Barnett Cohen

On the Instability of Brilliant Cresyl Blue.

The employment of this dye (dimethyl diamino toluphenazine chloride) in biochemical work warrants the publication of this note on some of its properties. The purified dry dye when extracted with dry ether, benzene or xylene gives no coloration to the solvent. Chloroform has the disadvantage that the dye salt is appreciably soluble in it. Addition of water to the dye-solvent mixture results within 10 minutes in coloration of the non-aqueous phase. This coloration increases progressively with time, with increased temperature and with extremes of pH. It is not a result of interaction of dye with the non-aqueous solvent, for independent oxidation-reduction titrations in aqueous solutions show the presence and accumulation of secondary systems as contact of dye with water is prolonged. The conclusion must be, therefore, that brilliant cresyl blue remains ‘pure only so long as it is kept out of contact with water. This instability seems to be of the same kind and degree as that found in methylene blue. The nature of the secondary products is unknown. It is suspected that they are demethylated and deaminated derivatives of the parent dye. At least 2 are present. One has the properties of an oxazone. It is a very weak base with dissociation constant less than 10-13 and its solutions from pH 1 up to normal KOH are colored red with a golden fluorescence. The second product has the properties of a stronger base, with a dissociation constant near 10-6.∗ Its salt is colored blue and the base orange. Brilliant cresyl blue itself is a stronger base with a dissociation constant of 10-3.

Brom cresol green, a sulfonphthalein substitute for methyl red:

The sulfonphthalein indicators of Clark and Lubs have shown themselves quite stable and reliable in biological fluids. Methyl red, which is not a sulfonphthalein, is not altogether reliable, but was included in the Clark and Lubs series because it was indispensable in covering a certain range of H-ion concentration. Methyl red is easily reduced irreversibly to a colorless compound—frequently by microbic action—thereby impairing its utility as an indicator under all conditions. A sulfonphthalein indicator has been synthesized which has an apparent dissociation constant almost identical with that of methyl red and which seems as stable and reliable as the rest of the sulfonphthaleins. This compound is tetra-brom m-cresol sulfonphthalein. It is made by the bromination in glacial acetic acid of m-cresol sulfonphthalein. The common name suggested for this compound is Brom Cresol Green. Its effective range as an acid-base indicator is between PH 4.0 and 6.0, with a color change from yellow to green to blue-green. Its apparent dissociation constant in terms of PH is 5.00 (that of methyl red is 4.95). Color standards of Brom Cresol Green were unaffected after several months exposure in test tubes to usual laboratory conditions, while similar standards of methyl red had faded irregularly and become totally useless. Brom Cresol Green may be used directly in a bacteriological culture medium, for instance, while this would be out of the question for methyl red.

Reduction Intensity in Anaerobic Ameba dubia

Lack of suitable indicators has hitherto prevented an examination of intracellular oxidation-reduction intensities by microinjection under anoxybiosis. Earlier attempts by Cohen, Chambers and Reznikoff 1 using phenosafranine were inconclusive because the dye underwent irreversible change during the time required for manipulation and observation. Stiehler, Chen, and Clark 2 and Stiehler 3 have recently made available some new indicators for the more negative regions of reduction potential which we have utilized. Some of these compounds undergo irreversible change but by quick operation (injections made within a few minutes after reduction) we have succeeded in obtaining definite indications. Observations were made in an improved hermetic chamber through which flowed moistened purified nitrogen or hydrogen. Injection of oxidizing agent or exposure to air were used to check the reversibility of the indicator in the cell interior. Results on immersion of the cells in oxidant or reductant confirmed those obtained by microinjection. The experiments were restricted to one type of unicellular organism, e. g., Ameba dubia. The results are given in the accompanying table. These preliminary observations are now being augmented by examination of other available indicators.

Some phases of the disinfection theory

Bact. typhosum and Bact. coli (communis) were suspended in distilled water, tap water, and M/500 buffer solutions, respectively, at constant temperature levels (0°, 10°, 20°, 30° C.); and the numbers of survivors were determined by means of decimal dilutions upon agar plates. The conditions imposed (moderate H-ion concentrations at moderate temperatures) permitted a closer study of the disinfection process than has been usually possible. It was found, of course, that coli was relatively more resistant than typhosum, but this greater resistance (at PH 3.5) decreased as the temperature level rose. At 0° C., coli was 67 times more resistant, and at 30° C. it was only 8 times more resistant than typhosum. There was a high inconstancy in results between duplicate tests carried out in tap or distilled water. This inconstancy could at times be correlated with comparatively insignificant fluctuations in PH of the water. When very dilute (M/500) Clark and Lubs buffers were used, this variability disappeared very largely. At 20° C., Bact. typhosum possesses the greatest tolerance within a narrow zone delimited by PH 5.0 and 6.4. A slight increase in acidity beyond the zone results in conditions of maximum mortality. For Bact. Coli the zone is wider and centered about neutrality. Cohen and Clark 1 found that the PH optima for growth and fermentation of bacteria may be different. It is now found that the optimum for tolerance may also be distinct. The logarithmic decline in numbers of bacteria may be modified by suitably chosen conditions. This applies also to some monomolecular chemical reactions. The logarithmic course in either case is merely a statistical integration and gives no information as to the mechanism of chemical decomposition or of bacterial disinfection. They both illustrate the operation of the law of mass action.

Discrepancies in blood oxygen analyses by the methods of Van Slyke and Henderson-Smith.1

Loosely bound oxygen is liberated from the hemoglobin in blood by the addition of potassium ferricyanide. In the Van Slyke method, 2 all the gases are exhausted by means of a Toricellian vacuum from a laked blood-ferricyanide mixture and measured directly. In the Henderson-Smith method, 3 the oxygen is evolved into a fixed volume of air, a portion of which is analyzed directly for oxygen by absorption with alkaline pyrogallate. After application of all the corrections suggested by the authors of these methods, the results are not identical,—analyses by the Van Slyke method yielding 4 to 10 volumes per cent. more oxygen than those by the Henderson-Smith method. The divergence between the results from each method may represent a variation of 17 to 64 per cent. A few typical figures are cited in Table 1. It is clear that there is a constant factor or factors at play, inherent in the methods of analysis employed, which ought to account for this discrepancy. The gas evacuated by the Van Slyke procedure is not all oxygen but probably contains in addition to nitrogen, minute amounts of carbon monoxide, hydrogen, methane and the rare atmospheric gases. Bohr 1 states that blood contains I .23 volumes per cent. of nitrogen (incorrectly quoted by Van Slyke as 0.9 vol. per cent.) and 0.22 volumes per cent. of the other gases-a total of about 1.45 volumes per cent. of gas not oxygen. We have absorbed with alkaline pyrogallate, the oxygen from the gas extracted in the Van Slyke procedure and have found in all cases a residue of 0.055 to 0.082 C.C. from 2 C.C. of blood-an average of about 3.3 volumes per cent. This residue does not contain CO2, and we have reason to believe that it is practically all nitrogen.

The influence of cooking and drying cabbage on its antiscorbutic properties for guinea pigs.1

The experimental scurvy induced in guinea pigs by a special soy bean-milk-yeast-paper pulp-salt diet 3 could be prevented by a daily addition of 10 gm. raw cabbage along with the ration. Cabbage cooked for thirty minutes at 100° C., subsequently incorporated with the rest of the food, and dried at 65-70° C. for two days lost its antiscorbutic power. Cabbage heated in an oven for two hours at 75-80° C., then dried at 65-70° C., ground, intimately mixed with the food, and the whole dried further for two days at 65-70° C. exhibited no potency as an antiscorbutic. Cabbage dried in a blast of air at 40-52° C. retained some of its antiscorbutic value in that it delayed markedly the onset of scorbutic symptoms. Furthermore it could be used as a therapeutic agent when the signs of scurvy were recognized early enough.

Observations on the production of experimental scurvy in the guinea pig

The customary method of producing experimental scurvy in the guinea pig with a monotonous cereal diet is open to several points of criticism. The protein may be inadequate, inorganic salts deficient and the physical texture unsatisfactory. Our work led to a search for some good food material capable of dietary analysis; and through the results obtained by Osborne and Mendel 1 and others the soy bean seemed most suitable. Its protein is adequate for maintenance and growth (of the rat) and the only inorganic constituents deficient are Na, Ca and Cl. We used soy bean flour autoclaved at 15 pounds for 30 minutes. To it was added Ca lactate and NaCl. Dried brewers yeast and fresh raw Jersey milk supplied the two recognized dietary accessories (vitamines). Young guinea pigs on this diet gain in weight faster than on the usual cabbage-carrots and oats ration. After 10 days, while still gaining in weight they develop swelling of the joints and general tendernes—symptoms considered indicative of experimental scurvy. Later occurs loss in weight followed by death. Stools were frequent and pasty; toward the end diarrheal. A marked polyuria was present from the beginning of the diet. Addition of over 7 per cent. cellulose as roughage did not change the result. Orange juice, 5 c.c. daily, or fresh cabbage prevented and cured these symptoms promptly with increase in weight.

Diet and roughage in relation to the experimental scurvy of guinea pigs

It has been repeatedly demonstrated that exclusive diets of cereals produce scurvy in the guinea pig. We have fed filter paper, sawdust and hay respectively, as supplements to an oat diet without averting the appearance of scurvy. Duration of the disease and decline were not appreciably different when these supplements were fed. The addition of 7, 10, or 18 per cent. of paper pulp to a special soy bean diet 1 failed to supply an antiscorbutic property. Feeding raw milk in addition to oats induces marked constipation with impaction of feces in the cecum. Animals fed 40 c.c. milk daily showed definite symptoms of scurvy in about a month. As the daily allowance of milk was increased, the symptoms seemed to recede in severity. Yet even when 80 c.c. milk were consumed daily, the animals became very constipated and died; but there were scarcely any signs of scurvy. Autopsy of such a case revealed absence of the typical macroscopic hemorrhages or of fragility of the bones. These observations appear to confirm the findings of Chick, Hume and Skelton, 2 which indicate that a sufficient amount of milk fed to guinea pigs will prevent scurvy. Such observations render debatable the hypothesis that the experimental scurvy of guinea pigs is attributable to failure of normal intestinal movement. Preliminary experiments on the nutritive qualities of descicated vegetables indicate that the drying of fresh cabbage does not entirely remove its antiscorbutic property. 1