AGITATION DURING INCUBATION REDUCES THE TIME REQUIRED FOR A LYSINE MICROBIOLOGICAL GROWTH ASSAY USING AN ESCHERICHIA COLI AUXOTROPHIC MUTANT

Abstract

Microbiological assays involving Escherichia coli lysine auxotrophs must be optimized to facilitate routine use. Our objectives in this study were to characterize growth of an auxotrophic E. coli lysine mutant (American Type Culture Collection strain #23812) and examine the effect of agitation on E. coli mutant growth. A defined minimal salts basal medium was used and supplemented with various lysine concentrations. The E. coli lysine auxotroph responded to increasing lysine concentration with increasing optical density. When maximum optical density (MOD) was determined for the auxotroph, a linear increase was obtained as lysine concentrations were increased (R2± 0.96) for both agitation and static cultures. Growth rates were not significantly (p > 0.05) affected by lysine concentrations, cultural conditions or their combined effect. However, growth with agitation significantly (p < 0.05) reduced the assay time by shortening the lag phase and causing stationary phase to occur earlier. The values of R2 (± 0.96) relatively remained constant over the range while the bacterial population were in the stationary phase. In conclusion, the lysine growth assay using the E. coli lysine auxotroph can be made more rapid by agitating the culture during incubation.

Development of a whole cell green fluorescent sensor for lysine quantification

“…Microbiological assays based on the growth response of an E. coli lysine auxotroph to external lysine, measured turbidimetrically, have been shown to be simple, inexpensive and specific (Payne et al 1977;Erickson et al 2002). The disadvantage of these methods is their relatively high level of time input ranging from 12 to 18 h (Li et al 2000;Prachaiyo and McLandsborough 2000;Erickson et al 1999). Even though some genetic alterations have been made to improve the lysine auxotrophic growth response (Li and Ricke 2003), and the OD-based growth assay can be completed in 8 h, the time required for the entire procedure still remains relatively inconvenient for a single day analysis.…”

Application of an Escherichia coli green fluorescent protein - based lysine biosensor under nonsterile conditions and autofluorescence background

“…The disadvantage of this method is that it is relatively time consuming, with 12–18 h required to complete the assay (Payne et al. 1977; Li et al. 2000).…”

“…A microbiological assay based on increasing turbidity as a result of growth of an Escherichia coli lysine auxotroph unable to synthesize lysine and consequently requiring external lysine supplementation is simple, inexpensive and specific (Payne et al 1977;Erickson et al 1999a). The disadvantage of this method is that it is relatively time consuming, with 12-18 h required to complete the assay (Payne et al 1977;Li et al 2000). Even though some improvements have been made (Li and Ricke 2003), and the completion of the growth assay can be accomplished in 8 h, the time required for the entire procedure still remains relatively long.…”

A cad-gfpmut3 plasmid construct in Escherichia coli for gene induction-based quantification of lysine in acid hydrolysates of feedstuffs

“…2006). In addition, the induction‐based approach eliminates the 10‐h lysine depletion procedure which is a required step for the growth‐based lysine assay (Li et al. 2000).…”