=Sugar Broths.=--Sugar broths are used for determining the action of bacteria on these carbohydrates, since this is a valuable means of differentiating certain forms, especially those from the intestinal tract. Broth _free from sugar_ must first be made. This is done by adding to broth prepared as already described, _just previous to final filtering and sterilization_, a culture of some sugar-destroying organism (_Bacterium coli_ is ordinarily used), and then allowing the organism to grow in the raw broth at body temperature for twenty-four hours. Any carbohydrate in the broth is destroyed by the _Bacterium coli_. This mixture is then boiled to kill the _Bacterium coli_, restandardized and then 1 per cent. by weight of required sugar is added. Dextrose, saccharose and lactose are the most used, though many others are used for special purposes. After the sugar is added the medium must be sterilized by _discontinuous heating_ at 100° for three or four successive days, because long boiling or heating in the autoclave splits up the di- and polysaccharids into simpler sugars and may even convert the simple sugars (dextrose) into acid. Various other _modified broths_ are frequently used for special purposes but need not be discussed here. =Dunham’s peptone solution=, frequently used to determine indol production, is a solution of 1 per cent. of peptone and 0.5 per cent. of salt in tap water. It does not need to be titrated, but should be boiled and filtered into tubes or flasks and sterilized. =Nitrate Broth.=--Nitrate broth for determining nitrate reduction is 1 per cent. of peptone, 0.2 per cent. of C. P. potassium nitrate dissolved in distilled water and sterilized. =Milk.=--Milk is a natural culture medium much used. It should be fresh and thoroughly skimmed, best by a separator or centrifuge to get rid of the _fat_. If the milk is not fresh, it should be titrated as for broth and the reaction adjusted. The milk should be sterilized discontinuously to avoid splitting up the lactose as well as action on the casein and calcium phosphate. _Litmus Milk._--Litmus milk is milk as above to which litmus has been added as an acid production indicator. The milk should show blue when the litmus is added or be made to by the addition of normal NaOH solution. It should be sterilized discontinuously. Frequently on heating litmus milk the blue color disappears due to a reduction of the litmus. This blue color will reappear on shaking with air or on standing several days, due to absorption of O and oxidation of the reduced litmus, provided the heating has produced no other change in the milk, as proper heating will not.