310 Biological Stains 



Biological Tests: Nigrosin is tested in Dorner's spore stain, using a 2 to 4 day old 

 culture of some rapid spore-former such as Bacillus cereus. 



A heavy suspension of the organism is made in 2 to 3 drops of distilled water in a 

 small test tube, and an equal quantity of freshly filtered Ziehl's carbol fuchsin is 

 added. This mixture is allowed to stand in a boiling water bath 10 minutes or longer. 

 On a cover slip or slide, one loopful of the stained preparation is mixed with a loopful 

 of a 5-10% aqueous nigrosin. (This solution must be filtered before use, and may 

 be kept indefinitely if preserved with a few drops of formalin). It is smeared as 

 thinly as possible and allowed to dry fairly rapidly. A good sample shows the 

 spores red, the vegetative cells unstained, and the background dark gray. 



THE DIPHENYL METHANE DERIVATIVES 

 AURAMINE O, C. I. No. 655. 



Identification: Auramine O has the empirical formula Ci7H22N3Cl. It is identified 

 by the following method: Dissolve 50 mg. in 250 ml. of distilled water. Dilute 5 ml. 

 of this solution to 200 ml. with distilled water. Read in Beckman spectrophoto- 

 meter. Absorption maximimi 430-434 mju; ratio P— 15/P+15 is from 1.02 to 1.04, 



Method of Analysis: Spectrophotometric. Percent dye = D-peak X 127. D-peak 

 (color density at peak) measured on dye solution described under "Identification". 

 Samples of this stain must contain not less than 80% anhydrous dye. 



Biological Tests: Auramine O is tested for acid-fast bacteria in sputum. Smears 

 from sputum are air-dried and are stained 2-3 min. in the staining solution (aura- 

 mine 0, 0.1 g.; liquified phenol, 3 ml.; distilled water, 97 ml.), washed in tap water, 

 destained 3-5 min. in freshly prepared solution containing 100 ml. 70% ethyl 

 alcohol, 0.5 ml. cone. HCl, 0.5 g. NaCl and dried. The smears are examined under a 

 monocular microscope, using 8 mm. dry objective and a 20X ocular. Illumination 

 should be a low voltage, high amperage microscope lamp, supplied with a blue 

 (ultraviolet transmitting) filter, and a complementary yellow filter for the ocular. 

 Acid-fast bacteria should be bright yellow, fluorescent, other organisms not visible- 

 and background nearly black. 



THE TRI-PHENYL METHANE DERIVATIVES 



Malachite Green, C. I. No. 657 



Identification: Malachite green is the chloride or oxalate of p, p -tetramethyldi- 

 amino-triphenylcarbinol anhydride, C23H25N2CI or 2C23H25N2 + 3C2H2O4. The 

 method of identification is as follows: Dissolve 50 mg. in 250 ml. of distilled water. 

 Dilute 3 ml. of this solution to 200 ml. with distilled water. Read in Beckman spec- 

 trophotometer. Absorption maximum 617-619 m^^; ratio P— 15/P+15 is from 1.05 

 to 1.11. To differentiate between the chloride and the oxalate, dissolve approxi- 

 mately 0.1 g. of the dye in 50 ml. of cold water and precipitate the dye base by the 

 addition of a slight excess of dilute NaOH. Filter off the base and divide the filtrate 

 into two parts. Heat one part to boiling and add dilute Ca(0H)2 solution. A fine 

 white crystalline precipitate indicates that the original dye was the oxalate. Make 

 .the other portion of the filtrate slightly acid with dilute HNO3 and if a copious 



