We thank Hisayoshi Nozaki (University of Tokyo), Takashi Nakada (Keio University), Yuka Marukawa Hashimoto (Japan Women&#39;s University), and Hiroyuki Sekimoto (Japan Women&#39;s University). We wish to thank the reviewers for their comments. This study was supported by a Grant-in-Aid for Scientific Research (No. 26440223 and 15H04413) from the Japan Society for the Promotion of Science, Japan, and by a grant from the New Technology Development Foundation to Yuki Tsuchikane.

1.&#160;Collection of a Water Sample Containing Algae
NOTE: Unicellular conjugatophytes grow in stagnant freshwater areas, such as lakes, marshes, and paddy fields. An algae-containing water sample is collected from one of these environments to produce a culture strain. The following items are needed before starting the process: a plankton net, a disposable pipette, and a bottle or a 50 mL tube for collecting water samples.
<ol>
	<li>Use a plankton net in a lake environment to collect algae from the water. Use an appropriate net, depending on the purpose. 
	NOTE: The upper part of the net is permeable to water. The algae caught by the net are concentrated into the bottom container of the plankton net. A coarse mesh allows small algae to pass through. A fine mesh gets clogged easily.</li>
	<li>Transfer the water (about 50 mL) to the 50 mL tube or a 100 mL plastic bottle. Bring the concentrated water sample to the laboratory.</li>
	<li>Unicellular conjugatophytes also grow in marshes or paddy fields. In these shallow water areas, water sample containing algae can be directly sampled with a dropper. Then, transfer the water (30 - 50 mL) to the 50 mL tube or a 50 mL plastic bottle. 
	NOTE: The water is carefully sampled above the soil surface so that the sample does not contain any soil.</li>
</ol>
2.&#160;Confirmation of Algae
NOTE: The following items are required: a loupe or portable microscope and a 60 mm dish for observation.
<ol>
	<li>To confirm the presence of algae in the sample while it is in the disposable pipette, use the loupe to observe the sample directly. 
	NOTE: Larger cells of about 400 - 1,000 &#181;m in length (e.g., Closterium ehrenbergii) can easily be observed using this method.</li>
	<li>To confirm algae under the portable microscope, pour the water sample in a plastic laboratory dish.
	<ol>
		<li>Put the sample (about 3 mL) on the inner part of the upper half (lid) of the 60 mm Petri dish, then place the lower half, with its bottom part facing the inner part of the lid, on top. 
		NOTE: This method keeps the sample from moving and makes the observation easier.</li>
	</ol>
	</li>
</ol>
3.&#160;Preparation of a Glass Capillary Pipette from a Pasteur Pipette for the Isolation
NOTE: Before starting the process, the following items are needed: sterilized Pasteur pipettes, a rack for the Pasteur pipettes, gloves, a rubber bulb, a spirit lamp, methanol for the spirit lamp, a lighter, forceps, a trash can for glass, and a clean room or laminar flow hood (if necessary).
<ol>
	<li>Ignite an alcohol burner.
	<ol>
		<li>To produce a fine glass capillary pipette for the isolation, make sure the flame is not flickering. Sharpen the flame and melt the Pasteur pipette at its tip.</li>
	</ol>
	</li>
	<li>Heat a sterile glass Pasteur pipette on the flame, supported on the side of the rubber bulb by hand and on the tip side by using forceps.</li>
	<li>Take the Pasteur pipette off the flame and stretch it.
	<ol>
		<li>When the glass is melting, quickly remove the Pasteur pipette from the flame and then pull. 
		NOTE: In this experiment, the Pasteur pipette was extended by 15 - 20 cm. Make sure that the pipette is off the flame during the pulling.</li>
	</ol>
	</li>
	<li>Break the tip of the glass capillary pipette to the proper length. Because the bowing part is the finest, it is ideal for the tip. 
	NOTE: In this study, a glass capillary pipette with a tip of 5 - 10 cm was prepared.
	<ol>
		<li>Be sure to discard the capillary after melting it, and confirm that the flame is put out.</li>
	</ol>
	</li>
	<li>The bubbles released from the glass capillary pipette indicate the size of the tip opening. Select the proper size for the cell to be isolated. About 1 mm of bubbles is proper for the isolation of Closterium species, with cell widths of 10 - 20 &#181;m.</li>
</ol>
4.&#160;Single-cell Isolation by a Glass Capillary Pipette
NOTE: The following items are needed before starting: an inverted light microscope, watch glasses, a plastic dish, and sterile threaded test tubes containing CA medium (Table 1) or conditioned CA medium7.
<ol>
	<li>Prepare a few watch glasses (22 mm in diameter and 1 mm in depth), each containing 1 mL of sterile CA medium.</li>
	<li>Isolate a target cell from the water sample collected in the field.
	<ol>
		<li>Pour the water sample (about 30 - 40 mL) containing the target cells into the plastic dish.</li>
		<li>While observing the sample in the plastic dish under an inverted light microscope, pick up single cells using the glass capillary pipette.</li>
		<li>Bring the glass capillary pipette near the cell to facilitate the aspiration of the cell by capillary action.</li>
	</ol>
	</li>
	<li>Transfer the cells into a watch glass containing 1 mL of sterile CA medium (Figure 1a).</li>
	<li>Pick up the cells again using a new glass capillary pipette from the sterile CA medium and transfer it to a second watch glass containing sterile CA medium (Figure 1b). 
	NOTE: This process is repeated until a single cell is isolated in the spot plate (Figure 1c).</li>
	<li>Pick up the single cell using a new glass capillary pipette, and finally transfer it to a test tube containing 14 mL of CA medium or conditioned CA medium (Figure 1d). Incubate the sample at 23 &#176;C under a 16-h light:8-h dark cycle for 2 weeks. 
	NOTE: The unknown factor(s) for cell proliferation, which are secreted from growing cells into the surrounding environment, are included in the conditioned medium to facilitate cell division4. If a clonal culture is established, prepare the conditioned CA medium from the culture medium4. The culture will turn green if successful.</li>
</ol>

<ol><li>Wickett, N. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phylotranscriptomic+analysis+of+the+origin+and+early+diversification+of+land+plants%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences%22%5BJournal%5D)">Phylotranscriptomic analysis of the origin and early diversification of land plants.</a> Proceedings of the National Academy of Sciences. 111, E4859-E4868 (2014).</li>
<li>Delwiche, C. F., Cooper, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+evolutionary+origin+of+a+terrestrial+flora%5BTitle%5D)+AND+%22Current+Biology%22%5BJournal%5D)">The evolutionary origin of a terrestrial flora.</a> Current Biology. 25, R899-R910 (2015).</li>
<li>Daste, P., Neuville, D., Victor-Baptiste, B. <a target="_blank" href="https://www.tandfonline.com/doi/abs/10.1080/00071618300650011">A simple procedure for obtaining clonal isolation of diatoms.</a> British Journal of Psychology. 18, 1-3 (1983).</li>
<li>Pringsheim, E. G. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aPringsheim%2C+EG+%22Pure+Cultures+of+Algae%3A+Their+Preparation+and+Maintenance%22">Pure Cultures of Algae: Their Preparation and Maintenance</a>. , Cambridge University Press. London, UK. 119(1946).</li>
<li>Sieracki, M., Poulton, N., Crosbie, N. Automated isolation techniques for microalgae. <a target="_blank" href="https://www.elsevier.com/books/algal-culturing-techniques/andersen/978-0-12-088426-1">Algal Culturing Techniques: Automated isolation techniques for microalgae</a>. Andersen, R. A. , Elsevier Academic Press. 101-116 (2005).</li>
<li>Andersen, R. A., Kawachi, M. Traditional microalgae isolation techniques. <a target="_blank" href="https://www.elsevier.com/books/algal-culturing-techniques/andersen/978-0-12-088426-1">Algal Culturing Techniques: Traditional micro algae isolation techniques</a>. Andersen, R. A. , Elsevier Academic Press. 83-100 (2005).</li>
<li>Abe, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stable+nuclear+transformation+of+the+Closterium+peracerosum-strigosum-littorale+complex%5BTitle%5D)+AND+%22Plant+and+Cell+physiology%22%5BJournal%5D)">Stable nuclear transformation of the Closterium peracerosum-strigosum-littorale complex.</a> Plant and Cell physiology. 52, 1676-1685 (2011).</li>
<li>Tsuchikane, Y., Tsuchiya, M., Kokubun, Y., Abe, J., Sekimoto, H. <a target="_blank" href="https://onlinelibrary.wiley.com/doi/abs/10.1111/j.1440-1835.2010.00602.x">Conjugation processes of Penium margaritaceum (Zygnemophyceae, Charophyta).</a> Phycological Research. 59, 74-82 (2011).</li>
<li>Kanda, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CRISPR%2FCas9-based+knockouts+reveal+that+CpRLP1+is+a+negative+regulator+of+the+sex+pheromone+PR-IP+in+the+Closterium+peracerosum-strigosum-littorale+complex%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">CRISPR/Cas9-based knockouts reveal that CpRLP1 is a negative regulator of the sex pheromone PR-IP in the Closterium peracerosum-strigosum-littorale complex.</a> Scientific Reports. 7, 17873(2017).</li>
<li>Ichimura, T. <a target="_blank" href="https://link.springer.com/article/10.1007/BF02493393">Mating types and reproductive isolation in Closterium-ehrenbergii Meneghini.</a> Botanical Magazine-Tokyo. 94, 325-334 (1981).</li>
<li>Sekimoto, H., Satoh, S., Fujii, T. <a target="_blank" href="https://link.springer.com/article/10.1007/BF02411384">Biochemical and physiological properties of a protein inducing protoplast release during conjugation in the Closterium peracerosum-strigosum-littorale complex.</a> Planta. 182, 348-354 (1990).</li>
<li>Ichimura, T., Watanabe, M. <a target="_blank" href="http://agris.fao.org/agris-search/search.do?recordID=US201302724192">The Closterium calosporum complex from the Ryukyu Islands-Variation and taxonomical problems.</a> Memoirs of the National Science Museum. 7, (1974).</li>
<li>Provasoli, L., Pintner, I. J. Artificial media for fresh-water algae: problems and suggestions . <a target="_blank" href="https://archive.org/details/ecologyofalgaesy00tryo">The Ecology of Algae, a symposium held at the Pymatuning Laboratory of Field Biology on June 18 and 19, 1959</a>. , Pymatuning Laboratory of Field Biology, University of Pittsburgh, . Pittsburgh. 84-96 (1960).</li>
<li>Provasoli, L. Media & prospects for the cultivation of marine algae. <a target="_blank" href="https://ci.nii.ac.jp/naid/10008776057/">Proceedings of U. S.-Japan Conference in Hakone, Japan</a>. Tryon, C. A. Jr, Hartmann, R. T. , Japanese Society of Plant Physiologists. 63-75 (1968).</li>
<li>Guillard, R. R. L. Purification methods for microalgae. <a target="_blank" href="https://www.elsevier.com/books/algal-culturing-techniques/andersen/978-0-12-088426-1">Algal Culturing Techniques: Purification methods for microalgae</a>. Andersen, R. A. , Elsevier Academic Press. 117-132 (2005).</li>
</ol>

Using the present method, 9 clonal culture strains of Closterium sp. were established from 15 cells isolated from the water sample (Inba1), representing a 60% success rate. Species identification will be carried out in the future by morphological observation as well as DNA analyses, such as molecular phylogenetic analysis8.
In the present method, the freshness of the water sample is important to the success rate. It is better to isolate the cells from the water samples as soon as possible after the field collection. Although cells of the Closterium species (e.g., C. ehrenbergii, C. peracerosum-strigosum-littorale complex) can be maintained in the water sample for about 7 d by storing it at 4 - 8 &#176;C, the isolation of the cells is desirable on the day of the collection or the next day. It is also important to remove any contaminants other than the target cells, so increasing the number of washing repetitions (i.e., &#62; 3x) may prevent a contamination of the cultures by microorganisms in the soil and cells.
A limitation of this technique is that the culture media used in this protocol (CA or conditioned CA medium) are not always appropriate for all conjugating algae. In some cases, it may be necessary to consider using another medium, as well as different light and temperature conditions. Also, as it is difficult to prove whether a culture has grown from one single cell, it is necessary to accurately pick up only 1 cell when transferring cells to a test tube. Therefore, the transferring should be done with a new glass capillary pipette each time.
Establishing a clonal culture with this pipette washing method is a classical/standard method and is the most reliable method to use to isolate the desired cells for a study. The clonal cultures of conjugating algae used in many studies8,9,10,11 were established using the present method. It is difficult to analyze conjugating algae without a clonal culture. Furthermore, in recent years, de novo whole genome sequences became easy to obtain (e.g., by using the MinION nanopore sequencer), and establishing clonal cultures will further facilitate de novo sequencing. In other words, this method is the first step in the future study of these algae to better understand their biodiversity.
In order to establish a stable culture strain, it is necessary to perform certain critical steps accurately within the protocol. The most important step is the preparation of a glass capillary pipette with an optimal aperture to permit the passage of a single cell only. The aperture of the glass capillary pipette must be slightly more than the minor axis length of the cell of interest. The optimum glass capillary pipette can aspirate a single cell by capillary action. However, if the diameter of the aperture is too large, the capillary will also draw in non-living contaminant materials or even (an)other organism(s), in addition to the target cell.
To obtain a glass capillary pipette with an optimum aperture, the following points are important to note. First, the pillar of flame must be narrow when heating the glass capillary pipette. Next, when pulling the Pasteur pipette, it has to be removed from the flame; otherwise, the aperture of the Pasteur pipette will collapse.
The equipment and containers shown in this protocol are basic and can be modified. For example, by using multi-well plates instead of watch glasses with one well, the cell washing can be made more efficient. In addition, the containers can be substituted for other similar ones. By transferring a single cell to the culture medium at the last step, a clonal culture can be established.
Preparing a sterilized container and working in a hood will establish axenic (uncontaminated) strains. To prevent contamination, it is necessary to repeat the washing step with fresh aliquots more than 3x. Sometimes, bacteria are also sterilized by adding antibiotics15.
This method focused on desmid (Zygnematales) isolation, but by changing the size of the glass capillary pipette aperture, it can be applied to the isolation of differently sized plankton.

Conjugating algae (of the order Zygnematales), also known as conjugatophytes, occupy a key phylogenetic position as the closest living relatives of the immediate ancestors of land plants1,2. Established clonal cultures of algae have led to a wide variety of taxonomic, physiological, and genetic studies over the past years. The isolation techniques of microorganisms from a natural field have a long tradition3,4. In recent years, automated isolation techniques using flow cytometry have also been established5. The most common method used to obtain a single cell for the establishment of a clonal culture is single-cell isolation using a glass capillary pipette6. This traditional method requires skill and a precise experimental protocol.
In this article, we demonstrate the sampling of algae from field samples and the establishment of clonal cultures of unicellular conjugating algae, such as the Closterium species, using&#160;a glass capillary pipette. A water sample containing vegetative cells is collected from a field site (e.g., a lake, pond, or paddy field). The cells are isolated from the water sample by using a glass capillary pipette and then washed. The cells are cultured in a test tube containing nitrogen-supplemented medium (CA medium) at 24 &#176;C under a 16-h light:8-h dark regime. This method is also suitable for isolating other microalgae to generate clonal cultures.

<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

It is essential to establish clonal cultures of microalgae for use in studies of various topics, such as physiology, genetics, taxonomy, and microbiology. Thus, it is extremely important to develop techniques to establish clonal cultures. In this article, we demonstrate the establishment of clonal cultures of a conjugating alga. Water samples are collected from the field. Subsequently, cells are isolated using a glass capillary pipette, placed in media, and grown under conditions suitable for generating a clonal culture.

A water sample (Inba1) of 50 mL was collected from one sampling point in Lake Inba-numa (pH 8.1, 24.7 &#176;C; 35&#176;44&#8242;30.2&#8243;N 140&#176;12&#8242;08.2&#8243;E) at Sakura-shi, Chiba, Japan, on June 25, 2016. The water sample was stored at 4 - 8 &#176;C. The next day after the collection, a specimen of Closterium sp. was isolated from the water sample containing vegetative cells. Fifteen vegetative cells of identical morphology (Inba1-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -13, -14, and -15) were isolated from the sample and then washed using the pipette washing method. Fifteen isolated single cells were cultured in independent test tubes containing 15 mL of conditioned CA medium. After 2 weeks of incubation, cell proliferation was observed in 9 test tubes [Inba1-1, -3, -4, -5 (Figure 2a), -6, -9, -10, -12 (Figure 2b), and -13; Table 2]. Nine clonal cultures were established from the Inba1 sample isolates (Figure 3).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57761/57761fig1.jpg" /> 
Figure 1: The flow of single-cell isolation. (a) Transfer the single target algal cell from the original water sample to the sterile medium in the first watch glass. (b, c) Transfer the cell again to the next watch glass to wash. This procedure should be repeated until there are no other cells/contaminants than the target in the medium; three watch glasses are shown in the diagram here as an example. (d) Finally, transfer the washed cell to a test tube of the appropriate medium for growth. <a href="https://www.jove.com/files/ftp_upload/57761/57761fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57761/57761fig2v2.jpg" /> 
Figure 2: Photographs of vegetative cells of Closterium sp. (a) Inba1-5 and (b) Inba1-12 are shown here. The scale bar = 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/57761/57761fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57761/57761fig3.jpg" /> 
Figure 3: Establishment of a clonal culture from the Inba1 sample. The test tubes cultured for 2 weeks were photographed from the bottom. An asterisk indicates cell proliferation. The scale bar = 2 cm. <a href="https://www.jove.com/files/ftp_upload/57761/57761fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Name of the media</td>
			<td>Reference</td>
			<td colspan="2">Comments</td>
		</tr>
		<tr>
			<td rowspan="13">CA&#160;</td>
			<td rowspan="13">Ichimura and Watanabe12</td>
			<td>Ca(NO3)2&#183;4H2O</td>
			<td>2 mg</td>
		</tr>
		<tr>
			<td>KNO3</td>
			<td>10 mg</td>
		</tr>
		<tr>
			<td>NH4NO3</td>
			<td>5 mg</td>
		</tr>
		<tr>
			<td>&#946;&#8211;Na2glycerophosphate&#183;5H2O</td>
			<td>3 mg</td>
		</tr>
		<tr>
			<td>MgSO4&#183;7H2O&#160;</td>
			<td>2 mg</td>
		</tr>
		<tr>
			<td>Vitamin B12</td>
			<td>0.01 &#956;g</td>
		</tr>
		<tr>
			<td>Biotin</td>
			<td>0.01 &#956;g</td>
		</tr>
		<tr>
			<td>Thiamine HCl</td>
			<td>1 &#956;g</td>
		</tr>
		<tr>
			<td>PIV metals</td>
			<td>0.1 mL</td>
		</tr>
		<tr>
			<td>Fe (as EDTA; 1:1 molar)&#160;</td>
			<td>0.1 mL</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>40 mg</td>
		</tr>
		<tr>
			<td colspan="2">Add water to make 100 mL</td>
		</tr>
		<tr>
			<td colspan="2">pH adjust with NaOH to 7.2</td>
		</tr>
		<tr>
			<td rowspan="7">P IV metals</td>
			<td rowspan="7">Provasoli and Pintner13</td>
			<td>Na2EDTA&#183;2H2O</td>
			<td>100 mg</td>
		</tr>
		<tr>
			<td>FeCl3&#183;6H2O</td>
			<td>19.6 mg</td>
		</tr>
		<tr>
			<td>MnCl2&#183; 4H2O</td>
			<td>3.6 mg</td>
		</tr>
		<tr>
			<td>ZnCl2*</td>
			<td>1.04 mg</td>
		</tr>
		<tr>
			<td>CoCl2&#183;6H2O</td>
			<td>0.4 mg</td>
		</tr>
		<tr>
			<td>Na2MoO4&#183;2H2O</td>
			<td>0.25 mg</td>
		</tr>
		<tr>
			<td colspan="2">Add water to make 100 mL</td>
		</tr>
		<tr>
			<td rowspan="3">Fe (as EDTA; 1:1 molar )</td>
			<td rowspan="3">Provasoli14</td>
			<td>Fe(NH4)2(SO4)2&#183;6H2O,&#160;</td>
			<td>70.2 mg&#160;</td>
		</tr>
		<tr>
			<td>Na2EDTA&#183;2H2O,&#160;</td>
			<td>66 mg</td>
		</tr>
		<tr>
			<td colspan="2">Add water to make 100 mL</td>
		</tr>
		<tr>
			<td>Conditioned CA medium</td>
			<td>Abe et al.7</td>
			<td colspan="2">Incubate cells in fresh CA medium for 14&#8211;20 days. Collect the cultured medium by filtration using qualitative filter paper and sterilize the filtered medium by autoclaving (121 &#176;C for 15 min).</td>
		</tr>
		<tr>
			<td colspan="4">*In the NIES, 1.04 mg ZnCl2 is replaced by 2.2 mg ZnSO4&#183;7H2O.</td>
		</tr>
	</tbody>
</table>
Table 1: Formulations of the media used in this study.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Water sample</td>
			<td>Name of cell</td>
			<td>Status after culture</td>
			<td>Strain name</td>
		</tr>
		<tr>
			<td rowspan="15">Inba1</td>
			<td>-1</td>
			<td>Cells proliferated</td>
			<td>Inba1-1</td>
		</tr>
		<tr>
			<td>-2</td>
			<td>Not changed</td>
			<td></td>
		</tr>
		<tr>
			<td>-3</td>
			<td>Cells proliferated</td>
			<td>inba1-3</td>
		</tr>
		<tr>
			<td>-4</td>
			<td>Cells proliferated</td>
			<td>inba1-4</td>
		</tr>
		<tr>
			<td>-5</td>
			<td>Cells proliferated</td>
			<td>inba1-5</td>
		</tr>
		<tr>
			<td>-6</td>
			<td>Cells proliferated</td>
			<td>inba1-6</td>
		</tr>
		<tr>
			<td>-7</td>
			<td>Not changed</td>
			<td></td>
		</tr>
		<tr>
			<td>-8</td>
			<td>Not changed</td>
			<td></td>
		</tr>
		<tr>
			<td>-9</td>
			<td>Cells proliferated</td>
			<td>inba1-9</td>
		</tr>
		<tr>
			<td>-10</td>
			<td>Cells proliferated</td>
			<td>inba1-10</td>
		</tr>
		<tr>
			<td>-11</td>
			<td>Not changed</td>
			<td></td>
		</tr>
		<tr>
			<td>-12</td>
			<td>Cells proliferated</td>
			<td>inba1-12</td>
		</tr>
		<tr>
			<td>-13</td>
			<td>Cells proliferated</td>
			<td>inba1-13</td>
		</tr>
		<tr>
			<td>-14</td>
			<td>Not changed</td>
			<td></td>
		</tr>
		<tr>
			<td>-15</td>
			<td>Not changed</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 2: The isolation trial summary.

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

In this article, we demonstrate the establishment of clonal cultures of unicellular conjugating algal species collected from a natural field site.

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

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Research

JoVE Journal

Biology

13.2K Views.  Japan Women's University. In dit artikel tonen we de oprichting van klonen culturen soorten eencellige conjugating algen verzameld uit een natuurlijke veld site.

Video: 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.

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

Environment

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.

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.

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.

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.

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.

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

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.

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

Developmental Biology

Enhancing Chlorophyll Production in Microalgae via Growth Factor Optimization

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.

Author Spotlight: Optimizing Growth Factors for Production of Biotechnologically Relevant Secondary Metabolites

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.

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

Biochemistry

Stable Gene Integration in Chlorella vulgaris Using Agrobacterium tumefaciens

Agrobacterium tumefaciens-Mediated Genetic Engineering of Green Microalgae, Chlorella vulgaris

Agrobacterium tumefaciens-mediated transformation (AMT) serves as a widely employed tool for manipulating plant genomes. However, A. tumefaciens exhibit the capacity for gene transfer to a diverse array of species. Numerous microalgae species lack well-established methods for reliably integrating genes of interest into their nuclear genome. To harness the potential benefits of microalgal biotechnology, simple and efficient genome manipulation tools are crucial. Herein, an optimized AMT protocol is presented for the industrial microalgae species Chlorella vulgaris, utilizing the reporter green fluorescent protein (mGFP5) and the antibiotic resistance marker for Hygromycin B. Mutants are selected through plating on Tris-Acetate-Phosphate (TAP) media containing Hygromycin B and cefotaxime. Expression of mGFP5 is quantified via fluorescence after over ten generations of subculturing, indicating the stable transformation of the T-DNA cassette. This protocol allows for the reliable generation of multiple transgenic C. vulgaris colonies in under two weeks, employing the commercially available pCAMBIA1302 plant expression vector.

Author Spotlight: Optimized Transformation Protocol for Chlorella vulgaris Using Agrobacterium tumefaciens 

This protocol outlines the utilization of Agrobacterium tumefaciens-mediated transformation (AMT) for integrating gene(s) of interest into the nuclear genome of the green microalgae Chlorella vulgaris, leading to the production of stable transformants.

Agrobacterium tumefaciens-mediated transformation (AMT) serves as a widely employed tool for manipulating plant genomes. However, A. tumefaciens exhibit ...

Bioengineering

Inducing Polyp Bail-out in Coral Colonies to Obtain Individualized Micropropagates for Laboratory Experimental Use

Corals are colonial animals formed by modular units called polyps. Coral polyps are physiologically linked and connected by tissue. The phenomenon of polyp bail-out is a process induced by acute stress, in which coral polyps digest the tissue connecting them to the rest of the colony and ultimately detach from the skeleton to continue living as separate individuals. Coral biologists have acknowledged the process of polyp bail-out for years, but only recently the micropropagates generated by this process have been recognized as a model system for coral biology studies. The use of polyp bail-out can create a high number of clonal units from a single coral fragment. Another benefit is that single polyps or patches of polyps can be easily visualized under a microscope and maintained in highly standardized low-cost environments such as Petri dishes, flasks, and microfluidic chips. The present protocol demonstrates reproducible methods capable of inducing coral micropropagation and different approaches for maintaining the single polyps alive in the long term. This methodology was capable of successfully cultivating polyps of the coral species Pocillopora verrucosa for up to 8 weeks after bail-out, exhibiting the practicality of using individual coral polyps for coral research.

Polyp bail-out is a process induced by acute stress, in which coral polyps digest the tissue connecting them to their colony and detach from it to live as individuals. The present protocol describes how to induce coral micropropagation by bail-out using hypersaline or calcium-free seawater treatments.

Corals are colonial animals formed by modular units called polyps. Coral polyps are physiologically linked and connected by tissue. The phenomenon of ...

Establishment of a Clonal Culture of Unicellular Conjugating Algae

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