The Effect of the Chemical Nature of a Decolorizer on its Functioning. i. the Gram Classification

Decolorizers which are distinctly acidic or basic in their chemical nature give abnormally high decolorization in the Gram stain for bacteria. Acidic substances yield more regular results. Ideally an “inert” decolorizer should be used, but ordinarily such substances will not dissolve the dye or dye-mordant precipitate from the smear. The most practical substances seem to be those so very slightly acidic in character as to be practically inert, such as acetone or alcohol, or a mixture of such substances.     

Progress in the Standardization of Stains the Standardization of Eosin and Related Dyes

Decolorizers which are distinctly acidic or basic in their chemical nature give abnormally high decolorization in the Gram stain for bacteria. Acidic substances yield more regular results. Ideally an “inert” decolorizer should be used, but ordinarily such substances will not dissolve the dye or dye-mordant precipitate from the smear. The most practical substances seem to be those so very slightly acidic in character as to be practically inert, such as acetone or alcohol, or a mixture of such substances.     

Effects of strong electrolytes on the iron alum-Alcian Blue-Safranin staining of mast cell granules of the rat

Summary Rat mast cells fixed in Carnoy's fluid were stained with iron alum-Alcian Blue-Safranin solution after pre-treatment with strong electrolyte solutions including acids, neutral salts and alkalis. Although both red and blue mast cells were observed without pre-treatment, most mast cells were stained blue and a few red when they were stained after the pre-treatment. Mast cell granules contain salt complexes formed between basic proteins and acidic polysaccharides through ionic linkages between protein basic groups and polysaccharide sulphate and carboxylic acid groups. It is suggested that when sections are treated with strong electrolyte solutions, complexes are broken by disruption of ionic linkages and sulphate and carboxylic acid groups of polysaccharides masked by basic proteins become available for binding Alcian Blue. This was confirmed by model experiments performed with smears of a heparin-lysozyme complex.When mast cells were fixed in aldehyde-containing fixatives, no effects of strong electrolyte solutions on the staining properties of mast cell granules were revealed.     

Cytochemical evaluation of the Guard procedure a regressive staining method for demonstrating chromosomal basic proteins. I. Effects of fixation, blocking reactions, selective extractions, and polyacid "differentiation".

Appropriately fixed preparations stained by a modification of the Guard (1959) reaction for "sex chromatin" display selective staining of interphase chromatin and mitotic or meiotic chromosomes. This is a regressive staining method which seems to depend on the selective displacement of an acidic dye from less basic structures, and retention of the dye at more basic sites. The results obtained with the reaction can be controlled by the length of time that the preparations are "differentiated" in solutions containing phosphomolybdic and phosphotungstic acids (polyacids). After three- or four-hour exposures to polyacid solutions, all chromatin is stained. However, with longer differentiation, "condensed" chromatin can be stained preferentially. Of a number of fixatives investigated, only 10% formalin, ethanol-acetic acid (3:1), and Bouin's solution proved useful. Others resulted in diminished specificity or a total loss of selectivity. The most intense staining was obtained after formalin fixation. Less intense dyebinding was observed after fixation in 3:1 - probably due to extraction of some histone fractions-and the least amount of dye was bound in Bouin's-fixed chromatin - probably due to blockage of arginine residues by picric acid. The reaction was not affected by enzymatic removal of nucleic acids or the extraction of lipids. It was diminished by treatment with trypsin or weak acetylation, and it was completely prevented by strong acetylation, deamination, or extraction of basic proteins with HCl. The results presented suggest that the modified Guard (1959) procedure selectively demonstrates basic nucleoproteins. Further, by the use of regressive differentiation in polyacid solutions, the retention of dye in more condensed chromatin can be favored.     

Detailed Differentiation of Bacteria by Means of A Mixture of Acid and Basic Dyes at Different pH-Values

As previously reported by the author (1927), a mixture of methylene blue and eosin Y can be used for the differential staining of bacteria. It gives a fairly deep staining of bacteria at about pH 3 and above. Below pH 3 the eosin Y stains bacteria only a very pale pink; at such high H-ion concentration, the eosin is present as undissociated color acid, and for this reason not enough eosin is in solution to stain bacteria. To improve the staining at such reactions, the eosin was replaced by a stronger acid dye, namely acid fuchsin. The mixture of methylene blue medicinal Merck and acid fuchsin can be successfully used at a pH-value as low as 0.8. The method of staining by this new mixture is entirely the same as with the old mixture. It is sensitive enough to detect the difference in the isoelectric points: (1) of the single bacteria from the same pure culture, (2) of different strains of the colon and typhoid organisms. Some strains of the colon organism were found by this method with an isoelectric point at a pH-value as low as that of the Staphylococcus. Others, on the contrary, have their isoelectric point as high in the pH-scale as that of the typhoid organism. The new mixture can also be used for the study of the chemical composition of the different parts of bacterial body. Applying it at a definite pH-value, the author was able to stain differentially polar bodies of the typhoid group and of the diphtheria organism. This new mixture can be recommended in staining of B. diphtheriae as a substitute for Neisser's stain. It is interesting to note that polar bodies of the colon group consist of more alkaline protein than the body of the bacteria itself, i. e., they are stained by acid fuchsin. The polar bodies of the B. diphtheriae on the contrary are composed of more acid protein than the bacterial body; i. e., they are stained by methylene blue. The impossibility of detecting the above mentioned variations in the isoelectric points of bacteria using the Gram method is explained by the absence of pH variations in the latter technic. The differentiation of bacteria by the Gram stain depends chiefly on the varying stability of the compound formed (Gram-positive or Gram-negative bacteria plus gentian violet and iodine) in the presence of organic solvents, such as alcohol, acetone, etc.     

Structural studies on a glycoprotein isolated from alveoli of patients with alveolar proteinosis.

A major glycoprotein 36 000 molecular weight) has been isolated from lung lavage of patients with alveolar proteinosis and found to contain five residues of hydroxyproline, fifty residues of glycine, three residues of methionine, 3 mol of sialic acid, 4.4 mol of mannose, 4.0 mol of galactose, 6.0 mol of glucosamine, and 1 mol of fucose. Cyanogen bromide (CNBr) treatment of the glycoprotein resulted, as expected, in four peptides of apparent molecular weights of 18 000, 12 000, 5000 and 1000, respectively. The chemical compositions of the CNBr peptides indicate the presence of hydroxyproline and high amounts of glycine in all but one of the peptides; two of the four CNBr peptides contain carbohydrate. Gel filtration, acrylamide gel electrophoresis and end-group analyses of the native glycoprotein and its CNBr peptides indicate that the peptides are homogeneous. End-group analyses of the CNBr cleavage products assign the 18 000 molecular weight peptide to the NH2-terminal portion and the 1000 molecular weight peptide to the COOH-terminal portion of the native glycoprotein molecule. Pronase digestion of the 36 000 molecular weight glycoprotein, followed by gel filtration and cation exchange chromatography, resulted in two fractions. One fraction was acidic and contained all the carbohydrate, a high content of aspartic acid and no hydroxyproline. The other fraction was basic and contained 8.4% hydroxyproline, 14% proline, 28% glycine and no carbohydrate, suggesting the presence of collagen-like sequence in the peptide chain. Paper electrophoresis of the basic fraction demonstrated two components, the amino acid compositions of which are identical to those of collagen. Partial amino-terminal sequence analysis of one of the CNBr peptides (18 000 molecular weight) indicated the presence of -Fly-Pro-HyP-Gly-sequence in the peptide chain, which confirms our suggestion that collagen-like regions are present in the native glycoprotein molecule. Limited acid hydrolysis of the acidic fraction and subsequent fractionation of the acid hydrolysate using Dowex column yielded a fraction which produced brown colour with ninhydrin reagent. Paper chromatography of this fraction demonstrated a large component which also stained brown with ninhydrin reagent. After acid hydrolysis, this component was found to consist of equal amounts of asparitic acid and glucosamine, indicating that the N-acetylglucosamine of the oligosaccharides is linked to the asparagine residue of the peptide. No serine or threonine linkages are present.     

The effect of amino acid substitutions at position 342 on the secretion of human alpha 1-antitrypsin from Xenopus oocytes

A glutamic acid to lysine change in the Z variant of human alpha 1-antitrypsin is associated with a failure to secrete the protein from synthesising cells. The block in export of the protein may be caused either by the loss of an acidic residue or the introduction of a basic one at this point in the polypeptide chain. Site-directed mutagenesis has been used to construct novel alpha 1-antitrypsin mutants which show that the side chain interactions from Glu-342 are not obligatory for protein export and it is rather the introduction of a basic residue at this point which produces the intracellular accumulation of the protein.     

Variables Influencing Results, and the Precise Definition of Steps in Gram Staining as a Means of Standardizing the Results Obtained

Results of a Gram staining procedure varied with modifications of each of the steps involved. The best Gram differentiation was obtained when crystal violet and iodine solutions of high concentrations were used, and when n-propyl alcohol was used as the decolorizer. The decolorization step must be carefully quantitated, and one of the most important variables observed was whether a slide was brought into the decolorizer wet, or dry. Dry slides took 6 to 12 times as long to decolorize as wet. Wash steps, following crystal violet, and following the decolorizer, also greatly influence results by causing Gram-positive organisms to appear to be Gram-negative. The results indicated that Gram-stain procedures should not be varied to suit the whims of individual operators, and that each step could be specifically defined both as to the reagent used, and the procedure to be followed.

The followng Gram procedure is recommended for heat-fixed bacterial smears on glass slides. Flood the slide with Hucker's crystal violet for 1 ruin. Wash for 5 sec by dipping into tap water running into a 250 ml beaker at a rate of 30 ml per sec Rinse off the excess water with Burke's iodine, flood the slide with this solution for 1 min, then wash 5 sec in tap water as above. Decolorize by passing the wet slide through 3 (75 × 25 mm) Coplin dishes containing n-propyl alcohol, decolorize 1 min in each dish for a total of 3 min. Wash 5 sec in tap water as above, rinse off the excess water with 0.25% safranin, then flood the slide with this solution for 1 min. Wash as above, blot dry, and examine. An alternate procedure for decolorization would be to use either 95% n-propyl alcohol or 95% ethyl alcohol, but shorten the decolorization time to 30 sec per dish for a total of 1.5 min. After 10 slides, the decolorizer in the first dish should be replaced by fresh. This dish is then placed last in the sequence, with dish No. 2 moved to the No. 1 position.     

Effects of ionizing radiation on nitric oxide myoglobin. Part 2. Effects on the globin moiety

Irradiation of nitric oxide myoglobin (NOMb) induces changes in the haem as well as protein moiety of NOMb, especially at doses of 400-800 krad. The changes in the protein include: Conformational changes, with apparent partial denaturation of globin alpha-helix as evidenced by circular dichroism. Preferential scission of the polypeptide chain and dimerization via covalent bond(s) as evidenced by SDS-polyacrylamide gel electrophoresis. Products with a spectrum of hydrodynamic volumes between those of the monomer and the dimer are also formed. The shift of NOMb pIs toward more acidic pHs (probably due to modification and/or destruction of basic amino acid residues by water radiolytic products) as evidenced by isoelectric focusing.     

Immunoperoxidase staining of cervicovaginal smears after radiotherapy.

Cervicovaginal smears from 2 women with postirradiation dysplasia, 4 women with postirradiation squamous cell carcinoma of the cervix, 30 women with irradiation atypia and 5 healthy, nonirradiated women were stained immunohistochemically with six keratin antibodies. For four of the antibodies--CK19 (BA17), EMA, PKK-1 and CAM 5.2--squamous cells showing irradiation atypia, postirradiation dysplasia or postirradiation squamous cell carcinoma were more likely to stain positively than were nonirradiated squamous cells. For three of the antibodies in which multiple squamous cells stained positively, the proportion of squamous cells showing postirradiation dysplasia or postirradiation squamous cell carcinoma staining strongly was equal to or greater than the corresponding overall proportion for squamous cells showing irradiation atypia. This was statistically significant with only one antibody, PKK-1. No statistically significant differences were seen in staining of irradiated and nonirradiated squamous cells by MAK-6 and AE1:AE3. The data show that some keratin antigens are more often expressed in the irradiated groups and that there may be differences in the degree of antigen expression between squamous cells showing postirradiation dysplasia or postirradiation squamous cell carcinoma and squamous cells showing irradiation atypia.     

Modifications of Kohn's chlorazol black E staining and Wheatley's trichrome staining for temporary wet mount and permanent preparation of Entamoeba histolytica.

Preparation of stained smears of Entamoeba histolytica has several drawbacks. We therefore tried to simplify the staining procedures by modifing Kohn's chlorazol black E staining and Wheatley's trichrome staining techniques. Trophozoites and cysts of axenically cultured E. histolytica and Entamoeba invadens, respectively, and trophozoites and cysts of E. histolytica in stools of patients were used. Karyosomes and peripheral chromatin of nuclei and chromatoid bodies became distinctly visible after amoebae were suspended in the basic solution of Kohn's stain. Amoebae fixed in suspension with either basic solution or Bouin's fixative were clearly stained with Kohn's and trichrome preparations, both as wet mounts directly and as permanent slides after processing for mounting. These procedures were easier when the basic solution was used as a fixative and trichrome stain was employed. Erythrocytes ingested by trophozoites, however, were not stained with either of these preparations after fixation in the basic solution but were clearly stained when Bouin's fixative was used. Cysts of E. histolytica in stools concentrated using basic solution (instead of formalin) and ether were also stained with these stains. Consequently, without employing highly toxic mercuric chloride, wet mounts and permanent smears can be prepared with permanent stains, and preserved cysts can be stained after concentration.     

Scanning Feulgen-deoxyribonucleic acid cytophotometry of Papanicolaou destained preparations.

Feulgen deoxyribonucleic acid cytophotometry of Papanicolaou destained specimens revealed a differential loss in Feulgen reactivity among human buccal and cervical smears, cultured embryonic lung fibroblasts and invasive cervical carcinoma cells. Loss in Feulgen reactivity in Papanicolaou destained fibroblasts and polyploid nuclei of malignant lesions was observed to result in underestimates of relative Feulgen deoxyribonucleic acid and nuclear area values using scanning integrating microdensitometry. Thus, Papanicolaou stained preparations may not be suitable for deoxyribonucleic acid quantification of high ploidy lesions since distributional absorption error is unpredictably influenced by such factors as ploidy level, nuclear size, chromatin dispersion and differential aldehyde loss during destaining. Feulgen deoxyribonucleic acid cytophotometry of Papanicolaou stained preparations can be useful for differentiating benign from malignant lesions if extent of aneuploidy (as reflected in abnormal deoxyribonucleic acid frequency distribution profile) is used as a diagnostic indicator.     

Immunoperoxidase staining of fine needle aspiration specimens previously stained by the Papanicolaou technique

The use of immunoperoxidase techniques was investigated in 21 fine needle aspiration (FNA) cytology smears that had been previously stained by the Papanicolaou technique. The retrospectively selected slides were destained before applying the immunostain, utilizing antisera to calcitonin, prostatic acid phosphatase (PrAP), prostate-specific antigen (PSA), alpha-lactalbumin (AL), S-100 protein (S-100), carcinoembryonic antigen (CEA), common leukocyte antigen (LA), epithelial membrane antigen (EMA) and alpha-fetoprotein (AFP). Positive results were obtained with six of nine small-cell carcinomas of the lung stained with EMA, all three colonic carcinomas stained with CEA, one of two prostatic carcinomas stained with PSA and PrAP, one of two lymphomas stained with LA and the one medullary thyroid carcinoma stained with calcitonin. Negative staining results were observed in the one melanoma stained with S-100, the two breast carcinomas stained with AL and the one hepatocellular carcinoma stained with AFP. These results indicate that immunostaining can be a helpful diagnostic tool in diagnosing some fine needle aspirates using smears previously stained with the Papanicolaou stain.     

A pH-dependent conformational change of NhaA Na(+)/H(+) antiporter of Escherichia coli involves loop VIII-IX, plays a role in the pH response of the protein, and is maintained by the pure protein in dodecyl maltoside.

Digestion with trypsin of purified His-tagged NhaA in a solution of dodecyl maltoside yields two fragments at alkaline pH but only one fragment at acidic pH. Determination of the amino acid sequence of the N terminus of the cleavage products show that the pH-sensitive cleavage site of NhaA, both in isolated everted membrane vesicles as well as in the pure protein in detergent, is Lys-249 in loop VIII-IX, which connects transmembrane segment VIII to IX. Interestingly, the two polypeptide products of the split antiporter remain complexed and co-purify on Ni(2+)-NTA column. Loop VIII-IX has also been found to play a role in the pH regulation of NhaA; three mutations introduced into the loop shift the pH profile of the Na(+)/H(+) antiporter activity as measured in everted membrane vesicles. An insertion mutation introducing Ile-Glu-Gly between residues Lys-249 and Arg-250 (K249-IEG-R250) and Cys replacement of either Val-254 (V254C) or Glu-241 (E241C) cause acidic shift of the pH profile of the antiporter by 0.5, 1, and 0.3 pH units, respectively. Interestingly, the double mutant E241C/V254C introduces a basic shift of more than 1 pH unit with respect to the single mutation V254C. Taken together these results imply the involvement of loop VIII-IX in the pH-induced conformational change, which leads to activation of NhaA at alkaline pH.     

Numerical calculations of the pH of maximal protein stability. The effect of the sequence composition and three-dimensional structure.

A large number of proteins, found experimentally to have different optimum pH of maximal stability, were studied to reveal the basic principles of their preference for a particular pH. The pH-dependent free energy of folding was modeled numerically as a function of pH as well as the net charge of the protein. The optimum pH was determined in the numerical calculations as the pH of the minimum free energy of folding. The experimental data for the pH of maximal stability (experimental optimum pH) was reproducible (rmsd = 0.73). It was shown that the optimum pH results from two factors - amino acid composition and the organization of the titratable groups with the 3D structure. It was demonstrated that the optimum pH and isoelectric point could be quite different. In many cases, the optimum pH was found at a pH corresponding to a large net charge of the protein. At the same time, there was a tendency for proteins having acidic optimum pHs to have a base/acid ratio smaller than one and vice versa. The correlation between the optimum pH and base/acid ratio is significant if only buried groups are taken into account. It was shown that a protein that provides a favorable electrostatic environment for acids and disfavors the bases tends to have high optimum pH and vice versa.     

A phosphorus-magnetic-resonance study of the interaction of Mg2+ with adenyl-5'-yl imidodiphosphate. Binding sites of Mg2+ ion on the phosphate chain.

The interaction of Mg2+ ions with adenyl-5'-yl imidodiphosphate, AMP-P(NH)P, has been studied at basic and acidic pH values by phosphorus magnetic resonance spectroscopy in aqueous solution. The results suggest that Mg2+ binds simultaneously to one (or both) of the two free oxygen atoms of the beta-phosphate moiety and to the nitrogen atom of the phosphate chain (P alpha-O-P beta-N-P gamma). The interaction arises from 1: 1 complexing of Mg2+ to AMP-P(NH)P. The mode of the Mg2+ binding on the phosphate chain remains the same at both basic and acidic pH values. As in the case of ATP and ADP, the association of Mg2+ reduces the pK by about 1.5 units. On the other hand phosphorus titration curves showed that when the phosphate chain does not possess the regular periodicity (O-P alpha-O-P beta-X-P gamma-O,X not equal to O) as in the case of ATP, protonation of the terminal phosphate group may induce a 31P chemical shift variation less important for this group than for the preceding one.     

Peptide-membrane interactions A fluorescence study of the binding of oligopeptides containing aromatic and basic residues to phospholipid vesicles

The binding of oligopeptides containing basic and aromatic residues to phospholipid vesicles has been studied by fluorescence spectroscopy. Tryptophan-containing peptide such as Lys-Trp-Lys or Lys-Trp(OMe) exhibit a shift of their fluorescence toward shorter wavelengths and an increased fluorescence quantum yield upon binding to phosphatidylinositol (PI) or phosphatidylserine (PS) vesicles. No binding was detected with phosphatidylcholine vesicles. The binding is strongly dependent on ionic strength and pH. Binding decreases when ionic strength increases indicating an important role of electrostatic interactions. The pH-dependence of binding reveals that the apparent $\text{p}\text{K}$ of the terminal carboxyl group of Lys-Trp-Lys is raised by ～3 units upon binding to PI and PS vesicles. The binding of tyrosine-containing peptides to PI and PS vesicles is characterized by an increase in the fluorescence quantum yield of the peptide without any shift in fluorescence maximum. A natural nonapeptide from the myelin basic protein which contains one tryptophan residue binds to PI and PS vesicles at low pH when the acidic groups are neutralized. This binding is accompanied by a shift of the tryptophyl fluorescence toward shorter wavelengths together with an enhancement of the fluorescence quantum yield. Dissociation of the complex is achieved at high ionic strength. These results indicate that aromatic residues of oligopeptides bound to the phospholipid polar heads by electrostatic interactions become buried in a more hydrophobic environment in the vicinity of the aliphatic chains of the lipids.     

Isoelectric-focusing behavior of acid hydrolases in rat kidney lysosomes. Effects of the pH gradient, autolysis and neuraminidase.

Isoelectric focusing was used to study the multiple forms of acid phosphatase, arylsulfatase, beta-glucuronidase and beta-N-acetylhexosaminidase in lysosomes isolated from rat kidney. The isoelectric points of the main protein and hydrolase peaks were 1-1.5 units lower when electrofocusing was done in a pH 3-10 gradient than in a pH 10-3 gradient, apparently because the lysosomal constituents aggregated strongly at their isoelectric points and tended to settle somewhat in the gradient due to gravity. In the extended pH gradient the acidic form of each hydrolase occurred as asingle, relatively discrete peak. However, when pooled acidic fractions were refocused in a restricted pH gradient (pH 6-3 or 3-5) multiple acidic enzyme and protein components were resolved with isoelectric points between 2.7 and 5.1. When autolysis was minimized by extracting lysosomal fractions at alkaline pH (0.2% Triton X-100, 0.1%p-nitrophenyloxamic acid, 0.1 M glycine buffer, pH9) and including 0.1%p-NITROPHENYLOXAMIC ACID, AN INHIBITOR OF LYSOSOMAL NEURAMINIDASE AND CATHEPSIN D, in the pH gradient, arylsulfatase, beta-glucuronidase and beta-N-acetylhexosaminidase occurred in two forms, an acidic form with an isoelectric point of about 4.4, and a basic form with an isoelectric point close to 6.2, 6.7 and 8.0, respectively. Acid phosphatase occurred in three forms with isoelectric points of 4.1, 5.6 and 7.4. When some autolytic digestion was permitted by extracting lysosomal fractions in an acidic medium (0.2% Triton X-100, 0.1 M sodium acetate buffer, pH 5.2) AT 0-4DEGREES C and omitting p-nitrophenyloxamic acid from the gradient, the acidic form of beta-glucuronidase and the intermediate form of acid phosphatase were lost, the isoelectric points of the acidic forms of acid phosphatase, arylsulfatase and beta-N-acetylhexosaminidase were increased 0.6-1.2 units, and the isoelectric point of the basic forms of acid phosphatase, arylsulfatase and beta-glucuronidase was increased 0.5 unit. When lysosomal extracts were incubated with bacterial neuraminidase before electrofocusing, the acidic forms of acid phosphatase, arylsulfatase and beta-glucuronidase were largely lost, the isoelectric point of the acidic form of beta-N-acetylhexosaminidase was increased from 4.5 to 6.4, and the isoelectric points of the basic forms of all four hydrolases were increased 0.5-1.5 units. Autoincubation of lysosomal extracts in vitro at pH 5.2 PRODUCED SIMILAR, THOUGH LESS MARKED, effects. cont'd     

Two Isolectins from Leaves of Winged Bean, Psophocarpus tetragonolobus (L.) DC.

Two isolectins were isolated from leaves of winged bean andcharacterized. They differed from each other in terms of theirimmunological properties, hemagglutinating activities, sugarinhibition patterns, and amino acid compositions. Both lectinswere acidic and one of them (L-I), which was inactive towardtrypsinized human type O erythrocytes, was similar to one ofgreen shell lectins (WGS-1); which resembled basic seed lectinin its immunological properties. The amino-terminal sequenceof L-I was homologous to that of WGS-1. The amino acid compositionof L-I was similar to that of basic seed lectin, but the extentof the homology between amino-terminal sequences was low whenL-I and basic seed lectin were compared. Examination by ELISArevealed that L-I and WGS-1 were distinct from the basic lectinsof seeds and tuberous roots. L-I had a disulfide bridge betweentwo subunits and it exhibited high hemagglutinating activitytoward human type A erythrocytes, as compared to its activitytoward other erythrocytes. By contrast, the properties of asecond acidic lectin from winged bean leaves (L-II) were verysimilar to those of acidic lectins from seeds and tuberous roots,and the similarities extended as far as the immunological properties. (Received January 6, 1994; Accepted August 15, 1994)     

The Effect of Preliminary Treatment (Fixing Fluids) on Staining Properties of the Tissues

As was reported in a previous paper,¹ staining properties depend on the chemical composition of the tissues and on the strength of the dyes themselves. Applying mixtures of basic and acid dye on tissues (methylene blue, eosin Y) at different pH-values, it is possible to find differences in the isoelectric points of the nuclei and cytoplasm of different tissues. For example, the nucleus of polymorphonuclear cells of the blood consists of the most acid protein, with an isoelectric point around pH 2.5, while the nucleus of lymphatic tissues has an isoelectric point of about pH 4.0, and that of connective tissue about pH 3.4.

With a knowledge of the above, a constant method of staining at various pH-values was used to study the effect of different fixing fluids on the staining properties of the tissues. In this way it was found that many fixing fluids gave very stable compounds with tissue proteins, and that they almost permanently change the chemical composition (i.e. the staining properties of the tissues). In some instances, these changes can be easily explained from the regular chemical standpoint. For example, formalin forms inert compounds with amino groups of the amino acids of proteins and in this way it makes the tissue proteins more acid, i.e. it moves the isoelectric point of the proteins toward a lower pH-value. The same is true in the case of the polivalent acids. The bivalent heavy metals such as mercury, on the contrary, it is assumed, combine with carboxyi groups of amino acids and in this way move the isoelectric point of the proteins toward a higher pH.