A Compact Co2 Chilling Device for Continuous Duty in Paraffin-Type Sectioning

To obtain uniform sections cut from some of the newer embedding materials of lower melting points, particularly the polyethylene glycols, it is necessary to cool the microtome knife. A cooling device constructed for this and other paraffin-type sectioning consists of a small brass wedge attached to the knife by means of an adjustable spring clip. The wedge is small enough to mount directly on the back of the knife and does not interfere with any movement of the microtome. Three small channels are drilled in the wedge to provide one entrance and two exits for gaseous CO₂. CO₂ from an upright tank is regulated by a modified Spencer Model 930 freezing attachment, which is connected to the brass wedge by copper capillary tubing.     

Cutting Unfixed Frozen Sections for Fluorescence Antibody Studies

A simplified method of section cutting, dispensing with guide attachment on microtome blade, and suitable for serial sections of unfixed frozen tissue of 4μ is described. Essential features are low temperature maintenance (—13°C), critical angle of knife and moistening of slides in cold alcohol or isopentane. This method has been found suitable for fluorescence antibody studies where maintenance of low temperatures is necessary.     

CO2浓度升高与氮添加对三江平原湿地小叶章生长的影响

利用开顶箱薰气室,设置正常大气CO₂浓度(350 μmol·mol^-1)、高CO₂浓度(700 μmol·mol^-1)2个CO₂水平和不施氮(0 g N·m^-2)、中氮(5 g N·m^-2)和高氮(15 g N·m^-2)3个氮素水平,研究CO₂浓度升高和氮肥施用对三江平原草甸小叶章生长的影响.结果表明：随着CO₂浓度升高,小叶章物候期提前,其中抽穗期提前1～2 d,成熟期提前3 d;不施氮、中氮和高氮水平下, CO₂浓度升高使小叶章的分蘖分别增加8.2%(P<0.05)、8.4%(P<0.05)和5.5%(P>0.05);在小叶章生长初期,CO₂浓度升高对其生物量的增加有促进作用,拔节期和抽穗期小叶章地上生物量分别增加12.4%和20.9%(P<0.05);生长后期则对小叶章地下生物量的促进作用增大,腊熟期和成熟期的地下生物量分别增加20.5%和20.9% (P<0.05).小叶章生物量对高浓度CO₂的响应与供氮水平有关,供氮充足条件下, 高浓度CO₂对生物量的促进效应更大.     

采伐对小兴安岭森林沼泽非生长季土壤温室气体排放的影响

中高纬度地区非生长季温室气体排放对生态系统碳、氮循环具有重要影响,但采伐干扰如何影响森林沼泽非生长季土壤温室气体排放尚不明确.本研究采用静态箱-气相色谱法,观测小兴安岭4种森林沼泽(毛赤杨沼泽、白桦沼泽、落叶松苔草沼泽、落叶松藓类沼泽)不同采伐方式下(对照、择伐45%、皆伐,试验处理已10年)非生长季土壤CO₂、CH₄、和N₂O通量及其相关环境因子(温度、湿度及碳氮含量等),分析采伐干扰对温带森林沼泽非生长季土壤温室气体排放的影响规律及主控因子.结果表明: 采伐干扰10年后,4种森林沼泽土壤CO₂、CH₄和N₂O非生长季平均通量分别在53.08～81.31 mg·m^-2·h^-1、0.09～3.07 mg·m^-2·h^-1和4.07～8.83 μg·m^-2·h^-1,其中,皆伐显著提高毛赤杨沼泽和落叶松藓类沼泽非生长季土壤CO₂、CH₄和N₂O排放量,择伐显著提高白桦沼泽、落叶松藓类沼泽及降低毛赤杨沼泽的CO₂排放量,且显著降低4种森林沼泽CH₄排放量及落叶松苔草沼泽的N₂O排放量;天然森林沼泽非生长季土壤CO₂排放受土壤温度、有机碳含量及C/N调控,CH₄受土壤温度、有机碳含量调控,N₂O受气温、土壤pH调控,采伐增加了CO₂排放与气温、土壤含水量及积雪深度的相关性,增加了CH₄排放与气温、土壤含水量、C/N的相关性,增加了N₂O排放与土壤全氮和C/N的相关性;温带天然森林沼泽非生长季土壤CO₂、CH₄和N₂O的年贡献率分别为33.2%～46.5%、6.3%～9.1%和61.5%～68.3%,皆伐提高了白桦沼泽和落叶松藓类沼泽CO₂年贡献率和除落叶松藓类沼泽外其他样地的N₂O年贡献率,择伐提高了落叶松苔草沼泽、落叶松藓类沼泽CO₂、CH₄和N₂O年贡献率,但降低了白桦沼泽3种气体年贡献率.温带天然森林沼泽非生长季土壤N₂O和CO₂的年贡献率相对较大,皆伐使两者年贡献率进一步提高,择伐却较大幅度提高了其CH₄的年贡献率.     

FROZEN THIN SECTIONS OF FRESH TISSUE FOR ELECTRON MICROSCOPY, WITH A DESCRIPTION OF PANCREAS AND LIVER

A simple method has been developed that allows frozen thin sections of fresh-frozen tissue to be cut on a virtually unmodified ultramicrotome kept at room temperature. A bowl-shaped Dewar flask with a knifeholder in its depths replaces the stage of the microtome; a bar extends down into the bowl from the microtome's cutting arm and bears the frozen tissue near its lower end. When the microtome is operated, the tissue passes a glass or diamond knife in the depths of the bowl as in normal cutting. The cutting temperature is maintained by flushing the bowl with cold nitrogen gas, and can be set anywhere from about -160°C up to about -30°C. The microtome is set for a cutting thickness of 540–1000 A. Sections are picked up from the dry knife edge, and are placed on membrane-coated grids, flattened with the polished end of a copper rod, and either dried in nitrogen gas or freeze-dried. Throughout the entire process the tissue is kept cold and does not come in contact with any solvent. The morphology seen in frozen thin sections of rat pancreas and liver generally resembles that in conventional preparations, although freezing damage and low contrast limit the detail that can be discerned. Among unusual findings is a frequent abundance of mitochondrial granules in material prepared by this method.     

Some Improvements in the Preparation of Fresh Frozen Sections

For the histochemical demonstration of sensitive enzymes it is necessary to use fresh unfixed tissue sections. With the following procedure one can constantly obtain such sections 10-20μ thick with relative ease. Schanze's sliding microtome is employed. The microtome knife is deeply cooled by placing blocks of dry ice on its surface, and is provided with a device for preventing the sections from rolling up. The microtome is operated in an ordinary refrigerator maintained at a temperature of 0-3°C. For this purpose, the door of the refrigerator is replaced by a wooden door provided with a glass window, gloved arm holes, and a small door.     

Improved Polyester Wax Embedding for Histology

Polyester waxes are fatty add esters of polyethylene glycol. Polyethylene glycol 400 distearate melts at 35°C, infiltrates tissues well, and sections readily at 2 μ to more than 30 μ. Sections 2 μ to 6 μ are more easily cut when a kitchen strainer full of solid CO₂ (dry ice) is mounted above the microtome to cool the block and the knife, and when the knife crosses the block very slowly. Ribbons are flattened in water at room temperature and are mounted conventionally. Polyester ribbons are somewhat stickier than paraffin ribbons. Polyethylene glycol 400 distearate is slightly hydrophilic; immediately after microtomy and before the ribbon is affixed to the microscope slide, sections in the wax ribbon may conveniently be stained with 0.05% toluidine blue in aqueous benzoate buffer, pH 4.4. Tissue structure is better preserved in polyester than in paraffin wax, probably because structural lipids are better retained and localized. However, this difference between waxes is slight if tissues are well fixed and dehydrated. Other advantages of polyester wax are that sections fragment less, hard tissues rarely split away from the wax ribbon, no static electricity is generated, and the microtome knife seems to remain sharp for a longer time.     

Techniques for Studying Adipocytes

Various fixatives as well as tissue and slide handling procedures have been evaluated in attempts to demonstrate adipocytes histochemically while maintaining cell and tissue integrity. The optimal procedure for analysis of immature adipose depots consists of the following steps: 1) fresh, unfixed tissues are frozen rapidly in isopentane quenched in a liquid nitrogen bath; 2) cryostat sections are cut, removed from the knife with a room temperature slide, and then air dried for 5-10 minutes; 3) slides can be stained directly with picro-Ponceau or toluidine blue procedures or with oil red O following fixation for 30 minutes in cold (4 C) 10% formalin-CaCl₂ (1.25%). For analysis of mature rat adipose depots steps 2 and 3 are modified as follows: 2) cryostat sections are removed from the knife with a cold slide (-20 C) and dried for 30 minutes at 4 C; 3) the mounted sections are stained with oil red O following fixation for 30 minutes in cold (4 C) 10% formalin-HgCl₂ (2.5%). When procedures described above for immature adipose depots are combined with esterase fining, adipocyte cytoplasm is clearly demonstrated. These procedures allow the routine use of fresh frozen, unfixed cryostat sections in studies of adipose cellularity.     

Thin Sections from Unfixed Tissues for Histochemical Staining

Sections were cut from fresh unfixed tissues by means of a microtome provided with an apparatus for the simultaneous cooling of the knife and freezing stage. These sections were of uniform thickness and were found to be very suitable for histochemical staining. Such sections were immersed while still frozen in the fluid which contained the necessary chemicals for a specific technic. After remaining in the fluid for an appropriate time, the sections were put on slides and dried in warm air. The remaining steps were carried out on the slides. Several histochemical procedures (phosphatase, esterase, glycogen) were found to give good results when this technic was used.     

Improved Paraffin Sectioning with Etched Microtome Knife Edges

Carbon steel microtome knives etched with 0.1 N HNO₃ for 2 min display a very sharp cutting edge. Over a period of 3 yr no damage to the steel has been detected. The effect on paraffin sectioning was observed by comparing acid-treated knives with nonetched but well-sharpened ones. Sections of whole eyes cut with an etched blade showed approximately 15% less compression of the parffin matrix than those sectioned with an untreated knife. Tissues selected from routine autopsy material presented approximately 9% reduction in compression. As a result, excellent ribbons of sections could be cut from 5-7 μ and floated on water at 42—46 C with a minimum of folds or distortion. Etching improved sectioning when knife edges having bevel angles in a range of 31-39° were used, and also when the bevel was decreased to 20°, but the 20° edge gave impractically short service.     

Effect of elevated CO2, temperature and drought on dry matter partitioning and photosynthesis before and after cutting of nodulated alfalfa

The rising atmospheric CO₂ concentration resulting from industrial development may enhance photosynthesis and plant growth. However, there is a lack of research concerning the effect of combined factors such as CO₂, temperature and water availability on plant regrowth following cutting or grazing, which represent the usual methods of managing forage legumes like alfalfa. Elevated CO₂, temperature and drought can interact with cutting factors (e.g. cutting frequency or height), and source-sink balance differences before and after defoliation can modify photosynthetic behaviour and dry matter accumulation, as well as dry matter partitioning between above- and belowground organs. The aim of our study was to determine the interactive effect of CO₂ (ambient, around 350 μmol mol⁻¹ versus 700 μmol mol⁻¹), temperature (ambient versus ambient + 4 °C) and water availability (well-irrigated versus partially irrigated) on dry matter partitioning and photosynthesis in nodulated alfalfa after vegetative normal growth and during regrowth. At the end of vegetative normal growth, CO₂ enhanced dry matter accumulation despite photosynthesis being down-regulated at the end of this period. Photosynthesis was stimulated by elevated CO₂ and resulted in greater dry matter accumulation during the regrowth period. Aboveground organs were affected more by drought than belowground organs during the entire experiment, particularly during vegetative normal growth. The higher drought tolerance (greater growth) observed during the regrowth period may be related to higher mass and greater reserves accumulated in the roots of plants.     

Epoxy Embedding, Sectioning and Staining of Plant Material for Light Microscopy

By using a formula which gives a relatively soft epoxy embedding medium, it is possible to cut sections of plant material with a sliding microtome equipped with a regular steel knife. Blocks having a cutting face of 10 × 10 mm, giving sections of 4-10 μm, can be used. Tissues are fixed in Karnovsky's fluid, postfixed in 1 or 2% OsO₄, embedded in Spurr's soft epoxy resin, Araldite, or Epon mixtures. 5% KMnO₄, followed by 5% oxalic acid, then neutralized in 1% LiCO₃, are used to mordant the sections. Some of the stains used are Mallory's phosphotungstic acid-hemotoxylin, acid fuchsin and toluidine blue, or toluidine blue. Mounting is done with whichever soft epoxy resin was used in casting the blocks.     

Dark reduction of QA in intact barley leaves as an effect of lowered CO2 concentration monitored by chlorophyll a luminescence and chlorophyll a F0 dark fluorescence

When the CO₂ concentration in the atmosphere above an intact barley leaf was lowered in the dark after illumination, chlorophyll a luminescence and chlorophyll a dark fluorescence were stimulated. The stimulation was induced by lowered levels of CO₂ in a wide concentration range including concentrations well above that saturating photosynthesis. The stimulation of luminescence by lowered CO₂ concentrations was more pronounced after far-red excitation than after white light excitation. The difference in response to lowered CO₂ concentrations after white/far-red excitation was less pronounced for fluorescence than for luminescence. Stimulation of luminescence was more pronounced when the CO₂ concentration was lowered in an O₂-containing atmosphere than under anaerobic conditions. It is concluded that lowering of the CO₂ concentration in the dark after illumination causes a partial reduction of the primary Photosystem II acceptor Q_A.     

水稻光合碳在植物-土壤系统中的分配及其对CO2升高和施氮的响应

采用¹³C-CO₂进行连续标记,研究水稻分蘖期和孕穗期光合碳在植株-土壤系统中的分配及其对大气CO₂浓度升高(800 μL·L^-1)和施氮(100 mg·kg^-1)的响应.结果表明: CO₂浓度升高显著提高分蘖期根系生物量和孕穗期地上部生物量,并使生物量根冠比在分蘖期增加,而在孕穗期减小.CO₂浓度升高条件下,施氮使水稻地上部分生物量增加,却显著降低了孕穗期水稻根系生物量.CO₂浓度升高使光合¹³C在孕穗期向土壤的输入显著增加,然而施肥并没有促进由CO₂浓度升高驱动的光合¹³C在土壤中的积累,而且还降低了土壤中的光合¹³C的分配比例.综上,CO₂浓度升高显著提高了稻田土壤光合碳输入,促进稻田有机碳周转;施氮促进了水稻地上部的生长,却降低了光合碳向地下的分配比例.     

粳稻生育后期剑叶光合日变化和光合色素对大气CO2浓度和温度升高的响应——FACE研究

为研究水稻叶片光合色素和光合日变化对大气CO₂浓度和气温升高的响应,我们采用在开放空气中控制升高CO₂浓度和温度的方法,以常规粳稻南粳9108为试验材料,设置了环境CO₂和高大气CO₂浓度(增加200 μmol·mol^-1)、环境温度和增温(增加1～2 ℃)交互的4个处理,测定了灌浆中期和后期水稻剑叶的光合日变化特征和光合色素含量.结果表明: 水稻剑叶净光合速率(P_n)为双峰曲线,发生了光合“午休”现象;大气CO₂浓度升高提高了剑叶P_n,灌浆中期和后期平均分别增加了47.6%和39.1%;高温有降低P_n的趋势,但相关性未达到显著水平.大气CO₂浓度和温度升高导致水稻剑叶生育后期气孔导度(g_s)平均分别降低了17.0%和11.8%.高CO₂浓度水稻剑叶生育后期蒸腾速率(T_r)、叶绿素a、叶绿素b、类胡萝卜素、总叶绿素和叶绿素a/b值显著降低,平均降幅分别为5.9%、50.4%、21.3%、41.4%、39.4%和21.4%,明显增加了剑叶水分利用率(WUE),平均增幅达47.9%.与之相反,生育后期增温使水稻剑叶T_r增加了10.2%,使WUE平均降低了20.4%.综上所述,大气CO₂浓度升高对粳稻生育后期剑叶P_n、g_s和光合色素含量的影响明显大于增温效应.因此,应重视大气CO₂浓度和温度对水稻光合作用和光合色素的综合效应,减弱增温的负效应.     

水稻光合碳在植物-土壤系统中的分配及其对CO2升高和施氮的响应

采用¹³C-CO₂进行连续标记,研究水稻分蘖期和孕穗期光合碳在植株-土壤系统中的分配及其对大气CO₂浓度升高(800 μL·L^-1)和施氮(100 mg·kg^-1)的响应.结果表明: CO₂浓度升高显著提高分蘖期根系生物量和孕穗期地上部生物量,并使生物量根冠比在分蘖期增加,而在孕穗期减小.CO₂浓度升高条件下,施氮使水稻地上部分生物量增加,却显著降低了孕穗期水稻根系生物量.CO₂浓度升高使光合¹³C在孕穗期向土壤的输入显著增加,然而施肥并没有促进由CO₂浓度升高驱动的光合¹³C在土壤中的积累,而且还降低了土壤中的光合¹³C的分配比例.综上,CO₂浓度升高显著提高了稻田土壤光合碳输入,促进稻田有机碳周转;施氮促进了水稻地上部的生长,却降低了光合碳向地下的分配比例.     

粳稻生育后期剑叶光合日变化和光合色素对大气CO2浓度和温度升高的响应——FACE研究

为研究水稻叶片光合色素和光合日变化对大气CO₂浓度和气温升高的响应,我们采用在开放空气中控制升高CO₂浓度和温度的方法,以常规粳稻南粳9108为试验材料,设置了环境CO₂和高大气CO₂浓度(增加200 μmol·mol^-1)、环境温度和增温(增加1～2 ℃)交互的4个处理,测定了灌浆中期和后期水稻剑叶的光合日变化特征和光合色素含量.结果表明: 水稻剑叶净光合速率(P_n)为双峰曲线,发生了光合“午休”现象;大气CO₂浓度升高提高了剑叶P_n,灌浆中期和后期平均分别增加了47.6%和39.1%;高温有降低P_n的趋势,但相关性未达到显著水平.大气CO₂浓度和温度升高导致水稻剑叶生育后期气孔导度(g_s)平均分别降低了17.0%和11.8%.高CO₂浓度水稻剑叶生育后期蒸腾速率(T_r)、叶绿素a、叶绿素b、类胡萝卜素、总叶绿素和叶绿素a/b值显著降低,平均降幅分别为5.9%、50.4%、21.3%、41.4%、39.4%和21.4%,明显增加了剑叶水分利用率(WUE),平均增幅达47.9%.与之相反,生育后期增温使水稻剑叶T_r增加了10.2%,使WUE平均降低了20.4%.综上所述,大气CO₂浓度升高对粳稻生育后期剑叶P_n、g_s和光合色素含量的影响明显大于增温效应.因此,应重视大气CO₂浓度和温度对水稻光合作用和光合色素的综合效应,减弱增温的负效应.     

CO2浓度升高、增温和冬小麦种植对土壤酶活性的影响

研究农田土壤酶活性对CO₂浓度升高和增温的响应,可为气候变化背景下农田生态系统养分管理提供科学依据。本研究在人工模拟气候室进行盆栽控制试验,设置了4种气候情景,分别为对照(CK,CO₂浓度400 μmol·mol^-1+正常环境温度)、CO₂浓度升高(ECO₂,CO₂浓度800 μmol·mol^-1+正常环境温度)、增温(ET,CO₂浓度400 μmol·mol^-1+增温4 ℃)及CO₂浓度和温度均升高(ECO₂+T,CO₂浓度800 μmol·mol^-1+增温4 ℃),研究有、无冬小麦生长下β--葡萄糖苷酶(βG)、β-N-乙酰葡糖苷酶(NAG)、碱性磷酸单脂酶(ALP)和多酚氧化酶(PPO)4种土壤酶活性在冬小麦拔节期(JS)、开花期(AS)、灌浆期(FS)和成熟期(MS)对CO₂浓度升高和增温的响应。结果表明:无冬小麦生长下,ECO₂与CK间4种土壤酶活性差异不显著,而ET和ECO₂+T处理对4种土壤酶活性有显著抑制作用。有冬小麦生长条件下,与CK相比,ECO₂和ECO₂+T处理对4种土壤酶活性均无显著影响;ET处理对土壤ALP和PPO活性有显著影响;ECO₂+T与ET间4种土壤酶活性有显著差异,与ET相比,ECO₂+T处理的土壤βG活性在JS期显著增加,NAG活性在JS期显著降低,ALP活性在AS和FS期显著增加,PPO活性在JS期显著降低,而在AS期显著增加。CO₂浓度升高与增温的交互作用在有、无冬小麦生长下均对土壤NAG和ALP活性有显著影响;无冬小麦生长下,增温和试验时段的交互作用对4种土壤酶活性有显著影响,而在有冬小麦生长下,增温和生育期的交互作用仅对ALP和PPO活性有显著影响;CO₂浓度升高、增温与试验时段的交互作用在无冬小麦生长下对土壤βG、ALP和PPO活性有显著影响,而在有冬小麦生长下CO₂浓度升高、增温与生育期对土壤NAG、ALP和PPO活性有显著影响。冬小麦生长对土壤βG、NAG和ALP活性在前两个生育期(JS+AS期)表现为显著抑制作用,在后两个生育期(FS+MS期)表现为显著促进作用,对土壤PPO活性在全生育期均表现为显著抑制作用。总体上,CO₂浓度升高对冬小麦土壤酶活性的影响不显著,而CO₂浓度与温度均升高对冬小麦土壤酶活性的影响在不同生育期因土壤酶种类不同而不同;此外,有、无冬小麦条件下4种土壤酶活性对CO₂浓度升高与增温的交互作用响应程度不一。     

透光抚育对温带帽儿山红松林非生长季土壤温室气体排放的影响

非生长季土壤温室气体排放在碳氮循环中具有重要作用,而采伐干扰对非生长季森林土壤温室气体排放具有何种影响并不明确.采用静态暗箱-气相色谱法,同步观测温带帽儿山50年生红松人工林在不同透光抚育方式下(次生林冠下栽植红松10年时设立为对照;半透光抚育: 伐除上层林木50%;全透光抚育: 伐除上层林木100%)非生长季土壤3种温室气体(CO₂、CH₄和N₂O)排放通量及其相关环境因子(土壤温度、含水量及碳氮含量等),研究采伐干扰对非生长季森林土壤温室气体排放的影响及主控因子.结果表明: 透光抚育会降低非生长季土壤CO₂、CH₄和N₂O的排放通量,全透光和半透光抚育显著降低了温带红松林非生长季土壤CO₂排放量21.0%和22.8%,并降低CH₄吸收量16.0%和16.4%,但差异不显著,全透光抚育显著降低N₂O排放量23.5%,而半透光抚育降低11.2%且差异不显著.温带红松林非生长季土壤CO₂、CH₄和N₂O的年贡献率分别为11.7%～14.2%、13.1%～17.0%和63.9%～72.6%,透光抚育降低了非生长季土壤CO₂(1.4%～2.5%)和CH₄(0.7%～3.9%)年贡献率,但提高了N₂O年贡献率(2.4%～8.7%).透光抚育增加了CO₂排放与土壤温度、含水量、硝态氮及铵态氮的相关性,降低其与有机碳的相关性,增加了CH₄排放与土壤含水量、酸碱度、有机碳和铵态氮的相关性,降低其与硝态氮的相关性,增加了N₂O排放与土壤温度相关性,降低其与硝态氮和铵态氮的相关性,改变了其与土壤酸碱度的正负相关关系.因此,透光抚育经营方式能够显著影响温带森林非生长季土壤温室气体排放,其中全透光抚育降低非生长季土壤N₂O排放能力要强于半透光抚育.     

Consumption of O2 in the light by Chlorella pyrenoidosa and Chlamydomonas reinhardtii

A mass spectrometer has been used to measure O₂ production and consumption and net CO₂ exchange near the CO₂ compensation point in Chlorella pyrenoidosa and Chlamydomonas reinhardtii grown in air and in 2% (v/v) and 5% (v/v) CO₂ in air. Some experiments were run in presence of -hydroxy-2-pyridinemethanesulfonic acid and, for others, data are presented from parallel assays for enzymic glycolate oxidation. Although the glycolate-oxidising enzyme was present in cells grown with and without CO₂ enrichment, the activities measured were not compatible with direct measurements of O₂ consumption in the light. This and other evidence argues against glycolate being the primary substrate for light-induced O₂ consumption by these organisms.