VOL 38

JULY 20. 1923

No. 29

Intensive Localized Distribution of the Spore of B. Botulinus and Probable Relation of Preserved Vegetables to Type Demonstrated.

By J. C. Geigeb, Epidemiologist, United States Public Health Service, and Harriet Benson, Department of Hygiene and Bacteriology, University of Chicago.

In a report to the Surgeon General,1 August, 1921, the writer gave a brief summary of an intensive investigation of outbreaks and distribution of the spore of B. botulinus in a mountain valley, the Yakima Valley, in the State of Washington. A recent investigation, also carried out in the State of Washington, adds further evidence to the localization of distribution of the spore of B. botulinus in a comparatively limited area.

In the southern part of Okanogan County, Wash., situated at an approximate elevation of 2,000 feet and near the Columbia River, there is a ranch of several hundred acres that offers, in the examinations of its soil and preserved food products, the best field evidence to substantiate this interesting epidemiological observation. The following table illustrates this particular point:

Table I.—Botulism, H. Ranch, Okanogan County, Wash.


1 It Is stated that several families of Colville Indians working on this ranch died under mysterious circumstances several years ago. The recent outbreaks have focused attention on theso cases, and it is now suspected that thev were botulism.

1 Several families of Indians.

'Taken from the dining table of a family living on the ranch by the county health officer.

• The q unlity of the beans was tested by feeding them to chickens. The chickens later died with typical symptoms of fowl botulism.

It will be noted that this ranch has had two outbreaks of botulism in human beings, which were undoubtedly botulism and in one of which the food involved (home-canned string beans) was proved to contain a toxin (M. L. D. for mice of 1/100,000 of a c. c.) which was neutralized with botulinus antitoxin, type A. The other outbreak was stated to have been due to home-canned beef; but this was not proved by laboratory examination. This is the first outbreak to be recorded in the United States as being caused by this food. An explanation of the high toxicity rate of the foods involved may be found in the distribution of the spores of B. botulinus in the soil of this ranch and in the method of preservation. The cold pack method was used, and it is not unlikely that the temperature at which the processing was done was totally inadequate. It was frankly stated that no attention was paid to either the "fill" or the relation of altitude to the processing temperatures (boiling point).

1 Annual Report of the Surgeon General of the Public Health Service for the Fisoal Year 1921, p. 17. 51376--23—1 (1611)

The bacteriological and toxicological examination of the soil yielded interesting and conflicting data. The garden soil was said to have been moderately manured at irregular intervals. Two samples of garden soil, when tested colorimetrically, showed a reaction of pH 7.1, and a third a reaction of pIT 7.3. The reaction of the uncultivated soil, or virgin soil, was pll 6.6. The following table shows briefly the results of the examination of the soil for the spores of B. botulinus, after enrichment in beef-heart media:

Table II.—Result* of soil examination for B. botulinus, IT. Ranch, Ohonogan

County, Wash.


There are to be noted two things: First, there was a uniformity of results in the various amounts of soil used. This was unexpected, as laboratory data in such experiments usually show variations, which have been explained by assuming that there was an uneven distribution of spores in the specimen; secondly, the same type of toxin was produced—type B.

The demonstration of type A toxin in the foods and type B toxin in the soil demands explanation. These anomalous results were obtained on three different occasions. It seems probable that both organisms are present in the soil, and that the food substances, corn and string beans, are more favorable for the growth of type A organisms and that those of type B are suppressed. This observation may have a bearing on the fact that nearly all human outbreaks of botulism in the United States have been due to type A.

Experimental evidence on the point referred to is shown by the following: 20 grams of commercial canned corn and string beans previously autoclaved in open petri dishes for 30 minutes at 17 pounds' pressure were added to 100 c. c. flasks of beef heart media, together with 1 gram of virgin soil from the ranch, heated to 80° C. for one hour, and overlaid with sterile vaseline. In all, 18 cultures so treated were incubated at 37° C. for 10 days. Two flasks containing soil but no vegetables served as controls. Tests for the presence of toxin and the determination of type were made by intraperitoneal inoculation into mice.

The results are shown in the following table:

Table III.—Teats for the presence of toxin.

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It will be seen that of two specimens of virgin soil alone in 1 gram amounts, one produced a toxin, type B, B. botvlinus. This same soil in 1 oram amounts (eight specimens) with the addition of autoclaved corn yielded 4 positives, type A. With the addition of beans, out of 8 specimens tested, 3 were positive. Two of these proved to contain type A and one type B toxin, B. botuHmis.

Obviously the soil contains both types. The results obtained when the soil alone is planted in ordinary beef-heart medium indicate that type B is predominant. When the soil, together with the corn or beans, is planted in the same medium, type A appears to predominate, indicating that the corn and beans have an enriching effect or favor the growth of type A. There is the other possibility that type B, being the less resistant type to heat, may be destroyed, although this is unlikely to have occurred in the above experiment, as all temperatures were the same, namely, 80° C, one hour.

The following experiment seems to prove that both types, A and B, were present in the soil. Some of the original cultures obtained by planting the soil in the beef-heart medium, which gave a positive test for the presence of type B toxin by the mouse test, were boiled for 30 minutes. Agar shake cultures were made from these and incubated. From these shake cultures, 37 colonies morphologically resembling those of B. botuiinus were fished with pasteur pipettes under the dissecting microscope and placed in beef-heart enrichment media. Ten of these, after 10 days incubation at 37° C, when tested, were positive for type A toxin. No type B colonies were isolated. There is, of course, the possibility that the liver agar may have, supplied something that favors the growth of type A: or that in the process of boiling, typa B organisms were destroyed; or that the writer unintentionally "fished" type A colonies only and missed those of type B.

To test further the effect of heat, the original soil samples in beefheart media, 12 in number, were detoxified by heating to 80° C. for one hour. Twenty grams of autoclaved corn were added to each of two flasks, and 20 grams of autoclaved beans to each of two other flasks. These flasks were then boiled for 30 minutes and incubated at 37° C. for 10 days. Four flasks similarly treated were not boiled. Two flasks without corn or beans were used as controls for each treatment, with and without boiling. The results are briefly shown in Table IV.

Table IV.—Experiments to determine the effect of boiling on the types of organisms.


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The above experiment does not offer any great amount of additional evidence as to the effect of temperature on types, but indicates rather definitely that with the addition of corn and beans type A toxin was produced in three out of eight instances in soil in which previously type B had been demonstrated.

It has been stated that type A colonies were fished from liver agar shake cultures made from the growth, in beef-heart medium, obtained from the soil which apparently contained only type B organisms. It was thought that by the addition of soil to beef-broth cultures of these type A colonies it might be possible to demonstrate type B toxin. One gram of the original soil was therefore added to four of these cultures. Two cultures without the addition of soil served as controls. All the cultures were subjected to a temperature of 80° C.

for one hour and wore then incubated 10 days, duction showed type A in all the cultures.

Table V.'

Tests for toxin pro



4 cultures from Brer agar" shake "colonies which yielded type A originally,

plus 1 gram original soil, heated to SO" C. 1 hour.
OnTol— 2 cultures from liver ai;ar 'shake" colonies which yielded type A

originally, heated to SO* C, 1 hour.

All proved positive type A toxin.

Both cultures proved positive type A toxin.

These results indicate the stability of the type A, once the toxin is formed in the food, even when there is the addition of soil in which type B toxin can generally be demonstrated. However, it would seem more probable that there were many more type A spores in the culture than there were type B spores in the soil and that typo A developed and type B was suppressed.


The above experiments are offered as evidence that probably many soils contain spores of both type A and type B, B. botulinus, which, when inoculated into suitable media, are capable of producing their selective type of poison. (The conditions that induce the formation of specific types in preserved foods arc far from being clearly understood.) It is, howrever, not unlikely that foods like corn and string beans, when contaminated with soil containing the spores of both type A antl type B, B. botidinus, are responsible in some manner for the production of one or the other type of toxin, and these experiments suggest a more frequent occurrence of type A toxin. We havo observed on this ranch curious and interesting anomalous data. Furthermore, the data render doubtful any dogmatic assertion as to any particular type of toxemia being limited to foods grown in any particular geographical region. The experimental and field data here presented do not furnish any conclusive evidence that one type is a mutant of the other, but rather surprisingly indicate that type B is the predominating type in both garden and virgin soil of this restricted area of a western State. Likewise, it is conclusive that this ranch has had a remarkable concentration of outbreaks and that home canning of vegetables grown on its soil under the present conditions is unsafe.

Acknowledgments—It is desired to acknowledge with thanks the cooperation of Dr. Paul West, county health officer of Okanogan County, Wash.

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