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.
