34 METHODS OF CULTIVATION OF BACTERIA 



exact method for making media to be used in growing organisms, 

 the growth characteristics of which are to be described for 

 systematic purposes. Such a method should also be used in 

 studying the changes in reaction produced in a medium by the 

 growth of bacteria. It, however, involves considerable 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 medium, and for 

 accurately making it of a definite degree of alkalinity. Normal l 

 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 alkali. 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 neutral methylated spirit 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. The 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 + or to indicate acid or 

 alkaline respectively, and a number to indicate the number of 

 cubic centimetres of normal acid or alkaline solution necessary 

 to make a litre of the medium neutral to phenol-phthaleine. 

 Thus, for example, "reaction = -15," will mean that the 

 medium is alkaline, and requires 15 c.c. of normal HC1 to make 

 a litre neutral. It has been found that when a medium such 

 as bouillon reacts neutral to litmus, its reaction to phenol- 



*A "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 Aveight of Na = 23, of Cl = 35'5). If the metal be 

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

 (e.g. calcium), an equivalent is the molecular weight in grammes divided 

 by two. Thus in the case of CaCl 2 an equivalent would be 55 '5 grammes 

 (atomic weight of Ca = 40, of C1 2 = 71 ). 



