An Easy Method of obtaining Methaemoglobin Crystals for Microscopic Examination

More recently, however, Hüfner and Külz³ have advanced a third theory concerning the constitution of methaemoglobin, and that is that it contains the same amount of oxygen as oxyhaemoglobin, only in a closer state of combination. They are able to make this assertion from actual analyses; and these analyses were possible, inasmuch as they succeeded in obtaining methaemoglobin in a crystalline form. The method of obtaining these crystals is as follows:⁴ —Three or four cubic centimetres of a concentrated solution of ferricyanide of potassium are added to a litre of concentrated solution of haemoglobin. A quarter of a litre of alcohol is added, and the mixture frozen. After one or two days’ exposure to this low temperature abundant crystals of a brown colour, which give the absorption spectrum of methaemoglobin, are deposited. They were obtained in this way from the haemoglobin of the dog, pig, and horse, and their form is the same as that of the oxyhaemoglobin crystals of the same animals, i. e. rhombic prisms. Dr. Gamgee¹ had prepared these crystals from dog’s blood many years previously, but their true nature was not at that time recognised. His method was much the same as Hüfner’s, the chief difference being that the nitrite of potassium or amyl was employed instead of ferricyanide of potassium.

Jäderholm² has also obtained these crystals from dog’s blood by the ferricyanide method, and confirms Hüfner’s statement that they are rhombic prisms. He also figures some crystals of methæmoglobin obtained by Profossor Hammarsten from the horse by the same method, which were regular six-sided plates, and showed no double refraction if lying flat; they therefore presumably belonged to the hexagonal system, and were more insoluble in water than the crystals of dogs’ methæmoglobin. I can find no previous reference to the methæmoglobin crystals of rodent animals.

Hüfner’s ferricyanide method is applicable when one wishes to obtain large quantities of the crystals for analysis. I now wish to describe a much simpler method of obtaining these crystals for purposes of microscopic observation. I have tried this method with the blood of the ox, dog, cat, rabbit, rat, guinea-pig, and squirrel, but only successfully in the three last-named animals. In other words, methæmoglobin crystals are obtained with ease from the same animals as yield oxyhæmoglobin crystals with readiness.

The method consists in taking a few cubic centimetres of the defibrinated blood of the animal, adding an equal number of drops of nitrite of amyl in a test-tube, and shaking the mixture vigorously for a minute or two. The colour changes to the dark chocolate tint of methæmoglobin, and spectroscopic observation shows the typical absorption bands of that compound. A drop of this liquid is then placed¹ on a slide and covered; in a few minutes crystals form, which observation with the spectroscope shows to be composed of methæmoglobin. The edges of the cover-glass may then be sealed, and the crystals kept unchanged for several months.

The crystals obtained from guinea-pig’s blood by this process are tetrahedra, which differ only in colour and spectroscopic appearances from those of oxyhæmoglobin from the same animal.

The crystals obtained from squirrel’s blood are perfectly regular hexagonal plates, which remain dark between crossed nicols.

The crystals obtained from rat’s blood are also perfectly regular hexagonal plates, which remain dark between crossed nicols, and which consequently are precisely similar to those of squirrel’s methæmoglobin. This remarkable fact helps to show that the difference between the oxyhæmoglobin of these two animals cannot be a very deep or essential one.

In the case of rat’s methæmoglobin there were, in addition to the hexagons, a few other plates of various shapes scattered in different parts of the field. These are depicted in the accompanying cuts.

Mr. Fletcher kindly examined these for me, and expressed it as his opinion that the triangular and rhombic forms were merely variations of the hexagons. In the case of B faces 1, 3, and 5, and in the case of D faces 3 and 6 have virtually disappeared.