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Extrusion of fluorescein diacetate by multidrug-resistant Candida albicans.

Over-expression of multidrug efflux transporters causes Candida albicans cells to be resistant to azole antifungal agents. There are several kinds of indicator for multidrug resistance (MDR) phenotype of higher eukaryotic cells. Calcein AM is a prefluorochrome that is known as a substrate for multidrug efflux transporters of mammalian cells. We investigated whether calcein AM was also extruded by the ATP-dependent multidrug transporter (cdr1p) of C. albicans. There was no significant difference in the accumulation of calcein AM between MDR cells and drug-susceptible cells of C. albicans even with sodium azide, suggesting that calcein AM may not be associated with the CDR1-gene-related multidrug efflux system of C. albicans. However, a structurally related prefluorochrome derivative, fluorescein diacetate (FDA), was shown to be extruded by the CDR1 mRNA-overexpressing yeast cells. In comparison with drug-susceptible cells, the resistant cells emitted very weak fluorescence when stained with FDA. Furthermore sodium azide increased the fluorescence of the resistant cells more than 20 times, whereas the fluorescence in the drug-susceptible cells with FDA and sodium azide was three to four times stronger. These results suggested that FDA might be extruded by the CDR1-related multidrug efflux transporter of C. albicans.

Antifungal Agents↗

Evaluation of recovery methods to detect faecal streptococci in polluted waters.

AIMS: This paper compares the faecal streptococci count on 25 samples of polluted waters obtained with three techniques: most probable number (MPN), membrane filtration (MF) and pour plate (PP) methods. Although the PP method is a simple technique, familiar to water bacteriologists, it is not recommended in the international methods. METHODS AND RESULTS: For the MPN method, azide dextrose broth and ethyl violet azide broth were employed. For the MF technique, Millipore filters were placed onto azide maltose agar (KF agar), while for the PP method, 1 ml of a decimal water dilution was added to (Kennel Faecal) KF medium. Regression analysis and Friedman's ANOVA were performed to determine the relationship between faecal streptococci counts obtained with the three techniques. Statistical analysis of the results showed that the MPN, MF and PP techniques were equally valid with respect to faecal streptococci enumeration in polluted waters. CONCLUSION: Since the PP method was found to be as good as the other techniques, it may be preferred in polluted waters. It is more economical in terms of both time and materials than the MPN count, and it is as accurate as the MF count. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates that the PP method, although not recommended internationally, is a reliable alternative to MF and MPN.

Bacteriological Techniques↗

Lateral diffusion of surface immunoglobulin, Thy-1 antigen, and a lipid probe in lymphocyte plasma membranes.

Fluorescence photobleaching recovery was used to measure the lateral diffusion coefficient and mobile fraction of surface immunoglobulin (sIg), Thy-1 antigen, and a lipid probe in the plasma membrane of mouse lymphocytes. The lipid probe (3,3'-dioctadecylindocarbocyanine) had a mean (+/-SD) diffusion coefficient of (1.7 +/- 0.3) x 10(-8) cm(2)/sec, with essentially all of the probe mobile in the membrane. We detected little or no effect on the diffusion of this probe due to the presence of microvilli. Its diffusion was slightly restricted in capped regions. No differences in lipid probe mobility were detected between T and B cells. Fifty to 90% of the detectable sIg and Thy-1 antigen was free to move in the plane of the membrane with diffusion coefficients of approximately 3 x 10(-10) cm(2)/sec; the remainder was immobile. Crosslinking of sIg with anti-Ig antibodies (in the presence of azide to inhibit capping) completely immobilized sIg at high concentrations but failed to do so at low concentrations. Thy-1 antigen could not be immobilized with an IgG rabbit anti-mouse brain reagent without an additional layer of crosslinking antibody. In parallel labelings (in the absence of azide), capping of sIg and Thy-1 antigen was observed only under crosslinking conditions sufficient to immobilize the membrane antigen. Sodium azide, colchicine, and cytochalasin B had no measurable effect on lipid probe, sIg, or Thy-1 diffusion.

Animals↗

X-ray structure of a vanadium-containing enzyme: chloroperoxidase from the fungus Curvularia inaequalis.

The chloroperoxidase (EC 1.11.1.-) from the fungus Curvularia inaequalis belongs to a class of vanadium enzymes that oxidize halides in the presence of hydrogen peroxide to the corresponding hypohalous acids. The 2.1 A crystal structure (R = 20%) of an azide chloroperoxidase complex reveals the geometry of the catalytic vanadium center. Azide coordinates directly to the metal center, resulting in a structure with azide, three nonprotein oxygens, and a histidine as ligands. In the native state vanadium will be bound as hydrogen vanadate(V) in a trigonal bipyramidal coordination with the metal coordinated to three oxygens in the equatorial plane, to the OH group at one apical position, and to the epsilon 2 nitrogen of a histidine at the other apical position. The protein fold is mainly alpha-helical with two four-helix bundles as main structural motifs and an overall structure different from other structures. The helices pack together to a compact molecule, which explains the high stability of the protein. An amino acid sequence comparison with vanadium-containing bromoperoxidase from the seaweed Ascophyllum nodosum shows high similarities in the regions of the metal binding site, with all hydrogen vanadate(V) interacting residues conserved except for lysine-353, which is an asparagine.

Amino Acid Sequence↗

Two sites of interaction of anions with cytochrome a in oxidized bovine cytochrome c oxidase.

An interaction between cytochrome a in oxidized cytochrome c oxidase (CcO) and anions has been characterized by EPR spectroscopy. Those anions that affect the EPR g = 3 signal of cytochrome a can be divided into two groups. One group consists of halides (Cl-, Br-, and I-) and induces an upfield shift of the g = 3 signal. Nitrogen-containing anions (CN-, NO2-, N3-, NO3-) are in the second group and shift the g = 3 signal downfield. The shifts in the EPR spectrum of CcO are unrelated to ligand binding to the binuclear center. The binding properties of one representative from each group, azide and chloride, were characterized in detail. The dependence of the shift on chloride concentration is consistent with a single binding site in the isolated oxidized enzyme with a Kd of approximately 3 mm. In mitochondria, the apparent Kd was found to be about four times larger than that of the isolated enzyme. The data indicate it is the chloride anion that is bound to CcO, and there is a hydrophilic size-selective access channel to this site from the cytosolic side of the mitochondrial membrane. An observed competition between azide and chloride is interpreted by azide binding to three sites: two that are apparent in the x-ray structure plus the chloride-binding site. It is suggested that either Mg2+ or Arg-438/Arg-439 is the chloride-binding site, and a mechanism for the ligand-induced shift of the g = 3 signal is proposed.

Animals↗

Myeloperoxidase potentiates nitric oxide-mediated nitrosation.

Nitrosation is an important reaction elicited by nitric oxide (NO). To better understand how nitrosation occurs in biological systems, we assessed the effect of myeloperoxidase (MPO), a mediator of inflammation, on nitrosation observed during NO autoxidation. Nitrosation of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ; 10 mum) to 2-nitrosoamino-3-methylimidazo[4,5-f]quinoline (N-NO-IQ) was monitored by HPLC. Using the NO donor spermine NONOate at pH 7.4, MPO potentiated N-NO-IQ formation. The minimum effective quantity of necessary components was 8.5 nm MPO, 0.25 mum H(2)O(2)/min, and 0.024 mum NO/min. Autoxidation was only detected at >/=1.2 mum NO/min. MPO potentiation was not affected by a 40-fold excess flux of H(2)O(2) over NO or less than a 2.4-fold excess flux of NO over H(2)O(2). Potentiation was due to an 8.8-fold increased affinity of MPO-derived nitrosating species for IQ. Autoxidation was inhibited by azide, suggesting involvement of the nitrosonium ion, NO(+). MPO potentiation was inhibited by NADH, but not azide, suggesting oxidative nitrosylation with NO(2)(.) or an NO(2)(.)-like species. MPO nonnitrosative oxidation of IQ with 0.3 mm NO(2)(-) at pH 5.5 was inhibited by azide, but not NADH, demonstrating differences between MPO oxidation of IQ with NO compared with NO(2)(-). Using phorbol ester-stimulated human neutrophils, N-NO-IQ formation was increased with superoxide dismutase and inhibited by catalase and NADH, but not NaN(3). This is consistent with nitrosation potentiation by MPO, not peroxynitrite. Increased N-NO-IQ formation was not detected with polymorphonuclear neutrophils from two unrelated MPO-deficient patients. Results suggest that the highly diffusible stable gas NO could initiate nitrosation at sites of neutrophil infiltration.

Chromatography, High Pressure Liquid↗

Bactericidal activity of a superoxide anion-generating system. A model for the polymorphonuclear leukocyte.

The acetaldehyde-xanthine oxidase system in the presence and absence of myeloperoxidase (MPO) and chloride has been employed as a model of the oxygen-dependent antimicrobial systems of the PMN. The unsupplemented xanthine oxidase system was bactericidal at relatively high acetaldehyde concentrations. The bactericidal activity was inhibited by superoxide dismutase (SOD), catalase, the hydroxyl radical (OH.) scavengers, mannitol and benzoate, the singlet oxygen (1O2) quenchers, azide, histidine, and 1,4-diazabicyclo[2,2,2]octane (DABCO) and by the purines, xanthine, hypoxanthine, and uric acid. The latter effect may account for the relatively weak bactericidal activity of the xanthine oxidase system when purines are employed as substrate. A white, carotenoid-negative mutant strain of Sarcina lutea was more susceptible to the acetaldehyde-xanthine oxidase system than was the yellow, carotenoid-positive parent strain. Carotenoid pigments are potent 1O2 quenchers. The xanthine oxidase system catalyzes the conversion of 2,5-diphenylfuran to cis-dibenzoylethylene, a reaction which can occur by a 1O2 mechanism. This conversion is inhibited by SOD, catalase, azide, histidine, DABCO, xanthine, hypoxanthine, and uric acid but is only slightly inhibited by mannitol and benzoate. The addition of MPO and chloride to the acetaldehyde-xanthine oxidase system greatly increases bactericidal activity; the minimal effective acetaldehyde concentration is decreased 100-fold and the rate and extent of bacterial killing is increased. The bactericidal activity of the MPO-supplemented system is inhibited by catalase, benzoate, azide, DABCO, and histidine but not by SOD or mannitol. Thus, the acetaldehyde-xanthine oxidase system which like phagocytosing PMNs generates superoxide (O.2-) and hydrogen peroxide, is bactericidal both in the presence and absence of MPO and chloride. The MPO-supplemented system is considerably more potent; however, when MPO is absent, bactericidal activity is observed which may be mediated by the interaction of H2O2 and O.2- to form OH. and 1O2.

Acetaldehyde↗

Respiratory metabolism of normal and divisionless strains of Candida albicans.

Respiration of a normal strain of Candida albicans was compared with that of a divisionless mutant which has a biochemical lesion such that metabolically generated hydrogen "spills over," during growth, for non-specific dye reduction. This waste is not at expense of growth, since both strains grow at essentially similar rates, nor at expense of respiration, since the mutant reduces oxygen more rapidly than the normal strain. Respiration in both strains is qualitatively similar, and seemingly unique among highly aerobic organisms in that it is not mediated by cytochrome oxidase. In resting cells of both strains, respiration is not only resistant to, but markedly stimulated by, high concentrations of cyanide, carbon monoxide, and azide. In contrast, growth of these yeasts is inhibited by low concentrations of cyanide and azide. Cytochrome oxidase could not be detected in cell-free preparations; reduced cytochrome c was not oxidized by such preparations. Cytochrome bands could not be observed in thick cell suspensions treated with reducing agents. However, incorporation of superoptimal levels of zinc and iron into the culture medium resulted in growth of cells possessing distinct cytochrome bands; respiration of these cells remained insensitive to cyanide, monoxide, and azide, and the bands were maintained in a reduced form on oxygenation. In the divisionless yeast, tetrazolium dyes compete with oxygen for reduction; this is not the case in the normal strain. The firmness with which hydrogen transfer is channeled in the latter for reduction of disulfide bonds (of importance in the division mechanism) and of oxygen, is contrasted with the lack of such control in the mutant.

Candida↗

Total morphine stability in urine specimens stored under various conditions.

The stability of total morphine in urine stored under various conditions was studied using control and experimental specimens. Samples in the control group were prepared using drug-free urine spiked with morphine at three concentration levels (300, 1000, and 2500 ng/mL), each with the pH adjusted to 5.5, 6.5, and 7.5. Samples in the experimental group came from 20 alleged heroin addicts (provided by Taipei Municipal Psychiatric Hospital). Samples in both groups were divided into two categories--one with and one without the precipitate (formed at 0 degrees C) removed. Samples in each of these two categories were further divided into two sub-groups--one with and one without sodium azide (0.05%) added. Total morphine contents in these samples were first determined by gas chromatography-mass spectrometry prior to storage and at 6, 12, 18, and 24 months following storage at -20, 4, 25, and 35 degrees C. Effects of sample treatment (azide addition and precipitate removal), pH, and storage temperature and length were evaluated by examining the percentage of total morphine remaining at the four time intervals following the initial determination. Major findings were as follows: (1) total morphine decomposition was minimal when stored for 12 months at -20 degrees C, which is a common current practice; (2) samples with lower initial sample pH had slower total morphine decomposition rates; and (3) azide addition appeared to have no detectable effect, whereas precipitate removal appeared to marginally reduce the decomposition rate, especially for samples with lower pH.

Drug Stability↗

Reaction Properties of the Ascorbic Acid Oxidase from Myrothecium verrucaria.

Ascorbic acid oxidase activity in Myrothecium verrucaria extracts resulted in O(2) uptake exceeding 0.5 mole per mole of ascorbic acid and in CO(2) evolution. Measurement of oxidized ascorbic acid at completion of the reaction demonstrated that an average of 10% of the oxidized product disappeared. A comparison of the gas exchange data with the amount of ascorbic acid not accounted for indicated that the reaction could not be explained by independent oxidase and oxygenase systems. Chromatographic examination of the reaction mixtures identified l-threonic acid. Experiments with ascorbic acid-1-(14)C showed that C-1 was partially decarboxylated during the oxidation. Test of the fungal extracts for enzymes that might explain the deviation from expected stoichiometry showed that phenolase, glutathione reductase, cytochrome oxidase, peroxidase and oxalic decarboxylase were not involved. Addition of azide in concentrations sufficient to block catalase increased excess O(2) consumption about 65%. No enzymes were found that could directly attack oxidized ascorbic acid. H(2)O(2) accumulated during oxidation in azide-blocked systems.The O(2) excess could be explained by assuming the enzyme had peroxidative capacity on a reductant other than ascorbic acid. An intermediate of ascorbic acid oxidation appeared to function as the substrate yielding CO(2) and l-threonic acid on degradation. The increase in excess O(2) utilized in azide-blocked systems and the H(2)O(2) accumulation also were explained by the proposed scheme.Another interpretation would involve production of free radicals during ascorbic acid oxidation. Evidence for this was the ability of extracts to oxidize DPNH in the presence of ascorbic acid. Oxygen radicals formed in such reactions were considered possible agents of degradation of ascorbic acid.

Journal Article↗

Properties of Higher Plant Mitochondria. III. Effects of Respiratory Inhibitors.

The effects of representative respiratory inhibitors were investigated on the coupled respiration of mung bean mitochondria using succinate and l-malate as substrates. The inhibitors studied were: (I) malonate, (II) amytal and rotenone, (III) antimycin A and 2-n-nonyl-4-hydroxyquinoline N-oxide (NOQNO), and (IV) cyanide and azide.Malonate inhibition of succinate oxidation follows a classical type of competitive inhibition with an inhibitor dissociation constant of 0.13 mm. There is no inhibition detectable when malate is used as substrate. In contrast to animal mitochondria, amytal is capable of inhibiting 20 to 40% of succinate oxidation and 90 to 100% of malate oxidation, but inhibition due to rotenone amounts to only 0 to 20% of succinate oxidation and 40 to 50% of malate oxidation. The half-maximal inhibition caused by amytal occurs at 2 to 2.5 mm and that by rotenone at 3 mmumoles/mg protein.The maximal inhibition caused by either antimycin A or NOQNO is 70 to 80% of the state 3 respiration. Very little inhibition was observed on the state 4 respiration, and both inhibitors were capable of titrating stoichiometrically with mitochondrial protein with identical titers, 0.22 mmumoles/mg protein for half-maximal inhibition. They differ, however, in that NOQNO does uncouple oxidative phosphorylation in mung bean mitochondria, but antimycin A does not do so. Both cyanide and azide inhibit the state 3 rate 65 to 80%. Inhibition of state 4 respiration can be up to 50% by cyanide, while almost none by azide. Uncoupling action was noted with cyanide, but very little with azide.It is concluded that the second state 3 rate of succinate oxidation includes 80% succinoxidase, the remaining 20% being contributed by the NADH pathway. Malate oxidation apparently does not involve succinoxidase. Malate oxidation is completely sensitive to amytal, but only 50% inhibited by rotenone. A difference between animal and plant mitochondria appears to be in the flavoproteins associated with NADH oxidation.From the observations that antimycin A, NOQNO, cyanide, and azide do not cause complete inhibition, it is suggested that a leakage of electrons to oxygen exists before the site of inhibition of antimycin A or NOQNO.

Journal Article↗

Di-mu-azido-bis{[1-(2-pyridylmethyliminomethyl)-2-naphtholato]zinc(II)}.

The title compound, [Zn2(C17H13N2O)2(N3)2], is an azide-bridged dinuclear zinc(II) complex which has inversion symmetry. The ZnII atom is five-coordinated in a square-pyramidal configuration by one O and two N atoms of one Schiff base ligand [Zn-O=1.902 (2) A and Zn-N=1.938 (2) and 2.002 (2) A] and by one terminal N atom [Zn-N=1.985 (2) A] of a bridging azide ligand defining the basal plane, and by another terminal N atom of another bridging azide ligand [Zn-N=2.554 (2) A] occupying the apical position.

Journal Article↗

Azidotetrakis(trimethylphosphine)nickel(II) tetrafluoroborate.

The title complex, [Ni(N3)(C3H9P)4]BF4, is a nearly perfect trigonal bipyramid with the azide group at an apical position. The metal-azide bond angle, Ni1--N1--N2, of 138.6(5) degrees is the largest observed for a terminal azide ligand.

Crystallography, X-Ray↗

Hidden receptors for nerve growth factor in PC12 cells.

The binding of nerve growth factor (NGF) to its receptors in PC12 cells was studied in two experimental conditions: (a) cell fixation with paraformaldehyde followed by permeabilization of the plasma membrane with methanol and (b) metabolic poisoning of living cells with sodium azide. Paraformaldehyde fixation of PC12 cells causes a 60-70% reduction of NGF binding capacity; the original binding capacity is restored following permeabilization with methanol. A kinetic analysis of NGF binding under these conditions reveals a single homogeneous population of receptors at variance with experiments performed in living cells where two kinetically distinct types of NGF receptors were demonstrated [Landreth, G. E. and Shooter, E. M. (1980) Proc. Natl Acad. Sci. USA, 77, 4751-4755; Schechter, A. L. and Bothwell, M. A. (1981) Cell, 24, 867-874]. Our results suggest that a proportion of the NGF receptors in PC12 cells is hidden, i.e. not available for binding to the ligand, and in a dynamic equilibrium with exposed receptors. The existence of hidden receptors is confirmed by treatment of PC12 cells with sodium azide, which causes a 50% reduction in NGF binding capacity and protection from trypsin digestion of the remaining pool of hidden receptors. The latter become exposed at the cell surface following removal of sodium azide. Our data provide an interpretation for the as yet unsatisfactorily explained data on NGF receptors.

Cell Line↗

Differentiation by hydroquinone of relaxations induced by exogenous and endogenous nitrates in non-vascular smooth muscle: role of superoxide anions.

1. The influence of hydroquinone on relaxations induced by nitric oxide (NO), nitrovasodilator drugs, and non-adrenergic, non-cholinergic (NANC) field stimulation has been investigated in three tissues in which endogenous nitrates have been implicated in the NANC response; the mechanism of action of hydroquinone was also studied. 2. In mouse anococcygeus, hydroquinone (10-100 microM) produced a concentration-dependent inhibition of relaxations induced by 15 microM NO. Hydroquinone, 100 microM, which reduced responses to NO by 85%, had no effect on relaxations induced by NANC field stimulation (10 Hz; 20s trains), hydroxylamine (10 microM), sodium nitroprusside (1 microM) or sodium azide (20 microM). 3. In guinea-pig trachea, 100 microM hydroquinone reduced relaxations to 150 microM NO by 75%, but had no effect on those to NANC stimulation (10 Hz; 30 s trains) or sodium azide (5 microM). 4. In rat gastric fundus, 100 microM hydroquinone reduced relaxations to 1 microM NO by 85%, but had no effect on those to NANC stimulation (0.5 Hz; 15 s trains) or sodium azide (2 microM). 5. Superoxide dismutase (SOD; 50 u ml-1) had no effect on relaxations of the mouse anococcygeus in response to 15 microM NO or 10 Hz NANC stimulation. Further, the inhibition of responses to NO by hydroquinone was unaffected in the presence of SOD. 6. Hydroquinone (10-100 microM) failed to generate superoxide anions, as detected by a chemiluminescent assay. However, 100 microM hydroquinone, like SOD (50 u ml-1), produced almost complete inhibition of superoxide anion chemiluminescence induced by xanthine (500 microM): xanthine oxidase (0.07 u ml-1). 7. It is concluded that, in our system, hydroquinone inhibits NO by acting as a free radical scavenger rather than by generating superoxide anions. The ability of hydroquinone to block relaxations to NO, but not NANC stimulation, may suggest that the endogenous nitrate substance released by these NANC nerves may not be free NO, but may be an NO-containing, or NO-generating, molecule.

Animals↗

Properties of cloned ATP-sensitive K+ currents expressed in Xenopus oocytes.

1. We have studied the electrophysiological properties of cloned ATP-sensitive K+ channels (KATP channels) heterologously expressed in Xenopus oocytes. This channel comprises a sulphonylurea receptor subunit (SUR) and an inwardly rectifying K+ channel subunit (Kir). 2. Oocytes injected with SUR1 and either Kir6.2 or Kir6.1 exhibited large inwardly rectifying K+ currents when cytosolic ATP levels were lowered by the metabolic inhibitors azide or FCCP. No currents were observed in response to azide in oocytes injected with Kir6.2, Kir6.1 or SUR1 alone, indicating that both the sulphonylurea receptor (SUR1) and an inward rectifier (Kir6.1 or Kir6.2) are needed for functional channel activity. 3. The pharmacological properties of Kir6.2-SUR1 currents resembled those of native beta-cell ATP-sensitive K+ channel currents (KATP currents): the currents were > 90% blocked by tolbutamide (500 microM), meglitinide (10 microM) or glibenclamide (100 nM), and activated 1.8-fold by diazoxide (340 microM), 1.4-fold by pinacidil (1 mM) and unaffected by cromakalim (0.5 mM). 4. Macroscopic Kir6.2-SUR1 currents in inside-out patches were inhibited by ATP with a Ki of 28 microM. Kir6.1-SUR1 currents ran down within seconds of patch excision preventing analysis of ATP sensitivity. 5. No sensitivity to tolbutamide or metabolic inhibition was observed when SUR1 was coexpressed with either Kir1.1a or Kir2.1, suggesting that these proteins do not couple in Xenopus ocytes. 6. Our data demonstrate that the Xenopus oocyte constitutes a good expression system for cloned KATP channels and that expression may be assayed by azide-induced metabolic inhibition.

Adenosine Triphosphate↗

Microbially Mediated Mn(II) Oxidation in an Oligotrophic Arctic Lake.

Rates of oxidation of Mn(II) were measured by an in situ incubation technique in the water column of Toolik Lake, Alaska. Measured rates were lower than those observed in other aquatic systems but were sufficient to oxidize all Mn(II) in the lake within a 3-month period. Measured rates compared favorably with rates estimated from a previous study of the geochemical cycling of Mn in Toolik Lake. The Mn(II) oxidation was largely microbially mediated, as indicated by inhibition of oxidation rates by sodium azide. Azide had been previously demonstrated to be a suitable microbial poison for studying Mn(II) oxidation in seawater. This study demonstrates that azide is also a suitable poison for freshwaters and that it inhibits microbial but not abiotic oxidation of Mn(II). Manganese(II) oxidation rates were similar during cold, under-ice conditions in early spring and during warmer summer conditions. This observation suggests that Mn(II) concentration, rather than temperature or oxygen concentration, is the most important factor regulating Mn(II) oxidation rates in Toolik Lake.

Journal Article↗

Comparison of enterococci and coliform microorganisms in commercially produced pecan nut meats.

Pecan nut meats in the unbroken shell are sterile for enteric microorganisms. Recovery of coliform microorganisms or enterococci from finished pecan nut meats indicated contact contamination, assuming the tempering procedures to be satisfactory. Results of specific studies, designed toward developing background data on the sanitary significance of enterococci and coliform microorganisms in the production of pecan meats are reported. Unbroken pecan nuts or nut meats from various stages of shelling operations were diluted with a phosphate-buffered diluent. Serial dilutions were inoculated into Lactose Broth and Azide Dextrose Broth. The lactose fermentors were carried through indole, methyl red, Voges-Proskauer, and citrate reactions; the positive Azide Dextrose cultures were confirmed in Ethyl Violet Azide Broth and microscopically. Viable plate counts were obtained. Enterococci were found resistant to many deterrent factors affecting coliforms. Recoveries of enterococci were detected long after pollution had occurred. Little correlation was found between enterococcal recovery and observed insanitary practices in commercial shelling operations. Using the coliaerogenes group and, specifically, Escherichia coli as a sanitation index, microorganisms allowed accurate appraisal of tempering, personnel practices, and contact surface contaminating factors. It is felt this was due, in part, to the more delicate growth characteristics of E. coli. The fact that other pathogenic microorganisms, capable of causing gastrointestinal upsets, are associated with the presence of E. coli introduces a health factor which is important to regulatory agencies concerned with consumer protection.

Carya↗