It can readily be seen that in each instance there was a marked increase in the number of leucocytes. This is also fairly good evidence that the milk never reached a temperature of 60° C. in the process of pasteurization.

TYPE OF BACILLI.

Throughout this work very little effort was made to identify the type of tubercle bacilli found except in one sample of the market milk where two rabbits were inoculated with a culture obtained from the milk. Both rabbits developed generalized tuberculosis in two months, which is considered strong evidence in favor of the culture being of the bovine type.

INOCULATION SUPERIOR TO MICROSCOPIC EXAMINATON.

With every sample of milk examined several smears were made from both the cream and the sediment. Each smear was examined carefully for the presence of tubercle bacilli, and kept until the death of the guinea pigs, when they could be reexamined in case the pigs developed tuberculosis. In no instance could tubercle bacilli be demonstrated in the milk by a microscopic examination of the smears. In 8 samples out of the 150 examined the smears were at the time of inoculation marked as being suspicious of containing tubercle bacilli, but the animals inoculated with the same milk on post-mortem examination showed no signs of tuberculosis.

The writer attaches very little importance to a microscopic examination of milk for determining the presence of tubercle bacilli unless it be from a well advanced case of udder tuberculosis where pus can be obtained in the milk.

CONCLUSIONS.

1. One hundred and thirty samples of Philadelphia's market milk were examined, and of these 13.8 per cent were found to contain tubercle bacilli.

2. The method of commercial pasteurization used in the trade can not be relied upon as a means of destroying disease-producing bacteria. 3. If pasteurization is to be used as a means of purifying milk, it should be done under legal regulation and official supervision.

4. A microscopic examination for determining the presence of tubercle bacilli in milk is of little value.

ACKNOWLEDGMENT.

The writer wishes to thank Dr. John R. Mohler, Chief of the Pathological Division of the Bureau of Animal Industry, Washington, D. C., and Dr. John Reichel, of the state live-stock sanitary board of Pennsylvania, for many valuable suggestions in this work.

60506-11- -12

THE VIABILITY OF TUBERCLE BACILLI IN BUTTER.

By JOHN R. MOHLER, HENRY J. WASHBURN, and LORE A. ROGERS. Since it has been shown that milk may frequently contain tubercle bacilli, it is evident that butter and other products manufactured from milk so contaminated must also contain these organisms, and unless they have been attenuated during the manufacturing process the products must offer to the persons consuming them the same danger of tuberculous infection that was possessed by the raw contaminated milk.

The claim having been made that tubercle bacilli, if mixed into butter at the time of manufacture, would gradually lose their virulence when the butter was placed in cold storage, it seemed desirable to prepare some butter by introducing a definite number of tubercle bacilli at the time of manufacture, and to then watch the effect of cold storage upon them while embedded in the fatty product. The length of time that tubercle bacilli would continue to live and retain their virulence while in butter under usual market conditions was therefore made a subject of cooperative investigation between the Dairy and Pathological divisions.

CREAMERY METHODS OF HANDLING CREAM AND BUTTER.

In the ordinary creamery practice the cream, when separated at the creamery from milk received in the morning or on the farms with hand separators, is allowed to ripen until it reaches an acidity equivalent to 0.4 to 0.6 per cent of lactic acid. This ripening is completed in the afternoon of the day on which the cream is received and is checked by cooling with iced water or by adding ice directly to the cream. In many of the larger creameries the ripening is preceded by pasteurization in a continuous machine, but this is not yet a common practice. The cream is churned on the following morning. The acidity of the cream will increase slightly during the night and may be at the time of churning 0.6 or 0.7 per cent. Shipments of butter from the smaller creameries are usually made once a week. The butter is held at the creamery in an ice-cooled refrigerator. The shipment is always in refrigerator cars and may be from two to six or seven days on the road, the time depending on the distance of the creamery from the market. In the larger plants, which receive only cream sepa

rated on the farm and frequently shipped long distances, the practice necessarily differs somewhat from that followed in the smaller creameries.

All butter not sold for immediate consumption is placed in storage at temperatures rarely above 20° F. and usually near zero. In the best warehouses the temperature for butter is from zero to -10° F.

BACTERIAL CHANGES IN RIPENING CREAM AND IN BUTTER.

The ripening of the cream is essentially the development of acidity through the growth of lactic-acid bacteria. This fermentation is usually hastened by the addition of a pure culture of bacteria in the form of a soured milk. These bacteria soon produce in the cream conditions unfavorable to the growth of other bacteria, and properly ripened cream contains lactic-acid bacteria almost exclusively. This is illustrated in Table 1, which is typical of the ripening of unpasteurized cream, excepting that the lactic-acid bacteria did not reach. the usual number.

TABLE 1.—Bacterial changes in unpasteurized cream during ripening.

At the time of churning, the bacteria have reached, or nearly reached, their maximum development. These bacteria are in the milk serum of the cream, which may be considered as a homogeneous fluid holding the globules of fat in an imperfect emulsion. When the cream is made into butter the small part of the milk serum which is retained in the butter becomes a nearly saturated salt solution.

The decided acidity and the high concentration of the salt of the milk contained in the average creamery butter make unfavorable conditions for the existence of bacteria, and there is usually a decrease in the number of bacteria of all kinds. The rapidity of this decrease is dependent on various factors, but chiefly on the temperature at which the butter is held. The decrease is more rapid at the higher temperatures and is retarded as the storage temperature is lowered. This is illustrated in the accompanying diagram (fig. 5), which shows the number of bacteria at three intervals (0, 6, and 9 months) in butter stored at -10, 10, and 32° F. The weaker bacteria, among which are the lactic-acid group, succumb first, and the butter finally contains only spores and a few of the most resistant bacteria.

In consideration of these facts it would be expected that the bacillus of tuberculosis or other pathogenic bacteria would not survive long in butter made by the usual creamery methods.

DESCRIPTION OF EXPERIMENTS.

This point was tested in making butter under conditions approximating as closely as possible to creamery conditions. The milk used in making this but

30

28

26

22

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20

$18

BACTERIA, HUNDREDS OF THOUSANDS PER GRAM.

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ter was inoculated in various ways with Bacillus tuberculosis, as later described. The cream was separated in a hand separator and about 12 per cent of pure culture starter added. The cream was ripened at 70 to 75° F. and cooled late in the afternoon of the day of churning or on the following morning. The acidity at time of churning varied from 0.5 to 0.7 per cent of lactic acid. Churning was done in a small barrel churn and the working on a hand worker. The butter packed in glass jars and sent by express to the storage rooms in Chicago. These rooms were arranged for experimental work and held very constantly at the indicated temperature. One jar at a time was sent to Washington for experimental tests as required.

was

6

A

~

2

-10°F

5 6

7

3 4
MONTHS IN STORAGE.

10%

32°F

FIG. 5.-Diagram showing decrease in bacterial content of butter stored at different temperatures.

Five samples of butter were prepared in the Dairy Division. The first sample, which will be referred to as Lot I, was inoculated with an artificial culture of tubercle bacilli just previous to its passage through the cream separator. This butter was tested in the salted condition, having received one ounce of salt to the pound of butter.