Application of a non-hazardous vital dye for cell counting with automated cell counters

Recent advances in automated cell counters enable us to count cells more easily with consistency. However, the wide use of the traditional vital dye trypan blue (TB) raises environmental and health concerns due to its potential teratogenic effects. To avoid this chemical hazard, it is of importance to introduce an alternative non-hazardous vital dye that is compatible with automated cell counters. Erythrosin B (EB) is a vital dye that is impermeable to biological membranes and is used as a food additive. Similarly to TB, EB stains only nonviable cells with disintegrated membranes. However, EB is less popular than TB and is seldom used with automated cell counters. We found that cell counting accuracy with EB was comparable to that with TB. EB was found to be an effective dye for accurate counting of cells with different viabilities across three different automated cell counters. In contrast to TB, EB was less toxic to cultured HL-60 cells during the cell counting process. These results indicate that replacing TB with EB for use with automated cell counters will significantly reduce the hazardous risk while producing comparable results.     

Rapid detection and counting of single bacteria in a wide field using a photon-counting TV camera

Using Escherichia coli as a model bacterium, we tested a photon-counting method for enumeration of bacteria. This method is based on the principle that microscopic sized luminous particles in a wide field can be directly detected and counted using a photon-counting TV camera without the use of a microscope. E. coli cells were labeled with peroxidase and luminescence induced by adding a luminol-based reaction mixture. The number of luminous spots in the TV images was in good agreement with the number of bacterial colonies grown from labeled cells. The results show that our method provides a rapid and easy microbial counting system for such purposes as clinical diagnosis, microbial analysis in food, and environmental assessment.     

Summary of the National Institute of Standards and Technology and US Food And Drug Administration cell counting workshop: Sharing practices in cell counting measurements

The emergence of cell-based therapeutics has increased the need for high-quality, robust and validated measurements for cell characterization. Cell count, being one of the most fundamental measures for cell-based therapeutics, now requires increased levels of measurement confidence. The National Institute of Standards and Technology (NIST) and the US Food and Drug Administration (FDA) jointly hosted a workshop focused on cell counting in April 2017 entitled “NIST-FDA Cell Counting Workshop: Sharing Practices in Cell Counting Measurements.” The focus of the workshop was on approaches for selecting, designing and validating cell counting methods and overcoming gaps in obtaining sufficient measurement assurance for cell counting. Key workshop discussion points, representing approximately 50 subject matter experts from industry, academia and government agencies, are summarized here. A key conclusion is the need to design the most appropriate cell counting method, including control/measurement assurance strategies, for a specific counting purposes. There remains a need for documentary standards for streamlining the process to develop, qualify and validate cell counting measurements as well as community-driven efforts to develop new or improved biological and non-biological reference materials.     

Preparing nuclei from cells in monolayer cultures suitable for counting and for following synchronized cells through the cell cycle

A procedure is described for preparing nuclei from cells in monolayer culture so that they may be counted using an electronic particle counter. It takes only 10 to 15 min, and consists of swelling the cells in hypotonic buffer and then lysing them with the quaternary ammonium salt, ethylhexadecyldimethylammonium bromide. The cells are completely lysed, yielding a suspension of clean single nuclei which is stable, free of debris, and easily counted. The method was developed for a cell line of epithelial origin (MCF-7), which is often difficult to trypsinize to single cells. It works equally well at all cell densities up to and beyond confluence, and has been used with a variety of cells in culture, including 3T3 cells, bovine macrophages, rat mammary epithelial cells, mouse mammary tumor cell lines, and human fibroblasts. The size of the nuclei produced by this procedure is related to their DNA content, and the method is thus suitable for following cultures of synchronized cells through the cell cycle, and for performing differential counts of cells with substantial differences in DNA content.     

The cleavage pattern of the axolotl egg studied by cinematography and cell counting

Summary The temporal pattern of cleavage in the egg of the axolotl,Ambystoma mexicanum, was studied 1. by time-lapse microcinematography, and 2. by counting the total number of blastomeres dissociated at successive stages.Eggs were filmed from the one-cell stage till the early gastrula either (A) simultaneously from above and below with a double-camera assembly, or (B) from the side with a single camera.The animal blastomeres divide synchronously from the 2nd up to and including the 10th cleavage. The cycle length is roughly constant from the 3rd till the 10th cleavage. The cycle from the 2nd to the 3rd cleavage is slightly longer, while that from the 1st to the 2nd cleavage is about 20% longer. After the 10th cleavage the synchrony of divisions is lost owing to variable lengthening of cell cycles in individual blastomeres. Gastrulation starts around the onset of the 15th cleavage in the animal blastomeres.The analysis of films taken in side view reveals seven recurring cleavage waves, from the 5th till the 11th cleavage. Cells in the animal, equatorial and vegetative regions in sequence repeatedly pass through the three successive phases of the cleavage cycle—rounding-up, division, and relaxation—but with a shift in phase. The start of the 10th cleavage division of the slowest vegetative cells more or less coincides with that of the 11th division of the animal cells; from then on the cleavage waves become increasingly obscured.Morulae and blastulae were dissociated by placing them in 1/15 M phosphate buffer (pH 7.8) for the duration of 2–3 cleavage cycles and then removing the vitelline membrane. In this solution cell divisions continued without disturbance of the temporal cleavage pattern. The dissociated cells were fixed either just prior to the onset of the next cleavage (up to the 10th cleavage) or at those times when cleavageswould have been expected, had there been no lenthening of cleavage cycles (beyond the 10th cleavage). The total cell number was counted, dividing cells being scored as two.Prior to the 11th cleavage the total cell number increased exponentially. Beyond the 10th cleavage the rate of increase was considerably lower. At the time when gastrulation would have started if the egg had not been dissociated, the total cell counts were 13,000–15,000, whereas the number anticipated without lengthening of cleavage cycles would be of the order of 130,000 (2¹⁷).The application of Balfour's rule to amphibian eggs is criticized.     

New technologies for automated cell counting based on optical image analysis `The Cellscreen'

A prototype of a newly developed apparatus for measuring cell growth characteristics of suspension cells in micro titre plates over a period of time was examined. Fully automated non-invasive cell counts in small volume cultivation vessels, e.g. 96 well plates, were performed with the Cellscreen system by Innovatis AG, Germany. The system automatically generates microscopic images of suspension cells which had sedimented on the base of the well plate. The total cell number and cell geometry was analysed without staining or sampling using the Cedex image recognition technology. Thus, time course studies of cell growth with the identical culture became possible. Basic parameters like the measurement range, the minimum number of images which were required for statistically reliable results, as well as the influence of the measurement itself and the effect of evaporation in 96 well plates on cell proliferation were determined. A comparison with standard methods including the influence of the cultured volume per well (25 l to 200 l) on cell growth was performed. Furthermore, the toxic substances ammonia, lactate and butyrate were used to show that the Cellscreen system is able to detect even the slightest changes in the specific growth rate.     

霉菌总数测定培养基的改进研究

在霉菌检测国标法GB4789.15-94中所采用的孟加拉红培养基的基础上,添加不同浓度的葡萄糖、胰蛋白胨、酵母膏等营养物和表面活性荆吐温80,分别对黑曲霉、青霉菌液和不同种类的食品进行霉菌总数测定,经大量对比试验已优选出A4培养基,即在孟加拉红培养基中,添加0。15％的吐温80和1.0％的葡萄糖的培养基。试验结果表明,A4培养基对霉菌检出率较国标孟加拉红培养基的检出率提高了57.9％,且茵落更清晰易数。     

Flow-cytometric cell counting and DNA estimation for the study of plant cell population dynamics

A one-step procedure is presented for simultaneous measurement of cell number and DNA content in cultured plant cells by flow cytometry. In order to obtain nuclei representative of the growth stadium of the culture and of all phases of the cell cycle, cells were carefully sampled and immediately fixed. Next, nuclei were isolated by enzymatic and mechanical maceration, and stained with a DNA-specific fluorescent dye. In the resultant preparation, cells can be counted at relative ease by means of a fluorescence microscope. However, flow-cytometric counting appeared to be superior to manual counting since the time needed for flow-cytometric counting was one-fourth that for manual counting and the variance between counts of the samples was significantly less. In addition, from the same routine, accurate DNA distributions were obtained as a second important parameter of the population dynamics.     

Feeding and egestion rates of individual zooplankton using Cerenkov counting

Cerenkov radiation was used to measure ingestion and release by Brachionus calyciflorus and Daphnia pulex that were fed ³²P-labeled Rhodotorula glutinus. A method is described that uses automatic pipet tips as cages, permitting measurement of ingestion rates and isotope loss by individual animals. Estimated gut renewal times were 16 min for B. calyciflorus and 25–28 min for D. pulex. Filtering and ingestion rates for both animals agree closely with previous estimates. B. calyciflorus release ³²P at high rates in several pulses of short duration. Loss of ³²P by D. pulex occurs at a slower rate and in a single pulse sustained over a longer period. 32P release by B. calyciflorus was not dependent on the presence of food, whereas food influenced the timing and quantity of ³²P release by D. pulex.Financial support to JFH was provided by the Alexander von Humboldt-Stiftung.Financial support to JFH was provided by the Alexander von Humboldt-Stiftung.     

烟粉虱成虫密度自动计数系统

为改善烟粉虱Bemisia tabaci(Gennadius)种群密度调查的手段,根据图像识别原理,形成了一套田间烟粉虱成虫密度自动计数系统,该系统通过成像装置采集烟粉虱成虫的图像,测量成像装置与成像对象之间的距离,对图像中的烟粉虱图像进行识别并计数,计算寄主植物叶面积,最后得出烟粉虱成虫密度。应用该系统对烟粉虱成虫密度进行调查,准确率在90%以上。     

Comparison of two pollen counting methods of slides from a hirst type volumetric trap

Two of the most frequently used methods of pollen counting on slides from Hirst type traps are evaluated in this paper: the transverse traverse method and the longitudinal traverse method. The study was carried out during June–July 1996 and 1997 on slides from a trap at Worcester, UK. Three pollen types were selected for this purpose: Poaceae, Urticaceae and Quercus. The statistical results show that the daily concentrations followed similar trends (p < 0.01, R-values between 0.78–0.96) with both methods during the two years, although the counts were slightly higher using the longitudinal traverses method. Significant differences were observed, however, when the distribution of the concentrations during 24 hour sampling periods was considered. For more detailed analysis, the daily counts obtained with both methods were correlated with the total number of pollen grains for the taxon over the whole slide, in two different situations: high and low concentrations of pollen in the atmosphere. In the case of high concentrations, the counts for all three taxa with both methods are significantly correlated with the total pollen count. In the samples with low concentrations, the Poaceae and Urticaceae counts with both methods are significantly correlated with the total counts, but none of Quercus counts are. Consideration of the results indicates that both methods give a reasonable approximation to the count derived from the slide as a whole. More studies need be done to explore the comparability of counting methods in order to work towards a Universal Methodology in Aeropalynology. This revised version was published online in June 2006 with corrections to the Cover Date.     

Estimating red deer Cervus elaphus populations: an analysis of variation and cost-effectiveness of counting methods

• 1 Different counting methods are currently used to estimate red deer populations in the open range in Scotland, but there are few data available to compare variation in estimates, or relative cost‐effectiveness.
• 2 While it is impossible to determine the accuracy of counts (as real numbers are unknown), variation within and between different methods can be measured by repeat counts of the same area within as short a period as possible.
• 3 This study aimed to quantify the variation observed from repeat counts using each of four methods (ground, helicopter, infrared helicopter and dung‐counting methods) at one of three study sites in late winters 2003, 2004 and 2005. Additional data from digital camera images of groups from counts in other areas of Scotland were also used to assess the accuracy of visual counts.
• 4 Coefficients of variation (CVs) within any method of between 5% and 16% were recorded, consistent with previous comparisons for red deer open range counts in Scotland. CVs were lowest for ground and helicopter counts. The infrequency of optimal conditions was likely to limit the applicability of infrared counts in Scotland.
• 5 In terms of cost‐effectiveness, helicopter counting was the least labour‐intensive, with costs of other techniques depending on the availability of existing manpower as an overhead cost.
• 6 It is concluded that helicopter counts are most likely to minimize errors while maximizing cost‐efficiency. Accuracy can be improved by the use of digital photography for counting larger deer groups. Estimates are likely to be improved further by increasing the frequency of counts and using the same methods, counters and routes for repeat counts.

     

An evaluation of the microscopical counting methods of the tape in hirst-burkard pollen and spore trap

Three different sampling units in current use and different sampling strategies were tested. Randomly placed microscope fields are good in estimating the daily mean concentration, but very big sample size is needed. Traverses across the slide in systematic order are best to estimate the shortterm concentrations and diurnal variation. A formula for the estimation of the error in one transverse traverse is given. Twelve transverse traverses in systematic order is also enough to estimate the daily mean concentration. One or two traverses along the length of the slide give often unreliable estimates because of the irregularities in the transverse variation of the particle concentrations on the slide. For the same reason it is not safe to choose an “effectively collecting area”. Instead the whole width of the tape should be studied.     

Improved particle counting and size distribution determination of aggregated virus populations by asymmetric flow field-flow fractionation and multiangle light scattering techniques

A method using a combination of asymmetric flow field-flow fractionation (AFFFF) and multiangle light scattering (MALS) techniques has been shown to improve the estimation of virus particle counts and the amount of aggregated virus in laboratory samples. The method is based on the spherical particle counting approach given by Wyatt and Weida in 2004, with additional modifications. The new method was tested by analyzing polystyrene beads and adenovirus samples, both having a well-characterized particle size and concentration. Influenza virus samples were analyzed by the new AFFFF-MALS technique, and particle size and aggregate state were compared with results from atomic force microscopy analysis. The limitations and source of possible errors for the new AFFFF-MALS analysis are discussed.