Calcium and magnesium in Mueller-Hinton agar and their influence on disk diffusion susceptibility results

Concentrations of calcium and magnesium were determined for 39 lots of media, including broth and agar media used for susceptibility tests and plain agar. In addition, the effect that media with and without physiological levels of these divalent cations would have on the disk diffusion susceptibility of 21 strains ofPseudomonas aeruginosa to four antimicrobics was also ascertained. Mueller-Hinton agar showed a wide variation in calcium and magnesium content. Mueller-Hinton broth contained lower concentrations of calcium and magnesium, and there was little lot-to-lot variation. Lots of Mueller-Hinton agar with higher concentrations of calcium and magnesium yielded smaller zone diamaters than those with lower concentrations. Even at equal cation concentration, zones of inhibition varied from lot to lot. Since the addition of calcium and magnesium to Mueller-Hinton agar to obtain a predetermined level did not result in equivalent zone diameters, performance testing of susceptibility media is recommended.     

Detection of sulfa drugs and antibiotics in milk

A disc assay method for testing sulfa drugs and antibiotics in milk was developed wherein Bacillus megaterium ATCC 9855 was used as the test organism and Mueller-Hinton agar was used as the test substrate. Incubation was at 37 C for 4 to 5 hr. The test procedure is an improvement over the Bacillus subtilis-Antibiotic Medium No. 1 method, as described in Standard Methods for the Examination of Dairy Products, in that it is sensitive to eight sulfa drugs and to bacitracin without a significant change in sensitivity to eight other antibiotics commonly used for mastitis therapy.     

Catecholamine-induced Regulation in Vitro and ex Vivo of Intralymphocyte Ionized Magnesium

Despite the importance of the adrenergic activity and of the metabolism of magnesium in some important cardiovascular pathologies, very little is known about how intracellular ionized magnesium (Mg _i ²⁺ ) is regulated by catecholamines. We made an in-vitro study of the variations in the concentration of ionized magnesium in human lymphocytes using the fluorescent probe furaptra in response to different catecholamines. We also made an ex-vivo study of the changes in intracellular ionized magnesium in lymphocytes in 20 subjects with essential arterial hypertension, 10 treated with 120 mg/d of propranolol and 10 with placebo. Norepinephrine and isoproterenol significantly decrease Mg _i ²⁺ and this effect is blocked by β-blockers but not by α-blockers. The EC ₅₀ of the effect of norepinephrine is within the range of concentrations physiologically present in plasma. The substitution of extracellular sodium with choline blocks the decrease in intracellular ionized magnesium induced by norepinephrine, which leads us to suppose that the magnesium-reducing effect of catecholamines is a result of the activation of a Na⁺-Mg²⁺ exchanger. We were not able to demonstrate any change in intracellular ionized magnesium after 1 and 17 days of active treatment in essential hypertensives. The impossibility of demonstrating ex vivo the mechanism of catecholamine-mediated regulation that is evident in vitro is perhaps due to our experimental conditions or to substances which in vivo inhibit the action of the catecholamines on magnesium, such as insulin and/or glucose.     

Effect of supplementall-tyrosine on pigment production in cultures of the Legionnaires' disease bacterium

The etiologic agent of Legionnaires' disease grows on certain agar media. Cultures of this organism on supplemented Mueller-Hinton agar are characterized by the appearance of brown pigment in and around areas of bacterial growth. The major peptone source in Mueller-Hinton agar is an acid hydrolysate of casein. Legionnaires' disease bacterium also grows on a medium in which the peptone source is 0.25% yeast extract, but no pigment is produced. If the yeast extract agar is enriched withl-tyrosine, pigment formation can occur. Pigmentation of cultures of Legionnaires' disease bacterium may be mediated by a phenolo-monooxygenase, or tyrosinase.     

Effect of medium composition on the susceptibility of oral streptococci to mercuric chloride

Although there is considerable interest in identifying mercury-resistant bacteria, no standardized assay exists for this purpose. In this study, the effect of the composition of the medium on the susceptibility of oral streptococci to HgCl₂ was investigated. The minimum inhibitory concentration (MIC) of HgCl₂ for 52 streptococcal strains and the reproducibility of MIC values for Hg-sensitive and Hg-resistant strains was determined with 11 different media. Addition of blood increased the MIC values, and some media (tryptone soya agar, with or without blood) could not discriminate between Hg-sensitive and Hg-resistant strains. The proportion of streptococci that appeared to be resistant to Hg was very high (>70%) on some media (mitis-salivarius, tryptone soya, Columbia), but not on others (Mueller-Hinton, Brain Heart Infusion, Isosensitest). The MICs of the control strains varied considerably on different testing occasions for tryptone soya agar (with and without blood), Isosensitest agar, and Columbia agar (with blood). Mueller-Hinton (without blood) appeared to be the most suitable medium for isolating Hg-resistant oral streptococci. Received: 21 December 2001 / Accepted: 17 January 2002     

Agar Underlay Method for Recovery of Sublethally Heat-Injured Bacteria

A method of recovering sublethally heat-injured bacteria was developed. The procedure (termed the agar underlay method) uses a nonselective agar underlaid with a selective medium. In a two-chambered petri dish, the Lutri plate (LP), a nonselective agar is inoculated with a population of sublethally heat-injured bacteria. After a 2-h repair incubation period, selective agar is added to the bottom chamber of the LP and incubated. By diffusing through the nonselective top agar, selective agents from the underlay medium impart selectivity to the system. By the agar underlay method, recovery rates of the heat-injured food-borne pathogens Escherichia coli O157:H7 and Salmonella typhimurium were not different (P > 0.05) from recovery rates determined with nonselective media. Sublethally heat-injured cells (60°C for 1.5 min in buffer or 80°C for 30 s on meat surfaces) grew and produced a typical colony morphology and color reaction when the agar underlay procedure was used with the appropriate respective selective agars. Unlike agar overlay methods for injury repair, the agar underlay procedure allows the typical selective-medium colony morphology to develop and allows colonies to be more easily picked for further characterization. Higher recovery rates of heat-injured fecal enterococci from bovine fecal samples and total coliforms from animal waste lagoons were obtained by the agar underlay method with selective agars than by direct plating on the respective selective media.     

A rapid microtechnique for the preparation of biological material for the simultaneous analysis of calcium,magnesium and protein

No simple documented method of sample preparation is available for the analysis of calcium and magnesium in biological samples despite increasing awareness of the biological roles of these cations. The technique described here is rapid, avoids the use of dangerous reagents or costly equipment, and allows accurate determination of protein, calcium, and magnesium content of the speciment after sample preparation. Tissue is solubilized in 1 n NaOH after which one aliquot is used for protein analysis by the method of Lowry et al. (1951) (J. Biol. Chem.193, 265–275), and another is used for determination of cations by atomic absorption spectroscopy. The method was used to analyze biological samples including adipocyte subcellular fractions, bovine liver, and orchard leaves. Results correlated well with those using reference wet ashing and low-temperature ashing techniques with correlation coefficients (r²) of 0.95 for calcium and 0.997 for magnesium. Intraassay coefficients of variation were 4–4.2%. The base-digestion technique is a simple, rapid, and precise method which avoids most of the limitations of currently available sample preparation techniques.     

Effect of Storage of Mueller-Hinton Agar Plates on Zone Sizes for Antimicrobial Testing

The length of time Mueller-Hinton agar plates can be stored at 4 C without affecting the size of zones of inhibition in susceptibility testing by the Bauer-Kirby method was studied. It was found that these plates can be stored for 3 weeks at 4 C without an appreciable affect on zone sizes. Storage of plates in sealed plastic bags did not alter the results significantly. The findings indicate that commercially prepared Mueller-Hinton agar plates, which may be several days old when received at the laboratory, are suitable for use in routine susceptibility tests by the Bauer-Kirby method.     

Effects of bovine milk lactoperoxidase system on some bacteria

Bovine lactoperoxidase (LPO) was purified from skimmed milk using amberlite CG-50-H⁺ resin, CM sephadex C-50 ion-exchange chromatography, and sephadex G-100 gel filtration chromatography. Lactoperoxidase was purified 20.45-fold with a yield of 28.8%. Purity of enzyme checked by sodium dodecyl sulphate-polyacrylamide gel electrophoresis method and a single band was observed. K _m was 0.25 mM at 20°C, V _max value was 7.95 μmol/ml min at 20°C (pH 6.0). Antibacterial study was done by disk diffusion method of Kirby-Bauer using Mueller-Hinton agar medium with slight modification. Bovine LPO showed high antibacterial activity in 100 mM thiocyanate-100 mM H₂O₂ medium for some bacteria (Brevibacillus centrosaurus, B. choshinensis, B. lyticum, Cedecea davisae, Chryseobacterium indoltheticum, Clavibacter michiganense pv. insidiosum, Kocuria erythromyxa, K. kristinae, K. rosea, K. varians, Paenibacillus validus, Pseudomonas syringae pv. populans, Ralstonia pickettii, Rhodococcus wratislaviensis, Serratia fonticola, Streptomyces violaceusniger, Vibrio cholerae-nonO1) respectively, and compared with well known antibacterial substances (levofloxacin, netilmicin). LPO system has inhibition effects on all type bacteria and concentration is really important such as LPO-100 mM thiocyanate-100 mM H₂O₂ system was proposed as an effective agent against many factors causing several diseases.     

THE COMPOSITION OF FLUIDS AND SERA OF SOME MARINE ANIMALS AND OF THE SEA WATER IN WHICH THEY LIVE

1. The electrolyte composition, the pH, and freezing points of the fluids of several invertebrates and one primitive chordate are reported. 2. Fluids of the worms, echinoderms, and the clam Venus were isotonic with sea water; fluids of the Arthropoda were hypertonic to sea water. 3. The pH of all fluids was below that of sea water. In the Arthropoda and Myxine less individual variation in pH appeared than in the echinoderms and worms. 4. Ratios of ionic concentrations in the fluid to those in the sea water indicated (1) uniform distribution of ions between the internal and external media for the echinoderms and Venus, (2) differential distribution of potassium and magnesium in the worms; (3) differential distribution of sulfate, magnesium, potassium, and calcium in the Arthropoda; and (4) differential distribution of calcium, magnesium, and sulfate in Myxine. 5. The unequal distribution of ions implies the expenditure of energy against a concentration gradient across the absorbing or excreting membranes, a capacity frequently overlooked in the invertebrates. 6. The sera of the Arthropoda from diluted sea water showed higher concentrations of sodium, potassium, calcium, and chloride ions relative to the respective concentrations in the external medium than in normal sea water, and also showed different orders for those ions. 7. The increase in osmotic pressure of the sera of the animals moving into brackish water is caused by unequal accumulation of sodium, potassium, calcium, and chloride ions. Sulfate and magnesium ionic ratios do not change.     

Relationship between gene expression of UDP-glucose pyrophosphorylase and agar yield in Gracilariopsis lemaneiformis (Rhodophyta)

UDP-glucose pyrophosphorylase (UGPase) is an enzyme involved in the biosynthesis of UDP-D-galactose, a subunit of agar in red seaweeds. The relationship between agar content and expression levels of the UGPase encoding gene (glugp) was studied in thalli under different treatment conditions using a quantitative real-time PCR-based method (qPCR). Moreover, this qPCR method for the measurement of glugp expression was also applied to commercial varieties of Gracilariopsis lemaneiformis, a red macroalga, in order to examine its reliability on material obtained from field cultivation. Both the agar content and glugp gene expression in G. lemaneiformis grown under low salinity (17?‰) conditions for 1 week showed a slight increase in comparison with the control group (33?‰ salinity, natural salinity of seawater), but the difference was not statistically significant (P?>?0.05). However, when the culture time was extended to 2 weeks, the increase in both the agar yield and glugp expression became significant (P?glugp expression (P?>?0.05). Our results suggest that glugp gene expression and agar content are highly positively correlated and that the measurement of glugp expression, using only a small amount of thalli material, may be an efficient approach to evaluate agar content. In addition, both the agar content and glugp expression in cultivars 981, 07-2, and ZC differed significantly from those of MT-18. The findings of this study suggest that UGPase may be involved in agar biosynthesis and indicate that glugp gene expression could be a fairly reliable molecular marker to reflect the agar content of strains during breeding and selection of G. lemaneiformis.     

Glucose-induced alterations of intracellular ionized magnesium in human lymphocytes

The intracellular ionic content of human erythrocytes may be altered by hyperglycaemia. Despite this, very little is known about the cellular mechanisms linking glucose and cellular magnesium homeostasis. We measured intracellular ionized magnesium in human lymphocytes, by means of a fluorimetric technique, total intracellular magnesium by means of atomic absorption spectrophotometry and intracellular ATP by means of HPLC. The incubation of lymphocytes with D-glucose in the absence of insulin was followed by a significant decrease in intracellular ionized magnesium; this effect did not occur when the cells were incubated with L-glucose. The effect of glucose on intracellular ionized magnesium was blocked by amphotericin B and the EC(50) of the effect of glucose on intracellular ionized magnesium was about 5 mmol/l of glucose. The increase of intracellular ionized magnesium in cells incubated in the absence of glucose was followed by a decrease in intracellular ATP. In a Na(+)-free medium the decrease of intracellular ionized magnesium in the presence of glucose was still present and the incubation of lymphocytes with glucose did not modify total intralymphocyte magnesium. By selective permeabilization of cell membranes, we established that glucose could not increase compartmentalized intracellular ionized magnesium. Our data supports the hypothesis that glucose per se induces a substantial decrease in intracellular ionized magnesium, which is probably due to an augmented binding of intracellular ionized magnesium to cellular ATP.     

Cyclic AMP-dependent and -independent protein kinases in HeLa cells

Malate saturation isotherms for the NAD⁺ malic enzyme determined at widely differing, but saturating, concentrations (8, 80, 160 mm) of magnesium show the same response to malate concentration only when velocity is plotted against the concentration of free malate^2?. This identification of the ionized malic acid as the true substrate for this enzyme, together with the observation that the complex of Mg with malate has no influence on the reaction rate even at very high concentrations, indicates that the metal ion activator of the enzyme must also bind in the ionized form. A kinetic analysis shows that, with respect to magnesium and malate, the malic enzyme catalyzes a rapid equilibrium reaction of the intersecting type. Either Mg²⁺ or malate^2? may bind first but the fact that the K_m's for both Mg²⁺ and malate^2? are smaller than the respective K_i's suggests that, when either metal ion or malate is present on the enzyme, the other is bound more tightly than when it binds to the free enzyme. This demonstration of the nature of the true substrates for this enzyme has implications for studies of the NAD⁺ malic enzyme in which conditions influencing the amount of free magnesium and malate, e.g., changes in pH, addition of weak acid effectors etc., are involved.     

Hair Sheep Blood,Citrated or Defibrinated,Fulfills All Requirements of Blood Agar for Diagnostic Microbiology Laboratory Tests

Background

Blood agar is used for the identification and antibiotic susceptibility testing of many bacterial pathogens. In the developing world, microbiologists use human blood agar because of the high cost and inhospitable conditions for raising wool sheep or horses to supply blood. Many pathogens either fail to grow entirely or exhibit morphologies and hemolytic patterns on human blood agar that confound colony recognition. Furthermore, human blood can be hazardous to handle due to HIV and hepatitis [1], [2]. This study investigated whether blood from hair sheep, a hardy, low-maintenance variety of sheep adapted for hot climates, was suitable for routine clinical microbiology studies.

Methods and Findings

Hair sheep blood obtained by jugular venipuncture was anticoagulated by either manual defibrination or collection in human blood bank bags containing citrate-phosphate-dextrose. Trypticase soy 5% blood agar was made from both forms of hair sheep blood and commercial defibrinated wool sheep blood. Growth characteristics, colony morphologies, and hemolytic patterns of selected human pathogens, including several streptococcal species, were evaluated. Specialized identification tests, including CAMP test, reverse CAMP test, and satellite colony formation with Haemophilus influenzae and Abiotrophia defectiva were also performed. Mueller-Hinton blood agar plates prepared from the three blood types were compared in antibiotic susceptibility tests by disk diffusion and E-test.

Conclusions

The results of all studies showed that blood agar prepared from citrated hair sheep blood is suitable for microbiological tests used in routine identification and susceptibility profiling of human pathogens. The validation of citrated hair sheep blood eliminates the labor-intensive and equipment-requiring process of manual defibrination. Use of hair sheep blood, in lieu of human blood currently used by many developing world laboratories and as an alternative to cost-prohibitive commercial sheep blood, offers the opportunity to dramatically improve the safety and accuracy of laboratory diagnosis of pathogenic bacteria in resource-poor countries.     

The effect of hyperbaric oxygenation on thein vitro growth ofEscherichia coli in environments with and without blood cells

The aim of this study was to investigate whether thein vitro presence of blood cells influences the anti-microbial activity of hyperbaric oxygen (HBO) againstEscherichia coli. FiftyE. coli isolates from clinical samples were used in the study. A small number of colonies belonging to each isolate from the nutrient media were transferred into two K₃EDTA tubes (the blood group) and two Mueller-Hinton broth tubes (the broth group). Then, both groups were divided into subgroups according to whether HBO was administered (HBO subgroup) or not (non-HBO subgroup). HBO treatment was applied for one hour at 2.5 absolute atmospheres. The tubes in the non-HBO subgroup were left at room temperature during this period. Subsequently, all the tubes were cultured on Mueller-Hinton and Eosin Methylene Blue agar using the quantitative counting technique. After 18 to 24 h incubation at 37 °C, the colonies formed in the plates were counted. In the blood group, compared with non-HBO subgroup samples, the number of colonies decreased in 56% of samples, increased in 32% of samples and did not change in 12% of samples in the HBO subgroup. Whereas, in the broth group the number of colonies decreased in only 32% of samples increased in 38% of samples and did not change in 30% of samples in the HBO subgroup compared with the non-HBO subgroup. The difference between the blood and the broth groups revealed a statistical significance using Pearson’s Chi-square test (P=0.025). We concluded that the antibacterial effect of HBO onE. coli increases in the cellular environment belonging to the host organism.     

Quantification of gentamicin in Mueller–Hinton agar by high-performance liquid chromatography

The aim of this study was to optimise a method for gentamicin determination in an agar matrix and to investigate if and how agar composition can affect the gentamicin diffusion kinetics during the agar diffusion tests for antibiotics sensitivity. Gentamicin was separated by RP-HPLC and detected at 365 nm after pre-column derivatization with 1-fluoro-2,4-dinitrobenzene. Recovery (≥79%), linearity (r²≥0.997) and sensitivity (1 μg/ml) were assessed using four different agar matrices. The kinetics of gentamicin diffusion tested on BioMerieux and DID manufacturers’ products showed in uninoculated agar plates significant differences that were even more pronounced in the presence of Pseudomonas aeruginosa metabolism.     

Evaluation of diffusion and dilution methods to determine the antibacterial activity of plant extracts

The aim of this study was to evaluate diffusion and dilution methods for determining the antibacterial activity of plant extracts and their mixtures. Several methods for measurement of the minimal inhibitory concentration (MIC) of a plant extract are available, but there is no standard procedure as there is for antibiotics. We tested different plant extracts, their mixtures and phenolic acids on selected gram-positive (Staphylococcus aureus, Bacillus cereus, and Listeria monocytogenes) and gram-negative bacteria (Escherichia coli O157:H7, Salmonella Infantis, Campylobacter jejuni, Campylobacter coli) with the disk diffusion, agar dilution, broth microdilution and macrodilution methods. The disk diffusion method was appropriate only as a preliminary screening test prior to quantitative MIC determination with dilution methods. A comparison of the results for MIC obtained by agar dilution and broth microdilution was possible only for gram-positive bacteria, and indicated the latter as the most accurate way of assessing the antimicrobial effect. The microdilution method with TTC (2,3,5-triphenyl tetrazolium chloride) or INT (2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride) to indicate the viability of aerobic bacteria was found to be the best alternative approach, while only ATP determination was appropriate for microaerophilic Campylobacter spp. Using survival curves the kinetics of bacterial inactivation on plant extract exposure was followed for 24 h and in this way the MIC values determined by the microdilution method were confirmed as the concentrations of extracts that inhibited bacterial growth. We suggest evaluation of the antibacterial activity of plant extracts using the broth microdilution method as a fast screening method for MIC determination and the macrodilution method at selected MIC values to confirm bacterial inactivation. Campylobacter spp. showed a similar sensitivity to plant extracts as the tested gram-positive bacteria, but S. Infantis and E. coli O157:H7 were more resistant.     

Enantioselective determination of sotalol enantiomers in biological fluids using high-performance liquid chromatography

A simple and sensitive high-performance liquid chromatograhic (HPLC) method for the determination of (+)-(S)-sotalol and (−)-(R)-sotalol in biological fluids was established. Following extraction with isopropyl alcohol from biological samples on a Sep-Pak C₁₈ cartridge, the eluent was derivatized with 2,3,4,6-tetra-O-acetyl-β-d-glucopyranosol isothiocyanate (GITC). The diastereoisomeric derivatives are resolved by HPLC with UV detection at 225 nm. Calibration was linear from 0.022 to 4.41 μg/ml in human plasma and from 0.22 to 88.2 μg/ml in human urine for both (+)-(S)- and (−)-(R)-sotalol. The lower limit of determination was 0.022 μg/ml for plasma and 0.22 μg/ml for urine. The within-day and day-to-day coefficients of variation were less than 7.5% for each enantiomer at 0.09 and 1.8 μg/ml in plasma and at 0.44 and 4.4 μg/ml in urine. The method is also applicable to other biological specimens such as rat, mouse and rabbit plasma.