Standardization of the Methylene Blue Reduction Test by the Use of Methylene Blue Thiocyanate.
SINCE the first use of methylene blue reduction as an indicator of bacteriological quality in milk, fluid milk supplies on this continent have undergone notable improvement. Moreover, during this period it has been shown that the inaccuracies in the test increase with increasing reduction times.6' 8, 9 An effort to minimize these inaccuracies is, therefore, worth while. Because of the extensive use of this test in milk control activities, it becomes necessary to duplicate frequently the standard tablets of methylene blue certified by the Commission on Standardization of Biological Stains and recognized by the American Public Health Association in Standard Methods of Milk Analysis.' This duplication involves not only the creation of standards but also the purification or, at least, the determination of purity of the dye. Clark, Cohen, and Gibbs 2 review the difficulty, if not the impossibility, either of purification or of determination of purity of methylene blue chloride, the methylene blue salt used in these tablets.
Studies on Oxidation-Reduction in Milk
Summary The mechanism of reduction is discussed. The work reported in this paper tends to confirm the theory of Barthel that the disappearance of methylene blue in milk takes place in two stages, viz.: 1.The removal of the dissolved oxygen by bacteria. 2.The reduction of the dye by constituents of the milk. The time taken for the first stage may be long. The time taken for the second stage is usually short. The oxygen relationship is of great importance. We were unable to find that a preliminary shaking of the milk materially affects reduction times. Evidence is presented that milks produced normally are in approximate oxygen equilibrium with the atmosphere. In milks to which this test is applicable, the variations in pH are insufficient to cause a measurable difference in reduction times. The importance of the role played by the leucocytes is not known. The plate counts of 38 samples of milk taken at the moment of reduction varied from 3.5million to 45million with an average of 22million. It is probable that the two most important sources of inaccuracy are :- 1.The different rates of oxygen uptake of different species of bacteria. 2.The sweeping of bacteria out of the body of the milk by the rising butterfat during the operation of the test. It is probable that the inaccuracies in this test follow the growth curve of the bacteria. Therefore, we do not consider the test reasonably accurate after the 5½-hour period as laid down in "Standard Methods of Milk Analysis." Since the methylene blue is, indirectly, an indicator for oxygen, different concentrations of the dye, within limits defined in a previous paper, give identical reduction times. A 10-cc. dipper is, therefore, sufficiently accurate for use in measuring samples for this test. It is sufficient that this dipper be thoroughly rinsed in the incoming milk or in water between each sample, if times are not to be read after the 5½-hour period. The use of any water fit for drinking purposes is warranted as the solvent for the dye. The methylene blue reduction test is as accurate a measure of the keeping quality of milk as any method yet available. It will divide milks into three or four classes with reasonable accuracy, as will any of a number of tests for milk quality. It is inexpensive and as nearly fool-proof as any method for this purpose available to the dairy bacteriologist. Methylene blue reduction times should not be reported in terms of the plate count.
LIMITING FACTORS IN THE LACTIC FERMENTATION
In the normal course of the lactic fermentation the bacterial growth passes through four stages: the lag period, the period of rapid or logarithmic growth, the period in which there is little or no increase in numbers, and the final period in which fermentatior ceasesand the cells slowly die. Ti-a-dianges from the first to the second and from the second to tL... ird period are comparatively rapid and are accompanied by mo )hological and physiological changes in the cells. The transit n from the third to the fourth period is gradual. In the third period the cells may be in a quiescent condition ina which there is little or no multiplication, or the multiplication and death rate may be so nearly balanced that the number counted remains constant. Counts made by the plate method would be likely to give the impression that the latter is the case since only the living cells are counted. If direct microscopic counts are made and the population remains constant it must be assumed either that the --cells are quiescent or that they lose their ability to take stain very soon after death. In our work on this problem we used a microscopic count in order to avoid as far as possible the error introduced into the plate method by the tendency of the lactic streptococci to grow in pairs and chains. The very large numbers of cells with which it was necessary to work militated against accurate counts, and it was difficult to obtain a series of counts which would give a reasonably smooth curve. However, the constancy with which uniform counts were repeatedly secured under similar conditions indicated that 211