Fundamental and Molecular Mechanisms of Mutagenesis ELSEVIER Mutation Research 358 (1996) 123-124 Current Issues in Mutagenesis and Carcinogenesis, No. 78 Presence of wlactose in some batches of proprietary LB broth: Relevance to the Muta’“Mouse ZacZ gene mutation assay H. Tinwell a* * , J. Ashby a, C.V. Williams b a zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Zeneca Central Toxicology Laboratory, Alderley Park, Cheshire. SK10 4TJ, UK b Zeneca Pharmaceuticals, Alderley Park, Cheshire. SKI0 4TJ, UK Received 3 July 1996; accepted 16 July 1996 The positive selection version of the MutaTMMouse ZacZ gene mutation assay [I] has been running suc- cessfully in our laboratory for three years [2]. How- ever, we recently encountered large numbers of plaques on the P-Gal containing selection plates. These plaques were usually diffuse and much larger than the true mutant plaques. However, some were identical to the mutant plaques. We therefore sus- pected a systemic contamination of the assay. We were aware of the likelihood of adventitious growth of non-mutated E. coli C lacZ- Gal E- bacteria around the edges of selective plates caused by the uneven distribution of P-Gal on the plates. However, the contamination we were encountering was clearly not due to this phenomenon. We had observed a similar problem several months earlier, and that was due to a bacterial contamination of the SM buffer. In the present case, however, stepwise replacement of the SM buffer, and of the other consumables, such as maltose and MgSO,, did not erradicate the contamination. In addition, both mass spectroscopy and NMR spectroscopy con- firmed the identity and purity of the P-Gal being used. Another indication of a problem with the func- tioning of the assay was provided by the short-term * Corresponding author. Fax: (44x0)1625 590249. growth characteristics of the bacteria. In general, although these bacteria grow best from an overnight culture, they will grow readily from a frozen glycerol stock (1 : 1 bacteria/glycerol) to give the required optical density in N 5 h. However, in recent months, there had been occasions when growth from a frozen stock was severely delayed. On the other hand, growth from an overnight culture appeared to be unaffected. This change in growth pattern often seemed linked to the presence of large numbers of plaques on the selective plates. These observations, coupled with the ineffective attempts described above to erradicate the contamination, left us with one of two further possible explanations. First, it was possi- ble that we had a contamination of our bacterial stocks. However, replacement with fresh stocks (courtesy of Coming Hazelton, UK) did not solve the problem. The second possibility was a problem with the Luria Broth (LB) base we had used to prepare the media and agars (Sigma). This possibility was strengthened by the fact that the assay performed satisfactorily when transferred to a separate labora- tory. The only difference between the test protocols used in the two laboratories was that the media was prepared from independently obtained stocks. In ad- dition, exchange of media between the two laborato- ries led to the respective reversal of assay perfor- mance. Inspection of the two batches of LB broth revealed that they had different lot numbers. 0027.5107/96/$15.00 Copyright 0 1996 Elsevier Science B.V. All rights reserved. PII SOO27-5107(96)00167-4