Quantitative studies of a mycobacterial phage‐host system II. Electron microscopy

Abstract

Quantitative studies of a mycobacterial phage-host system 11. Electron microscopy 8 3 1968 187-194 .HANA MOHELSKA and ANEiIiA MAKOVCOVA (Einyeganyen w m 21. 6. 1967) The morphology of some rnycobacterial phagcs was already studied by ORTALI and PEESO (1949), WHITTAKER (1950), SELLERS and co-workers (1957), BOWMAN (1959) and others. Detailed studies on particle structures using negative staining techniques are less frequent (MOHELSK~ and MAKOVCOV~ 1968).From these papers conclusions can be drawn, that mycobacterial phages are not different from phages of other bactcrial groups. The morphological study of interaction of one mycophage with its host is reported by TAKEYA and coworkers (1 961 ). In this study attention was paid to the morphological pattern of the phage 11.lyF,PISYa-reproduction cycle on Mycobacterium fortuitum niinetti. &!aterial and methodsH o s t s t r a i n . Mycobacterium fortuitum minetti from Prof. PENSO (Istituto Superiore di Sanith, Rome) was used. A series of single colony isolations was carried out. All final isolates displayed t'he same colony morphology, as well a8 the same sensitivity to the phage. Isolate No. 2 was chosen and checked by differential diagnosis. The strain was freeze-dried in 1.5 per cent Na-glutamate and used as standard host cnlture. A culture met,hod appropriate for quantit'ative experiments with this strain will be described elsewhere.Baet.eriophage. Phage l12yF3P/59a (~U L A and S U L O V~ 1959) was nsed. Its characteristic properties as well as the preparation of freeze-dried stock \$:ere described elsewhere (MOHELSKB and MAKOVCOVA 1968).T h e b a c t e r i a l g r o w t h c u r v e a n d t h e p h a g e r e p r o d u c t i o n cycle. The st'rain was cultivated in glycerine brot'h according to VANURA (1960). Under conditions of aerated cultivat'ion the culture was displaying a diffuse growth, and from the third to the twenty second honr was in the logarithmic growt'h phase. w k r e in the populat'ion less than 10 per cent dead cells were present'. The 18-hour old culture was chosen for phage infection.I n the first 30 minutes aft'er infection the intensive adsorption period occured (70 per cent of phage particles adsorbed), then till 3 honrs the latency t'ook place being covered with slow adsorption (in 3 hours 96 per cent, phages adsorbed). From this moment. the rise period began, ending 10 hours after infection, the average burst size being 117.Samples for t.he electron microscopy were t,a.kcn from the cwlt.iirc before and after infection every hoiir till the coniplet.ion of the Iysis.E l e c t r o n m i v r o s c o p i c p r e p a r a t . i o n . The samples were processed according t.0 thc agarr filtration technique (KELLENBERGEH 1957). The specimens were shadowed with P t a t an angle of tan 1/3.:For ultrathin sections the samc samples were fixed wit'h 1 per ccnt OsO,, in SJ~STEASI) buffer pH 7,2 (ZAPF and I A~7 1 1 1 7 i~ 1961) for 3 hoiirs at 4 "C. The embedding in Vestopal was done according to I,UDV~K (l9li3). The intervals of penetration of t'he...