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.
Concerning the Accuracy of the Methylene Blue Reduction Test
Summary 1.The methylene blue reduction test is based upon the rate of oxygen consumption in the milk by the bacteria present during incubation. This rate cannot be measured with reasonable accuracy where varying proportions of the bacteria are removed from the main body of the milk during the creaming process. 2.The accuracy of the test is greatly increased by periodical inversion of the tubes during incubation. In addition, reduction time is generally shortened and decolorization is more uniform, especially with the better grades of milk. 3.It is believed that these advantages more than compensate for the slight inconvenience entailed by this modification. 4.Milks showing similar reduction times by the modified technic sometimes show wide differences in standard reduction times. These appear to be associated with differences in the degree and rate of creaming. 5.Reduction time is prolonged by approximately 20 per cent with the proposed stronger dye concentration. This is more than compensated for in all but the poorer grades of milk by the shortening of reduction time when inversion is practised.
OBSERVATIONS ON THE OXIDATION-REDUCTION PROPERTIES OF STERILE BACTERIOLOGICAL MEDIA
Sterile plain broth contains an active oxidation-reduction system, the characteristics of which have been analyzed. 1. Intensity factor: Under vaseline seal, the lot of broth used in these experiments reaches a reduction potential corresponding to reduced indigo disulfonate (rH = 10). All the indicators with a more positive E0' are reduced, the others are not affected. It seems probable that fresh broth, which has not undergone oxidation by molecular oxygen, would give a higher reduction potential. 2. Capacity factor: The maximum amounts of different indicators that can be reduced correspond to equimolecular concentrations. This seems to indicate either (a) that the broth does not contain several "independent" reducing systems (at least in appreciable amounts), or (b) that these hypothetical "independent" systems all have about the same reduction potential. 3. Time factor: The different indicators of oxidation-reduction potentials are reduced in the order of the electromotive series. 4. Nature of the system: The system seems to be reversible (this not excluding the possibility of irreversible autoxidations) and does not appear to be of the nature of a sugar. The relation of these phenomena to the cultivation of different species of bacteria will be reported later.