264 PHYSIOLOGICAL CHEMISTRY 



reaction very carefully. They suggest a new procedure in preparing the 

 reagent 1 and in conducting the test. 



The test follows : Into a perfectly clean dry test-tube introduce 1.4 c.c. 

 benzidine solution, 1 add 0.2 c.c. of water or glacial acetic acid, then i c.c. of the 

 fluid to be tested and finally 0.4 c.c. of 3 per cent hydrogen peroxide. Note the 

 appearance of a blue color, which reaches its maximum in five to six minutes. 



The acetic acid keeps the benzidine in solution. An excess dimin- 

 ishes the delicacy of the reagent. 



Hydrogen peroxide supplies oxygen for the reaction and also bleaches 

 the blue color. An excess of peroxide interferes with the reaction by 

 destroying the catalytic power of the blood and by reacting with the 

 benzidine itself, with the formation of products which appear to have 

 an inhibitory action. It is very essential that the peroxide be added 

 last. 



The benzidine solution should be dilute. Such solutions are exceed- 

 ingly sensitive and permit the detection of blood when present in ratio 

 i : 5,000,000. 



15. Hemin Test. (a) Teichmann's Method. Place a very small drop of 

 blood on a microscopic slide, add a minute grain of sodium chloride 2 and care- 

 fully evaporate to dryness over a low flame. Put a cover-glass in place, run 

 underneath it a drop of glacial acetic acid and warm gently until the formation of 

 gas bubbles is noted. Add another drop of glacial acetic acid, cool the prepara- 

 tion, examine under the microscope and compare the crystals with those shown in 

 Figs. 78 and 79. 



The hemin crystals result from the decomposition of the hemoglobin 

 of the blood. What are the steps involved in this process ? The hemin 

 crystals are also called Teichmann's crystals. Is this an absolute test 

 for blood? Is it possible to differentiate between human blood and the 

 blood of other species by means of the hemin test? 



(b) Nippe's Method. 3 Spread a small drop of blood on a slide in the form of 

 a film and evaporate to dryness over a low flame. Now add 2 drops of a solution 

 containing o.i gram each of potassium chloride, iodide and bromide hi 100 c.c. 

 of glacial acetic acid. Place a cover-glass in position and heat gently over a low 

 flame until gas bubbles form and the solution boils. Run 1-2 drops of the 

 reagent underneath the cover-glass and examine under a microscope. Compare 

 the crystals with those shown in Figs. 78 and 79. 



This method is more rapid than Teichmann's method and crystals 



1 Benzidine solution may be prepared as follows: Place 4.33 c.c. of glacial acetic acid in 

 a small Erlenmeyer flask, warm to 50 and add 0.5 gram of benzidine. Heat the flask for 

 eight to ten minutes in water at 50. To the resultant solution add 19 c.c. of distilled 

 water. This solution may be kept for several days without deterioration. 



2 Buckmaster considers the use of potassium chloride preferable. 

 3 Nippe: Deut. med. Woch., 38, 2222, 1912. 



