Lycette, R. M. (Illinois Institute of Technology, Chicago) and L. R. Hedrick. Adsorption and fluorescence of fat-soluble fluorescent dyes on class I and class III Saccharomyces cerevisiae. J. Bacteriol. 85:1-6. 1963.-Class III cells of the brewery yeast Saccharomyces cerevisiae flocculate, but class I cells do not, when cultured in an organic YM medium. Class III cells adsorb the fat-soluble stilbyl-triazole fluorescent dye (STC) more readily and produce more fluorescence than do class I cells. This suggests that cells of the class III yeasts have more lipids or more phospholipids on their surface than do cells of class I yeasts. Fluorescence of STC in the absence of cells is pH-independent, but adsorption of the compound on the surface of cells, with the associated fluorescence, is pH-dependent. As the pH level was decreased within the range of pH 9 to 0, and as the temperature was elevated within the range of 0 to 40 C, there was an increase in adsorption and a corresponding increase in fluorescence. Maximal adsorption was attained in less than 15 min when the cells and dye were shaken at 26 C. Cells of class III adsorbed 2.5 mug of STC per 3.25 mg (dry wt) of cells. The kinetics of adsorption follow the Langmuir monomolecular model.

The ‘ceiling temperature’ of a pox virus has been defined as the maximum temperature (to the nearest 0·5° C.) of incubation at and below which that virus will grow and produce pocks on the chorioallantois of 12-day-old chick embryos, and above which no pocks appear.Ceiling temperatures have been estimated for: alastrim (2 strains), 37·5° C.; variola major (2 strains), 3·8° C.; ectromelia (3 strains) and monkey pox (1 strain), 39°C.; cowpox (2 strains), 40°C. Five strains of vaccinia and two of rabbit pox were all capable of pock formation at 40·5° C. Above this temperature difficulty was encountered because many embryos died. But the ceiling temperature for two strains of vaccinia and one strain of rabbit pox was probably 41° C. The Utrecht strain of rabbit pox produced some lesions at 41·5° C.—the highest temperature used.The ceiling temperatures of the viruses used were not correlated with their thermal stabilities at 55°C.in vitro. Thus vaccinia strain, Lederle-7 N, had a high ceiling temperature and a low thermal stability, while variola major had a low ceiling temperature and a high thermal stability. For this reason ceiling temperatures and thermal stability are regarded as distinct characters.In experiments with twelve of the seventeen viruses of which the ceiling temperatures had been determined, the virulence for the chick embryo was then measured. It was found that, in general, the higher the ceiling temperature of a virus the greater was its virulence for the chick embryo.The presentation of these results is followed by a brief discussion of their significance and potential use.

The characterization of 16 clones of virus derived from mixed infections with variola major and cowpox has been described. This work has involved the description of the plaques produced by variola major in chick embryo monolayers, and a more detailed study of the effects of raised temperature on pock production by pox viruses on the C.A.M.Two of the variola major—cowpox clones were found to correspond to cowpox virus, while the other 14 shared various combinations of parental characters. Among them there were 10 distinct new types of virus. Evidence of the stability of these viruses has been presented.Analysis of the combination of characters encountered among the hybrids has given good evidence that all the eight marker characters used segregate independently. A particular instance of this was the character of chick embryo virulence which, among the hybrid viruses, was shown to be unrelated either to ceiling temperature or speed of plaque formation.The significance of these findings in relation to the general problem of virulence has been briefly discussed. The possibility that vaccinia may have originated as a hybrid of variola and cowpox has lso been considered.

The characters of seven clones of virus derived from mixed infections with alastrim and rabbit pox have been described. One clone was shown to behave as rabbit pox in respect of all its markers. The other six were found to be distinct new types of virus, each having a different combination of the parental characters. The reasons for accepting these viruses as hybrids have been discussed. An analysis of the pairwise crosses between individual markers suggested that each of the nine markers was capable of segregating independently.

A simple method for the isolation of pox virus hybrids on the C.A.M. has been described. One parental virus was used as a heat-inactivated suspension. The other parent was used in the active state, but at a temperature higher than its ceiling temperature. Under these conditions the inactive parent was reactivated so that pocks resulted only from the cells infected with both parental viruses. Many of these pocks were unlike those of either parent. Such lesions were found to contain a high proportion of hybrids. In these experiments, alastrim was crossed with rabbit pox and variola major with cowpox.The term ‘heat-tethered’ has been used to describe virus whose intracellular cycle of development has been arrested by incubation at too high a temperature. Heat-tethered virus has interesting properties and two of these have been described. When the temperature is lowered, heat-tethered virus will start to grow again. Its reactivating potential has been mentioned above. A more detailed account of the properties of heat-tethered virus is being prepared.