It was stated that one of the outstanding characteristics of viruses is the fact that no one had ever succeeded in making them grow in a nutritive broth or on an agar “slope” after the manner of bacteria and then examined under a high power microscope. If this is the case, how then can we grow the viruses for further study and evaluation? One idea to consider is that: viruses must have a living cell in which to multiply. The cell is the nurse to the virus, not the whole organism. We can say therefore that if we can grow a few cells in a nutritive medium, then we can also grow an appropriate virus in these cells. We thus make use of the technique of tissue culture, whereby a piece of tissue can be made to grow for varying periods in a sterile medium, to cultivate viruses, and then observing them using a high power microscope. This procedure is not an easy task because some viruses are very particular and will multiply only in certain kinds of cells belonging to certain kinds of animals. For example, the virus of poliomyelitis (infantile paralysis) will multiply only in the living nerve cells of man and some types of monkeys.

The growth of viruses in tissue cultures, however, is a very important technique, because it not only affords good opportunity for the study of viruses but has also yielded most important practical results in the control of some virus diseases. For instance, when the virus of yellow fever is cultivated in mouse tissue for long periods, it loses much of its virulence for man and can therefore be used for vaccination. Over a million human vaccinations against yellow fever have been carried out with this modified virus. Evidence shows that if the yellow fever virus is cultivated for long periods in chick embryo tissue from which the nervous tissue has been removed, the virus loses its affinity for nervous tissue. We can say then that cultivating viruses in this manner has the effect in some cases of modifying the virus by educating it to attack tissues, which normally it does not affect. There is another way of growing viruses which involves a technique having some of the properties of tissue culture and some of those of animal inoculation. The discovery of this technique was made possible by developing hen’s egg which was a remarkably good medium for growing viruses. Surprisingly a large number of viruses could be cultivated in this way. The method involves the cutting of a small triangle of shell from the side of the egg, taking care that the membrane is not injured. At the same time a hole is made in the air space at the blunt end of the egg. Then a little slit is made in the fibrous membrane just beneath the shell and suction is applied to the hole in the air space. This draws away the sensi¬tive membrane from the fibrous membrane. The contents of the egg are then shifted slightly, so that an artificial air space is formed about the living membrane. Drops of the virus fluid, which must be free of all contaminating organisms, are then placed in position and the virus will multiply in the cells of the membrane. Burnett has applied this method to the cultivation of influenza virus with remarkable results. He used a strain of virus which had been kept going in living ferrets and succeeded in making it grow on the egg membrane and the observed it using a high power microscope. For a time the virus continued to multiply, although no visible effect was produced on the membrane or on the chick embryo. The virus still retained its infectivity for ferrets. After many transfers, lesions or pocks started to develop on the membrane but the embryo itself was still unaffected. More serial transfers led to an increase in virulence so that now the virus commenced to attack the embryo. Finally the adaptation was complete and the virus would kill the chick embryo in two or three days. This is an interesting and important point as the influenza virus increased in virulence for the chick, so its virulence towards ferrets, mice and men dwindled. This gives us the hope, then, that before long we may have some such modified influenza virus which can be used for immunization in the same way as the modified yellow fever virus. The viruses which can be used as immunizations to prevent if not totally eradicate the various diseases can be scrutinized more clearly under the microscope.

The culture of virus on the egg membrane served as the beginning many other viruses that have been grown in this way including the virus of yellow fever. Attempts have also been made to cultivate the virus of tobacco mosaic on the developing egg, but so far these have proved unsuccessful.

Another way of cultivating virus is through tissue culture. It is quite different from the egg membrane culture. Trager conceived the idea of culturing an insect¬ borne virus in the tissues of the insect which transmits it. The virus used was the one attacking horses known as equine encephalomyelitis. It is transmitted by a mosquito. To perform this kind of work involves great technical difficulties. All tissue-culture work must be performed under absolutely aseptic conditions. It means that it must be in the complete absence of any bacteria or other organisms. How could one obtain a mosquito or its larva which was free of all contaminating organisms? The problem was solved by giving careful attention to the upbringing of the mosquitoes. First of all the eggs were sterilized on the outside and then dropped into a special nutritive broth which had also been carefully sterilized by heat. Under these conditions it was possible to rear bacterio¬logically sterile larvae and adult mosquitoes. It is known that the mosquito larvae are aquatic, and this fact makes it possible to grow them in a sterile medium. It would not be possible to obtain in a bacteriologically sterile state for such insects as aphides or leaf-hoppers, because the young forms of these are not aquatic or semi-aquatic. The growth of the virus in tissue culture can be observed better with the aid of the microscope.

The result of the sterile mosquito tissue culture showed that the virus grew better in some types of tissue than in other tissue cultures. In the end the best results were obtained by growing the virus for 7 days in tissue from the thorax of the larva, transferring it for 7 days to tissue from the head of the pupa, then back for 7 days to larval thoracic tissue and finally back for another 7 days to papal head tissue. After this period of 28 days in tissue culture the virus was found to have increased 100,000 times. Although this multiplication is greater than what takes place normally in the living mosquito, it is much less than that obtained by the developing egg technique. Incidentally, this experiment also proves that the horse virus does actually multiply in its insect vector.
It is noteworthy to mention one experiment which was carried out to cultivate plant viruses using the tissue culture. The procedure involved getting small pieces of root from a tomato plant which was infected with tobacco mosaic virus. They were sterilized on the outside by dipping them in a weak disinfectant. Next they were washed in sterile water and dropped into a nutritive liquid which, of course, was also free of all organisms. It was found that the pieces of root grew well, increasing their length .many times over, while the virus in them also continued to multiply. This experiment could be carried on indefinitely as small pieces of root could be broken off the original piece and sub-cultured in another bottle or flask of fresh medium. The virus continued to multiply in these detached pieces of root. There was no evidence, however, that the virus was in any way changed or modified by this treatment as compared to those cases performed with some animal viruses.