Nutrition, Cultivation and Isolation of Viruses

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Chapter: Pharmaceutical Microbiology : Nutrition, Cultivation and Isolation of microorganisms : Bacteria-Actinomycetes-Fungi-Viruses

The world has broadly witnessed by 1900 and accepted generally that severlal of the recognized dreadful human ailments were duly caused by various microorganisms.



The world has broadly witnessed by 1900 and accepted generally that severlal of the recognized dreadful human ailments were duly caused by various microorganisms. However, the first and foremost evidence of viruses responsible for causing human disease came into notice in 1892 when Iwanowski rightly demonstrated that the cell-free extracts of the diseased tobacco leaves passed through the bacteria-proof filters may ultimately cause disease in the ‘healthy plants’. Furthermore, such cell-free filtrates when cultured upon the bacterial growth media they eventually exhibited practically little growth thereby suggesting that the said filtrates contained the actual disease causing agents that are other than microorganisms. Martinus Beijernick, another scientist reconfirmed the excellent epoch making findings of Iwanowski.


Twort and d’Herrelle (1915) individually showed the ‘glassy phenomenon’ present very much in the microorganisms when it was observed clearly and distinctly that the bacterial cells might be adequately infected with and duly destroyed by the filterable agents, which in turn caused various serious diseases both affecting the animal kingdom and the plant kingdom. Later on, these disease producing filterable agents are known as bacteriophages (i.e., the bacteria-eaters).


Wendell M Stanley (1935), an American Chemist, first and foremost isolated the tobacco mo-saic virus (genus Tobamovirus) thereby making it possible to perform the chemical as well as struc-tural studies on a purified virus. Interestingly, almost within the same time, the invention of the elec-tron microscope took place which eventually made it quite possible to visualize the said viruses for the first time.


The galloping advancement and progress in the in-depth studies on the viruses across the globe based duly upon the latest molecular biology techniques in the 1980s and 1990s have remarkably led to the discovery of the new dreadful human viruses. In the year 1989, the world has duly acknowledged the discovery of Hepatitis C virus, and Pestivirus, which specifically causes acute pediatric diarrhoea. The year 1993, critically observed the outbreak of a Hantavirus infection occurring exclusively in the South-western USA, which essentially possesses the potential for new infections to emerge at any time. Hantavirus disease refers to the acute ailment related to respirator disease and may even prove fatal.


1. Bacteriophages


Bacteriophages designated the ‘last group of viruses’ which were duly recognized and best characterized. As to date one may have the evidence for the presence of such disease producing agents that are found to be even smaller in size than the viruses, and termed as viriods. They usually consist of the nucleic acids (i.e., DNA and RNA) exclusively.


Example : The spindle tuber disease of potatoes is a glaring example of a specific disease invariably caused by the viriods.


A good number of bacteriophages infecting various microorganisms have now been duly iso-lated, characterized, and recognized. The following Table 5.2. records the variuos bacteriophages, host(s), particle dimensions (viz., head and tail in nm), structure, and composition adequately.

*DS = Double Stranded ; **SS = Single Stranded ;


[Adapted From : Tauro P et al., An Introduction to Microbiology, New Age International, New Delhi, 2004].


Viral Species : A viral species may be defined as ‘a group of viruses essentially sharing the same genetic information and ecological niche.


It is, however, pertinent to state here that the particular epithets for viruses have not yet been established completely, thereby logically and emphatically the viral species are duly designated by such common descriptive nomenclatures as : human immunodeficiency virus (HIV), with subspecies duly indicated by a number (HIV-1).


Standardization of the ‘viral nomenclature’ is now in an active and progressive stage ; and as such the following specific criteria are being adopted in the latest textbooks and literature alike, namely :


·        New viral family


·        Genus names


·        Common species names


·        Common names are expressed in regular type viz., herpes simplex virus


·        Genus names are now usually capitalized and italicized viz., Simplexvirus.


Table 5.3. records a comprehensive summary of the latest classification of viruses that invari-ably infect the human beings.

Table 5.3. Latest Classification of Human Viruses


2. Growth of Bacteriophages in the Laboratory


It is practically possible to grow the bacteriophages in two different manners, namely :


(a) In suspensions of organisms in liquid media, and


(b) In bacterial cultures on solid media.


Advantages of using Solid Media : In actual practice, the use of solid media makes it feasible and possible the plaque method for the easy detection and rapid counting of the viruses.


Methodology (Plaque Method) : The various steps that are involved in the ‘plaque method are as enumerated under :


(1) Sample of bacteriophage is duly mixed with the host bacteria and molten agar.


(2) The resulting agar countaining the various bacteriophages as well as the host bacteria is then poured carefully into a Petri-plate adequately containing a hardened layer of the agar growth medium.


(3) In this manner, the ensuing mixture of virus-bacteria gets solidified into a thin top-layer that invariably comprises of a layer of organisms nearly one-cell thick. This specific step allows each virus to infect a bacterium, multiplies subsequently, and helps to release several hundred altogether new viruses.


(4) Nevertheless, these newly generated viruses in turn duly infect other organisms that are present in the immediate close vicinity ; and hence, more new crop of viruses are produced ultimately.


(5) Thus, several accomplished virus multiplication cycles, all the organisms duly present in the area surrounding the original virus are destroyed finally. In this way, a good number of ‘clearings’ or plaques are produced, which may be seen against a “lawn” of bacterial growth upon the surface of the agar ; whereas, the plaques are observed to form uninfected microorganisms elsewhere in the Petri dish (or Petri plate) undergoing rapid multiplication and giving rise to a turbid background finally.


Note : Each plaque correspond theoretically to a single virus in the initial suspension. Hence, the concentra-tions of viral suspensions measured by the actual number of plaques are invariably expressed in terms of plaque-forming units (pfu).


3. Bacteriophage Lambda : The Lysogenic Cycle


In a broader and precise perspective the bacteriophage may conveniently exist in three phages, namely :


(a) As a free particle virion,


(b) In a lysogenic state as a prophage, and


(c) In the vegetative state i.e., lytic cycle.


One may, however, observe that virion is inert in nature ; and hence, cannot reproduce. Salient Features : The various salient features of the bacteriophage lambda are as stated

under :


(1) In the critical ‘lysogenic state’, the DNA of the phage is duly integrated very much within the bacterial DNA. It usually exists in a non-infectious form known as the prophage, and adequately replicates in synchrony with the bacterial DNA.


(2) In the corresponding ‘lytic cycle’, the phage particle infects the susceptible host, undergoes multiplication, and ultimately causes the lysis of the bacterial cell with the concomitant release of the progeny virus particles.


(3) In a situation when the integrated phage is carefully induced to become the corresponding vegetative phage, the lytic cycle comes into being.


(4) Such phages which specifically give rise to the phenomenon of ‘lysis’ are normally termed as the virulent phages, as opposed to such phages that may exist in a lysogenic state and are usually called as the ‘temperate phages’.


(5) The microorganisms that particularly carry the ‘temperate phages’ are invariably termed as the ‘lysogenic bacteria’, which are observed to be absolutely immune to the ensuing superinfection caused by the same phage.


Figure 5.8 diagramatically illustrates the lysogenic cycle of bacteriophage λ in E. coli.


However, it is pertinent to state here that whether decisively the ‘lytic’ or the ‘lysogenic’ response takes place immediately following infection by a temperate phage will solely depend upon both the bacterium and the phage.


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