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Desiccation and Extraction with Fat Solvents

bacteria, dried, filter, alcohol and luminous

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DESICCATION AND EXTRACTION WITH FAT SOLVENTS.

Luminous animals may be divided into two classes—those in which the luminous material is burnt within the living cell (firefly, fungi) and those in which it is secreted by the cell outside (many worms, crus tacea, and myriapods). As first shown by Molish (8) the luminous substance must be burnt within the bacterial cell, since a dense emul sion of the luminous bacteria may be separated from the medium by a Chamberland or Berkefeld filter and a clear dark filtrate with no trace of phosphorescence obtained. I have repeated Molish's experi ment and can confirm him. An alundum filter crucible was used. Can this photogenic substance in the cell be freed of other cell material and obtained in a more or less pure state? We know that the bacteria can be dried, and when moistened again will phosphoresce, even though the majority are not living, and will give rise to no new growth if inocu lated in a suitable culture medium.' These dried bacteria form the material used for extraction purposes. The organisms are best grown in bulk in a thin layer of peptone (1 per cent), glycerine (1 per cent), sea-water nutrient fluid covering the bottom of a white enameled pie-plate and covered by another pie plate, the whole readily sterilized and serving as a large Petrie dish. The medium must be faintly alkaline to phenolphthalein. The bac teria are easily collected by centrifuging. The dense mass of centri fuged bacteria is then spread in a thin layer on filter paper or on glass wool, placed in a desiccator, and dried over in a vacuum. Spreading on glass wool has the advantage that the glass wool may be ground up in a mortar and a powder obtained which phosphoresces when moistened, but the powder does not give so brilliant a light as does the filter paper containing dried bacteria. It is well to wash the glass wool with several changes of water to remove alkali. Such strips of dried bacteria-impregnated filter paper can be extracted with boiling ether or cold absolute alcohol for 12 hours without losing their power to phosphoresce when the solvent has been removed and they are again moistened. Colonies of luminous bacteria sometimes appear when the

filter paper is placed on a nutrient medium, even after such rigorous treatment with ether and alcohol and other fat solvent& Buchner and Gaunt (24) obtained similar results with the acetic-acid-forming bac teria of beer (Mycoderma The dried acetic bacteria are not all killed by extraction with acetone; moist bacteria are invariably killed.' In testing the solubility in fat solvents, strips of filter paper contain ing the dried bacteria were placed in the solvents in sterile tubes for a definite time at a definite temperature, the solvent completely removed, and the filter paper tested for light-production by adding sterile sea water. A plus mark ( -1-) indicates light, a minus mark ( — ) indicates no light. Controls, untreated with any solvent, always gave a good light. Table 13 gives the results. In the last column are similar results obtained with the dried powdered luminous organs of the firefly. The amyl alcohol and ethyl butyrate were completely removed by washing with ether.

The great majority of fat solvents extract nothing which is essential to light-production, as can be seen from the table. Chloroform might have extracted something, as the material glows only faintly after chloroform treatment. I have, however, evaporated the chloroform extract to dryness in vacuo and added water as well as a water extract of luminous bacteria (in itself non-luminous; possibly containing a second necessary substance) to the residue without obtaining light production. The same result was obtained with the residue of the boiling alcohol extract, so that we must conclude that the chloroform and boiling alcohol extract nothing, but rather destroy the photogenic material. The temperature of boiling alcohol, 78.4°, is not destructive to the photogen. These results are very similar to my previous results on firefly material, as may be seen by inspecting the last column. The photogen of the firefly is not weakened by chloroform or acetone or a boiling mixture of equal parts alcohol and ether, but does suffer from carbon disulphide. Otherwise the results are the same.

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