Treated with antiserum 3 4 virus in disrupted 3 Diluted 1:

In document The use of inhibitors in the analysis of the growth cycle of vaccinia virus (Page 36-39)

TIME AFTER INFECTION (HOURS)

2. Treated with antiserum 3 4 virus in disrupted 3 Diluted 1:

suspension

Total virus content 1. Disrupted 77 128 2. Diluted 1:100

of suspension

Cells were infected and incubated in growth medium. After

26 and 48 hours, aliquots of cells were removed, washed and treated prior to assay as shown above.

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b ) 0.02^6 v e rsen e c ) rem ained u n tr e a te d , f o r 30 m inutes a t 3 7 °. The c e l l s were th e n washed tw ice w ith g e l a t i n e s a l i n e and f r o z e n . On t i t r a t i o n no d if f e r e n c e c o u ld he d e te c te d betw een c e l l s

exposed to t r y p s i n o r v e rse n e and th o s e n o t t r e a t e d .

Both e x p e rim e n ts i n d i c a t e t h a t m ost of th e v ir u s produced rem ains i n t r a c e l l u l a r .

Absence o f Secondary I n f e c t i o n i n th e Growth Tube

I t was im p o rta n t t o d e cid e w h eth er seco n d ary i n f e c t i o n o c c u rre d under th e c o n d itio n s d e s c r ib e d . C e lls were in f e c te d w ith s e v e r a l m u l t i p l i c i t i e s o f v ir u s and a f t e r a d s o r p tio n each p r e p a r a t io n was d iv id e d i n t o two p a r t s . One o f each o f th e s e was d i l u t e d w ith an e q u a l volume of u n in f e c te d c e l l s so t h a t th e f i n a l c o n c e n tr a tio n o f a l l p r e p a r a t io n s was 10^ c e l l s p e r m l. A ll th e sam ples were th e n in c u b a te d f o r 24 h o u rs . E x am in atio n o f s ta i n e d sm ears made a t th e end o f t h i s p e rio d showed t h a t only h a l f as many c e l l s i n th e p r e p a r a t io n s mixed w ith f r e s h c e l l s were i n f e c t e d a s i n th e c o n t r o l s . W ith m u l t i p l i c i t i e s o f a b o u t 1, 60-7O/o o f c e l l s showed f lu o r e s c e n c e by th e 24th h o u r and no more f lu o r e s c e d on p ro lo n g ed in c u b a tio n . I t was

p o s s ib le t h a t c e l l s n o t i n f e c t e d i n i t i a l l y became in s u s c e p t i b l e due to t h e i r m aintenance i n s u s p e n s io n . However, i f c e l l s were m a in ta in e d i n s u s p e n s io n i n a grow th tu b e , a t a

FIG, 1,6. Effect of prior incubation of KB cells in

suspension for twenty-four hours on the time of production

of virus.

o Cells incubated for 24 hours before infection.

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to 10° per ml in an adsorption tube and infected in parallel with a freshly prepared cell suspension they were equally susceptible to infection (Fig. 1.6).

Both types of experiment indicate that secondary infection in the growth tube does not occur to a significant extent.

In document The use of inhibitors in the analysis of the growth cycle of vaccinia virus (Page 36-39)