Revival and Growth of Lactic Acid Bacteria

Abstract

Source: Ayivi, R. et al., The Cultivation, Growth, and Viability of Lactic Acid Bacteria: A Quality Control Perspective. J. Vis. Exp. (2022)

This video demonstrates the method for recovering cold-stressed lactic acid bacteria from a frozen glycerol stock, restoring bacterial viability through anaerobic incubation, and isolating active colonies for culture expansion.

Protocol

1. Protocol for the activation and quality control of Lactic acid bacteria (LAB) cultures

1. Take the prepared glycerol stock of LAB strains (in 2 mL centrifuge tubes) from the -80 °C ultra-low freezer, and do not allow them to thaw before use.
2. Clean and disinfect the opening of the centrifuge tubes with 70% alcohol, and gently vortex before use.
3. Pipette about 250 µL (0.25 mL) of the stock LAB culture from the centrifuge tubes into fresh 2 mL MRS (deMan, Rogosa, and Sharpe media) test tubes.
4. Gently vortex, parafilm the test tubes, and anaerobically incubate them overnight at 42 °C for 12-16 h.
5. Take about 500 µL (0.5 mL) of 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 °C for 12-16 h.
6. Assess the microbial growth by measuring the optical density (OD) or growth of the cultures at 610 nm with a UV-visible spectrophotometer and recording acceptable results between 0.7 and 0.9.
7. Streak the overnight cultures from the 7 mL MRS tubes onto MRS and MRCM-PYR (modified reinforced clostridial medium-pyruvate) agar plates and incubate them anaerobically for 72 h at 42 °C.
8. Isolated colonies are picked from the agar plates, transferred into fresh 7 mL MRS test tubes, gently vortexed, and anaerobically incubated overnight at 42 °C for 12-16 h.
9. Store the agar plates containing the isolated strains at 4 °C in the refrigerator for a week.
10. 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.
11. To obtain a 1:10 ratio, perform ten-fold dilutions (serial dilutions) of the grown LAB cultures from the final 7 mL MRS test tubes using 9 mL of peptone water (a physiological buffer).
12. Finally, take about 250 µL (0.25 mL) from appropriate serial dilutions for all fermentation experiments.
13. Activate the broth containing strains (250 µL) from step 2.12 by transferring them into fresh 7 mL MRS broth and incubating them anaerobically at 42 °C for 16 h.
    NOTE: All L. bulgaricus strains were activated in the freshly prepared MRS broth and were then incubated anaerobically at 42 °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.

Materials

List of materials used in this article

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