The authors have nothing to disclose.

1. Preparing the Growth Medium
NOTE: The chemical composition of minimal medium M63 is as follows: 62 mM K2HPO4, 39 mM KH2PO4, 15 mM (NH4)2SO4, 1.8 &#181;M FeSO4, 15 &#181;M thiamine-HCl, 0.2 mM MgSO4, and 22 mM glucose. M63 is made by mixing three stock solutions: Five X solution, 20% glucose and MgSO4 thiamine solution. Store all solutions at 4 &#176;C.
<ol>
	<li>Preparing the Five...

<ol>
	<li>Kovarova-Kovar, K., Egli, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+kinetics+of+suspended+microbial+cells:+from+single-substrate-controlled+growth+to+mixed-substrate+kinetics.">Growth kinetics of suspended microbial cells: from single-substrate-controlled growth to mixed-substrate kinetics.</a> Microbiol Mol Biol Rev. 62 (3), 646-666 (1998).</li><li>Soupene, E., 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=Physiological+studies+of+Escherichia+coli+strain+MG1655:+growth+defects+and+apparent+cross-regulation+of+gene+expression.">Physiological studies of Escherichia coli strain MG1655: growth defects and apparent cross-regulation of gene expression.</a> J Bacteriol. 185 (18), 5611-5626 (2003).</li><li>Sezonov, G., Joseleau-Petit, D., D'Ari, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escherichia+coli+physiology+in+Luria-Bertani+broth.">Escherichia coli physiology in Luria-Bertani broth.</a> J Bacteriol. 189 (23), 8746-8749 (2007).</li><li>Egli, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+growth+and+physiology:+a+call+for+better+craftsmanship.">Microbial growth and physiology: a call for better craftsmanship.</a> Front Microbiol. 6, 287 (2015).</li><li>Kurokawa, M., Seno, S., Matsuda, H., Ying, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+between+genome+reduction+and+bacterial+growth.">Correlation between genome reduction and bacterial growth.</a> DNA Res. 23 (6), 517-525 (2016).</li><li>Matsumoto, Y., Murakami, Y., Tsuru, S., Ying, B. W., Yomo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+rate-coordinated+transcriptome+reorganization+in+bacteria.">Growth rate-coordinated transcriptome reorganization in bacteria.</a> BMC Genomics. 14, 808 (2013).</li><li>Nahku, 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=Specific+growth+rate+dependent+transcriptome+profiling+of+Escherichia+coli+K12+MG1655+in+accelerostat+cultures.">Specific growth rate dependent transcriptome profiling of Escherichia coli K12 MG1655 in accelerostat cultures.</a> J Biotechnol. 145 (1), 60-65 (2010).</li><li>Dai, X., 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=Reduction+of+translating+ribosomes+enables+Escherichia+coli+to+maintain+elongation+rates+during+slow+growth.">Reduction of translating ribosomes enables Escherichia coli to maintain elongation rates during slow growth.</a> Nat Microbiol. 2, 16231 (2016).</li><li>Madrid, R. E., Felice, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+biomass+estimation.">Microbial biomass estimation.</a> Crit Rev Biotechnol. 25 (3), 97-112 (2005).</li><li>Harris, C. M., Kell, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+estimation+of+microbial+biomass.">The estimation of microbial biomass.</a> Biosensors. 1 (1), 17-84 (1985).</li><li>Yates, G. T., Smotzer, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+lag+phase+and+initial+decline+of+microbial+growth+curves.">On the lag phase and initial decline of microbial growth curves.</a> J Theor Biol. 244 (3), 511-517 (2007).</li><li>Kargi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Re-interpretation+of+the+logistic+equation+for+batch+microbial+growth+in+relation+to+Monod+kinetics.">Re-interpretation of the logistic equation for batch microbial growth in relation to Monod kinetics.</a> Lett Appl Microbiol. 48 (4), 398-401 (2009).</li><li>Peleg, M., Corradini, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+growth+curves:+what+the+models+tell+us+and+what+they+cannot.">Microbial growth curves: what the models tell us and what they cannot.</a> Crit Rev Food Sci Nutr. 51 (10), 917-945 (2011).</li><li>Sprouffske, K., Wagner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growthcurver:+an+R+package+for+obtaining+interpretable+metrics+from+microbial+growth+curves.">Growthcurver: an R package for obtaining interpretable metrics from microbial growth curves.</a> BMC Bioinformatics. 17, 172 (2016).</li><li>Hall, B. G., Acar, H., Nandipati, A., Barlow, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+rates+made+easy.">Growth rates made easy.</a> Mol Biol Evol. 31 (1), 232-238 (2014).</li><li>Hermsen, R., Okano, H., You, C., Werner, N., Hwa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+growth-rate+composition+formula+for+the+growth+of+E.coli+on+co-utilized+carbon+substrates.">A growth-rate composition formula for the growth of E.coli on co-utilized carbon substrates.</a> Mol Syst Biol. 11 (4), 801 (2015).</li><li>Engen, S., Saether, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=r-+and+K-selection+in+fluctuating+populations+is+determined+by+the+evolutionary+trade-off+between+two+fitness+measures:+Growth+rate+and+lifetime+reproductive+success.">r- and K-selection in fluctuating populations is determined by the evolutionary trade-off between two fitness measures: Growth rate and lifetime reproductive success.</a> Evolution. 71 (1), 167-173 (2017).</li></ol>

We thank Kohei Tsuchiya for providing the CFU assay example. This work was partially financially supported by a Grant-in-Aid for Scientific Research (C) no. 26506003 (to BWY) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

Critical steps in the protocol include the preparation of a common stock of exponentially growing cells and the replication of the same samples in multiple wells at various positions on the microplate. Previously, microbiologists started the culture from an overnight pre-culture. While this method may reduce the lag time of bacterial growth, it is difficult to achieve reproducible growth curves. As shown in Figure 2, the independent measurements using the common glycerol stocks resulted in n...

Microbiological studies often start with the culture of bacterial cells and the assessment of the bacterial growth curves, which represent a fundamental phenomenon of bacterial physiology1,2,3. Basic culture principles are widely available in the published research literature and textbooks because bacterial culture is a fundamental methodology. At the bench level, substantial attention has traditionally been focused on optimizing growth media and culturing conditions, but controlling the growth rate, which would likely provide even greater understanding of microbial physiology, has not been extensively studied4. For exponentially growing bacteria, a key parameter of the cellular state is the growth rate, which has been reported to be coordinated with the genome, transcriptome, and proteome5,6,7,8. Thus, quantitative evaluation of bacterial growth is crucial for understanding microbial physiology.
To evaluate bacterial growth, the experimental methods used to estimate biomass are well established9,10 and are based on the detection of biochemical, physical, or biological parameters, such as optical turbidity. In addition, the analytical methods used to capture the dynamic properties of growth changes are commonly based on established nonlinear models11,12,13, for example, logistic equations. Growth dynamics are generally acquired by timed sampling of cell growth in culture by either measuring optical turbidity or performing colony-forming unit (CFU) assays. The limitation of these culturing and detection methods is that the data points are not a true reflection of population dynamics because the measurement intervals are often in hours and because the culture condition (e.g., changes in temperature and aeration) is disturbed at the time of sampling. Culture and analysis techniques must be updated using recent developments in technology and understanding. Recent advances in microplate readers allow the real-time observation of bacterial growth and significantly decrease labor costs. Using these advanced devices, the latest studies on bacterial growth have reported analytical methods for high-throughput measurements14,15.
The purpose of this protocol is to evaluate the precise growth dynamics in a high-throughput manner, which will be valuable for quantitative studies that ultimately address the questions of how the growth rate is determined and what factors affect the growth rate. The protocol addresses all factors that should be taken into account for the repeatable and precise quantitation of bacterial growth. The experimental method and analysis are described in detail in the main text. This method permits the precise and reproducible analysis of bacterial growth in a high-throughput manner. Microbiologists can use this protocol to derive additional quantitative results from their experimental evidence. This protocol can also be used for studies in systems biology that attempt to draw conceptual conclusions or to achieve a theoretical overview of growth.

<table>
	<tbody>
		<tr>
			<td>K2HPO4</td>
			<td>Wako</td>
			<td>164-04295</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Wako</td>
			<td>166-04255</td>
			<td></td>
		</tr>
		<tr>
			<td>(NH4)2SO4</td>
			<td>Wako</td>
			<td>019-03435</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4-7H2O</td>
			<td>Wako</td>
			<td>138-00415</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiamine-HCl</td>
			<td>Wako</td>
			<td>201-00852</td>
			<td></td>
		</tr>
		<tr>
			<td>glucose</td>
			<td>Wako</td>
			<td>049-31165</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Wako</td>
			<td>080-01066</td>
			<td></td>
		</tr>
		<tr>
			<td>Iron (II) sulfate heptahydrate (FeSO4-7H2O)</td>
			<td>Wako</td>
			<td>094-01082</td>
			<td></td>
		</tr>
		<tr>
			<td>KOH</td>
			<td>Wako</td>
			<td>168-21815</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Wako</td>
			<td>075-00611</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tube (50 mL, sterilized)</td>
			<td>WATSON</td>
			<td>1342-050S</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips, 200 &#181;L</td>
			<td>WATSON</td>
			<td>110-705Y</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips, 1000 &#181;L</td>
			<td>WATSON</td>
			<td>110-8040</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtube (1.5 mL)</td>
			<td>WATSON</td>
			<td>131-715C</td>
			<td></td>
		</tr>
		<tr>
			<td>8 multichannel-pipette</td>
			<td>WATSON</td>
			<td>NT-8200</td>
			<td></td>
		</tr>
		<tr>
			<td>PASORINA STIRRER</td>
			<td>AS ONE</td>
			<td>2-4990-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass cylinder (200 mL)</td>
			<td>AS ONE</td>
			<td>1-8562-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision pH mater</td>
			<td>AS ONE</td>
			<td>AS800 / 1-054-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetman P-200</td>
			<td>GILSON</td>
			<td>1-6855-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetman P-1000</td>
			<td>GILSON</td>
			<td>1-6855-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Serolocical Pipettes (10 mL)</td>
			<td>SANPLATEC</td>
			<td>SAN27014</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Serolocical Pipettes (25 mL)</td>
			<td>SANPLATEC</td>
			<td>SAN27015</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtube stand</td>
			<td>BM Bio</td>
			<td>801-02Y</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>BM Bio</td>
			<td>BM-V1</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Costar 96-well microplate with lid (Flat bottom, Clear)</td>
			<td>Sigma-Aldrich</td>
			<td>Corning, 3370</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Costar reagent reservoir (50 mL)</td>
			<td>Sigma-Aldrich</td>
			<td>Corning, 4870</td>
			<td></td>
		</tr>
		<tr>
			<td>Stericup GV PVDF (250 mL, 0.22 &#181;M)</td>
			<td>Merck Millipore</td>
			<td>SCGVU02RE</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet-Aid XP</td>
			<td>DRUMMOND</td>
			<td>4-000-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioshaker (BR-23UM MR)</td>
			<td>TAITEC</td>
			<td>0053778-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposal cell (1.5 mL)</td>
			<td>Kartell</td>
			<td>1938 / 2-478-02</td>
			<td></td>
		</tr>
		<tr>
			<td>DU 730 Life Science UV/Vis Spectrophotometer</td>
			<td>Beckman Coulter</td>
			<td>A23616</td>
			<td></td>
		</tr>
		<tr>
			<td>EPOCH2</td>
			<td>BioTek</td>
			<td>2014-EP2-002 / EPOCH2T</td>
			<td></td>
		</tr>
		<tr>
			<td>Beaker (500 mL)</td>
			<td>IWAKI</td>
			<td>82-0008</td>
			<td></td>
		</tr>
		<tr>
			<td>BIO clean bench</td>
			<td>Panasonic</td>
			<td>MCV-B131F</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass tubes</td>
			<td>NICHIDEN RIKA GLASS</td>
			<td>P-10M~P-30 /101019</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone rubber stoppers</td>
			<td>ShinEtsu Polymer</td>
			<td>T-19</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacterial strains</td>
			<td>Strain bank organization; National Bio Resource Project (NBRP) in Japan</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

precise, high-throughput analysis of bacterial growth

Bacterial growth is a central concept in the development of modern microbial physiology, as well as in the investigation of cellular dynamics at the systems level. Recent studies have reported correlations between bacterial growth and genome-wide events, such as genome reduction and transcriptome reorganization. Correctly analyzing bacterial growth is crucial for understanding the growth-dependent coordination of gene functions and cellular components. Accordingly, the precise quantitative evaluation of bacterial growth in a high-throughput manner is required. Emerging technological developments offer new experimental tools that allow updates of the methods used for studying bacterial growth. The protocol introduced here employs a microplate reader with a highly optimized experimental procedure for the reproducible and precise evaluation of bacterial growth. This protocol was used to evaluate the growth of several previously described Escherichia coli strains. The main steps of the protocol are as follows: the preparation of a large number of cell stocks in small vials for repeated tests with reproducible results, the use of 96-well plates for high-throughput growth evaluation, and the manual calculation of two major parameters (i.e., maximal growth rate and population density) representing the growth dynamics. In comparison to the traditional colony-forming unit (CFU) assay, which counts the cells that are cultured in glass tubes over time on agar plates, the present method is more efficient and provides more detailed temporal records of growth changes, but has a stricter detection limit at low population densities. In summary, the described method is advantageous for the precise and reproducible high-throughput analysis of bacterial growth, which can be used to draw conceptual conclusions or to make theoretical observations.

The described method provides a means to capture dynamic bacterial growth in a continuous, high-throughput manner by utilizing a 96-well format reader that takes multiple optical density measurements at various time intervals (from minutes to hours to days). The growth curves of an assortment of E. coli strains expressing various genomes can be precisely acquired in a single experiment (Figure 1A). In comparison to the described method, the tradition...

Watch this Scientific Journal Video about Precise, High-throughput Analysis of Bacterial Growth at JoVE.com

Precise, High-throughput Analysis of Bacterial Growth

Quantitative evaluation of bacterial growth is essential to understanding microbial physiology as a systems-level phenomenon. A protocol for experimental manipulation and an analytical approach are introduced, allowing for precise, high-throughput analysis of bacterial growth, which is a key subject of interest in systems biology.

Bacterial growth is a central concept in the development of modern microbial physiology, as well as in the investigation of cellular dynamics at the ...

The overall goal of this experiment is to analyze bacterial growth precisely in a high throughput manner. Using this procedure, either multiple media or varied strains can be tested simultaneously. This method can help answer key questions in the systems biology and the microbiology fields such as, what determination factors of cell growths are.The main advantages of this technique is that it allows researchers to precisely evaluate global parameters of maximal growth rate and maximal population density in growth curves of bacteria cells. Though this method can provide insight into e. Coli growth, it can also be applied to other micro-organisms such as, the growth assay and screening of environmental bacteria.To begin, place five sterilized glass tubes with silicone rubber stoppers, pipettes and the target strains on a clean bench. Expose the mouth of the glass tube to a bunsen burner before opening the silicone rubber stopper. Expose the silicone rubber stopper to the flame after the tube is opened and then lightly place the cap back on the glass tube.Next, use the electronic pipette and disposable serological pipette to add five milliliters of M63 to one of the glass tubes and 4.5 milliliters of M63 to other four tubes. Use the p200 tip to pick a colony and inoculate it in the glass tube containing five milliliters of M63. Vortex the tube to make a suspension.Then, dilute the solution 10 fold by transferring 0.5 milliliters of this solution to one of the four tubes containing 4.5 milliliters of M63. Repeat this process for the remaining tubes. After sterilizing the mouths of the glass tubes and the silicone rubber stoppers, cap the tubes with the stoppers.Then, place the five tubes in a pre-warmed shaking incubator at 37 degree celsius and shake it 200 rpm. Incubate the culture overnight or for 10 to 30 hours. Following incubation, add 1, 000 microliters of M63 to a disposable cuvette with the p1000.Place the disposable cuvette in a spectrophotometer. Start the program at a fixed wavelength of 600 nanometers and measure the blank. Move the five glass tubes from the shaking incubator to the clean bench.Discard M63 from the disposable cuvette and add 1, 000 microliters of culture to the same disposable cuvette with the p1000. Then, measure the optical turbidity of the cell culture. Next, add 250 microliters of sterilized 60%glycerol solution and 750 microliters of the selected cell culture to a 1.5 milliliter microtube and mix by pipetting.Place the remaining nine microtubes in the microtube stand and dispense 100 microliters of the prepared mixture to each tube. Store the stocks in the deep freezer at 80 degrees celsius. To set up the microplate reader, first open the software, open protocols in the task manager and choose create new.Then, select standard protocol. Open procedure and adjust the settings. Open set temperature and select, incubator on.Then, set temperature to 37 degree celsius and gradient to zero degree celsius. Check preheat before continuing with the next step. Open start kinetic and set run time for 24 or 48 minutes, then, set the interval 30 minutes or one hour.Open shake and set shake mode as linear. Check continuous shake and set frequency at 567 cpm. Next, open read, check absorbance, endpoint/kinetic and monochromators.Set the wavelength to 600. Finally, click validate to confirm that the procedure is correct. And click save to save it as a new program for future use.For real time recording of growth, add approximately 25 milliliters of M63 to a sterilized reagent reservoir. Then, add 900 microliters of M63 to the microtubes in preparation for making serial dilutions. Next, add 900 microliters of M63 to the thawed glycerol stock and vortex.Transfer 100 microliters of the 10 fold dilution to another microtube containing 900 microliters of M63 and vortex. Repeat this step until the desired number of dilutions are achieved. Now, fill the wells at the edge of a sterilized 96 well flat bottom microplate with 200 microliters of M63, using an eight channel pipette.Load 200 microliters of each diluted sample to the microplate wells according to the reference table. To avoid allocational layers due to the heating and the seeding efficiency, never use the wells at edge of the microplate for your samples and load the same sample into multiple wells at varied locations on the microplate. Place the 96 well microplate onto the plate reader.Open read now and task manager and choose the program. Click okay to start measuring. Finally, save the recording as a new experimental file for data analysis.Shown here is an example of a reference table that was made before performing the experiment. A 96 well plate pictogram was drawn as an eight by 12 table to indicate the positions of the inoculated culture samples on the 96 well plate. The growth rate is calculated by applying the equation for all pairs of consecutive values of OD600.The mean and the standard deviation of five consecutive growth rates were calculated to estimate the maximal growth rate. The growth rates of a total of 60 cell cultures are indicated as the displayed heat map. Degradation from white to red indicates the growth rates from low to high.Once mastered, this technique can be done within a few minutes if it is performed properly and in order. After watching this video, you should have a good understanding of how to prepare and load samples to the microplate for highly precise measurements of bacterial growths in a high throughput manner. While attempting this procedure, it's important to remember to make a reference table before performing the experiment to avoid cell cultures of low density and to make sure the cells start growing within 24 hours.Following this procedure, other methods like automation of experimental manipulation and the computational analysis can be performed in order to answer additional questions like what determination factors for population propagation and the dynamics are. After its development, this technique paved the way for researchers in the fields of systems biology and microbiology. To explore the fundamental principles in cell growth and to optimize compositions of culture media.

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23.5K vistas.  University of Tsukuba. The overall goal of this experiment is to analyze bacterial growth precisely in a high throughput manner. Using this procedure, either multiple media or varied strains can be tested simultaneously. This method can help answer key questions in the systems biology and the microbiology fields such as, what determination factors of cell growths are.The main advantages of this technique is that it allows researchers to precisely evaluate global parameters of maximal growth rate and maximal ...