ON THE NATURE OF THE DYE PENETRATING THE VACUOLE OF VALONIA FROM SOLUTIONS OF METHYLENE BLUE

When uninjured cells of Valonia are placed in methylene blue dissolved in sea water it is found, after 1 to 3 hours, that at pH 5.5 practically no dye penetrates, while at pH 9.5 more enters the vacuole. As the cells become injured more dye enters at pH 5.5, as well as at pH 9.5. No dye in reduced form is found in the sap of uninjured cells exposed from 1 to 3 hours to methylene blue in sea water at both pH values. When uninjured cells are placed in azure B solution, the rate of penetration of dye into the vacuole is found to increase with the rise in the pH value of the external dye solution. The partition coefficient of the dye between chloroform and sea water is higher at pH 9.5 than at pH 5.5 with both methylene blue and azure B. The color of the dye in chloroform absorbed from methylene blue or from azure B in sea water at pH 5.5 is blue, while it is reddish purple when absorbed from methylene blue and azure B at pH 9.5. Dry salt of methylene blue and azure B dissolved in chloroform appears blue. It is shown that chiefly azure B in form of free base is absorbed by chloroform from methylene blue or azure B dissolved in sea water at pH 9.5, but possibly a mixture of methylene blue and azure B in form of salt is absorbed from methylene blue at pH 5.5, and azure B in form of salt is absorbed from azure B in sea water at pH 5.5. Spectrophotometric analysis of the dye shows the following facts. 1. The dye which is absorbed by the cell wall from methylene blue solution is found to be chiefly methylene blue. 2. The dye which has penetrated from methylene blue solution into the vacuole of uninjured cells is found to be azure B or trimethyl thionine, a small amount of which may be present in a solution of methylene blue especially at a high pH value. 3. The dye which has penetrated from methylene blue solution into the vacuole of injured cells is either methylene blue or a mixture of methylene blue and azure B. 4. The dye which is absorbed by chloroform from methylene blue dissolved in sea water is also found to be azure B, when the pH value of the sea water is at 9.5, but it consists of azure B and to a less extent of methylene blue when the pH value is at 5.5. 5. Methylene blue employed for these experiments, when dissolved in sea water, in sap of Valonia, or in artificial sap, gives absorption maxima characteristic of methylene blue. Azure B found in the sap collected from the vacuole cannot be due to the transformation of methylene blue into this dye after methylene blue has penetrated into the vacuole from the external solution because no such transformation detectable by this method is found to take place within 3 hours after dissolving methylene blue in the sap of Valonia. These experiments indicate that the penetration of dye into the vacuole from methylene blue solution represents a diffusion of azure B in the form of free base. This result agrees with the theory that a basic dye penetrates the vacuole of living cells chiefly in the form of free base and only very slightly in the form of salt. But as soon as the cells are injured the methylene blue (in form of salt) enters the vacuole. It is suggested that these experiments do not show that methylene blue does not enter the protoplasm, but they point out the danger of basing any theoretical conclusion as to permeability on oxidation-reduction potential of living cells from experiments made or the penetration of dye from methylene blue solution into the vacuole, without determining the nature of the dye inside and outside the cell.     

METHYLENE BLUE INHIBITION OF PHOTOSYNTHESIS IN RHODOPSEUDOMONAS PALUSTRIS

1. Photosynthesis in Rhodopseudomonas palustris was found tobe completely inhibited by the reduced form of methylene blueat the concentration below 1x10^–5 M, provided that thecells had been in contact with the dye in the dark in anaerobiosis.The inhibition could be eliminated by washing the poisoned cellsor by replacing the anaerobic atmosphere with an aerobic one. 2. Other thiazine or oxazine dyes with E¹₀ near 0 volt, suchas thionine, brilliant cresyl blue and toluidine blue, in theirreduced forms, were also almost equally effective. 3. The mechanism of this dye-inhibition of bacterial photosynthesiswas discussed. ¹Present address: Laboratory of Biological Chemistry, TokyoInstitute of Technology, Meguro-ku, Tokyo. (Received August 21, 1961; )     

THE PHOTODYNAMIC INACTIVATION OF PHAGE PRECURSOR BY METHYLENE BLUE

Methylene blue added to suspensions of activated staphylococci in amounts sufficient to furnish 1 x 10⁵ molecules of dye/bacterium inactivates the phage precursor content of the cells without causing cell death when the mixtures are exposed to strong light of 4000–8000 Å. There is a lag phase of approximately 15 minutes in the photodynamic inactivation of phage precursor by methylene blue. This delay seems to be due to a primary reaction between the cell and methylene blue after the completion of which exposure to light brings about the inactivation of precursor quite promptly.     

EXPERIMENTS WITH THE METHYLENE BLUE REDUCTION TEST FOR MILK

SUMMARY: The effects of shaking milk samples before the addition of methylene blue, delaying the time of addition of the dye, adding a second quantity of dye with a hypodermic syringe after the initial charge had been decolourized and of increasing the storage time or varying the testing time have been studied in five separate experiments. Vigorous shaking at the time of dye addition lengthened the reduction time. This effect was more pronounced when the milk was stored in a tube than in a bottle. Delayed addition of the dye resulted in a shorter reduction time than expected for inverted samples and longer than expected for samples which were not inverted between the time they were put into the 37° bath and the addition of the dye. Second additons of dye did not usually have reduction times exceeding 1 hr. Methylene blue reduction times did not necessarily decrease with successive tests carried out over a period of time on one sample: a time of 30 min. might persist over a series of tests for 4 hr. or more and small increase and decrease in reduction time with successive tests were also noted.     

THE RATE OF REDUCTION OF METHYLENE BLUE BY BACILLUS COLI

This paper deals with the relation between substrate concentration and velocity in the case of the reduction of methylene blue and of the other oxidation-reduction indicators of Clark by B. coli in the presence of succinic acid and glucose. This system is compared with starch and barley amylase. Reasons are given for considering the mechanism as an adsorption phenomenon.     

THE EFFECT OF PENICILLIN ON METHYLENE BLUE ADSORPTION ONTO ACTIVATED CHARCOAL

Penicillin inhibits the adsorption of methylene blue onto activated charcoal in concentrations as low as 100 units/ml. Penicillin in the low inhibitory concentration of 100 units/ml, antagonizes to a small extent the strong inhibition of MB adsorption by 0.1, 0.2, and 1 per cent peptone.     

DETERMINATION OF METHYLENE BLUE BIOSORPTION BY Rhizopus arrhizus IN THE PRESENCE OF SURFACTANTS WITH DIFFERENT CHEMICAL STRUCTURES

Methylene blue (MB) biosorption properties of Rhizopus arrhizus were investigated in the presence of surfactants. The effects of cationic and anionic surfactants on MB removal by dead biomass (1 g L^?1) were determined. MB removal was tested as a function of initial pH (2–12), contact time (5–1440 min), and dye (37.4–944.7 mg L^?1) and surfactant (0–10 mM) concentrations. The opposite charged anionic surfactant dodecylbenzenesulfonic acid sodium salt (DBS) enhanced sorption of cationic MB by biomass dramatically. Maximum biosorption capacity was 471.5 mg g^?1 at pH 8 with 0.5 mM DBS at 944.7 mg L^?1 MB concentration. The surfactant-stimulated fungal decolorization method may provide a highly efficient, inexpensive, and time-saving procedure in biological wastewater treatment technologies.     

CORRELATION OF OXIDATION AND PHOSPHORYLATION IN HEMOLYZED BLOOD IN PRESENCE OF METHYLENE BLUE AND PYOCYANINE

1. The system:hemolyzed blood + glucose never exhibits glycolysis or, in the air, oxidation of glucose. When glucose is replaced by hexosephosphate ester, addition of methylene blue causes oxidation in air. 2. When cozymase is added also, the oxidation is increased, and a synthesis of hexosephosphate esters takes place. 3. When pyocyanine is used instead of methylene blue, the rate of oxidation is the same as with methylene blue, but a synthesis of phosphate esters takes place without addition of cozymase. 4. There is never a phosphate ester synthesis without oxidation going on, but oxidation does not necessarily go hand in hand with phosphate synthesis. 5. In order to couple the oxidation process with phosphate synthesis, two methods are available: either to start oxidation by methylene blue and to add coenzyme from yeast cells; or to start oxidation by pyocyanine, in which case coenzyme is unnecessary, though it improves the effect. 6. Iodoacetate always suppresses synthesis, but only under certain conditions decreases oxidation. Cyanide has no effect upon either process.     

THE DETERMINATION OF COLI-AEROGENES ORGANISMS IN RAW MILK BY SURFACE SMEARS ON DRIED EOSIN METHYLENE BLUE AGAR PLATES

SUMMARY: The determination of the coli-aerogenes content of raw milk by smearing 0·1 or 0·2 ml of suitable dilutions on dried Levine's eosin methylene blue agar plates and incubating at 37° or 30°, was found too unselective as a routine technique. Only about two-thirds of the colonies showing the characteristic appearance of coli-aerogenes bacteria were confirmed as such by the formation of acid and gas in MacConkey's broth. Appreciable proportions of the numerous rose coloured colonies and of the very small dark colonies with metallic sheen, which are not considered to be coliaerogenes bacteria, formed acid and gas in MacConkey's broth.