小体积的生物测定评估细菌/浮游植物共培养使用水脉冲幅度调制（WATER-PAM）荧光
Summary
The goal of this procedure is to demonstrate the reproducibility and adaptability of using a microtiter plate format for microalgal screening. This rapid screen combines WATER-Pulse-Amplitude-Modulated (WATER-PAM) fluorometry to measure photosynthetic yield as an indicator of Photosystem II (PSII) health with small volume bacterial-algal co-cultures.
Abstract
常规方法实验操作微藻雇用大量培养物（20毫升至5升），以使培养能在整个实验1-7进行二次取样。大容量二次抽样可能会产生问题，原因如下：1）它引起的变化的总体积与表面积：在实验过程中培养的体积比; 2）伪复制（ 即复制从相同的待遇烧瓶8个样本）经常采用而不是真正的复制（ 即从重复治疗抽样）; 3）在实验的持续时间是有限的总体积;和4）纯性培养物或通常的细菌微生物是困难的，因为污染子采样期间通常发生在长期实验来维持。
使用微量滴定板的使1毫升培养体积要用于每个复制，与内多达48个独立的处理一12.65 X 8.5×2.2厘米板，从而降低了实验体积并允许广泛的复制子采样未经任何处理。此外，这种技术可以被修改以适应多种实验格式，包括：细菌藻共培养，藻类的生理测试，以及毒素筛选9-11。各孔用藻类，细菌和/或共培养物可以被采样为众多的实验室程序，包括但不限于：水 – 脉冲幅度调制（WATER-PAM）荧光，显微镜检查，细菌菌落形成单位（cfu）的计数和流式细胞术。微量滴定板格式和水的PAM荧光的组合允许光化学产量与样品中，高再现性之间的低变异性等光化学参数的多个快速测量，并避免了子采样一个大玻璃瓶或锥形瓶中在实验的过程中的许多陷阱。
Introduction
浮游植物生理学历来研究中尺度实验范围从20毫升锥形瓶中，以5升大玻璃瓶中1-7。这个试验规模需要二次抽样实验监测，作为牺牲重复样品对每个时间点创建一个无法控制的实验装置。
的能力，同时使用相同的昼夜培养箱空间通过小型化的实验量为藻类生理学实验将减少或消除二次抽样和伪复制的限制从大量增加的独立实验的数目。微孔板格式已经开发出用1毫升培养体积的实验操作藻可变的条件藻类生物测定。这种小规模的试验体积允许要增加重复的次数，增加实验重复性由于重复样品之间降低的变异性实验中，并且允许真复制，同时保持试验对照（ 即无菌藻类培养物）140天（ 图2）12。
这种微量滴定板形式很容易适应于多种实验问题，如：没有细菌具有与其藻宿主共生，中性或致病相互作用？是另外一种化合物刺激或有毒的藻类？这些和其它问题，可在使用这种新格式9-11快速高通量的方式加以解决。
48孔微量滴定培养皿允许每个1毫升井是一个独立的实验装置所采样的单个时间点。各种参数可以从这个1毫升体积包括被采样，但不限于：使用水-脉冲幅度调制（WATER-PAM）荧光（参见材料和设备表 ）1叶绿素荧光和光化学参数3。WATER-PAM荧光是一种快速，非侵入性的技术，可用于监测与藻类13进行的实验。它允许光合效率和PSII健康从小培养体积测量（150 – 300μl的文化在培养基中稀释到2 – 4毫升体积WATER-PAM）14,15。除了水-PAM荧光，这种设置可以用来测量各种其它参数，包括但不限于：显微镜可视化附着到的藻细胞和变化，藻细胞形态的细菌;细菌菌落形成单位（CFU）计数;和流式细胞仪对藻类细胞计数和识别的亚群。
Protocol
Representative Results
Discussion
藻类生长在一个小型化的格式。
藻培养物在微量滴定板1毫升培养物体积的小型化允许一个实验中的复制增加。以确保藻是在整个实验的健康是非常重要的;执行一个生长曲线（ 图2），使用微量滴定板形式，以评估各种藻类介质，以确保满足所述藻类的营养需求。此外，为了优化昼夜周期（亮和暗期）和温度可能是重要的。适当的优化对于给定的藻类可以允许用于?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by Natural Sciences and Engineering Research Council of Canada (grant 402105), Canadian Foundation for Innovation (grant 129087) and Alberta Education and Training (grant AAETRCP-12-026-SEG) to RJC.
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| 10 cu. ft. Diurnal Incubator (6012-1) | Caron Corporate | 112310-6012-1-11 | www.caronproducts.com |
| Nunc EasYFlask 25cm2, Vent/Close Cap, 7mL working volume, 200/Cs | Thermo Fisher Scientific | N156340 | www.fishersci.ca |
| Multiwell TC Plates – 48 Well | BD Biosciences Discovery Labware | 353078 | www.bdbiosciences.com |
| P1000 Gilson The Pipetting Standard—Gilson's Pipetman | Mandel Scientific Company Inc. | GF-F123602 | www.mandel.ca |
| P10mL Gilson The Pipetting Standard—Gilson's Pipetman | Mandel Scientific Company Inc. | GF-F161201 | www.mandel.ca |
| Wide Orifice Tips nonsterile [100–1250 µL] | VWR International | 89079-468 | www.ca.vwr.com |
| Ultrafine Tips nonsterile [100–1250 µL] | VWR International | 89079-470 | www.ca.vwr.com |
| Finntip 10mL [Vol: 1-10mL] | Thermo Fisher Scientific | 9402151 | www.fishersci.ca |
| WATER-Pulse Amplitude Modulation (Water-ED) | Heinz Walz GmbH, Effeltrich, Germany | EDEE0232 | www.walz.com |
| 15 mm diameter quartz glass cuvette (WATER-K) | Caron Corporate | www.caronproducts.com | |
| Sodium Chloride (Crystalline/Certified ACS), Fisher Chemical | Thermo Fisher Scientific | Thermo Fisher Scientific | www.fishersci.ca |
| BD Difco Marine Broth 2216 | BD Biosciences Discovery Labware | BD Biosciences Discovery Labware | www.bdbiosciences.com |
| BD Bacto Agar | BD Biosciences Discovery Labware | BD Biosciences Discovery Labware | www.bdbiosciences.com |
| L1 Medium Kit, 50L | NCMA [National Center for Marine Algae and Microbiota | NCMA [National Center for Marine Algae and Microbiota | www.ncma.bigelow.org |
References
- Scarratt, M. G., Marchetti, A. Assessing microbial responses to iron enrichment in the Subarctic Northeast Pacific: Do microcosms reproduce the in situ condition?. Deep Sea Res Part II Top. Stud. Oceanogr. 53 (20-22), 2182-2200 (2006).
- Bidle, K. D., Haramaty, L., Barcelos E Ramos, J., Falkowski, P. Viral activation and recruitment of metacaspases in the unicellular coccolithophore, Emiliania huxleyi. Proc. Natl. Acad. Sci. U. S. A. 104 (14), 6049-6054 (2007).
- Moore, L. R., Goericke, R., Chisholm, S. W. Comparative physiology of Synechococcus and Prochlorococcus: influence of light and temperature on growth, pigments, fluorescence and absorptive. Mar. Ecol. Prog. Ser. 116, (1995).
- Iglesias-Rodriguez, M. D., Halloran, P. R. Phytoplankton calcification in a high-CO2 world. Science. 320 (5874), 336-340 (2008).
- Chen, M., Tang, H., Ma, H., Holland, T. C., Ng, K. Y. S., Salley, S. O. Effect of nutrients on growth and lipid accumulation in the green algae Dunaliella tertiolecta. Bioresour. Technol. 102 (2), 1649-1655 (2011).
- Lv, J. -. M., Cheng, L. -. H., Xu, X. -. H., Zhang, L., Chen, H. -. L. Enhanced lipid production of Chlorella vulgaris by adjustment of cultivation conditions. Bioresour. Technol. 101 (17), 6797-6804 (2010).
- Geider, R., Graziano, L., McKay, R. M. Responses of the photosynthetic apparatus of Dunaliella tertiolecta (Chlorophyceae) to nitrogen and phosphorus limitation. Eur. J. Phycol. 33 (4), 315-332 (1998).
- MacIntyre, H. L., Cullen, J. J. Using Cultures to Investigate the Physiological Ecology of Microalgae. Algal Cult. Tech. , 287-326 (2005).
- Blaise, C., Vasseur, P. Algal microplate toxicity test. Small-scale Freshw. Toxic. Investig. Vol. 1 Toxic. Test Methods. , 137-179 (2005).
- Skjelbred, B., Edvardsen, B., Andersen, T. A high-throughput method for measuring growth and loss rates in microalgal cultures. J. Appl. Phycol. 24, 1589-1599 (2012).
- Nagai, T., Taya, K., Annoh, H., Ishihara, S. Application of a fluorometric microplate algal toxicity assay for riverine periphytic algal species. Ecotoxicol. Environ. Saf. 94, 37-44 (2013).
- Seyedsayamdost, M. R., Case, R. J., Kolter, R., Clardy, J. The Jekyll-and-Hyde chemistry of Phaeobacter gallaeciensis. Nat. Chem. 3 (4), 331-335 (2011).
- Schreiber, U., Schliwa, U., Bilger, W. Continuous recording of photochemical and non-photochemical chlorophyll fluorescence quenching with a new type of modulation fluorometer. Photosynth. Res. 10 (1-2), 51-62 (1986).
- Jones, R. J., Ward, S., Amri, A. Y., Hoegh-Guldber, O. Changes in quantum efficiency of photosystem II of symbiotic dinoflagellates of corals after heat stress, and of bleached corals sampled after the 1998 Great Barrier Reef mass bleaching event. Mar. Freshw. Res. 51 (345), 659-668 (1998).
- Beer, S., Larsson, C., Poryan, O., Axelsson, L. Photosynthetic rates of Ulva (Chlorophyta) measured by pulse amplitude modulated fluorometry. Eur. J. Phycol. 35 (1), 69-74 (2000).
- . . WATER-PAM Chlorophyll Fluorometer. Instrument Description and Information for Users. , (2013).
- Maxwell, K., Johnson, G. M., Heers, J. Chlorophyll fluorescence–a practical guide. J. Exp. Bot. 51 (345), 659-668 (2000).
- Herigstad, B., Hamilton, M., Heersink, J. How to optimize the drop plate method for enumerating bacteria. J. Microbiol. Methods. 44 (2), 121-129 (2001).
- Kooten, O., Snel, J. The use of chlorophyll fluorescence nomenclature in plant stress physiology. Photosynth. Res. 25 (3), 147-150 (1990).
- Maxwell, K., Johnson, G. N. Chlorophyll fluorescence–a practical guide. J. Exp. Bot. 51 (345), 659-668 (2000).
- Schreiber, U. Pulse-Amplitude-Modulation (PAM) Fluorometry and Saturation Pulse Method: An Overview. Chlorophyll a Fluoresc. A Signat. Photosynth. , 279-319 (2004).
- Roháček, K., Barták, M. Technique of the modulated chlorophyll fluorescence: basic concepts, useful parameters, and some applications. Photosynthetica. 37 (3), 339-363 (1999).
- Da Silva, J. M., da Silva, A. B., Pádua, M. Modulated chlorophyll a fluorescence: a tool for teaching photosynthesis. J. Biol. Educ. 41 (4), 178-183 (2007).
- Vieira, S., Ribeiro, L., Jesus, B., Cartaxana, P., da Silva, J. M. Photosynthesis assessment in microphytobenthos using conventional and imaging pulse amplitude modulation fluorometry. Photochem. Photobiol. 89 (1), 97-102 (2013).
