i8 



of sterilizing large quantities of the nutrient material. 

 These dangers recur with the institution of every new 

 culture ; for in order to secure isolation of the organisms 

 from the blood or tissue which accompanied them into 

 the first flask, many transplantations must be made. 

 Successful induction of infection by inoculation from the 

 first culture is of course, met by the objection that the 

 liquids contains solid or fluid unorganized ingredients 

 from the animal diffused through it, and that the effect 

 cannot be attributed to the contained bacteria alone. 



With every transfer to fresh culture fluid the possibili- 

 ties of adulteration by intrusion of other organisms con- 

 tained in the liquid, the flask, the air, etc., are encoun- 

 tered ; moreover, the last culture, however long the 

 series may be, is theoretically, at least, a dilution of the 

 animal juices present in the first flask, although by many 

 successive generations the dilution becomes homoeopathic 

 and may be practically disregarded. That this danger 

 of the intrusion by other bacteria during transfer, how- 

 ever carefully done, is not trivial is known to every ex- 

 perienced observer. Mr. Cheyne ("Antiseptic Surgery," 

 page 261) says : " In. the room in which [ work I have 

 never been able, without the aid of the spray, to transfer 

 micrococci from one flask to another. For in the latter 

 flask bacteria " (in the generic sense) "almost invaria- 

 bly developed." 



The chief objection to flask- or tube-cultivations, how- 

 ever, one which renders them utterly unsatisfactory as at- 

 tempts at isolation, is the impossibility of detecting with 

 certainty the presence of foreign organisms. Some va- 

 rieties, it is true, indicate their presence macroscopic- 

 ally, but the absence of them does not prove the absence 

 of others. One is compelled to remove the cotton, with- 

 draw a drop of the liquid, and submit it to microscopic 

 examination a proceeding perilous to the purity of the 

 culture. But even in this way no certainty can be as- 

 sured, for the one drop may be free from intruding or- 

 ganisms, which may nevertheless be present in the flask. 

 More than that ; since many varieties, at least many 



