Isolation of previously uncultured rumen bacteria by dilution to extinction using a new liquid culture medium

A new anaerobic medium that mimics the salts composition of rumen fluid was used in conjunction with a dilution method of liquid culture to isolate fermentative bacteria from the rumen of a grass-fed sheep. The aim was to inoculate a large number of culture tubes each with a mean of < 1 culturable cell, which should maximize the number of cultures that develop from a single bacterium. This minimizes the effort that has to be put into purifying the resultant cultures. Of 1000 tubes, 139 were growth positive. Of the 93 that were able to be subcultured, 54 (58%) appeared to be pure cultures. The phylogenetic placements of these 54 cultures, together with another 6 cultures obtained from a preliminary study, were determined. Using a criterion of < 93% 16S rRNA gene sequence identity to a previously named bacterium as a proxy for defining a new genus, 27 (45%) of the 60 cultures belonged to 14 potentially novel genera. Many of these had 16S rRNA genes that shared > 97% sequence identity to genes of uncultured bacteria detected in various gastrointestinal environments. This strategy has therefore allowed us to cultivate many novel rumen bacteria, opening the way to overcoming the lack of cultures of many of the groups detected using cultivation-independent methods.     

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.     

Basic principles of quantitative PCR

The polymerase chain reaction (PCR) is an extremely sensitive method owing to the repetitive multiplication of template molecules. This property is a drawback for quantitative measurements because small differences in the multiplication factor lead to large differences in the amount of product. Two methods can be used to solve the problem of quantification: kinetic methods based on the determination or comparison of the amplification factor; and coamplification methods, which compare the amount of product to that of a simultaneously amplified standard template. An overview of the theoretical background of both methods is presented. For selection of a suitable method, both theoretical and practical considerations are important. Kinetic methods are the most convenient if PCR can be performed without opening the tubes, as in some apparatus using fluorescence detection. Coamplification methods can be done without expensive equipment but requires the parallel running of several PCR tubes. When the number of initial template molecules is close to one, as in the limiting dilution technique, statistical considerations become important.     

A simplified method for estimating ammonium oxidising bacteria

Summary Most probable number of ammonium oxidising bacteria in soil has been estimated without the need to carry out chemical tests to determine the production of nitrite or nitrate in dilution tubes. The medium used contained phenol red which changes colour from pink to yellow as the oxidation of ammonium to nitrate decreases the pH. This makes it possible to determine growth visually. An incubation period of 8 weeks at 25°C was sufficient to obtain maximum estimates of ammonium oxidising bacteria in the soils tested.     

A technique for using hair tubes beneath the snowpack to detect winter-active small mammals in the subnivean space

The study of winter-active small mammals beneath the snowpack has proved challenging for researchers because of the relative inaccessibility. We present a technique using hair tubes that permits the detection of small mammals active in the subnivean space. Hair tubes are cylindrical or funnel-shaped structures containing suitable bait and an adhesive surface that harvests hairs from small mammals as they attempt to reach the bait. Hair tubes eliminate many of the difficulties often associated with live trapping and permit the expansion of systematic sampling to larger scales than allowed by conventional live-trapping methods. The technique was used successfully to detect five small mammal species in the subnivean space in Kosciuszko National Park (KNP) in southeastern Australia. These included the common bush-rat, Rattus fuscipes; the dusky and agile antechinus, Antechinus swainsonii and A. agilis; the broad-toothed rat, Mastacomys fuscus; and the mountain pygmy possum, Burramys parvus. Although hair tubes have a number of limitations, such as not providing a measure of abundance or allowing the identification of individual animals, we believe that these limitations are balanced by the fact that the technique can be used at any spatial scale. Hair tubes are particularly suited to studies of animal distribution at the landscape-scale, because many hair tubes can be deployed and dispersed over large areas, and monitored on a regular basis by a small team of researchers. The technique also makes use of readily available, low-cost materials and could be easily adapted to a range of conditions and different target species.     

Estimation of microbial densities from dilution count experiments

Although dilution counts have been widely used in quantitative microbiology, their interpretation has always been widely discussed both in microbiology and in applied statistics. Maximum-likelihood (most-probable-number) methods hae generally been used to estimate densities from dilution experiments. It has not been widely recognized that these methods are intrinsically and statistically biased at the sample sizes used in microbiology. This paper presents an analysis of proposed method for correction of such biases, and the method was found to be robust for moderate deviations from Poisson behavior. For analyses at greater variance with the Poisson assumptions, the use of the Spearman-Karber method is analyzed and shown to yield an estimate of density of lesser bias than that produced by the most-probable-number method. Revised methods of constructing confidence limits proposed by Loyer and Hamilton (M.W. Loyer and M.A. Hamilton, Biometrics 40:907-916, 1984) are also discussed, and charts for the three- and four-decimal dilution series with five tubes per dilution are presented.     

Investigations on Some Attributes of the Likelihood Function for MPN Determination

The paper investigates the likelihood function to determine the MPN (Most Probable Number as a measure of concentration in the virology), dependent on several parameters. Taking only ‘probable’ result schemes into the MPN-tables they would become much shorter. There are suggested two conditions for the selection of the result schemes, given and compared by some tables together with a mathematical foundation of the number of the selected schemes. Some tables and a theorem about success probabilities of the result schemes point out their changes under different conditions (number of tubes, number of dilutions, dilution ratio), and make it possible to estimate success probabilities of result schemes not selected by the more restricted condition.     

Estimation of microbial densities from dilution count experiments.

Although dilution counts have been widely used in quantitative microbiology, their interpretation has always been widely discussed both in microbiology and in applied statistics. Maximum-likelihood (most-probable-number) methods hae generally been used to estimate densities from dilution experiments. It has not been widely recognized that these methods are intrinsically and statistically biased at the sample sizes used in microbiology. This paper presents an analysis of proposed method for correction of such biases, and the method was found to be robust for moderate deviations from Poisson behavior. For analyses at greater variance with the Poisson assumptions, the use of the Spearman-Karber method is analyzed and shown to yield an estimate of density of lesser bias than that produced by the most-probable-number method. Revised methods of constructing confidence limits proposed by Loyer and Hamilton (M.W. Loyer and M.A. Hamilton, Biometrics 40:907-916, 1984) are also discussed, and charts for the three- and four-decimal dilution series with five tubes per dilution are presented.     

真水狼蛛纺器的扫描电镜观察

通过室内饲养,获得了不同龄期的真水狼蛛。对其纺器扫描电镜观察表明：真水狼蛛的纺器表面分布许多纺管,纺管的数目随着龄期的增大面增加。１龄幼蛛纺器分化没有完成。因而不能纺丝。雌蛛和雄蛛在纺器的排列位置、形态、纺管类型和纺管数目上都存在较大差异。纺器的形态、结构与纺器纺丝、结网的功能相适宜。     

最大或然数法在光合细菌计数中的应用及效果研究

1引言光合细菌(photosynthetic bacteria,PSB)是能进行不放氧光合作用的一大类细菌的总称,属水圈微生物,广泛分布于地球生物圈,无论江、河、湖、海,水田、旱地都有存在[3].光合细菌在水体自净、调节微生态平衡、促进动植物生长、增加产量和提高产品质量、防病、固氮等方面具有重要作用[3,22].近年来,光合细菌菌剂在水产养殖、家畜养殖、污水处理及植物生产上的应用日益广泛[3,13,19,27,28,31],相继推出多种产品,包括单菌菌剂、复合菌剂,剂型上有液体菌剂、浓缩菌剂和固体菌剂(粉剂)等[20].为保证产品质量和应用效果,有必要对光合细菌菌剂…     

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.     

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.     

A modified capillary assay for chemotaxis

Summary A modification is described of the capillary assay for chemotaxis. It employs a 96-well dilution plate and its cover. Capillary tubes are inserted through the cover and are supported by small rubber collars. The method is faster and less tedious and gives more precise results than earlier methods.     

ANTIGENS IN EGGS AND DEVELOPMENTAL STAGES OF THE SEA URCHIN : I. Immunological and Physicochemical Properties

A number of antigens in unfertilized eggs and embryos of the sea urchin Paracentrotus lividus were characterized with respect to both immunological and physicochemical properties. Experiments involved single diffusion in agar (Oudin technique) combined with mutual dilution, serial dilution, and heating of antigenic extracts, as well as immunoelectrophoresis with normal and heated extracts and agar electrophoresis followed by staining of the antigenic spots with protein specific dyes. The gradual transition in migration rates of bands of precipitates in Oudin tubes following mutual dilution of either extracts or antisera allowed the identification of 6 immunologically identical antigens in eggs and embryonic stages. Similarities with respect to diffusion coefficients, sensitivity to heat, electrophoretic mobility, and reaction to protein specific dyes indicated that the antigens in extracts of eggs and various developmental stages also had certain physicochemical properties in common. Such knowledge is of importance for an understanding of antigenic changes occurring during ontogenesis.     

Effect of pre-analytical handling on haematological variables in minipigs

Pre-analytical handling may be an important determinant of haematological variables, if analysis is delayed. We investigated the effect of anticoagulants, i.e. tripotassium ethylenediamine-tetraacetic acid (EDTA) and citric acid, theophylline, adenosine, dipyridamole (CTAD), storage time (0.5, 1.5, 3.5, 5.5, 7.5, 25.5 and 27.5 h after blood sampling), and storage temperature (5 degrees C and 20 degrees C) on the variation in haemoglobin (HGB), red blood cell count (RBC), haematocrit (HCT), white blood cell count (WBC), and platelet count (PLT) in minipigs. Medians of HGB, RBC, HCT, WBC and PLT were significantly higher in EDTA tubes than in CTAD tubes due to the dilution effect of the anticoagulant. We found a minor significant increase in HCT after 25.5 h in blood stored at 20 degrees C, and at the same time a minor significant increase in WBC in EDTA tubes stored at 20 degrees C. We found a significant decrease in PLT in blood stored at 5 degrees C, especially in EDTA tubes. Minor variations were also observed in HGB and RBC. Our results indicate that PLT should only be measured in tubes placed at room temperature. If HCT or WBC analyses are to be performed on the day after blood sampling, the samples must be stored in a refrigerator until analysis. Our studies underline that time delay before analysis of haematological variables can cause increased variation, and should therefore be limited as far as possible in order to reduce the number of animals needed to make reliable conclusions.     

Measurement of Larval Activity in the Drosophila Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals’ positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.     

Modification of the standard most-probable-number procedure for fecal coliform bacteria in seawater and shellfish.

To determine whether or not presumptive tubes which take 48 h to become positive need to be transferred to EC medium during the fecal coliform multitube most-probable-number procedure, 572 seawater and shellfish samples were considered. When the 24-hour positive tubes alone were transferred to EC, it appeared that incorrect dilution data would be rare.     

Fast solid support detection of PCR amplified viral DNA sequences using radioiodinated or hapten labelled primers.

Oligonucleotides with novel modifications have been synthesized and incorporated into enzymatically amplified DNA sequences. They allow the fast detection of viral DNA sequences after two rounds of amplification. The hybrids formed are immobilized by affinity on coated tubes and detected by direct beta (32P) or gamma (125I) counting or by colorimetric revelation. The effect of a dilution step between the two amplifications is studied to obtain optimal sensitivity and specificity. This test is used to detect Human Papillomavirus types 16 and 18 in cells and biopsies and for the specific colorimetric detection of HIV1 in extracted DNA.     

Estimation of the Number of Polyhydroxyalkanoate (PHA)-Degraders in Soil and Isolation of Degraders Based on the Method of Most Probable Number (MPN) Using PHA-Film

A new method to estimate the number of polyhydroxyalkanoates (PHA)-degraders in soil and to isolate degraders, called the film-MPN method, is proposed. The incubation time was measured by the first order reaction (FOR) model. This method was used to estimate numbers of poly (3-hydroxybutyrate-co-3-hydroxyvalerate)[P(3HB-co-3HV)]- and poly(3-hydroxyvalerate-co-4-hydroxybutyrate)[P(3HB-co-4HB)]-degraders in garden soil (4.30 × 10⁵ and 2.15 × 10⁵ aerobic degraders per gram of dry soil, respectively). The number of P(3HB-co-3HV)-degraders in paddy field soil was 5.06 × 10⁵ aerobic degraders per gram dry soil. Also, several P(3HB-co-3HV)-degraders were isolated directly from positive-growth tubes of high dilution.     

Counting human neural stem cells

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as plating cells for immunocytochemistry or for cell transfections. This protocol describes a straightforward and fast method for differentiating between live and dead cells and quantifying the cell concentration and total cell number using a hemacytometer. This procedure first requires detaching cells from a growth surface and resuspending them in media. Next, the cells are diluted in a solution of Trypan blue (ideally to a concentration that will give 20-50 cells per quadrant) and placed in the hemacytometer. Finally, averaging the counts of viable cells in several randomly selected quadrants, dividing the average by the volume of one 1 mm(2) quadrant (0.1 microl) and multiplying by the dilution factor gives the number of cells per l. Multiplying this cell concentration by the total volume in microl gives the total cell number. This protocol describes counting human neural stem/precursor cells (hNSPCs), but can also be used for many other cell types.