THE RELATION BETWEEN THE OXYGEN CONCENTRATION AND RATE OF REDUCTION OF METHYLENE BLUE BY MILK

The rate of reduction of methylene blue by milk and acetaldehyde is proportional to the concentration of oxygen in the milk. This fact may be made the basis of a method of determining oxygen in gaseous mixtures.     

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.     

INHIBITORY ACTION OF PARATYPHOID BACILLI ON THE FERMENTATION OF LACTOSE BY BACILLUS COLI. I

Bacteria of the paratyphoid group may be divided into two classes according to the behavior of 4 day cultures in lactose bouillon after a second inoculation with certain types of Bacillus coli. One class includes all true hog-cholera bacilli, the other nearly all true paratyphoid and enteriditis types. Under the imposed conditions Bacillus coli produces the usual amount of gas in the presence of the first group. In the presence of the second no gas or only a bubble appears. The production of acid is not interfered with. The significance of the inhibition was investigated in a variety of ways suggested by the particular hypothesis entertained at the time. Two main possibilities presented themselves; first, the direct association of the inhibition with living paratyphoid bacilli, and, second, the existence of a ferment or other product of growth as the inhibiting agent. The theory that the living bacilli or those killed at the lowest possible temperature are responsible was favored by a number of experiments. Thus the complete removal of bacteria by filtration, or by centrifugation combined with the use of kaolin to produce a clear fluid restored gas production. The presence of a fine cloud of bacteria was sufficient to inhibit. On the other hand, the addition of large numbers of living bacteria from agar slants or from lactose bouillon after the requisite incubation period to fresh lactose bouillon failed to inhibit gas production when Bacillus coli was added simultaneously. When the inhibiting culture was heated at 62°C. for 35 minutes to sterilize it, gas production was still largely inhibited. But it was restored when higher temperatures were used, completely at 100°C. and above. It was also gradually restored by exposing the heated culture to 37°C. for 3 or more days. The presence of variable amounts of lactose, or even the complete absence of lactose did not interfere with the development of the inhibitory factor. The activity of the inhibition factor presents itself in the form of a curve, beginning at 0 when both paratyphoid and colon bacilli are inoculated simultaneously and rising as Bacillus coli is inoculated at longer intervals from the paratyphoid bacilli. The maximum of inhibition is reached at about the 4th day; thereafter it remains at the same level for a few days and then gradually falls until it is lost within 3 or 4 weeks. The curve of the hog-cholera group is delayed in that the maximum inhibition is reached at the end of 3 weeks. These curves have not been accurately determined. Taking into consideration all the accumulated data the writers tentatively present the hypothesis that the inhibitory factor is some metabolic product of the paratyphoid bacillus, possibly an enzyme, which is destroyed at a temperature somewhat above the thermal death point of the bacilli and which more gradually disappears from incubated cultures. The substance fails to pass Berkefeld filters. It is carried down mechanically with substances clearing the culture fluid. The experiments support current theories which hold that the acid-producing and gas-producing entities in cultures are distinct.     

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.     

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.     

FURTHER STUDIES ON THE INHIBITION OF CYPRIDINA LUMINESCENCE BY LIGHT,WITH SOME OBSERVATIONS ON METHYLENE BLUE

1. Eosin, erythrosin, rose bengale, cyanosin, acridine, and methylene blue act photodynamically on the luminescence of a Cypridina luciferin-luciferase solution. In presence of these dyes inhibition of luminescence, which without the dye occurs only in blue-violet light, takes place in green, yellow, orange, or red light, depending on the position of the absorption bands of the dye. 2. Inhibition of Cypridina luminescence without photosensitive dye in blue-violet light, or with photosensitive dye in longer wave-lengths, does not occur in absence of oxygen. Light acts by accelerating the oxidation of luciferin without luminescence. Eosin or methylene blue act by making longer wave-lengths effective, but there is no evidence that these dyes become reduced in the process. 3. The luciferin-oxyluciferin system is similar to the methylene white-methylene blue system in many ways but not exactly similar in respect to photochemical change. Oxidation of the dye is favored in acid solution, reduction in alkaline solution. However, oxidation of luciferin is favored in all pH ranges from 4 to 10 but is much more rapid in alkaline solution, either in light or darkness. There is no evidence that reduction of oxyluciferin is favored in alkaline solution. Clark''s observation that oxidation (blueing) of methylene white occurs in complete absence of oxygen has been confirmed for acid solutions. I observed no blueing in light in alkaline solution.     

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.     

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; )