The strips of filter paper moistened with sterile sea-water were then transferred under sterile conditions to nutrient agar to see if colonies of luminous bacteria would result. It was found that not in every case, but in at least one experiment out of several tried, luminous colonies were obtained after extraction of the material with ether, alcohol, toluol, acetone, and benzol.
I have already mentioned the fact that dried bacteria will glow if moistened after extraction with cold (20°) absolute alcohol, and also the fact that if 50 volumes of absolute alcohol are added to a mass of moist centrifuged bacteria and they are then shaken for 10 minutes, the alcohol removed, and bacteria quickly dried, no phosphorescence is obtained on moistening this dry powder. Neither will dried bacteria phosphoresce if extracted with 95, 80, 70, 50, or 37 per cent alcohol. What is the explanation of this? The alcohol does not dissolve out a luminous substance. Water appears to exert some influence. Perhaps we may explain the above result as merely an example of the effect of alcohol on dry albumins as opposed to albumin solutions. Powdered egg albumin can be extracted with absolute alcohol (or acetone) for 6 hours and is still readily soluble in water. But if a concentrated aqueous solution of albumen is precipitated by a large excess of alcohol (or acetone), even though the alcohol (or acetone) be removed within 15 minutes, the precipitate of albumen is found to be practically insol uble in water. It would be futile to discuss the matter without further experimental results, but the results seem to indicate that in photo genesis there is involved a substance which in the moist state is irre versibly precipitated (or changed) by alcohol and that it is probably protein in nature.
At any rate, the experiments show (1) that the photogenic material is not a fat or a fatlike body soluble in fat solvents, and (2) that phos phorescence does not depend on the living cell, since many of the dried bacteria which can still phosphoresce when moistened will give rise to no new colonies.
The question may now be raised as to whether bacteria whose struc ture has been completely destroyed by grinding in the dry state will phosphoresce on moistening. Oxidation can not take place so long as water is absent. Experiment shows that they will not phosphoresce, as the following procedure indicates: Luminous bacteria dried on glass wool are powdered in a porcelain mortar and divided into two equal parts, A and B. A was then ground in the porcelain mortar for 20 minutes with pure quartz sand; B was thoroughly mixed in another mortar with an equal volume of sand, previously ground for 20 minutes, and exposed to the air during the time of grinding A. In this way the effect of quartz powder or the possibility of absorbing moisture from the air would be the same for the unground bacteria B or the ground bacteria A. On moistening B with sterile sea-water, a good phosphor escence appeared, while the ground material gave no light with sea water. I have repeated the experiment with the same result and feel that there are no possible sources of error. Microscopic examination shows the sand to be ground to the size of the bacteria or smaller and it is well known that even the smallest cells may be broken up by grinding with sand. I find that the dried luminous organs of the fire fly likewise lose their power to phosphoresce if thoroughly ground with sand. This result differs from that of McDermott (21), who finds that firefly tissue can be frozen and ground in liquid air without losing its power to phosphoresce. Both A and B were inoculated on agar nutrient medium. The ground bacteria, A, gave rise to no luminous colonies, while the unground bacteria, B, did develop several luminous colonies—further proof that the ground bacteria were wholly broken up and destroyed.