice is the one that is least toxic to the virus. Virus particles migrate to an equilibrium position where the density of the solution is equal to their buoyant density and form a visible band. Virus bands are harvested by puncture through the bottom of the plastic centrifuge tube and assayed for infectivity.

Additional methods for purification are based on the chemical properties of the virus surface.

As shown by column chromatography, virus is bound to a substance such as DEAE or phosphocellulose, then eluted by changes in pH or salt concentra­tion. Zone electrophoresis permits the separation of virus particles from contaminants on the basis of charge.

Identification of a Particle as a Virus. When a characteristic physical particle has been obtained, it should fulfill the following criteria before it is identified as a virus particle.

(1) The particle can be obtained only from in­fected cells or tissues.

(2) Particles obtained from various sources are identical, regardless of the cellular species in which the virus is grown.

(3) The degree of infective activity of the virus varies directly with the number of particles present.

(4) The degree of destruction of the physical par­ticle by chemical or physical means is associated with a corresponding loss of virus activity.

(5) Certain properties of the particles and infec­tivity must be shown to be identical, such as their sedimentation behaviour in the ultracentrifuge and their pH stability curves.

(6) The absorption spectrum of the purified physical particle in the ultraviolet range should coincide with the ultraviolet inactivation spectrum of the virus.

(7) Antisera prepared against the infective virus should react with the characteristic particle, and vice versa. Direct observation of an unknown virus can be accomplished by electron microscopic examination of aggregate formation in a mixture of antisera and crude virus suspension.

(8) The particles should be able to induce the characteristic disease in vivo (if such experiments are feasible).

(9) Passage of the particles in tissue culture should result in the production of progeny with biologic and serologic properties of the virus.

 

REACTION TO PHYSICAL & CHEMICAL AGENTS

Heat and Cold. Virus infectivity is generally destroyed by heating at 50-60 °C for 30 minutes, although there are some notable exceptions (eg, hepatitis virus, adenoassociated satellite virus, scrapie virus).

Viruses can be preserved by storage at subfreezing temperatures, and some may withstand lyophilization and can thus be preserved in the dry state at 4 °C or even at room temperature. Viruses that withstand lyophilization are more heat-resistant when heated in the dry state. Enveloped viruses tend to lose infectivity after prolonged storage even at —90 °C and are particu­larly sensitive to repeated freezing and thawing; how­ever, in the presence of dimethyl sulfoxide (DMSO) at concentrations of more than 5%, these viruses are stabilized.

Stabilization of Viruses by Salts. Many viruses can be stabilized by molar concen­trations of salts, i.e., they are not inactivated even by heating at 50 °C for 1 hour. The mechanism by which the salts stabilize virus preparations is not known. Viruses are preferentially stabilized by certain salts. Molar MgCl₂ stabilizes picorna- and reoviruses, molar MgSO₄ stabilizes orthomyxo- and paramyxo-viruses, and molar NazSO₄ stabilizes herpes-viruses.

The stability of viruses is important in the prepa­ration of vaccines. The ordinary nonstabilized polio-vaccine must be stored at freezing temperatures to preserve its potency. However, with the addition of salts for stabilization of the virus, potency can be maintained for weeks at ambient temperatures, even in the high temperatures of the tropics.

Heating of some virus preparations in the pres­ence of high salt concentrations can be used to remove adventitious agents. For example, heating poliovirus suspensions in molar MgCI₂ will inactivate such sim­ian contaminants as SV40, foamy virus, and herpes B virus but has no deleterious effect on the infectivity and potency of poliovirus.

PH. Viruses are usually stable between pH values of 5.0 and 9.0. In hemagglutination reactions, variations of less than one pH unit may influence the result.

Radiation. Ultraviolet, x-ray, and high-energy particles inac­tivate viruses. The dose varies for different viruses.

Vital Dyes. Viruses are penetrable to a varying degree by vital dyes such as toluidine blue, neutral red, and proflavine. These dyes bind to the viral nucleic acid, and the virus then becomes susceptible to inactivation by visible light. Impenetrable viruses like poliovirus, when grown in the dark in the presence of vital dyes, incorporate the dye into their nucleic acid and are then susceptible to photodynamic inactivation. The coat antigen is unaffected by the process.

Ether Susceptibility. Ether susceptibility can distinguish viruses that possess a lipid-rich envelope from those that do not. The following viruses are inactivated by ether: herpes-, orthomyxo-, paramyxo-, rhabdo-, corona-, retro-, arena-, toga-, and bunyaviruses. The following viruses are resistant to ether: parvo-, papova-, adeno-, picorna-, and reoviruses. Poxviruses vary in sensitiv­ity to ether.

Antibiotics. Antibacterial antibiotics and sulfonamides have no effect on viruses. However, rifampin can inhibit pox virus replication.

Antibacterial Agents. Quaternary ammonium compounds are not effec­tive except for a few viruses. Organic iodine com­pounds are also ineffective. Larger concentrations of chlorine are required to destroy viruses than to kill bacteria, especially in the presence of extraneous pro­teins. For example, the chlorine treatment of stools adequate for typhoid bacilli is inadequate to destroy poliomyelitis virus present in feces. Formalin destroys resistant poliomyelitis and coxsackieviruses. Alcohols such as isopropanol and ethanol are relatively ineffec­tive against certain viruses, especially picornaviruses.

 

REPLICATION OF VIRUSES

Viruses multiply only in living cells. The host cell must provide the energy and synthetic machinery and also the low-molecular-weight precursors for the syn­thesis of viral proteins and nucleic acids. The viral nucleic acid carries the genetic specificity to code for all the virus-specific macromolecules in a highly organised fashion. In some cases, as soon as the viral nucleic acid enters the host cell, the cellular metabo­lism is redirected exclusively toward the synthesis of new virus particles. In other cases the metabolic processes of the host cell are not altered significantly, although the cell synthesises viral proteins and nucleic acids.

During the replicative cycle, viruses transfer ge­netic information in several ways from one generation to another. The essential theme, however, is that spe­cific mRNAs must be transcribed from the viral nu­cleic acid for successful expression and duplication of genetic information. Once this is accomplished, vi­ruses use cell components to translate the mRNA. Various classes of viruses use different pathways to synthesize the mRNAs depending upon the structure of the viral nucleic acid. Some viruses (eg, rhabdo-viruses, myxoviruses) carry RNA polymerases to synthesise mRNAs. RNA viruses of this type are called negative-strand viruses, since their single-strand RNA genome is complementary to messenger RNA, which is conventionally designated positive-strand. Table 3 summarises the various pathways of transcrip­tion (but not necessarily those of replication) of the nucleic acids of different classes of viruses.

  Most of the viral mRNAs possess a sequence of polyadenylic acid [Poly (A)] at their 3′-end and an unusual blocked, methylated structure at the 5′-end called a cap. The precise function of these features is yet to be elucidated, but the capped structure appears to enhance initiation of translation. Viral mRNA is not always an exact copy of the genome template, since some mRNAs are processed or spliced to delete certain sequences.

 

Table 3. Pathways of nucleic acid transcription for various virus classes.

 

DS = double-stranded; SS = single-stranded; “–” indicates negative strand;  “+” indicates positive strand;

“+” indicates a helix containing a positive and a negative strand

Virus multiplication was first studied suc­cessfully in bacteriophages. For animal vi­ruses, some of the steps of the interaction between the infecting virus and susceptible cells have now been elucidated.

The following sections describe the replication of an RNA and a DNA virus.

 

RNA Virus Replication (Fig 4). Poliovirus contains a single-stranded RNA as its genome. All of the steps are independent of host DNA and occur in the cell cytoplasm. Polioviruses adsorb to cells at specific cell receptor sites (step 1), losing in the process one virus polypeptide (VP4), which may, therefore, be important in adsorption. The sites are specific for virus coat-cell interactions. Whereas in­tact poliovirus infects only primate cells in culture, the isolated RNA also infects nonprimate cells (rabbit, guinea pig, chick) and completes one cycle of multi­plication. Multiple cycles of infection are not observed ionprimate cells because the resulting progeny pos­sess protein coats and will again infect only primate cells. After attachment, the virus particles are taken into the cell by viropexis (similar to pinocytosis) (step 2), and the viral RNA is uncoated (step 3). The single-stranded RNA then serves as its own messenger RNA. This messenger RNA is translated (step 4), resulting in the formation of an RNA polymerase that catalyzes the production of a replicative intermediate (RI), a par­tially double-stranded molecule consisting of a com­plete RNA strand and numerous partially completed strands (step 5). At the same time, inhibitors of cellular RNA and protein synthesis are produced. Synthesis (+) and (—) strands of RNA probably occurs by similar mechanisms; this is completely elucidated only (+) strands. Here the RI consists of one complete ( strand and many small pieces of newly synthesized ( strand RNA (step 6). The replicative form (RF) consists of 2 complete RNA strands, one (+) and one (-).

The single (+) strand RNA is made in large amounts and may perform any one of 3 functions: serve as messenger RNA for synthesis of structural proteins, (b) serve as template for continued RI replication, or (c) become encapsidated, resulting mature progeny virions. The synthesis of viral cap proteins (step 7) is initiated at about the same time RNA synthesis.

The entire poliovirus genome acts as its own mRNA, forming a polysome of ~350S, and is translated to form a single large polypeptide that is processed during and after translation to form the various viral polypeptides. Thus, the poliovirus genome serves as a polycistronic messenger molecule. The giant polypeptide is cleaved to form a capsid precursor protein and 2 noncoat proteins one of which undergoes further processing. The capsid precursor protein cleaved into coat proteins VPO, VP 1, and VP3. During encapsidation, VPO is cleaved into coat proteins VP2 and VP4.

Completion of encapsidation (step 8) produces mature virus particles that are then released when cell undergoes lysis (step 9).

 

DNA Virus Replication (Fig 5). In pox virus replication, synthesis of virus components and assembly of virus particles occur wit the cytoplasm of the infected cell. The replication of other DNA viruses (including the adeno-, herpes-, and papovavirus families) differs in that viral DNA is replicated in the nucleus, where viral proteins are synthesized in the cytoplasm, 1 lowed by their migration to and assembly withiucleus. Fig.5 shows the steps in the replication adeno virus, a double-stranded DNA virus. Adsorption (step 1) and penetration (step 2) of the virus into cell are similar to steps described for poliovirus. addition to viropexis, enveloped viruses penetrate fusion of the virus envelope with the plasma membrane, releasing the nucleocapsid into the cytoplasm.

After the virus enters the cell, the protein coat removed (step 3), presumably by cellular enzymes and the viral DNA is released into the nucleus. One both DNA strands are transcribed (step 4) into spec mRNA, which in turn is translated (step 5) to synthesize virus-specific proteins, such as a tumor anti, and enzymes necessary for synthesis of virus DNA. This period encompasses the early virus functions. Host cell DNA synthesis is temporarily elevated an then suppressed as the cell shifts over to the manufacture of viral DNA (step 6). As the viral DNA continues to be transcribed, Iate virus functions become apparent. Messenger RNA transcribed during the later phase of infection <step 6) migrates to the cytoplasm and is translated (step 7). Proteins for virus capsids are synthesized and are transported to the nucleus to be: incorporated into the complete virion (step 8). The migration of some structural proteins of certain viruses from the cytoplasm to the nucleus can be inhibited when arginine is absent from. the growth medium. Assembly of the: protein subunits around the viral DNA results in the formation of complete virions (step 9), which are released after cell lysis.

Summary of Viral RepIication. The molecular events that have been discussed above are summarized in Fig. 6. Virus genomes containing double-stranded (ds) nucleic acid proceed along most of the: steps shown in this figure.

Viruses with single-stranded (ss) nucleic acid only some of the steps. For the orthomyxovirus, the  RNA template is utilized for the synthesis of complementary RNA strand that produces the replicative form of the nucleic acid. This in turn serves template for the synthesis of the progeny viral RNA. For the retroviruses, the ssRNA acts as a template for the RNA-dependent DNA polymerase (revertase transcriptase) to synthesize dsDNA. The dsDNA molecules are then used as templates for the transcription and synthesis of ssRNA molecules that serve either as viral mRNA molecules or as viral genomes for encapsidation by the viral structural proteins.

Figure 4.  Replication of poliovirus, which containing an RNA genome

 

 

Figure 5. Steps in the replication of adenovirus, which contains DNA in its genome

 

Control Mechanisms of Virus Replication. In the course of virus replication, all the virus-specified macromolecules are synthesized in a highly organized sequence, although virus components are usually made in excess. In some virus infections, early viral proteins are synthesized soon after infection and late proteins are made only late in infection, after viral DNA synthesis. Early genes may or may not be shut off when late products are made. In addition to these temporal controls, quantitative controls also exist, since not all virus proteins are made in the same amounts. Virus-specific proteins may regulate the ex­tent of transcription of genome or the translation of viral messenger RNA. .Although the exact mechanism of these controls is unknown, we do know something about the mechanism of mRNA synthesis. Small ani­mal viruses and bacteriophages are good models for study of gene expression. Their small size has enabled the total nucleotide sequence of a few small DNA phages and SV40 to be elucidated. This led to the discovery that some pieces of DNA are expressed twice by mRNA, being read off either in 2 reading frames or by 2 mRNA molecules with different starting points being read in the same frame.

One surprising discovery has been the observa­tion that animal “virus mRNA molecules (at least for adeno virus and SV40) are not direct copies of their DNA genomes. In these viruses, the mRNA sequences coding for a given protein are preceded in the mRNA molecule by short sequences from farther “upstream”  on the DNA template, with intervening sequences spliced out. This suggests the possibility that modifica­tion and control of virus gene expression could occur at the level of mRNA construction or “splicing”.

Laboratory diagnosis of viral infections is based on either detect­ing a causative agent or demonstrating specific antibodies in the blood.

Virus isolation from a patient is performed, using cell cultures, chicken embryos, or experimental animals. Isolated viruses are identified with such serological tests as virus neutralization, haemagglutination inhibition, and others.

Specific antiviral antibodies in patients’ blood are studied over time, using paired sera. An increase in the serum antibody titre becomes diagnostically significant only when it is of at least a four­fold magnitude. Rapid diagnostic techniques (immunoelectron mi­croscopy, immunofluorescence test, radioimmunoassay, enzyme-linked immunosorbent assay, etc.) are increasingly gaining in impor­tance.

 

VIRUS DETECTION AND IDENTIFICATION IN CELL CULTURES

There are primary, diploid, and continuous cell cultures. Primary cultures are obtained directly from animal or human tissue by breaking the intercellular substance with proteolytic en­zymes (trypsin, collagenase, pronase). Dissociated (dispersed) cells placed in a culture medium are capable of adhering to the surface of a culture vessel and of proliferating there. Since cells of most primary cultures remain viable for several generations, they may be repeated­ly subcultured (passaged). Several passages may produce a diploid culture, i.e., a population of fibroblast-like cells which can be rapidly reproduced and endure 30 to 60 passages still retaining their initial sets of chromosomes. Human diploid cells are highly sensitive to numerous viruses and are extensively used in virology. Both human (WI-38, MRC-5, MRC-9, IMR-90, etc.) and animal (cow, swine, sheep, and lamb) diploid cell cultures have been obtained.

Continuous cell cultures can be subcultured endlessly. They are derived from the primary cultures of cells due to their genetic vari­ability during the growing process, rapidly become dominant in the cell population, and have chromosomal sets typical of all contin­uous cell lines. Continuous (stable) cell cultures have been obtained from various normal and neoplastic human tissues: amnion (A-0, A-1, FL), kidneys (Rh), cervical carcinoma (HeLa), laryngeal carci­noma (Hep-2), bone marrow from patients with lung cancer (Detroit-6), human embryo rhabdomyosarcoma (RD), etc.

Continuous cell lines are stored in liquid nitrogen and thawed before use.

Cells are cultivated in glass or plastic vessels of various size and shape, preferably disposable, with sterility strictly observed at all stages of cultivation. Nutrient media for cell cultures contain the whole range of amino acids, vitamins, and growth factors. Com­mercially available are fluid (medium 199, Eagle’s medium, lactic albumin hydrolysate) and dry media or concentrates which are dissolved before use.

There are growth and maintenance culture media. Cell cultures are grown, using growth media enriched with human or animal sera, e.g., bovine or foetal (embryonic) cow serum. The serum makes up 2 to 30 per cent of the medium, depending on the properties of the cell culture and composition of the medium.

The maintenance media are used to preserve the established cell monolayers during virus inoculation. These media contain a smaller amount of serum, or they are added to the culture without it. Before the medium is used, antibiotics are added to it in order to prevent the growth of possible extraneous microorganisms. Culture media are sterilized; if they contain unstable constituents, filtration is carried out. Using buffer systems (commonly, bicarbonate buffer), the pH of the medium is maintained at 7.2-7.6. An indicator is added to the media, e.g., phenol red which becomes orange-yellow in acid medium or crimson in alkaline medium.

Obtaining Cell Culture

Primary cell cultures are obtained from any animal or human embryonic tissue, since embryonic cells have a high ability for growth and proliferation. Cultures are commonly prepared of a mixture of several tissues, such as skin, bone, and muscle. Commonly used are human embryonic fibroblasts, chicken embryonic fibroblast, and hu­man kidney cells. Human embryonic tissues from aborted pregnan­cies and 8-12-day-old chicken embryos are used for cell cultures.

Preparing cell suspension. The tissue is washed in Hanks’ solution or antibiotic-containing phosphate buffer to remove blood, fat, cell detritus or other admixtures, chopped with scissors, and washed once again until the solution is clear. Then, it is immersed in trypsin (200-300 ml per 100 g of tissue) and dispersed with a magnetic mixer or pipette. The supernatant containing trypsin-treated cells is decanted and stored in a refrigerator at 4 °C.

Trypsinization is repeated several times. The cell suspension is centrifuged at 600 X g for 5-10 min, resuspended in the medium, and stained with fuchsine, methylene blue, or other dyes. Cell concentra­tion is determined in a Goryaev counting chamber. The suspension is diluted with nutrient medium to a concentration of 400 000-800 000 cells per ml, dispensed into culture flasks, tightly stoppered with rubber plugs, and incubated at 35-37 °C for 48-96 hours (flasks are tilted at a 5 degree angle); thereafter, cultures with well-formed monolayers are harvested.

Passage of continuous (stable] cell cultures. After pipetting off the nutrient medium, pour solution of 0.25 per cent trypsin or 0.02 per cent versene warmed to 37 °C into cell-containing flasks and place them in an incubator for 3-5 min. Then, remove trypsin or versene, add a small amount of the nutrient medium into the vessel, and make the cells suspend in the medium by vigorous shaking. Count the cells, adjust the cell concentration to the required level, and dispense the continuous culture into new flasks.

Suspension cultures. Most continuous cell cultures can proliferate in the medium in a suspended state, which is achieved by automatic rotation in a special drum or cultivation in fermenters.

For virus isolation, the medium is decanted from test tubes with established monolayers, and cells are washed several times with Hanks’ solution to remove serum antibodies and inhibitors. A 0.1-0.2-ml portion of the tested material, examined for sterility and appropriately pretreated for virological examination, is placed in every test tube (see p. 189). At least two tubes are used for one test. Some 30-60 min after inoculation, 1 ml of maintenance medium is added to each tube which is then placed in a 37 °C incubator.

If the examined material (e.g., faeces) has a toxic effect on the cell monolayer, it is initially diluted with 1 ml of nutrient medium and, after a 30-60-min exposure to the cell culture, is removed and replaced with the maintenance medium.

 

Virus Detection

Detection of viruses in the cell culture is based on their cytopathic effect, electron microscopic identification of intracellular inclusions, immunofluorescence, and haemadsorption and also on interference and the “plaque” formation phenomena. A positive haemagglutination test indicates the presence of a virus in the culture fluid.

A cytopathic effect (CPE) represents degenerative cell alterations resulting from intracellular virus reproduction. It is manifested within the first days after cell culture inoculation with some viruses ‘(variola, polio, etc.) and much later (in 1-2 weeks) when others (adenoviruses, parainfluenza viruses, etc.) are used. The nature of CPE primarily depends on a virus species.

Monolayer cell degeneration is subdivided into total and partial. Total degeneration due to such viruses as polio, Coxsackie and ECHO significantly affects monolayer cells, with great numbers of them sloughing off the slide. The remaining separate cells are shrunken (nuclear and cytoplasmic pyknosis) and characterized by double refraction, i.e., strong fluorescence on microscopy.

Partial degeneration of cultured cells falls into several types:

(1) Racemose formation: rounding, enlargement, and partial conflu­ence of cells producing characteristic racemose aggregates. Degenera­tion of this type is caused by adenoviruses.

(2) Focal degeneration: local cell injuries (microplaques) appearing against the background of a largely intact monolayer. This type of degeneration is induced by certain strains of variola, variola vacci­nes, and influenza viruses.

(3) Symplast formation: virus-induced cell aggregation resulting in the formation of giant multinuclear cells (symplasts or syncytia). Symplast formation is caused by measles, mumps, parainfluenza, respiratory-syncytial, and herpes viruses.

Certain oncogenic viruses cause malignant transformation of cells provoking their intense proliferation, in other words changes of a proliferative type.

If the CPE in infected cell cultures is absent or mild, “blind passages” are performed, i.e., new cell cultures are inoculated with the culture fluid.

Intracellular inclusions occur when certain viruses are reproduced in cell nuclei and cytoplasm (variola, rabies, influenza, herpes viru­ses, etc.). They are detected by light microscopy after staining a monolayer-carrying slide with the Romanowsky-Giemsa solution or with other dyes, or by the luminescent microscopy, using acridine orange (1:20000).

Depending on a virus type, solitary virions or their crystalloid clusters can. be visualized in cell nuclei and cytoplasm with the electron microscope.

A specific virus antigen can be detected in virus-infected cell cul­tures. using the direct immunofluorescence test.

Plaque formation. Plaques, or negative virus colonies, are sites of virus-destroyed cells in the agar-coated monolayer. Infective virus activity is quantified by counting these colonies.

 

 

To obtain the plaques, different dilutions of virus suspension are streaked onto one-layer tissue cultures in Hat vials or Petri dishes and overlaid with a layer of nutrient agar; virus reproduction and CPE are thus confined to initially infected and adjacent cells. Sites of cell degeneration, i.e., plaques, are identified by staining the culture with neutral red which is either included in the composition of the agar layer or added immediately before reading the results. Con­sisting of dead cells, the plaques are not stained with neutral red and, therefore, are recognized as light spots on a pink-red cell monolayer.

Other techniques of detecting virus plaques in cell cultures are also available, e.g., demonstration of plaque formation under a bentonite layer. Finely dispersed purified bentonite is added to a fluid nutrient medium, and the infected cell monolayer is immersed with this mixture. Because of adsorption of bentonite particles on cell surfaces, the monolayer becomes milk-coloured. At sites of virus reproduction (plaques), cells are not covered with bentonite, and are partially or completely stripped off the slide.

Virus plaques are identified under the bentonite nutrient layer, using mono-layer cultures of continuous human or animal cells sensitive to the tested virus; 1-2-day-old thin cell monolayers are also suitable for this purpose.

Ten-fold dilutions of the material to be tested are prepared, and at least two culture matrasses (Erienmeyer flasks or penicillin vials) are inoculated with every dilution. After virus adsorption (30-40 min) the monolayers arc washed 3-4 times with sterile sodium chloride solution and coated with a bentonite nutrient layer: bidistilled water (415ml), 5-fiper cent bentonite gel (5 ml), Earle’s solution, ten-fold concentrate (50ml), native bovine serum (15 ml), 7.5 per cent sodium hydrocarbonate solution (15 ml), penicillin (200 U/ml), streptomy­cin or lincomycin (100 U/ml).

An infected cell monolayer in 50-ml Erienmeyer flasks is covered with 20-30 ml and that on bottoms of penicillin vials with 5-6 ml o[ bentonite.

Bentonite gel is obtained from dry mineral. Sorbent properties of bentonite are improved by impregnating it with sodium cations. Finally, it is sterilized for 40 min at 111 °C. Bentonite gel may be stored at room temperature for years without any changes in its sorbent properties.

Time of plaque formation under a bentonite layer varies with different viru­ses. Enterovirus plaque formation is evaluated, for example, in 36-48 hours. Culture flasks are inverted with the monolayer upward, washing degenerated cells away with the medium. Plaques formed by different enteroviruses vary in size, development, and margin patterns.

Since one infective viral particle (virion) produces one plaque, the plaque formation test accurately measures both the number of infective units in the specimen and the neutralizing activity of virus antibodies.

Haemagglutination test is based on the ability of certain viruses to clump (agglutinate) red blood cells obtained from animals of definite species. Influenza and some other viruses with supercapsid membrane contain the surface antigen haemagglutinin responsible for the erythrocyte agglutination.

The HA test is performed in test tubes, on special plexiglass plates, and in a Takata apparatus. A virus-containing specimen is double-diluted in 0.5 ml of isotonic saline. Half a millilitre of 1°n erythrocyte suspension thrice washed in isotonic saline is added into all test tubes, and 0.5 ml of erythrocyte suspension is mixed with an equal volume of virus-free isotonic sodium chloride solution, to be used as control. The mixture may be incubated at 37°, 20° or 4 °C, depending on the properties of the tested virus.

Test results are assessed at 30-60 rain after complete erythrocyte sedimentation in the control, with the findings reading as follows:

(++++), intense and rapid erythrocyte agglutination with a star-like, marginally festooned sediment (“umbrella”}; (++4-), residue of erythrocytes has clearings; (++), a less marked residue; (+), a floccular sediment surrounded with lumps of agglutinated erythro­cytes, and (–), a markedly localized erythrocyte sediment (“rou­leaus”), as in the control.

Using HA, one can detect the presence of an agglutinating virus in the specimen and determine its titre. The virus titre is defined as the maximum virus dilution at which erythrocyte agglutination still occurs. This dilution is accepted as containing one haemagglutinating unit of the virus.

Results of the haemagglutination test are influenced by several factors: species and individual sensitivity of erythrocytes, tempera­ture and pH of the medium, etc. Furthermore, erythrocyte haemagglutination may be induced by certain microorganisms, such as staphylococci, Escherichia, salmonella, shigella, cholera vibrio El Tor. Therefore, in cases where viruses are detected in bacteria-contaminated material, caution should be exercised while interpret­ing the results obtained.

Haemadsorption test makes it possible to reveal the virus before the onset of CPE due to the appearance of the virus-specific antigen (haemagglutinin) on the surface of an infected cell. After a peri­od of incubation appropriate for a virus, 0.2 ml of 0.5 per cent erythro­cyte suspension is added to the cell culture (both control and virus-infected) so that the monolayer is covered, and the culture is stored for 15-20 min at 4°, 20° or 37 °C (depending on virus properties). Then, the test tubes are shaken in order to remove unadsorbed erythrocytes, and erythrocyte clusters are counted on single cells or throughout the monolayer by low-magnification microscopy. Uninfected cells should carry no erythrocytes.

 

VIRUS DETECTION AND IDENTIFICATION IN CHICKEN EMBRYOS

Virus demonstration in chicken embryos. Viruses are cultivated in 6-15-day-old chicken embryos. The material to be assayed is introduced with a syringe onto the chorio-allantoic membrane (CAM), into the yolk sac, and into the amniotic and allantoic cavity.

To infect the CAM (Fig. 7, 2), the eggshell is treated with iodine and alcohol, punctured above the air sac and a 7×2 mm opening is made laterally at the place of the greatest vascular ramification. Without destroying the shell-underlying membrane, 0.1-0.2 ml of virus-containing material is placed onto the CAM with a short thieedle. The damaged sites of the shell are coated with sterile paraffin or collodion. Then, the embryos are put into an incubator, putting the eggs horizontally.

 

 

Figure 7. Different methods of chicken embryo inoculation

 

For inoculation into the allantoic cavity the tested material is introduced through the lateral opening of the shell ID-15 mm deep.

When the amniotic cavity  is to be inoculated, the virus-containing specimen is injected through the opening at the obtuse end of the egg; the needle should be directed toward the embryonic body so as to ensure penetration of the virus into different organs and tissues of the embryo. Puncture sites are sealed with paraffin or collodion.

The infected embryos are stored in an incubator at 35-37 °‘C for 48-72 hrs, depending on the species of the assayed virus. Then, the eggs are cooled at 4 °C for 18 hrs for maximum constriction of the embryonic blood vessels and opened under sterile conditions. The amniotic and allantoic fluid is aspirated with a syringe, and the membranes and embryo are transferred into sterile Petri dishes.

Virus reproduction in chicken embryos is evidenced by character­istic changes on the CAM. Viruses in the amniotic and allantoic fluid can be recovered by means of HA.

Variola, variola vaccine, and herpes simplex viruses produce plaques on the CAM, which look like whitish convex spots 1 to 2 mm in diameter, with their number corresponding to the number of infectious particles.

Erythrocyte agglutination induced by the allantoic and amniotic fluid of infected embryos is a marker of orthomyxovirus and paramyxovirus accumulation. Quantitatively, the virus is determined by the haemagglutination litre (maximum dilution of virus-containing fluid causing erythrocyte agglutination).

Viruses can be titrated on pieces of the chorio-allantoic membrane. Pieces of the shell from an 11-12-day-old chicken embryo with the intact CAM are placed in wells on sterile plates, 0.5-1 ml of virus-containing fluid diluted ten-fold with a buffer (NaCl (8.0 g), KC1 (0.6 g), glucose (0.3 g), MgCl₂x6H₂O (0.05 g), CaCl₂-6H₂O (0.8 ml), phenol red 1:100 (10.0 g), NaOH 1M (0.2 ml), gelatine (2.0 g), antibiotics) is added, and the plates are covered with foil and incubated at 35-37 °C. The shell is re­moved from the wells in 24-72 hrs and 0.5 per cent chicken erythrocyte suspension is added to the remaining medium. A positive HA test indicates virus reproduction.

Viruses cultivated in chicken embryos are identified with the aid of the neutralization test which is interpreted as positive if plaque formation is inhibited on the CAM and haemagglutination is absent; other identification techniques include the HAI, PG, CF, and IF tests.

 

According to the nature of the material to be tested and the proce­dures utilized, the methods for diagnosing viral infections may be categorized into rapid, viroscopic, virological, and serological (Table).

Table

 Methods of the Diagnosis of Viral Infections

 

 

 

viruses of bacteria (bacteriophages). Morphology, chemical composition, phase of interaction of bacteriophage AND bacterial CELL. Phage conversion. Practical IMPORTANCE of THE phenomenon of bacteriophagia

 

Bacteria are host to a special group of viruses called bacteriophage, or «phage», which pass through fine-porous filters and develop at the expense of reproducing bacteria (N. Gamaleya, 1892; F. Twort, 1915; F. d’Herelle, 1917). Although any given phage is highly host-specific, it is probable that every known type of bacterium serves as host to one or more phages. Phages have not been successfully used in therapy. They are important, however, because they furnish ideal materials for studying host-parasite rela­tionships, virus multiplication, and molecular ge­netics.

LIFE CYCLES OF PHAGE AND HOST

Figure 1 summarizes the potential life cycles of bacterial cells infected with double-stranded DNA phages. Single-stranded DNA phages and RNA phages are discussed in later sections. Fig 1 shows the following:

 

(1) Life cycle of uninfected bacterium. An uninfected bacterium may reproduce by binary fission, showing no involvement with phage.

(2) Adsorption of free phage. When an uninfected bacterium is exposed to free phage, infection will take place if the cell is sensitive. Bacteria may also be genetically resistant to phage infection; such cells lack the necessary receptors on their surfaces.

When infection takes place, the phage is adsorbed onto the cell surface and the nucleic acid of the phage penetrates the cell. In this state phage nucleic acid is called “vegetative phage”.

(3) Lytic infection. The injected vegetative phage material may be reproduced, forming many replicas. These mature be acquisition of protein coats, following which the host cell lyses and free phage is liberated.

(4) Reduction of vegetative phage to prophage. Many phages, termed “temperate”, are capable of reduction to prophage as alternative to producing a lytic infection. The bacterium is now lysogenic; after an indeterminate number of cell division, one of its progeny may lyse and liberate invective phage.

(5) Loss of prophage.  Occasionally a lysogenic bacterium may lose its prophage, remaining viable as an uninfected cell.

Figure 1.  Phage-host life cycles

 

METHODS OF STUDY

Assay. Since phages (like all viruses) multiply only within living cells, and since their size precludes direct observation except with the electron microscope, it is necessary to follow their activities by indirect means. For this purpose, advantage is taken of the fact that one phage particle introduced into a crowded layer of di­viding bacteria on a nutrient agar plate will produce a more or less clear zone of lysis in the opaque film of bacterial growth (Figure 2). This zone of lysis is called a «plaque»; it results from the fact that the initially infected host cell bursts (lysis) and liberates dozens of new phage particles, which then infect neighbouring cells. This process is repeated cyclically until bacterial growth on the plate ceases as a result of exhaustion of nutrients and accumulation of toxic products. When handled properly, each phage particle produces one plaque; any material containing phage can thus be titrated by making suitable dilutions and plating mea­sured samples with an excess of sensitive bacteria. The plaque count is analogous to the colony count for bacterial titration.

 

Isolation and Purification. In order to study the physical and chemical properties of phage, it is necessary to prepare a large batch of purified virus as free as possible of host cell material. For this purpose, a liquid culture of the host bacterium is inoculated with phage and incubated until the culture is completely lysed. The now clear culture fluid, or lysate, contains in suspension only viral parti­cles and bacterial debris. These materials are easily separated from each other by differential centrifugation. The centrifuged pellet of phage material can be resuspended and washed in the centrifuge as often as needed and may then be used for chemical and physical analysis in the laboratory or for electron microscopy.

 

 

Figure 2. Different phage plaque types (1 – large colonies; 2 – small colonies)

 

PROPERTIES OF PHAGE

One group of phages has been studied more ex­tensively than any other: certain phages that attack Escherichia coli strain B (coliphages). Of the numer­ous coliphages, 7 have been selected for intensive study. Unless otherwise noted, the information given below applies to this group, which has beeumbered Tl through T7.

Morphology. A typical phage particle consists of a “head” and a “tail”. The head represents a tightly packed core of nucleic acid surrounded by a protein coat, or capsid. The protein capsid of the head is made up of identical subunits, packed to form a prismatic structure, usually hexagonal in cross section. The smallest known phage has a head diameter of 25 nm; others range from 55 x 40 nm up to 100 X 70 nm.

The phage tail varies tremendously in its com­plexity from one phage to another. The most complex tail is found in phage T2 and in a number of other coli and typhoid phages. In these phages, the tail consists of at least 3 parts: a hollow core, ranging from 6 to 10 nm in width; a contractile sheath, ranging from 15 to 25 nm in width; and a terminal base-plate, hexagonal in shape, to which may be attached prongs, tail fibers, or both. Electron micrographs of phage preparations embedded in electron-dense material such as phosphotungstate show the phages to exist in 2 states: in one, the head contrasts highly with the medium, the sheath is expanded, and the base-plate appears to have a series of prongs. In the second state, the head is of low contrast, the sheath is contracted, and the base­-plate is now revealed to have 6 fibers attached to it. The former state presumably represents active phage, con­taining nucleic acid; the latter state presumably repre­sents phage that has ejected its nucleic acid (eg, into a host cell). These 2 states are diagrammed in Fig 3.

 

Figure 3.  Diagrams of phage T2 on electron micrographic observation

 

A number of other tail morphologies have been reported. In some of these, sheaths are visible but the contracted state has not been observed; and in one case no sheath can be seen. The phages also vary with respect to the terminal structure of the tail: some have base-plates, some have «knobs,» and some appear to lack specific terminal structures.

The phage tail is the adsorption organ for those phages that possess them. Some phages lack tails al­together; in the RNA phages, for example, the capsid is a simple icosahedron.

Although most phages have the head-and-tail structure described above, some filamentous phages have been discovered that possess a very different morphology. One of these, called «fd,» has been characterized in some detail. It is a rod-shaped struc­ture measuring 6 nm in diameter and 800 nm in length. It contains DNA and protein, which are complexed in a manner that is not completely understood. The DNA may be intertwined with the protein, rather than form­ing a core.

Chemistry. Phage particles contain only protein and one kind of nucleic acid. Most phages contain only DNA; how­ever, phages that contain only RNA are also known. In the T-even phages, the nucleic acid makes up about 50% of the dry weight and consists of a single molecule (called the phage chromosome) with a molecular weight of 1.3 x 10⁸, sufficient to code for about 200 different proteins of molecular weight 30,000. In phages T2, T4, and T6, a unique base (hy-droxymethylcytosine) is present to which are attached short chains of glucose units. This pyrimidine has never been found in the nucleic acid of the uninfected bacterial host.

The proteins that make up the head, the core, the sheath, and the tail fibers are distinct from each other; in each case, the structure appears to be made of repeating subunits.

An unusual phage called PM2 has been isolated from a culture of a marine pseudomonad. PM2 is a double-stranded DNA phage in which the virion is surrounded by a lipoprotein  membrane and contains 2 enzymes: an endonuclease that converts the phage DNA to the linear form within the host, and a DNA-dependent RNA polymerase.

The resistance of phages to physical and chemical factors is greater than that of the corresponding microbes. Phages withstand high pressures (up to 6000 atmospheres), are resistant to the action of radiant energy, and maintain their activity in a pH range from 2.5 to 8.5. In sealed ampoules phages do not lose their lytic properties for 5-6 and even 12-13 years and can be preserved for relatively long periods in glycerin. Phages perish quickly under the effect of boiling, acids, ultraviolet rays and chemical disinfectants. In relation to resistance phages are intermediate between the vegetative forms of bacteria and spores. Some substances (thymol, chloroform) and enzyme poisons (cyanide, fluoride, dinitrophenol) have no effect on phages, but cause bacteria to perish or inhibit their growth. These preparations are used for maintaining phages in cultures and for destroying bacteria, actinomycetes and fungi.

The specific action of phages. Phages possess both species and type specificity. On solid nutrient medium types Tl, T3, and T7 E. coli phages form large colonies, types T2, T4, and T6 produce small colonies. These types also differ morphologically.

The classification of phages is based on morphology, chemical structure, type of nucleic acid, and their interaction with the bacterial cell. All phages are divided into DNA- and RNA-containing. The relation of phages to sex differentiation of bacteria is taken into account in determination of additional taxonomic signs. It has been established that one group of phages affects only male bacteria (F⁺), another group only female bacteria (F^–), while a third group of phages is indifferent in respect to sex differentiation of cells. Phages are marked by a specific effect on the corresponding bacterial species. Each phage has its own host in which it lives as a parasite and reproduces. Staphylococci have 40 phage types, E coli 50, S. typhi 56, S. paratyphi A 11, S. schottmueleri B 7, Corynebacterium diphtheriae 19, Vibrio cholerae 9, etc.

 

PHAGE REPRODUCTION

The bacteriophage phenomenon depends on the age of the culture, the concentration of bacteria, phage activity, bacterial phage resistance, composition of the nutrient medium, temperature and other factors. It is manifested in four main phases occurring in succession: adsorption, penetration into the cell, intracellular development, and liberation of phages.

Adsorption. The kinetics of phage adsorption have been thoroughly analyzed, and the process has been shown to be a first-order reaction; the rate of adsorption is proportional to the concentration of both the phage and the bacterium. Under optimal conditions, the ob­served rates are compatible with the assumption that almost every collision between phage and host cell results in adsorption. If the bacteria are mixed with an excess of phage, adsorption will continue until as many as 300 particles are adsorbed per cell.

Before the phage can be adsorbed onto the host cell, the phage surface must be modified by attachment of positively charged cations (the nature and number of cations varying from one phage to another, for example, bivalent cations Ca ⁺⁺, Mg⁺⁺) and, in some cases, the amino acid tryptophan. Each phage is quite specific with regard to the cofactors required for adsorption.

The bacterial surface, i.e., the cell wall, is complex and heterogeneous. In gram-negative bacteria, there appear to be 3 distinct layers: an inner layer composed of peptidoglycan, the outer membrane, and lipopolysaccharide. Different bacterial strains are highly specific with regard to the phages that they will adsorb. For example, a strain able to adsorb phages T2, T4, and T6 can give rise to mutants unable to adsorb one or another of these viruses. This specificity has been found to reside in the cell wall; when cell walls are isolated and purified, they exhibit the same adsorption patterns as the cells from which they are prepared. The factors in the cell wall responsi­ble for adsorption appear to be discrete, localized «re­ceptors»; the receptors for phages T3, T4, and T7 reside in the lipopolysaccharide layer, whereas the receptors for phages T2 and T6 reside in the outer membrane. Ability to adsorb phage is obviously a factor in the determination of bacterial sensitivity to infection.

In certain phages (eg, phages T2, T4, T6), the attachment of phage particles (or of empty phage capsids) causes a profound change in the cell membrane: at low phage multiplicities, the membrane becomes permeable to small molecules; and at high multi­plicities the cell lyses («lysis from without»). Even a single phage or ghost particle will affect the mem­brane, causing not only a permeability change but also the inhibition of host DNA and protein synthesis.

Penetration. Phages with contractile tails, such as the T-even phages (Fig 9-3), behave as hypodermic syringes, injecting the phage DNA into the cell. In phage T4, it has been found that the triggering of DNA injection requires the maintenance of a membrane potential by the host cell.

The filamentous DNA phages penetrate the host cell by a different mechanism. The entire phage struc­ture penetrates the cell wall; the major protein of the phage coat is then deposited on the cell membrane, which is penetrated by the phage DNA. A minor coat protein enters the cytoplasm along with the DNA.

Intracellular Development of DNA Phages. Some phages always lyse their host cells shortly after infection, generally in a matter of minutes and usually before the host cell can divide again. (See «lytic infection» cycle in Fig 1.) The process of intracellular development is as follows:

(1) For several minutes following infection (eclipse period), active phage is not detectable by artificially induced premature lysis (eg, by sonic oscil­lation). During this period, a number of new proteins («early proteins») are synthesized. These include cer­tain enzymes necessary for the synthesis of phage DNA: a new DNA polymerase, new kinases for the formation of nucleoside triphosphates, and a new thymidylate synthetase. The T-even phages (T2, T4, T6), which incorporate hydroxymethylcytosine in­stead of cytosine into their DNA, also cause the ap­pearance of a series of enzymes needed for the synthe­sis of hydroxymethylcytosine, as well as an enzyme that destroys the deoxycytidine triphosphate of the host. Later on in the eclipse period, «late proteins» appear, which include the subunits of the phage head and tail as well as lysozyme that degrades the peptidoglycan layer of the host cell wall. All of these enzymes and phage proteins are synthesized by the host cell using the genetic information provided by the phage DNA.

(2) During the eclipse period, up to several hundred new phage chromosomes are produced; as fast as they are formed, they undergo random exchanges of genetic material (see below).

In many phages, the linear DNA molecule that enters the cell has cohesive ends consisting of short complementary base sequences. Base pairing of these cohesive ends converts the DNA from the linear to the circular form; circularization is completed by a ligase-catalyzed sealing of the single-stranded gaps. Replication then occurs in the circular state, by either a simple-circle or a rolling-circle mechanism.

(3) The protein subunits of the phage head and tail aggregate spontaneously (self-assemble) to form the complete capsid. In the case of a complex capsid such as that of phage T4, capsid formation results from the coming together of 3 independent subassembly lines: one each for the head, the tail, and the tail fibers. Each subassembly proceeds in a defined sequence of protein additions.

(4) Maturation consists of irreversible combina­tion of phage nucleic acid with a protein coat. The mature particle is a morphologically typical infectious virus and no longer reproduces in the cell in which it was formed. If the cells are artificially lysed late in the eclipse period, immature phage particles are found in which the DNA and protein are not yet irreversibly attached, so that the DNA is easily removed.

Lysis and Liberation of New Phage. Phage synthesis continues until the cell disintegrates, liberating infectious phage. The cell bursts as a result of osmotic pressure after the cell wall has been weakened by the phage lysozyme. (The ex­ceptions are the filamentous DNA phages, in which the mature virus particles are extruded through the cell wall without killing the host).

 

REPLICATION OF RNA PHAGES

When a molecule of viral RNA enters the cyto­plasm of the host cell, it is immediately recognized as messenger RNA by the ribosomes, which bind to it and initiate its translation into viral proteins. One such viral protein is a complex enzyme, RNA polymerase. This enzyme brings about the replication of the viral RNA: it polymerizes the ribonucleoside triphosphates of adenine, guanine, cytosine, and uracil, using viral RNA as template.

The first step in the process of RNA replication is the formation of double-stranded intermediates, in which the entering viral RNA strand (called the “plus” strand) is hydrogen-bonded to the complemen­tary «minus» strand synthesized by the polymerase. The polymerase now uses the double-stranded molecule as a template for the repeated synthesis of new plus strands, each new plus strand displacing the previous one from the double-stranded intermediate.

As the newly synthesized plus strands are re­leased from the replicative intermediate, they are either used by the polymerase to form a new double-stranded intermediate or are assembled into mature virions by the attachment of coat protein subunits.

The complete nucleotide sequence of one RNA phage, MS2, has been determined. It is a single molecule, 3566 nucleotides in length, and contains 3 functional genes coding respectively for the RNA polymerase, the coat protein, and a second protein called the «A protein.» The single-stranded RNA molecule is capable of folding back on itself and form­ing double-stranded regions by base-pairing; the sec­ondary structure that results appears to play a major role in the regulation of viral RNA replication and translation.

 

PHAGE GENETICS

Phage particles exhibit the same 2 fundamental genetic properties that are characteristic of organized cells: general stability of type and a low rate of herita­ble variation.

Phage Mutation. All phage properties are controlled by phage genes and are subject to change through gene muta­tion. Most of our knowledge concerning the chemical basis of mutation comes from studies on phage genetics.

Phage Recombination. If a bacterium simultaneously adsorbs 2 related but slightly different DNA phage particles, both can infect and reproduce; on lysis, the cell releases both types. When this occurs, many of the progeny are observed to be recombinants. Recombination takes place between pairs of phage DNA molecules and is repeated many times between different, random pairs of replicating phage DNA before maturation. Three-way recombinants are therefore possible in a cell si­multaneously infected with 3 parental phage types.

Genetic Maps. The relative positions on the phage chromosome of mutant loci involved in phage structure or phage reproduction can be determined by a combination of genetic and physical mapping procedures. In genetic mapping, 2 different mutants are propagated simulta­neously in the same host cell, and the frequency of their recombination is measured: the lower the fre­quency, the shorter the distance between the 2 loci. In physical mapping, heteroduplexes are made between single strands of DNA from normal phage and deletion mutants; examination in the electron microscope re­veals the location of the deletion in the form of a non-base-paired region.

Some procedures have been used to produce de­tailed maps of phage chromosomes. An example of such a map is given in Fig 9-5 for the phage X. The genes lettered A-W on the map were originally iden­tified as conditional lethal mutations that were sup­pressed in a host strain carrying a particular suppressor gene; their functions were later identified by electron microscopy and biochemical analyses.

Phage genomes vary widely in size. The smallest known phage genome, that of the RNA phage MS2, has only 3 genes, as described above. In contrast, the largest phages contain sufficient DNA to code for about 200 proteins of average size; genes are present for coat proteins, morphogenesis, enzymes and regu­lators of phage replication, glycosylation of phage DNA, inhibitors of host restriction enzymes, enzymes that degrade host DNA, DNA repair enzymes, recom­bination enzymes, and proteins involved in integration and excision of prophage DNA.

 

LYSOGENY

Prophage. Earlier in this chapter it was mentioned that some phages (“temperate phages”) fail to lyse the cells they infect and then appear to reproduce synchronously with the host for many generations. Their presence can be demonstrated, however, because every so often one of the progeny of the infected bacterium will lyse and liberate infectious phage. To detect this event it is necessary to use a sensitive indicator strain of bac­terium, i.e., one that is lysed by the phage. The bacteria that liberate the phage are called “lysogenic”; when a few lysogenic bacteria are plated with an excess of sensitive bacteria, each lysogenic bacterium grows into a colony in which are liberated a few phage parti­cles. These particles immediately infect neighbouring sensitive cells, with the result that plaques appear in the film of bacterial growth; in the centre of each plaque is a colony of the lysogenic bacterium.

A culture of lysogenic bacteria can also be centrifuged, removing the cells and leaving the temperate phage particles in the supernatant. Their number can be measured by plating suitable dilutions of the super­natant on a sensitive bacterial indicator strain and counting typical plaques.

The release of infectious phage in a culture of lysogenic bacteria is restricted to a very few cells of any given generation. For example, in one bacterial type about 1 in 200 lyse and liberate phage during each generation; in another type it may be 1 in 50,000. The remainder of the cells, however, retain the potentiality to produce active phage and transmit this potentiality to their off spring for an indefinite number of genera­tions.

With the rare exceptions mentioned, lysogenic bacteria contaio detectable phage, either as mor­phologic, serologic, or infectious entities. However, the fact that they carry the potentiality to produce, generations later, phage with a predetermined set of characteristics means that each cell must contain one or more specific noninfectious structures endowed with genetic continuity. This structure is termed «prophage.»

The Nature of Prophage. Two entirely different prophage states are found in different phages. In one state, discovered in phage X, the prophage consists of a molecule of DNA inte­grated with the host chromosome. The chromosomes of E coli and of phage X are circular; the length of the phage chromosome is about one-fiftieth that of the bacterial chromosome. Both the phage and bacterial chromosomes carry a specific attachment site. The bacterial attachment site is immediately adjacent to the gal locus (see Fig 4); the phage attachment site is similarly located at a specific point on the phage ge­netic map. When l infects a cell of E coli, recombina­tion between the 2 attachment sites occurs, with the result that the 2 circles are integrated (Fig 4). This integration process requires the action of a phage gene product: phage mutants defective in this gene (the int locus) are unable to lysogenize the cell.

A number of coliphages are of the l type: their prophages integrate with the host chromosome at spe­cific attachment sites. One phage, called Mu, is un­usual in that it is capable of integrating at totally random sites on the chromosome, including sites within bacterial genes. Such integrations result in the inactivation of the gene in question and produce the appearance of mutations.

In the other state, discovered in phage PI, the phage chromosome circularizes and enters a state of «quiescent» replication that is synchronous with that of the host; no phage proteins are formed. The prophage in the «PI type» of system is not integrated with the chromosome; its replication is analogous to that of plasmids.

Further Properties of the Lysogenic System

A. Immunity. Lysogenic bacteria are immune to infection by phage of the type already carried in the cell as prophage. Wheonlysogenic cells are exposed to temperate phage, many permit phage multiplication and are lysed, while other cells are lysogenized. Once a cell carries prophage, however, neither it nor its progeny can be lysed by homologous phage. Adsorp­tion takes place, but the adsorbed phage simply per­sists without reproducing and is quickly “diluted out” by continued cell division.

 

 

Figure 4. The integration of prophage and host chromosome. (1) The phage DNA is injected into the host. (2) The ends of the phage DNA are covalently joined to form a circular element. (31 Pairing occurs between a sequence of bases adjacent to the ga/ locus and a homologous sequence on the phage DNA. (4) Breakage and reciprocal rejoining («crossing over») within the region of pairing integrates the 2 circular DNA structures. The integrated phage DNA is called prophage. The length of l DNA has been exaggerated for diagrammatic purposes. It is actually 1 -2% of the chromosomal length.

 

It has been shown that temperate phages cause the appearance in the cytoplasm of a repressor substance that inhibits multiplication of vegetative phage. Repressor also blocks the detachment of prophage (which otherwise would occur by the reversal of the integra­tion process described above) as well as the expression of other phage genes (eg, formation of phage proteins). The establishment of the lysogenic state is thus depen­dent on the production and action of repressor. The X repressor has been isolated and characterized as a pro­tein that specifically binds to l DNA.

The immunity of a lysogenic cell to homologous phage, mediated by a repressor, is clearly different from the phenomenon of «resistance» to virulent phage exhibited by certain bacteria. In the latter case, resistance is caused by failure to adsorb the phage.

B. Induction. «Vegetative phage» is defined as rapidly reproducing phage on its way to mature infec­tive phage, whereas «prophage « reproduces synchro­nously with the host cell. On rare occasions prophage «spontaneously» develops into vegetative (and later into mature) phage. This accounts for the sporadic cell lysis and liberation of infectious particles in a lysogenic culture. However, the prophage of practi­cally every cell of certain lysogenic cultures can be induced by various treatments to form and liberate infectious phage. For example, ultraviolet light will induce phage formation and liberation by most of the cells in a lysogenic culture at a dose that would kill very few nonlysogenic bacteria.

Induction requires the inactivation or destruction of repressor molecules present in the cell. Phage mu­tants have been obtained that produce thermolabile repressors: these phages can be induced simply by raising the temperature to 44 °C. Agents such as ul­traviolet light that damage host DNA induce prophage development by the following series of reactions the end result of which is the inactivation of phage repressor: (1) The DNA lesions are recognized by specific endonucleases that digest a short segment of one strand. (2) The single-stranded regions thus formed bind a protein called the recA protein (product of the recA gene), which acts as a protease. (3) The recA protein cleaves the phage repressor molecules, which have also bound to the single-stranded regions of DNA. Oligonucleotides, produced in the first step, are required to activate repressor cleavage.

C. Mutation to Virulence. When virulent phage is mixed with bacterial cells, all of the infected cells lyse. When temperate phage is mixed with non­lysogenic bacteria, some of the cells reproduce the phage and are lysed, while others are lysogenized.

Temperate phage can mutate to the virulent state. Two types of virulent mutants have been found. In one type, the mutation has made the phage resistant to the repressor, so that it can multiply even in lysogenic cells that are otherwise immune; in the other type, the phage has lost the ability to produce repressor. Virulent mu­tants of temperate phages are quite different from the naturally virulent phages such as T2. The latter cause the appearance of enzymes that degrade host DNA and stop the synthesis of ribosomal RNA, whereas the former do not interfere with the normal metabolism of the host in this manner.

D. Effect on Genotype of Host. When a lysogenic phage, grown on host «A,» infects and lysogenizes host “B”  of a different genotype, some of the cells of host “B” may acquire one or more closely linked genes from host “A”. For example, if the phage is grown in a lactose-nonfermenting host, about 1 in every million cells infected becomes lactose-fermenting. The transferred property is heritable. This phenomenon, called «transduction».

In other instances, phage genes may themselves determine new host properties. For example, the toxin of Corynebacterium diphtheriae and the toxins of many clostridia are determined by genes carried in prophage DNA. In Salmonella, phage infection con­fers a new antigenic surface structure on the host cell. The acquisition of new cell properties as the result of phage infection is called «phage conversion.» Phage conversion differs from transduction in that the genes controlling the new properties are found only in the phage genome and never in the chromosome of the host bacterium.

Genetic Regulation of Phage Reproduction. The vegetative and prophage modes of temperate phage reproduction are regulated by a complex series of genes that govern the transcription of different seg­ments of the phage DNA.

It should be recalled that l DNA is circularized immediately after penetration of the cell membrane and that the circular DNA may be replicated and ulti­mately combined with coat proteins to form mature virions, or alternatively may be integrated into the host chromosome by a recombinational event.

A. Regulation of Vegetative Replication and Maturation. Gene activity involved in productive phage growth occurs in 3 phases: (1) In the «immediate-early» phase, transcription initiates at promoters Pl and Pr proceeds left and right respec­tively, and terminates at the ends of the N and cro genes. These termination sites are designated t[, and tri; some rightward transcripts extend further, to ter­mination site trz. (2) In the «delayed-early « phase, the N gene product (protein) acts as an antitermination factor, allowing the above transcriptions to extend further through the genes for replication, recombina­tion, and regulation. (3) In the «late» phase, the cro protein acts at operators ol and or to reduce the initiation of early mRNA transcription from promoters pl and pr respectively. Also, Q protein activates rightward transcription from the promoter P’r, which continues through the lysis, head, and tail genes. (Re­member that the genome is circular, the m and m’ ends being joined during this phase.)

B. Regulation of Lysogenic Development. Dif­ferent genes are involved in the establishment and maintenance of lysogeny: (1) In the «establishment» phase, the ell and cIII proteins activate leftward tran­scription from the promoters Pe and P₁, thus transcrib­ing the cl and int genes; the ell and cIII proteins also inhibit rightward transcription of the lysis genes. (2) In the «maintenance» phase, the cl protein acts at operators Ol and Jr to repress nearly all transcription from promoters Pl and Pr. The cl protein also regu­lates its own synthesis by controlling leftward tran­scription from the promoter Pm. Transcription from this site is stimulated by low cl protein concentrations and inhibited by high cl protein concentrations.

The choice between the lytic and lysogenic modes of phage development depends on the relative concen­trations of the cl protein (the «l repressor») and the cro protein; the former is required for lysogeny and the latter for lytic growth. Both proteins bind to 3 repressor binding sites within the Or operator; whether tran­scription from the adjacent Pr promotor is inhibited or stimulated depends on their patterns of binding.

 

C. DNA Replication. Replication starts at the site marked ori and requires the activities of the pro­teins coded by phage genes 0 and P.

D. Integration and Excision of Prophage. The a-a’ attachment site is recognized by the int protein, catalyzing integration by crossing over at a specific attachment site on the host chromosome. Excision, brought about by a second crossover event, requires the activities of both the int and xis proteins.

E. Cleavage of the Circular DNA. Prior to its packaging in virions the circular DNA must be cleaved at a specific site (m-m’) to form linear molecules. This requires the activity of the A protein as well as the presence of phage head precursors.

Restriction and Modification. The phenomena of restriction and modificationwere discovered as a result of their effects on phage multiplication. It was ob­served that if phage l is grown in E coli strain K12, only about 1 in 10⁴ particles can multiply in strain B. The few that succeed, however, liberate progeny that infect B with an efficiency of 1.0 but infect strain K12 with an efficiency of 10^-4.

It was shown that DNA of particles formed in K 12 is modified by a K12 enzyme so as to be immune to degradation in K12. In strain B, however, the DNA of such particles is rapidly degraded by the restricting enzyme of the host. The few particles that escape restriction are modified by the specific modification enzyme of strain B; the progeny formed are now sus­ceptible to degradation in K12 but not in B. The mod­ifying enzymes have been shown to act by methylating bases at specific sites in the DNA.

Certain temperate phages carry genes that govern the formation of new modification and restriction en­zymes in the host. Thus, E coli cells carrying PI prophage will degrade all DNA not modified in a Pi-containing cell.

A given restricting enzyme recognizes a particular site on DNA and causes cleavage at that site unless the site has already been protected by the homologous modifying enzyme. Restriction appears to be a mechanism by which a cell protects itself against invasion by foreign DNA. Some phages have been found to have mechanisms for resist­ing restriction: Phages T3 and T7, for example, pro­duce an early protein that inhibits the host restriction endonuclease; in other cases, the phage codes for en­zymes that modify its DNA (eg, by glycosylation) so as to block the action of the restriction enzymes.

Distribution of phages iature. Phages are wide-spread iature. Wherever bacteria are   found – in the animal body, in body secretions, in water, drainage waters and in museum cultures, conditions may be created for the development of phages. Specific phages have been found in the intestine of animals, birds, humans, and also in galls of plants. and in nodules and legumes. Phage has been isolated from milk, vegetables and fruits.

River water, sea water and drainage waters quite frequently contain an abundance of phages in relation to various microbes including pathogenic (cholera vibrio, bacteria of enteric fever, paratyphoid, dysentery) organisms.

Sick people and animals, carriers and convalescents serve as the main source of phages against pathogenic microbes. In sick people the phage can be found not only in the intestinal contents, but in the urine, blood, sputum, saliva, pus, nasal exudate, etc. It is extremely easy to isolate the phage during the period of convalescence. The phage is employed for the determination of species and type specificity of the isolated cultures.This method has beeamed phage diagnostics.

The discovery of different phages against pathogenic microbes in the environment (water, soil) illustrates the presence in a given area of sick people and animals which excrete the corresponding agents or phages. This can be employed in giving an additional characteristic of the sanitary-epidemic state of water sources and the soil.

The isolation of the phage from the material under investigation has been carried out by a special direct method and an accumulation method. F. Sergienko. G. Katsnelson and M. Sutton. V. Timakov and D. Goldfarb devised a method for the rapid discovery of pathogenic bacteria in the environment with the help of the reaction of successive growth of the titre of the specific phage.

Production of a phage and the determination of its activity. The phage is obtained by adding a special maternal phage into vats with broth cultures, which are kept for one day at 37 C and then filtered. The filtrate is checked for purity, sterility, harmlessness and activity (potency).

Practical importance of the phage in medicine. Arising from the data obtained on the  mechanism of phage activity, phages have been used in prophylaxis and medical treatment against dysentery, enteric fever, paratyphoid, cholera, plague, anaerobic. staphylococcal, streptococcal, and other diseases. Bacteriophagia is used in the diagnosis of certain infectious diseases. With the help of special phages the species and types of isolated bacteria of the typhoid-dysentery group, staphylococci. causative agents of plague, cholera, etc.. are determined.

Phages are often very harmful in the manufacture of antibiotics and sour milk products as a result of inhibiting beneficial micro-organisms.

At present due to the introduction of antibiotics into practice phage therapy and prophylaxis of infectious disease are used to a limited extent.

Lysogenic bacteria are most suitable biological models for studying the interaction of the virus and cell. the mechanisms of toxigenicity, the biological efficacy of ionizing radiation, and other problems.

The phage is now used widely as a model in genetic research. The structure and function of the gene may be determined more exactly by means of this model.

 

 

 

Microflora of the environment

Microbes are distributed everywhere in the environment surrounding us. They are found in the soil, water, air, in plants, animals, food products, various utensils, in the human body, and on the surface of the human body.

The relationship of micro-organisms with the environment has beeamed ecology (Gr.  oikos home, native land, logos idea, science). This is an-adaptive relationship. Micro-organisms have a remarkable ability to adapt themselves to certain environmental conditions. Individual ecology, population ecology, and association community ecology are distinguished. The study of micro-organism ecology forms the basis for understanding parasitism, zoonotic diseases, and particularly diseases with natural nidality, as well as for elaborating measures for the control of various infectious diseases.

 

Soil Microflora

Soil science was founded by V. Dokuchaev, P. Kostychev. S. Vinogradsky, V. Williams, and others. Soil fertility depends not only on the presence of inorganic and organic substances, but also on the presence of various species of micro-organisms which influence the qualitative composition of the soil. Due to nutrients and moisture in the soil the number of microbes in 1 g of soil reaches a colossal number — from 200 million bacteria in clayey soil to 5 thousand million in black soil. One gram of the ploughed layer of soil contains 1-10 thousand million bacteria.

 

 

Soil microflora

Soil microflora consists algae nitrifying nitrogen-fixing, denitrifying, cellulose-splitting  and sulfur bacteria, pigmented microbes fungi, protozoa, etc.

The blue-green algae play an important part in enrichment of the soil with nitrogen. The extent to which the soil is contaminated with microbes depends on its nature and chemical composition (Table 1).

The greatest amount of microbes (1 000000 per cu cm) is found in the top layer of soil at a depth of 5-15 cm. In deeper layers (1.5-5 m) individual microbes are found. However, they have been discovered at a depth of 17.5 m in coal, oil, and artesian water.

Table 1

Total Amount of Microbes in Different Soils according to the Direct Counting Method

 

Oil’ bacteria live in oil wells. Using paraffins (distillates of oil) as nutrients, they turn part of the oil into a thick asphalt-like mass with the formation of which natural oil reservoirs become clogged up. It has been calculated that in the ploughed layer of cultivated soil over an area of 1 hectare there may be from 5 to 6 tons of microbial mass.

The number of microorganisms in the soil depends on the extent of contamination with faeces and urine, and also on the nature of treating and fertilizing the soil. For example, ploughed soil contains 2.5 times more microbes than forest soil.

Saprophytic spores (B. cereus. B, meguterium, etc.) survive for long periods in the soil.

Pathogenic bacteria which do not produce spores due to lack of essential nutrients, and also as a result of the lethal activity of light, drying, antagonistic microbes, and phages do not live long in the soil (from a few days to a few months) (Table 2).

Usually the soil is an unfavourable habitat for most pathogenic species of bacteria, rickettsiae, viruses, fungi, and protozoa. The survival period of some pathogenic bacteria is shown in Table 2. However, the soil as a factor of transmitting a number of causative agents of infectious diseases is quite a complex substrate. Thus, for example, anthrax bacilli after falling on the soil produce spores which can remain viable for many years. In favourable conditions (in dark brown soil and chernozem) they pass through the whole cycle of development: during the summer months the spores germinate into the vegetative forms and then this cycle is repeated.

Table 2

Survival Period of Pathogenic Bacteria in the Soil

 

As is known, the spores of clostridia causing tetanus, anaerobic infections, and botulism, and of many soil microbes survive for long periods in the soil. The soil is the habitat for various animals (rodents) which are parasitized by the carriers of the causative agents of plague, tularaemia, the viruses of mosquito fever, haemorrhagic fever, encephalitis, agricultural leishmaniasis, etc. The cysts, of intestinal protozoa (amoeba, balantidium, etc.) spend a certain stage in the soil. The soil plays an important role in transmitting worm invasions (ascarids, hook-worms, nematode worms, etc.). Some fungi live in the soil. Entering the body they cause fusariotoxicosis, ergotism, aspergillosis, penicilliosis mucormycosis, etc.

Taking into consideration the definite epidemiological role played by the soil in spreading some infectious diseases of animals and man, sanitary-epidemiological practice involves measures directed at protecting the soil from pollution and infection with pathogenic species of microorganisms.

S. Vinogradsky, V. Omeliansky, N. Kholodny and others devised a method of  investigating soil microbes and used the results obtained in agriculture.

A valuable index of the sanitary condition of the soil is the discovery of the colibacillus and related bacteria, also enterococci, and Clostridium perfringens. The presence of the latter indicates an earlier faecal contamination.

 

Microflora of the Water

Pseudomonas fluorescens, Micrococcus roseus, etc., are among the specific aquatic  aerobic microorganisms. Anaerobic bacteria are very rarely found in water.

The microflora of rivers depends on the degree of pollution and the quality of purification of sewage waters flowing into river beds. Micro-organisms are widespread in the waters of the seas and oceans. They have been found at different depths (3700-9000 m).

The degree of contamination of the water with organisms is expressed as saprobity which designates the total of all living matter in water containing accumulations of animal and plant remains. Water is subdivided into three zones. Polysaprobic zone is strongly polluted water, poor in oxygen and rich in organic compounds. The number of bacteria in 1 ml reaches 1 000000 and more. Colibacilli and anaerobic bacteria predominate which bring about the processes of  putrefaction and fermentation. In the mesosaprobic zone (zone of moderate pollution) the mineralization of organic substances with intense oxidation and marked nitrification takes place. The number of bacteria in 1 ml of water amounts to hundreds of thousands, and there is a marked decrease in the number of colibacilli. The ohgosaprobic zone is characteristic of pure water. The number of microbes is low, and in 1 ml there are a few tens or hundreds; this zone is devoid of the colibacillus.

Depending on the degree of pollution pathogenic bacteria can survive in reservoirs and for a certain time can remain viable. Thus, for example, in tap water, river, or well water  salmonellae of enteric fever can live from 2 days to 3 months, shigellae — 5-9 days, leptospirae — from 7 to 150 days. The cholera, vibrio El Tor lives in water for many months, the causative agent of tularaemia — from a few days to 3 months.

Tap water is considered clean if it contains a total amount of 100 microbes per ml, doubtful if there are 100-150 microbes, and polluted if 500 and more are present. In well water and in open reservoirs the amount of microbes in 1 ml should not exceed 1000. Besides, the quality of the water is determined by the presence of E. coli and its variants.

The degree of faecal pollution of water is estimated by the colititre or coli-index. The colititre is the smallest amount of water in millilitres in which one E. coli bacillus is found. The coli-index is the number of individuals off. coli found in 1 litre of water. Tap water is considered good if the colititre is within the limits of 300-500. Water is considered to be good quality if the coli-index is 2-3.

Due to the fact that Str. faecalis (enterococci) are constant inhabitants only of the intestine in man and warm-blooded animals, and are highly resistant to temperature variations and other environmental factors, they are taken into account with the colititre and coli-index for the determination of the degree of faecal pollution of water, sewage waters,  soil, and other objects. At present new standards of enterococcus indices are being worked out.

Water is an important factor for the transmission of a number of infectious diseases (enteric fever, paratyphoids, cholera, dysentery, leptospiroses, etc.).

Due to the enormous sanitary-epidemiological role of water in relation to the intestinal group of diseases, it became necessary to work out rapid indicator methods for revealing colibacillus and pathogenic bacteria in water.

These include the methods of luminescent microscopy for the investigation of water for the presence of pathogenic microbes and the determination of the increase of the titre of the phage. Upon the addition of specific phages to liquids containing a homologous microbe in 6-10 hours a considerable increase in the amount of phage particles can be observed.

For a more complete and profound study of the microflora of the soil and water capillary microscopy is used. The principle is that very thin capillary tubes are placed in the soil or water reservoirs after which their contents are exposed to microscopic investigations. This method reveals those species of micro-organisms which do not grow in ordinary nutrient media, and which for many years were unknown to microbiologists.

 

Microflora of the Air

The composition of the microbes of the air is quite variable. It depends on many factors: on the extent to which air is contaminated with mineral and organic suspensions, on the temperature, rainfall, locality, humidity, and other factors. The more dust, smoke, and soot in the air, the greater the number of microbes. Each particle of dust or smoke is able to adsorb on its surface numerous microbes. Microbes are rarely found on the surfaces of mountains, in the seas of Arctic lands covered with snow, in oceans, and in snow.

The microflora of the air consists of very different species which enter it from the soil, plants, and animal organisms. Pigmented saprophytic bacteria (micrococci, various sarcinae), cryptogams (hay bacillus, B. cereus, B. megaterium), actinomycetes, moulds, yeasts, etc., are often found in the air.

 

 

The number of microbes in the air vanes from a few specimens to many tens of thousands per 1 cu m. Thus, for example, the air of the Arctic contains 2-3 microbes per 20 cu m. In industrial cities large numbers of bacteria are found per 1 ml of air. In the forests, especially coniferous forests, there are few microbes because the volatile plant substances, phytoncides, have bactericidal properties which cause a lethal effect.

According to the investigations of E. Mishustin, 1 cu m of air in Moscow at an altitude of 500 m contains from 1100 to 2700 microbes while at an altitude of 2000 m only from 500 to 700. Some microbes (sporulating and moulds) were found at an altitude of 20 km, others at an altitude of 61 to 67 km. One gram of dust contains up to 1 million bacteria. Pathogenic species of microbes (pyogenic cocci, tubercle bacilli, anthrax bacilli, bacteria of tularaemia, rickettsiae of Q-fever, etc.) may be found in the surroundings of sick animals and humans, infected arthropods and insects, and in dust.

At present Streptococcus viridans serves as sanitary indices for the air of closed buildings, and haemolytic streptococci and pathogenic staphylococci are a direct epidemiological hazard.

Depending on the time of the year, the composition and the amount of microflora change. If the total amount of microbes in winter is accepted as 1, then in spring it will be 1.7, in summer— 2 and in autumn — 1.2.

The total amount of microbes in an operating room before operation should not exceed 500 per 1 cu m of air, and after the operatioot more than 1000. There should be no pathogenic staphylococci and streptococci in 250 litres of air. In operating rooms of maternity hospitals before work the number of saprophyte microflora colonies isolated from the air by precipitating microbes on meat-peptone agar within 30 minutes should not exceed 20. In 1 gram of dust in hospitals, there are up to 200000 pyogenic (haemolytic) streptococci.

The number of microbes in factories and homes is associated closely with the sanitary hygienic conditions of the building. In overcrowding,  poor ventilation and natural lighting and if the premises are not properly cleaned, the number of microbes increases. Dry cleaning processes, infrequent floor washing, the use of dirty rags and brushes, and drying them in the same room make the conditions favourable for the accumulation of microbes in air. The causative agents of influenza, measles, scarlet fever, diphtheria, whooping cough, meningococcal infections, tonsillitis, acute catarrhs of the respiratory tract, tuberculosis, smallpox, pneumatic plague, and other diseases can be transmitted through the air together with droplets of mucus and sputum during sneezing, coughing, and talking.

Microbes can be spread by air currents, by aerial dust and aerial droplets. During sneezing, coughing and talking, a sick person can expel pathogenic bacteria together with droplets of mucus and sputum into the surrounding environment within a radius of 1.0-1.5 m or more. Microorganisms contained in air can remain in three phases of the bacterial aerosol — droplet, droplet-nuclei, and dust. An aerosol is the physical system of solid or liquid particles suspended in a gaseous medium.

On the average a person breathes about 12000-14000 litres of air daily, while 99.8 per cent of the microbes contained in air are held back in the respiratory tract. The bacterial aerosol produced naturally in the nasopharynx, during sneezing and coughing is thrown into the air — up to 60000 droplets of different size. Among them almost 60 per cent consist of large droplets (100 mcm), 30 per cent — of average sized droplets (50 mcm), and 10 per cent — of small (5 mcm) droplets.

The greatest amount of bacteria is discharged during sneezing, less — during coughing, and still less — during talking. With each sneeze a man expels from 10000 to 1 000000 droplets. In one cough from 10 to 1000 droplets containing bacteria are discharged into the environment, and when a person utters 10-20 words — up to 80 droplets are expelled. The nature of the bacterial aerosol depends on the viscosity of the secretion excreted from the respiratory tract. A liquid secretion is dispersed into more minute droplets more easily than a viscous one. Near the person expelling the bacteria a more concentrated aerosol of bacterial droplets from 1 to 2000 mcm in size is produced. Most of the droplets are from 2 to 100 mcm in size. Large droplets from 100 to 2000 mcm in size are thrown out to a distance of 2-3 m and more and quickly settle on the ground. Small drops of the bacterial aerosol (1-10 mcm) can remain in a suspended condition for a long time (for hours or days).

The air is an unfavourable medium for microbes. The absence of nutrient substances, the presence of moisture, optimal temperature, the lethal activity of sunlight, and desiccation do not create conditions for keeping microbes viable and most of them perish. However, the relatively short period during which the microbes are in air is quite enough to bring about the transmission of pathogenic bacteria and viruses from sick to healthy persons, and to cause extensive epidemics of diseases such as influenza.

For the purpose of prophylaxis various methods are used in protecting humans from infection via air-borne dust. Thus, the sputum of tuberculosis patients is burned or  decontaminated, the room is frequently ventilated, and cleaned by mopping, the streets are sprayed, drainage and absorbers are used, and masks are used during sorting of wool and rags, etc. The air of operating rooms, isolating rooms, wards, and bacteriological laboratories is decontaminated by ultraviolet radiation (mercury-quartz, uviol lamps, etc.).

The laboratory investigation of air is carried out to determine the qualitative and quantitative composition of its microflora. This is achieved by using simple and complex methods. For a more accurate investigation of microbial contents of the air special apparatus are used (Rechmensky’s bacterial absorber, Krotov’s apparatus, Kiktenko’s apparatus (Fig. 1), and others).

 

 

Figure 1. Kiktenko’s apparatus for bacteriological testing of the atmosphere

At present rapid methods for the indication of microbes in the environment are being devised which will allow quick determinations of the presence of micro-organisms in the soil, the water and air.

 

Microflora of Food Products

Proteins, carbohydrates, vitamins and other nutrient substances contained in food products have a favourable effect not only on the preservation of different micro-organisms but also on their multiplication.

Products of sour milk and foodstuff’s produced by fermentation contain a great number of microbes which lend them flavour and consistency (specific microflora). Besides, micro-organisms or their spores may get into foodstuff’s from the environment (non-specific  microflora).

The reproduction of some micro-organisms may cause spoiling of food products which become unsuitable for eating. In some cases the foodstuffs may be seeded with Salmonella and Shigella organisms, staphylococci, Clostridium botulinun, Escherichia coli. Bacillus cereus, Clostridium perfringens, and other microbes which cause food toxicoinfections and other diseases among humans.

Milk may be contaminated with Mycobacterium bovis, Brucellae, Coxiella burnetti, pathogenic streptococci, and encephalitis viruses from sick animals. During transportation or when it is being bottled or treated milk may be infected with Salmonella and Shigella organisms, pathogenic streptococci and staphylococci, Corynebacterium diphtheriae. Vibrio cholerae, and other microbes by personnel who are sick or are microbe carriers.

Meat may have been contaminated when the animals or poultry were still alive but sick or it may be infected when they are slaughtered, cut, or when the carcasses are improperly stored and transported. Cl. perfringens, B. cereus, enteric bacteria. Streptococcus faecalis, Proteus, and other bacteria are usually found in meat. Meat and meat products, minced meat in particular, are most frequently contaminated during treatment when pathogenic microbes are found on the surface of the meat chopper, on the hands, and on the kitchen utensils (cutting board, etc.).

 

 

Bacteria from meat

 

 

The flesh of fish is infected with a wide variety of microbial species found in water, the scales and guts of fish, on the hands of persons involved in processing the fish products, and on various objects (knives, tables, boards used in preparing the fish, the deck of a fishing boat, etc.). The most dangerous micro-organisms are Cl. botulinum which produce an exotoxin in canned fish products and Vibrio parahaemolytica. When sanitary regimens are not observed, S. typhi, Sh. flexneri, and in some cases the El Tor vibrio are found in the flesh of fish and oysters.

Vegetables and fruit may be seeded with Shigella and Salmonella organisms, Vibrio cholerae, and microflora found in the soil and on the hands of persons who take part in their harvesting, packing, transportation, and those who sell them. Improperly canned vegetables (tomatoes, mushrooms, etc.) may sometimes be the cause of botulism.

Various microflora, pathogenic species among others (Salmonella organisms, fungi, actinomycetes), penetrate eggs quite often; egg powder may be contaminated with staphylococci.

Baker’s products are a relatively rare source of infection of man with pathogenic micro-organisms. Only those baked from grain left in the field the whole winter cause fusariotoxicosis due to pathogenic Fusarium genus moulds.

Among all food poisonings encountered among humans, 70 per cent are due to pathogenic bacteria. Salmonella organisms, staphylococci, and streptococci are most dangerous; they multiply and accumulate in the foodstuff’s without causing changes in the organoleptic properties.

In the different countries the quality norms of most foodstuffs are set by the All- State Standard (GOST) or Provisional Technical Specifications (VTU).

Microbiological methods for testing foodstuffs. Foodstuffs are tested in the following cases: (1) as a planned measure to control the observance of the sanitary and hygienic regimen in the preparation, storage, and realization of food, particularly those foodstuffs which are not subjected to treatment at high temperature; (2) when there is doubt concerning the quality of the food; (3) when food toxicosis or diseases due to the intake of food occur.

The main task of microbiological testing of a food product is the determination of the total content of microbes and the model sanitary microbes. The model sanitary microbe for most foodstuffs and water is E. coli.

Some foodstuffs are tested for the presence of Proteus vulgaris. Salmonella organisms, aerobic and anaerobic organisms and for the toxins of these microbes.

The technique of collecting the samples and the sanitary and bacteriologic examination are fixed strictly by instructions in the corresponding State Standard. It, for instance, specifies the methods for collecting samples and all stages in testing milk, cream, ice-cream, butter, koumiss (fermented mare’s milk), yoghurt, sour clotted milk, sour cream, acidophilin (sour fermented milk), cottage cheese and food prepared from it, dried dairy products, condensed milk, and cold beverages prepared from milk. For testing, liquid foodstuffs are diluted with sterile isotonic solution 1:10. Compact products are melted or ground in the mortar and diluted in sterile water 1:10.

Sanitary and bacteriologic tests of milk and dairy products consist in determination of the total microbial content (microbial count) and the coli titre. In sour dairy products (yoghurt, cottage cheese, cheese, etc.) the microbial count is not determined. The microbial count in milk is determined by a direct count and by inoculating nutrient media with 1.0 ml of different dilutions of the product that is tested. The dairy products should only contain microorganisms specific for the given food, e. g. lactic streptococci and lactobacilli in sour clotted milk, lactobacilli and yeasts in koumiss, etc.

The permissible microbial count in various dairy products ranges between 500 (children’s mixtures subjected to pasteurization and cooking) and 300000 (cow’s milk in cans and cisterns). The microbial count for pausterized milk kept in bottles and packets is 75 000, for ice-cream 250000, for condensed milk 50000, for dried cow’s milk 50000 per one millilitre.

The coli titre of dairy products is determined by a three-stage fermentation method and for most of them it ranges from 0.3 to 3; only for children’s milk mixtures (pausterized and cooked) it is above 11.1. Since  milk and dairy products may be vehicles of the causative agents of certain infectious diseases (typhoid fever, paratyphoid fevers, brucellosis, tuberculosis, Q fever, etc.), these agents are identified by special methods discussed in the corresponding sections of the special part of this textbook. If pathogenic micro-organisms are detected in dairy products it is unquestionable that these products are not fit to be eaten.

The sanitary and bacteriological testing of meat and meat products comprises  determination of the total amount of microbes per 1.0 g of the product and the presence of E. coli, Proteus vulgaris. Salmonella organisms, and anaerobes.

No stable norms have been fixed to date for the sanitary and bacteriological assessment of meat and meat products. According to the accepted provisional norms, the permissible microbial count for roast meat should be less than 500, for boiled sausage and meat jelly less than 1000. The coli titre for roast meat should be above 1 g, for boiled sausage and meat jelly more than 10. The presence of pathogenic and putrefactive microbes indicates that these products are not suitable for use.

Canned foodstuffs, such as canned meat, lard, beans, fish, vegetables and juices are also subjected to sanitary and bacteriological testing.  Canned food is tested microbiologically for aerobic and anaerobic micro-organisms and for the botulinum toxin. When there are epidemiological indications, canned food is tested for Salmonella organisms, pathogenic staphylococci, and Proteus vulgaris; the presence of these microbes shows that the canned food is spoiled and cannot be eaten. It is permissible for canned food to contaion-pathogenic sporulating microbes provided there is no bulging of the can and the organoleptic properties of the food are normal.

Fish, vegetables, and eggs are tested microbiologically usually in cases of food poisoning or diseases among humans. Tests are performed for detecting pathogenic and conditionally pathogenic microbes or their toxins by the commonly accepted methods.

Remnants of food and foodstuffs, vomit, lavage waters, faeces, blood, mucus, washings and scrapings, and autopsy material may be subjected to bacteriological testing according to the epidemiological indications or on instruction of a health officer.

The prevention of food poisoning and other diseases acquired through foodstuffs consists in the observance of sanitary and hygienic measures in preparation of food products, their storage, transportation, and realization. It is also necessary to observe strictly the rules for processing foodstuffs, especially for canning them. Since foodstuffs may be infected by the service staff among whom there may be sick persons or carriers of pathogenic microorganisms, all personnel of food-supplying establishments must be examined regularly. Control of the vectors of the causative agents of intestinal infections and health education among the population are extensively carried out.

Microbiological investigation of soil. For this purpose it is necessary to select most typical area not more then 25 m². The samples are taken from different places of the are field along the diagonal, the angles and the center 10 — 20 cms deep. The weight of each sample must be 100 – 200 g. The total weight of the soil 0,5 – 1 kg.

After careful mixing take an average sample of weight 100 – 200 g. Put the samples of soil in the sterile banks, mark and deliver to the laboratory. The soil specimens for plating are grinded in sterile mortar, make serial dilutions in an isotonic solution of sodium chloride 1: 10, 1:100, 1: 1000 etc. Plate 0,1 – 1 ml of specimens into special media for aerobic and anaerobic microbes. After incubation at optimal temperature count the  colonies on the plates.

 

 

Collection of soil

 

 

Examination of the general microbe number

 

Microbiological investigation of water. The sanitary – bacteriological investigation of water includes determination of total number of microbes in 1 ml of water, determination of a coli-index or coli-titer, and detection of pathogenic microbes, their toxins and Е. соli bacteriophages.

There are quantitative parameters of faecal pollution: a coli-titre and coli-index. Coli-titre is an index, which characterise  an amount of water in millilitres which contain  one E.coli. Coli-index characterises a number of E. coli in one litre of water.

The important part of investigation is taking of water samples. This procedure can do only by special persons (sanitary doctor or his assistant).

The samples of water for investigation are taken in 0,5 L sterile bottles.

 

Water collection

 

Water cocks, pumps, tubes previously must be sterilize by a flame using a cotton plug moistened before with alcohol. Then the water on for 10 min to wash off the bacteria which are on their surface inside. From the open reservoirs water for investigation is taken with the bathometer. This instrument is metal frame with a sterile bottle inside. It gives the possibility to take the water samples from any depth. Usually water is taken from the depth of 15 cms. The bottles with samples of water should have labels, which should mark the source, place  of sample,  time (day, hour), surname of the person who has taken the sampleassay, and also purpose of research.

Water is delivered to the laboratory immediately after its receival. The transportation should be done within 2 hours.  When doctor examines chlorinated water, it is taken in sterile bottles with 2 mL of 1,5 %  of solutions of Sodium hyposulphitum (Na₂S₂Оз • 5Н₂0). Such quantity of Sodium hyposulfite binds chlorine at its concentration up to 2 mg/L  in 500 ml. In summer water transports in ice, and in winter in special hot-water bottle (temperature  must be 1–5 °C). After delivery of samples to the laboratory the investigation begins. It  includes determination of total number of microbes and determination of a coli-titre and coli-index.

Quantitative Analysis. Bacteria cannot be accurately counted by microscopic examination unless there are at least 100 million (10⁸) cells per mililiter Natural bodies of water, however, rarely contain more than 10⁵ cells per millilitre. The method employed is therefore the plate count. A measured volume of water is serially diluted (see below), following which 1 mL from each dilution tube is plated iutrient agar and the resulting colonies counted. Since only cells able to form colonies are counted, the method is also known as the “viable count”.

A typical example of serial dilution would be the following. One millilitre of the water sample is aseptically transferred by pipette to 9 mL of sterile water. The mixture is thoroughly shaken, yielding a 1:10 dilution (For obvious reasons, this is also known as the “10^–1” dilution). The process is repeated serially until a dilution is reached that contains between 30 and 300 colony-forming cells per millilitre, at which point several 1-mL samples are plated in a nutrient medium. Since the original sample may have contained up to 1 million (10⁶) viable bacteria, it is necessary to dilute all the way to 10^–5, plate 1-mL samples from each dilution tube, and then count the colonies only on those plates containing 30-300 colonies. The reasons for these numerical limits are that with over 300 colonies the plate becomes too crowded to permit each cell to form a visible colony, whereas with below 30 colonies the percent counting error becomes too great. (The statistical error of sampling can be calculated as follows’ The standard deviation of the count equals the  square root of N, where N equals the average of many samples. Ninety-five percent of all samples will give counts within 2 standard deviations of the average. For example, if the average count is 36, then 95% of all samples will lie between 24 and 48 [36 ± 12]. In other words, within 95% confidence limits a sample count of 36 has an error of plus or minus 33%.) Assume that the above procedure has been carried out with the results shown in Table 3. The 10^–3 dilution has a suitable number of colonies, the others being either too high or too low for accuracy. The original water sample is calculated to have contained 72,000 (72 x 10³) viable cells per millilitre.

Qualitative Analysis. The methods of plating and enrichment culture are used to obtain a picture of the aquatic bacterial population. Although such methods are satisfactory for general biologic studies, they are inadequate for the purpose of sanitary water analysis; this involves the detection of intestinal bacteria in water, since their presence indicates sewage pollution and the consequent danger of the spread of enteric diseases. Since any enteric bacteria would be greatly outnumbered by other types present in the water samples, a selective technique is necessary in order to detect them. Two widely used procedures for sanitary water analysis are as follows:

Table.

Example of a viable count

 

              *Each count is the average of 3 replicate plates

 

1.Tube method. Dilutions of a water sample are inoculated into tubes of a medium which is  elective for coliform bacteria and in which all coliform bacteria but few noncoliform bacteria will form acid and gas. Such media include MacConkey’s medium, which contains bile salts as inhibitors of noncoliform bacteria; lactose-containing media; and glutamatecontaining media. Cultures showing both acid and gas may then be subjected to further tests to confirm the presence of Escherichia coli or closely related enteric gram-negative rods. Such tests include streaking cultures on a lactose-peptone agar containing eosin and methylene blue (EMB agar), on which E coli forms characteristic blue-black colonies with a metallic sheen; subculturing at 44 °C; and a series of diagnostic biochemical tests

2. Membrane filtration method. A large measured volume of water is filtered through a sterilized membrane of a type that retains bacteria on its surface while permitting the rapid passage of smaller particles and water (fig.1). The membrane is then transferred to the surface of an agar plate containing a selective differential medium for coliform bacteria (fig. 2). Upon incubation, coliform bacteria give rise to typical colonies on the surface of the membrane. The advantages of this method are speed (the complete test takes less than 24 hours) and quantitation, the number of coliform cells being determined for a given volume of water.

 

 

 

Figure. Water samples (100 ml)  are passed through bacteriological filters (0,2 to 0,45 mm pore size) to trap bacteria The filters with trapped bacteria are placed on a medium containing lactose as a carbon source, an inhibitor to suppress growth of noncoliforms and indicator substances to facilitate differentiation of coliforms. Coliform bacteria form distinct colonies on Endo medium

 

 

Figure. Colonies of Е. coli on membranous  filters.

 

The drinking water should not have more than 100 microbes in 1 ml. The microbic number in water of wells and open reservoirs can be up 1000.

During determination of a coli-index and coli-titre of water it is necessary to take into consideration the ability of Е. coli  of the man and animal to grow at 43 °C

 

Microbiological investigation of the air. The sanitary – hygienic investigation of the microflora of the air includes determination both the total number of microbes in 1 m³ of the air and revealing of pathogenic staphylococci and streptococci. For taking the samples sedimentation and aspiration methods are used.

Plate method (sedimentation method). The Petry’s dishes with meat-peptone agar or another special nutrient media for staphylococci and streptococci, for example blood agar, yolk-salt agar are used. They stay in open form at various height from a floor. It is recommend to take one sample on every 20 m² of a premises. Term of exposition depends on prospective quantity of microbes in the air. With a plenty of microorganisms a cup is opened for 5 – 10 minutes, with a little – for 20 — 40 minutes.

Place the dishes into 37 °C incubator for 24 hrs and then incubate for 48 hrs at room temperature (18-20 °C). Study colonies, count them,  and  isolate  pure culture of different microbes.

According to Omeliansky data on a surface of medium by 100 cm² sedimentate in 5 minutes as so many microbes, as they present in 10 L of air. For example, on the dish surface with MPA after 5 minute exposure 32 colonies have grown. It is necessary to calculate amount of microbes which are present in 1 m³ of the air, applying the Omeliansky’s formula. The plate has 78,5 cm² (S = pr² =3,14 • 5² = 78,5 см²). Thus,  it is possible to determine, what quantity of microbes (х) would grow at the given exposure on a surface of medium in 100 см²,

x = (32 • 100) : 78,5 = 40

This quantity of microbes contains in 10 L of the air, and in 1 m³ (1000 л) there will be  –                  (40 • 1000) : 10 = 4000. 

There is a special table for determination of total number of microbes in 1 m³ of the air (Table.).

Table

Account of bacteria number in 1 m³ of the air at a 10-minute exposure

 

For determination of  microbial dissemination degree quantity of the colonies on the dish surface which have been counted should be multiplied with one of multiplier.

If on  a Petry’s dish (78,5 cm²) at a 10-minute exposure 40 colonies have grown, the  quantity of microbes in 1 m³ of the air will be equal 40 • 60 = 2400.

Aspiration method. It  is based on a shock action of an air jet about a surface of a medium. Krotov’s apparatus is used for this purpose. It give us the possibility to let pass 50 –100 L of air with a speed of 25 L per minute through clinoid chink in the special glass above the open dish MPA. The rotation of Petry’s dish (1 rotation/sec) provides uniform dispersion of microorganisms on all surface of a medium. Then dish is incubated in a thermostat at 37 °C for 18-24 hrs.

For example, 250 colonies are revealed  on the surface of dish after 2-minutes exposure with a 25 l/min speed. Thus the number of microbes (x) in 1 l of the air is: x = (250 • 1000) : 50 = 5000.

There are temporary standards       of a sanitary – hygienic state of the air: in operating room the   total number of microbes prior to the beginning of the operation must be no more than 500 in 1 m³, after the operation – 1000.

In preoperative and dressing rooms limiting number of microbes prior before the beginning of work – 750 microbes in 1 m³, after work  – 1500. In birth wards the total number of microbes is about 2000 in 1 m³ of the air, and staphylococci and streptococci  are not higher then 24 in 1 m³, and iewborn rooms  – about 44 in 1 m³.

 

SUPPLEMENT

http://interactive.usask.ca/Ski/agriculture/soils/soilliv/soilliv_micfl.html

http://www.google.com/search?hl=en&q=soil+microflora

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=647419&dopt=Abstract

http://jac.oxfordjournals.org/cgi/content/abstract/46/suppl_1/41

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=3103209&dopt=Abstract

www.gsbs.utmb.edu/microbook/ch006.htm

www.pedresearch.org/cgi/content/full/54/5/739