Abstract
Determination of metabolically active cell count is an important step in designing, operating and controlling fermentation processes. It’s particularly relevant in processes involving mixed cultures, where multiple species contribute to the total growth. The motivation for the current study is to develop a methodology to estimate metabolically active cell counts for the individual species in a mixed culture with approximate equal numbers. Further, the methodology should indicate the presence of a contaminant in short time periods since in the agar plate methods used frequently it takes about 24 h. We present a methodology based on the rate of Methylene blue (MB) reduction to evaluate total count of metabolically active cells. The standard curve relating the slope of MB reduction and CFU of the individual species could be used to measure the metabolic activity of each species in the mixed culture. The slope of MB reduction could also be used to obtain the growth rate of individual species in a mixed culture and that of the total cell count. These measurements were achieved in less than 6 minutes during the growth of the cells. Evaluating the metabolic activity of individual species in a mixed culture is tedious, difficult and time consuming. The Methylene Blue dye Reduction Test (MBRT) presented here is capable of quickly estimating colony forming units (CFU) of individual species in a mixed culture if the ratio of the numbers of cells is known. The method was used to dynamically detect the occurrence of a contaminating microorganism during fermentation. The protocol developed here can be adapted to applications in processes involving mixed cultures.
Highlights
Quantification of metabolic active cell is essential for design, operation and control of processes involving mixed cultures
Firstly, we established a linear relationship between the optical density measurement (OD) and the Methylene Blue dye Reduction Test (MBRT) slope obtained from a mixed culture of E. coli and B. subtilis
Beyond which the two ODs do not match, since MBRT is related to the metabolic active cells, while OD measures the turbidity
Summary
Quantification of metabolic active cell is essential for design, operation and control of processes involving mixed cultures. Several methods have been used in the past to measure CFU including agar plating, imaging using microscope and fluorescence techniques, membrane filtration and flow cytometry. Most of these methods are labor intensive, error prone, time consuming, instrumentation intensive and expensive (Auty et al 2001, Davey and Kell 1996, Walley and Germida 1995, Wright et al 1993). There is a need for fast and accurate method to enumerate metabolic active cell count of organisms present in a mixed culture. The rate of disappearance of the color is correlated to the cell count of any aerobic organisms This method can be used to obtain colony forming units in about 3 minutes (Bapat et al 2006)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
