CELL AND GROWTH CHARACTERISTICS OF TYPES A AND B OF EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE) AS DETERMINED BY FLOW CYTOMETRY AND CHEMICAL ANALYSES1

Two morphotypes of Emiliania huxleyi (Lohmann 1902) Hay et al. 1967, types A and B, known to be unequally distributed in the oceans, were grown in dilution cultures at a range of photon flux densities (PFDs) (1.5–155 μmol photons·m^?2·s^?1) and two temperatures (10° and 15° C). Calcite carbon and organic carbon content of the cells as well as instantaneous growth rate, cell size, chlorophyll fluorescence, and light-scatter properties clearly depended on growth conditions and differed considerably for the two morphotypes. The ratio between calcite carbon and organic carbon production showed an optimum of 0.65 in E. huxleyi type A cells at PFD = 17.5. The ratio increased slightly with a temperature increase from 10° to 15°C but remained < 1.0 at both temperatures in light-limited cells. In contrast, calcite carbon production exceeded organic carbon production (ratio: 1.4–2.2) in phosphate-deprived cultures. Emiliania huxleyi type B generally showed a higher calcite carbon/organic carbon ratio than E. huxleyi type A, but the relation with PFD was similar. The content of calcite carbon and organic carbon as well as the instantaneous growth rate, cell size, chlorophyll fluorescence, and light-scatter properties showed large diel variations that were closely related to the division cycle. Our results show the importance of mapping the structure of any sampled cell population with respect to the phase in the cell division cycle, as this largely determines the outcome of not only “per cell” measurements but also short time (less than 24 h) flux measurements. For instance, dark production of calcite by E. huxleyi was negatively affected by cell division. Slowly growing (phosphate-stressed) cultures produced calcite in the light and in the dark. In contrast, rapidly growing cultures at 10°C produced calcite only in the light, whereas in the dark there was a significant loss of calcite due to dissolution.     

MEASUREMENT OF MICROALGAL CELL VOLUME BY FLOW CYTOMETRY

Single cell analysis by flow cytometry is a powerful tool that has been employed to identify many different characteristics of phytoplankton populations. Cell volume is an important physiological component of many cellular processes. We have used a Coulter EPICS XL flow cytometer to measure cell volume in the spheroid dinoflagellate Amphidinium operculatum as a function of forward scatter. Cell volume measurements of this alga were quantified as equivalent spherical diameters from a standard curve obtained with latex beads of known diameter. This parameter was used to monitor cell diameter throughout the cell division cycle. In log phase cultures, A. operculatum showed increasing cell volumes throughout the light phase and a maximum cell volume concurrent with the onset of cell division late in the light phase. The maximum equivalent spherical diameter measured 14 μm, while the minimum equivalent spherical diameter was 10 μm that occurred late in the dark phase. Stationary phase cultures of A. operculatum did not exhibit oscillating cell volumes throughout the diel cycle. Chemical inhibition of the cell cycle using 100 μM olomoucine diminished cell volume changes during the light phase. These results suggest a coupling of size control to the cell division cycle.     

A COMPARISON OF NUCLEAR DRY WEIGHTS DETERMINED BY CHEMICAL AND BY INTERFEROMETRIC METHODS

1. The nuclei of cells from the thymus of the calf were isolated by three different techniques; the citric acid, the sucrose-calcium chloride, and the non-aqueous. 2. The mean dry weights of the nuclei were determined by chemical methods and by microscopic interferometry. There was a close correlation between the results from the interferometric and chemical methods. 3. The range of values about that mean was determined in each sample: the nuclei isolated in aqueous media contained approximately 45 per cent less material than those isolated in non-aqueous media. 4. The variations in dry weight with varying nuclear type are discussed. 5. The possible relationship between DNA content and dry weight is discussed.     

CELL-PRODUCTION RATES ESTIMATED BY THE USE OF VINCRISTINE SULPHATE AND FLOW CYTOMETRY

A new method for the evaluation of cell production rates combining flow cytometry (FCM) and the stathmokinetic method using vineristine sulphate (VS) has been used for the analysis of three aneuploid ascites tumours at different stages of growth. Using this technique it was possible to estimate the well-known decrease in cell production rates of ageing ascites tumours. The percentage of normal host cells in the aneuploid tumours studied was easily determined by FCM prior to the calculation of the tumour cell-production rates. A correlation was found between the percentage of tumour cells in the S phase and the tumour cell-production rate. This correlation is probably explained by the gradual transfer of proliferating cells in S phase to resting G₁ and G₂ phases with increasing tumour age.     

小儿急性白血病细胞DNA、蛋白质含量及其临床意义

用流式细胞术（ＦＣＭ）测定了４５例不同病期急性白血病患儿和１３例非白血病患儿骨髓细胞ＤＮＡ及蛋白质含量,结果显示：１．急性白血病患儿的ＤＮＡ非整倍体检出率为４１．２％,其中ＡＬＬ为３３．３％,ＡＮＬＬ为６０％;２．ＡＬＬ组和ＡＮＬＬ组的ＤＮＡ合成期细胞百分数（Ｓ％）显著低于非白血病组,而ＣＲ组与非白血病组之间的Ｓ％差异无显著性;３．ＡＮＬＬ组的ＰｒＩ显著高于ＡＬＬ组、非白血病组及ＣＲ组,ＡＬＬ的ＰｒＩ显著低于ＣＲ组和非白血病组,而非白血病组与ＣＲ组的ＰｒＩ差异无显著性。四组之间ＬＰＣ－Ｆ差异无显著性;４．在对１１例ＣＲ病人进行的连续观察过程中,５例病人检出ＤＮＡ非整信体,其中４例于检出ＤＮＡ非整倍体后３３～７４天复发。     

MOLECULAR STRUCTURE OF PLANT FIBERS DETERMINED BY X-RAYS

It has been shown that the wall of the plant fiber is probably built up of unit groups of atoms which have assumed the form of a space lattice. The elementary cell of the lattice is an orthorhombic structure with the dimensions 6.10 x 5.40 x 10.30 Å.u., and contains two unit groups equal in size to two C₆H₁₀O₅ groups. The crystallographic unit cell would contain 4 of these elementary cells and would be represented by Fig. 9 rather than by Fig. 3. The groups of atoms, C₆H₁₀O₅, are arranged in parallel chains running lengthwise of the fiber. In each chain the odd numbered groups have a different orientation from the even numbered. The chains, parallel to one another are spaced 6.10 Å.u. in one direction and 5.40 Å.u. at right angles to that. In these two directions the odd numbered chains also would have a different orientation from the even numbered. On account of the cylindrical shape of the fiber, the elementary cells are arranged in the form of concentric cylinders or layers. The dimensions of the fibers are such that the fiber wall is about 40,000 elementary cells in thickness, or in other words, the fiber is composed of that many concentric layers. If it could be magnified sufficiently, a cross-section of a fiber would show the end view of each cylinder as a dotted circle. The dots, representing the unit groups of atoms, would have considerable uniformity of spacing in both the tangential and the radial directions, 6.10 Å.u. in one and 5.40 Å.u. in the other. The structure could not be as rigidly exact as might be inferred, since the wall is deposited more or less rhythmically during a period of several days or weeks* in which adjustments in the arrangement of the unit groups undoubtedly occur. It is common knowledge that the fibers, under the microscope, rarely appear as true circles on cross-section; usually they appear as irregular, many-sided polygons and the wall thickness is normally uneven. For our purpose it is simpler to think of the fiber as composed of concentric cylinders with diameters so large in proportion to the size of the unit groups that in relatively large segments they closely approach the parallelism of the planes of a rectangular lattice, sufficiently close to be capable of producing diffraction patterns. Although these conclusions seem to be in agreement with the diffraction patterns obtained from various positions of a bundle of approximately parallel fibers, the fact must not be overlooked that the structure cannot be proved with as great certainty as can the structure of a well formed crystal. The very nature of the fiber, its cylindrical shape, and the many internal adjustments which must take place, militate against a clean-cut demonstration. Models, made more or less to scale, were used in working out this structure. The unit group was constructed according to Irvine''s suggestion that all the groups are glucose residues. An intensive study is now under way in which an attempt is being made to bring the models into agreement with the chemical and physical properties of the cellulose fibers and with the diffraction patterns. A report on that part of the work will soon be submitted for publication.     

CHARACTERIZATION OF OCEANIC PHOTOSYNTHETIC PICOEUKARYOTES BY FLOW CYTOMETRY1

To interpret flow cytometric data that are routinely obtained on natural oceanic communities, 23 strains of photosynthetic picoeukaryotes belonging to four classes (Prasinophyceae, Chlorophyceae, Pelagophyceae, and Prymnesiophyceae) and six pigment types were investigated for their light scattering in the forward and right-angle directions, chlorophyll fluorescence, and DNA content as measured by flow cytometry. Cell she was assessed by Coulter counter, and pigment composition was measured by reverse-phase high-performance liquid chromatography. The size and GC% of the nuclear genome of cultured picoeukaryotes was measured from the fluorescence of DNA-specific dyes. Using these two parameters, we could discriminate species within pigment groups. DNA staining of preserved natural samples may also prove useful in discriminating cooccurring populations in situ as long as the communities are not too complex. Using the relationships that we established between size and light-scattering properties of the cells, we estimated equivalent diameters of picoeukaryotes in natural populations to be between 1.3 and 2 μm. Chlorophyll a content was between 6 and 16 fg·cel^?1 as calculated from relationships that we established between chlorophyll a content and red fluorescence of the cultured strains. With respect to size, chlorophyll a content, and pigment composition, Pelagomonas sp. strains (Pelagophyceae) appeared to be the most representative of the natural communities in subtropical ocean waters. In contrast, green coccoid strains, which often outcompete other strains in culture, might only be minor contributors to these communities.     

流式细胞光度术DNA分析在非何杰金氏淋巴瘤预后中的意义

用流式细胞光度术对一组非何杰金氏淋巴瘤(NHL)存档石蜡标本进行DNA分析。DNA异倍体肿瘤占总例数的47.4％,高、中度恶性组异倍体肿瘤的比例(76.8％和51.9％)分别高于低度恶性组(17.6％)(P<0.05)。S期细胞比例(synthetic phase fraction,SPF)随组织学恶性程度升高而增大,低、中和高度恶性淋巴瘤平均SPF值分别是6.5(0.6—18.8)％,13.5(3.2—37.3)％和23.4(4.4—41.4)％。二倍体和异倍体肿瘤患者的生存率无差别。全部病例中高SPF值肿瘤患者具有生存短的趋向(P=0.1)。低度恶性组内也存在此种趋向(P=0.08)。上述结果表明流式细胞光度术DNA分析对估价非何杰金氏淋巴瘤生物学行为是一种不依赖形态学的有用方法。     

流式细胞术分析强声波对植物细胞周期的影响

应用流式细胞术分析烟草细胞在交变应力作用下细胞周期的变化。用特制的强声波发生装置产生频率和强度可调的交变应力场,研究不同频率和强度的交变应力作用后烟划细胞周期的变化,实验结果表明,在交变应力作用下直接影响细胞或细胞分裂的同步化,促进S期的DNA合成,有助于细胞有丝分裂,声波频率在400Hz至800Hz,强度在90dB到110dB内,随频率和强度的增加,交变应力使S期细胞明显增加,但频率或强度达大,反而使S期细胞大大减少。     

DETERMINATION OF NON-PROLIFERATING CELLS IN CULTURE BY COMBINING FLOW CYTOMETRY WITH STATHMOKINETICS

Colcemid was added to the growth medium of L-cells in monolayer culture. The proliferating cells continued their progression through the cycle up to metaphase, where they were arrested. At different times after Colcemid addition the cells were trypsinized. suspended immediately in a solution of the DNA-specific fluorescent dye Hoechst 33258 and analysed with a flow cytometer. The histograms were evaluated to give the fraction of cells in the 2c peak as a function of time after Colcemid addition. The flux into the 2c compartment being interrupted, the peak content decreased until all proliferating (G₁) cells had entered S-phase. With increasing cell density or with increasing time after serum deprivation an increasing fraction of cells remained in the 2c peak at times greater than the normal G₁ duration. The possibility of applying this method to the determination of non-proliferating cells in a population is discussed.     

CD11b/DNA双参数流式细胞术检测HL-60细胞分化的细胞周期

目的采用双参数流式细胞术研究全反式维甲酸(alltransretinoidacid,ATRA)诱导人类急性早幼粒白血病细胞HL-60细胞分化的细胞周期。方法HL-60细胞经分化诱导剂ATRA(终浓度为1μmol/L)诱导不同时间点后,利用CD11b/DNA双参数流式细胞术同时检测分化细胞表面抗原CD11b的表达及分化细胞DNA含量。结果HL-60细胞经ATRA诱导后,细胞表面分化抗原CD11b表达明显升高,细胞阻滞于G0/G1期,且CD11b阳性细胞主要位于G0/G1期。结论CD11b/DNA双参数流式细胞术能简便,快速,直观地检测细胞分化的细胞周期。     

应用单抗和流式细胞仪分析淋球菌的表面抗原

报告了利用流式细胞仪测定分析10株抗淋球菌脂寡糖单克隆抗体所识别的抗原分子表达的特点,定量地显示了这些抗原分子表达的稳定性和数量的多少,评价了单克隆抗体与不同血清型淋球菌的反应性。     

FLOW CYTOMETRY AND THE SINGLE CELL IN PHYCOLOGY

Flow cytometers measure light scattering and fluorescence characteristics from individual particles in a fluid stream as they cross one or more light beams at rates of up to thousands of events per second. Flow cytometrically detectable optical signals may arise naturally from algae, reflecting cell size, structure, and endogenous pigmentation, or may be generated by fluorescent stains that report the presence of otherwise undetected cellular constituents. Some flow cytometers can physically sort particles with desired optical characteristics out of the flow stream and collect them for subsequent culture or other analyses. The statistically rigorous, cell‐level perspective provided by flow cytometry has been advantageous in experimental investigations of phycological problems, such as the regulation of cell cycle progression. The capacity of flow cytometry to measure large numbers of cells in large numbers of samples rapidly and quantitatively has been used extensively by biological oceanographers to define the distributions and dynamics of marine picophytoplankton. Recent work has shown that flow cytometry can be used to elucidate relationships between the optical properties of individual cells and the bulk optical properties of the water they live in, and thereby may provide an explicit link between algal physiology and global biogeochemistry. Unfortunately, commercially available flow cytometers that are optimized for biomedical applications have a limited capacity to analyze larger phytoplankton. To circumvent these limitations, many investigators are developing flow cytometers specifically designed for analyzing the broad range of sizes, shapes, and pigments found among algae. These new instruments can perform some novel measurements, including simple fluorescence excitation spectra, detailed angular scattering measurements, and in‐flow digital imaging. The growing accessibility and power of flow cytometers may allow the technology to be applied to a wider array of problems in phycology, including investigations of nonplanktonic and multicellular algae, but also presents new challenges for effectively analyzing the large quantity of multiparameter data produced. Ultimately, the detection of molecular probes by flow cytometry may allow single‐cell taxonomic and physiological information to be garnered for a variety of algae, both in culture and in nature.     

RAPID DETECTION of STAPHYLOCOCCUS AUREUS IN FOOD BY FLOW CYTOMETRY

This report describes the detection of Staphylococcus aureus in buffer and in several kinds of food by flow cytometry. Fluorescein isothiocyanate conjugated anti-protein A antibodies were used in a 4-h procedure to label cells, 10⁵-10⁶ cells/mL are needed. the use of single parameter, green fluorescence, enabled specific differentiation of S. aureus from other bacteria including 11 Staphylococcus species. the flow cytometric method can detect S. aureus in food samples after 48-h enrichment in trypticase soy broth with 10% NaCl. As low as 2 S. aureus cells present in 10 mL enrichment broth could grow to a population density detectable by the flow cytometric method after enrichment. This method was faster and less laborious than the conventional BAM (Bacteriological Analytical Manual) or AOAC (Association of Official Analytical Chemists) methods, and could be automated for analysis of S. aureus in food.     

A COMPARED STUDY ON THE PURIFICATION OF FETAL AND ADULT RAT HEPATOCYTE SUSPENSIONS BY BIOMAGNETIC ISOLATION TECHNIQUES AND FLOW CYTOMETRY ANALYSIS

An analysis of liver cell populations from both adult and 21 day pregnancy rat fetuses (E21) was carried out. The results show that E21 hepatocytes express OX-43, as do endothelial cells but not adult hepatocytes. OX-43 could be used in future as a cell marker for the hepatocyte maturation.     

应用流式细胞仪定量评估人—鼠杂交瘤株细胞抗体生成功能稳定性的研究

用FACS分析和Luria-Delbrǔck方程计算淋巴细胞杂交瘤株细胞丧失抗体生成功能的突变机率(μ),以评估杂交瘤的稳定性。结果表明μ值大,则杂交瘤不稳定,易丧失抗体生成功能,反之则杂交瘤比较稳定。实验证明所建立的方法能反映杂交瘤稳定性的程度,具有实验少人为主观因素干扰,统计意义强,适用范围广等优点。     

He—Ne激光引起DNA突变的量子模式

根据Lǒwdin提出,在DNA分子中,碱基对之间的氢键体系X—H…y中存在一个对称的双势阱。本文研究了DNA分子受He-Ne激光影响后,使质子在双势阱小的隧道效应极大增加,从而引起了对于激光育种有实用意义的DNA突变。并且提出了He-Ne激光促使DNA突变的量子模式。