真菌孢子三种计数方法相关性的探讨

以对数期生长旺盛的黑曲霉、白地霉和康宁木霉孢子为试验材料,分别用血球计数法、比浊法和平板菌落计数法进行计数,用所测得的孢子数分别与其对应的OD值建立回归方程,方程分别为y=2.193×10-2 8.714×10-8x、y=6.472×10-2 7.251×10-8x和y=5.571×10-2 7.163×10-8x。3种真菌孢子计数方法所测得的孢子数与其对应OD值均有极显著的直线回归关系(P<0.01),计数真菌孢子时用比浊法测得OD值带入方程便可快速准确地得出孢子数目。因此,可用比浊法替代血球计数法和平板菌落计数法,作为实验室计数真菌孢子的首选方法。     

基于目标颜色基及梯度方向匹配的菌落分割计数算法

【目的】通过菌落测试片提取菌落并计数,在农业、食品业、医疗卫生等领域中是一项常用且重要的工作。目前,菌落自动计数算法大都是以菌落培养皿为主要工作对象,对菌落测试片适用性较差。另外,目前相关技术在常规的粘连物体分割中有着较好的效果,但在菌落分割计数中,由于菌落本身的形态特征,对粘连菌落分割计数的效果尚不够精准。【方法】为解决此类问题,本文提出一种基于目标颜色基及梯度方向匹配的菌落分割计数算法。首先利用图像中菌落的颜色特征作为基,将图像转换到基空间内,以增强菌落与背景之间的差异,其次利用菌落图像的梯度幅值特征对梯度方向进行滤波,然后通过梯度方向进行匹配,进而将粘连的菌落分割,最后利用非极大值抑制的方法筛选出菌落并计数。【结果】经试验,本研究算法的计数精度可达98.00%,能够满足实际需求。【结论】在针对菌落的目标分割计数中,本研究算法不仅计数精度高,而且具有较好的鲁棒性,在对不同厂家的菌落总数测试片菌落分割计数中均有优异效果;然而在对大面积目标的检测分割中算法的准确率会有所下降,因此,该算法更适合于菌落等小目标的检测分割。     

滴水湖沉积物中可培养优势微生物种群初探

于滴水湖湖心采集底泥样品,对底泥中可培养优势菌种进行分离、纯化,并利用Biolog微生物自动分析系统进行鉴定。结果显示,滴水湖沉积物中菌落总数为2.43×104CFU/g,分离纯化后的8株优势菌种中,革兰氏阴性菌占87.5%,其中7株为GN-NENT(革兰氏阴性非肠道菌)、1株为GP-ROD SB(革兰氏阳性芽孢杆菌)。鉴定结果显示,8株菌种分别为:鳗鱼气单孢菌(Aeromonas encheleia)、乙酸钙不动杆菌/基因型1(Acinetobacter calcoaceticus/genospecies1)、舒氏气单胞菌(Aeromonas schubertiiDNA group12)、腐败希瓦氏菌B(Shewanella putrefaciens B)、维罗纳/温和气单胞菌(Aeromonas veronii/sobria DNA group8)、坎氏弧菌(Vibrio campbelli)、蕈状芽孢杆菌(Bacillus mycoides)和梅氏弧菌(Vibrio metschnikovii)。     

流式细胞术检测胞内重组耻垢分枝杆菌的活力研究

目的建立一种快速检测胞内分枝杆菌活力的方法。方法将一定量培养至对数生长期的含pMV-eis的重组耻垢分枝杆菌感染U937巨噬细胞,以含空质粒的耻垢分枝杆菌为对照,吞噬作用2 h后洗去胞外细菌,再分别培养4、12、24和48 h后收集细胞并裂解之。获得的胞内细菌用FDA荧光染料染色后用流式细胞仪检测死亡率,并与平板菌落计数法进行比较。结果流式细胞仪检测出感染12 h后重组耻垢分枝杆菌胞内死亡率较对照组均有显著下降（P〈0.05）,流式细胞仪检测法与平板菌落计数法相比差异无统计学意义（P〉0.05）。结论流式细胞术与传统的平板计数法相比具有快速、敏感、方便的特点,可用于分枝杆菌活菌快速检测。     

The effect of sample composition and vial type on Ĉerenkov counting in a liquid scintillation counter

The effect of sample vial type and sample composition on the ?erenkov count rate detected from ³²P and ³⁶Cl was studied using a liquid scintillation counter. When counting was done in the noncoincident mode, glass vials allowed higher counting efficiency than plastic vials. In the coincident mode light scattering caused by polyethylene and polyproplyene vials allowed higher counting efficiency than glass vials. Highest coincident counting efficiency was from plastic minivials in a glass carrier vial. Increased solute concentration in samples caused increased counting efficiency due to changes in the refractive index of the solution. This can cause significant counting efficiency changes with no sample channel ratio change in density gradient fractions. The use of wavelength shifters is shown to be inappropriate when the sample pH varies, as this can change the fluorescent properties of the shifters and thereby the observed count rate.     

Disaggregation of colonies of Microcystis (Cyanobacteria): efficiency of two techniques assessed using an image analysis system

Disaggregation of colonies of Microcystis (Cyanobacteria) into smalleraggregates or single cells is a usefulprocess for improving the accuracy andprecision of cell abundance estimates innatural populations. An image analysissystem was therefore used to assess theefficiency of two techniques,heating/vortexing and grinding, fordisaggregating fixed and live colonies. Eight environmental samples, consisting ofeither colonial or non-colonial strains,were tested in time course experimentsusing each technique by analysing thenumber and total area of the objects (i.e.,cells, clumps of cells, colonies) acquired. The results show that i) image analysis isan efficient tool to compare the efficiencyof disaggregation techniques on colonies ofcyanobacteria, ii) the grinding techniqueis more efficient in disaggregating alltypes of Microcystis colony, withoutsignificant loss, and iii) the heatingtechnique does not work for all types ofcolonies, in particular, compact coloniesfixed with Lugol's iodine solution.     

Morphogenetic evolution of hydroid colony pattern

A scheme of evolution of hydrozoan colony pattern is proposed based upon the consideration of macro-morphogenesis. Four main processes play decisive roles(1) hard skeleton formation by soft tissues, (2) changes in duration of the growth phase relative to the transition to differentiation in interdependent zones of growth, (3) ratio in growth rates between adjacent zones of growth within the rudiment, the shoot, or the whole colony, and (4) spatial relationships among growth zones. The main tendency in morphological evolution of the hydroids is an increasing integration of the colony as revealed by increasing complexity of its structure. That is from a temporary colony towards the permanent one with highly organised shoots, as hydranths and branches are localised in a strictly arranged manner. An analysis of diverse data allows one to state that the main morphogenetic mechanism of increasing complexity in the hydroid colony is convergence, then fusion, of adjacent growth zones, a variant of heterochrony.     

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.     

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.

     

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.     

A METHOD FOR THE QUANTITATIVE AND QUALITATIVE DETERMINATION OF PLANKTONIC DIATOMS

SUMMARY

A method for the quantitative and qualitative determination of planktonic diatoms was developed. The method uses the Utermöhl counting technique (in which an inverted microscope is employed) as a basis but also involves the calculation of the relative density of each species in the association (as determined with the ordinary light microscope). A combination of the results thus obtained ensures that even the smallest diatoms (2,5 μm) are accounted for. These often constitute a significant proportion of the population, but are often overlooked or may not even be resolved using Utermöhl's method.     

The calculating cormorants

Traditional fishing with captive cormorants was observed on the Li-Kiang River. After catching seven fish the birds were allowed to eat the eighth. Without this reward they refused to fish again. These birds could apparently count up to seven.     

截头堆砂白蚁原始繁殖蚁形成的周期

采用马尾松(Pinusmassonianalamb)、东北松(PinusSpp.)、北美黄杉(花旗松)(Pseudotsugamenziesii)、青岗(CyclobalanopsisSpp.)、荷木(SchimaSpp.)作为截头堆砂白蚁Cryptotermesdomesticus(Haviland)的食料,研究其原始繁殖蚁配对后新建群体的形成和发展,从饲养的新建群体中培养产生了下一代的原始繁殖蚁,完成了1个世代的生活周期观察。试验证实,截头堆砂白蚁在广州市室温下,产生下一代原始繁殖蚁(完成1个生活周期)为7年;在湛江市室温下6、7年;而在恒温27℃,相对湿度80%的条件下只需2～3年。     

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.     

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.