ioo METHODS OF PURE CULTURE 



sterilised by intermittent heat, being left for twenty to thirty minutes in the 

 steamer on three consecutive days, as explained in the preceding chapter. In 

 bacteriological treatises frequent mention is made of " nutrient gelatin " pure 

 and simple without any qualifying term ; in such cases the peptonised bouillon 

 gelatin referred to above is always meant. The tubes spoken of in the colloquial 

 language of the bacteriological laboratory as " gelatin tubes " are ordinary test- 

 tubes containing 5 to 10 c.c. of nutrient gelatin. The preparation of wort 

 gelatin is very simple, the unhopped or hopped wort (according to the purpose 

 it is intended for) being mixed with 10 per cent, of gelatin, melted, boiled for 

 half an hour in the steamer, filtered hot, filled into vessels, and sterilised by the 

 intermittent process. Must gelatin requires a little care in preparation, the 

 high acidity (= 0.7 to i.o per cent, of tartaric acid) of the must having to be 

 previously almost exactly neutralised by caustic potash, since otherwise the 

 setting power of the gelatin is impaired. After the acid has been neutralised, 

 10 per cent of gelatin is added, liquefied, cooled down to between 30 and 40 C., 

 and mixed with the white of an egg beaten up to a froth ; then boiled for half 

 an hour in the steamer, filtered off, filled into the recipients and sterilised as pre- 

 scribed. During storage, numerous crystalline concretions (up to the size of millet 

 seed) of potassium tartrate separate out in the solid medium, and by presenting 

 the appearance of colonies, give rise to the supposition that the medium has been 

 imperfectly sterilised. Attention is therefore now called to this phenomenon. 



As already stated, nutrient gelatin liquefies above 30 C., and therefore also 

 at the usual temperature prevalent in the incubator, viz., 38 39 C., on which 

 account it is unsuitable for use in the separation of such organisms as require 

 higher temperatures for their development. In such cases a medium tempered 

 not with gelatin, but with agar-agar, is used. This substance, obtained from 

 Eastern Asia, and fully described in a treatise by N. K. SCHULTZ (I.), is a dried 

 vegetable jelly prepared from various marine algse and put on the market in the 

 form of thin strips or as a powder. Its manipulation being more conveniently 

 effected in the latter condition, the use of agar-agar powder is recommended as 

 preferable. In French literature this gelatin is generally known as " gelose." 

 For use, not more than 2 parts of agar-agar per ioo of nutrient solution should 

 be taken. It dissolves very slowly and with great difficulty. For the prepara- 

 tion of peptonised bouillon agar-agar Hans Buchner recommends the following 

 process : The meat-broth (bouillon), prepared in the usual manner, is qualified 

 with peptone, Nad, and I or 2 per cent, of agar-agar, and boiled underpressure 

 at about 105 0.; neutralised after cooling down to 100 C. ; then boiled up 

 again, filtered hot, and filled into vessels for use. It is sterilised by exposure to 

 120 C., under pressure, for a quarter of an hour. The agar-agar media do not 

 readily adhere to the glass walls of the vessels, a circumstance which in many 

 operations may be very troublesome, but may be obviated if the adherent 

 properties be increased by adding to the agar-agar employed (i to 2 per cent.) 

 the same amount of gelatin or gum. For the study of the lactic acid bacteria of 

 the distillery, which thrive best at about 48 to 50 0., a i per cent, unhopped 

 wort agar-agar medium containing 2 per cent, of gelatin is used. Moreover, 

 these agar-agar media do not lose their power of solidification if stored for a long 

 time at 100 to 120 C. They are liquefied only at temperatures exceeding 

 40 C., and since this last-named temperature is for many organisms the highest 

 supportable maximum, the agar-agar is used in the following way when designed 

 for the separation of a bacterial mixture : The recipient tubes ai-e immersed in 

 boiling water to induce liquefaction of the contents, which are then cooled down 

 to 40 C. (at which temperature they are still just fluid), inoculated quickly, 

 shaken up and mixed thoroughly, and poured out on to the aforesaid glass plates, 

 which rest on a support warmed to 40 C. 



