This study was undertaken with the object of determining the part played by the several component elements of the tissue method of anaerobic cultivation in the establishment of anaerobic conditions in the culture tube. Data have been presented to show the suitability of methylene blue as an indicator of reduction processes in culture media by which the removal of the last traces of oxygen may be demonstrated. With methylene blue as the indicator, the elements subjected to experiment included the choice of a seal for culture tubes, the activity and requisite size of the kidney tissue fragment, the chemical and physical characters of the medium which promote or retard deoxygenation, the length of the column of medium, and the advantages of external aids such as the McIntosh and Fildes anaerobic jar.

As a result of our experiments, we have come to the following conclusions:

1. Liquid paraffin oil, used extensively as a seal for anaerobic cultures and in gas analysis, has very little value in inhibiting the access of oxygen. Solid vaseline, on the other hand, forms an effective oxygen-resisting seal. The difference is due to the physical states of the substances at incubator temperature.

2. Fresh kidney tissue is an active reducing agent and quickly decolorizes methylene blue in its vicinity. The reducing effect of fresh kidney tissue is relative to the amount used. As a reducing agent, at least 0.6 gm. per tube is required for the establishment of an adequate oxygen-free zone.

3. Culture media may be classified as reducing or non-reducing. Those containing dextrose or peptone in a faintly alkaline solution belong to the former class. Ascitic fluid and dilute serum belong to the latter class, for their content of reducing substances is practically insignificant. For the prompt establishment of strictly anaerobic conditions these media require the addition of reducing substances such as dextrose, peptone, or kidney tissue aided by an effective seal or an anaerobic jar.

4. Semisolid media effectively inhibit the penetration of oxygen to the depths of the tube, but they likewise limit the diffusion of reducing substances and presumably of nutrient substances from imbedded kidney tissue.

5. The length of the column of medium is of minor importance under a vaseline seal.

We clearly recognize the impracticability of standardizing a biological technique which by its very nature must be subject to wide modifications for special purposes. Such variations from a standard are especially necessary in the search for unknown organisms, and in work with hitherto uncultivated microbes in which the tissue technique has been successfully applied by Noguchi.

We wish, therefore, to present the results of our studies simply as guides in the variation and control of the elements examined and to make certain suggestions relative to the establishment of strictly anaerobic conditions in the culture tube. The numerous other factors of equal importance which must be taken into account—hydrogen ion concentration, source and character of nutritive elements, temperature, time, etc.—are outside the limited scope of the present report.

For the establishment of strictly anaerobic conditions in the culture tube, we would suggest (1) the substitution of solid vaseline for liquid paraffin oil as an oxygen-resisting seal; (2) the use of large pieces of fresh kidney, the standard size to be upwards of 0.6 gm. unless other reducing substances are present in the medium; (3) the addition of peptone or dextrose or both in the form of peptone dextrose broth in fractional percentages to non-reducing media such as ascitic fluid or serum to aid in the prompt establishment of anaerobic conditions; (4) the use of the McIntosh and Fildes jar as a further aid to the prompt deoxygenation of the medium; (5) for reasons of economy the use of smaller amounts of culture medium, for example, 7 to 8 cc., under a vaseline seal; and (6) in dealing with anaerobes which may be injured by exposure to oxygen it might be advisable to prepare the medium a day or two in advance and to incubate it under a vaseline seal so that sterility is assured and the anaerobic conditions are already established when inoculation is made. The infected material is then introduced with a capillary pipette in the vicinity of the kidney tissue and the seal restored by gentle heating to melt a portion of the superposed vaseline.

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