Trypan Blue

TRYPAN BLUE, trypan blue, a water-soluble and lipid-insoluble blue dye, chemically the sodium salt of o-tolidine-diazobi-1,8-amidonaphthol-3,6 disulfuric acid NH2OH

OHNH2 NaO3S ^/4^ SO3Na NaO3S CH CH NaO3S SO3Na In water it gives a colloidal solution approaching in its physicochemical properties the solutions of so-called semicolloids (Schulemann) (see Dextrins, Peptones and other semicolloids). Some authors classify it with the group of hydrophilic colloids, others with the group of hydrophobic colloids. The trypan blue particle is negatively charged in water; its ability to diffuse is very low, and it as a rule does not pass through animal membranes. The dye does not show surface activity at the water/air interface, but is strongly active at the interfaces of two immiscible liquids, such as water/benzene, water/olive oil (Okunev). The trypan blue particle consists of two components—blue and red (Mollendorff). This accounts for the violet tint of the dye solutions in water and their blue color in the presence of another colloid (gelatin, protein) that adsorbs the blue component (Okunev). The red component, compared to the blue, has a lesser ability to adsorb on proteins and cellulose, but its diffusibility is higher. Spectrophotometrically, the absorption maximum for an aqueous solution of trypan blue is at a wavelength of 580 mμ, and for the dye solution in the presence of gelatin—at a wavelength of 600 mμ (Okunev). The significance of trypan blue in biology and medicine is connected with the following properties: 1) its ability to kill certain microorganisms (trypanosomes), 2) its property to give rise to so-called vital staining phenomena, 3) its peculiarities as a colored colloid, making it possible to trace the fate of a colloidal substance introduced into the organism. As a trypanocidal substance, trypan blue was first used in 1906 (Nicolle and Mesnil), but was soon replaced by more effective agents. The aforementioned researchers noted the ability of this dye to produce so-called vital staining phenomena (see 'Vital Staining'). This question was more thoroughly developed by Goldmann in the morphological aspect and by Schulemann in the chemical and physicochemical aspects. Since then, this dye has found the widest application for purposes of vital staining. The advantageous properties of trypan blue as a vital stain are its colloidal properties, comparative harmlessness to the organism, and blue color. Trypan blue has the ability to both diffusely stain membranes and protoplasm and to deposit in the protoplasm in the form of grains. The latter property of the dye, apparently connected with its colloidal nature, is especially valuable, as it allows the study of the ability of certain cells to accumulate colloidal substances in the protoplasm in the form of grains (Speicherung in German authors). With the use of vital staining with trypan blue, a number of interesting and important facts were established in the field of cytology (granule formation, Golgi apparatus, etc.) as well as in the field of pathological physiology (reticuloendothelial apparatus, inflammation, etc.). Finally, the peculiarities of trypan blue as a colored colloid have found application in two directions. First, this dye was used to determine the total amount of blood in the organism (Seyderhelm and Lampe, see Blood, total blood volume) as a substance that, due to its colloidal properties, slowly disappears from the blood when administered intravenously. Second, trypan blue was used to study the regularities of the disappearance of a colloid introduced into the blood, as well as to study the absorption of colloids from the peritoneal cavity, subcutaneous tissue, and intestinal cavity (Okunev). The advantage in these experiments is that the absorption of the colloid is determined directly by its accumulation in the blood by means of colorimetry of the serum.

N. Okunev. Trypan blue has found wide application in veterinary medicine for pyroplasmosis in cattle and dogs and for canine distemper (with less success). In pyroplasmosis, trypan blue is used as a therapeutic and prophylactic agent. Dosage: for dogs subcutaneously 2-15 cm³ of a 1% aqueous solution, for cattle intravenously 100-200 cm³ of the same concentration. Trypan blue kills paramecia within an hour in a 1:80,000 solution; if the dilution of trypan blue reaches 1:280,000, the paramecia die after 6 hours. Over time, infusoria develop a habituation to trypan blue. White mice, with subcutaneous administration of trypan blue, tolerate it in the amount of 10 mg without signs of poisoning; from 20 mg they lose weight, become exhausted, and with repeated injections die; from 36 mg they die within the first three days. With subcutaneous administration of 10 mg of trypan blue, vital staining of tissues, tear fluid, urine, and feces is observed, however, the coloring substance does not penetrate the central nervous system, not only with subcutaneous but even with intravenous administration. If, however, trypan blue is introduced directly into the area of the central nervous system, the animal, with convulsive phenomena, quickly dies from even very small doses. For experimental purposes, trypan blue is used in the same dosage for determining the total amount of blood, vital staining of tissues, etc. Locally, abscesses at the injection site have been occasionally noted.