716 A MANUAL OF PHYSIOLOGY 



(c) Plunge another muscle into boiling physiological salt solution. 

 It becomes harder than in (b), and its reaction becomes acid to litmus - 

 paper. 



(d) Stimulate another muscle with an interrupted current from 

 an induction machine (Fig. 81, p. 184), till it no longer contracts. 

 The reaction is now acid to litmus -paper. Brown turmeric paper 

 may also be turned yellow. 



(e) To demonstrate the formation of lactic acid in muscle in heat 

 rigor or fatigue, perform the following experiment : Pith a frog, and 

 afterwards leave it for half an hour at rest, so that the lactic acid 

 produced in the movements connected with the pithing operation 

 may disappear from the muscles. See that the circulation in the 

 hind-limbs is not interfered with by pressure or flexion. Then 

 remove both hind -limbs. Carefully, but rapidly, remove the muscles 

 of one from the bones with as little manipulation as possible. Im- 

 mediately place them in a small mortar cooled in ice, and containing 

 some sand and 20 or 30 c.c. of ice-cold 95 per cent, alcohol, and 

 quickly grind them up. Produce heat rigor (p. 674) of the muscles of 

 the other hind -limb, or fatigue them with induction shocks, and then 

 grind them up under alcohol in the same way. Filter the alcoholic 

 extracts, and then evaporate them to dryness on the water-bath. 

 Rub up the residues with a few c.c. of hot water. Add to each 

 aqueous extract a small quantity (say a decigramme) of finely 

 powdered charcoal. Then heat each extract to boiling in a test- 

 tube, and filter. Evaporate the filtrates to dryness, and apply 



Hopkins's Reaction for Lactic Acid. The reagents required are 

 (i) a very dilute alcoholic solution of thiophene (10 to 20 drops in 

 100 c.c.) ; (2) a saturated solution of copper sulphate ; and (3) ordinary 

 strong sulphuric acid. 



Have ready a glass beaker containing water briskly boiling. 

 Place about 5 c.c. of strong sulphuric acid in a test-tube, with i drop 

 of the copper sulphate solution.* Add to the mixture a few drops of 

 the solution to be tested, and shake well.f 



(In the case of the muscle extracts the dry residues are dissolved in 

 the 5 c.c. of strong sulphuric acid, the acicf transferred to test-tubes, 

 and the test proceeded with by the addition of the copper sulphate 

 solution, etc.) 



Now place the test-tube in the boiling water for one to two 

 minutes. Then cool it well under the cold-water tap, and add 2 or 

 3 drops of the thiophene solution from a pipette. Replace the tube 

 in the boiling water, and immediately observe the colour. If lactic 

 acid is present the liquid rapidly takes on a bright cherry-red colour, 

 which is only permanent if the test-tube be cooled immediately after 

 its appearance. The tube should always be cooled, as described, before 

 addition of the thiophene, as the gradual appearance of the colour en 

 re -warming makes the test more delicate. 



(The extract of the resting limb generally gives a negative, that of 

 the other a strongly positive, reaction.) 



* The copper sulphate is added to hasten the oxidation that follows. 



f For practice use a i per cent, alcoholic solution of lactic acid. The 

 test cannot be applied directly to material which chars with the strong 

 sulphuric acid used. In this case preliminary extraction of the lactic 

 acid is necessary. Alcohol should be used as the solvent, or if ether is 

 employed it must first be well washed to remove aldehyde-yielding 

 products, since the colour change is due to an aldehyde reaction with 

 thiophene. 



