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Singlet excited oxygen as a mediator of the antibacterial action of leukocytes.

Human polymorphonuclear leukocytes kill a colorless mutant strain of Sarcina lutea much more readily than a carotenoid-containing strain. A similar protective effect has been reported in the organism during photodynamic inactivation, where it is attributable to the quenchintg of singlet excited oxygen by carotenoids. The findings with leukocytes support the suggestion that singlet excited oxygen acts as one of the mediators of their bactericidal action.

Bacteriolysis↗

Glutamine-selective membrane electrode that uses living bacterial cells.

A novel bioselective membrane electrode for L-glutamine has been constructed by coupling living bacteria of the strain Sarcina flava to a potentiometric ammonia gas sensor. Tests in aqueous standards and human serum show that the electrode combines excellent sensitivity and selectivity with rapid response and a useful lifetime of at least 2 weeks.

Ammonia↗

Target substances of some antifungal agents in the cell membrane.

Copiamycin, an antifungal antibiotic, exhibits antimicrobial activity against a few bacteria in addition to a wide variety of fungi. The methanol extract of Sarcina lutea, one of the most susceptible bacteria, was found to reverse the antimicrobial activity of copiamycin. The reversing activity was associated with the phospholipid fraction of the bacteria. The S. lutea phospholipids also reversed the activities of azalomycin F and miconazole, but not that of clotrimazole. The effects of authentic phospholipids and fatty acids were also investigated. As the antimicrobial activities of copiamycin and azalomycin F were most strongly reversed in the same manner by phospholipids with unsaturated fatty acids and basic hydrophilic groups, the sites on the cell membrane sensitive to both antibiotics are assumed to be identical. On the other hand, the activity of miconazole was affected by different phospholipids from those which affected these two antifungal antibiotics, and the activity of clotrimazole was not affected by any of the phospholipids and fatty acids. It was postulated that the sites on the cell membrane sensitive to miconazole and clotrimazole are different from those sensitive to copiamycin and azalomycin F.

Antifungal Agents↗

Simple assay for clindamycin in the presence of aminoglycosides.

The presence of aminoglycoside antibiotics interferes with the determination of clindamycin levels by standard microbiological assay. A new bioassay method of quantitation of clindamycin in the presence of aminoglycosides is presented. This technique is based on the fact that incorporation of calcium chloride into assay agar blocks the antimicrobial activity of aminoglycosides against Sarcina lutea but allows the assay of as little as 0.31 mug of clindamycin per ml. The error of the system is less than 10%. This method is simple and adaptable to existing assay systems for clindamycin.

Aminoglycosides↗

Inhibitory effects of citrates in the determination of trace amounts of penicillin.

In the microbiological assay for the determination of trace amounts of penicillin, it has been shown that sodium citrate and penicillin act synergistically against Sarcina lutea. The inhibitory action of citrate appears to involve a sequestering of divalent cations essential to the growth of the organism as shown by minimal inhibitory concentration and agar diffusion studies. The addition of calcium or magnesium to broth or agar at various concentrations overcomes this inhibition, and 0.02 M magnesium was found to be the most effective concentration. On agar supplemented with magnesium chloride, penicillin dose-response curves with and without citrate present were practically superimposable, and larger inhibition zones against a greater amount to total growth resulted in improved assay susceptibility and an interference-free method. Misleading results can be obtained when pharmaceutical products containing citrates are tested by conventional methods for penicillin cross-contamination.

Biological Assay↗

Ecophysiological adaptations of anaerobic bacteria to low pH: analysis of anaerobic digestion in acidic bog sediments.

The dynamics of anaerobic digestion were examined in the low-pH sediments of Crystal Bog in Wisconsin. The sediments (pH 4.9) contained 71% organic matter and the following concentrations of dissolved gases (micromoles per liter): CO2, 1,140; CH4, 490; and H2, 0.01. The rate of methane production was 6.2 mumol/liter of sediment per h, which is slower than eutrophic, neutral sediments. Microbial metabolic processes displayed the following pH optima: hydrolysis reactions, between 4.2 and 5.6; aceticlastic methanogenesis, 5.2; and hydrogen-consuming reactions, 5.6. The turnover rate constants for key intermediary metabolites were (h-1): glucose, 1.10; lactate, 0.277; acetate, 0.118; and ethanol, 0.089. The populations of anaerobes were low, with hydrolytic groups (10(6)/ml) several orders of magnitude higher than methanogens (10(2)/ml). The addition of carbon electron donors to the sediment resulted in the accumulation of hydrogen, whereas the addition of hydrogen resulted in the accumulation of fatty acids and the inhibition of hydrogen-producing acetogenic reactions. Strains of Lactobacillus, Clostridium, and Sarcina ventriculi were isolated from the bog, and their physiological attributes were characterized in relation to hydrolytic process functions in the sediments. The present studies provide evidence that the pH present in the bog sediments alter anaerobic digestion processes so that total biocatalytic activity is lower but the general carbon and electron flow pathways are similar to those of neutral anoxic sediments.

Acetates↗

Survival of bacteria on metal surfaces.

Survivor curves were determined for Serratia marcescens, Sarcina lutea, Pasteurella tularensis, and P. pestis deposited from the airborne state onto metallic surfaces and subsequently stored at various humidities and temperatures. Cells of all species tested remained alive longest in a dry atmosphere, except that cells of S. marcescens survived best in a saturated atmosphere. Survival decreased most rapidly at the intermediate humidity level for three of the test organisms, yet P. tularensis died most rapidly in a saturated atmosphere. An increase in temperature decreased survival of P. pestis and P. tularensis.

Aerosols↗

Independent measurement of ampicillin and cloxacillin in mixtures.

A simplified method of assaying mixtures of ampicillin and cloxacillin is presented. The method consists of cylinder-plate assays of appropriately diluted samples in three assay systems in which: (i) cloxacillin is measured, utilizing agar seeded with a penicillinase-producing Staphylococcus aureus; (ii) ampicillin is measured after the greatest dilution practicable, by use of agar seeded with Sarcina lutea; and (iii) the proportion of activity found in the second assay that remains after treatment with staphylococcal penicillinase is determined on agar seeded with S. lutea. The error was 4% or less when each system was run in quadruplicate, except when there was 10 or more times as much cloxacillin as ampicillin, in which case the greatest error was 12%. Mixtures of methicillin with benzylpenicillin or with ampicillin can also be quantitated by this method.

Ampicillin↗

Ultraviolet bactericidal irradiation of ice.

We investigated the germicidal activity of 2,537 A ultraviolet (UV) radiation on bacteria in ice cubes of varying thickness and in aqueous suspensions beneath an ice layer. The test bacteria used were Escherichia coli, Serratia marcescens, Bacillus subtilis, and Sarcina lutea; aqueous suspensions of the selected organisms were frozen into ice cubes, 2 mm to 30 mm thick, at -20 C. The cubes were irradiated for 1 min, whereas the suspensions of bacteria were placed beneath an ice block (19 cm thick) and were irradiated for 0.5 to 15 min. In both groups of experiments, the standard plate count method was used to compare the number of bacteria surviving the UV treatment with the number of bacteria in the untreated controls. The results showed that 1 min of UV treatment killed as many as 97% of the gram-negative and at least 60% of the gram-positive test bacteria (freezing survivors) frozen in ice cubes 30-mm thick. Within 15 min, UV light transmitted through a 19-cm thick ice block inactivated 98% of the bacteria suspended in the buffer solution. We concluded that the UV rays were able to penetrate at least 19 cm of ice and still retain enough energy to kill bacteria. However, the UV penetration depended greatly on the optical quality of the ice. Although it was not the purpose of these experiments to find a practical method for sanitizing ice, the results of this study and of our other unpublished experiments indicate that UV light has adequate penetrating power to be considered practical in certain selected applications.

Bacillus subtilis↗

Measurement of three antibiotics (penicillin, cephalothin, and chloramphenicol) when present together in mixtures.

Benzylpenicillin, cephalothin, and chloramphenicol were measured individually and quantitatively when present together in mixtures at concentrations found in patients. The assay depended on the selective inactivation of two of the antibiotics, permitting the third to be assayed as if present alone. Agar seeded with a chloramphenicol-resistant, penicillinase-negative Staphylococcus aureus was used for a cylinder-plate assay of appropriately diluted fluid to determine the activity of either benzylpenicillin or cephalothin after either beta-lactamase inactivation of the former or ultraviolet light inactivation of the latter. Chloramphenicol was assayed with Sarcina lutea after appropriate beta-lactamase inactivation of both cephalothin and benzylpenicillin. Fluids containing various amounts of the three antibiotics were assayed by this method with less than 8% error. Procedures that fail to measure each antibiotic individually may give larger errors.

Biological Assay↗

Disruption of bacterial cells by a synthetic zeolite.

The use of a synthetic zeolite (type 4A, Union Carbide Corp., Linde Div., New York, N.Y.) in a procedure for the preparation of pure cell wall fractions proved successful for many gram-positive, gram-negative, and acid-fast bacteria, as well as for some fungi. The technique, however, was found to be limited in effectiveness for Rhodospirillum rubrum, Gaffkya tetragena, and Sarcina lutea, and not applicable to preparations of heat killed microorganisms. The possible mechanisms of zeolite action, together with the effect of the disruptive procedure on the chemical composition of cell wall fragments, were investigated also.

Aluminum Silicates↗

Scanning electron and phase-contrast microscopy of bacterial spores.

The three-dimensional immages of free and intrasporangial spores produced by scanning electron microscopy show surface structures not visible by phase-contrast microscopy. Although fine surface detail is not elucidated by scanning electron microscopy, this technique does afford a definitive picture of the general shape of spores. Spores of Bacillus popilliae, B. lentimorbus, B. thuringiensis, B. alvei, B. cereus, and Sarcina ureae have varying patterns of surface ridge formation, whereas spores of B. larvae, B. subtilis, and B. licheniformis have relatively smooth surfaces.

Bacillus↗

Inhibition of the plaquing efficiency of T4r+ bacteriophage by subtilisin.

The mechanism by which the replicative cycle of T4r(+) phage is inhibited by certain nonhost bacterial systems was investigated. Some Bacillaceae, especially Bacillus subtilis, decreased the plaquing efficiency of this virus more than 95% within 24 hr of exposure. Sarcina lutea and Micrococcus sp. both failed to cause any significant change in the infectivity of T4r(+) phage. Preliminary investigations into the nature of the inhibitory substance(s) suggested that an extracellularly elicited protein was at least partially responsible for this effect. Further analysis has implicated subtilisin, an exoprotease from B. subtilis, as the cause of some, if not all, of the observed decrease in plaquing efficiency. Gel-filtration chromatography of control and treated (14)C-labeled T4r(+) phage showed a wide dispersal of phage-specific material of these particles after 24 hr of exposure to pure subtilisin or to expended medium exoprotease from B. subtilis. It was concluded that B. subtilis exoprotease is capable of chemically altering the structure of the phage capsid, thus causing a decrease in its plaquing efficiency.

Amylases↗

Tentative method for the qualitative detection and quantitative assessment of air contamination by drugs.

A method for detecting and measuring air contamination by drugs is described which uses an electrostatic bacterial air sampler, sprayers for micronizing drugs, and Mueller-Hinton medium seeded with a highly susceptible strain of Sarcina lutea. Three antibiotics (penicillin, tetracycline, aminosidine) and a sulfonamide (sulfapyrazine) were identified by pretreating portions of medium, showing no bacterial growth, with penicillinase or p-aminobenzoic acid solution and subsequently determining how both drug(-) susceptible and drug-resistant strains of Staphylococcus aureus were affected by this pretreatment. Quantitative determinations were also attempted by measuring the size of the inhibition zones.

Air↗

Survival of airborne bacteria in a high urban concentration of carbon monoxide.

Vegetative cells of Serratia marcescens 8UK, Sarcina lutea, and spores of Bacillus subtilus var. niger were held in aerosols, with and without an urban concentration of CO (85 muliters per liter or ppm), for up to 6 hr at 15 C and a relative humidity (RH) of approximately 0, 25, 50, 75, and 95%. It was found that CO enhanced the death rate of S. marcescens 8UK at least four- to sevenfold at low RH (ca. 1 to 25%), but protected the cells at high RH (ca. 90%). Death rates of S. lutea, with or without added CO, were comparatively low over the entire RH range. However, in the first hour, airborne S. lutea held in CO-containing air were more stable than those in air without added CO (i.e., CO protection). A marked increase in the death rate (up to 70-fold) occurred in the subsequent 5 hr within the RH range of approximately 0 to 75%. Statistical analysis indicated that aerosol decay rates of B. subtilus var. niger spores decreased significantly, when held in a CO-containing as compared to a non-CO-containing atmosphere, in the 0 to 85% RH range. Thus, the data presented indicate that CO in the urban environment may have a protective or lethal effect on airborne bacteria, dependent upon at least the microbial species, aerosol age, and relative humidity. A mechanism for CO death enhancement and protection of airborne S. marcescens 8UK is suggested to involve CO uncoupling of an energy-requiring death mechanism and an energy-requiring maintenance mechanism at high and low RH, respectively.

Aerosols↗

Stomach fermentation in East African Colobus monkeys in their natural state.

The microbial fermentation in the stomachs of two monkeys, Colobus polykomos, collected in Kenya, was studied. The gas accumulated within the stomach contained H(2) but no CH(4). Volatile fatty acid concentrations were high, but accumulated acid prevented determination of the fermentation rate in untreated, incubated stomach contents. Upon addition of bicarbonate, a very rapid rate could be demonstrated. Some D- and L-lactate were in the stomach contents. Starchy seeds or fruits rather than leaves appeared to have been consumed. Microscopically, the most prominent microorganisms seen were large, very refringent cocci, possibly Sarcina ventriculi, and various smaller cocci and rods. The 28 cultured strains of bacteria included 14 Staphylococcus, 2 Streptococcus, 10 Propionibacterium, and 2 Peptostreptococcus. The culture count constituted 10 to 20% of the direct count. No protozoa or cellulolytic bacteria were found.

Animals↗

Use of antibody to membrane adenosine triphosphatase in the study of bacterial relatioships.

An antiserum to Ca(2+)-activated adenosine triphosphatase from membranes of Micrococcus lysodeikticus cross-reacted in agar gels with membrane adenosine triphosphatases from other pigmented micrococci and related species. Species of Micrococcus and Sarcina showed different levels of inhibition of adenosine triphosphatase activities in heterologous reactions with antiserum. Inter- and intraspecific relationships based on the inhibition reaction were compared with an independent parameter, namely the quantitative and qualitative composition of the bacterial membrane phospholipids and fatty acids. The guanine plus cytosine contents in the deoxyribonucleic acid of the species studied correlated well with the serological cross-reactivity of adenosine triphosphatases from their membranes. The types of cross-bridges found in the peptidoglycans of these cocci were also compared with the other properties. The results suggest that an antiserum specific for a major membrane protein may be a reliable and most useful adjunct in studying bacterial serotaxonomy.

Adenosine Triphosphatases↗