Modified bacterial mutation test procedures for evaluation of peptides and amino acid-containing material

Abstract

Biological materials can release amino acids during the course of bacterial mutation testing. Low levels of released amino acids from soluble materials can cause moderate increases in the number of revertant colonies on the plate, whereas higher levels lead to overgrowth of the background lawn, making counting of revertant colonies impossible. For poorly soluble material, the released amino acids can be present at high levels in localized spots on the plate, leading to the growth of 'pseudorevertant' colonies. The 'treat and wash' modified preincubation method employed here is an adaptation of the treat and plate method (used for evaluation of antibiotics) and involves washing the bacteria free of test compound after a 90 min exposure prior to plating out on minimal plates. The MC overlay method is a modified version of the standard plate incorporation assay, in which a top overlay containing 4% high viscosity methylcellulose is used in place of agar to stabilize the test compound in solution, preventing precipitation and subsequent localized amino acid release. Both modified methods produce the expected results for negative and positive controls. Peptides [synthetic curtailed analogs of human parathyroid hormone, PTH(1-34) and Ostabolin-C] that produced false positive results or could not be evaluated owing to overgrowth of the background lawn using standard methods, showed no artifacts and no evidence of genotoxicity using the modified methods. It is concluded that the treat and wash and MC overlay methods are valid versions of the bacterial mutation test for avoiding complications associated with released amino acids.

Dietary Exposure of Nigerians to Mutagens and Estrogen-Like Chemicals

“…The treat-and-wash assay was conducted according to the method described by Thompson et al [39]. The protocol applied was as per the standard plate incorporation assay with the exception that the S9 mix, bacteria and sample extract were incubated for 90 min prior to the addition of molten top agar.…”

“…Methylcellulose overlay assay was performed as previously described [39]. Briefly, a 500 µL aliquot of S9 mix/phosphate buffer (0.2 M, pH 7.4) was combined with 100 µL of late-log bacterial culture in a sterile 15 mL tube.…”

Composition characterization of Myctophids (Benthosema pterotum): Antioxidation and safety evaluations for Myctophids protein hydrolysates

“…Mutagenicity assay. The Ames test of BPH was conducted by the standard plate incorporation method (Maron & Ames, 1983) with minor modification (Thompson et al, 2005). The ability of the test subject to cause mutation in S. Typhimurium TA98 was evaluated in the presence/absence of the S9 metabolic activation system mix.…”

“…In order to avoid false positive responses and overgrowth of background bacterial lawn, the genotoxicity potential of BPH was conducted with modified treatment and washing steps (Thompson et al, 2005) as well as a bacterial reverse mutation test (Ames test; using S. Typhimurium TA98 as a testing strain). Table 1 shows that BPH did not have toxicity to S. Typhimurium TA98, whereof there was no significant difference in the numbers of colonies between the control and test material.…”

Bacterial Mutation Assay

“…The washed bacterial pellet is collected by centrifugation at 2000 g for 30 min. Supernatant is discarded and the bacterial pellet is resuspended in the residual supernatant ( ∼ 0.7 mL of the supernatant) prior to plating [28] .…”