ESTIMATION OF HEMOGLOBIN. 25 



then gradually to thaw ; pour the lake-coloured blood into a plate, until it 

 forms a stratum not more than 1^ m.m. in thickness, and allow it to evaporate 

 slowly in a cool place, when crystals will separate. 



Method Of Hoppe-Seyler. Mix defibrinated blood with ten volumes of a 20 

 per cent, salt solution, and allow it to stand for two days. Remove the clear 

 upper fluid with a pipette, wash the thick deposit of blood-corpuscles with 

 water, and afterwards shake it for a long time with an equal volume of ether, 

 which dissolves the blood-corpuscles. Remove the ether, filter the lake-coloured 

 blood, add to it of its volume of cold (0) alcohol, and allow the mixture 

 to stand in the cold for several days. The numerous crystals can be collected in 

 a filter and pressed between folds of blotting-paper. 



Method of Gscheidlen. Crystals several centimetres in length were obtained 

 by taking defibrinated blood which had been exposed for twenty-four hours to the 

 air, and keeping it in a closed tube of narrow calibre for several days at 37C. 

 When the blood is spread on glass, the crystals form rapidly. [Vaccine tubes 

 answer very well.] 



[Method of Stirling and BritO- It is in many cases sufficient to mix a drop 

 of blood with a few drops of water on a microscopic slide, and to seal up the 

 preparation. After a few days beautiful crystals are developed. The addition of 

 water to the blood of some animals, such as the rat and guinea-pig, is rapidly 

 followed by the formation of crystals of haemoglobin. Very large crystals may 

 be obtained from the stomach of the leech several days after it has sucked blood.] 



13. Quantitative Estimation of Haemoglobin. 



(a.) From the Amount Of Iron- As djy (100C.) haemoglobin contains 0'42 

 per cent, of iron, the amount of iron may be calculated from the amount of 

 haemoglobin. If m represents the percentage amount of metallic iron, then the 

 percentage of haemoglobin in blood is 



_ 1 50w 

 ~ "0*42" 



The procedure is the following : Calcine a weighed quantity of blood, and exhaust 

 the ash with HC1 to obtain ferric chloride, which is transformed into ferrous 

 chloride. The solution is then titrated with potassic permanganate. 



(b.) ColorimetriC Method. Prepare a dilute watery solution of haemoglobin 

 crystals of a known strength. With this compare an aqueous dilution of the 

 blood to be investigated, by adding water to it until the colour of the test 

 solution is obtained. Of course, the solutions must be compared in vessels with 

 parallel sides and of exactly the same width, so as to give the same thickness of 

 fluid (Hoppe-Seyler). [In the vessel with parallel sides, or, hcematinometer, the sides 

 are exactly one centimetre apart. Instead of using a standard solution of oxyhae- 

 moglobin, a solution of picro-carminate of ammonia may be used (Rajewsky, 

 Malassez. ) ] 



(c.) By the Spectroscope. Preyer found that a 0'8 per cent, watery solution 

 (1 c.m. thick), allowed the red, the yellow, and the first strip of green to be seen 

 (Fig. 11, 1). Take the blood to be investigated (about 0*5 c.m.), and dilute it with 

 water until it shows exactly the same optical effects in the spectroscope. If k is 

 the percentage of Hb, which allows green to pass through (0'8 per cent.), b, the 

 volume of blood investigated (about 0'5 c.m.), to, the necessary amount of water 

 added to dilute it, then x = the percentage of Hb in the blood to be investi- 

 gated 



_ k (w + b) 



