Stanley’s discovery gave a great impetus to chemical research on viruses. It was not long before other viruses were isolated in a crystalline state some of which gave rise to very beautiful crystals. The appearance of the virus in the crystalline form is best viewed with the use a high power microscope such as the electron microscope.
How do we know that the crystals are really the virus and not just carrying the virus contained within them? This was the question that people may ask, but there is now such a large body of evidence which suggests that virus and crystal are one. Much of this evidence, however, is highly technical. First, this unusual virus protein can only be obtained from virus-diseased plants and not from healthy ones. In the case of the tobacco mosaic virus, the protein can be isolated from plants unrelated to the tobacco plant, provi¬ded it is infected with the virus, but not otherwise. Any process, which destroys or denatures this protein, also destroys the virus activity. It is not possible to dissociate one from the other. Lastly, it has been shown by physical and chemical tests that these proteins are actually pure protein. As such the presence of any other contaminating substance is ruled out unless it is a substance which has the same physical and chemical properties as the virus itself. It seems unnecessary to postulate the existence of two such substances when one will fulfill all the conditions. The study of the virus in the crystalline state can be best conducted with the aid of a high power microscope.
Aside from isolating the virus through chemical means, there is still another method that can be employed. This method is a purely physical one, by which viruses can be isolated. This is made possible by the fact that the virus particle is a very large molecule which can be sedimented in a centrifugal field. In plain English this means that if a virus solution is spun at great speed in an ultracentrifuge, the virus collects at the bottom of the tube. The speeds obtained with the ultracentrifuge are very high and may be 30,000 revolu¬tions per minute or higher. At these speed the operation has to take place in a vacuum otherwise the friction of the air would heat up the virus till it was destroyed. By means of the ultracentrifuge, similar crystalline preparations of viruses have been prepared, and they do not differ in composition from those pre¬pared by chemical methods. To further compare the appearances and shapes of the virus derived from the chemical and physical processes, it is necessary to use a high power microscope.
The purification and concentra¬tion of the viruses, which are involved in their crystalli¬zation, increases their infectiousness to a very great extent. The amount necessary to give infection varies with different viruses. It may be stated that as far as tobacco mosaic virus is concerned, from one ten-¬millionth to one thousand-millionth of a gram of purified virus is sufficient to infect a plant.
It seems that to try and measure the size of the actual virus particle is an impossible task. However, the nature of the virus itself allows methods of study which could not be applied to most biological material. The state of “suspended animation” of the viruses outside their hosts enables them to be exposed to various forms of rough treatment and yet retain their power of multi¬plication when returned to their respective hosts. We can, therefore, apply some of the exact methods of the physicist to the measurement of the virus particle using a high power microscope.
There are at least five or six methods which can be used for this put-pose. Since most of them are highly technical only the brief outlines of the methods are mentioned. Let us discuss largely the results achieved.
One method of measuring the particle size of viruses is by means of the ultracentrifuge, an apparatus which is used in the purification of viruses. By measuring the rate at which the virus particles sediment, it is possible by means of a formula to calculate the size of individual particles. This has been done with several viruses. The results compare well with those achieved by other methods. The production of a pure crystalline virus has enabled the X-ray crystallographer to apply his methods to the measurement of particle sizes, and at least two viruses have been photographed with X-rays and measured in this way. In the last section of this chapter which deals with how viruses can be seen, there have been evidences that show not only of the size but also of the shape of viruses.
Another method of measurement is what is known to be ultra¬filtration. As what the name implies, ultrafiltration simply means the passage of viruses through filter membranes of which the pore size is accurately known. The virus in question is filtered through a graded series of collodion membranes until a membrane is reached in which the pores are just too small for the virus to pass, and so it is retained upon the surface of the filter. It is then possible to calculate, from the pore size of the retaining membrane, the particle size of the virus so retained. Ultrafiltration sounds a simple business, but in reality it is a very difficult and exacting technique.
The brief description of the methods of virus measurement serves only to show that the sizes of virus particles can actually be measured. How then can the results be achieved by means of these highly technical methods? First of all we cannot obvious¬ly measure such things as virus particles in fractions of an inch. Let us study the new units of measurement for this purpose and show their relationship to other more familiar units. We start then, with the millimetre which, as you may or may not know, is .039 of an inch. How¬ever, a millimetre is still far too big. We need to use a unit which is one-thousandth of a millimetre and is known as a micron, generally indicated by the Greek letter. This is still too big for most viruses, so we have to reduce it by a thousand times and call it a millimicron, indicated as - mµ. Here, then, is our measuring unit, the milli¬micron, a thousandth part of a micron or a millionth of a millimetre, whichever you prefer.
It is common knowledge that various viruses differ greatly in size, although each kind of virus is itself very uniform in size. Let us start by applying our new unit of measurement, the millimicron, to some fairly familiar visible particles. A red blood cell, for example, measures 7,500 mµ and a common bac¬terium, known as B. prodigiosus, measures 750 mµ. Compare these with two of our smallest viruses, those of tomato bushy stunt and foot-and-mouth disease, which measure 37 and 10 mµ respectively. See how these last two compare with the size of the hemoglobin molecule which is about 6 mµ, and you will realize that it needs no great stretch of the imagination to think of these viruses as molecules. We had enough evidence to show that the virus of tobacco mosaic was rod-shaped, and this has been confirmed by the methods briefly discussed. The particle is, in fact, a rod about 20 times as long as broad, 300 mµ long and 15 mµ in width. We must, however, bear in mind the possibility that this rod may not be the ultimate limit of size for the particle. The shape of the tobacco mosaic virus is best viewed under a high power microscope.
The viruses present a graded series of sizes. The Rickettsiae are the largest. Psittacosis or parrot fever, for instance, measures 275 mµ. Vaccinia virus gives protection from disease known as smallpox. It measures 200 mµ. Herpes virus on the other hand measures 50 mµ. The virus causing tumors in chickens measures 70 mµ, while foot-and-mouth and polio¬myelitis viruses are 15 and 12 mµ respectively. The smallest plant virus so far measured is that causing a mosaic disease of Lucerne (alfalfa) which is computed to be only 16 mµ in diameter. The more detailed comparison of the appearances of these different viruses can be best viewed with the use a high power microscope.