NILE BLUE, ANILINE BLUE AND NEUTRAL RED AS INDICATORS OF LIPOLYSIS

SUMMARY: Dyes which have been used to detect lipolysis (Nile Blue, Aniline Blue and Neutral Red) and others (Methylene Blue, Toluidine Blue and Thionin) were critically examined for their inhibitory effects in both liquid and solid media. It was confirmed that Nile Blue would inhibit the Gram-positive bacteria tested, even at 2 × 10⁻⁵. Gram-negative bacteria were not inhibited at 1 × 10⁻⁴ and the colour change in fat media was good. Aniline Blue and Neutral Red did not inhibit Gram-positive organisms at 1 × 10⁻⁴ and the colour change was moderately good. The other three dyes were not toxic at 2·5 × 10⁻⁵, but there was no colour change. When butter fat saturated with the precipitated bases of Nile Blue, Basic Fuchsin, Crystal Violet or Malachite Green was incorporated in peptone-Yeastrel agar, the resulting media were toxic to Gram-positive bacteria. Fat saturated with the bases of Aniline Blue or Neutral Red permitted growth, but the colour change with lipolytic species was not striking, being one of intensity only. Satisfactory growth and colour changes were obtained using butter fat stained with the inert dye Waxolene Green in medium containing Neutral Red in the aqueous phase, and with butter fat stained with the oxazine base of Nile Blue in agar containing Aniline Blue.     

Evaluation of the lipolytic activity of starter cultures for meat fermentation purposes

A modified pork fat based agar method was specially developed to determine the lipolytic activity of starter strains used for meat fermentations. The lipolytic ability of strains of lactobacilli, pediococci, staphylococci and micrococci, was examined on agar plates using different substrates and in a lipid broth model system. The screening results indicate that lipase activity was confined to strains of Staphylococcus and Micrococcus . None of the lactic acid bacteria tested showed lipolytic activity. A strain of Staphylococcus xylosus , which displayed high lipolytic activity during the screening process was examined to find its optimum conditions for lipase activity regarding pH, temperature and NaCl concentration. The lipolytic activity of seven bacterial strains was determined under optimum conditions (0% salt, pH 7 and 30 °C) and sausage conditions (3·5% salt, pH 5 and 20 °C) using fat buffer model systems. High lipolytic activity was determined for all seven strains under optimal conditions, whereas no activity was detected under fermented sausage conditions.     

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.     

Lipase production byAspergillus andPenicillium species

Forty each of aspergilli and penicillia were screened for extracellular lipase production on agar plates and in liquid medium containing olive oil as substrate. Twenty-nine aspergilli and twenty-six penicillia produced lipase. Out of these, 19 aspergilli and 22 penicillia showed activity both on Nile blue sulfate and glycerol tributyrate agar plates while only 10 aspergilli and 4 penicillia showed a positive response to glycerol tributyrate agar alone. The screening revealed 11Aspergillus spp. and 15Penicillium spp. as new lipase producers. Pig fat as an economic substrate for lipase production was also investigated.     

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.     

The vitamin requirements of Staphylococcus cohnii

In heat-resistance studies with spores of Clostridium sporogenes BC-2, an improved recovery medium was needed for severely heat-damaged spores as the used previously--Wynne medium in Miller-Prickett tubes--did not allow accurate counts of spores because of gas formation and disruption of agar. Initial test with pour plates of Viande-Leyure medium containing egg-yolk gave much increased counts for spores previously heated for 50 min at 112 degrees C; this increase was attributed to the presence of egg-yolk. Addition of egg-yolk to Reinforced Clostridial Agar, All-Culture Medium and Trypticase Soy Agar showed that Trypticase Soy Agar with egg-yolk was the best recovery medium. For the final formulation, the value of supplementation with cysteine-HCl and methylene blue was also shown. The resultant Egg-yolk Trypticase Soy Agar is conveniently prepared from BBL Trypticase Soy Agar (40 g) with the addition of 0.4 g/l cysteine-HCl, 4 mg/l of methylene blue and 2% Oxoid egg-yolk emulsion aseptically to the melted basal medium. For optimal spore counts, pour plates are incubated anaerobically for 5--7 d at 30 degrees C.     

产H_2O_2的乳酸杆菌分离培养基的改良

对MRS培养基的使用方法进行了改良。将MRS(含0.25 mg/ml TMB)融化后倾倒入平皿,待其凝固后,在琼脂表明加入200μl(0.01 mg/ml)HRP,涂匀后进行乳酸杆菌划线分离。37℃、厌氧培养48~72 h。结果表明,产生H2O2的乳酸杆菌呈蓝色菌落,和原来的培养方法相比,改良的方法简便经济、HPR的使用量为原来的1%,分离产H2O2的乳酸杆菌的效果良好。     

高灵敏假单胞菌铁载体的平板检测方法

CAS蓝色检测平板是一种筛选、检测各类细菌铁载体的常用方法,而蔗糖-天冬酰氨培养基被用于假单胞菌产铁载体规律的研究。用天冬氨酸替代天冬酰氨,将CAS蓝色检测液与蔗糖-天冬氨酸培养基（MSA培养基）相结合,得到一种改进的MSA-CAS检测平板。通过对假单胞菌属7个种8个株进行荧光与非荧光铁载体检测方面的比较研究,结果表明MSA-CAS检测平板假单胞菌铁载体的检测灵敏度比通用CAS检测平板高,而且在检测荧光铁载体方面具有荧光背景低、荧光铁载体晕圈明显和晕圈与背景的对比度大的优点。     

Characterization of Esterases Produced by a Ruminal Bacterium Identified as Butyrivibrio fibrisolvens

A method of obtaining clones of Tetrahymena pyriformis on solid medium has been developed. The medium consists of a basal layer of 1.5% agar topped with 2 ml of 0.3% agar in sterile, plastic petri plates (100 by 15 mm). Both agar layers contain either 2% proteose peptone and 0.1% liver extract (complex medium) or defined medium supplemented with proteose peptone. After drying, 0.5 ml of liquid culture is spread evenly over the top agar, and the plates are then sprinkled lightly and evenly with autoclaved dry Sephadex G-25 (fine). Cell colonies can be observed after 5 days of incubation either by viewing with a microscope or without the aid of a microscope after staining. Plating efficiency is high on either complex or defined medium with a number of strains of Tetrahymena, both micronucleate and amicronucleate. Colonies can be picked and transferred to liquid culture for further growth. The existence of clones was demonstrated by plating a mixture of two different drug-resistant mutants. The method should prove useful in selective procedures for the isolation of mutants and for determining survival after treatments such as ultraviolet irradiation.     

Induction of the mar operon by miscellaneous groceries

AIM: To investigate the potential of non-antibacterial consumer products to act as inducers of the multiple antibiotic resistance (mar) operon of Escherichia coli SPC105. METHODS AND RESULTS: Wells were cut into chemically defined agar medium (CDM) contained within Petri dishes. Molten agar slurries were prepared by mixing known quantities of 35 consumer products with molten CDM and these were pipetted into each well. Plates were overlaid with molten CDM (5 ml), containing 40 microg ml(-1) X-gal and approx. 1000 CFU ml(-1) of an overnight culture of E. coli SPC105 containing a chromosomal marOII::lacZ fusion. After incubation (37 degrees C, 24 h), plates were examined for zones of growth inhibition and the presence of a blue coloration, indicative of mar (marOII::lacZ) induction. Of the 35 products tested (nine herbs and spices, 19 food and drinks and seven household products), 24 (69%) of the items produced inhibitory zones and 22 (63%) of the items induced mar expression. Apple puree was inhibitory but did not induce marOII::lacZ. Mustard, chilli and garlic were shown to be powerful inducers of marOII::lacZ. Overall six products were shown to be powerful marOII::lacZ inducers. None of these made hygiene claims. CONCLUSIONS: In addition to induction by specific biocides and antibiotics, mar is induced by the exposure of bacteria to natural substances, many of which are common to a domiciliary setting. SIGNIFICANCE AND IMPACT OF THE STUDY: Concern that the overuse of antibacterials within consumer products might select for mar-mediated resistance is shortsighted and fails to recognize the ubiquity of inducers in our environment.     

Capacity of Irpex lacteus and Pleurotus ostreatus for decolorization of chemically different dyes

The rate and efficiency of decolorization of poly R-478- or Remazol Brilliant Blue R (RBBR)-containing agar plates (200 μg g⁻¹) were tested to evaluate the dye degradation activity in a total of 103 wood-rotting fungal strains. Best strains were able to completely decolorize plates within 10 days at 28 °C. Irpex lacteus and Pleurotus ostreatus were selected and used for degradation of six different groups of dyes (azo, diazo, anthraquinone-based, heterocyclic, triphenylmethane, phthalocyanine) on agar plates. Both fungi efficiently degraded dyes from all groups. Removal of RBBR, Bromophenol blue, Cu-phthalocyanine, Methyl red and Congo red was studied with I. lacteus also in liquid medium. Within 14 days, the following color reductions were attained: RBBR 93%, Bromophenol blue 100%, Cu-phthalocyanine 98%, Methyl red 56%, Congo red 58%. The ability of I. lacteus to degrade RBBR spiked into sterile soil was checked, the removal being 77% of the dye added within 6 weeks. The capacity of selected white rot fungal species to remove efficiently diverse synthetic dyes from water and soil environments is documented.     

STUDIES ON PENETRATION OF DYES WITH GLASS ELECTRODE : V. WHY DOES AZURE B PENETRATE MORE READILY THAN METHYLENE BLUE OR CRYSTAL VIOLET?

Glass electrode measurements of the pH value of the sap of cells of Nitella show that azure B in the form of free base penetrates the vacuoles and raises the pH value of the sap to about the same degree as the free base of the dye added to the sap in vitro, but the dye salt dissolved in the sap does not alter the pH value of the sap. It is concluded that the dye penetrates the vacuoles chiefly in the form of free base and not as salt. The dye from methylene blue solution containing azure B free base as impurity penetrates and accumulates in the vacuole. This dye must be azure B in the form of free base, since it raises the pH value of the sap to about the same extent as the free base of azure B dissolved in the sap in vitro. The dye absorbed by the chloroform from methylene blue solution behaves like the dye penetrating the vacuole. These results confirm those of spectrophotometric analysis previously published. Crystal violet exists only in one form between pH 5 and pH 9.2, and does not alter the pH value of the sap at the concentrations used. It does not penetrate readily unless cells are injured. A theory of "multiple partition coefficients" is described which explains the mechanism of the behavior of living cells to these dyes. When the protoplasm is squeezed into the sap, the pH value of the mixture is higher than that of the pure sap. The behavior of such a mixture to the dye is very much like that of the sap except that with azure B and methylene blue the rise in the pH value of such a mixture is not so pronounced as with sap when the dye penetrates into the vacuoles. Spectrophotometric measurements show that the dye which penetrates from methylene blue solution has a primary absorption maximum at 653 to 655 mµ (i.e., is a mixture of azure B and methylene blue, with preponderance of azure B) whether we take the sap alone or the sap plus protoplasm. These results confirm those previously obtained with spectrophotometric measurements.     

Use of Coomassie blue-polyurethane interaction inscreening of polyurethanase proteins andpolyurethanolytic bacteria

The interaction of the dye Coomassie blue with intact polyurethane provides the basisfor a rapid and sensitive assay system for bacterial strains posing polyurethanase activity. Thepotential advantage of this system in enumerating and characterizing polyurethanolytic bacteriaderives largely from the intense color of the dye-substrate complex, which allows the use of verylow substrate concentrations and a corresponding decrease in the time required to detect lowerlevels of enzyme activity. The method entails incubation of agar plates containing thepolyurethane substrate, followed by immersion in 0.1% Coomassie blue then destaining with 10%acetic acid-40% methanol. Resulting agar plates contain transparent bands corresponding toenzymatic activity against a blue background.     

New method for detecting rhamnolipids excreted byPseudomonas species during growth on mineral agar

Summary A new semi-quantitative agar plate test for the detection of extracellular rhamnolipids has been developed. These biological anionic tensides (biosurfactants) form an insoluble ion pair with the cationic tenside cetyltrimethylammonium bromide and the basic dye methylene blue which was included in mineral agar plates. On the light blue agar, productive colonies ofPseudomonas spec. were surrounded by dark blue halos. The test is specific for anionic biosurfactants and can be applied to other glycolipid producing microorganims.     

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.     

Free fatty acids as feedback regulators of adenylate cyclase and cyclic 3':5'-AMP accumulation in rat fat cells.

Rat fat cells incubated with lipolytic agents released substances to the medium which acted as feedback regulators of cyclic adenosine 3':5'-monophosphate (cyclic AMP) accumulation. The feedback regulators were not removed by adenosine deaminase. Dialyzed medium that had previously been incubated with fat cells in the presence of norepinephrine markedly inhibited cyclic AMP accumulation by fresh cells, whereas dialyzed medium from control cells did not inhibit cyclic AMP accumulation. The effects of lipolytic agents could be mimicked by adding dialyzed medium previously incubated with fat cells in the presence of oleic acid. This suggested that free fatty acids were the nondialyzable and adenosine deaminase-insensitive inhibitors of cyclic AMP accumulation released to the medium by fat cells incubated with lipolytic agents. The regulatory function of free fatty acids was related to the molar ratio of fatty acid to albumin. Profound inhibition of both lipolysis and cyclic AMP accumulation was seen as the free fatty acid/albumin ratio exceeded 3. The inhibition of cyclic AMP accumulation by oleate was seen as soon as there was a detectable increase in cyclic AMP due to lipolytic agents. Protein kinase activity (in the presence of cyclic AMP) of the infranatant obtained after centrifugation of fat cell homogenates at 48,000 x g was inhibited by medium from cells incubated with lipolytic agents or added oleate. Adenylate cyclase activity of rat fat cell ghosts was also inhibited by dialyzed or nondialyzed medium that previously had been incubated with lipolytic agents or added fatty acids. The direct addition of oleate markedly inhibited adenylate cyclase activity as the free fatty acid/albumin ratio exceeded 2. These data suggest that the prolonged drop in cyclic AMP accumulation seen during the incubation of rat fat cells with lipolytic agents is due to the inhibition of adenylate cyclase. This occurs when the free fatty acid/albumin ratio exceeds 3.     

P-Aminodimethylaniline Monohydrochloride as an Indicator of Microbial Action on Fats

Flooding p-aminodimethylaniline monohydrochloride on fat emulsion agar inoculated with certain types of microorganisms frequently results in marked color changes in the fat globules. It is shown in this paper that the colors result from the increased solubility in fat and fatty acids of this dye as it becomes oxidized. Some of the acids oxidize the dye on contact and therefore color very quickly; fats become colored only when some other agent oxidizes the dye. The characteristic color reactions with certain fats and fatty acids are described for various degrees of oxidation of the dye; this suggests the explanation for the colors observed in the inoculated globules flooded with this dye. A table is included showing the colors in globules of oil that were inoculated with 39 pure cultures of bacteria.     

CULTIVATION OF A FILAMENTOUS ALGA IN QUANTITY ON AGAR PLATES1,2

A method for the cultivation of Zygnema in quantity on agar plates is described. Axenic filaments were chopped with razor blades, and 4 ml of the suspension of short filaments in nutrient medium were transferred with a syringe to 1% agarized nutrient medium plates. Filamentous growth uniformly covered agar plates in 10 days or less.     

A Note on the Inhibition of Pseudomonas aeruginosa by a Modification of Brilliant Green Agar for Improved Salmonella Isolation

A strain of Pseudomonas aeruginosa having colonies that resemble those of salmonellas on brilliant green agar is almost totally inhibited by the addition of 1.0 mg/ml of sulphacetamide to the medium. Low numbers of Ps. aeruginosa grew equally well on brilliant green and nutrient agar, but 10⁶–10⁷ organisms were needed before any growth appeared on the medium containing sulphacetamide. During 12 months of routine use of the sulphacetamide medium, involving almost 3000 plates, Ps. aeruginosa has been isolated as a contaminant only once. Forty-seven salmonella serotypes were grown on the sulphacetamide brilliant green agar in the same period.