Freda M. Bachmann
1916 Journal of Industrial & Engineering Chemistry  
A laboratory study of the preservative action of spices was first made by Hoffman and Evans in 191 I . They reported t h a t as a result of their experiments they found certain spices t o have considerable antiseptic properties. They found cinnamon most effective in inhibiting the growth of organisms; cloves and allspices next in effectiveness; and t h e other spices of little value as preservatives. They used pure cultures of some of the bacteria and yeasts, but depended on chance inoculation
more » ... chance inoculation for mold growth. Apple sauce, t o which varying amounts of spice were added, was exposed for several days. The yeasts and bacteria were grown in sterile broth t o which cinnamic aldehyde, eugenol, or benzoic acid was added. The present work was undertaken t o confirm the results of Hoffman and Evans and t o collect more data on the subject. N E TI3 0 D For the greater part of the work, pure cultures of organisms were used. Species of the common molds, Rhizopus, Penicillium, Aspergillus, and Alternaria, which are frequently found on spoiled preserves, and of the bacteria, B . coli, B. prodigiosus, and B. subtilis, mere the organisms used in most of the experiments. The Aspergillus was obtained from tomato butter which had spoiled; the Penicillium. from a growth on canned peaches. For the greater part of the work, agar has been used as a nutrient medium. The molds grow best on Thaxter's potato hard agar, which consists of potato broth with 3 per cent agar and z per cent glucose. The bacteria were grown on ordinary nutrient agar. T h e spice in some form was introduced into the agar before it solidified. T o compare readily the growth on a spiced medium with t h a t on a medium without the spice, it was found convenient t o use some kind of a double plate. The double plate described b y FrostZ in his studies on antagonism of organisms was first used. Here the Petri dish is divided through the center by a glass rod which is sealed t o the bottom of the dish by means of collodion. It is then sterilized. The one objection to this method for the present studies is that in sterilizati.on the collod' 'on sometimes shrivels t o such an extent t h a t the rod is no longer well sealed t o the plate, so t h a t some of the agar poured in on one side is likely t o run underneath the rod into the opposite side of the d k h . A modification of this method seemed desirable.3 For studying the amount of diffusion, it was found satisfactory to cover one-half of the bottom of the Petri dish with a piece of cheese-cloth and then sterilize the dishes. Agar containing no spice was then poured into the dish and when solid, the cloth with the ad-1 Published with permission of the Director, Wisconsin Experiment Station. (1914), 641. 8 W. D Frost and Freda M. Bachmann, "A4nother Use of the Double-Plate Method," Science, N. s., 43 (1916). herent agar removed by means of a sterile forceps, thus leaving agar on only one-half of the plate. The spiced agar mas then poured into the side opposite the plain agar. I n such plates, whatever inhibition of groyth there is on the side where there is no spice is due t o the amount of spice which diffuses through the medium and to whatever volatilizes. T o distinguish between diffusion and rolatilization in inhibiting growth, it was necessary t o use still another kind of double plate, one in which there could be no diffusion through the meiium. Small, thin glass watch crystals were sterilized in the Petri dishes and later the spiced agar poured into the watch crystal and the plain agar in the Petri dish. I n plates prepared in this way the inhibition of growth on the plain agar could be due only t o the amount of spice which volatilizes. The organisms have also been grown on spiced agar slants and in spiced bouillon. The latter is not alvays very desirable because t h e spice may cause a cloudiness which makes a very small amount of growth difficult to detect. Cooked apples to which spice, or vinegar, or both, were added, have also been used as a medium. Spice was introduced in the form of ground spice, the alcoholic extracts, the essential oils, and the active principles. The ground spice and the alcoholic extracts were introduced directly into a definite amount of agar, The essential oils and the active principles were diluted with water. Amounts of this emulsion were introduced into the liquefied agar. The emulsion was very thoroughly shaken each time before transferring any of it t o the agar. To inoculate the plates with molds, a suspension of the mold spores in sterile water was streaked across the agar with the platinum loop. I n this way a very even amount oE growth was obtained all along the streak in the control plates. However, in order t o make certain t h a t a greater amount of growth on t h e side farthest from the spiced agar would not be due t o heavier inoculation, the streak was made by drawing the loop from the center t o the circumference, thus placing the greater number of spores near the spice. All the cultures have been grown a t room temperature. EFFECT 0 P ALCOHOLIC EXTRACTS O P SPICE The alcoholic extracts were prepared b y the laboratory of pharmacy. The spices were extracted with alcohol and the extract evaporated so t h a t x cc. of the extract represented I g. of the spice. Controls in which the same amount of 95 per cent alcohol was used were made for comparison. The results given in Table I are from plates where the spiced and u nspiced agar were in contact and were taken one week after inoculation. There is considerable difference in the amount of spice which prevents mycelial growth and the amount pres-enting germination of spores. Frequently in plates in which the agar in one-half of the dlsh contained suEcient alcoholic extract of a spice t o prevent germination of the spores, the mycelium which had covered the side without spice grew readily across
doi:10.1021/i500007a014 fatcat:hpzoipuujngx7c7k6flpzp6yeu