毒素的產(chǎn)生需要碳、氮、磷以及其他元素。

首先碳是細(xì)胞生長、毒素合成所必需的基本元素，對于有毒甲藻來說，大多數(shù)是光合自養(yǎng)生物，甲藻通過光合作用吸收空氣中的CO2來利用碳，碳元素進(jìn)入藻內(nèi)首先參與和生長有關(guān)的初級代謝，然后再參與和毒素合成有關(guān)的次級代謝。

如果碳元素供應(yīng)不足，產(chǎn)毒就會減少。Boyer et al.（1987）和Anderson et al.（1990b）都發(fā)現(xiàn)往Alexandriun tamarense培養(yǎng)液里添加碳酸氫鹽可以增加生長，而Wang et al.（2002a）發(fā)現(xiàn)往A. tamarense培養(yǎng)液中充氣（CO2）可以增加細(xì)胞生物量和毒素產(chǎn)量。

氮元素在毒素組成中占有重要地位，麻痹性貝毒本身就是氮豐富的化合物，氮在麻痹性貝毒分子中占到17-35%的重量，而在A. tamarense（MacIntyre et al., 1997）、A. minutum（Flynn et al., 1994）、以及Gymnodinium catenatum（Flynn et al., 1996b）中，麻痹性貝毒中的氮占到整個細(xì)胞總氮的5-10%。氮缺乏會造成甲藻細(xì)胞毒素含量下降（Anderson et al., 1990a; Flynn et al., 1994; MacIntyre et al.,1997; John et al., 2000; Wang et al., 2002b, 2005b）。

盡管麻痹性貝毒分子中并不含有磷元素，但培養(yǎng)液中的磷卻顯著影響毒素的合成，磷限制下毒素的含量會顯著增加已經(jīng)被很好的闡述（Boyer et al., 1987;Anderson et al., 1990b; Flynn et al., 1994; Bechemin et al., 1999; John et al.,2000; Wang et al., 2002b, 2005b; Matsuda, et al.,2006）。

考慮到細(xì)胞分裂過程中富磷的DNA的情況，Anderson et al.（1990b）提出：磷限制造成胞內(nèi)精氨酸的增加，因為減少了細(xì)胞分裂過程中磷依賴途徑，而這些途徑是競爭利用精氨酸的，而精氨酸被認(rèn)為是麻痹性貝毒合成的前體（Shimizu, 1996）。Flynn et al.（1996）指出在A. minutum中毒素合成是和胞內(nèi)氨基酸，特別是精氨酸的濃度成比例。磷限制抑制了細(xì)胞分裂，但能使細(xì)胞繼續(xù)合成毒素或者將胞內(nèi)其他組分轉(zhuǎn)化為毒素。有關(guān)微量金屬在毒素合成中的作用現(xiàn)在還知之甚少，一個研究是將A. tamarense培養(yǎng)在鐵限制條件下，細(xì)胞毒素含量在穩(wěn)定期比對照組含量有稍微的增高（Boyer et al., 1985），影響可能是非直接的，而是通過光合作用過程中的某些中間體，如鐵蛋白，鐵氧化還原蛋白，細(xì)胞色素等來作用的。微量金屬對毒素產(chǎn)生的影響還需進(jìn)一步的研究去證實(shí)。

This enriched seawater medium was designed specifically for oligotrophic (oceanic) marine phytoplankters that are poisoned by higher levels of trace metals. The medium uses a 10 fold higher EDTA chelation than most common marine media, and a substantial number of trace elements are included. The necessity of Tris is questionable, and it may be omitted. If organisms do not require silica, the silicate solution should be omitted because it enhances precipitation.

To prepare, begin with 950 mL of filtered natural seawater, add the following components and then bring the final volume up to 1 liter with filtered natural seawater. Autoclave.

Trace Metal Solution

To prepare, dissolve the following components to 950 mL of dH₂O (heat if necessary) and adjust the pH up with sodium hydroxide until all the components are in solution, approximately 20 pellets per liter of trace metal solution.? Bring the final volume to 1 liter using dH₂O.

f/2 Vitamin Solution

(Guillard and Ryther 1962, Guillard 1975)

First, prepare primary stock solutions. To prepare final vitamin solution, begin with 950 mL of dH₂O, dissolve the thiamine, add the amounts of the primary stocks as indicated in the quantity column below, and bring final volume to 1 liter with dH₂O.At the NCMA we autoclave to sterilize. Store in refrigerator or freezer.

Guillard, R.R.L. 1975. Culture of phytoplankton for feeding marine invertebrates. pp 26-60.?In?Smith W.L. and Chanley M.H (Eds.)?Culture of Marine Invertebrate Animals. Plenum Press, New York, USA.

Guillard, R.R.L. and Ryther, J.H. 1962. Studies of marine planktonic diatoms. I.?Cyclotella nana?Hustedt and?Detonula confervacea?Cleve.?Can. J. Microbiol.?8:?229-239.

Keller, M.D. and Guillard, R.R.L. 1985. Factors significant to marine diatom culture. pp. 113-6.?In?Anderson, D.M., White, A.W. and Baden, D.G. (eds.)?Toxic Dinoflagellates. Elsevier, New York.

Keller, M.D., Selvin, R.C., Claus, W. and Guillard, R.R.L. 1987. Media for the culture of oceanic ultraphytoplankton.?J. Phycol.?23:?633-638.

*please note that Na₂?b-glycerophosphate is sold as the disodium X hydrate (each batch having a different hydrate level) or as 5 hydrate. The 1987 paper used an x hydrate chemical even though the concentration of the stock? was based on an anhydrous weight. Bob Guillard confirmed this for me 10Mar2011.? We here at the CCMP have used an x hydrate chemical preparing a 2.16 gram per liter phosphate stock.