Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Patent
1984-02-27
1987-06-23
Marantz, Sidney
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
435 29, 435 6, C12Q 104, C12Q 102, C12Q 168
Patent
active
046752889
DESCRIPTION:
BRIEF SUMMARY
The present invention is concerned with a method for the performance of a mutagenicity test so that a cell population is subjected to a mutagen, as a result of which a part of the population mutates, whereat the originally uniform cell population is differentiated into sub-populations.
Mutagenicity testing is used for rapid preliminary testing of substances suspected as carcinogens, for in bacterial mutagenicity tests it has been established that at least 90% of known carcinogens are also mutagens. Out of biological samples, it is possible to establish, e.g. in the form of mutagenic activity of urine, that a person concerned has perhaps been exposed to mutagenic substances, which, thus, potentially cause the risk of cancer.
It is possible to determine mutagens present in the samples of air, water and foods and, e.g., possible traces of mutagenic pesticides etc. agents in foods.
Mutagenicity testing is to an ever increasing extent a part of the statutory toxicological analysis of a product or equivalent. Some of the most important users of these tests are at present the pharmaceutical and chemical industries. A specific field of their own is formed by the authorities dealing with environmental protection and with occupational safety, who must usually examine complex samples containing several different chemicals. Along with the increasing legislation for the protection of consumers, e.g., foods and food additives are also being included in the scope of mutagenicity studies. The testing of these samples may become problematic, because of the growth factors contained in the said products may cause false positive results.
In prior art, a microbiological fluctuation test has been used for the testing of mutagenicity. The fluctuation test is a two-stage test of bacterial mutagenicity, which is suitable for the testing of low, non-toxic concentrations of mutagenic substances. In the first test stage, which takes 18 to 20 hours, the mutation, if any, takes place. After that a selection of 3 days takes place in a growth medium free from growth-factors.
Since the test takes place in a liquid medium in which attempts are made to isolate each original mutant, it follows from the above requirement that the performance of the test requires a great number of test tubes. The more test tubes are available per sample, the higher is the probability that the original mutants produced can be isolated each of them in its own tube.
The observation of mutant growth is based on the use of a pH-indicator, for the mutated cells excrete acid fermentation products when growing in the selection medium.
The microbiological fluctuation test is one of the most sensitive mutagenicity tests that have been developed by now. The Ames test, which is the most common bacterial mutagenicity test in use, requires about 10 to 100 times higher concentrations of mutagens to give a similar positive response as compared with the fluctuation test.
A drawback of the fluctuation test is its sensitivity to the toxic and growth-factor effects of the substance, and that is why the test requires lots of work. Per one sample, at least 50 test tubes are used and, if the sample is unknown in respect of its toxic properties, up to eight different dilutions of the said sample may be required in order to reach non-toxic, but still mutagenic concentration ranges. In such a case, the total number of tubes may be up to 400 per sample. Including all the working steps, the performance of the test takes about 30 min. per dilution (50 tubes), which restricts the number of samples to be analyzed considerably. The mutagenization and selection to be performed on subsequent days restrict the starting days of the test to Mondays and Thursdays only, if working during the weekends is to be avoided. A drawback of the mutageneity test performed manually is the high cost.
The method in accordance with the present invention is mainly characterized in that the variation of turbidity caused by the change in the density of the differentiated cell populations is measured as a change in
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Foley Shawn P.
Labsystems Oy
Marantz Sidney
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