Azure Stains

Two uses of methylene azure are suggested. This dye gives a very good nuclear stain after most fixations when preceded by weak NaOH; but eosin Y cannot be used as a counter-stain. Methylene azure also proves very useful in the Mallory eosinemethylene-blue technic, in which it can be substituted to advantage for polychrome methylene blue. The following three schedules are recommended:

2.5% aqueous phloxined˙ 15 minutes

0.1% aqueous azured˙ 1-30 minutes

2.5% aqueous phloxined˙ 15 minutes

Mixture in equal parts of 0.1% azure and 0.1% methylene blued˙ 30 minutes

2.5% aqueous phloxined˙ 1 minute

1.0% aqueous azured˙ 1-2 minutes

Of these the first two give rather better results; but when time is lacking the third is quite satisfactory.     

Progress in the Standardization of Stains

The Commission must acknowledge the assistance recently given by two commercial concerns in developing two new products, namely, a pure type of hematein and a special grade of acid fuchsin.     

Stains recently certified

The micronucleus test (Schmid 1975) is widely used as an indicator of cytogenetic damage induced in vivo by clastogens and spindle poisons. Yamamoto and Kikuchi (1980) have recently showed that by comparing the relative size of micronuclei it is possible to determine whether an agent acts as a clastogen or a spindle poison. This finding will undoubtedly increase the use of the technique in routine protocols for the testing of chemicals. Besides, the test is quite sensitive and much simpler and faster than chromosome analysis. One obstacle, however, hinders several laboratories: the increasing unavailability of fetal calf serum. Recently, Das and Kar (1980) have proposed sodium citrate as a substitute for fetal calf serum. However, 1% sodium citrate solution is hypotonic, which fact may pose difficulties for an experimenter having to sacrifice several animals within a short time, a common situation in routine tests. We were able to overcome the problem of hypotonia by replacing fetal calf serum with the isotonic solutions 0.9% NaCl and Ringer's saline for mammals (9.00 g NaCl, 0.42 g KCl, 0.24 g CaCl₂, 0.20 g NaHCO₃, 1000 ml distilled. H₂O) at room temperature.     

Stains recently certified

For staining in toto, planarians are fixed in a mixture of 10 ml of commercial formalin, 45 ml of 95% ethanol and 2 ml of glacial acetic acid. After treatment with 70% ethanol 3-10 days, they are washed in distilled water and immersed in 10% CuSO₄. 5H₂O for 3 hr at 50° C, transferred without washing to 1% AgNO₃ for 1.0-1.5 hr at 50° C; and then developed in: 10 ml of 1% pyrogallol, 100 ml of 56% ethanol and 1 ml of 0.2% nitric acid. Gold toning, 5% Na₂S₂O₃ and dehydration follow as usual. For staining sections, material is fixed in the same fixative, embedded in paraffin and sectioned at 10 μ. After bringing sections to water, they are immersed in 20% CuSO₄. 5H₂O for 48 hr at 37° C; then rinsed briefly in distilled water and placed in 7% AgNO₃ for 24 hr at 37° C. They are washed briefly in distilled water and reduced in: hydroquincne, 1 gm; Na₂SO₃, 5 gm and distilled water 100 ml. Gold toning, followed by 5% Na₂S₂O₃ and dehydration completes the process. Any counterstaining may follow.