This publication was made possible by grant number NC.X-267-5-12-170-1 from the National Institute of Food and Agriculture (NIFA) and in part by NIZO Food Research BV, The Netherlands, Jarrow Formulas, USA, and the Department of Family and Consumer Sciences and the Agriculture Research Station at North Carolina Agriculture and Technical State University (Greensboro, NC, USA 27411). This work was also supported, in part, by 1890 Capacity Building Program grant no. (2020-38821-31113/project accession no. 021765). This work was also partially supported by the Bulgarian Ministry of Education and Science under the National Research Programme &#8216;Healthy Foods for a Strong Bio-Economy and Quality of Life&#8217; approved by DCM # 577 / 17.08.2018.

1. General materials and methods
<ol>
	<li>Source of Lactobacillus delbrueckii subsp. bulgaricus
	<ol>
		<li>Obtain L. bulgaricus strains from reliable sources. 
		NOTE: In this study, a total of five (5) L. bulgaricus strains were used in the quality control study (Table 1). Two strains of freeze-dried L. bulgaricus for the industrial production of fermented milk products were provided by Dr. Albert Krastanov, Department of Biotechnology at the University of Food Technologies, Plovdiv, Bulgaria. Two strains isolated from commercial yogurt products available in the US market were obtained from the -80 &#176;C stock of the Food Microbiology and Biotechnology laboratory at North Carolina A&#38;T State University and one freeze-dried strain was supplied by a vendor C. All LAB strains were kept at -80 &#176;C until further use. Another bacterial strain (Limosilactobacillus reuteri) was obtained from the -80 &#176;C stock of the Food Microbiology and Biotechnology laboratory at North Carolina A&#38;T State University was also evaluated.</li>
	</ol>
	</li>
	<li>Standard L. MRS fermentation broth medium
	<ol>
		<li>Prepare deMan, Rogosa Sharpe (MRS) medium by completely dissolving 55 g of MRS, and 0.5 g of L-Cysteine in 1 L of deionized water (DW).</li>
		<li>Dispense 7 mL and 2 mL of the prepared MRS solution into test tubes, respectively, autoclave at 121 &#176;C for 15 min, and then cool at room temperature (RT).</li>
	</ol>
	</li>
	<li>MRS agar medium
	<ol>
		<li>Prepare MRS agar medium by completely dissolving 55 g of MRS and 0.5 g of L-Cysteine in 1 L of DW. Add agar powder (15 g), sterilize the agar medium at 121 &#176;C for 15 min, and then cool it in a water bath.</li>
		<li>Pour all freshly-prepared media into sterile Petri dishes and store them at 4 &#176;C until further needed.</li>
	</ol>
	</li>
	<li>Modified reinforced clostridial medium-pyruvate (mRCM-PYR)
	<ol>
		<li>Optimize a reinforced clostridial medium according to Oyeniran et al.13 and Nwamaioha and Ibrahim18, for selectivity and accurate enumeration of L. bulgaricus by dissolving 10 g of peptone #3, 10 g of beef extract, 5 g of yeast extract, 5 g of sodium chloride, 3 g of sodium acetate, 2 g of potassium phosphate dibasic, 0.1 g of uracil, 0.25 g of calcium chloride, 5 g of dextrose, 5 g of fructose, 10 g of maltose, 2 g of sodium pyruvate, 0.2% Tween 80, and 0.5 g of L- Cysteine in 1 L of DW.</li>
		<li>Adjust the final pH (6.0 &#177; 0.2) of the solution using 6 N HCl before the addition of 0.008% aniline blue and 15 g of agar. Autoclave the medium at 121 &#176;C for 15 min, cool in a water bath, and pour into sterile Petri dishes. Store all freshly-prepared media in sterile Petri dishes at 4 &#176;C until needed.</li>
	</ol>
	</li>
	<li>Glycerol stock of LAB cultures
	<ol>
		<li>Prepare glycerol stocks (50% glycerol) by diluting 100% glycerol in the same volume of DW and sterilize at 100 &#176;C for 30 min using a dry sterilization cycle. Cool the glycerol stocks to RT and aseptically store them for further use.</li>
		<li>Use bacterial growth (a single colony of L. bulgaricus obtained using the developed QC protocol) from overnight cultures.</li>
		<li>Pipette 500 &#181;L of the overnight cultures into 50% glycerol in a 2 mL centrifuge tube and gently mix them together. Freeze and store the glycerol stock containing the LAB cultures at an ultra-low temperature of -80 &#176;C until needed. 
		&#8203;NOTE: A graphical scheme of the protocol for the quality control and activation of LAB cultures is shown in Figure 1.</li>
	</ol>
	</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>No</td>
			<td>Product Code</td>
			<td>Sample</td>
			<td>Source</td>
			<td>Bacterial Composition as labeled1</td>
		</tr>
		<tr>
			<td>1</td>
			<td>S9</td>
			<td>Pure Industrial Strain</td>
			<td>Bulgaria</td>
			<td>Lb. bulgaricus</td>
		</tr>
		<tr>
			<td>2</td>
			<td>LB6</td>
			<td>Pure Industrial Strain</td>
			<td>Bulgaria</td>
			<td>Lb. bulgaricus,</td>
		</tr>
		<tr>
			<td>3</td>
			<td>ATCC 11842</td>
			<td>Pure Industrial Strain</td>
			<td>ATCC</td>
			<td>Lb. bulgaricus</td>
		</tr>
		<tr>
			<td>4</td>
			<td>DAW</td>
			<td>Yogurt</td>
			<td>USA</td>
			<td>Lb. bulgaricus, other live culture</td>
		</tr>
		<tr>
			<td>5</td>
			<td>E22</td>
			<td>Yogurt</td>
			<td>USA</td>
			<td>Lb. bulgaricus, other live culture</td>
		</tr>
		<tr>
			<td>6</td>
			<td>Reuteri</td>
			<td>Yogurt</td>
			<td>USA</td>
			<td>Limosilactobacillus reuteri</td>
		</tr>
		<tr>
			<td colspan="5">1Lb. =Lactobacillus</td>
		</tr>
	</tbody>
</table>
Table 1: Probiotic strains. The table lists the probiotic strains used in this study.
2. Protocol for the activation and quality control of LAB cultures
<ol>
	<li>Take the prepared glycerol stock of LAB strains (in 2 mL centrifuge tubes) from the -80 &#176;C ultra-low freezer, and do not allow them to thaw before use.</li>
	<li>Clean and disinfect the opening of the centrifuge tubes with 70% alcohol, and gently vortex before use.</li>
	<li>Pipette about 250 &#181;L (0.25 mL) of the stock LAB culture from the centrifuge tubes into fresh 2 mL MRS test tubes.</li>
	<li>Gently vortex, parafilm the test tubes, and anaerobically incubate them overnight at 42 &#176;C for 12-16 h.</li>
	<li>Take about 500 &#181;L (0.5 mL) from the overnight grown cultures from the 2 mL MRS test tubes into fresh 7 mL MRS test tubes, vortex, and anaerobically incubate them overnight at 42 &#176;C for 12-16 h.</li>
	<li>Assess the microbial growth by measuring the optical density (OD) or growth of the cultures at 610 nm with a UV- visible spectrophotometer and record acceptable results between 0.7 and 0.9.</li>
	<li>Streak the overnight cultures from the 7 mL MRS tubes onto MRS and MRCM-PYR agar plates and incubate them anaerobically for 72 h at 42 &#176;C.</li>
	<li>Pick isolated colonies from the agar plates, transfer them into fresh 7 mL MRS test tubes, gently vortex, and anaerobically incubate them overnight at 42 &#176;C for 12-16 h.</li>
	<li>Store the agar plates containing the isolated strains at 4 &#176;C in the refrigerator for a week.</li>
	<li>Measure and confirm the OD (between 0.7 and 0.9) from the 7 mL MRS test tubes of the LAB cultures isolated from the streaked plates at 610 nm and use them as working cultures for all related experiments.</li>
	<li>Perform ten-fold dilutions (serially dilute) of the grown LAB cultures from the final 7 mL MRS test tubes using 9 mL of peptone water (a physiological buffer) to obtain a 1:10 ratio.</li>
	<li>Finally, take about 250 &#181;L (0.25 mL) from appropriate serial dilutions for all fermentation experiments.</li>
	<li>Activate the broth containing strains (250 &#181;L) from step 2.12 by transferring them into fresh 7 mL MRS broth and incubating them anaerobically at 42 &#176;C for 16 h.</li>
	<li>Continue by repeating steps 2.6-2.12 to ensure viable and superior cell growth from LAB cultures. 
	NOTE: All L. bulgaricus strains were activated in the freshly prepared MRS broth and were then incubated anaerobically at 42 &#176;C for 16 h in order to reach an optical density (OD610 nm) of bacterial growth between 0.7 and 0.9. Bacterial growth was measured with a UV-visible spectrophotometer at 610 nm. The pH values of the overnight cultures were in the range of 3.5 to 5.3 as a result of the production of lactic acid, an organic end product of LAB fermentation. Safety procedures such as proper airflow circulation in the biosafety hood and avoiding burns during the use of the bunsen burner were adhered to and observed.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63314/63314fig01.jpg" /> 
Figure 1: A graphical scheme of the protocol for the activation of lactic acid bacteria (LAB) cultures. The scheme provides details and the basic instruments required for the handling and activation of LAB culture strains. <a href="https://www.jove.com/files/ftp_upload/63314/63314fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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The authors have nothing to disclose.

The results of all strains evaluated with the quality control protocol and without the use of the protocol were the same, and as such, results linked to only strains (S9, and LB6) were presented. The activated LAB strains had superior cell growth that was characterized by a high intensity of cell biomass, therefore, causing a turbid appearance of the MRS fermentative broth in the test tube11. The observed cell growth after culture activation was evident between 12 h and 16 h at an anaerobic fermen...

Lactic acid bacteria (LAB) are a group of uniquely diverse bacteria that have industrial potential. Strains belonging to Lactobacillus delbreuckii subsp. bulgaricus and Streptococcus thermophilus are mostly used as dairy starter cultures for fermented dairy food products such as yogurt1. Selected LAB strains are also classified as probiotics as they confer health benefits to humans when dosages are adequately administered2. Lactic acid bacteria are also gram-positive, non-spore-forming, non-respiring but aerotolerant microorganisms that are generally characterized by the production of lactic acid as a key fermentation product. LAB also synthesizes essential metabolites, for example, organic acids, bacteriocins, and other antimicrobial compounds3 that can inhibit a broad spectrum of foodborne pathogens4. Lactic acid, a major end product of carbohydrate catabolism and a by-product of LAB fermentation, is an organic metabolite that possesses antimicrobial properties and is potentially useful for food biopreservation applications3,5,6. Furthermore, the organic acids produced by LAB impart the flavor, texture, and aroma of foods, thus consequently enhancing their overall organoleptic properties5,6. The distinct nutritional requirements of LAB coupled with their ubiquitous nature, ultimately enable the bacteria to easily thrive in different environments such as dairy-based foods, fermented foods, vegetables as well as in the human gut7.
There is a growing demand for starter cultures from LAB for yogurt production and many diverse dairy applications8,9, hence critical attention and established scientific techniques should be adhered to, in LAB strains cultivation, as well as in the activation of both lyophilized and isolated strains as this activity is vital for enhanced fermentation performance. The Food Microbiology and Biotechnology laboratory, therefore, actively engages in suitable technology development geared toward the activation, superior growth, and fermentation characteristic of LAB strains isolated from fermented dairy products as well as from industrial starter cultures employed for yogurt production. Furthermore, it is noteworthy that LAB culture strains industrially produced undergo preservative activities such as freeze-drying and frozen storage, causing cell stress and injury, as a result of the cold shock process they are subjected to10. In limiting, the viability challenges and improving the functionality of LAB strains obtained from either isolated food products or freeze-dried products, it is important to properly activate these cultures as a form of quality control to enhance their fermentative characteristic8. In this study, the objective was to develop an in-house quality control protocol for the activation and growth of L. delbrueckii subsp. bulgaricus culture strains that ultimately promoted viable LAB growth, as well as enhanced the fermentation performance and the metabolic functionality of LAB strains. This protocol could ultimately be adapted (using optimal growth media and appropriate culturing conditions) for the cultivation of other LAB strains for fermentation research, as well as for industrial purposes or bioprocessing operations. This LAB activation and quality control protocol will therefore ensure superior viable dairy starter cultures are obtained and potentially functional for diverse applications in the global dairy and food industry.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Aniline Blue</td>
			<td>Thermo Scientific</td>
			<td>R21526</td>
			<td>25 g</td>
		</tr>
		<tr>
			<td>Beef extract</td>
			<td>Research Products International</td>
			<td>50-197-7509</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Fisher Scientific</td>
			<td>BP1422-500</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>Calcium Chloride dihydrate</td>
			<td>Fisher Scientific</td>
			<td>C79-500</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>Dextrose Anhydrous</td>
			<td>Fisher Scientific</td>
			<td>BP350500</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>D-Fructose</td>
			<td>ACROS Organics</td>
			<td>AC161355000</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>Difco agar powder</td>
			<td>Difco</td>
			<td>DF0812-07-1</td>
			<td>2 kg</td>
		</tr>
		<tr>
			<td>TPY agar</td>
			<td>Difco</td>
			<td>211921</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>Eppendorf microcentrifuge tube (Snap-Cap Microcentrifuge Safe-Lock)</td>
			<td>Fisher Scientific</td>
			<td>05-402-12</td>
			<td>2 mL</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Thermo Scientific</td>
			<td>PI17904</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Infrared CO2 Incubator</td>
			<td>Forma Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lactobacillus delbrueckii subsp. bulgaricus</td>
			<td>American Type Culture Collection (ATCC)</td>
			<td>ATCC 11842</td>
			<td></td>
		</tr>
		<tr>
			<td>Lactobacillus delbrueckii subsp. bulgaricus</td>
			<td>Bulgaria</td>
			<td>S9</td>
			<td></td>
		</tr>
		<tr>
			<td>Lactobacillus delbrueckii subsp. bulgaricus</td>
			<td>Bulgaria</td>
			<td>LB6</td>
			<td></td>
		</tr>
		<tr>
			<td>Lactobacillus delbrueckii subsp. bulgaricus</td>
			<td>Food Microbiology and Biotechnology Laboratory (NCATSU)</td>
			<td>DAW</td>
			<td></td>
		</tr>
		<tr>
			<td>Lactobacillus delbrueckii subsp. bulgaricus</td>
			<td>Food Microbiology and Biotechnology Laboratory (NCATSU)</td>
			<td>E22</td>
			<td></td>
		</tr>
		<tr>
			<td>Lactobacillus reuteri</td>
			<td>Biogai, Raleigh / Food Microbiology and Biotechnology Laboratory (NCATSU)</td>
			<td>RD2</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine hydrochloride monohydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C6852-25G</td>
			<td>25 g</td>
		</tr>
		<tr>
			<td>Maltose monohydrate</td>
			<td>Fisher Scientific</td>
			<td>M75-100</td>
			<td>100 g</td>
		</tr>
		<tr>
			<td>MRS broth</td>
			<td>Neogen</td>
			<td>50-201-5691</td>
			<td>5 kg</td>
		</tr>
		<tr>
			<td>Peptone No. 3</td>
			<td>Hach</td>
			<td>50-199-6719</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic (K2HPO4)</td>
			<td>Research Products International</td>
			<td>50-712-761</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>Sodium acetate trihydrate</td>
			<td>Fisher Scientific</td>
			<td>S220-1</td>
			<td>1 kg</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Fisher Scientific</td>
			<td>BP358-1</td>
			<td>1 kg</td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Fisher Scientific</td>
			<td>BP356-100</td>
			<td>100 g</td>
		</tr>
		<tr>
			<td>Test Tubes with Rubber-Lined Screw Caps</td>
			<td>Fisher Scientific</td>
			<td>FB70125150</td>
			<td>25 x 150 mm</td>
		</tr>
		<tr>
			<td>Tween 80</td>
			<td>Fisher Scientific</td>
			<td>T164-500</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Ultra low freezer</td>
			<td>So-Low</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Uracil</td>
			<td>ACROS Organics</td>
			<td>AC157301000</td>
			<td>100 g</td>
		</tr>
		<tr>
			<td>UV- visible spectrophotometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>Evolution 201</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Genie 2</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Fisher Scientific</td>
			<td>BP1422-500</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fisher Scientific</td>
			<td>T08204K7</td>
			<td>4 L</td>
		</tr>
		<tr>
			<td>Hydrochloric Acid (6N (Certified), Fisher Chemical)</td>
			<td>Fisher Scientific</td>
			<td>&#160;SA56-500</td>
			<td>500 mL</td>
		</tr>
	</tbody>
</table>

the cultivation, growth, and viability of lactic acid bacteria: a quality control perspective

Lactic acid bacteria (LAB) are essential dairy starter cultures that are significantly employed for the manufacture of fermented dairy products such as yogurt and cheese. LAB predominantly produce lactic acid as a major end product of fermentation, and they synthesize important metabolites that impart the organoleptic characteristics of fermented food products. LAB are fastidious bacteria that thrive in many environments when adequate nutritional requirements are fulfilled. The demand for superior LAB dairy starter cultures for fermentation applications in the food and dairy industry, has resulted in the need to provide viable and active cultures for all bioprocessing operations. The development of a standard protocol for ensuring the viability and enhanced functionality of LAB cultures in the laboratory as well as dairy processing environments is thus very critical. In addressing concerns linked to resuscitating weak, stressed, and injured LAB culture cells, a protocol that vividly outlines salient steps to recover, enhance cell regeneration, and improve metabolic functionality of LAB strains is of the utmost importance. The maintenance of culture purity, functionality, and viability for LAB starter cultures is likewise critical. Therefore, adherence to a unique protocol guideline will result in the promotion of fermentation performance for many LAB strains dedicated to fermentation and biotechnology processes. As a result, the Food Microbiology and Biotechnology Laboratory at North Carolina Agriculture and Technical State University has developed a standard protocol for the activation and quality control of selected LAB strains that has resulted in highly functional and viable LAB culture strains employed for fermentation research. The adaptation and recommendation of a protocol such as this for use in the dairy and food industry&#160;will help to ensure LAB viability and functionality for many applications.

Cell growth of the evaluated LAB strains cultivated with the quality control protocol was significantly different (P &#60; 0.05) than the strains cultivated without this standard protocol. The QC protocol for both L. bulgaricus and L. reuteri employed a multi-subculturing approach (subculturing three times before streaking on agar plates), whereas the control procedure had subculturing done only once with all other conditions kept constant. The colony growth was also higher and well defined on the growt...

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The Cultivation, Growth, and Viability of Lactic Acid Bacteria: A Quality Control Perspective

The quality control assessment of lactic acid bacteria (LAB) cultures has been confirmed as an effective way to enhance the viability and functionality of LAB strains for fermentation procedures. To buttress this assertion, we developed a protocol that elucidates how LAB cultures are activated and cultivated for fermentation and bioprocessing procedures.

Lactic acid bacteria (LAB) are essential dairy starter cultures that are significantly employed for the manufacture of fermented dairy products such as ...

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Research

JoVE Journal

Biology

7.7K Views.  North Carolina A&T State University, Greensboro, NC, USA. The quality control assessment of lactic acid bacteria (LAB) cultures has been confirmed as an effective way to enhance the viability and functionality of LAB strains for fermentation procedures. To buttress this assertion, we developed a protocol that elucidates how LAB cultures are activated and cultivated for fermentation and bioprocessing procedures.

Video: The Cultivation, Growth, and Viability of Lactic Acid Bacteria: A Quality Control Perspective

Counting and Determining the Viability of Cultured Cells

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A hemacytometer is a thick glass slide with a central area designed as a counting chamber. Cell suspension is applied to a defined area and counted so cell density can be calculated. 

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. In this video, we demonstrate how cells are counted using a hemacytometer.

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A ...

DNA Microarrays: Sample Quality Control, Array Hybridization and Scanning

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, different physiological or pathological states, response to therapy, and, in general, any condition that is being experimentally tested1. Expression profiling of neural tissues requires isolation of high quality RNA, amplification of the isolated RNA and hybridization to DNA microarrays. In this article we describe protocols for reproducible microarray experiments from brain tumor tissue2. We will start by performing a quality control analysis of isolated RNA samples with Agilent's 2100 Bioanalyzer "lab-on-a-chip" technology. High quality RNA samples are critical for the success of any microarray experiment, and the 2100 Bioanalyzer provides a quick, quantitative measurement of the sample quality. RNA samples are then amplified and labeled by performing reverse transcription to obtain cDNA, followed by in vitro transcription in the presence of labeled nucleotides to produce labeled cRNA. By using a dual-color labeling kit, we will label our experimental sample with Cy3 and a reference sample with Cy5. Both samples will then be combined and hybridized to Agilent's 4x44 K arrays. Dual-color arrays offer the advantage of a direct comparison between two RNA samples, thereby increasing the accuracy of the measurements, in particular for small changes in expression levels, because the two RNA samples are hybridized competitively to a single microarray. The arrays will be scanned at the two corresponding wavelengths, and the ratio of Cy3 to Cy5 signal for each feature will be used as a direct measurement of the relative abundance of the corresponding mRNA. This analysis identifies genes that are differentially expressed in response to the experimental conditions being tested.

We demonstrate the use of DNA microarrays for expression profiling of the nervous system. We describe RNA quality control, sample labeling, and array hybridization and scanning.

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, ...

Assessment of GFP Expression and Viability Using the Tali Image-Based Cytometer

Single-cell and population information are commonly obtained either by flow cytometry or fluorescence microscopy. However, these two methods provide different information. Flow cytometry gives quantitative multi-parametric information about physical characteristics and staining or expression, but doesn't allow for visualization. Stand-alone fluorescence microscopy provides visual data, but doesn't allow for straightforward quantitative measurements1.
Image-based cytometry bridges the gap between these two methods, enabling the quick visualization and simultaneous quantitative analysis of thousands of cells in heterogeneous populations2. Here, we present a method for performing cell viability and green fluorescent protein (GFP) expression assays using the Tali Image-Based Cytometer3. The Tali instrument is a 3-channel (bright field, green fluorescence, red fluorescence) benchtop assay platform that offers several advantages over flow cytometry and fluorescence microscopy. The Tali cytometer is less expensive, takes up less bench space, requires less maintenance, and the work flow has been simplified so that the operation and analysis is much simpler and quicker. The Tali cytometer is capable of performing a range of suspension cell-based assays, including GFP and red fluorescent protein (RFP) expression, apoptosis4-6 and cell viability analysis with propidium iodide (PI)7-11.
Here, we demonstrate the use of the Tali instrument in performing a cell viability assay in cells expressing GFP. GFP-transduced cells are stained using the Tali Viability Kit - Dead Cell Red. The cells are then pipetted into a Tali Cellular Analysis Slide and loaded into the cytometer. Bright field, red fluorescence and green fluorescence images are captured and analyzed using assay specific algorithms. Histograms are then generated to display cell size, PI fluorescence intensity, and GFP fluorescence intensity. These parameters can then be thresholded to home in on a specific cell population.
A side-by side comparison of the Tali Image-Based Cytometer and traditional flow cytometry demonstrates that the two methods provide comparable data regarding cell viability and protein expression. However, the Tali instrument provides additional visual information about the cell population that cannot be obtained using a flow cytometer.

This protocol describes how to perform cell viability and fluorescence expression assays using the Tali Image-Based Cytometer.

Single-cell and population information are commonly obtained either by flow cytometry or fluorescence microscopy. However, these two methods provide ...

Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of Massachusetts (USA). Since then MTB have been discovered in stratified water- and sediment-columns from all over the world2. One feature common to all MTB is that they contain magnetosomes, which are intracellular, membrane-bound magnetic nanocrystals of magnetite (Fe3O4) and/or greigite (Fe3S4) or both3, 4. In the Northern hemisphere, MTB are typically attracted to the south end of a bar magnet, while in the Southern hemisphere they are usually attracted to the north end of a magnet3,5. This property can be exploited when trying to isolate MTB from environmental samples.
One of the most common ways to enrich MTB is to use a clear plastic container to collect sediment and water from a natural source, such as a freshwater pond. In the Northern hemisphere, the south end of a bar magnet is placed against the outside of the container just above the sediment at the sediment-water interface. After some time, the bacteria can be removed from the inside of the container near the magnet with a pipette and then enriched further by using a capillary racetrack6 and a magnet. Once enriched, the bacteria can be placed on a microscope slide using a hanging drop method and observed in a light microscope or deposited onto a copper grid and observed using transmission electron microscopy (TEM).
Using this method, isolated MTB may be studied microscopically to determine characteristics such as swimming behavior, type and number of flagella, cell morphology of the cells, shape of the magnetic crystals, number of magnetosomes, number of magnetosome chains in each cell, composition of the nanomineral crystals, and presence of intracellular vacuoles.

We demonstrate a method to collect magnetotactic bacteria (MTB) that can be applied to natural waters. MTB can be isolated and enriched from sediment samples using a relatively simple setup that takes advantage of the bacteria's natural magnetism. Isolated MTB can then be examined in detail using both light and electron microscopy.

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of ...

Deferred Growth Inhibition Assay to Quantify the Effect of Bacteria-derived Antimicrobials on Competition

Competitive exclusion can occur in microbial communities when, for example, an inhibitor-producing strain outcompetes its competitor for an essential nutrient or produces antimicrobial compounds that its competitor is not resistant to. Here we describe a deferred growth inhibition assay, a method for assessing the ability of one bacterium to inhibit the growth of another through the production of antimicrobial compounds or through competition for nutrients. This technique has been used to investigate the correlation of nasal isolates with the exclusion of particular species from a community. This technique can also be used to screen for lantibiotic producers or potentially novel antimicrobials. The assay is performed by first culturing the test inhibitor-producing strain overnight on an agar plate, then spraying over the test competitor strain and incubating again. After incubation, the extent of inhibition can be measured quantitatively, through the size of the zone of clearing around the inhibitor-producing strain, and qualitatively, by assessing the clarity of the inhibition zone. Here we present the protocol for the deferred inhibition assay, describe ways to minimize variation between experiments, and define a clarity scale that can be used to qualitatively assess the degree of inhibition.

The deferred growth inhibition assay can be used to assess the competition effect by one bacterial isolate on another. Inhibition is quantified by measuring the zone of clearing around the inhibitor-producing isolate, or qualitatively assessed by determining the visible extent of inhibition.

Competitive exclusion can occur in microbial communities when, for example, an inhibitor-producing strain outcompetes its competitor for an essential ...

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we showed that D. discoideum is remarkably resilient to protein aggregation and can be used to gain insights into the cellular protein quality control system. However, the use of D. discoideum as a model system poses several challenges to microscopy-based experimental approaches, such as the high motility of the cells and their susceptibility to photo-toxicity. The latter proves to be especially challenging when studying protein homeostasis, as the phototoxic effects can induce a cellular stress response and thus alter to behavior of the protein quality control system. 
Temperature increase is a commonly used way to induce cellular stress. Here, we describe a temperature-controllable imaging protocol, which allows observing temperature-induced perturbations in D. discoideum. Moreover, when applied at normal growth temperature, this imaging protocol can also noticeably reduce photo-toxicity, thus allowing imaging with higher intensities. This can be particularly useful when imaging proteins with very low expression levels. Moreover, the high mobility of the cells often requires the acquisition of multiple fields of view to follow individual cells, and the number of fields needs to be balanced against the desired time interval and exposure time.

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The social amoebae Dictyostelium discoideum has recently been established as a system to study protein misfolding and proteostasis. Here, we describe a new imaging-based methodology to study temperature-induced protein aggregation and the cellular stress response in D. discoideum.

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we ...

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we know about C. elegans is limited to laboratory cultivation of the nematodes that may not necessarily reflect the environments they normally inhabit in nature. Cultivation of C. elegans in a 3D habitat that is more similar to the 3D matrix that worms encounter in rotten fruits and vegetative compost in nature could reveal novel phenotypes and behaviors not observed in 2D. In addition, experiments in 3D can address how phenotypes we observe in 2D are relevant for the worm in nature. Here, a new method in which C. elegans grows and reproduces normally in three dimensions is presented. Cultivation of C. elegans in Nematode Growth Tube-3D (NGT-3D) can allow us to measure the reproductive fitness of C. elegans strains or different conditions in a 3D environment. We also present a novel method, termed Nematode Growth Bottle-3D (NGB-3D), to cultivate C. elegans in 3D for microscopic analysis. These methods allow scientists to study C. elegans biology in conditions that are more reflective of the environments they encounter in nature. These can help us to understand the overlying evolutionary relevance of the physiology and behavior of C. elegans we observe in the laboratory.

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

We present a simple method to construct 3D nematode cultivation systems called NGT-3D and NGB-3D. These can be used to study nematode fitness and behaviors in habitats that are more similar to natural Caenorhabditis elegans habitats than the standard 2D laboratory C. elegans culture plates.

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we ...

A Hydroponic Co-cultivation System for Simultaneous and Systematic Analysis of Plant/Microbe Molecular Interactions and Signaling

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. Arabidopsis thaliana-Agrobacterium tumefaciens provides an excellent model system to study bacterial pathogenesis and plant interactions. Previous studies of plant-Agrobacterium interactions have largely relied on plant cell suspension cultures, the artificial wounding of plants, or the artificial induction of microbial virulence factors or plant defenses by synthetic chemicals. However, these methods are distinct from the natural signaling in planta, where plants and microbes recognize and respond in spatial and temporal manners. This work presents a hydroponic cocultivation system where intact plants are supported by metal mesh screens and cocultivated with Agrobacterium. In this cocultivation system, no synthetic phytohormone or chemical that induces microbial virulence or plant defense is supplemented. The hydroponic cocultivation system closely resembles natural plant-microbe interactions and signaling homeostasis in planta. Plant roots can be separated from the medium containing Agrobacterium, and the signaling and responses of both the plant hosts and the interacting microbes can be investigated simultaneously and systematically. At any given timepoint/interval, plant tissues or bacteria can be harvested separately for various &#34;omics&#34; analyses, demonstrating the power and efficacy of this system. The hydroponic cocultivation system can be easily adapted to study: 1) the reciprocal signaling of diverse plant-microbe systems, 2) signaling between a plant host and multiple microbial species (i.e.&#160;microbial consortia or microbiomes), 3) how nutrients and chemicals are implicated in plant-microbe signaling, and 4) how microbes interact with plant hosts and contribute to plant tolerance to biotic or abiotic stresses.

The described hydroponic cocultivation system supports intact plants with metal mesh screens and cocultivates them with bacteria. Plant tissue, bacteria, and secreted molecules can then be separately harvested for downstream analyses, simultaneously allowing for the molecular responses of both plant hosts and interacting microbes or microbiomes to be investigated.

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. ...

Anaerobic Growth and Maintenance of Mammalian Cell Lines

Most mucosal surfaces along with the midpoints in tumors and stem cell niches are geographic areas of the body that are anoxic. Previous studies show that the incubation in normoxic (5% CO2 in air) or hypoxic (low oxygen levels) conditions followed by an anoxic incubation (an absence of free oxygen) results in limited viability (2&#8211;3 days). A novel methodology was developed that enables an anoxic cell cultivation (for at least 17 days; the maximum time tested). The most critical aspect of this methodology is to ensure that no oxygen is introduced into the system. This is obtained by the degassing of media, and by flushing tubes, dishes, flasks, and pipettes with an anaerobic gas mixture (H2, CO2, N2) followed by permitting the materials to equilibrate to the anoxic (non-oxygen) environment prior to usage.&#160;Additional care must be exercised when acquiring photomicrographs to ensure that the micrographs obtained do not contain artifacts. In the absence of oxygen, cell morphology is significantly altered. Two distinct morphotypes are noted for all anaerobically-grown cells. The ability to grow and maintain mammalian cells in the absence of oxygen can be applied to the analysis of cell physiology, polymicrobial interactions, and the characterization of biosynthetic pathways for novel cancer drug development.

Here, we describe a new method that enables the anaerobic long-term cultivation of established cell lines. The maximum survival time that was tested is 17 days. This method is suitable for the testing of cytotoxic agents and exploring the physiology of anoxically replicating cells.

Most mucosal surfaces along with the midpoints in tumors and stem cell niches are geographic areas of the body that are anoxic. Previous studies show that ...

Growing Magnetotactic Bacteria of the Genus Magnetospirillum: Strains MSR-1, AMB-1 and MS-1

Magnetotactic bacteria are Gram-negative, motile, mainly aquatic prokaryotes ubiquitous in freshwater and marine habitats. They are characterized by their ability to biomineralize magnetosomes, which are magnetic nanometer-sized crystals of magnetite (Fe3O4) or greigite (Fe3S4) surrounded by a lipid bilayer membrane, within their cytoplasm. For most known magnetotactic bacteria, magnetosomes are assembled in chains inside the cytoplasm, thereby conferring a permanent magnetic dipole moment to the cells and causing them to align passively with external magnetic fields. Because of these specific features, magnetotactic bacteria have a great potential for commercial and medical applications. However, most species are microaerophilic and have specific O2 concentration requirements, making them more difficult to grow routinely than many other bacteria such as Escherichia coli. Here we present detailed protocols for growing three of the most widely studied strains of magnetotactic bacteria, all belonging to the genus Magnetospirillum. These methods allow for precise control of the O2 concentration made available to the bacteria, in order to ensure that they grow normally and synthesize magnetosomes. Growing magnetotactic bacteria for further studies using these procedures does not require the experimentalist to be an expert in microbiology. The general methods presented in this article may also be used to isolate and culture other magnetotactic bacteria, although it is likely that growth media chemical composition will need to be modified.

Growing Magnetotactic Bacteria of the Genus Magnetospirillum: Strains MSR-1, AMB-1 and MS-1

We present a procedure for growing several strains of Magnetospirillum in two different types of growth media. Magnetospirillum gryphiswaldense strain MSR-1 is grown in both liquid and O2 concentration gradient semi-solid media while M. magneticum strain AMB-1 and M. magnetotacticum strain MS-1 are grown in liquid medium.

Magnetotactic bacteria are Gram-negative, motile, mainly aquatic prokaryotes ubiquitous in freshwater and marine habitats. They are characterized by their ...

The Cultivation, Growth, and Viability of Lactic Acid Bacteria: A Quality Control Perspective

Summary

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Chapters in this video

Counting and Determining the Viability of Cultured Cells

DNA Microarrays: Sample Quality Control, Array Hybridization and Scanning

Assessment of GFP Expression and Viability Using the Tali Image-Based Cytometer

Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment

Deferred Growth Inhibition Assay to Quantify the Effect of Bacteria-derived Antimicrobials on Competition

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

A Hydroponic Co-cultivation System for Simultaneous and Systematic Analysis of Plant/Microbe Molecular Interactions and Signaling

Anaerobic Growth and Maintenance of Mammalian Cell Lines

Growing Magnetotactic Bacteria of the Genus Magnetospirillum: Strains MSR-1, AMB-1 and MS-1