page 2

Materials and Methods

The preparation of material for paraffin embedding (collection of animals and their maintenance in the laboratory; anaesthetization; dehydration and embedding) has been described elsewhere (Wineera, 1969). For paraffin embedding in the present study animals were fixed in Lillie's alcohol-acetic acid-formalin (Pearse, 1960, p.788), and in buffered 4% formaldehyde (Pease, 1964, p.52) for 18 hrs. Sagittal, transverse and frontal sections were cut at 5μ on a rotary microtome. For the study of lipids, some worms were fixed in Bakers formol-calcium (Pearse, 1960, p.787), embedded in 20% gelatin and cut at 8μ and 5μ on a freezing microtome. The following histochemical tests were applied:

Tests for Protein:

1.	The mercury/bromphenol blue test (Pearse, 1960). Staining times of ½ hr. and 2 hrs. were employed.
2.	The Ninhydrin/Schiff test for protein bound NH2 (Pearse, 1960).
3.	The Sakaguchi reaction for arginine; Baker's 1947 modification (Pearse, 1960).
4.	The Millon reaction for tyrosine (Casselman, 1959). The Millon reaction as given by Pearse (1960, p.791) differs from that given by Casselman, although both authors claim it to be the Baker modification of this test. Both authors give the same instructions for the preparation of 200 ml. mercuric sulphate reagent. Pearse then instructs that 0.5 ml. 0.25% sodium nitrate is to be added to the mercuric sulphate reagent, while Casselman says to add 0.5 ml. 0.25% sodium nitrite to 5 ml. of the mercuric sulphate reagent for use. In the present study a positive reaction for tyrosine in tissue sections was obtained with Casselman's method but not with Pearse's.
5.	The DMAB-nitrate reaction for tryptophan (Adams, 1957, cited in Pearse, 1960).

Tests for Lipid:

1.	Staining with Sudan black B. The stain was used as a saturated solution in 60% isopropyl alcohol (Casselman, 1959). The position of Sudan staining tissue components in some sections were recorded by means of a microscope stage micrometer and the sections were then decoloured by soaking (1 hr. to 16 hrs.) in 60% isopropyl alcohol. They were then restained with Sudan black to test whether tissue components which took the stain initially exhibited "true sudanophilia" (Casselman, 1959, p.74).
2.	Some section were extracted with pyridine at 60°C for 24 hrs. and then stained in Sudan black.
3.	Unstained frozen sections were examined by phase contrast microscopy.

Tests for Carbohydrates:

1.	The periodic acid-Schiff reaction (Casselman, 1959). Oxidation was for 9 min. at room temperature in 0.5% periodic acid. The Schiff's reagent used was a variant of Lillie's, cited in Casselman (p.36). Some sections were treated with malt diastase (1% in distilled water at 37°C for ½ hr.) prior to the PAS test, and others were treated with Schiff's page 3 reagent without periodic acid oxidation. Nuclear counterstains used were Delafield's haematoxylin, and 0.1% Azure A in 30% ethanol.
2.	Metachromasia. Sections were stained for 5 min. in 0.01% Azure A in 30% ethanol (Kramer and Windrum, 1953) or in 0.1% toluidine blue in 30% ethanol for 1 min. All sections were examined in distilled water before being dehydrated, cleared and mounted in DPX.
3.	Sulphation/metachromasia. The low temperature sulphation of Moore & Schoenberg (1957) was employed. Sections were then stained and examined as in 2 above.
4.	Mowry's colloidal iron method for acid mucopolysaccharides (Mowry, 1958).
5.	Alcian blue staining for acid mucopolysaccharides (Wagner and Shapiro, 1957).
6.	A combination of sulphation/Mowry's colloidal iron technique.
7.	Sulphation followed by Alcian blue staining.

Tests for Nucleic Acids:

1.

The Feulgen reaction (Pearse, 1960) for deoxyribosenucleic acid. Hydrolysis was for 10 minutes in N. HCl at 60°C; the "Schiff" reagent used was Azure A-Schiff prepared according to Himes and Moriber (1956).

2.

The methyl green/pyronin Y method of Kurnick (cited in Pearse, 1960) for deoxyribosenucleic acid and ribosenucleic acid. The proportions of methyl green to pyronin Y, and the staining time were modified. Serial sections were treated with RNase (Sigma(r)), 1 mg./ml. in glass distilled water for 1½ hrs. and 3hrs. at 37°C prior to staining. Controls were placed in distilled water at 37°C for corresponding times.

Combination Methods:

1.	The Allochrome procedure (Lillie, 1951). Although not strictly a histochemical technique, this test successfully distinguished between certain tissue constituents. The picromethyl blue solution employed was 40 mg. methyl blue per 100 ml. saturated aqueous picric acid. The staining time in this solution was 6 min.
2.	Feulgen-PAS technique for demonstration of DNA and polysaccharide in the same section.
3.	The Himes & Moriber (1956) method for DNA, protein and polysaccharide. This method is a Feulgen-PAS technique followed by staining in naphthol yellow S, a dye which Deitch (1955) has shown is specific for basic protein within a wide range of dye concentrations and staining times.
4.	Sudan black B/PAS. Sections were stained in Sudan black as described above (Tests for Lipid, 1). The positions of sudanophil structures were recorded using a microscope stage micrometer, and the sections were then decoloured. The PAS test was then applied, and the positions page 4 of the previous sudanophilia were examined. Some sections were placed in Schiff's reagent without periodic acid oxidation, as controls.
5.	Sudan black B/Naphthol yellow S. As for 4 above, but the PAS routine was replaced by staining in naphthol yellow S, 0.02% in 1% acetic acid for 5 min.
6.	Mowry colloidal iron method/PAS, for demonstration of acidic and neutral polysaccharides in the same section (Mowry, 1958).

Additional Methods:

1.	Bleaching of pigment. Sections were placed in 10% hydrogen peroxide, and in peracetic acid (Pearse, 1960, p.860) until the pigment was bleached.
2.	Whole worms were placed in dilute (5%) hydrochloric acid in an attempt to extract pigment.
3.	The ultraviolet/fluorescence method of MacRae (1961) for demonstrating the presence of porphyrins in planarians.
4.	Falg technique (Gurr, 1965).