CLASSIFICATION BASED ON CHANGES PRODUCED IN BLOOD 565 



superiority of poiired plates, and the observation of deep colonies, over plates 

 which have been inoculated by surface spreading only. 



The medium recommended by Brown consists of veal peptone agar containing 5 per 

 cent, of horse blood. The agar is stored in tubes in 12 ml. amounts ; when required 

 for use a tube is melted and cooled to 45° C. ; 0-66 ml. of horse blood is added and evenly 

 mixed with the agar, the medium is moculated with a loop or two of a 24-hours' broth 

 culture that has been so diluted as to give about 100 colonies ; and is then poured into 

 a Petri dish 9 cm. in diameter, thus giving a layer about 2 mm. thick. The plates, after 

 prehminary drying in the inverted position with the plate tilted on the lid, are incubated 

 at 37° C. for 24 hours. They are then examined and the appearances noted. They are 

 re-exammed after another 24 hours' incubation at 37° C, and finaUy after a further 

 24 hours in the ice-chest. For the recognition of hsemolytic and non-hsemolytic strains 

 in primary mixed cultures the use of blood agar plates with a surface inoculation usually 

 suffices, and has certain advantages ; but for the critical determination of the type of 

 haemolysis given by any strain, once it has been isolated in pure culture, the technique 

 recommended by Brown should be strictly adhered to, at least in regard to the medium 

 used and the examination of deep colonies. The use of horse blood is particularly impor- 

 tant. It is well known that the red corpuscles of different animal species vary widely 

 in their resistance to different hsemolytic agents. In spite of this fact, many workers 

 have used the blood of the rabbit or ox or some other animal, in testing the hsemolytic 

 activity of various species, or strains, of streptococci ; and it is probable that some at 

 least of the discrepancies met with in the literature are due to this variable factor. It 

 may, at times, be desirable to use the blood or red ceUs of some particular species in a 

 given series of observations on streptococcal hsemolysin ; but, if the results are to be used 

 for purposes of identification or classification, they should always be controlled by parallel 

 tests made with horse blood. 



Brown records four different types of reaction in blood agar plates, which he 

 designates as follows : 



a. A somewhat greenish discoloration and partial hsemolysis of the blood corpuscles 

 immediately surrounding the colony, forming a rather indefinitely bounded zone 1-2 mm. 

 in diameter, outside of which is a second, narrow, clearer, not discoloured zone. Under 

 the microscope many corpuscles are seen in the inner zone, and these are obviously dis- 

 coloured, the discoloration varying in degree with dififerent strains of streptococci. Very 

 few corpuscles remain in the outer, clearer zone ; and these are never discoloured. These 

 typical appearances may fail to appear after 24 hours', or even after 48 hours' incubation, 

 at the end of which time the narrow outer zone of hsemolysis may not have developed. In 

 such cases this zone makes its appearance during the subsequent 24 hours in the ice-chest. 

 If a plate, which has developed the typical appearances, is reincubated for 24 to 48 hours, 

 and then placed in the ice-chest for a further 24 hours, a double series of rings will frequently 

 develop, so that the colony is surrounded by a hazy discoloured ring, a clear hsemolysed 

 ring, a second hazy ring, and a second clear ring. By repeating the whole process it is 

 sometimes possible to develop three or more series of such rings. 



p. The colonies are surrounded by sharply defined, clear, colourless zones of hsemolysis, 

 2-4 mm. in diameter. Under the microscope no corpuscles can be seen within this zone. 

 The zones of /S-hsemolysis develop more rapidly than those of the a type. They are often 

 well developed after 18 hours' incubation. They extend slightly between the 24th and 

 48th hour, but show no qualitative changes. They undergo no alteration or extension 

 during the subsequent 24 hours in the ice-chest. 



a' (a prime). The colonies are surrounded by a zone of hsemolysis, which is slightly 

 hazy, and less sharply hmited than in the case of true /S-hsemolysis. The colony itself is 

 not sharply defined, and examination with the microscope shows that the hsemolysed zone 

 contains, throughout, a moderate number of unaltered corpuscles, which are most numerous 



