Samples of sugar cane (Saccharum oflcinarum), cane juice, raw sugar and refinery samples were screened for osmophilic yeasts able to ferment sucrose. One of the isolates showed properties bordering on the original definition of an obligate osmophil. Therefore, diagnostic tests were adapted to fit ecological considerations. These modifications in technique were adopted for all isolates when it was found that more consistent results could thus be obtained. As a result of these tests relevant organisms were identified as Torulopsis apicola, T. globosa, T. lactis-condensi, T. bacillaris, Candida guilliermondii, Saccharomyces Jlorentinus and a new species, T. kestoni.

SUMMARY: Osmophilic yeasts are found in the f i l m of molasses on the crystals of raw sugars and in intermediate sugar-refining products of a wide range of concentration. They are allied to yeasts found in similar situations such as concentrated fruit juices, etc., and can be made to spore more or less freely under dry conditions, usually with conjugation. They are unable to destroy sucrose in strongly buffered solutions; some are able to produce sufficient acid from traces of invert sugar present gradually to invert the sucrose. During this process multiplication takes place. Previous workers have shown that the majority of organisms of this group is highly acid resistant.Organisms which grow in a wide range of concentration of dissolved solids (salts, sugar) show, at acid pH values, an adaptation to growth in higher concentrations accompanied by a diminished metabolic rate. This is lost after a period of [6][7][8] weeks in dilute media, but can be revived in many cases by increasing the sugar concentration in not less than two steps. Temperature resistance is also increased in concentrated media.According to Henrici's monograph (1941) the term osmophilic was first applied by Richter in 1912 to micro-organisms which multiply in solutions of high osmotic pressure. Apart from the brine yeasts, this term is most frequently applied to the yeasts tolerant of high sugar concentration to be found in a wide range of natural and manufactured products. Nussbaumer (1910) was the first to find that osmophilic yeasts were a factor in honey spoilage, and Lochhead & Heron (1929) traced these to the honey of various flowers. Kroemer & Krumbholtz (1931) studied similar organisms in fermenting wine musts produced from grapes infected with Botrytis cinerea; Baker & Mrak (1938) found organisms from the same group on crystallized fruit; Sacchetti (1932, 1939) studied the microflora of fruit juices, Italian wine musts and honey; Owen (1948) has reported the presence of Torulopsis spp. in sugars and molasses.More detailed diagnostic work on honey by Lochhead & Farrell (1931) and Lochhead & McMasters (1931), on honey and wine musts by Sacchetti (1932, 1939), and on fruit juices by Ingram (1949a, b) showed that the majority of organisms involved belonged to the sub-genus Zygosaccharomyces Barker and many were osmophilic strains of known species, Kroemer & Krumbholtz (1931) divide the osmophilic zygosaccharomyces on physiological grounds into two groups :(1) Those which will only grow in solutions of high concentration are able to ferment disaccharides and produce little volatile acid. fig. 4). Asci are formed with or without previous anisogamous conjugation, and 1 to 4 (usually 2) ascospores are produced. A sediment is produced in wort and in some cases also a surface ring. Pseudomycelium when formed is normally in the sediment, so that lack of oxygen may stimulate this formation (Pl. 1, All forty yeasts appear to belong to Kroemer & Krumbholtz's second group, as they are tolerant of a wide range of sugar concentrations s...

sugars, and it has been shown repeatedly that these are altered to some extent when media are cooked. Particular care must be taken with media containing glucose, since this is affected more than most other sugars. So long as the pH does not exceed about 4-5 the destruction of glucose is slow, but at higher pH it is accelerated, so much so that a medium which was originally clear and colourless may be muddy brown after sterilisation. The products of decomposition of the sugars are toxic to fungi, and may suppress growth completely.With species which grow very poorly on ordinary media it is sometimes useful to use parts of plants-small bits of stems, leaves, seeds, etc.-and sterilise these in the cold by means of propylene oxide. The method used is to put the materials in a preserving jar with I ml. of propylene oxide per litre, screw down the lid and set aside overnight. The lid is partially loosened, when most of the gas escapes. The pieces of material are then lifted out with the usual precautions and dropped into Petri dishes containing freshly-poured, and still warm, plain agar, i.e., agar without any nutrients whatsoever. By this means the last traces of propylene oxide are driven off, and the half-immersed specimens may be inoculated.At the present time there is a considerable amount of work in progress on the physiology of fungi. I t is only from studies of this type that we are likely to understand the requirements of fungi sufficiently for the curators of culture collections to feel the satisfaction of having a range of really suitable media at their disposal.The selective media found useful in the examination of sugar products are : (a) Osnzophilic medium, consisting of a synthetic wort agar made in a 45" Brix, (b) A modified Sabaroztd's broth for the estimation of yeasts on membrane filters. (c) Dextrose tryptone agar, liver broth and iron sulphite agar for thermophils. I n the third medium reduced sulphite concentration would probably be more accurate in the estimation of Cl. nzgrijcans.There is no satisfactory selective medium for capsulatd bacteria in sugar products.partially inverted syrup, for the examination of osmophilic yeasts.

SUMMARY Osmophilic yeasts growing in concentrated sugar solutions vary in cell density with age and activity. Microscopic observations with phase contrast by Barer & Joseph's (1958) technique have confirmed this with all strains examined. Examination of syrups containing a culture of Saccharomyces rouxii indicates that growth is limited to these high concentrations owing to the physiological dryness of the habitat. However, some growth does take place, even in the film of molasses surrounding the sucrose crystal in stored raw sugar. At c. 60% (w/w) of dissolved solids it is possible to identify active cells microscopically. Therefore the examination of raw sugar consignments for such cells is a rapid way of indicating whether the sugar is likely to deteriorate on storage.

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