Detection by replica plating of false revertant colonies induced in the Salmonella-mammalian microsome assay by hexavalent chromium.

The replica plating method as developed by Lederberg has been used to differentiate between "true" and "false" histidine-requiring revertant bacterial colonies which develop on minimal agar plates in the Ames test. Strains of S. typhimurium LT2, TA 100, when exposed to either sodium dichromate or the fumes from the welding of stainless steel, develop colonies whose apparent numbers are directly in proportion to the Cr(VI) content per plate in both cases, over a wide dose range. Replica impressions of the resulting colonies were transferred to Vogel Bonner minimal agar plates and incubated for 48 hr at 37 degrees C. It was then observed that considerable numbers of "false" revertant colonies were obtained at those Cr(VI) doses which resulted in a pronounced toxic effect, albeit with an acceptable level of the bacterial background lawn. No morphological distinction between "true" and "false" revertant colonies could be made. Although it would appear that at low doses (i.e., low toxicity) the true mutagenicity of stainless steel welding fumes can be completely accounted for by the presence of Cr(VI), the dose range over which the mutagenicity assay is reliable cannot be estimated from examination of the background lawn or from an estimate of the degree of survival of the treated cultures. Thus there is raised a serious question concerning the reliability of quantitative data published in bacterial mutagenicity testing where replica testing of the histidine requirement of the resulting "revertant" colonies is not routinely made. It is suggested that the replica technique can easily be developed as a simple and useful tool for the control of histidine requirement and ampicillin resistance in routine mutagenicity testing.(ABSTRACT TRUNCATED AT 250 WORDS)


Introduction
The Ames test is widely used to assess the mutagenic potential of chemicals (1). The principle of this assay is based on the properties of inducing reverse mutations in histidine requiring auxotrophe Salmo-neUa typhimusium LT2 strains. The reverted bacteria are detected as colonies on minimal agar plates. Although current attempts at standardization of this assay do not include a procedure for controlling prototrophy of colonies developed on minimal agar * Department of Wood Technology, Technological Institute, 2630 Tastrup, Denmark. t Chemical Analytical Laboratory, Technological Institute, 2630 Tastrup, Denmark. t The Danish Welding Institute, 2600 Glostrup, Denmark.
Author to whom reprint requests should be sent. plates, the use of a replica technique to confirm the lack of histidine requirement has been previously recommended (2).
Dichromates, chromates and chromium trioxide have all been found mutagenic in different bacterial mutagenicity assays (3,4), and in the Ames test they induce base-pair substitutions as well as frameshift mutations (5). It is also well documented that fume particles from metal arc welding on stainless steel have mutagenic activity (6, 7) primarily due to their Cr(VI) content (8). In this study, the replica technique, as developed by Lederberg (9), is used to differentiate between "true" and "false" revertant colonies on minimal agar plates in the Ames test, for a series of test materials containing Cr(VI), in order to determine if the replica technique might be a useful tool in verifying histidine requirement when substances with a pronounced toxic effect are tested.

Test Materials
The welding fumes -Na2Cr2O7, MMA/SS (Manual Metal Arc/Stainless Steel) welding fume and MIG/SS (Metal Inert Gas/Stainless Steel) welding fumefrom a series described by Stern and Pigott (10), were produced and collected as described by Stern (11) and subsequently dissolved in sterile demineralized water and exposed to ultrasound for 5 mins. The Cr(VI) content is assumed identical to the water-soluble Cr content measured by AAS.

Mutagenesis Assay
The Salmonella/mammalian microsome mutagenicity test (plate incorporation assay), using the tester strain TA100 of Salmonela typhimurium, was performed as described by Ames et al. (12) with the following modification: 0.4 mL of an overnight culture of TA 100 was transferred to 20 mL nutrient broth and incubated 6 hr at 37°C before use. The addition of S-9 mix was not included in the test series. Upper dose levels were chosen as 50 l.g of Na2Cr2O7 and 50 I*g water-soluble chromium in the welding fumes.

Replica Technique
The replica technique as developed by Lederberg (9) was used to transfer copies of one plate of each dose to Vogel-Bonner Medium E plates. Sterilized velvet with a dense nap and mounted on a wooden block was used as contact material.

Results
The number of revertant colonies per plate (average of three plates) induced by suspensions of Na2Cr2O7 and of MMA/SS and MIG/SS welding fume particles, as a function of weight of Na2Cr2O7 and of water-soluble chromium, per plate is shown in Fig-ures 1-3. The number of colonies on one of the master plates and on each of the respective replicas as a function of dose is listed in Table 1  The background of bacterial growth was clearly observed to be inhibited at 50,ug/plate of all sub-2 1L 0. k tn z ,pg Nc2Cr2O7 FIGURE 1. The average number of colonies per plate (from three plates ± 1 SD) vs. the concentration of Na2Cr2O7. The number of colonies on replica plates transferred from one of the master plates is shown (see Table 1). Note that although the initial slope of the dose revertant curves indicates a response to Cr(VI) from dichromate which is three times that from MMA/SS fumes (Fig. 2), the corresponding slopes of "true" revertants vs. Cr(VI) from replica plating are almost identical for dichromate, MIG/SS (Fig. 3) and MMA/SS (Fig. 2). All Cr(VI)containing substances tested exhibit a clear dose-related induction of revertant colonies. On the average, 10 ,g Na2Cr2O7, 9 Mg water-soluble chromium in MMA/SS fume and 5 ,g (water-soluble) chromium in MIG/SS fume per plate account for a doubling in the number of "true" revertant colonies.
On the replica plates, 65-85% of the number of colonies on the master plates could be recognized as separate colonies at dose levels up to 15,g/plate. A considerable drop in the number of replicated colonies was found at 25 and 50 Jg/plate.

Discussion
Metals such as chromium form electrophilic ions in solution and are able to react with nucleophile materials such as the N-bases in DNA. This characteristic appears to be an essential feature of substances with mutagenic properties. It is well documented that materials containing Cr(VI) have mutagenic activity in many different short-term mutagenicity tests, and the present investigation is a further confirmation of the dose-related induction of mutations in bacterial assays (13). It is, however, often rather difficult to compare data from different investigations on complex materials such as welding fumes, because the a-600c 500-  results are only rarely expressed as a function of specific active substances (such as amount of water-soluble chromium per plate) and collecting techniques are often very different in different laboratories.
Dichromates and welding fume particles have a pronounced toxic effect on the tester strains of Salmonella typhimurium. An examination of the background lawn of bacterial growth is routinely used to determine the presence of toxic effects and the limit of toxicity. By using this practice in the present investigation the limit of toxicity would have been fixed at 25pg Na2Cr2O7 or 25 ,ug water-soluble chromium per plate: Stern et al. (8)  It is well known that massive killing of bacteria on the minimal agar plates permits the development of small colonies of histidine-requiring bacteria because of the large amount of histidine available per vital bacterium on the media (12).
On the master plates of this study it was not possible to make a morphological distinction between different (i.e., false and real) revertant colonies at 25 pg 229 C, 0. Cr(VI); at this dose the background lawn was not inhibited to any appreciable extent.
The results of these preliminary investigations suggest that it might be necessary to recommend that prototrophy of the colonies developed on the minimal agar plates be controlled in standard procedures, especially when dealing with test materials with a distinct toxic effect on the tester strains. Examination of the background lawn of bacterial growth might not always be a reliable method of evaluating the influence of toxic effects on the test system, and an estimation of the number of surviving bacteria should at least be included in all routine analyses. The application of the replica technique has a limitation in the practical difficulties of transferring separate colonies from densely packed plates, and the results at high colony counts yield only qualitative information. With some experience in handling the technique it should be a simple and useful tool in the control of histidine requirement and ampicillin resistance in bacterial mutagenicity testing.