311 Observations on histological methods involving the use of phosphotungstic and phosphomolybdic acids, with particular reference to staining with phosphotungstic acid / haematoxylin By D. BULMER (From the Anatomy Department, The University, Manchester 13) Summary An attempt has been made to elucidate some of the factors involved in differential staining of tissues with Mallory's phosphotungstic acid / haematoxylin, and to find out how far these factors are applicable to other histological methods in which phos- photungstic or phosphomolybdic acid is used. Sections have been subjected to various pretreatments to find whether specific chemical groupings are responsible for any of the staining reactions, but the results obtained are not always easy to interpret. The most important single factor in determining the staining reaction of a tissue material with PTAH appears to be its relative permeability to the two colour-complexes of the PTAH mixture. The red complex, of larger molecular size, penetrates collagen; muscle-fibres are penetrated mainly by the smaller blue complex, while red blood- corpuscles fixed with formaldehyde are not penetrated by either. The staining reactions of muscle and red blood-corpuscles can be altered by a methylation pro- cedure, by treatment with performic acid or formic acid, or by mild alkaline hydro- lysis ; but this appears to be due to alteration of permeability rather than to chemical alteration of any specific dye-binding groups. The effects of blocking reactions indicate that the binding of both complexes is to basic groups in the tissues, though it is possible that hydroxyl groups and carboxyl groups may also be involved. It appears that similar factors control the differential staining with other techniques which involve the use of complex acids (Baker, 1958), and that the chemical speci- ficities which have been claimed for some of them are not well founded. Introduction DESPITE the extensive use of phosphotungstic and phosphomolybdic acids in histological practice there has been little agreement on the mechanism of the several methods in which they are employed. Baker (1958), referring to the publications of earlier workers, considered that differential staining with acid dyes, as in trichrome methods, is dependent upon differences in the permeability of proteins to dye molecules of differing molecular sizes, but other recent workers appear to have largely ignored this viewpoint. Thus, Monne and Slautterback (1951), using the Azan technique on sea-urchin eggs, sug- gested that the aniline blue was bound by the amino-sugars of mucopoly- saccharides and the azocarmine and orange G by the amino-groups of proteins. Hrsel (1957) introduced a staining technique employing treatment with phosphomolybdic acid, eosin, and light green, after previous mordanting of the section with chromic acid. From the use of blocking techniques he deduced that the light green stained protein-bound amino-groups while the [Quart. J. micr. Sci., Vol. 103, pt. 3, pp. 311-23, 1962.] 312 Buhner—Phosphotungstic acid j haematoxylin eosin was specific for proteins containing tryptophane. Jones (i960), however, made an observation which appears to support Baker's view. He found that tanned proteins stained with the orange G of Mallory's phosphotungstic acid / aniline blue / orange G mixture. After destruction of the tanning bonds with diaphanol the orange G stainability was replaced by staining with the aniline blue, but this process could be reversed by re-tanning the protein with quinone. Landing, Uzman, and Whipple (1952) described the use of phospho- molybdic acid in a histochemical method for the demonstration of choline. They believed that the complex acid was bound to choline residues in the tissues, and could subsequently be demonstrated in situ by reduction to molybdenum blue. Treatment of formalin-fixed frozen sections with various reagents increased the number of structures binding phosphomolybdic acid, and Landing and his colleagues supposed that this was due to the freeing of bound choline, which had previously been unable to react. Pearse (i960), quoting the method, comments on the increased number of reacting sites in paraffin sections compared with frozen sections, but does not appear to accept that these are necessarily associated with the presence of choline. The reaction of phosphomolybdic acid with collagen was investigated by Puchtler and Isler (1958). They found that the binding of the complex acid, which they demonstrated by the molybdenum blue reaction, resulted in an intense basiphilia of the collagen. They believed that the phosphomolybdic acid became bound to basic groups of the tissue proteins, leaving several of its own acidic groups free for staining with basic dye, and that in trichrome methods the amphionic aniline blue acts as a basic dye and is held by the free acidic groups of bound complex acid. A related problem which has received little recent attention is that of the so-called metachromatic staining produced by Mallory's phosphotungstic acid / haematoxylin mixture. Tissues which take the fibre stain of a trichrome method are usually red in a phosphotungstic acid / haematoxylin preparation, while those which take the plasma stain of a trichrome method are usually blue. This paper records an attempt to elucidate some of the factors which may be concerned in the differential staining with phosphotungstic acid / hae- matoxylin and to find how far these are also applicable to other staining methods which employ the complex acids. Methods and results The phosphotungstic acid / haematoxylin mixture (PTAH) contains 2% phosphotungstic acid (PTA) and o-i% haematoxylin. It is ripened either naturally or, as in the present investigation, by the addition of 17-7 mg % of potassium permanganate. Mallory's technique (Mallory and Parker, 1929) prescribes fixation in Zenker's fluid and removal of mercury from the section with alcoholic iodine. The section is then exposed to 0.5% permanganate for 5-10 min and 5% oxalic acid for 10-20 min(reduced by Lillie(i954)to 5 min), stained in the PTAH mixture for several hours, and taken straight to 95 % Buhner—Phosphotungstic acid j haematoxylin 313 alcohol and dehydrated, cleared, and mounted. It has been pointed out by Peers (1941) that formalin-fixed sections produce satisfactory results after preliminary mordanting with mercuric chloride, while Earle suggested (Lillie, 1954) that mercurial treatment could be omitted. Sections of rat uterus were employed as test material and blocks were fixed in 4% neutral formaldehyde or in pure acetone for 24 h. Formalin-fixed sections of rat striated muscle and tendon were also used. Adequate staining was obtained in the formalin-fixed sections without any pretreatment, though smooth muscle tended to have a deep purple rather than a pure blue colour. Nucleoli were stained intensely blue, while the rest of the nuclear material was sometimes blue and sometimes red. This may correspond with the different staining of whole and sectioned nuclei with a modified Azan tech- nique (Lison, 1955). The purple staining reaction of smooth muscle appears to be due to the presence of both blue and red colours, and it is interesting that the blue binds much more slowly than the red. After a few minutes in the staining mixture collagen is stained strongly red and muscle less markedly so—only after about 15 min does the blue stain become more prominent in muscle. Mordanting in a saturated solution of mercuric chloride at 5 8° C for 3 h (Peers, 1941), followed byremoval of the mercury, intensified the blue staining of the muscle-fibres and the staining of collagen, though the latter become orange rather than red. Treatment with permanganate and oxalic acid after the mercurial mordanting, as prescribed by Mallory and Parker (1929), reduced the staining to a level rather less than that obtained in the untreated formalin-fixed sections. Treatment of the formalin-fixed sections with per- manganate and oxalic acid, without previous mercurial mordanting, produced weak staining of collagen and patchy, irregular blue staining of muscle. There was no apparent difference in the behaviour of formalin-fixed and acetone- fixed sections, except in the staining of red blood-cells. With formalin fixation the majority of the red blood corpuscles were unstained, while in the acetone- fixed material they were a deep blue. The PTAH mixture continues to ripen further for several months after the initial artificial oxidation. With an old mixture the staining of formalin- fixed sections is intense, with a deep blue colour in the muscle-fibres. Mer- curial mordanting has much less effect on the staining of muscle than with a fresh PTAH mixture, and red cells usually stain blue without any pretreat- ment. In an attempt to elucidate the mechanism of staining, sections were sub- mitted to a variety of pretreatments before exposure to PTAH. For this purpose formalin-fixed sections were usually employed, without mercurial mordanting or permanganate-oxalic acid treatment, and staining was carried out in a PTAH mixture within one month of artificial ripening. Deamination. Treatment with van Slyke's reagent for 24 h before exposure for several hours to PTAH produced slight reduction in the staining of 314 Buhner—Phosphotungstic acid j haematoxylin collagen and rather more marked impairment in the staining of muscle. The muscle was, however, a pure blue, in contrast to the purple of the control. When a nitrosated section was treated for a short period with PTAH (about 30 min) and compared with a control section exposed to PTAH for the same period there was complete abolition of staining in the muscle while the stain- ing of collagen was only slightly impaired. There are various possible ex- planations of this late appearance of muscle staining after nitrosation. In particular, the effect of nitrous acid on some basic groups may be in part reversible by exposure to the staining mixture, or the groups which still bind the blue stain maybe ones that are resistant to nitrosation but in which staining is normally delayed.