Name: the cultivation, growth, and viability of lactic acid bacteria: a quality control perspective
Uploaded: 2022-06-16T12:40:00
Description: Watch this Scientific Journal Video about The Cultivation, Growth, and Viability of Lactic Acid Bacteria: A Quality Control Perspective at JoVE.com

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 Biotechnolo...

<ol>
	<li>Karakas-Sen, A., Karakas, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+identification+and+technological+properties+of+lactic+acid+bacteria+from+raw+cow+milk.">Isolation, identification and technological properties of lactic acid bacteria from raw cow milk.</a> Bioscience Journal. 34 (2), 385-399 (2018).</li><li>Martin, R., Langella, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+health+concepts+in+the+probiotics+field:+Streamlining+the+definitions.">Emerging health concepts in the probiotics field: Streamlining the definitions.</a> Frontiers in Microbiology. 10, 1047 (2019).</li><li>Sadishkumar, V., Jeevaratnam, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+probiotic+evaluation+of+potential+antioxidant+lactic+acid+bacteria+isolated+from+idli+batter+fermented+with+Piper+betle+leaves.">In vitro probiotic evaluation of potential antioxidant lactic acid bacteria isolated from idli batter fermented with Piper betle leaves.</a> International Journal of Food Science &amp; Technology. 52 (2), 329-340 (2017).</li><li>Ayivi, R. D., 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=Lactic+acid+bacteria:+Food+safety+and+human+health+applications.">Lactic acid bacteria: Food safety and human health applications.</a> Dairy. 1 (3), 202-232 (2020).</li><li>Quinto, E. J., 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=Probiotic+lactic+acid+bacteria:+A+review.">Probiotic lactic acid bacteria: A review.</a> Food and Nutrition Sciences. 5 (18), 1765-1775 (2014).</li><li>Hayek, S. A., Gyawali, R., Aljaloud, S. O., Krastanov, A., Ibrahim, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultivation+media+for+lactic+acid+bacteria+used+in+dairy+products.">Cultivation media for lactic acid bacteria used in dairy products.</a> Journal of Dairy Research. 86 (4), 490-502 (2019).</li><li>Bintsis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lactic+acid+bacteria+as+starter+cultures:+An+update+in+their+metabolism+and+genetics.">Lactic acid bacteria as starter cultures: An update in their metabolism and genetics.</a> Aims Microbiology. 4, 665-684 (2018).</li><li>Shao, Y., Gao, S., Guo, H., Zhang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+culture+conditions+and+preconditioning+on+survival+of+Lactobacillus+delbrueckii+subspecies+bulgaricus+ND02+during+lyophilization.">Influence of culture conditions and preconditioning on survival of Lactobacillus delbrueckii subspecies bulgaricus ND02 during lyophilization.</a> Journal of Dairy Science. 97 (3), 1270-1280 (2014).</li><li>Aryana, K. J., Olson, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+100-year+review:+Yogurt+and+other+cultured+dairy+products.">A 100-year review: Yogurt and other cultured dairy products.</a> Journal of Dairy Science. 100 (12), 9987-10013 (2017).</li><li>Kandil, S., El Soda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+freezing+and+freeze-drying+on+intracellular+enzymatic+activity+and+autolytic+properties+of+some+lactic+acid+bacterial+strains.">Influence of freezing and freeze-drying on intracellular enzymatic activity and autolytic properties of some lactic acid bacterial strains.</a> Advances in Microbiology. 5 (6), 371-382 (2015).</li><li>Malairuang, K., Krajang, M., Sukna, J., Rattanapradit, K., Chamsart, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+cell+density+cultivation+of+Saccharomyces+cerevisiae+with+intensive+multiple+sequential+batches+together+with+a+novel+technique+of+fed-batch+at+cell+level+(FBC).">High cell density cultivation of Saccharomyces cerevisiae with intensive multiple sequential batches together with a novel technique of fed-batch at cell level (FBC).</a> Processes. 8 (10), 1321 (2020).</li><li>Jeanson, S., Floury, J., Gagnaire, V., Lortal, S., Thierry, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+colonies+in+solid+media+and+foods:+A+review+on+their+growth+and+interactions+with+the+micro-environment.">Bacterial colonies in solid media and foods: A review on their growth and interactions with the micro-environment.</a> Frontiers in Microbiology. 6, 1284 (2015).</li><li>Oyeniran, 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=A+modified+reinforced+clostridial+medium+for+the+isolation+and+enumeration+of+Lactobacillus+delbrueckii+ssp.+bulgaricus+in+a+mixed+culture.">A modified reinforced clostridial medium for the isolation and enumeration of Lactobacillus delbrueckii ssp. bulgaricus in a mixed culture.</a> Journal of Dairy Science. 103, 5030-5042 (2020).</li><li>Iguchi, 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=Effects+of+repeated+subculturing+and+prolonged+storage+at+room+temperature+of+Enterohemorrhagic+Escherichia+coli+O157:+H7+on+pulsed-field+gel+electrophoresis+profiles.">Effects of repeated subculturing and prolonged storage at room temperature of Enterohemorrhagic Escherichia coli O157: H7 on pulsed-field gel electrophoresis profiles.</a> Journal of Clinical Microbiology. 40 (8), 3079-3081 (2002).</li><li>Hayek, S. A., Ibrahim, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+limitations+and+challenges+with+lactic+acid+bacteria:+a+review.">Current limitations and challenges with lactic acid bacteria: a review.</a> Food and Nutrition Sciences. 4 (11), 73-87 (2013).</li><li>Ahmed, S. A., Ibrahim, S. A., Kim, C., Shahbazi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Significance+of+bile+salt+tolerant+Lactobacillus+reuteri.">Significance of bile salt tolerant Lactobacillus reuteri.</a> Proceedings of the 2007 National Conference on Environmental Science and Technology. , 17-23 (2009).</li><li>Gyawali, 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=A+comparative+study+of+extraction+techniques+for+maximum+recovery+of+&#946;-galactosidase+from+the+yogurt+bacterium+Lactobacillus+delbrueckii+ssp.+bulgaricus.">A comparative study of extraction techniques for maximum recovery of &#946;-galactosidase from the yogurt bacterium Lactobacillus delbrueckii ssp. bulgaricus.</a> Journal of Dairy Research. 87 (1), 123-126 (2020).</li><li>Nwamaioha, N. O., Ibrahim, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+selective+medium+for+the+enumeration+and+differentiation+of+Lactobacillus+delbrueckii+ssp.+bulgaricus.">A selective medium for the enumeration and differentiation of Lactobacillus delbrueckii ssp. bulgaricus.</a> Journal of Dairy Science. 101 (6), 4953-4961 (2018).</li></ol>

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...

Watch this Scientific Journal Video about The Cultivation, Growth, and Viability of Lactic Acid Bacteria: A Quality Control Perspective at JoVE.com

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 ...

Our protocol is significant because ensures purity as a quality control check. It's also promotes severe fermentation performance and consistency of yield in lactic acid bacteria fermentation. The main advantage of this technique is that it can enhance the fermentation performance of lactic acid bacteria via cell purity, viability, and functionality.This technique is user-friendly and can be easily performed by any person due to the use of only basic equipment without many technical requirements. Philip Yeboah is our graduate research assistant and he will demonstrate the procedure. To begin, take the prepared glycerol stock of lactic acid bacteria, or LAB strains, in two milliliters centrifuge tubes from the minus 80 degree Celsius ultralow freezer.Do not allow them to thaw before use. Clean and disinfect the opening of the centrifuge tubes with 70%alcohol and gently vortex before use. Pipette about 250 microliters of the stock LAB culture from the centrifuge tubes to fresh two milliliter MRS test tubes.Gently vortex the test tubes, parafilm them, and anaerobically incubate them overnight at 42 degrees Celsius for 12 to 16 hours. Next, take about 500 microliters from the overnight grown cultures from the two milliliter MRS test tubes to fresh seven milliliter MRS test tubes, vortex them, and anaerobically incubate them overnight at 42 degrees Celsius for 12 to 16 hours. Assess the microbial growth by measuring the optical density or growth of the cultures at 610 nanometers with a UV visible spectrophotometer and record acceptable results between 0.7 and 0.9.Streak the overnight cultures from the seven milliliter MRS tubes onto MRS and MRCMPYR agar plates and incubate them anaerobically for 72 hours at 42 degrees Celsius. Pick the isolated colonies from the agar plates, transfer them into fresh seven milliliter MRS test tubes, gently vortex, and anaerobically incubate them overnight at 42 degrees Celsius for 12 to 16 hours. Store the agar plates containing the isolated strains at four degrees Celsius in the refrigerator for a week.Next, measure and confirm the optical density from the seven milliliter MRS test tubes of the LAB cultures isolated from the streaked plates at 610 nanometers and use them as working cultures for all related experiments. Use nine milliliters of peptone water to perform tenfold pollutions of the grown LAB cultures from the final seven milliliter MRS test tubes to obtain a 1 to 10 ratio. Take about 250 microliters from the appropriate serial delusions for all fermentation experiments and activate the broth containing the strains by transferring them into fresh seven milliliter MRS broth and incubating them anaerobically at 42 degrees Celsius for 16 hours.Repeat the steps to ensure viable and superior cell growth from LAB cultures. Cell morphology growth of S9 and LB6 lactobacillus bulgaricus strains cultivated with the quality control protocol and S9 lactobacillus bulgaricus strains cultivated without the quality control protocol are shown here. The strains were streaked in triplicates and were anaerobically incubated at 42 degrees Celsius for 72 hours.When attempting this procedure, make sure to disinfect the stock culture tubes from the freezer with 70%alcohol to prevent cross-contamination. Optical density measurement of growth cultures should always be between 0.7 and 0.9. Following this protocol, efficient LAB fermentations or bioprocessing operations with superior use of culture can be achieved.

the-cultivation-growth-viability-lactic-acid-bacteria-quality-control

Research

JoVE Journal

Biology

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, ...

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 ...

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 ...

Characterizing Mechanical Properties of Primary Cell Wall in Living Plant Organs Using Atomic Force Microscopy

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth and, therefore, the future size and shape of the plant. Many sophisticated techniques have been developed to measure these properties; however, atomic force microscopy (AFM) remains the most convenient for studying cell wall elasticity at the cellular level. One of the most important limitations of this technique has been that only superficial or isolated living cells can be studied. Here, the use of atomic force microscopy to investigate the mechanical properties of primary cell walls belonging to the internal tissues of a plant body is presented. This protocol describes measurements of the apparent Young&#39;s modulus of cell walls in roots, but the method can also be applied to other plant organs. The measurements are performed on vibratome-derived sections of plant material in a liquid cell, which allows (i) avoiding the use of plasmolyzing solutions or sample impregnation with wax or resin, (ii) making the experiments fast, and (iii) preventing dehydration of the sample. Both anticlinal and periclinal cell walls can be studied, depending on how the specimen was sectioned. Differences in the mechanical properties of different tissues can be investigated in a single section. The protocol describes the principles of study planning, issues with specimen preparation and measurements, as well as the method of selecting force-deformation curves to avoid the influence of topography on the obtained values of elastic modulus. The method is not limited by sample size but is sensitive to cell size (i.e., cells with a large lumen are difficult to examine).

Studies of cell wall biomechanics are essential for understanding plant growth and morphogenesis. The following protocol is proposed to investigate thin primary cell walls in the internal tissues of young plant organs using atomic force microscopy.

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth and, therefore, the future size and shape of the ...

Optimizing Spray-Dried Probiotic Product Quality Through Process Development

Process Development for the Spray-Drying of Probiotic Bacteria and Evaluation of the Product Quality

Probiotics and prebiotics are of great interest to the food and pharmaceutical industries due to their health benefits. Probiotics are live bacteria that can confer beneficial effects on human and animal wellbeing, while prebiotics are types of nutrients that feed the beneficial gut bacteria. Powder probiotics have gained popularity due to the ease and practicality of their ingestion and incorporation into the diet as a food supplement. However, the drying process interferes with cell viability since high temperatures inactivate probiotic bacteria. In this context, this study aimed to present all the steps involved in the production and physicochemical characterization of a spray-dried probiotic and evaluate the influence of the protectants (simulated skim milk and inulin:maltodextrin association) and drying temperatures in increasing the powder yield and cell viability. The results showed that the simulated skim milk promoted higher probiotic viability at 80 &#176;C. With this protectant, the probiotic viability, moisture content, and water activity (Aw) reduce as long as the inlet temperature increases. The probiotics&#39; viability decreases conversely with the drying temperature.&#160;At temperatures close to 120 &#176;C, the dried probiotic showed viability around 90%, a moisture content of 4.6% w/w, and an Aw of 0.26; values adequate to guarantee product stability. In this context, spray-drying temperatures above 120 &#176;C are required to ensure the microbial cells&#39; viability and shelf-life in the powdered preparation and survival during food processing and storage.

Author Spotlight: Process Development for the Spray-Drying of Probiotic Bacteria and Evaluation of the Product Quality  

This protocol details the steps involved in the production and physicochemical characterization of a spray-dried probiotic product.

Probiotics and prebiotics are of great interest to the food and pharmaceutical industries due to their health benefits. Probiotics are live bacteria that ...

Selection, Expansion, and Quality Control of hiPSCs Generated from Dermal Fibroblasts

To begin, take the plate of electroporated fibroblasts and observe the formation of hiPSC colonies under a 10x to 20x objective phase contrast microscope. The hiPSC colonies with 300 micrometer diameter, distinct borders, and a high nuclei to body ratio are suitable for picking. Before picking, coat the wells of a 96 well plate suitable for hiPSC culture with 100 microliters of coating solution.And incubate for one hour at 37 degrees Celsius. During incubation, mark the colonies of interest at the bottom of the cell culture dish with a pen. For the final preparation of the 96 well plate, remove the coating solution and add 100 microliters of pre-warmed hiPSC culture medium, containing 10 micromoles of ROCK inhibitor Y-27632 to each well.Next, wash the cells with pre-warmed PBS and add fresh pre-warmed hiPSC culture medium containing 10 micromoles of ROCK inhibitor Y-27632 before picking to remove dead cells. Pick the hiPSC colonies using a gauge needle and divide the colonies into small pieces by drawing a grid into each colony. Afterward, use a phase contrast microscope to check that the colonies are successfully divided into pieces.Finally, transfer the colonies with a 100 microliter pipette to the prepared 96 well plate, holding the pipette upright over the colony. Allow the cells to attach overnight at 37 degrees Celsius and 5%carbon dioxide without disturbance. The following day, replace the medium with 200 microliters of fresh hiPSC culture medium to remove the ROCK inhibitor Y-27632 and place the plates back in the incubator.Monitor the 96 well plate and mark the wells with successfully picked clones. The marked clones can be expanded and frozen at this point. Phase contrast images have successfully reprogrammed hiPSC colonies, selected hiPSCs, spontaneously differentiated colonies, non-hiPSC colonies, and a too dense hiPSC culture are presented.

Rabbit Eye Dissection and Cryosectioning for Immunohistochemistry Studies

Obtaining High-Quality Cryosections of Whole Rabbit Eye 

This protocol describes how to obtain high-quality retinal cryosections in larger animals, such as rabbits. After enucleation, the eye is briefly immersed in the fixative. Then, the cornea and iris are removed and the eye is left overnight for additional fixation at 4 &#176;C. Following fixation, the lens is removed. The eye is then placed in a cryomold and filled with an embedding medium. By removing the lens, the embedding medium has better access to the vitreous and leads to better retinal stability. Importantly, the eye should be incubated in embedding medium overnight to allow complete infiltration throughout the vitreous. Following overnight incubation, the eye is frozen on dry ice and sectioned. Whole retinal sections may be obtained for use in immunohistochemistry. Standard staining protocols may be utilized to study the localization of antigens within the retinal tissue. Adherence to this protocol results in high-quality retinal cryosections that may be used in any experiment utilizing immunohistochemistry.

Author Spotlight: Optimizing Retinal Tissue Processing for Immunohistochemistry in Rabbit Models

This protocol describes a reliable method for obtaining high-quality cryosections of whole rabbit eyes. It details rabbit eye dissection, fixation, embedding, and sectioning procedures, which may be easily adapted for use in any study utilizing immunohistochemistry in larger eyes.

This protocol describes how to obtain high-quality retinal cryosections in larger animals, such as rabbits. After enucleation, the eye is briefly immersed ...