Penicillium is a plant which is distributed widely and apparently is able to grow wherever organic matter is found, though flourishing best when the material contains acid. It causes loss in canneries, breweries, distilleries, etc., the only use made of it being in the manufacture of Roquefort cheese, the immature cheese being inoculated with the conidia for the effect the mold produces in the maturing process.

PENICILLIUM.

CULTURES FROM KETCHUP PRESERVED WITH SODIUM BENZOATE.

DEVELOPMENT.

In developing, the mold forms a white felt-like mass, covering the medium on which it is growing; then as development proceeds, it changes to bluish-green, and finally to a darker, duller color. The change in color is accompanied by a change in structure, the surface becoming powdery in appearance, a slight current of air being sufficient to dislodge a cloud of fine dust. This fine dust is formed of small, spherical bodies, the spores or conidia (from the Greek meaning dust). These need no resting period, but are able to develop at once. When the conidia lodge on a moist substance they swell to a much greater size and then send out a tube from some part of their surface. The tube lengthens and septa form, dividing the tube into sections, or cells. At the same time branches are sent out, which again form other branches. The original conidium sends out a second branch shortly after the first one, and usually from the opposite side, and may even send out a third one. The formation of the septa and the subbranching goes on in all, so that in a short time the branches mat together and form a felt-like cover.

REPRODUCTION.

After a shorter or longer period of development, dependent on the conditions, branches are sent perpendicularly from the substratum, and into the air. These branches cease their growth in length, sending out branches near the tip, which take the same general direction as the original branch. Each of these subbranches is called a sterigma (from the Greek word meaning support). In vigorous development the sterigmata may form secondary branches, the whole forming a tassel-like arrangement. The tip of a sterigma enlarges, a septum forms around the enlargement, cutting it off from the sterigma, and forming a conidium. The sterigma develops to the original length and another conidium is formed, the operation being repeated many times, thus forming a chain of spores. As the other sterigmata are also forming conidia in the same manner, a series of these chains is formed close together. After the cessation of conidial development, the filament below the sterigmata is disorganized, setting free the conidia. The filament and head together are called the conidiophore (Greek, dust-bearer).

Penicillium forms spores sexually, but the conditions for their formation are unknown. Brefeld obtained them by growing the mold on damp bread placed between two glass plates, and excluding the air. Lindner obtained carpospores on a wort gelatin culture in a petri dish, from which the air was excluded. The writer has tried various methods for obtaining carpospores, but so far without success. Moist chambers were used with various media, excluding the air. The development of the mold is seemingly dependent on the amount of air in the chamber at the time of sealing. After the air is exhausted, the conidiophores assume fantastic forms, developing only one or a few sterigmata, and on these one or few conidia. In other cases the conidiophores are fascicled, in no cases, however, forming the conidia as luxuriantly as when air is supplied. The hyphÆ become clear, much vacuolated, and develop more septa, and some of the cells become much enlarged. An enlarged cell will often contain two or three septa, thus forming cells that are not larger than disks. In cultures from which the air was excluded from the start, no development took place. In test-tube cultures sealed with paraffin after twenty-four hours, the mold developed on the surface of the gelatin, forming a felted white mass, but no conidia nor carpospores were formed.

GROWTH IN KETCHUP.

The form of Penicillium which was used in the experiments was isolated from ketchup in which it grew luxuriantly. When conidia are first formed on the ketchup, they are a delicate blue in color; they then become bluish green, then green, and finally olive. The development of the color of mold growing on ketchup is practically the same as when grown in wort, tomato bouillon, pea bouillon, or gelatin made with these solutions as a basis. In ketchup containing sodium benzoate, the blue color appearing first remains for a long time, and in old cultures the mold is a dull drab, not olive, as in normal development.

In ordinary ketchup made without a preservative, the mold forms a heavy, wrinkled mycelium, showing a large development of conidia. In the bottles of ketchup, the mold pushes down into the ketchup, becoming entirely submerged, a clear liquid covering the mold and separating it from the ketchup. This occurred in more than one hundred bottles. No secondary mycelium formed on the surface of the liquid, a method of development which frequently occurs in ordinary media when a mass of mold is submerged.

An exception to this was shown in ketchup which had developed the mold in the laboratory. The bottles were then put in the refrigerator for two weeks. During this time scarcely any development took place; but after they were again placed in the laboratory, the mycelium pushed down into the ketchup and a new, very thin mycelium developed on the surface. The filaments when seen under the microscope were swollen, had irregular outlines, and a comparatively smaller number of septa, and were filled with a coarsely granular protoplasm. The ends were blunt and misshapen and the sterigmata were irregular, tending more toward a fasciculated arrangement, and forming fewer conidia. The filaments from the vinegar and acetic acid media had the same appearance as those developed on ketchup, but had a smoother outline.

TEMPERATURE TESTS.

The limits for the germination of Penicillium, as given by W. J. Sykes,[E] are 2° to 43° C. (35° to 110° F.), and the most favorable temperature 22° to 26° C. (72° to 79° F.). This author states also that according to Pasteur the dry spores retained their vitality at 108° C. (226° F.), but that they were soon killed when immersed in boiling water. KlÖcker[F] quotes Pasteur as saying that the conidia are killed if exposed to a temperature of 127° to 132° C. for half an hour, but that they retain life at 119° to 121° C.

A series of tests was made to determine the thermal death point of the moist and dry conidia of the Penicillium used in the experiments, a young, vigorous development on ketchup being used. The flasks were kept under observation for a month after the tests were made, as in many cases a development does not occur in the usual time. The high temperatures applied for longer periods of time were tried first, but both temperature and time were reduced as results from the series were obtained. Only the conditions obtaining in the final tests are given in the table. It was found that the Penicillium used did not have the high resistance supposed.

The tests were made in small flat-bottomed 10-cc flasks, tomato bouillon being used for the tests on moist conidia. The bouillon was used so as to have the conidia in a nutritive medium after the test was made, without transferring. The time for those at 100° C. was estimated from the time of ebullition. At the end of the specified time, the flasks were cooled promptly under running water. As the flat bottoms gave comparatively large surface, the heating and the cooling could be effected in a short time. For the tests below 100° C. a vessel of water was heated to the desired temperature, and the flasks were immersed in it and shaken constantly. The dry conidia were placed in test tubes which were immersed in boiling water for the desired time and cooled under running water, after which 10 cc of sterilized tomato bouillon was added. After determining the death point in this manner and finding it to be much lower than had been supposed, it was decided to make the test again, but using ketchup as the medium. Ten grams of ketchup were sterilized, then inoculated from a vigorous growth of mold, and tested with a set in which the tomato bouillon was used. For those below 100° C. the two flasks which were to receive the same temperature were held in the vessel of water at the same time, so that as nearly as possible the treatment would be identical. The following results were obtained:

Thermal death point of moist and dry conidia of Penicillium.

The moist heat was very effective in destroying the vitality of the conidia of Penicillium, the death point being 27° C. higher than the maximum temperature for germination as given by Sykes. The heating was more effective in destroying germs when applied to bouillon than to ketchup, no development taking place for any temperature above 65° C., even when applied for a short time.

In the ketchup the lower temperatures for the longer periods of time were more effective in checking the development, even though they did not destroy the vitality. In the ketchup, with the exception of Nos. 9 and 10, the colonies started invariably along the sides of the flasks. The greater access of air to those on the sides would account for this. The conidia on the sides of flasks Nos. 9 and 10 must have been destroyed, as no development took place in either case except in the center of the surface.

The dry conidia were destroyed at 100° C. when heated for thirty-five minutes; they did not reach a normal development in any case, even when heated for only ten minutes, many of the conidia being destroyed by this treatment. Where development failed to take place, the conidia were stained with a water solution of eosin, so as to be sure that the effect was death, and not an arrested development.

The results of the tests do not agree with those obtained in factory practice, where the ketchup is cooked at 100° C. for at least forty minutes and sometimes for fifty or fifty-five minutes, depending on the consistency of the pulp.