Automatic diluter for bacteriological samples.

The described apparatus, carrying 190 tubes, allows automatic and aseptic dilution of liquid or suspended-solid samples. Serial 10-fold dilutions are programmable from 10(-1) to 10(-9) and are carried out in glass tubes with screw caps and split silicone septa. Dilution assays performed with strains of Escherichia coli and Bacillus stearothermophilus permitted efficient conditions for sterilization of the needle to be defined and showed that the automatic dilutions were as accurate and as reproducible as the most rigorous conventional dilutions.     

非洲钝缘蜱感染Q热立克次体敏感度的测定

Q热立克次体以10^-1—10^-12不同稀释度,用血腔注射法感染非洲钝缘蜱,感染35天,取蜱血淋巴液,分别用Gimenez染色法及免疫荧光技术检查,在10^-1—10^-5的稀释度,两种方法检出率基本相似。10^-6以下各组,免疫荧光尚可检出少数阳性标本,而Gimcnez法则无阳性,前者总阳性率为48.28%,后者为40.68%。感染的蜱悬液接种豚鼠,12组动物血清做Q热补体结合试验全部阳性。动物发病情况如潜伏期长短、发烧天数等似与蜱内立克次体的含量有关。     

Improved efficiency in determining the titer of the Autographa californica baculovirus nonoccluded virus.

An economical and time-efficient method for titering the Autographa californica multiple-embedded nuclear polyhedrosis virus (AcMNPV) by the endpoint method is described. The method uses an electronic pipetting device to perform dilutions in the same 60-well microplate as is used for the assay, thus eliminating the need for test tubes or vials for making dilutions. Additionally, since small volumes are used for the dilutions, a substantial savings in medium is achieved. The effect of using three different lepidopteran cell lines in this assay for AcMNPV is also described. This test revealed that one line (the Trichoplusia ni IPLB-TN-R2 line) is at least 1.5 logs more sensitive to AcMNPV when using occlusion body formation as the measure of infection. The titer was about 6- to 12-fold higher in the IPLB-TN-R2 cell line than the other two lines when plaque assay procedures were used. The titer of a recombinant baculovirus with a bacterial beta-galactosidase gene was also measured in the three cell lines using X-gal as an indicator and showed the IPLB-TN-R2 line to be fourfold more sensitive to this virus.     

A new microtitration method for the enumeration of contagious bovine pleuropneumonia (CBPP) vaccines.

A new method is proposed for the microtitration of CBPP vaccines by performing eight parallel endpoint two-fold dilutions. When performed on a 96-well plate, it gives a titre with a precision of +/- 0.2 log10. By comparison, the established method, performed in tubes, gives a precision of only +/- 0.6 log10. The adaptation to microtitre plates allows much more economical culture of larger numbers of samples. Statistical analysis permits determination of vial-to-vial homogeneity of a batch and calculation of a titre with a precision of +/- 0.09 log10. The new method may be applied for the titration of mycoplasmas.     

THE ESTIMATION OF NUMBERS OF BACTERIA BY TENFOLD DILUTION SERIES

Results obtained by dilution series are sometimes so unlikely that doubt is cast on the validity of the method. Criteria for the rejection of results are discussed and a Table is given of acceptable results for tests with five tubes for each of three tenfold dilutions. Methods of estimating the concentration of bacteria for other numbers of tubes or of dilutions are suggested. The inherent lack of precision of the dilution series method is stressed.     

Inorganic pyrophosphate as an energy source for ruminal bacteria

The ability of the predominant ruminal flora to utilize inorganic pyrophosphate as an energy source was examined. Rumen contents from steers fed diets containing pyrophosphate from 0%–0.13% of the diet dry matter were serially diluted and inoculated into (a) basal medium, no carbohydrates (BM); (b) basal medium plus pyrophosphate (0.05%) (BP); (c) basal medium plus carbohydrates (CM); and (d) basal medium plus carbohydrates plus pyrophosphate (0.05%) (CP). Growth was measured as increase in optical density. Addition of pyrophosphate to the CM medium reduced the maximum optical density by 36% and 98% in tubes inoculated with 10^–1 and 10^–3 dilutions of rumen contents, repectively. No growth occurred in pyrophosphate-containing media at dilutions higher than 10^–3. In vitro enrichment of bacteria growing on pyrophosphate did not increase the growth yield. These result indicate that the predominant ruminal bacteria do not utilize pyrophosphate as an energy source.     

Isolation and characterization of novel iron-oxidizing bacteria that grow at circumneutral pH.

A gel-stabilized gradient method that employed opposing gradients of Fe2+ and O2 was used to isolate and characterize two new Fe-oxidizing bacteria from a neutral pH, Fe(2+)-containing groundwater in Michigan. Two separate enrichment cultures were obtained, and in each the cells grew in a distinct, rust-colored band in the gel at the oxic-anoxic interface. The cells were tightly associated with the ferric hydroxides. Repeated serial dilutions of both enrichments resulted in the isolation of two axenic strains, ES-1 and ES-2. The cultures were judged pure based on (i) growth from single colonies in tubes at dilutions of 10(-7) (ES-2) (ES-2) and 10(-8) (ES-1); (ii) uniform cell morphologies, i.e., ES-1 was a motile long thin, bent, or S-shaped rod and ES-2 was a shorter curved rod; and (iii) no growth on a heterotrophic medium. Strain ES-1 grew to a density of 10(8) cells/ml on FeS with a doubling time of 8 h. Strain ES-2 grew to a density of 5 x 10(7) cells/ml with a doubling time of 12.5 h. Both strains also grew on FeCO3. Neither strain grew without Fe2+, nor did they grow with glucose, pyruvate, acetate, Mn, or H2S as an electron donor. Studies with an oxygen microelectrode revealed that both strains grew at the oxic-anoxic interface of the gradients and tracked the O2 minima when subjected to higher O2 concentrations, suggesting they are microaerobes. Phylogenetically the two strains formed a novel lineage within the gamma Proteobacteria. They were very closely related to each other and were equally closely related to PVB OTU 1, a phylotype obtained from an iron-rich hydrothermal vent system at the Loihi Seamount in the Pacific Ocean, and SPB OTU 1, a phylotype obtained from permafrost soil in Siberia. Their closest cultivated relative was Stenotrophomonas maltophilia. In total, this evidence suggests ES-1 and ES-2 are members of a previously untapped group of putatively lithotrophic, unicellular iron-oxidizing bacteria.     

Problems in the measurement of zinc using heparin as an anticoagulant

Although several reports imply that anticoagulants and preservatives contain zinc, the quantity of zinc in heparin, if any, has not been documented. Zinc concentration was determined by flame atomic absorption spectroscopy in varying dilutions of multiple commercially obtained samples of purified sodium heparin N = 15 (microgram Zn/1000 Units heparin). Rubber stoppers of sterile heparin vials and of blood evacuation tubes were incubated in pre-analyzed water or saline on a mechanical shaker with fluid aliquots obtained up to 27 hours and analyzed for zinc content (microgram Zn/0.1 ml). Heparin, with contact or without contact with rubber stoppers, recorded identical zinc concentrations. Zinc concentrations varied from 0.222 +/- 0.01 (mean +/- SE) to 3.49 +/- 0.005 microgram Zn/1000 Units heparin. Leaching of zinc from rubber stoppers of vacutainer tubes (N = 9) was noted only with those containing known chelators of zinc. These results indicate that zinc is present in certain lots of sodium heparin and that caution must be exercised when reporting zinc concentrations of blood samples that contain sodium heparin as the anticoagulant.     

Nitrogen removal and recycling by Scenedesmus obliquus in semicontinuous cultures using artificial wastewater and a simulated light and temperature cycle

Semicontinuous cultures of Scenedesmus obliquus in artificial wastewater were maintained with 30% and 40% daily dilutions and under a 14:10 h light-dark cycle, with temperatures of 25.5 and 17 degrees C during light and dark hours. Under this regime, the production of organic biomass was 39.3 and 25.2 mg l(-1)d(-1) for the 30% and 40% dilutions, and 24.9 and 16.7 mg l(-1)d(-1) of single-cell proteins. Most of the nitrogen removal took place during the light hours, with daily totals of 9.27 and 8.45 mg l(-1) for the 30% and 40% dilutions. With the former, 43.7% of the nitrogen removed was recycled by the microalgae into proteins and other organic nitrogen cell contents, but this efficiency decreased to 26.4% when the dilutions were raised to 40%.     

Ecological factors determining establishment of cellulolytic bacteria and protozoa in the rumens of meroxenic lambs

Ecological factors that control the establishment of cellulolytic bacteria and ciliate protozoa in the lamb rumen were studied in meroxenic animals. Axenic lambs received dilutions of rumen liquor from either conventional lambs and sheep (pool A) or meroxenic lambs (pool B). The total number of bacteria established in the rumen was between 10(9) and 5 x 10(10) g-1. In lambs inoculated with dilutions (10(-6), 10(-7), 10(-8)) of pool A, cellulolytic bacteria did not become established. However, subsequent inoculation with Bacteroides succinogenes, resulted in colonization in lambs that had received 10(-6) and 10(-7) dilutions of pool A. However, B. succinogenes became established in only one of three lambs that received the 10(-8) dilution. Similar results were obtained for the protozoan Entodinium sp. With pool B, lambs were inoculated earlier and cellulolytic bacteria were established directly from the 10(-6) and 10(-7) inocula. Polyplastron multivesiculatum establishment occurred readily when inoculated into the lambs which had received the 10(-6) dilution of pool B. Results obtained in this study suggest that establishment of cellulolytic bacteria and protozoa requires an abundant and complex flora and is favoured by early animal inoculation.     

Preliminary study on the cryopreservation of tropical bagrid catfish (Mystus nemurus) spermatozoa; the effect of extender and cryoprotectant on the motility after short-term storage

The effects of different extenders, and cryoprotectants on the motility of tropical bagrid catfish (Mystus nemurus) spermatozoa were evaluated after short-term storage. Three extenders, physiological saline, Ringer or saline at three levels of sperm to extender dilutions (1:20, 1:30, or 1:40) and four cryoprotectants (DMSO, ethanol, glycerol or methanol) at three concentrations (5, 10, or 15%) were examined in two separate experiments. In the first experiment, milt was suspended in the respective extender at the three milt to extender dilution ratios in two sets of tubes. Extended milt in the first set of tubes was stored at -4 degrees C, and motility assessed after 24h, while the second set was kept at 23 degrees C and sperm motility was assessed immediately and at 30-min intervals thereafter. Ringer retained sperm motility better than the other extenders at all dilution levels at temperatures of 23 and -4 degrees C respectively. At 23 degrees C, the sperm motility was almost completely lost after 150 min except for those in Ringer at 1:20 dilution level which still had a motility of 18% (compared to those kept at -4 degrees C for 24, which had motility from 39 to 71%, regardless of extender). In the second experiment, various cryoprotectants were added to solutions of milt (that was diluted in Ringer at 1:20 ratio and cryopreserved in liquid nitrogen for 15 days). Sperm cryopreserved in 10% methanol had the highest motility (58%) compared with those in the other cryoprotectants at all concentrations.     

Molecular basis of the biological activity of substances and homeopathic remedies at zero concentrations

While getting ultra los dosages in serial dilutions interactions between a substance and impurities of a solvent occur parallel with diminishing of concentrations. The concentration of impurities in high dilutions prevails the calculated concentration of substrate. Experiments show that different concentrations of compounds of impurities in a solvent correspond to different substances in different dilutions, including < 10(-24) mol/l. While dissolving three components--a solvent, impurities and a substance--participate in two processes: changing of concentration and changing of composition. Changed chemical composition of impurities of a solvent reflects specificity of physicochemical properties of a dissolving substance and is the molecular basis for biological activity of solutions with a concentration lower than 10(-24) mol/l.     

Wine as a digestive aid: comparative antimicrobial effects of bismuth salicylate and red and white wine.

OBJECTIVE--To test whether red and white wines are as potent as bismuth salicylate against the bacteria responsible for traveller''s diarrhoea to try to explain wine''s legendary reputation as a digestive aid. DESIGN--Red and white wine, bismuth salicylate, two solutions containing ethanol (diluted absolute ethanol and tequila), and sterilised water were tested against suspensions of salmonella, shigella, and Escherichia coli to determine relative antibacterial activity. Suspensions of 10(7) colony forming units of shigella, salmonella, and E coli were added to the test solutions and plated on standard nutrient agar at 0, 10, 20, 30, 60, and 120 minutes and 24 hours. Dilutions of wine and bismuth salicylate were then tested with E coli as the test bacterium, and the experiment repeated. MAIN OUTCOME MEASURES--Exposure times necessary for eradication of organisms for the different solutions; decreases in colony counts at the different exposure times for dilutions of wine and bismuth salicylates. RESULTS--Undiluted wine and bismuth salicylate were both effective in reducing the number of viable organisms (by 10(5)-10(6) colony forming units) after 20-30 minutes. Dilutions of wine were much more effective in decreasing colony counts than were similar dilutions of bismuth salicylate. CONCLUSION--The antibacterial property of wine is largely responsible for wine''s reputation as a digestive aid.     

Possible use of N2O in MPN tubes for enumeration of denitrifiers

N₂O when used in most probable number tubes as the only electron acceptor was not consumed at dilutions, down to 10^–3 per gram. Additions of fresh carbon source and NO₃ after growth did not stimulate N₂O reduction. Since pure cultures of denitrifiers grew well under the same conditions this result was unexpected, the explanation has not been found. This approach, if successful, has the advantage that only denitrifiers can reduce N₂O to N₂. Further work in designing a method to use N₂O as an electron acceptor in MPN tubes is suggested.     

Growth stimulation effects of smoke-water and vermicompost leachate on greenhouse grown-tissue-cultured ‘Williams’ bananas

A common problem often experienced by both small and large scale banana farmers is the reduction in quality and yield of bananas due to soil moisture deficits and declining soil fertility. To overcome this, mechanisms of improving yield without compromising environmental integrity or public health are being embraced. Globally, researchers have continued to evaluate the competence of environmental-friendly organic fertilizers and smoke solutions to improve the growth, yield and quality of various crops. In the present study, the effect of smoke-water (1:1,000; 1:500 and 1:250 dilutions) and vermicompost leachate (1:20; 1:10 and 1:5 dilutions) treatments on the growth of greenhouse grown bananas was evaluated. Soil drenching (smoke-water and vermicompost leachate) and foliar application (smoke-water) were tested on 1-month-old tissue-cultured banana seedlings under greenhouse conditions. After 3 months, growth parameters such as number and length of roots and shoots; number of leaves and leaf area as well as their fresh and dry weights were analyzed. Soil drenching with smoke-water significantly increased the root length (1:1,000 and 1:500 dilutions) as well as fresh and dry weight (1:1,000; 1:500 and 1:250 dilutions) when compared to foliar application. Vermicompost leachate (1:10 and 1:5 dilutions) significantly enhanced the shoot length, root length, leaf area and dry weights. Vermicompost leachate (1:20; 1:10 and 1:5 dilutions) also significantly increased the number of off-shoots. The positive effect on rooting is beneficial for acclimatization and establishment of tissue-cultured banana plantlets in nurseries and subsequent transfer to the field. Moreover, the high cost of inorganic fertilizers as well as environmental safety concerns makes the use of smoke-water and vermicompost leachate potential alternatives to improving the growth and cultivation of bananas.     

Estimation of the thermal coefficient in the decline of a bacterial population under heat stress.

The "most probable number" (MPN) technique for estimating numbers of bacteria in suspensions is well known and has been used for decades by microbiologists, food researchers, and other laboratory scientists. A related procedure, involving an infinite number of serial dilutions at each of times ti, i = 0,1,...,n, is considered, and the joint probability law of the index numbers of the last tubes showing growth at these times is derived through use of probability-generating functions. Maximum likelihood estimates of the initial density lambda and the thermal death rate mu are computed. Another estimate, mu, of mu is given, using a simple weighting scheme. Finally, the thermal death time (TDT) is estimated by D = (1n 10)/mu.     

Immunization with viruslike particles induces long-term protection of rabbits against challenge with cottontail rabbit papillomavirus.

Rabbits were immunized with recombinant baculovirus-produced virus-like particles (VLPs) of cottontail rabbit papillomavirus (CRPV) to determine whether these antigens could induce long-term protection against experimental challenge with CRPV. Infectious CRPV and human papillomavirus type 11 L1 VLPs were used as positive and negative control immunogens, respectively. Three groups of immunized animals were challenged with 10-fold serial dilutions of infectious CRPV at 2 weeks, 6 months, and 12 months after immunizations. Antibody titers in serum reached 1:10,000 immediately after the final booster immunization and then decayed to 1:150 at 6 months and 1:100 at 12 months in unchallenged rabbits. Serum neutralization titers followed similar kinetics. Papillomas grew on control-immunized rabbits at sites challenged with 10(-1) (100% of sites), 10(-2) (96% of sites), 10(-3) (63% of sites), and 10(-4) (13% of sites) dilutions of virus. At 2 weeks after CRPV L1 VLP immunizations, the rabbits were completely protected against virus challenge. At both 6 and 12 months after CRPV L1 VLP immunizations, strong protection was also observed. In the last two groups, three of seven rabbits were completely protected and only 4 of 14 or 29% of sites challenged with 10(-1 dilution of virus grew papillomas. Papillomas growing at these four sites were also reduced in size (3.5 +/- 0.7 mm) at 50 days postchallenge compared with sites challenged with 10(-1) dilution on control-immunized rabbits (13.2 +/- 4.2 mm). The results demonstrate that strong and long-lasting protection against experimental challenge with papillomaviruses can be achieved with VLP immunogens.     

A mutant human T-cell line producing immunosuppressive factor(s)

6T-CEM-20, a subclone of a 6-thioguanine-resistant mutant derived from the human-T-cell line CEM, secreted into the medium, a high titered immunosuppressive factor specific for T cells. The cell-free supernatant was very potent in suppressing, via a noncytotoxic mechanism, mitogen-activated T-cell proliferation, cytotoxic T-cell functions, and pokeweed mitogen (PWM)-stimulated plaque-forming cells (PFC). Lower dilutions of the supernatant (10(-1)-10(-2] abrogated T-cell functions within 24 hr whereas higher dilutions (10(-3)-10(-7] required a culture period of up to 4 days with lymphocytes to arrest T-cell activities. The suppressive activity was most pronounced when the factor was added in the early part of the culture period. The factor was sensitive to heat treatment and both low and high pH (most stable at physiological pH). Preliminary purification with column chromatography indicates that the active moiety was contained in the high-molecular-weight fraction (MW greater than 200,000). Data from coculture experiments suggested that T lymphocytes, which were exposed for 5-12 hr to the active supernatant or the partially purified material, suppressed allogeneic T-cell proliferation in a dose-related manner. This suppressor factor which we called suppressor-activating factor (SAF) might have activated a suppressor population or induced the production of a suppressor factor which in turn mediated the observed suppression. Both the molecular structure and the detailed mechanism of action are under investigation.     

Effects of aurothiomalate and gold(III) complexes on spontaneous motility of isolated human oviduct

Organic gold complexes have different biological activity, depending on their potential for interactions with key functional molecules.The aim of this study was to investigate potential of several newly synthesized organic gold complexes to influence spontaneous motility of the Fallopian tubes.The effects of [Au(bipy)Cl(2)](+) (dichloride(2,2'-bipyridyl)aurate(III)-ion), aurothiomalate, [Au(DMSO)(2)Cl(2)]Cl and DMSO on spontaneous motility of Fallopian tubes were tested on the isolated tube segments in vitro. Aurothiomalate (from 2.9?×?10(-9) to 4.9?×?10(-4)?M/l), [Au(bipy)Cl(2)]Cl (from 3.3?×?10(-9) to 4.2?×?10(-5)?M/l) and DMSO (from 1.9?×?10(-8) to 1.0?×?10(-5)?M/l) did not affect spontaneous contractions of the isolated Fallopian tube ampulla, while [Au(DMSO)(2)Cl(2)]Cl (from 2.9?×?10(-9) to 4.2?×?10(-5)?M/l) showed concentration-dependent increase (stimulation) of spontaneous contractions of the isolated Fallopian tube isthmus, and remained without effect on the isolated ampulla.The drugs designed as organic gold complexes with weaker bonds between the gold itself and organic part of a molecule could adversely affect motility of the Fallopian tubes, and theoretically fertility of women taking such drugs in their reproductive age.