36 METHODS OF CULTIVATION OF BACTERIA. 



Standardisation of Reaction of Media. — While the above pro- 

 cedure of dealing with the reaction of a medium is sufficient 

 for ordinary work, it has been thought advisable to have a 

 more exact method for making media to be used in growing 

 organisms, the growth characteristics of which are to be de- 

 •scribed for systematic purposes. Such a method should also 

 be used in studying the changes in reaction produced in a me- 

 dium by the growth of bacteria. It, however, involves consid- 

 erable difficulty, and should not be undertaken by the beginner. 

 It entails the preparation of solutions of acid and alkali which 

 may be used for determining the original reaction of the me- 

 dium, and for accurately making it of a definite degree of alkalin- 

 ity. Normal -^ and decinormal solutions of sodium hydrate and 

 hydrochloric acid are used. 



Preparation of Standard Solutions. — The first requisites here are normal 

 solutions of acid and allcali. The latter is prepared as follows : 85 grammes 

 of pure sodium bicarbonate are heated to dull redness for ten minutes in a 

 platinum vessel and allowed to cool in an exsiccator ; just over 54 grammes 

 of sodium carbonate should now be present. Any excess is quickly removed, 

 and the rest being dissolved in one litre of distilled water, a normal solution 

 is obtained. A measured quantity is placed in a porcelain dish, and a few 

 drops of a .5 per cent solution of phenol-phthaleine in 50 per cent alcohol is 

 added to act as indicator. The alkali produces in the latter a brilliant rose 

 pink, which, however, disappears on the least excess of acid being present. 

 The mixture is boiled and a solution of hydrochloric acid of unknown strength 

 is run into the dish from a burette till the colour goes, and does not return 

 after very thorough stirring. Tlie strength of the acid can then be calculated, 

 and a normal solution can be obtained. From these two solutions any 

 strength of acid or alkali (such as the decinormal solution of NaOH mentioned 

 below) may be derived. 



As Eyre has suggested, the reaction of a medium may be 

 conveniently expressed by the sign -j- or — to indicate acid or 

 alkaline respectively, and a number to indicate the number of 

 cubic centimetres of normal acid or alkaline solution necessary 



lA "normal" solution of any salt is prepared by dissolving an "equivalent" 

 weight in grammes of that salt in a litre of distilled water. If the metal of the salt 

 be monovalent, i.e. if it be replaceable in a compound by one atom of hydrogen {^e.g. 

 sodium), an equivalent is the molecular weight in grammes. In the case of NaCl, it 

 would be 58.5 grammes (atomic weight of Na = 23, of = 35.5). If the metal be 

 bivalent, i.e. requiring two atoms of H for its replacement in a compound {e.g. cal- 

 cium), an equivalent is the molecular weight in grammes divided by two. Thus in 

 the case of CaCl2 an equivalent would be 55.5 grammes (atomic weight of Ca=40, 

 ofCl2 = 7l). 



