228 . CAPE WINE-LEVURES. 



a subsequent inoculation with pure levures, because mannite is 

 un fermentable by levures. 



i. Selection and Cultivation of the Puke Levures. 



The levures PA 1-4, HeAi-4. GA1-2 were obtained by 

 bringing some fresh Pontac. Hermitage and Greengrape lees 

 from Air. G. A. Retief's farm at Paarl into some sterilised 

 must, taking the fresh lees formed by this last fermentation 

 and pouring must-gelatine plates in the usual way. Some, well 

 isolated and simple colonies were inoculated into sterile must in 

 test-tubes taking the usual precautions. So as to make quite 

 sure that the cultures were really pure, that is. contained only 

 descendants from one original cell, plates were once more 

 poured of these cultures. They alway- gave the same colonies 

 showing their purity. One of these colonies was then inocu- 

 lated into sterilised must as before, and constituted the pure 

 culture worked with. Since several colonies were originally 

 inoculated into sterilised must, there are up to four cultures 

 from one kind of lees. Thus PAt. PA2, PA3, PA4 are four 

 cultures from Pontac lees. Since the<e studies are still incom- 

 plete it is at present impossible to say much about the identitv 

 of the different cultures. Still, it is certain that several of them 

 are the same. 



In the same way as above, PB1-2, GB1-3, and HaBi-3 were 

 obtained from the lees of the second crop grapes of Pontac. 

 Greengrape and Hanepoot at the Oeimlogical Station, Paarl. 

 HaCi-3 were similarly obtained from a Hanepoot wine sent in 

 for examination, and that had suffered from a mannitic fermen- 

 tation. These cultures were made during 1910 and have been 

 used in wine-making during the 191 1 and 1912 vintages. 



2. Fermentation Experiments with these Pure 

 Cultures. 



For these fermentations a sterilised, filtered must was used, 

 whose sugar was 25. 8° Balling, and total acidity equal to 4.0 \. 

 per mille Tartaric acid. Of this must 400 ccm. was introduced into 

 500 ccm. bottles, which were plugged with cotton-wool and then 

 sterilised in steam in the usual way. In a Hansen sterile case 

 streak-cultures on must-gelatine of the various selected cultures 

 all of the same age were taken to inoculate the different flasks, 

 taking the necessary precautions to avoid foreign infections. In 

 the Hansen case and immediately after inoculation, the cotton 

 wool plugs were replaced by sterilised, perforated corks contain- 

 ing fermentation-tubes into which had been poured some dilute 

 sulphuric acid to act as an air-valve and exclude any ulterior 

 infection. The charged, inoculated bottles were then weighed 

 and placed in a thermostat, which was at 23-25 C. weighed 

 daily at the same hour and again put into the thermostat. 



