CERTAIN OTHER BIOLOGICAL TESTS EMPLOYED IN CLASSIFICATION 571 



Certain Other Biological Tests Employed in Classification. — Before discussing 

 the important problem of the antigenic structure of the streptococci, we may deal 

 briefly with certain other biological tests that have been found useful in identifica- 

 tion and classification within this group. 



It was shown by Neufeld ( 1900) that pneumococci are soluble in bile, while other species 

 of streptococci are not. 



The mechanism of the bile-solubility test has been studied in some detail by many 

 subsequent observers. The pneumococcus is an organism that readily undergoes autolysis 

 in culture ; and Avery and Cullen ( 1923) have shown that extracts of washed pneumococci 

 contain an enzyme that lyses the bacterial cells. This enzyme is inactivated by heating 

 at 60° C. for half an hour ; but in its active state it is able to act on heat-killed pneumococci, 

 though not on haemolytic streptococci or other organisms. There seems little doubt (see 

 Mair 1929) that the effect of bile is simply to accelerate this natural autolytic process. In 

 regard to the active substance in the bile, many samples of ordinary commercial sodium 

 taurocholate induce lysis, but the action is very variable, and Mair (1917) has shown that 

 the most actively lytic bile constituent is sodium deoxycholate, a pure solution of which 

 forms the most satisfactory test reagent (see also White 1929, Downie, Stent and White 

 1931). Other factors, besides the presence of bile-acids, are concerned in this test. It 

 is usually stated (see Avery, R. C, and Cullen 1923, Mair 1929) that the optimum pH for 

 autolysis hes between pH 6 and pH 8, and that the range for bile solubility is about the 

 same, with the limitation that, since the bile-acids are thrown out of solution at pH 6-5, 

 the test must be carried out in a solution more alkaline than this. It is, however, cus- 

 tomary to employ a reaction of pH 7-6, or thereabouts ; and it is also customary if a negative 

 bile-solubility test is obtained, to add a drop of dilute alkali before regarding the test as 

 negative. It seems doubtful whether this solubility in alkaline solutions is entirely 

 dependent on the presence of bile-acids. Dr. Edith Straker (unpublished), working in 

 the authors' laboratory, has found that simple addition of alkali to a suspension of pneumo- 

 cocci lyses the great majority of recently isolated strains, though old laboratory strains 

 are often more resistant. 



The salt content, apart from acidity or alkalinity, also exerts a pronounced effect. 

 NicoUe and Adil-Bey (1907) reported that the addition of magnesium sulphate favoured 

 the lysis of pneumococci by bile salts, while Falk and Yang (1926) state that chlorides of 

 monovalent cations inhibit lysis in low concentrations (0004-1 per cent.), but may accelerate 

 it in high concentrations (20-40). Anderson and Hart (19346) studied this phenomenon 

 in more detail. They found that there was a reciprocal relation between the concentration 

 of sodium deoxycholate required to induce lysis and the concentration of sodium chloride 

 in the suspending fluid. There was no evidence of any inhibition by the salt. Lysis 

 occurred in the absence of sodium chloride, but as the concentration increased up to about 

 5 per cent., less and less deoxycholate was required to produce lysis. 



Bile salts are not the only reagents that cause, or accelerate, the lysis of pneumococci ; 

 Downie, Stent and White (1931) noted that sajionin had a similar effect. Klein and Stone 

 (1931) noted the same fact and Klein (1933, 1935) has extended the study of this reaction. 

 He has found that, in order to induce the lysis of pneumococci by saponin it is essential 

 that there should be added to the medium in which the organism is grown either a sterol 

 (cholesterol) or an animal fluid such as blood or serum. Under these conditions he has 

 found all pneumococci to be saponin-soluble. Eight cultures of Str. hcemolyticus or Str. 

 viridans were found to be saponin-insoluble. 



The question arises as to whether the " bile-solubiUty " test is to be regarded as a 

 completely rehable criterion, bile-insoluble strains being excluded from the species Str. 

 pneumonicB. Neufeld originally regarded bile solubility as being characteristic only of 

 recently isolated pneumococcal strains ; but when the test is made with sodium deoxy- 

 cholate and with due precautions as to the reaction of the medium, etc., the consensus 

 of opinion is that all, or almost all, strains of normal, smooth pneumococci undergo lysis. 



