Work in the authors&#39; lab was supported by the Deutsche Forschungsgemeinschaft (http://www.dfg.de; CO 1139/1-1), the Fonds der Chemischen Industrie (http://www.vci.de/fonds), and the G&#246;ttingen Centre for Molecular Biology (GZMB). The authors would like to acknowledge J&#246;rg St&#252;lke for helpful comments and critical reading of the manuscript.

1. Preparation of Agar Plates, Culture Media, Cryostocks, and Precultures
<ol>
	<li>Prepare growth media and required reagents (see table of materials and reagents).</li>
	<li>Streak the B. subtilis strains (e.g.&#160;BP40 (rocG+ gudBCR amyE::PgudB-yfp) and BP52 (rocG+ gudB+ amyE::PgudB-cfp) expressing one and two active GDHs, respectively)2 that will be used in the competition experiment on SP medium agar plates to get single colonies. Incubate the plates overnight at 37 &#176;C.</li>
	<li>Take single colonies and inoculate sterile culture tubes containing 4 ml LB liquid medium. Grow the bacteria overnight at 28 &#176;C and 220 rpm.</li>
	<li>Grow overnight cultures at 28 &#176;C to avoid that the cells lyse or sporulate.</li>
	<li>Measure the optical density of the cultures at a wavelength of 600 nm (OD600) using a standard spectrophotometer.</li>
	<li>If the cultures have reached an OD600 of 2.0, mix 0.75 ml of the cultures with 0.75 ml of a sterile 50% glycerol solution in 1.5 ml reaction tubes. The final OD600 should be 1.0 to obtain cryostocks containing about 108 cells/ml.</li>
	<li>Store the tubes at -80 &#176;C.</li>
	<li>Make three precultures of each strain labeled with the cfp and yfp encoding fluorophore genes. Inoculate the precultures (sterile culture tubes containing 4 ml LB liquid medium) with 1 &#956;l cells from -80 &#176;C cryostocks. Incubate the cultures overnight at 28 &#176;C and 220 rpm.</li>
</ol>
2. Cocultivation of Bacteria, Sample Collection, and Sample Storage
<ol>
	<li>Freshly prepare 100 ml C-Glc and CE-Glc minimal medium (see table of reagents and materials), and transfer 20 ml of each medium into sterile 100 ml shake flasks.</li>
	<li>Take 0.1 ml of the precultures that were grown overnight, dilute them with 0.9 ml LB medium in a 1.5 ml cuvette, and determine the OD600.</li>
	<li>For the competition experiment, take those precultures of the different strains that have a similar OD600 between 1.0-1.5.</li>
	<li>To obtain mixed cell populations, dilute the cells of the precultures that had the appropriate OD600 to an OD600 of 0.05 in 20 ml C-Glc and CE-Glc minimal medium supplemented in 100 ml shake flasks. For the competition experiment the two strains should be mixed in a 1:1 ratio.</li>
	<li>Take 10 ml samples from the flasks, transfer them to 15 ml plastic tubes, harvest the cells by centrifugation for 10 min at 4,000 x g in a standard table top centrifuge, and discard the supernatant.</li>
	<li>Resuspend the cells in 0.5 ml fresh LB medium, and transfer the cells to a sterile 1.5 ml reaction cup. Add 0.5 ml of 50% sterile glycerol, mix the suspension by rigorous vortexing, and store the samples in an -80 &#176;C freezer until further treatment.</li>
	<li>Incubate the cells that are left in the shake flasks for up to 24 hr at 37 &#176;C and 220 rpm. Keep the shake flasks in the dark to prevent photo bleaching of the fluorophores.</li>
	<li>Take further samples of 0.1 ml after 7 hr and 24 hr of growth. Measure and note the OD600 of 1:10 dilutions (in C-Glc or CE-Glc minimal medium) of the samples.</li>
	<li>Take the appropriate amount of cells from each culture to make cryostocks that have an OD600 of 1.0. Store the samples at -80 &#176;C until further treatment.</li>
</ol>
3. Sample Treatment, Plating, and Incubation for Quantitative Analyses
<ol>
	<li>After collecting all samples, thaw the cryostocks, and dilute the cells in a 0.9% saline solution (see table of reagents and materials) up to 10-3.</li>
	<li>Plate 0.1 ml of the 10-3 dilutions on SP medium agar plates and distribute the cells using sterile glass pipettes.</li>
	<li>Incubate the plates overnight at 37 &#176;C in the dark until single colonies have appeared.</li>
</ol>
4. Counting the Survivors by Stereo Fluorescence Microscopy for Quantitative Analysis
<ol>
	<li>Divide the bottom of the agar plate with a black pen in four parts for a better orientation while counting the survivors under the stereo fluorescence microscope.</li>
	<li>Place the plate upside down under the microscope and bring the colonies into focus using the cold light source.</li>
	<li>Once the colonies are in focus, switch to the appropriate filter set for CFP to visualize the surviving cells of the cfp-labeled strain. Count the survivors of this strain by labeling the colonies with a pen and note the number.</li>
	<li>Remove the labels with ethanol and switch to the YFP filter set to visualize the surviving cells of the yfp-labeled strain. Count the survivors again by labeling the colonies with a pen and note the number.</li>
</ol>
5. Sample Treatment and Microscopy for Semiquantitative Analyses
<ol>
	<li>For illustrative figures, spot 10 &#181;l of the 10-4 dilution (approximately 100 cells) from step 3.1&#160;on a SP agar plate (see Figure 3).</li>
	<li>Incubate the plates overnight at 37 &#176;C in the dark until single colonies have appeared.</li>
	<li>Place the Petri dish without lid under the microscope and bring the spot into focus using the cold light source.</li>
	<li>Take pictures of the spots for illustrative figures. Choose an appropriate exposure time for taking the pictures of the colonies with the cold light source.</li>
	<li>Without moving the plate, change to the CFP filter set, adjust the exposure time and take a picture. Do the same with the YFP filter set and save the pictures for further analyses.</li>
</ol>
6. Data Analysis
<ol>
	<li>Use software such as Excel for the quantitative data analyses. Based on the counted colonies, calculate the percentage of yellow and blue colonies with respect to the whole number of colonies, which are set to 100%.</li>
	<li>Use the calculated numbers to create a stacked bar diagram (see Figure&#160;4 and&#160;Figure&#160;5). Use an image-processing program such as Adobe Photoshop to construct merged pictures of the pictures taken from the different spots. Alternatively, the freely available software ImageJ, downloadable from <a href="http://rsbweb.nih.gov/ij/">http://rsbweb.nih.gov/ij/</a> can be used for image processing.</li>
	<li>Open the pictures from one spot that were taken with the CFP filter set and the YFP filter set. Optimize the contrast and the brightness to reduce any background fluorescence from the media.</li>
	<li>Go to one of the pictures and select all. Copy the picture and paste it onto the other picture.</li>
	<li>Either use the function &#8220;color dodge&#8221; to merge the pictures or overlay the fluorescent pictures using the channels tab. Merged pictures of the colonies from different media and different time points represent the growth of the different strains within the liquid culture.</li>
</ol>
7. Specific Tips: Dye Switch Experiment and Cocultivation of Isogenic Strains Labeled with cfp and yfp
The expression of either of the two fluorophore-encoding genes in B. subtilis might influence fitness and thus the growth rate of the bacteria. Therefore, it is recommended to perform the following experiments in order to exclude that the elimination of one competitor strain from the cell population during cultivation is simply due to a negative effect of the fluorophore:
<ol>
	<li>Repeat the whole experiment and cocultivate inversely labeled strains (e.g.&#160;the earlier cfp-labeled strain is now labeled with the yfp gene and vice versa). Although inverse, the obtained results should be comparable to the previous observation that one strain has a selective growth advantage over the other strain.</li>
	<li>Repeat the whole experiment and cocultivate isogenic strains labeled with yfp and cfp (e.g.&#160;BP40 (rocG+ gudBCR amyE::PgudB-yfp) and BP41 (rocG+ gudBCR amyE::PgudB-cfp)). Estimate the negative effect of either of the two fluorophores on fitness of the bacteria by following the composition of the cell population over time.</li>
</ol>

<ol>
	<li>Buescher, F. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+network+reorganization+during+dynamic+adaptations+of+Bacillus+subtilis+metabolism.">Global network reorganization during dynamic adaptations of Bacillus subtilis metabolism.</a> Science. 335, 1099-1103 (2012).</li><li>Gunka, K., Stannek, L., Care, R. A., Commichau, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection-driven+accumulation+of+suppressor+mutants+in+Bacillus+subtilis:+the+apparent+high+mutation+frequency+of+the+cryptic+gudB+gene+and+the+rapid+clonal+expansion+of+gudB++suppressors+are+due+to+growth+under+selection.">Selection-driven accumulation of suppressor mutants in Bacillus subtilis: the apparent high mutation frequency of the cryptic gudB gene and the rapid clonal expansion of gudB+ suppressors are due to growth under selection.</a> PLoS One. 8, (2013).</li><li>Al Mamum, A. A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identity+and+function+of+a+large+gene+network+underlying+mutagenic+repair+of+DNA+breaks.">Identity and function of a large gene network underlying mutagenic repair of DNA breaks.</a> Science. 338, 1344-1348 (2012).</li><li>Koeppel, A. F., Wertheim, J. O., Barone, L., Gentile, N., Krizanc, D., Cohan, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Speedy+speciation+in+a+bacterial+microcosm:+new+species+can+arise+as+frequently+as+adaptations+within+a+species.">Speedy speciation in a bacterial microcosm: new species can arise as frequently as adaptations within a species.</a> ISME J. 7, 1080-1091 (2013).</li><li>Maughan, H., Nicholson, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+fitness+and+alteration+of+metabolic+pathways+during+Bacillus+subtilis+evolution+in+the+laboratory.">Increased fitness and alteration of metabolic pathways during Bacillus subtilis evolution in the laboratory.</a> Appl. Environ. Microbiol. 77, 4105-4118 (2011).</li><li>Burkholder, P. R., Giles, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+biochemical+mutations+in+Bacillus+subtilis.">Induced biochemical mutations in Bacillus subtilis.</a> Am. J. Bot. 34, 345-348 (1947).</li><li>McLoon, A. L., Guttenplan, S. B., Kearns, D. B., Kolter, R., Losick, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracing+the+domestication+of+a+biofilm-forming+bacterium.">Tracing the domestication of a biofilm-forming bacterium.</a> J. Bacteriol. 193, 2027-2034 (2011).</li><li>Zeigler, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origins+of+168,+W23,+and+other+Bacillus+subtilis+legacy+strains.">The origins of 168, W23, and other Bacillus subtilis legacy strains.</a> J. Bacteriol. 190, 6983-6995 (2008).</li><li>Gunka, K., Tholen, S., Gerwig, J., Herzberg, C., St&#252;lke, J., Commichau, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-frequency+mutation+in+Bacillus+subtilis:+requirements+for+the+decryptification+of+the+gudB+glutamate+dehydrogenase.">A high-frequency mutation in Bacillus subtilis: requirements for the decryptification of the gudB glutamate dehydrogenase.</a> 194, 1036-1044 (2012).</li><li>Commichau, F. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Bacillus+subtilis+mutants+with+carbon+source-independent+glutamate+biosynthesis.">Characterization of Bacillus subtilis mutants with carbon source-independent glutamate biosynthesis.</a> J. Mol. Microbiol. Biotechnol. 12, 106-113 (2007).</li><li>Beckwith, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+suppressors+and+recovery+of+repressed+biochemical+memory.">Genetic suppressors and recovery of repressed biochemical memory.</a> J. Biol. Chem. 284, 12585-12592 (2009).</li><li>Gunka, K., Commichau, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+glutamate+homeostasis+in+Bacillus+subtilis:+a+complex+interplay+between+ammonium+assimilation,+glutamate+biosynthesis+and+degradation.">Control of glutamate homeostasis in Bacillus subtilis: a complex interplay between ammonium assimilation, glutamate biosynthesis and degradation.</a> Mol. Microbiol. 85, 213-224 (2012).</li><li>Yan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protection+of+the+glutamate+pool+concentrations+in+enteric+bacteria.">Protection of the glutamate pool concentrations in enteric bacteria.</a> Proc. Natl. Acad. Sci. U.S.A. 104, 9475-9480 (2007).</li><li>Belitsky, B. R., Sonenshein, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+and+regulation+of+Bacillus+subtilis+glutamate+dehydrogenase+genes.">Role and regulation of Bacillus subtilis glutamate dehydrogenase genes.</a> J. Bacteriol. 180, 6298-6305 (1998).</li><li>Commichau, F. M., Herzberg, C., Tripal, P., Valerius, O., St&#252;lke, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+regulatory+protein-protein+interaction+governs+glutamate+biosynthesis+in+Bacillus+subtilis:+the+glutamate+dehydrogenase+RocG+moonlights+in+controlling+the+transcription+factor+GltC.">A regulatory protein-protein interaction governs glutamate biosynthesis in Bacillus subtilis: the glutamate dehydrogenase RocG moonlights in controlling the transcription factor GltC.</a> Mol. Microbiol. 65, 642-654 (2007).</li><li>Gordo, I., Perfeito, L., Sousa, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fitness+effects+of+mutations+in+bacteria.">Fitness effects of mutations in bacteria.</a> J. Mol. Microbiol. Biotechnol. 21, 20-35 (2011).</li><li>Rabatinov&#225;, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+&#948;+subunit+of+RNA+polymerase+is+required+for+rapid+changes+in+gene+expression+and+competitive+fitness+of+the+cell.">The &#948; subunit of RNA polymerase is required for rapid changes in gene expression and competitive fitness of the cell.</a> J. Bacteriol. 195, 2603-2611 (2013).</li><li>Capra, E. J., Perchuk, B. S., Skerker, J. M., Laub, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptive+mutations+that+prevent+crosstalk+enable+the+expansion+of+paralogous+signalling+protein+families.">Adaptive mutations that prevent crosstalk enable the expansion of paralogous signalling protein families.</a> Cell. 150, 222-232 (2012).</li><li>Garc&#237;a-Betancur, J. C., Yepes, A., Schneider, J., Lopez, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+analysis+of+Bacillus+subtilis+biofilms+using+fluorescence+microscopy+and+flow+cytometry.">Single-cell analysis of Bacillus subtilis biofilms using fluorescence microscopy and flow cytometry.</a> J. Vis. Exp. (15), (2012).</li></ol>

The authors declare that they have no competing financial interests.

Several methods have been developed to analyze competitive fitness of bacteria16. In many cases the bacteria were labeled with different antibiotic resistance cassettes17. Similar to our approach, labeling of the cells with antibiotic resistance cassettes allows the evaluation of competitive fitness of the bacteria during cocultivation under defined growth conditions. Moreover, this method can be used to determine competitive fitness of cells that differ from each other in a specific locus on the ch...

Soil bacteria are usually endowed with flexible regulatory networks and broad metabolic capacities. Both features enable the cells to adjust their catabolic and anabolic pathways to compete with their fellows and other microorganisms for the nutrients, which are available in a given ecological niche1. However, if the bacteria are unable to adapt to their environment other mechanisms may account for survival of a species. Indeed, as many bacteria proliferate fast and the populations can reach high cell densities subpopulations may have spontaneously accumulated beneficial mutations that provide the cells with a selective growth advantage and therefore increase their fitness. Moreover, mutational hotspots and stress-induced adaptive mutagenesis can facilitate the evolution of a maladapted bacterium2,3. Thus, accumulation of mutations and growth under continuous selection is the origin for the enormous microbial diversity, even within the same genus4,5. As in nature, shaping of bacterial genomes does also occur in the laboratory due to continuous cultivation under selection. This is exemplified by the domestication of the Gram-positive bacterium B. subtilis, which is used worldwide in basic research and in industry. In the 1940s B. subtilis was treated with DNA-damaging X-rays followed by cultivation under a specific growth condition6. The mutations that have accumulated in the bacteria during their domestication account for the loss of many growth characteristics, i.e.&#160;the B. subtilis laboratory strain 168 lost the ability to form complex colonies7,8.
Nowadays, for the best-studied model bacteria Escherichia coli and B. subtilis, a variety of powerful tools is available to genetically manipulate their genomes in order to address specific scientific questions. Sometimes the inactivation of a gene of interest causes a severe growth defect, which is then clearly visible on standard growth medium9. By contrast, mutations that cause a weak growth defect and thus only slightly affect fitness of the strain are often ignored. However, in both cases prolonged incubation and passaging of the mutant strains for several generations usually result in the accumulation of suppressor mutants that have restored the phenotype of the parent strain2,9. The characterization of suppressor mutants and the identification of the mutations that have restored the growth defect of the parent mutant strain is a very helpful approach that allows elucidation of important and often novel cellular processes10,11.
We are interested in the control of glutamate homeostasis in B. subtilis12. Similar to E. coli, B. subtilis responds to perturbation of glutamate homeostasis (i.e.&#160;block in glutamate degradation2) by the accumulation of suppressor mutants. The genomic alterations in these suppressor mutants that were acquired by spontaneous mutation were shown to rapidly restore glutamate homeostasis9,13. Therefore, it is not surprising that adaptation of B. subtilis to a specific growth condition during domestication of the bacterium is mirrored in enzyme synthesis and in the evolved enzymatic activities, which are involved in glutamate metabolism12. It has been suggested that the lack of exogenous glutamate in the growth medium during the domestication process was the driving force for the emergence and fixation of the cryptic glutamate dehydrogenase (GDH) gudBCR gene in the laboratory strain 1682,14. This hypothesis is supported by our observation that the reduced amount of GDH activity in the laboratory strain provides the bacteria with a selective growth advantage when exogenous glutamate is scarce2. Moreover, cultivation of a B. subtilis strain, synthesizing the GDH GudB, in the absence of exogenous glutamate results in the accumulation of suppressor mutants that have inactivated the gudB gene2. Obviously, the presence of a catabolically active GDH is disadvantageous for the cell because endogenously produced glutamate that could otherwise be used for anabolism is degraded to ammonium and 2-oxoglutarate (Figure 1). By contrast, when glutamate is provided by the medium, a B. subtilis strain equipped with high-level GDH activity has a selective growth advantage over a strain that synthesizes only one functional GDH. It is reasonable to assume that high-level GDH activity allows the bacteria to utilize glutamate as a second carbon source in addition to other carbon sources provided by the medium2 (see Figure 1). Thus, GDH activity strongly affects fitness of bacteria, depending on the availability of exogenous glutamate.
Here we present a very illustrative method to monitor and to visualize intraspecies competition between two B. subtilis strains that differ in a single locus on the chromosome (Figure 2). The two strains were labeled with the yfp and cfp genes encoding the fluorophores YFP and CFP, and cocultivated under different nutritional conditions. By sampling over time and by plating appropriate dilutions on agar plates the survivors in each of the cultures could be easily monitored using a common stereo fluorescence microscope. The procedure described in this paper is easy to perform and suitable to visualize the rapid clonal expansion and elimination of beneficial and detrimental mutations, respectively, in a cell population over time.

<table border="0" cellpadding="0" cellspacing="0" width="814">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">(NH4)2SO4&#160;</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">3746</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Agar</td>
			<td style="width:175px;">Difco, USA</td>
			<td style="width:111px;">214010</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="40">
			<td height="61" rowspan="2" style="height:61px;width:363px;">Ammonium ferric citrate (CAF)</td>
			<td rowspan="2" style="width:175px;">Sigma-Aldrich, Germany</td>
			<td rowspan="2" style="width:111px;">9714</td>
			<td rowspan="2" style="width:167px;">-</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">CaCl2&#160;</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">5239</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Glucose</td>
			<td style="width:175px;">Applichem, Germany</td>
			<td style="width:111px;">A3617</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Glycerol</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">4043</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">K2HPO4 x 3 H2O</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">6878</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">KCl</td>
			<td style="width:175px;">Applichem, Germany</td>
			<td style="width:111px;">A3582</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">KH2PO4</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">3904</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">KOH</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">6751</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">MgSO4 x 7 H2O&#160;</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">P027</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">MnCl2&#160;</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">T881</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="40">
			<td height="61" rowspan="2" style="height:61px;width:363px;">MnSO4 x 4 H2O</td>
			<td rowspan="2" style="width:175px;">Merck Millipore, Germany</td>
			<td rowspan="2" style="width:111px;">102786</td>
			<td rowspan="2" style="width:167px;">-</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">NaCl</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">9265</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Nutrient broth</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">X929</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Potassium glutamate</td>
			<td style="width:175px;">Applichem, Germany</td>
			<td style="width:111px;">A3712</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Tryptone</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">8952</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Tryptophan</td>
			<td style="width:175px;">Applichem, Germany</td>
			<td style="width:111px;">A3445</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Yeast extract</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">2363</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">1.5 ml Reaction tubes</td>
			<td style="width:175px;">Sarstedt, Germany</td>
			<td style="width:111px;">72,690,001</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">2.0 ml Reaction tubes</td>
			<td style="width:175px;">Sarstedt, Germany</td>
			<td style="width:111px;">72,691</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">15 ml Plastic tubes with screw cap</td>
			<td style="width:175px;">Sarstedt, Germany</td>
			<td style="width:111px;">62,554,001</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Petri dishes</td>
			<td style="width:175px;">Sarstedt, Germany</td>
			<td style="width:111px;">82.1473</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">1.5 ml Polystyrene cuvettes</td>
			<td style="width:175px;">Sarstedt, Germany</td>
			<td style="width:111px;">67,742</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">15 ml Glass culture tubes&#160;</td>
			<td rowspan="2" style="width:175px;">Brand, Germany</td>
			<td rowspan="2" style="width:111px;">7790 22</td>
			<td rowspan="2" style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">with aluminium caps</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">100 ml Shake flasks with aluminium caps</td>
			<td style="width:175px;">Brand, Germany</td>
			<td style="width:111px;">928 24</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Sterile 10 ml glass pipettes</td>
			<td style="width:175px;">Brand, Germany</td>
			<td style="width:111px;">278 23</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Incubator (28 and 37 &#176;C)</td>
			<td style="width:175px;">New Brunswick</td>
			<td style="width:111px;">M1282-0012</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="42">
			<td height="85" rowspan="3" style="height:85px;width:363px;">Standard pipette set (2-20 &#956;l, 10-100 &#956;l, 100-1000 &#956;l)</td>
			<td rowspan="3" style="width:175px;">Eppendorf, Germany</td>
			<td style="width:111px;">4910&#160;000.034, 4910 000.042,&#160;&#160;</td>
			<td rowspan="3" style="width:167px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:111px;">4910 000.042,</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:111px;">4910 000.069&#160;</td>
		</tr>
		<tr height="80">
			<td height="101" rowspan="2" style="height:101px;width:363px;">Table top centrifuge for 1.5 and 2 ml reaction tubes</td>
			<td rowspan="2" style="width:175px;">Thermo Scientific, Heraeus Fresco 21, Germany</td>
			<td rowspan="2" style="width:111px;">75002425</td>
			<td rowspan="2" style="width:167px;">-</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="60">
			<td height="81" rowspan="2" style="height:81px;width:363px;">Table top centrifuge for 15 ml plastic tubes</td>
			<td rowspan="2" style="width:175px;">Heraeus Biofuge Primo R, Germany</td>
			<td rowspan="2" style="width:111px;">75005440</td>
			<td rowspan="2" style="width:167px;">-</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="80">
			<td height="101" rowspan="2" style="height:101px;width:363px;">Standard spectrophotometer</td>
			<td rowspan="2" style="width:175px;">Amersham Biosciences Ultrospec 2100 pro, Germany</td>
			<td rowspan="2" style="width:111px;">80-2112-21&#160;</td>
			<td rowspan="2" style="width:167px;">-</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:363px;">Stereofluorescence microscope&#160;</td>
			<td style="width:175px;">Zeiss SteREO Lumar V12, Germany</td>
			<td style="width:111px;">495008-0009-000</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Freezer (-20 and -80 &#176;C)</td>
			<td style="width:175px;">-</td>
			<td style="width:111px;">-</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Fridge (4 &#176;C)</td>
			<td style="width:175px;">-</td>
			<td style="width:111px;">-</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:363px;">Autoclave</td>
			<td style="width:175px;">Zirbus, LTA 2x3x4, Germany</td>
			<td style="width:111px;">-</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:363px;">pH meter</td>
			<td style="width:175px;">pH-meter 766, Calimatic, Knick, Germany</td>
			<td style="width:111px;">766</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="40">
			<td height="61" rowspan="2" style="height:61px;width:363px;">Vortex</td>
			<td rowspan="2" style="width:175px;">Vortex&#160; 3, IKA, Germany</td>
			<td rowspan="2" style="width:111px;">3340000</td>
			<td rowspan="2" style="width:167px;">-</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="21">
			<td height="64" rowspan="3" style="height:64px;width:363px;">Balance</td>
			<td rowspan="3" style="width:175px;">CP2202S, Sartorius, Germany</td>
			<td style="width:111px;">replaced by</td>
			<td rowspan="3" style="width:167px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:111px;">CPA2202S</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:111px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Black pen (permanent marker)</td>
			<td style="width:175px;">Staedler, Germany</td>
			<td style="width:111px;">317-9</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Powerpoint program</td>
			<td style="width:175px;">Microsoft, USA</td>
			<td style="width:111px;">-</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:363px;">Office Excel program</td>
			<td style="width:175px;">Microsoft, USA</td>
			<td style="width:111px;">-</td>
			<td style="width:167px;">Program for data processing</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:363px;">Adobe Photoshop CS5</td>
			<td style="width:175px;">Adobe, USA</td>
			<td style="width:111px;">replaced by CS6, download</td>
			<td style="width:167px;">Computer program for image processing</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Computer</td>
			<td style="width:175px;">PC or Mac</td>
			<td style="width:111px;">-</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;">ZEN pro 2011 software for the stereofluorescence microscope</td>
			<td style="width:175px;">Zeiss, Germany</td>
			<td style="width:111px;">410135 1002 110</td>
			<td style="width:167px;">AxioCam MRc Rev. Obtained through Zeiss</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:22px;width:815px;">Specific solution recipes</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">SP medium</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">8 g Nutrient broth</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">0.25 mg MgSO4 x 7 H2O</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">1 g KCl</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">if required, add 15 g agar for solid SP medium</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">ad 1 l with H2O, autoclave for 20 min at 121 &#176;C</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">1 ml CaCl2 (0.5 M), sterilized by filtration</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">1 ml MnCl2 (10 mM) sterilized by filtration</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:22px;width:815px;">2 ml ammonium ferric citrate (CAF, 2.2 mg/ml), sterilized by filtration</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">LB medium</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">10 g Tryptone</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">5 g Yeast extract</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">10 g NaCl</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">if required, add 15 g agar for solid LB medium</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">ad 1 l with H2O, autoclave for 20 min at 121 &#176;C</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">C-Glc minimal medium</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">200 ml 5 x C salts</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">10 ml L-Tryptophan (5 mg/ml), sterilized by filtration</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">10 ml ammonium ferric citrate (CAF, 2.2 mg/ml), sterilized by filtration</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">10 ml III&#8217; salts</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">25 ml Glucose (20%), autoclaved for 20 min at 121 &#176;C</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">ad 1 l with sterile H2O</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">CE-Glc minimal medium</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">200 ml 5 x C salts</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">10 ml L-Tryptophan (5 mg/ml), sterilized by filtration</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">10 ml ammonium ferric citrate (CAF, 2.2 mg/ml), sterilized by filtration</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">10 ml III&#8217; salts</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">20 ml Glutamate (40%)</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">25 ml Glucose (20%), autoclaved for 20 min at 121 &#176;C</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">ad 1 l with sterile H2O</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">5 x C salts&#160;</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">20 g KH2PO4</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">80 g K2HPO4 x 3 H2O</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">16.5 g (NH4)2SO4</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">ad 1 l with sterile H2O, autoclave for 20 min at 121 &#176;C</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">III&#8217; salts</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">0.232 g MnSO4 x 4 H2O</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">12.3 g MgSO4 x 7 H2O</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">ad 1 l with sterile H2O, autoclave for 20 min at 121 &#176;C</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">40% Glutamate solution</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">200 g L-Glutamic acid</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">adjust the pH to 7.0 by adding approximately 80 g KOH</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">ad 0.5 l with sterile H2O, autoclave for 20 min at 121 &#176;C</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">0.9% Saline (NaCl) solution</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">ad 1 l with sterile H2O, autoclave for 20 min at 121 &#176;C</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">50% Glycerol solution</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">295 ml Glycerol (87%)</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">ad 0.5 l with sterile H2O, autoclave for 20 min at 121 &#176;C</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:22px;width:815px;">Bacteria (All strains are based on the Bacillus subtilis strain 168)</td>
		</tr>
		<tr height="21">
			<td colspan="3" height="21" style="height:21px;width:648px;">Bacillus subtilis BP40 (rocG+ gudBCR amyE::PgudB-yfp)&#160;</td>
			<td rowspan="4" style="width:167px;">Laboratory strain collection</td>
		</tr>
		<tr height="21">
			<td colspan="3" height="21" style="height:21px;width:648px;">Bacillus subtilis BP41 (rocG+ gudBCR amyE::PgudB-cfp)&#160;</td>
		</tr>
		<tr height="21">
			<td colspan="3" height="21" style="height:21px;width:648px;">Bacillus subtilis BP52 (rocG+ gudB+ amyE::PgudB-cfp)</td>
		</tr>
		<tr height="21">
			<td colspan="3" height="21" style="height:21px;width:648px;">Bacillus subtilis BP156 (rocG+ gudB+ amyE::PgudB-yfp)</td>
		</tr>
	</tbody>
</table>

monitoring intraspecies competition in a bacterial cell population by cocultivation of fluorescently labelled strains

Many microorganisms such as bacteria proliferate extremely fast and the populations may reach high cell densities. Small fractions of cells in a population always have accumulated mutations that are either detrimental or beneficial for the cell. If the fitness effect of a mutation provides the subpopulation with a strong selective growth advantage, the individuals of this subpopulation may rapidly outcompete and even completely eliminate their immediate fellows. Thus, small genetic changes and selection-driven accumulation of cells that have acquired beneficial mutations may lead to a complete shift of the genotype of a cell population. Here we present a procedure to monitor the rapid clonal expansion and elimination of beneficial and detrimental mutations, respectively, in a bacterial cell population over time by cocultivation of fluorescently labeled individuals of the Gram-positive model bacterium Bacillus subtilis. The method is easy to perform and very illustrative to display intraspecies competition among the individuals in a bacterial cell population.

The method described here was successfully applied to visualize intraspecies competition in a cell population consisting of B. subtilis strains that were labeled with the cfp and yfp genes encoding the fluorophores CFP and YFP, respectively. As shown in Figure 3, the method can be used to visualize intraspecies competition in a very illustrative manner. By spotting the samples on small areas, the clonal composition of the cell population was made visible at a glance. Although n...

Watch this Scientific Journal Video about Monitoring Intraspecies Competition in a Bacterial Cell Population by Cocultivation of Fluorescently Labelled Strains at JoVE.com

Monitoring Intraspecies Competition in a Bacterial Cell Population by Cocultivation of Fluorescently Labelled Strains

Bacteria may accumulate either detrimental or beneficial mutations during their lifetime. In a population of cells individuals that have accumulated beneficial mutations may rapidly outcompete their fellows. Here we present a simple procedure to visualize intraspecies competition in a bacterial cell population over time using fluorescently labeled individuals.&#160;

Many microorganisms such as bacteria proliferate extremely fast and the populations may reach high cell densities. Small fractions of cells in a ...

monitoring-intraspecies-competition-bacterial-cell-population

Research

JoVE Journal

Biology

8.4K Views.  Georg-August University. Bacteria may accumulate either detrimental or beneficial mutations during their lifetime. In a population of cells individuals that have accumulated beneficial mutations may rapidly outcompete their fellows. Here we present a simple procedure to visualize intraspecies competition in a bacterial cell population over time using fluorescently labeled individuals. 

Video: Monitoring Intraspecies Competition in a Bacterial Cell Population by Cocultivation of Fluorescently Labelled Strains

Title Cell Encapsulation by Droplets

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA2, PKB3, PKC4, PKD5, ERK6, JNK7, Cdk18, Aurora B9 and Plk19. Given the modular design, additional probes are likely to emerge in the near future10. 
Progression through the cell cycle is affected by stress signaling pathways 11. Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress12.Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle8,9,13,14.
Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.

FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy (FCS)

In the past fifteen years the notion that cell membranes are not homogenous and rely on microdomains to exert their functions has become widely accepted. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. They play a role in cellular physiological processes such as signalling, and trafficking1,2 but are also thought to be key players in several diseases including viral or bacterial infections and neurodegenerative diseases3.
Yet their existence is still a matter of controversy4,5. Indeed, lipid raft size has been estimated to be around 20 nm6, far under the resolution limit of conventional microscopy (around 200 nm), thus precluding their direct imaging. Up to now, the main techniques used to assess the partition of proteins of interest inside lipid rafts were Detergent Resistant Membranes (DRMs) isolation and co-patching with antibodies. Though widely used because of their rather easy implementation, these techniques were prone to artefacts and thus criticized7,8. Technical improvements were therefore necessary to overcome these artefacts and to be able to probe lipid rafts partition in living cells.
Here we present a method for the sensitive analysis of lipid rafts partition of fluorescently-tagged proteins or lipids in the plasma membrane of living cells. This method, termed Fluorescence Correlation Spectroscopy (FCS), relies on the disparity in diffusion times of fluorescent probes located inside or outside of lipid rafts. In fact, as evidenced in both artificial membranes and cell cultures, probes would diffuse much faster outside than inside dense lipid rafts9,10. To determine diffusion times, minute fluorescence fluctuations are measured as a function of time in a focal volume (approximately 1 femtoliter), located at the plasma membrane of cells with a confocal microscope (Fig. 1). The auto-correlation curves can then be drawn from these fluctuations and fitted with appropriate mathematical diffusion models11.
FCS can be used to determine the lipid raft partitioning of various probes, as long as they are fluorescently tagged. Fluorescent tagging can be achieved by expression of fluorescent fusion proteins or by binding of fluorescent ligands. Moreover, FCS can be used not only in artificial membranes and cell lines but also in primary cultures, as described recently12. It can also be used to follow the dynamics of lipid raft partitioning after drug addition or membrane lipid composition change12.

Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy FCS

A technique to probe the lipid raft partitioning of fluorescent proteins at the plasma membrane of living cells is described. It takes advantage of the disparity in diffusion times of proteins located inside or outside of lipid rafts. Acquisition can be performed dynamically in control conditions or after drug addition.

In the past fifteen years the notion that cell membranes are not homogenous and rely on microdomains to exert their functions has become widely accepted. ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Using Digital Image Correlation to Characterize Local Strains on Vascular Tissue Specimens

Characterization of the mechanical behavior of biological and engineered soft tissues is a central component of fundamental biomedical research and product development. Stress-strain relationships are typically obtained from mechanical testing data to enable comparative assessment among samples and in some cases identification of constitutive mechanical properties. However, errors may be introduced through the use of average strain measures, as significant heterogeneity in the strain field may result from geometrical non-uniformity of the sample and stress concentrations induced by mounting/gripping of soft tissues within the test system. When strain field heterogeneity is significant, accurate assessment of the sample mechanical response requires measurement of local strains. This study demonstrates a novel biomechanical testing protocol for calculating local surface strains using a mechanical testing device coupled with a high resolution camera and a digital image correlation technique. A series of sample surface images are acquired and then analyzed to quantify the local surface strain of a vascular tissue specimen subjected to ramped uniaxial loading. This approach can improve accuracy in experimental vascular biomechanics and has potential for broader use among other native soft tissues, engineered soft tissues, and soft hydrogel/polymeric materials. In the video, we demonstrate how to set up the system components and perform a complete experiment on native vascular tissue.

We describe the use of digital image correlation to characterize the local surface strain field on vascular tissue samples subjected to uniaxial tensile testing. These measurements facilitate precise quantification of the sample mechanical response and the generation of constitutive stress-strain relations.

Characterization of the mechanical behavior of biological and engineered soft tissues is a central component of fundamental biomedical research and ...