R. KARNAD                                      353

milk alone, but do so on the addition of citric acid [Joshi and Ram Ayyar,
1936]. The organism described in this paper does not produce a high volatile
acidity in milk and produces a considerable amount of acetyl-methyl-carbinol
and diacetyl without the addition of citric acid to milk.

The organism can be considered to be a new species, its chief characteris-
tics being the production of aroma, mainly by the formation of diacetyl in the
culture.

II.—PRODUCTION OF AROMA IN MILK CULTURE INOCULATED WITH THE NEW
                    STRAIN OF DIACETYL AROMATICUS

Experimental.— Estimation of total diacetyl.—[Barnicoat, 1936 ; Schmal.
fuss and Barthmeyer, 1929, 1932].

(Acetyl-methyl-carbinol and diacetyl). Diacetyl is condensed with
hydroxylamine hydrochloride to form dimethyl glyoxime which can be esti-
mated gravimetrically as the nickel compound. It is always desirable to use
an all glass apparatus with no rubber connexions. One litre of the milk cul-
ture, mixed with ferric chloride solution is distilled in a three litre flask on an
oil-bath maintained at 110°C.—120°C. into a receiver containing excess of
diacetyl reagent (two parts of 20 per cent neutral hydroxylamine hydrochloride,
two parts of 20 per cent sodium acetate and one part of 10 per cent nickel sul-
phate)*. For the conversion of all the acetyl-methyl-carbinol to diacetyl
4 to 5 per cent ferric chloride is used. The adapter of the Liebig's condenser
is dipped in the diacetyl reagent in the receiver. The nickel dioxime is formed
in the receiver at the boundary between the reagent and distillate as red pris-
matic needles. Fractions of 50 c. c. of the distillate are collected until all the
diacetyl is distilled. The precipitation of the dioxime is completed by heating
on the water bath at 95°C. for one hour and leaving at the laboratory tempera-
ture for 24 hours. The nickel compound was filtered through a tared sintered
Jena glass crucible (Ig₃), washed several times with cold distilled water, dried
at 110°C. and weighed. The weight of the precipitate multiplied by 0.596 is
the weight of diacetyl. The method is quite accurate and trustworthy.

Volatile acids (Hammer's method)—

The volatile acidity was determined by steam-distilling a 250 gm. portion
of the milk culture after the addition of 15 c. c. N/10 sulphuric acid, collecting
the first one litre of the distillate and titrating against standard sodium hydro-
xide. The distillation was performed at such a rate that approximately two
hours were required to secure one litre of the distillate. The results are re-
corded in terms of number of c. c. of standard alkali (N/10) required to titrate

* The hydroxylamine hydrochloride solution should be freshly prepared and the
nickel sulphate filtered from any precipitate before use.