The Normal Limits of Variation of the Methy- Lene-blue Reduction Test '

The methylene-blue reduction or reducíase test as a means of judging the sanitary quahty of milk has met with great popularity in this country. This method of analysis has the advantages of being very simple to operate, easy to interpret, and requires very little expense for materials. Many of the small milk plants not equipped with elaborate facilities necessary to make agar-plate counts are using the methylene-blue reduction test as a means of detecting undesirable sources of milk supply. In many cities it has been adopted as apart of the system of regulatory control of the milk supply. Milk inspectors in the larger cities frequently use this test for general survey work and supplement it with other more elaborate tests. The wide use of the test under many conditions has led to many modifications of the procedure, which for the most part are designed to render it even more simple and practical. Many of those who use this test are interested only in a rather rough approximation of quality ; hence, extreme accuracy in measuring the ingredients for the test and strict aseptic precautions have not been deemed essential. In some dairy plants the 10 c. c. of milk used in the test is measured by means of a dipper instead of a pipette; in others, graduated test tubes are used. Instead of using bacteriologically sterile glassware, as is necessary in the plate count, the test tubes are placed in boiling water a few minutes, and the measuring dippers when not in use are kept either in hot water or in a disinfectant solution. Although these practices are admittedly inaccurate, the errors are commonly regarded as of no practical significance. The preparation of the methylene-blue solution has been simpUfied by the use of specially prepared tablets that contain a standard amount of the dye, thus eliminating the necessity for expensive chemical balances to weigh small quantities of the powder. One tablet is dissolved in 200 c. c. of water which has been partially sterilized by boiling for a few minutes. The present investigation was designed primarily to determine the expected limits of normal variation of the methylene-blue test in order to afford more concrete evidence of its accuracy and rehability. If a sample of milk reduces the méthylène blue in two and one-half hours, may a second determination be expected to give the same result? If variations are observed, are they likeljr to be a few minutes or one or two hours? In addition to answering these questions an effort was made to measure the effect of the modifications of technic that have been adopted as a means of simphfying the test.