reinforces this conclusion. None of these three curves shows linearity and, in total contrast to the previous result, 0i shows slightly greater resemblence to 08 than 07 in its inactivation rate. The reason why these curves are not linear is unknown. Perhaps the rate of inactivation is affected by debris present in the diluted lysate.
At this stage all that can be concluded from these experiments is that the three phages are closely related in the size of their genome. Because there is variation from one experiment to another and because there is not a significant difference in the inactivation rates, it is impossible to differentiate these phages on the grounds of UV inactivation kinetics.
The data in Figure 5.7 show that after 50-60 seconds treatment 07 and 08 had only decreased in titre by lj logs when titred on Su297. This is a significant difference from the rate of inactiv ation of both phages as titred on Nul8. There could be several reasons for this. Su297 may "repair" UV-induced damaged phage more efficiently than Nul8 can. Alternatively, the amount of
interfering debris in the lysate produced on Su297 could be greater than that in the Nul8 lysates. The reason for this difference was unknown and was not investigated further.
From the thermal inactivation curves (Figure 5.8) all three phages show a very rapid inactivation of plaque forming ability. Even though their original titres varied, each lysate lost 2j-3 logs activity after only five seconds. However, after this initial dramatic decrease in activity there was very little decrease in titre on continued heat exposure. Why should the thermal inactiv ation curves show such a dramatic biphasic response? Barnet and Vincent (1970) suggested that anomalies in the UV and thermal inactivation curves of 07 could be due to the reversible inhibition
of 07 by adsorption to host debris. According to their report these effects could be reversed by using L.S.B. as the medium in which the lysate was generated. However when this was tested no obvious difference in the thermal inactivation curves was noted (data not presented). Presumably, according to Barnet and Vincent, the rapid inactivation phase could be due to the inactivation of free phage in the lysate whereas the subsequent slow phase of inactivation could be due to the inactivation of debris-protected phage.
Whatever the reason for the biphasic kinetics of inactivation, it becomes clear that these phages cannot he differentiated b y the criterion of thermal inactivation kinetics. It must be concluded, therefore, that the differences between these three phages are so small that such crude methods as UV and thermal inactivation are totally inadequate as methods for the differentiation of one phage from another. Hence methods which are more sensitive to very slight differences in phage nucleic acid and coat proteins need to be employed here. Such methods include electron microscopy to examine and compare the phages morphologically as well as to examine heteroduplexes of cross-hybridised nucleic acid. Alternative methods of analysis would be to examine the coat proteins of each phage and determine the molecular weights of the phage genomes. To do such analyses high titres of phage are required and, to be
strictly comparative, they must all be grown on the same host, Nul8. Finally, comparative restriction enzyme analysis of the DNA from the three phages revealed:
1) The DNA from all three phages co-migrates.
2) There are no Eeo PI sites in any of these phages DNA.
3) The molecular weight of the DNA of these phages is 1 - 2 x 10 and thus cannot have a large coding capacity.
of 07 by adsorption to host debris. According to their report these effects could be reversed by using L.S.B. as the medium in which the lysate was generated. However when this was tested no obvious difference in the thermal inactivation curves was noted (data not presented). Presumably, according to Barnet and Vincent, the rapid inactivation phase could be due to the inactivation of free phage in the lysate whereas the subsequent slow phase of inactivation could be due to the inactivation of debris-protected phage.
Whatever the reason for the biphasic kinetics of inactivation, it becomes clear that these phages cannot be differentiated by the criterion of thermal inactivation kinetics. It must be concluded, therefore, that the differences between these three phages are so small that such crude methods as UV and thermal inactivation are totally inadequate as methods for the differentiation of one phage from another. Hence methods which are more sensitive to very slight differences in phage nucleic acid and coat proteins need to be employed here. Such methods include electron microscopy to
examine and compare the phages morphologically as well as to examine heteroduplexes of cross-hybridised nucleic acid. Alternative methods of analysis would be to examine the coat proteins of each phage and determine the molecular weights of the phage genomes. To do such analyses high titres of phage are required and, to be
strictly comparative, they must all be grown on the same host, Nul8. Finally, comparative restriction enzyme analysis of the DNA from the three phages revealed:
1) The DNA from all three phages co-migrates.
2) There are no Eeo RI sites in any of these phages DNA.
. 7
3) The molecular weight of the DNA of these phages is 1 - 2 x 10 and thus cannot have a large coding capacity.