Identification of Mycoplasmatales: Characterization Procedures

A large collection of avian Mycoplasma cultures was used in studies to improve and develop biological and biochemical characterization techniques. Differential patterns among 11 avian serotypes were shown by carbohydrate fermentation, tetrazolium- and methylene blue-reduction reactions, breakdown of arginine, and the formation of film on egg yolk-agar. Some cultures fermented as many as 14 carbohydrates. Polyhydric alcohols and pentoses were among the compounds fermented. An improved procedure for determining methylene blue reduction by Mycoplasma was developed. These simple, rapid procedures are reproducible and should be useful in grouping Mycoplasma isolates prior to definitive identification by serological or other means.     

Effect of pH on Growth and Survival of Three Avian and One Saprophytic Mycoplasma Species

The ability of Mycoplasma meleagridis, M. laidlawii, an unnamed, nonpathogenic avian species, and three isolates of M. gallisepticum to grow and survive in liquid media of various pH values was investigated. All species grew over a pH range that was generally about two units. The most significant finding was that cultures initiated in media within a pH range of approximately 8.5 to 8.9 remained viable for 45 to 60 days at 37 C, whereas cultures initiated at lower values survived for shorter periods.     

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.     

Phospholipid-deacylating enzymes of rat stomach mucosa

1. Rat stomach mucosa exhibited three distinguishable phospholipid-deacylating enzyme activities: lysophospholipase, phospholipase A1 and phospholipase A2. 2. The lysophospholipase hydrolyzed 1-palmitoyl lysophosphatidylcholine to free fatty acid and glycerophosphorylcholine. This enzyme had an optimum pH of 8.0, was heat labile, did not require Ca2+ for maximum activity and was not inhibited by bile salts or buffers of high ionic strength. 3. Phospholipase A2 and phospholipase A1 deacylated dipalmitoyl phophatidylcholine to the corresponding lyso compound and free fatty acid. The specific activity of phospholipase A2 was 2--4-fold higher than that of phospholipase A1 under all the conditions tested. Both activities were enhanced 4--7.5-fold in the presence of bile salts at alkaline pH and 11-18-fold at acidic pH. 4. In the absence of bile salts, phospholipase A1 exhibited pH optima at 6.5 and 9.5 and phospholipase A2 at pH 6.5, 8.0 and 9.5. The pH optima for phospholipase A1 were shifted to pH 3.0, 6.0 and 9.0 in presence of sodium taurocholate; the activity was detected only at a single pH of 9.5 in the presence of sodium deoxycholate and at pH 10.0 in the presence of sodium glycocholate. Phospholipase A2 optimum activity was displayed at pH 3.0, 6.0 and 8.0 in presence of taurocholage, pH 7.5 and 9.0, in presence of glycocholate and only at pH 9.0 in presence of deoxycholate. 5. Ca2+ was essential for optimum activity of phospholipases A1 and A2. But phospholipase A1 lost complete activity in presence of 0.5 mM ethyleneglycolbis-(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA) at pH 6.0, whereas phospholipase A2 lost only 50%. 6. Phospholipases A1 and A2 retained about 50% of their activities by heating at 75 degrees for 10 min. At 100 degrees, phospholipase A1 retained 22% of its activity, whereas phospholipase A2 retained only 7%.     

The Atmospheric Oxidation, or Dealkylation, of Aqueous Solutions of Methylene Blue

At room temperatures (approximately 25° C) dilute aqueous solutions of representative samples of methylene blue are stable below pH 9.5. Oxidation, or dealkylation, begins between pH 9.5 and 9.8 and increases in both rate and degree with increasing alkalinity. Below pH 11 the rate is slow and the total oxidation relatively limited. At pH 12, however, oxidation is rapid and is practically complete within a few days.     

The purification of methylene blue and azure B by solvent extraction and crystallization.

Detailed schemes are described for the preparation of purified methylene blue and azure B from commercial samples of methylene blue. Purified methylene blue is obtained by extracting a solution of the commercial product in an aqueous buffer (pH 9.5) with carbon tetrachloride. Methylene blue remains in the aqueous layer but contaminating dyes pass into the carbon tetrachloride. Metal salt contaminants are removed when the dye is crystallized by the addition of hydrochloric acid at a final concentration of 0.25 N. Purified azure B is obtained by extracting a solution of commercial methylene blue in dilute aqueous sodium hydroxide (pH 11-11.5) with carbon tetrachloride. In this pH range, methylene blue is unstable and yields azure B. The latter passes into the carbon tetrachloride layer as it is formed. Metal salt contaminants remain in the aqueous layer. A concentrated solution oa azure B is obtained by extracting the carbon tetrachloride layer with 4.5 X 10(-4)N hydrobromic acid. The dye is then crystallized by increasing the hydrobromic acid concentration to 0.23 N. Thin-layer chromatography of the purified dyes shows that contamination with related thiazine dyes is absent or negligible. Ash analyses reveal that metal salt contamination is also negligible (sulphated ash less than 0.2%).     

Zinc Chloride Methylene Blue. I. Biological Stain History, Physical Characteristics and Approximation of Azure B Content of Commercial Samples

Zinc chloride methylene blue appeared on the market almost contemporaneously with the zinc-free medicinal form. The former has rarely been reported as being used in blood stains. Recent suspension of manufacture of medicinal methylene blue by it. principal American producer has excited interest in the use of the zinc chloride form for the preparation of blood stains. According to Lillie (1944a,b) the azure B content of zinc chloride methylene blue may have varied from 5 to 30% in the samples studied. Taking the Merck Index (1968, 1976) figures for the spectroscopic absorption maximum (λmax) of 667.8 and 668 nm as standard, recent samples of zinc chloride methylene blue are calculated to contain 6-8% azure B. These figures are baaed on 1) the shift of λmax after exhaustive pH 9.5 chloroform extraction, 2) evaluation of the actual ratio of the observed TiCl₂ dye content to the theoretical for pure zinc chloride methylene blue, 3) comparison of spectroscopic and staining effects of graded hot dichromate oxidation products with those of highly purified azure B-methylene blue mixtures of known proportions.

As far as can be found, medicinal methylene blue is almost the exclusive source of cosin polychrome methylene blue blood stains. Lillie (1944c) included a short series comparing 5 zinc chloride methylene blues with a dozen medicinal methylene blue samples; all were oxidized with hot dichromate to produce successful Wright stains. No effort was made to remove the zinc Exhaustive pH 9.5 chloroform extraction of zinc chloride methylene blue (lot MCB 12-H-29) yielded a small amount of red dye which when extracted into 0.1 N HCI gave λmax = 650. The extraction moved the absorption peak of the zinc chloride methylene blue from 667 to 668 nm and the midpoint of the 90% maximum absorption band, 18 nm wide, from 666.5 to 667.5 nm.     

Tolerance to challenges miming gastrointestinal transit by spores and vegetative cells of Bacillus clausii

AIMS: To study Bacillus clausii from a pharmaceutical product (Enterogermina O/C, N/R, SIN, T) and reference strains (B. clausii and Bacillus subtilis) for eco-physiological aspects regarding the gut environment. METHODS AND RESULTS: Spores and vegetative cells were challenged in vitro miming the injury of gastrointestinal transit: pH variations, exposure to conjugated and free bile salts, microaerophilic and anaerobic growth. No relevant differences were found studying the growth at pH 8 and 10, whereas at pH 7 the yields obtained for O/C and SIN were higher than those obtained for N/R and T strains. The spores were able to germinate and grow in the presence of conjugated bile salts (up to 1%, w/v) or free bile salts (0.2%) and also exhibited tolerance for the combined acid-bile challenge. As evidenced by lag-time, growth rate and cell yield the tolerance of Enterogermina isolates for conjugated salts was comparable with that of B. clausii type strain (DSM 8716(T)), and resulted higher than that observed for B. subtilis (ATCC 6051(T)). All the considered B. clausii strains demonstrated microaerophilic growth, but only some grew anaerobically in a nitrate medium. CONCLUSIONS: The ability of B. clausii spores to germinate after an acid challenge and grow as vegetative cells both in the presence of bile and under limited oxygen availability is consistent with the beneficial health effects evidenced for spore-forming probiotics in recent clinical studies. SIGNIFICANCE AND IMPACT OF THE STUDY: The experimental evidence from this study emphasizes some functional properties of B. clausii strains regarding their use as probiotics.     

Purification, characterization, and expression of rat intestinal alkaline sphingomyelinase

Intestinal alkaline sphingomyelinase (SMase) has physiological roles in the digestion of sphingomyelin (SM) and clinical implications in colonic carcinogenesis. In the present work, the enzyme from rat has been purified 1,589-fold with 11% recovery by elution of the intestine with bile salt, precipitation of the proteins by acetone, and several types of chromatographies. Its molecular mass was 58 kDa and optimal pH was 9 to 9.5. Under the optimal conditions, the V(max) was 930 micromol/h/mg and K(m) was about 1.25 mM. The enzyme could hydrolyze phosphatidylcholine at pH 7.4 in the presence of Ca2+; the rate was about 8% of that for SM. The activity against SM was dependent on bile salt. Taurine conjugated bile salts were much more effective than glycine conjugated ones, and the most effective bile salts were taurocholate and taurochenodeoxycholate. 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) and Triton X100 (TX100) had no stimulatory effects. Unlike neutral SMase, intestinal alkaline SMase was not Mg2+ dependent, not inhibited by EDTA, and not inhibited by glutathione. The enzyme was stable during incubation with temperatures up to 50 degree C and in pHs from 7 to 10. Trypsin and chymotrypsin had no effects on its activity, and 10 mM dithiothreitol reduced its activity by 25%. A specific antibody against the enzyme was developed, and Western blot showed that the enzyme was expressed in the intestine but not in other organs. In conclusion, we purified a potentially important SMase in the intestine with several properties different from neutral SMase.     

Characterization of a Newly Identified Mycoplasma (Mycoplasma orale Type 3) from the Human Oropharynx

Six mycoplasma strains, isolated under anaerobic conditions from the human oropharynx, were studied by biologic and serologic means. The strains produced nippled colonies with weak hemolytic activity for guinea pig erythrocytes on agar medium. In addition, the strains metabolized arginine with a concomitant alkaline shift in the pH of the medium but did not produce a pH shift when grown in the presence of glucose or urea. The strains failed to reduce 2-3-5 triphenyl tetrazolium and were inhibited by 0.001% methylene blue. In addition, they required fresh yeast extract for growth. When compared by several serologic methods, the strains were found to be related to each other but distinct from 23 serotypes of human, animal, and avian origin. However, one-way serologic relationships between one of the new strains and Mycoplasma orale type 1 and M. salivarium were observed when they were tested by complement fixation. Furthermore, partial relationship of one of the new strains to all of the arginine-utilizing mycoplasma species of human origin was demonstrated with the agar gel diffusion technique. Thus, the new strains appear to constitute a new mycoplasma species, for which the name M. orale type 3 is tentatively proposed. M. orale type 3 accounted for 1.4% of 437 mycoplasma isolates from the oropharynx of adults. The new species probably is a rare member of the normal mycoplasmal flora of man.     

Concentration of rotavirus and enteroviruses from blue crabs (Callinectes sapidus).

A simple method for concentration and detection of rotavirus and enteroviruses in the blue crab is described. Virus was separated from tissue homogenates at pH 9.5, concentrated by adsorption to protein precipitates at pH 3.5, and recovered by elution of precipitates at pH 9.2. Test samples of 12 to 15 ml were produced from an initial 100 g of crab tissues. Cat-floc precipitation was used to remove sample toxicity for cell cultures. Recovery effectiveness averaged 52% with poliovirus 1, echovirus 7, and coxsackievirus B5 and 23% with rotavirus SA11.     

Concentration of rotavirus and enteroviruses from blue crabs (Callinectes sapidus)

A simple method for concentration and detection of rotavirus and enteroviruses in the blue crab is described. Virus was separated from tissue homogenates at pH 9.5, concentrated by adsorption to protein precipitates at pH 3.5, and recovered by elution of precipitates at pH 9.2. Test samples of 12 to 15 ml were produced from an initial 100 g of crab tissues. Cat-floc precipitation was used to remove sample toxicity for cell cultures. Recovery effectiveness averaged 52% with poliovirus 1, echovirus 7, and coxsackievirus B5 and 23% with rotavirus SA11.     

Borax Methylene Blue: A Spectroscopic And Staining Study

Borax methylene blue is quite stable at room temperatures of 22-25 C. At 30 C polychroming is slow; during 50 days in a water bath at this temperature the absorption peak moves from 665 to 656 nm. At 35 C, the absorption peak reaches 660 nm in 7 days, 654 nm in 14. At 60 C polychroming is rapid, the absorption peak reaching 640-620 nm in 3 days. When the pH of the borax methylene blue solutions, normally about 9.0, is adjusted to pH 6.5, the absorption peak remains at 665 nm even when incubated at 60 C for extended periods.

When used as a blood stain 0.4 ml borax methylene blue (1% methylene blue in 1% borax), 4 ml acetone, 2 ml borax-acid phosphate buffer to bring the solution to pH 6.5, and distilled water to make 40 ml, with 0.2 ml 1% eosin added just before using, an excellent Nocht-Giemsa type stain is achieved after 30 minutes staining. The material plasmodia P. falciparum, P. vivax, and P. berghei stain moderate blue with dark red chromatin and green to black pigment granules.

The study confirms Malacnowski's 1891 results and explains Gautier's 1896-98 failure to duplicate it.     

Borax methylene blue: a spectroscopic and staining study.

Borax methylene blue is quite stable at room temperatures of 22-25 C. At 30 C polychroming is slow; during 50 days in a water bath at this temperature the absorption peak moves from 665 to 656 nm. At 35 C, the absorption peak reaches 660 nm in 7 days, 654 nm in 14. At 60 C polychroming is rapid, the absorption peak reaching 640-620 nm in 3 days. When the pH of the borax methylene blue solutions, normally about 9.0, is adjusted to pH 6.5, the absorption peak remains at 665 nm even when incubated at 60 C for extended periods. When used as a blood stain 0.4 ml borax methylene blue (1% methylene blue in 1% borax), 4 ml acetone, 2 ml borax-acid phosphate buffer to bring the solution to pH 6.5, and distilled water to make 40 ml, with 0.2 ml 1% eosin added just before using, an excellent Nocht-Giemsa type stain is achieved after 30 minutes staining. The material plasmodia P. falciparum, P. vivax, and P. berghei stain moderate blue with dark red chromatin and green to black pigment granules. The study confirms Malachowski's 1891 results and explains Gautier's 1896-98 failure to duplicate it.     

Metal Contaminants in Commercial Thiazine Dyes

The iron, potassium, sodium and zinc contents of commercial samples of the thiazine dyes azure A (C.I. 52005), azure B (C.I. 52010), azure C (C.I. 52002), methylene blue (C.I. 52015), new methylene blue (GI. 52030), polychrome methylene blue, thionine (C.I. 52000) and toluidme blue (C.I. 52040) have been determined by atomic absorption spectrophotometry.

The metal concentrations varied widely in the 38 samples examined—iron, potassium, sodium and zinc together comprised between 0.02% and 25.35% of individual samples.     

The role of bile acids in the reduction in lipopolysaccharide uptake by cultured rat Kupffer cells

The influence of bile salts on the binding and uptake of Salmonella abortus equi lipopolysaccharide by cultured Kupffer cells was studied. In control preparations, the percentage of cell-associated lipopolysaccharide increased with time and reached a plateau after about 2 h incubation at 37 degrees C. About 1.2 micrograms lipopolysaccharide was associated with 10(6) Kupffer cells at this time interval. In the presence of 0.3, 0.6 and 1 mumol bile salts/ml the cell-associated lipopolysaccharide was respectively, about 5%, 13% and 29% lower than in control cultures. In the presence of 1 mumol bile salts/ml, the association of lipopolysaccharide to cells at 0 degrees C was about 25% lower than in controls. Preincubation of Kupffer cells with 1 mumol bile salts/ml, with or without lipopolysaccharide, did not affect cell-associated lipopolysaccharide after removal of the bile salts. The rate of secretion of radioactivity by Kupffer cells was not influenced by the presence of bile salts during the uptake or the secretion periods. Bile acids proved to inactivate lipopolysaccharide. From these observations it was concluded that low concentrations of bile salts influence the binding and uptake of lipopolysaccharide by Kupffer cells. It was, therefore, considered likely that, in patients with obstructive jaundice, the high serum bile acid level accounts for spill-over of portal lipopolysaccharide into the systemic blood.     

Metal contaminants in commercial thiazine dyes.

The iron, potassium, sodium and zinc content of commercial samples of the thiazine dyes azure A (C.I. 52005), azure B (C.I. 52010), azure C (C.I. 52002), methylene blue (C.I. 52015), new methylene blue (C.I. 52030), polychrome methylene blue, thionine (C.I. 52000) and toluidine blue (C.I. 52040) have been determined by atomic absorption spectrophotometry. The metal concentration varied widely in the 38 samples examined--iron, potassium, sodium and zinc together comprised between 0.02% and 25.35% of individual samples.     

The effect of estradiol benzoate on liver function, cholesterol metabolism and bile acids in the quail]

Immature female quails were treated for 6 days with estradiol benzoate at daily 0.01-, 0.02-, 0.01-, and 1-mg dosages. At the end of treatment, bile outflow, biliary cholesterol (CST), and bile acid (BA) secretory rates and liver, bile, and serum CST and BA levels were determined. Some quails were used to measure the ratio (R) of the rates of intravenously injected [2-14C]acetate radioactivity incorporation in cholic (C) and chenodeoxycholic (CDC) acids excreted in bile. The oestrogenic treatments at doses greater than 0.01 mg/day caused a marked disturbance in hepatic function and in CST and BA metabolism: they induced an increase in relative liver weight, liver CST stores, serum CST, C, CDC, and SGOT levels and in choleresis (respectively up to 56, 57, 650, 6000, 700, 42, and 235% increase at the daily 0.1-mg dosage) and they decreased bile total BA and CDC levels, bile and serum CDC to C level ratios, and R ratio (by 71, 82, 69, 84, and 58%, respectively). An increase in the bile salts independent fraction of bile was responsible for hypercholeresis, whether alone at low dosage or in conjunction with other factors at higher dosage. These results are compared with those obtained in mammals, particularly in the rat.     

Enzymatic microtiter plate-based nitrate detection in environmental and medical analysis

Our microtiter plate assay is based on the enzymatic reduction of nitrate by dissimilatory nitrate reductase from Pseudomonas stutzeri [EC 1.7.99.4]. Exogenous redox mediators like methyl viologen, methylene blue, and cibachron blue were applied to reduce nitrate reductase. Concentrations of 0.02-0.9 mM nitrate can be detected with +/-6% standard deviation, by using a photometric Griess reaction for the formed nitrite. Nitrate reductase is stable in the pH range 6.5-9.0 and works in the temperature range 4-76 degrees C. The assay shows no interferences with salt content up to 1 M chloride or 11 mM chlorate, and serum albumin content up to 50 mg/ml. The time demand of our two-step procedure is 20 min/100 samples. Nitrate reductase could be conserved on site of the wells of microtiter plates for at least 6 months at room temperature. The nitrate assay was applied in environmental and consumer goods analysis, and for medical diagnostics in human plasma samples.     

Zinc Chloride Methylene Blue, Ii. Preparation of Giesma and Wright Type Low Azure B Blood Stains from Dichromate Oxidized Commercial Dye

TO determine the amount of K₂Cr₂O₇ required to produce optimal Giemsa type staining, six 1 g amounts (corrected for dye content) of zinc methylene blue were oxidized with graded quantities of K₂Cr₂O₇ to produce 4, 8, 12, 16, 20 and 24% conversion of methylene blue to azure B. These were heated with a blank control 15 minutes at 100 C in 60-65 ml 0.4 N HCI. cooled, and adjusted to 50 ml to give 20 mg original dye/ml. Aliquots were then diluted to 1% and stains were made by the “Wet Giemsa” technic (Lillie and Donaldson 1979) using 6 ml 1% polychrome methylene blue, 4 ml 1% cosin (corrected for dye content), 2 ml 0.1 M pH 6.3 phosphate buffer, 5 ml acetone, and 23 ml distilled water. The main is added last and methanol fixed blood films are stained immediately for 20-40 min.

For methylene blue supplied by MCB 12-H-29, optimal stains were obtained with preparations containing 20 and 24% conversion of methylene blue to azure B. With methylene blue supplied by Aldrich (080787), 16% conversion of methylene blue to azure B was optimal. Eosinates prepared from a low azure B/methylene blue preparation selected in this way give good stains when used as a Wright stain in 0.3% methanol solution. However, when the 600 mg eosinate solution in glycerol methanol is supplemented with 160 mg of the same azure B/methylene blue chloride the mixture fails to perform well. The HCI precipitation of the chloride apparently produces the zinc methylene blue chloride salt which is poorly soluble in alcohol. It appears necessary to have a zinc-free azure B/methylene blue chloride to supplement the probably zinc-free eosinate used in the Giemsa mixture.