SUMMARYThe ability to produce opacity in horse serum is a characteristic of certain M types of group A streptococci. The types that produce the opacity factor (0 factor) are generally those for which it is difficult to produce good anti-M sera. M-positive (M+) and true M-negative (M-) variants of strains that belong to serotypes in which the serum opacity reaction (s.0.r.) is positive both possess the 0 factor, but there is a difference in binding of the factor to other cellular components in the two variants. The 0 factor is closely bound to the wall-membrane fraction of M-cells, whereas in M + cells it is easily extracted by Lancefield's method or I yo sodium deoxycholate.It is detectable in broth culture supernatants, in the cytoplasm and in the areas surrounding colonies in pour plates of M+ but not of M-cultures. The 0 factor is poorly antigenic, but when it is possible to obtain a good antiserum the inhibitory action is type specific. The 0 factor produced by several M types appears to be inhibited by normal rabbit serum. I N T R O D U C T I O NThe production of opacity in horse serum by group A streptococci is believed to be mediated by a lipoproteinase which acts upon the a,-lipoprotein fraction of the serum (Krumwiede, 1954;Rowen & Martin, 1963). Streptococci of groups other than A apparently do not produce opacity in serum, and within group A the serum opacity reaction (s.0.r.) is positive only in certain serotypes. Gooder (1961) and Kohler (1963) grew streptococci in a liquid medium consisting of 3 parts horse serum and I part Hartley digest broth, and recorded the presence of opacity in the culture supernatant as a positive s.0.r. Consistently s.0.r.-positive serotypes were those in which no M antigen was detectable, or for which satisfactory anti-M sera were difficult to prepare; on the other hand, members of a number of easily recognizable M serotypes were almost invariably s.0.r.-negative. Both workers suggested that there was an inverse relationship between a positive s.0.r. and the production of M antigen. Top & Wannamaker (1968a) incubated killed cells or deoxycholate extracts of fractions consisting predominantly of cell walls and cell membranes (' wall-membrane fractions') in horse serum. They confirmed that opacity was frequently produced by members of 'difficult' M types, but could find no true inverse relationship between M antigen and opacity production, except among type 1 2 strains.Most of the evidence concerning the supposed inverse relationship was obtained by examining collections of M-typable and M-untypable strains with common T-antigen

SUiMARYA streptococcal antigen that is closely associated with the M-antigen, but is not type specific can be detected by means of a complement-fixation test in extracts of M-positive, but not of M-negative, variants of group A streptococci. Purification of acid extracts results in a concomitant increase in the purity both of the typespecific M-antigen and of the M-associated protein (MAP). Antibody to MAP is present in the sera of patients who have had streptococcal infection. The highest titres are found in patients with rheumatic fever.

SOME group-A streptococci cause the appearance of opacity in horse serum (Ward and Rudd, 1938). The production of the opacity factor (OF) is confined to members of certain serotypes (Keogh and Simmons, 1940;Gooder, 1961), and the OF of each serotype is antigenically specific (Top and Wannamaker, 1968a). Widdowson, Maxted and Grant (1970) confirmed the serological specificity of the OF and showed that it corresponded exactly to that of the M type of the streptococcus that produces it. The examination of a limited number of strains of each M type (Widdowson et al., 1970; and our unpublished observations) suggested that OF is produced by all members of 16 welldefined M types, and by a number of other types not so far established by international agreement.We have now made a more sytematic examination of the distribution of OF among strains of group-A streptococci sent to us for typing, and have investigated the use of the serum opacity reaction (SOR), and of its neutralisation by specific antisera, in routine typing and as a means of indentifying hitherto unrecognised streptococcal serotypes. MATERIALS AND METHODSStreptococci Strains used to produce antisera to OF in rabbits were the standard type strains used in the Streptococcus Reference Laboratory for the production of M antisera. Other strains sent to us for type identification from numerous laboratories were used in studies of the distribution of OF.Cultural methods Oxoid Todd-Hewitt Broth with the addition of 1 per cent. Neopeptone was seeded with a loopful of growth from solid medium and incubated overnight at 37°C. These cultures were used for the detection both of OF and of M antigen. Typing techniqueThe methods used in the Streptococcus Reference Laboratory for the preparation of typing sera and for M and T typing are those described by Williams (1958). M antigen was ~

SUMMARY: A powerful lytic factor has been obtained in phage lysates of group C streptococci, which is active against streptococci of groups A, C and E and under some conditions group H. It is the factor responsible for 'nascent' phage lysis. The lytic activity remains unaltered by the removal of the phage by high-speed centrifuging, and also in the presence of phage antiserum. It is active against young and old cell suspensions, live, or killed by chloroform. The activity diminishes in the absence of reducing agents and it is destroyed by proteolytic enzymes. Heat-killed cocci when attacked by the lytic factor become Gram-negative but do not lyse. The addition of a proteolytic enzyme completes lysis. Efforts to demonstrate the release of proteinases from streptococcal suspensions have failed. After lysis the group polysaccharide is free as a hapten and some cell-wall structure remains. M antigen is also present in group A lysates.In 1934 Evans reported that a phage, ordinarily lytic for group C but not for group A streptococci, lysed the group A cocci when sensitive group C streptococci were also present in the culture. No phage active on group A cocci could be recovered from the lysate. Evans considered that a t the time of liberation the group C phage had a wider range of activity than later and referred to the phenomenon as 'nascent lysis'. This paper reports a re-investigation of the cause of nascent lysis, using the same phage as Evans. A powerful lytic agent was obtained from the lysates and this was studied to establish its origin and role in a 'nascent phage' system. METHODSThe phage used was Evans's original phage B563, specifically active for group C streptococci. It proved to be very active on a stock group C strain Azgazardah (Griffith's original type 7) and this strain was used for propagation throughout.Most of the group A strains employed were Griffith's original type strains kept lyophilized in this laboratory. Strains of other groups were from our stock collection originating from many sources. The proteolytic enzymes used were: crystalline trypsin (Armour and Co. Ltd.); ficin and papain supplied in powder form (L. Light and Co. Ltd.); crystalline streptococcal proteinase generously supplied by Dr S. D. Elliott.Phage propagation. A warmed 50 ml. volume of nutrient broth was seeded with 0-5 ml. of an 18 hr. broth culture of strain Azgazardah. After 2 hr. incubation, 1 ml. of stock phage suspension was added and incubation continued. The turbidity which had developed was cleared by phage action within 2& hr.

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