Inhibition of EDTA of growth of Lactobacillus casei in the folate microbiological assay and its reversal by added manganese or iron.

When we used plasma containing EDTA as an anticoagulant, the growth of Lacto bacillus casei was inhibited in the folate microbiological assay involving use of 300 p.L of assay mixture in a 96-well microtiter plate (1-3). We postulated that this inhibition was caused by EDTA chelation of manganese, iron, and magnesium ions, all of which are essential for growth of L. casei (4, 5). The singlestrength medium manufactured by Difco Laboratories (Detroit, MI) contains manganese, iron, and magnesium in concentrations of 25, 20, and 450 /LmolIL, respectively. Therefore, we tested the effect of adding either manganese, iron, or magnesium to the medium to reverse the growth inhibition of L. casei caused by EDTA. Figure 1 (top) shows L. casei response curves to (6S)-5HCO-H4PteGlu with various concentrations of EDTA. Inhibition of L. casei growth became obvious at an EDTA concentration of 0.65 mmollL. We considered that 2.6 mmol of EDTA per liter would be the maximum concentration possible if we used 50 tL of plasma per assay from 4.0 mL of whole blood drawn into a tube containing 10.5 mg of disodium EDTA. Addition of manganese or iron to the assay mixture restored the full growth of L. casei in the