Name: establishment of a clonal culture of unicellular conjugating algae
Uploaded: 2018-07-14T14:00:00.000+00:00
Duration: 10 min 53 s
Description: Watch this Scientific Journal Video about Establishment of a Clonal Culture of Unicellular Conjugating Algae at JoVE.com

我们感谢 Hisayoshi 野崎 (东京大学)、隆 Nakada (京庆大学)、原田 Marukawa 桥本 (日本女子大学) 和博行 Sekimoto (日本女子大学)。我们要感谢评价员的意见。这项研究得到了日本科学促进协会 (26440223 号和 15H04413) 资助的资助, 并得到了来自新科技发展基金会的赠款, Tsuchikane。

1. 收集含有藻类的水样
注: 单细胞 conjugatophytes 生长在停滞的淡水地区, 如湖泊、沼泽和稻田。从这些环境之一中收集含藻类的水样, 以产生一种培养菌株。在开始该过程之前需要下列项目: 浮游生物网, 一次性吸管, 和一个瓶子或50毫升管收集水样。
<ol><li>在湖泊环境中使用浮游生物网从水中收集藻类。根据用途, 使用适当的网。 注意: 网的上部对水是透水的。被网捕获的藻类集中在浮游生物网的底部容器中。一个粗糙的网格允许小藻类通过。细网容易被堵塞。</li>
	<li>将水 (约50毫升) 转移到50毫升管或100毫升塑料瓶。把浓缩的水样带到实验室。</li>
	<li>单细胞 conjugatophytes 也生长在沼泽或稻田。在这些浅水区域, 含有藻类的水样可以直接用吸管取样。然后, 将水 (30-50 毫升) 转移到50毫升管或50毫升塑料瓶。 注: 水仔细取样在土壤表面之上, 以便样品不包含任何土壤。</li>
</ol>2. 藻类的确认
注意: 以下项目是必需的: 放大镜或便携显微镜和60毫米的盘子观察。
<ol><li>为了确认样品中藻类存在于一次性吸管中, 请使用放大镜直接观察样品。 注意: 使用此方法可以很容易地观察到大约 400-1000 µm 的较大细胞 (例如,新月 ehrenbergii)。</li>
	<li>在便携式显微镜下确认藻类, 将水样倒入塑料实验盘中。<ol>
<li>将样品 (约3毫升) 放在60毫米培养皿的上半部分 (盖子) 的内侧, 然后将下半部分放在下部, 其底部朝向盖子的内部, 上面。 注: 此方法使样品不动, 使观察更容易。</li>
	</ol>
</li>
</ol>3. 从巴斯德吸管中制备玻璃毛细管吸管隔离
注意: 在开始该过程之前, 需要下列项目: 灭菌的巴斯德吸管, 一架为巴斯德吸管, 手套, 橡胶灯泡, 精神灯, 甲醇为精神灯, 打火机, 镊子, 垃圾桶为玻璃, 和一个干净的房间或层流流动罩 (如有必要)。
<ol><li>点燃酒精燃烧器。<ol>
<li>要生产一个细玻璃毛细管隔离, 确保火焰没有闪烁。锐化火焰, 并在其尖端熔化巴斯德吸管。</li>
	</ol>
</li>
	<li>用镊子在火焰上加热无菌玻璃巴斯德吸管, 用手和笔尖在橡胶灯泡一侧支承。</li>
	<li>把巴斯德吸管脱下火焰, 伸展它。<ol>
<li>当玻璃融化时, 迅速从火焰中取出巴斯德吸管, 然后拉。 注: 在本实验中, 巴斯德吸管的扩展为 15-20 厘米. 在拉拔过程中, 要确保吸管脱离火焰。</li>
	</ol>
</li>
	<li>将玻璃毛细管吸管的尖端折断到适当的长度。因为鞠躬的部分是最好的, 它是理想的尖端。 注: 在本研究中, 制备了一个 5-10 厘米尖端的玻璃毛细管吸管。<ol>
<li>在熔化后一定要丢弃毛细管, 并确认火焰熄灭。</li>
	</ol>
</li>
	<li>从玻璃毛细管吸管释放出来的气泡表明尖端开口的大小。选择要隔离的单元格的适当大小。大约1毫米气泡是适当的分离新月物种, 与细胞宽度 10-20 µm。</li>
</ol>4. 用玻璃毛细管吸管分离单细胞
注: 在启动前需要下列物品: 倒置光镜、手表眼镜、塑料碟和含钙介质的无菌螺纹试管 (表 1) 或条件 ca 中等7。
<ol><li>准备一些手表眼镜 (直径22毫米和1毫米深度), 每只含有1毫升的无菌钙培养基。</li>
	<li>将目标单元格与在该字段中收集的水样样本隔离。<ol>
<li>将含有靶细胞的水样 (约 30-40 毫升) 倒入塑料盘中。</li>
		<li>在用倒置的光镜观察塑料碟中的样品时, 用玻璃毛细管吸管提取单细胞。</li>
		<li>在细胞附近带上玻璃毛细管吸管, 用毛细管作用来促进细胞的吸入。</li>
	</ol>
</li>
	<li>将细胞转移到含有1毫升无菌 CA 培养基的手表玻璃上 (图 1a)。</li>
	<li>再用新的玻璃毛细管从不育的钙介质中提取细胞并将其转移到含有无菌 ca 介质的第二个手表玻璃上 (图 1b)。 注意: 此过程是重复的, 直到单个单元格被隔离在专版中 (图 1c)。</li>
	<li>使用新的玻璃毛细管吸管拾取单细胞, 最后将其转移到包含14毫升 ca 介质或条件 ca 介质的试管中 (图 1d)。在16小时 light:8-h 黑暗周期的2周内, 将样品孵化23摄氏度。 注: 细胞增殖的未知因素, 从生长细胞分泌到周围环境中, 包括在条件培养基中以促进细胞分裂4。如果建立克隆文化, 则从培养基4中制备条件 CA 培养基。如果成功, 文化将变成绿色。</li>
</ol>

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采用现有的方法, 从水样中分离出的15个细胞 (Inba1) 中, 建立了9株新月无性系培养菌株, 成功率达60%。今后将通过形态学观察和 DNA 分析等方法对物种进行鉴定, 如分子系统发育分析8。
在本方法中, 水样的新鲜度对成功率有重要意义。在野外采集后, 最好尽快将细胞从水样中分离出来。虽然新月物种的细胞 (例如, ehrenbergii, peracerosum-strigosum 护胸复合物) 可以保存在水样品约 7 d, 通过储存在 4-8 摄氏度, 细胞的隔离是可取的在收集的当天或第二天。除去目标细胞以外的任何污染物也很重要, 因此增加洗涤重复次数 (即> 3x) 可以防止土壤和细胞中的微生物对培养物造成污染。
这种技术的一个局限性是, 本协议中使用的培养基 (ca 或条件 ca 培养基) 并不总是适合所有的共轭藻类。在某些情况下, 可能需要考虑使用另一种介质, 以及不同的光和温度条件。此外, 由于很难证明某种文化是否从一个细胞中生长出来, 所以在将细胞转移到试管时, 必须准确地提取出1个细胞。因此, 每次都要用新的玻璃毛细管吸管进行转移。
用这种吸管洗涤法建立克隆文化是一种经典/标准方法, 是用来分离所需细胞进行研究的最可靠的方法。采用本方法建立了多项研究8、9、10、11共轭藻类的无性系培养。没有克隆文化, 很难分析共轭藻类。此外, 近年来,从头开始的整个基因组序列变得容易获得 (例如, 通过使用奴才纳米排序器), 并建立克隆文化将进一步促进新的排序。换言之, 这种方法是今后研究这些藻类的第一步, 以更好地了解它们的生物多样性。
为了建立稳定的文化张力, 必须在协议中准确地执行某些关键步骤。最重要的步骤是制备一个具有最佳孔径的玻璃毛细管吸管, 仅允许单个细胞通过。玻璃毛细管吸管的孔径必须略大于所感兴趣细胞的小轴长度。最佳的玻璃毛细管吸管可以通过毛细管作用吸入单个细胞。然而, 如果孔径的直径太大, 毛细管也将绘制非生物污染材料, 甚至 (a) 其他有机体, 除了目标细胞。
要获得最佳孔径的玻璃毛细管吸管, 必须注意以下几点。首先, 在加热玻璃毛细管时, 火焰的柱必须是狭窄的。其次, 当拔出巴斯德吸管时, 必须从火焰中除去它;否则, 巴斯德吸管的光圈就会崩溃。
本协议中显示的设备和容器是基本的, 可以修改。例如, 通过使用多孔板而不是用一个井观察眼镜, 可以使细胞洗涤更有效率。此外, 集装箱可以替代其他类似的。在最后一步, 通过将单个细胞转移到培养基上, 可以建立克隆培养。
准备一个灭菌容器和工作在一个罩将建立无菌 (无污染) 菌株。为了防止污染, 有必要重复清洗步骤与新鲜整除数超过3x。有时, 细菌也通过添加抗生素15消毒。
该方法侧重于 desmid (Zygnematales) 隔离, 但通过改变玻璃毛细管孔径的大小, 可以将其应用于不同大小的浮游生物的分离。

共轭藻类 (顺序 Zygnematales), 也被称为 conjugatophytes, 占据关键的系统发育地位, 作为最接近的生活亲属的直接祖先的土地植物1,2。在过去的几年里, 建立了藻类的克隆文化, 导致了各种各样的分类学、生理和遗传学研究。微生物的分离技术从一个自然领域有一个长的传统3,4。近年来, 采用流式细胞仪的自动隔离技术也建立了5。最常用的方法, 以获得一个单一的细胞建立克隆文化是单细胞隔离使用玻璃毛细管吸管6。这种传统的方法需要技巧和精确的实验协议。
在本文中, 我们展示了从田间样品中藻类的取样和建立单细胞共轭藻类的克隆文化, 如新月物种, 使用玻璃毛细管吸管。一个含有营养细胞的水样本是从一个田野遗址 (如湖泊、池塘或稻田) 收集的。细胞与水样分离, 使用玻璃毛细管吸管, 然后清洗。细胞是培养在一个试管中含有氮补充培养基 (CA 培养基) 在24摄氏度下, 在一个16小时 light:8-h 黑暗政权。该方法也适用于分离其他微藻生成无性系培养。

<table><tbody><tr><td>&lt;strong&gt;Sampling&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Plankton net</td><td>RIGO, Japan</td><td>N-NO380T</td><td>Mesh size: 32 &amp;micro;m</td></tr><tr><td>Portable microscope (e.g., Nature Scope FABRE)</td><td>Nikon, Japan</td><td>JAN: 4960759 206725</td><td>Magnification: 20&amp;times;</td></tr><tr><td>Loupe (e.g., Peak 1985 Steinheil System Magnifier)</td><td>Tohkai Sangyo Co. Ltd., Japan</td><td>1985-20</td><td>Magnification: 20&amp;times;</td></tr><tr><td>Spuit</td><td>EIKEN CHEMICAL CO. LTD. Japan</td><td>Sterile spuit No.4</td><td></td></tr><tr><td>50 mL Tube (50 mL centrifuge tubes with screw caps)</td><td>Labcon, USA</td><td>3181-345-008</td><td></td></tr><tr><td>Bottle (100 mL Polycarbonate bottle)</td><td>AS ONE Corporation, Japan</td><td>1-7403-01</td><td></td></tr><tr><td>60 mm dish</td><td>Asahi glass Co. Ltd., Japan</td><td>1010-060</td><td>&amp;nbsp;For observation of algae.&amp;nbsp; 60 mm/non-treated dish</td></tr><tr><td>&lt;strong&gt;Preparation of a micropipette&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Pasteur pipettes (cotton plugged 9&amp;rdquo; Pasteur Pipettes)</td><td>Fisher Scientific, USA</td><td>13-678-8B</td><td>7 &amp;times; 225 mm</td></tr><tr><td>Rubber bulb (SPOID SILICONE, 1 cc)</td><td>AS ONE Corporation, Japan</td><td>5-5669-01</td><td>10 &amp;times; 40 mm</td></tr><tr><td>Stainless forceps</td><td>AS ONE Corporation, Japan</td><td>PT-09</td><td>110 mm</td></tr><tr><td>&lt;strong&gt;Single-cell isolation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Watch glass (Blood reaction board)</td><td>Sekiya Rika Co., Ltd, Japan</td><td>F14-155-030</td><td>to rinse cells during isolation. 22 mm diameter and 1 mm depth</td></tr><tr><td>Threaded test tubes&amp;nbsp;</td><td>Fujimotorika, Co., Ltd, Japan</td><td>XX142</td><td>18 &amp;Oslash; &amp;times; 170 mm</td></tr><tr><td>Inverted light microscope</td><td>Olympus, Tokyo, Japan</td><td>CKX41N</td><td>for isolation of algae.</td></tr><tr><td>Plastic dish</td><td>Asahi glass Co. Ltd., Japan</td><td>SH90-15E&amp;nbsp;</td><td>&amp;nbsp;90 &amp;times; 15 mm</td></tr></tbody></table>

establishment of a clonal culture of unicellular conjugating algae

必须建立微藻的克隆文化, 用于研究各种主题, 如生理学、遗传学、分类学和微生物学。因此, 建立克隆文化的技术极为重要。本文展示了共轭藻无性系培养的建立。从田间采集水样。随后, 细胞被隔离使用玻璃毛细管吸管, 放置在媒体, 并生长在条件适合生成克隆文化。

在 2016年6月25日, 日本千叶樱花石 (Inba) 的一个取样点 (pH 8.1, 24.7 °c; 35°44′30.2″N 140°12′08.2″E) 采集了50毫升的水样 (Inba1)。水样品被存放在 4-8 °c。第二天收集后,新月的标本从含有营养细胞的水样中分离出来。十五相同形态的营养细胞 (Inba1-1,-2、-3、-4、5、-6、-7、-8、-9、-10、-11、-12、-13、-14、15、) 与样品分离, 然后使用吸管洗涤法进行清洗。在含有15毫升条件的钙培养基的独立试管中培养出十五株孤立单细胞。经过2周的孵化, 在9试管中观察到细胞增殖 [Inba1-1,-3,-4,-5 (图 2a),-6,-9,-10,-12 (图 2b), 和-13;表 2]。从 Inba1 样本分离株中建立了九种克隆培养物 (图 3)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57761/57761fig1.jpg"> 图 1: 单细胞分离的流动.(a) 将单一目标藻类细胞从原水样品转移到第一个观察玻璃中的无菌介质。(b、 c)再次将细胞转移到下一个手表玻璃上清洗。该程序应重复, 直到没有其他细胞/污染物比目标的媒介;三手表眼镜显示在图这里作为一个例子。(d) 最后, 将冲洗过的电池转移到适当培养基的试管中进行生长。<a href="https://www.jove.com/files/ftp_upload/57761/57761fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57761/57761fig2v2.jpg"> 图 2: 植物细胞的照片新月sp.(a) Inba1-5 和 (b) Inba1-12 显示在这里。刻度条 = 50 µm.<a href="https://www.jove.com/files/ftp_upload/57761/57761fig2v2large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57761/57761fig3.jpg"> 图 3: 从 Inba1 样品中建立克隆文化.从底部拍摄了2周培养的试管。星号表示细胞增殖。刻度条 = 2 厘米.<a href="https://www.jove.com/files/ftp_upload/57761/57761fig3large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>媒体名称</td>
			<td>参考</td>
			<td colspan="2">评论</td>
		</tr>
<tr>
<td rowspan="13">约</td>
			<td rowspan="13">市村和渡边12
</td>
			<td>Ca (NO3)2· 4H2O</td>
			<td>2毫克</td>
		</tr>
<tr>
<td>3
</td>
			<td>10毫克</td>
		</tr>
<tr>
<td>NH4NO3
</td>
			<td>5毫克</td>
		</tr>
<tr>
<td>β–Na2glycerophosphate·5H2O</td>
			<td>3毫克</td>
		</tr>
<tr>
<td>MgSO4· 7H2O</td>
			<td>2毫克</td>
		</tr>
<tr>
<td>维生素 B12</td>
			<td>0.01 微克</td>
		</tr>
<tr>
<td>素</td>
			<td>0.01 微克</td>
		</tr>
<tr>
<td>盐酸硫胺</td>
			<td>1微克</td>
		</tr>
<tr>
<td>PIV 金属</td>
			<td>0.1 毫升</td>
		</tr>
<tr>
<td>Fe (EDTA; 1:1 摩尔)</td>
			<td>0.1 毫升</td>
		</tr>
<tr>
<td>HEPES</td>
			<td>40毫克</td>
		</tr>
<tr>
<td colspan="2">加水使100毫升</td>
		</tr>
<tr>
<td colspan="2">pH 值与氢氧化钠调节为7。2</td>
		</tr>
<tr>
<td rowspan="7">P IV 金属</td>
			<td rowspan="7">Provasoli 和 Pintner13
</td>
			<td>Na2EDTA·2H2O</td>
			<td>100毫克</td>
		</tr>
<tr>
<td>FeCl3· 6H2O</td>
			<td>19.6 毫克</td>
		</tr>
<tr>
<td>MnCl2·4H2O</td>
			<td>3.6 毫克</td>
		</tr>
<tr>
<td>ZnCl2*</td>
			<td>1.04 毫克</td>
		</tr>
<tr>
<td>CoCl2· 6H2O</td>
			<td>0.4 毫克</td>
		</tr>
<tr>
<td>Na2MoO4· 2H2O</td>
			<td>0.25 毫克</td>
		</tr>
<tr>
<td colspan="2">加水使100毫升</td>
		</tr>
<tr>
<td rowspan="3">Fe (EDTA; 1:1 摩尔)</td>
			<td rowspan="3">Provasoli14
</td>
			<td>Fe (NH4)2(如此4)2· 6H2O,</td>
			<td>70.2 毫克</td>
		</tr>
<tr>
<td>Na2EDTA·2H2O,</td>
			<td>66毫克</td>
		</tr>
<tr>
<td colspan="2">加水使100毫升</td>
		</tr>
<tr>
<td>条件 CA 介质</td>
			<td>亚伯等7
</td>
			<td colspan="2">在新鲜的钙培养基中孵化细胞14–20天。用质滤纸过滤培养基, 通过热处理 (121 °c 15 分钟) 对过滤介质进行消毒。</td>
		</tr>
<tr>
<td colspan="4">* 在新兴工业化中, 1.04 毫克 ZnCl2被2.2 毫克 ZnSO4· 7H2O 取代。</td>
		</tr>
</tbody></table>表 1: 本研究使用的媒介配方。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>水样</td>
			<td>单元格名称</td>
			<td>文化后的状态</td>
			<td>应变名称</td>
		</tr>
<tr>
<td rowspan="15">Inba1</td>
			<td>-1</td>
			<td>细胞增殖</td>
			<td>Inba1-1</td>
		</tr>
<tr>
<td>-2</td>
			<td>未更改</td>
			<td></td>
		</tr>
<tr>
<td>-3</td>
			<td>细胞增殖</td>
			<td>inba1-3</td>
		</tr>
<tr>
<td>-4</td>
			<td>细胞增殖</td>
			<td>inba1-4</td>
		</tr>
<tr>
<td>-5</td>
			<td>细胞增殖</td>
			<td>inba1-5</td>
		</tr>
<tr>
<td>-6</td>
			<td>细胞增殖</td>
			<td>inba1-6</td>
		</tr>
<tr>
<td>-7</td>
			<td>未更改</td>
			<td></td>
		</tr>
<tr>
<td>-8</td>
			<td>未更改</td>
			<td></td>
		</tr>
<tr>
<td>-9</td>
			<td>细胞增殖</td>
			<td>inba1-9</td>
		</tr>
<tr>
<td>-10</td>
			<td>细胞增殖</td>
			<td>inba1-10</td>
		</tr>
<tr>
<td>-11</td>
			<td>未更改</td>
			<td></td>
		</tr>
<tr>
<td>-12</td>
			<td>细胞增殖</td>
			<td>inba1-12</td>
		</tr>
<tr>
<td>-13</td>
			<td>细胞增殖</td>
			<td>inba1-13</td>
		</tr>
<tr>
<td>-14</td>
			<td>未更改</td>
			<td></td>
		</tr>
<tr>
<td>-15</td>
			<td>未更改</td>
			<td></td>
		</tr>
</tbody></table>表 2: 隔离试验摘要。

Watch this Scientific Journal Video about Establishment of a Clonal Culture of Unicellular Conjugating Algae at JoVE.com

Establishment of a Clonal Culture of Unicellular Conjugating Algae

单细胞共轭藻类无性系培养的建立

在这篇文章中, 我们展示了建立的克隆文化的单细胞共轭藻类物种收集从一个自然的现场。

It is essential to establish clonal cultures of microalgae for use in studies of various topics, such as physiology, genetics, taxonomy, and microbiology. ...

establishment-of-a-clonal-culture-of-unicellular-conjugating-algae

Universidad San Sebastian

Research

JoVE Journal

Biology

13.2K 次观看.  Japan Women's University. 在这篇文章中, 我们展示了建立的克隆文化的单细胞共轭藻类物种收集从一个自然的现场。

视频: Establishment of a Clonal Culture of Unicellular Conjugating Algae

The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression

Barnacles are marine crustaceans with a sessile adult and free-swimming, planktonic larvae. The barnacle Balanus (Amphibalanus) improvisus is particularly relevant as a model for the studies of osmoregulatory mechanisms because of its extreme tolerance to low salinity. It is also widely used as a model of settling biology, in particular in relation to antifouling research. However, natural seasonal spawning yields an unpredictable supply of cyprid larvae for studies. A protocol for the all-year-round culturing of B. improvisus has been developed and a detailed description of all steps in the production line is outlined (i.e., the establishment of adult cultures on panels, the collection and rearing of barnacle larvae, and the administration of feed for adults and larvae). The description also provides guidance on troubleshooting and discusses critical parameters (e.g., the removal of contamination, the production of high-quality feed, the manpower needed, and the importance of high-quality seawater). Each batch from the culturing system maximally yields roughly 12,000 nauplii and can deliver four batches in a week, so up to almost 50,000 larvae per week can be produced. The method used to culture B. improvisus is, probably, to a large extent also applicable to other marine invertebrates with free-swimminglarvae. Protocols are presented for the dissection of various tissues from adults as well as the production of high-quality RNA for studies on gene expression. It is also described how cultured adults and reared cyprids can be utilized in a wide array of experimental designs for examining gene expression in relation to external factors. The use of cultured barnacles in gene expression is illustrated with studies of possible osmoregulatory roles of Na+/K+ ATPase and aquaporins.

The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression

The barnacle Balanus (Amphibalanus) improvisus is a model for studying osmoregulation and antifouling. However, natural seasonal spawning yields an unpredictable supply of cyprid larvae. Here, a protocol for the all-year-round culturing of B. improvisus is described, including the production of larvae. The use of cultured barnacles in gene expression studies is illustrated.

藤壶Balanus improvisus作为海洋模型的培养和基因表达

Barnacles are marine crustaceans with a sessile adult and free-swimming, planktonic larvae. The barnacle Balanus (Amphibalanus) improvisus is particularly ...

科研

环境科学

Cultivation of Green Microalgae in Bubble Column Photobioreactors and an Assay for Neutral Lipids

There is significant interest in the study of microalgae for engineering applications such as the production of biofuels, high value products, and for the treatment of wastes. As most new research efforts begin at laboratory scale, there is a need for cost-effective methods for culturing microalgae in a reproducible manner. Here, we communicate an effective approach to culture microalgae in laboratory-scale photobioreactors, and to measure the growth and neutral lipid content of that algae. Instructions are also included on how to set up the photobioreactor system. Although the example organisms are species of Chlorella and Auxenochlorella, this system can be adapted to cultivate a wide range of microalgae, including co-cultures of algae with non-algae species. Stock cultures are first grown in bottles to produce inoculum for the photobioreactor system. Algae inoculum is concentrated and transferred to photobioreactors for cultivation in batch mode. Samples are collected daily for the optical density readings. At the end of the batch culture, cells are harvested by centrifuge, washed, and freeze dried to obtain a final dry weight concentration. The final dry weight concentration is used to create a correlation between the optical density and the dry weight concentration. A modified Folch method is subsequently used to extract total lipids from the freeze-dried biomass and the extract is assayed for its neutral lipid content using a microplate assay. This assay has been published previously but protocol steps were included here to highlight critical steps in the procedure where errors frequently occur. The bioreactor system described here fills a niche between simple flask cultivation and fully-controlled commercial bioreactors. Even with only 3-4 biological replicates per treatment, our approach to culturing algae leads to tight standard deviations in the growth and lipid assays.

Here, we present a protocol to construct lab-scale bubble column photobioreactors and use them to culture microalgae. It also provides a method for the determination of the culture growth rate and neutral lipid content.

气泡柱光生物反应器中绿色微藻的培养及中性脂质的测定

There is significant interest in the study of microalgae for engineering applications such as the production of biofuels, high value products, and for the ...

Cultivation of the Marine Pelagic Tunicate Dolioletta gegenbauri (Uljanin 1884) for Experimental Studies

Gelatinous zooplanktons play a crucial role in ocean ecosystems. However, it is generally difficult to investigate their physiology, growth, fecundity, and trophic interactions primarily due to methodological challenges, including the ability to culture them. This is particularly true for the doliolid, Dolioletta gegenbauri. D. gegenbauri commonly occurs in productive subtropical continental shelf systems worldwide, often at bloom concentrations capable of consuming a large fraction of daily primary production. In this study, we describe cultivation approaches for collecting, rearing, and maintaining D. gegenbauri for the purpose of conducting laboratory-based studies. D. gegenbauri and other doliolid species can be captured live using obliquely towed conical 202 &micro;m mesh plankton nets from a drifting ship. Cultures are most reliably established when water temperatures are below 21 &deg;C and are started from immature gonozooids, maturing phorozooids, and large nurses. Cultures can be maintained in rounded culture vessels on a slowly rotating plankton wheel and sustained on a diet of cultured algae in natural seawater for many generations. In addition to the ability to establish laboratory cultures of D. gegenbauri, we demonstrate that the collection condition, algae concentration, temperature, and exposure to naturally conditioned seawater are all critical to the culture establishment, growth, survival, and reproduction of D. gegenbauri.

Cultivation of the Marine Pelagic Tunicate Dolioletta gegenbauri Uljanin 1884 for Experimental Studies

Doliolids, including the species Dolioletta gegenbauri, are small gelatinous marine zooplankton of ecological significance found on productive subcontinental shelf systems worldwide. The difficulty of culturing these delicate organisms limits their investigation. In this study, we describe cultivation approaches for collecting, rearing, and maintaining the doliolid Dolioletta gegenbauri.

用于实验研究的海洋可点多利奥莱塔·格根巴里(Uljanin 1884)的培养

Gelatinous zooplanktons play a crucial role in ocean ecosystems. However, it is generally difficult to investigate their physiology, growth, fecundity, ...

Streamlined Sampling and Cultivation of the Pelagic Cosmopolitan Larvacean, Oikopleura dioica

Oikopleura dioica is a planktonic chordate with exceptional filter-feeding ability, rapid generation time, conserved early development, and a compact genome. For these reasons, it is considered a useful model organism for marine ecological studies, evolutionary developmental biology, and genomics. As research often requires a steady supply of animal resources, it is useful to establish a reliable, low-maintenance culture system. Here we describe a step-by-step method for establishing an O. dioica culture. We describe how to select potential sampling sites, collection methods, target animal identification, and the set-up of the culturing system. We provide troubleshooting advice based on our own experiences. We also highlight critical factors that help sustain a robust culture system. Although the culture protocol provided here is optimized for O. dioica, we hope our sampling technique and culture setup will inspire new ideas for maintaining other fragile pelagic invertebrates.

Streamlined Sampling and Cultivation of the Pelagic Cosmopolitan Larvacean, Oikopleura dioica

Oikopleura dioica is a tunicate model organism in various fields of biology. We describe sampling methods, species identification, culturing setup, and culturing protocols for the animals and algal feed. We highlight key factors that helped strengthen the culture system and discuss the possible problems and resolutions.

优化取样和种植的佩拉吉大都会拉瓦切安，奥科普拉迪奥卡

Oikopleura dioica is a planktonic chordate with exceptional filter-feeding ability, rapid generation time, conserved early development, and a compact ...

Coupling Carbon Capture from a Power Plant with Semi-automated Open Raceway Ponds for Microalgae Cultivation

In the United States, 35% of the total carbon dioxide (CO2) emissions come from the electrical power industry, of which 30% represent natural gas electricity generation. Microalgae can biofix CO2 10 to 15 times faster than plants and convert algal biomass to products of interest, such as biofuels. Thus, this study presents a protocol that demonstrates the potential synergies of microalgae cultivation with a natural gas power plant situated in the southwestern United States in a hot semi-arid climate. State-of-the-art technologies are used to enhance carbon capture and utilization via the green algal species Chlorella sorokiniana, which can be further processed into biofuel. We describe a protocol involving a semi-automated open raceway pond and discuss the results of its performance when it was tested at the Tucson Electric Power plant, in Tucson, Arizona. Flue gas was used as the main carbon source to control pH, and Chlorella sorokiniana was cultivated. An optimized medium was used to grow the algae. The amount of CO2 added to the system as a function of time was closely monitored. Additionally, other physicochemical factors affecting algal growth rate, biomass productivity, and carbon fixation were monitored, including optical density, dissolved oxygen (DO), electroconductivity (EC), and air and pond temperatures. The results indicate that a microalgae yield of up to 0.385 g/L ash-free dry weight is attainable, with a lipid content of 24%. Leveraging synergistic opportunities between CO2 emitters and algal farmers can provide the resources required to increase carbon capture while supporting the sustainable production of algal biofuels and bioproducts.

A protocol is described to utilize the carbon dioxide in natural gas power plant flue gas to cultivate microalgae in open raceway ponds. Flue gas injection is controlled with a pH sensor, and microalgae growth is monitored with real time measurements of optical density.

将发电厂的碳捕获与半自动开放式管道池相结合，用于微藻养殖

In the United States, 35% of the total carbon dioxide (CO2) emissions come from the electrical power industry, of which 30% represent natural gas ...

Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis

The unicellular green alga Chlamydomonas reinhardtii (Chlamydomonas) has become a popular organism for research in diverse areas of cell biology and genetics because of its simple life cycle, ease of growth and manipulation for genetic analysis, genomic resources, and transformability of the nucleus and both organelles. Mating strains is a common practice when genetic approaches are used in Chlamydomonass, to create vegetative diploids for analysis of dominance, or following tetrad dissection to ascertain nuclear vs. organellar inheritance, to test allelism, to analyze epistasy, or to generate populations for the purpose of map-based cloning. Additionally, genetic crosses are routinely used to combine organellar genotypes with particular nuclear genotypes. Here we demonstrate standard methods for gametogenesis, mating, zygote germination and tetrad separation. This protocol consists of an easy-to-follow series of steps that will make genetic approaches amenable to scientists who are less familiar with Chlamydomonas. Key parameters and trouble spots are explained. Finally, resources for further information and alternative methods are provided.

Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis

Mating and tetrad separation are required for genetic analysis in Chlamydomonas reinhardtii. Here we demonstrate standard methods for gametogenesis, mating, zygote germination and tetrad dissection. This protocol consists of an easy-to-follow series of steps that will make genetic approaches amenable to scientists who are less familiar with Chlamydomonas.

交配和四分体分离衣藻遗传分析

The unicellular green alga Chlamydomonas reinhardtii (Chlamydomonas) has become a popular organism for research in diverse areas of cell biology and ...

生物学

A Multi-Omics Extraction Method for the In-Depth Analysis of Synchronized Cultures of the Green Alga Chlamydomonas reinhardtii

Microalgae have been the focus of research for their applications in the production of high value compounds, food and fuel. Moreover, they are valuable photosynthetic models facilitating the understanding of the basic cellular processes. System wide studies enable comprehensive and in-depth understanding of molecular functions of the organisms. However, multiple independent samples and protocols are required for proteomics, lipidomics and metabolomics studies introducing higher error and variability. A robust high throughput extraction method for the simultaneous extraction of chlorophyll, lipids, metabolites, proteins and starch from a single sample of the green alga Chlamydomonas reinhardtii is presented here. The illustrated experimental setup is for Chlamydomonas cultures synchronized using 12 h/12 h light/dark conditions. Samples were collected over a 24 h cell cycle to demonstrate that the metabolites, lipids and starch data obtained using various analytical platforms are well conformed. Furthermore, protein samples collected using the same extraction protocol were used to conduct detailed proteomics analysis to evaluate their quality and reproducibility. Based on the data, it can be inferred that the illustrated method provides a robust and reproducible approach to advance understanding of various biochemical pathways and their functions with greater confidence for both basic and applied research.

A Multi-Omics Extraction Method for the In-Depth Analysis of Synchronized Cultures of the Green Alga Chlamydomonas reinhardtii

System-wide analysis of multiple biomolecules is crucial to gain functional and mechanistic insights into biological processes. Hereby, an extensive protocol is described for high throughput extraction of lipids, metabolites, proteins and starch from a single sample harvested from synchronized Chlamydomonas culture.

一种对绿色藻原体同步文化进行深入分析的多表率提取方法

Microalgae have been the focus of research for their applications in the production of high value compounds, food and fuel. Moreover, they are valuable ...

发育生物学

<meta charset="utf8"></head><body>通过生长因子优化提高微藻中叶绿素的产生

Evaluation of the Effect of Growth Factors on Chlorophylls a and b Production from Microalgae

Microalgae contain two main groups of pigments: chlorophylls and carotenoids. Chlorophyll is a green pigment that absorbs light energy and transforms it into chemical energy to facilitate the synthesis of organic compounds. This pigment serves as a valuable primary source for biotechnological input products in the food, pharmaceutical, and cosmetic industries due to its high antioxidant properties and coloring capabilities. The objective of this research was to evaluate the effect of growth factors (CO2 concentration, light color, and light intensity) through a Taguchi L4 experimental design on cell growth and the cellular content of chlorophyll a and b in Chlorella sorokiniana, followed by validation of the method using Haematococcus pluvialis microalgae as an additional study model. Cell growth was quantified using the optical density spectrophotometric technique at a wavelength of 550 nm. For the quantification of chlorophylls, a cell extract was obtained using a 90% pure acetone solution, and subsequently, the concentrations of chlorophylls a and b were quantified using spectrophotometric techniques at wavelengths of 647 nm and 664 nm, according to the method described by Jeffrey and Humphrey. The experimental results indicated that controlling conditions of low CO2 addition, purple light, and low light intensity increases both cell growth and the concentration of chlorophylls a and b within the cells. The implementation of this chlorophyll quantification method allows for quick, simple, and precise determination of chlorophyll content, as the wavelengths used are at the absorbance peaks of both types of chlorophylls, making this technique easily reproducible for any microalgae under study.

<meta charset="utf8"></head><body>作者聚焦:优化生长因子以生产生物技术相关的次生代谢物

The present study highlights the advantages of employing the method developed by Jeffrey and Humphrey for extracting and quantifying fat-soluble pigments from microalgae. This method serves as a valuable tool for assessing the influence of growth factors on chlorophyll production and cellular content in these organisms.

生长因子对微藻叶绿素 a 和 b 生产的影响评价

Microalgae contain two main groups of pigments: chlorophylls and carotenoids. Chlorophyll is a green pigment that absorbs light energy and transforms it ...

单细胞共轭藻类无性系培养的建立

摘要

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一种对绿色藻原体同步文化进行深入分析的多表率提取方法

生长因子对微藻叶绿素 a 和 b 生产的影响评价

生长因子对微藻叶绿素 a 和 b 生产的影响评价

生长因子对微藻叶绿素 a 和 b 生产的影响评价