An easy test for bacteria in milk and cream (Barthel's reductase test improved)

Front matter

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An Easy Test for Bacteria in Milk and Cream

(Barthel's Reductase Test Improved)

Introduction

The problem of improving the sanitary quality of a milk supply is incapable of solution so long as there is not a simple test available for determining approximately the bacterial contents in the country. No amount of testing done in the city, at the point of consumption, can ever prevent the most serious pollution of the milk, which is that occurring previously, in the country, at the point of production. Much of the testing must be done in the country. If it is omitted, the creamery manager does not know which of the farmers that furnish milk to him, is to be held responsible whenever the bacteria run high in the mixed milk which he ships to the city. Before the cause of pollution can be removed, it must of necessity first be located. This cannot easily be done without a test. It is true that the inspectors sent out by the creameries to score the dairies discover some causes of pollution. But the inspectors need a test to determine beforehand which of their numerous dairies require inspection the most. Furthermore, experience shows that the inspectors are frequently deceived by the farmers. A farmer may have his dairy in first class condition when the inspector is around and yet deliver milk which runs excessively high in bacteria, without being discovered by the inspector. He may, for instance, feed sour ensilage during the milking, or keep his evening's milk at 75° F. instead of 45° F. over night in summer. So long as a few pieces of ice are put between the cans just before delivery, the inspector is easily cheated. His thermometer certainly fails to detect the excessive number of bacteria in such milk. The color test is the only practical way to locate the trouble. Actual experience proves this.

Over ten thousand samples of milk were tested, at receiving stations in the country, by the color test after my directions, during a period covering two years. These tests have been so successful for the purpose of locating the causes of high bacteria that milk dealers who had not used the test, but who had heard of it, quickly adopted the color test for their own creameries.

Furthermore, well known sanitarians and authorities on milk hygiene made inquiries concerning the test, as improved by me.

Under these conditions, I am sure I am complying with a general demand in publishing a detailed description of the test as improved, and directions for making it.

These directions, as will be noted, are so specific and so simple that every creamery man, including those who never had any previous experience in laboratory work, will be able to make the test without further instructions.

To the bacteriologists and chemists who may read my directions, I beg to apologize for using so simple a language and making statements of facts which are most elementary and well known to them. As thousands of creamerymen, including those who are without experience, are expected to use my directions, I believe I am right in giving elementary instructions, even at the risk of making those directions tiresome to the professional laboratory men. As the latter are comparatively few in number, the harm done by excessive simplicity will not be serious.

It will be noticed that this publication is designed to meet the needs of the milk dealers, creamery employees, condensary employees and others interested, not only as regards contents, but also as regards form.

My statement that the color test, or reductase test, is the only practical test for locating the cause of high bacteria in the country, is amply corroborated by authorities whose judgment cannot be questioned. For instance. Dr. H. C. Campbell of the pathological division of the U. S. Bureau of Animal Industry, an expert in milk hygiene, in speaking of the reductase, or reduction test and other biochemic tests, in the annual report of the bureau, for 1911, after experimenting with them, comments on these tests as follows:

"The reduction test agreed in most instances with the bacterial count ; that is, whenever the sample was reduced in a short time, the bacterial count was high."

"The biochemic tests used are simple in their technique. The results obtained are practically as good as those shown by plating, so far as estimating the number of bacteria is concerned."

"These tests, if applied at the dairy or site of milk production, would greatly aid the health officials in controlling a milk supply."

"In all pathological processes of the udder the reductase properties are increased."

Dr. Wm. Ernst, director of the milk control station at Munich; Dr. John R. Mohler, chief of the pathological division, U. S. Bureau of Animal Industry, and Dr. A. Eichhorn, senior bacteriologist of the same division, in their "Text Book of Milk Hygiene", published in 1915, make the following statements regarding the reductase test:

"Very valuable results are obtained with the reductase test when conducted in connection with the microscopic examination of the sediment."

"As already mentioned, for practical control work the counting of bacteria may be omitted. In this work the reductase test offers a quicker determination of the spoiled condition of the milk."

"... If (in Miiller's experiments) sour milk or cow manure was added to fresh milk, the time of reduction was hastened. He therefore proved that all factors which favorably influence bacterial growth, hasten the reduction."

"The methylene reductase test gives very good information relative to bacterial multiplication."

Manifestly, the extraordinary usefulness of the reductase test is recognized by the leading authorities on milk hygiene. There is a pressing need for a simple, easy, and rapid test for determining the sanitary quality of milk at all creameries, condensaries, dairies and similar places in the country, and the reductase test answers the purpose much better than any other test in existence.

In sharp contrast with the favorable comment of the experienced laboratory men is the comment which comes from superficial and inexperienced observers. Young students and others who had little or no experience in laboratory work, naturally failed to get correct results, chiefly because they did not prepare the methylene blue solution properly. Their technique was faulty because they could not devise their own technique,, while the published directions failed to mention the necessary points of technique in making the solution and the test. Their failure to get correct results has evidently no weight in judging the merits of the test. The test can certainly not be held responsible for the mistakes of beginners who misused it at a time when elementary instructions for making it were not yet available. This does not say, however, that the test could not be improved in other respects. Both, the instructions and the test needed improvement. The proper thing to do was evidently to give elementary instructions for making the test. This is what I have undertaken to do. The test itself needed improvement as regards the amount of methylene blue used. In this respect, criticism of the old form of the test hitherto in use may have been justifiable to a certain extent. Realizing that the reductase test is the only test in existence which is simple enough to be used in the country, and that it was badly needed, I undertook to improve it, and the thousands of results obtained with the improved form show that I was more or less successful.

It may be surmised that the improvements I made on the test are superfluous because experienced laboratory men could get satisfactory results without such improvements. This is, however, not the case. For instance, without a chemical standardization, the strength of the methylene blue solution was bound to vary, even in the hands of an expert, so long as it was based on saturation in alcohol or a similar solvent. A saturated solution made in summer is apt to be stronger than one made in winter, since warm alcohol dissolves more methylene blue than cold alcohol. Furthermore, the admixture of foreign substances sometimes found in methylene blue may also produce a variation. The chemical standardization is therefore necessary even if errors in technique are avoided. It secures more accurate results.

The "Double Time Test" and the bacteriological regulation of the amount of methylene blue used will also be found helpful in obtaining more accurate results.

Thus the improvements and the elementary directions will enable both, the experts and the beginners, to get better results hereafter.

The plating test with agar is a failure in the country for more reasons than one. Aside from the excessive first expense for the complex equipment used in this test, the current expenses are high for the reason that only a professional laboratory man can be relied on as furnishing correct results. Beginners who believe the results they obtain to be correct, are nearly always mistaken. The test has sometimes failed to give correct results in the hands even of the most expert bacteriologists, as Dr. H. W. Conn, the well known "specialist in agricultural bacteriology, has shown. This test is even more in need of an improvement than the reductase test had been. With all the money spent on this test, little is accomplished.

In most creameries it would be impossible anyway to use the test successfully even for a professional bacteriologist, since the equipment is never complete. It is for instance difficult to see how an incubator could be kept at constant temperature in a village where neither gas nor an electric current is available. An alcohol lamp would have to be used the same as in the case of the reductase test, with this difference, however, that it is practical to use it in day time for the reductase test, but quite impractical to use it at night for the incubator.

Even this is not yet the last of the disadvantages connected with the plating test. If the creamerymen had to wait 48 hours for the results, as in the plating test, they would fail to locate the causes of those pollutions which disappear in less than that time. They could not reject polluted milk delivered to them on the strength of the plating test, since the milk has to be either rejected or accepted in much less time than 48 hours.

Furthermore, the plating test lost much of its former value as an index of the sanitary quality of raw milk from the time on that pasteurization of milk became general. As the bacteria are killed anyway by pasteurization, the real pollution in the finished pasteurized milk does not consist in bacteria, but in the metabolic compounds left behind by the bacteria. The amount of these metabolic compounds should be determined by exact methods of quantitative chemical analysis and not by any of the biological methods, each of which is more or less inexact. The number of live bacteria, even if it were determined accurately, fails to measure the amount of metabolic compounds correctly for the further rea.son that these compounds are largely derived from the dead bacteria which in winter are so numerous, and which, evidently, do not show up at all in the plating test. The number of live bacteria, in other words, is out of proportion with the real pollution.

I do not see how any intelligent manager or director of a milk company can seriously consider the plating test for general adoption in the country stations. I know well that it is desirable to use the same test which the officials use. However, the days of the plating test, as the exclusive official test for raw milk to be pasteurized, are numbered.

For that insignificant portion of market milk which is sold raw to the consumer, the plating test may remain a valuable index of the sanitary quality, but not for raw milk to be pasteurized. Conscientious officials will admit that the plating test is not fit to be made the sole basis of legal prosecution for such milk, since it is misleading in several ways.

If the number of spore bearers is to be determined, the plating test again fails to give correct results, since the ratio of the spore bearers to the total number of live bacteria fluctuates constantly.

The direct microscopic count of the bacteria in milk after Skar's or Brew's method is a little more accurate than the plating test. It cannot be made entirely accurate, chiefly because only an extremely small amount, viz.: much less than a drop, can be examined. Such a small amount gives inaccurate results for the reason that it does not represent the number of bacteria in the milk supply correctly.

Furthermore, the microscopic test, like the plating test, is expensive and requires the services of a professional laboratory man. A microscope costing at least $75.00 cannot be turned over to a creameryman who has had no previous experience in such work. The time may come when every creamery manager will be required by law to pass an examination in bacteriology, chemistry, and milk hygiene before he is allowed to put up milk for infants, just as a pharmacist has to pass an examination in materia medica, chemistry, and dispensing before he is allowed to put up prescriptions. Such laws, in fact, are already in force in certain sections in Europe. For the present, however, we must take the creamerymen as they are and we cannot expect them to do scientific work of a high order, or at least not all of them. Therefore we must use a simple test, such as the reductase test, for the next twenty or thirty years to come, for this reason, and probably thereafter for other reasons.

The reductase test measures accurately the extent of bacterial activity, in the "Double Time Test," if the exact length of time required for decolorization is determined. This is a more valuable criterion to go by, in judging raw milk to be pasteurized, than the mere number of bacteria, since it shows the capacity of the bacteria for decomposing milk and for forming harmful decomposition products. Furthermore, the fact that hydrogen, hydrogen sulphid, and mcrcaptans generated by such organisms as the members of the coli-aerogenes group and the bacteria of putrefaction, decolorize the methylene blue in the reductase test, plainly shows that the reductase test indicates harmful decomposition, besides the mere extent of decomposition. That certain diseased conditions of the udder can be detected by the reductase test, is known. Under these circumstances, I am satisfied that the reductase test is a much more valuable index of the sanitary quality of raw milk to be pasteurized than the plating test. If the results of the plating test are in conflict with those of the reductase test properly carried out, it is merely one of the numerous confirmations of the fact that the plating test is unreliable as an index of the sanitary quality of such milk.

On agar, the milk bacteria can never de\-clnp those decomposition products which I hey would develop in milk. Consequently it is useless to employ agar as a culture medium if the nature and amount of those decom])osition products are to be determined, as in the case of raw milk to be pasteurized. The only proper medium for milk bacteria is milk, in such a case. This is another reason why the reductase test is valuable.

The reductase test never fails to detect any lack of cooling in summer, in raw milk. It also detects any admixture of dirt. To determine whether a rapid decolorization is due to lack of cooling or to dirt, the acidity test is made. If the acidity is considerably above normal, therefore considerably above 0.17 per cent, the rapid decolorization is due to lack of cooling. If not. it is due to dirt. It is understood, however, that these tests are made on fresh milk. If there is an interruption of transportation facilities and the milk is kept in the country several days, it is apt to show a high number of bacteria and a rapid decolorization when there was neither dirt nor lack of cooling. These abnormal and rare conditions are to be taken into consideration, if they occur, in distinguishing between lack of cooling and dirt, as above stated.

For pasteurized milk and pasteurized cream the reductase test is not recommended. The heat of pasteurization interferes with the test.

Cheese makers and others who desire to make the fermentation test will find that the apparatus furnished for the reductase test can just as well be used for the fermentation test, the only difference being that the blue solution is omitted and the temperature is lower, viz: 98° F. or 75° F., as may be desired.

It will be found convenient to have the standard methylene blue solution made in the city and to send it to the milk-receiving stations in the country. Likewise, any bacteriological tests for the purpose of determining how much of the standard solution is to be used for a given milk supply, may conveniently be made in the city. It will be noticed that the scientific work may easily be separated from the elementary work of making the tests on the milk samples, as the solution made in the city or the results of the plating test can easily be sent anywhere. This enables the creamerymen to get all the accurate results which scientific laboratory work may afiford, without having to do the scientific part of the work themselves. Under these circumstances anybody can make the test in the country and expensive help is unnecessary, so far as the country is concerned.

It is obvious that the reductase test affords the greatest advantages in summer. Whenever the number of bacteria is excessive either from lack of cooling, or from dirt, or from any other cause, this test never fails to detect it. Milk dealers who are shipping milk to cities or towns where the number of bacteria is limited by law, will have no trouble in locating, by this test, those farmers whose milk exceeds the limit. This will enable them to live up to the bacterial limits in the cities and towns, since they can exclude the objectionable milk from their shipments. '

The results of the reductase test generally agree better with the plating results in summer than in winter.

Officials would do well to adopt the reductase test as the official test. They cannot expect the milk dealers to use the plating test, since it is impractical and misleading. Some kind of a test will have to be adopted because without such a test, the milk dealers cannot live up to bacterial standards.

Creamerymen who ship milk to New York City arc reminded that the present limit (1915) for Grade B milk, raw, is lyi million bacteria per cubic centimeter, if the milk is to be pasteurized in the city. This corresponds to three hours in the "Double Time Test," just as it corresponds to 1^^ hours in the "Single Time Test."

All creamerymen and others making the test arc urged to study those sections in the pamphlet which arc printed in bold type, and to post the tables which go with the pamphlet in the testing room for daily use.

The color of the tubes should be observed in bright daylight, not at gas light or other artificial light, and the blank tube for comparison should not be omitted. The color can be seen best on a white background.

Bacteriologists who are called upon to regulate the amount of the standard methylene blue solution in accordance with their plating results, are reminded that the "Single Time Test" and the "Double Time Test" want to be regulated each independently of the other. It is not safe merely to use five times as much blue solution for the "Doube Time Test" as was found necessary for the "Single Time Test." Each test must be compared with plating results directly.

The reductase test, as improved, will be found to give correct results. In summer, the results agree so well with the plating results that the plating test itself, if it were made in the country, could not furnish any better results. While excessive numbers of bacteria are always detected, a moderate number of bacteria can escape detection only, if the test is not performed properly. Generally, whenever an inaccurate result is obtained, it is due to insufficient mixing of the milk sample. It is to be remembered that only a very violent agitation of the milk will break up the clumps of bacteria. If the sample is placed in a sterilized bottle, half filled, and the bottle shaken very vigorously 25 or 30 times, correct results are obtained every time. Can milk must be stirred vigorously before sampling.

Bacteriologists who compare plating results with the results of the reductase test, will find it convenient to use for the milk samples sterilized bottles of about six ounces capacity. Having the bottle only half filled, to make shaking easy, a good representative and well mixed sample is secured for both tests, and enough is left to repeat the tests, if necessary.

As the color test affords an easy means for detecting excessive numbers of bacteria, milk dealers need no longer run the risk of having their milk condemned by inspectors and of having their reputation ruined. If the color test shows that a given can of milk is above the legal limit for bacteria, all they have to do, is to send it to the churn instead of selling it as sweet milk. Or better still, they can make a contract with their farmers specifying that the milk furnished to them must not be decolorized, by the "Double Time Test," in less than 2^/2 hours, or 3 hours, or 4 hours, as the case may be. As they are then at liberty to return the milk to the farmer, without loss to them, if it runs high in bacteria, they can make sure that their milk will always pass inspection. Milk dealers, in other words, need not dread the consequences of official inspection any longer.

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Barthel's Reductase Test

Improved by Hugo Jone

Directions for Making the Color Test (Methylene Blue Test)

for Bacteria in Milk

Copyright 1915 by Hugo Jone

Most bacteria living in milk separate an enzym or body juice known scientifically by the name of reductase. The reductase has the property of destroying the blue color of the aniline dye known as methylene blue. If there are many millions of bacteria in a sample of milk, the large amount of reductase they separate is sufficient to decolorize at once the measured amount of methylene blue solution usually added to the milk for the test. If there are few bacteria, they do not separate enough reductase for decolorizing the methylene blue completely. However, if the bacteria are allowed to multiply by putting the milk in a warm place (usually 112' Fahrenheit), they will be so numerous in a few hours, that the reductase they separate is finally sufficient to decolorize the methylene blue completely. Manifestly, under these circumstances, a milk sample having many millions of bacteria in it, will be decolorized rapidly, while some samples having at the start only a few thousand bacteria in it, will require several hours for decolorization. Thus, we can tell the number of bacteria by the time required for decolorization. The samples must be kept at 112° F. A convenient apparatus for keeping the samples warm is a double boiler provided with an alcohol lamp and a tube holder. The tubes with the samples remain in the warm water until decolorized. The inner pot of the double boiler may be used alone.

Apparatus used for the Improved Color Test.

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(1) The inner pot is seen standing on the tripod, with a test tube holder, test tubes, and a bulb thermometer on the inside of it.

(2) The outer pot is seen standing on the table.

(3) The methylene blue solution in use is contained in the wide-mouth bottle near the corner of the table next to the glass cap. (4) The glass cap is taken off only for the purpose of making both, the bottle and the cap, better visible. In the actual testing, the cap is never removed from the bottle except at the moment when solution is taken out of the bottle with the pipette. All dust is to be kept out of the blue solution that way. This style bottle is called an English oil bottle. It is especially well adapted for holding a pipette besides a liquid without polluting either.

(5) The wire basket with empty tubes, (6) the glass tumbler with decolorized tubes, (7) the pint bottle with mushroom glass stopper containing methylene blue solution ready for shipment, and other objects will be recognized without further specification.

The pipette fitted into the English oil bottle is a short nozzle pipette measuring 2 cubic centimeters of blue solution.

The other pipette (8) shown standing in one of the glass tumblers is a short nozzle pipette measuring 10 cubic centimeters of milk. As 20 cubic centimeters of milk are used for each test, the pipette must be dipped into the milk twice, instead of once, when sampling. This is done on purpose to secure a good average sample of the milk.

The test is made as follows : — Enough warm water is placed in the outer pot of the double boiler to nearly fill the jacket formed when the inner pot is put inside, to insure uniform heating of the inside pot. Furthermore, in the inner pot enough water is put to submerge completely the milk samples in the test tubes, thereby insuring uniform heating of the milk samples. It would not do to have the milk in the tube stand higher than the surrounding water, because that portion of the milk column which sticks out above the water would cool off too much and in that way require more time than it should for decolorization.

The water in the inner as well as in the outer pot is brought to a temperature of 112 F. The water in both pots is then continued at 112 F. by means of the alcohol lamp.

The test tubes which contain 20 cubic centimeters of the different milk samples, each, and which are identified by the numbers on them, are then put into the water until they are as warm as the water, which is 112 F. Then 2 cubic centimeters of the blue solution are dropped into each tube and mixed very thoroughly with the milk sample. A rubber stopper may be used to close the tube, to facilitate mixing. This can be cleaned by wiping it with a clean towel. However, the rubber stopper must be cleaned each time after use. If the towel is not strictly clean, it may spoil the succeeding tests. After mixing, each tube is closed with a cotton stopper.

After dropping the 2 cubic centimeters of blue solution into each of the tubes, the time is noted down, the color is observed, at fixed intervals thereafter, and the number of bacteria estimated by the time required for complete decolorization. The blue color must be turned into a plain white completely, except that at the top a faint blue ring may remain. There must be absolutely no blue color left at the bottom. If there is any, the mixing was not done thoroughly.

When observing the color of the tubes, they may be lifted out of the warm heater and placed into a wire basket having a layer of cotton at the bottom. However, they must be put back into the water immediately to prevent their cooling down too much.

The number of bacteria is estimated as follows: If less than ' i hour is required for decolorization, over 5 million bacteria, if ■ _. hour, 3 to 5 million, if '4 hour, 2 to 3 million, if V/2 hours, 1 to 2 million, if 2'j hours, 300,000 to 1 million, if 3'j hours, 100,000 to 300,000, if over 4 hours, less than 200,000.

The tubes must be rinsed and then soaked in boiling hot water for at least 15 minutes after each use. If any milk adheres to a cotton stopper, it must be soaked off or washed off with hot water and the stopper dried thereafter.

Great care should be taken to keep the tubes at the proper temperature during the test. It must be 112 F. A deviation of as much as 5 degrees will influence the result perceptibly. The color must never be dropped in until the temperature of the milk is 112 F. or nearly so. If the milk is still cold, the time required for the decolorization will be unduly increased.

For the cotton stoppers, non-absorbent cotton should be used. It keeps clean longer than the absorbent cotton.

In testing high grade milk which is intended for infant feeding, it is generally necessary to keep the tubes in the warm water about twice as long as in the case of common market milk, since it takes longer to decolorize the clean milk.

The Health Department of New York City allows not more than 200,000 bacteria (determined by plating on agar) per cc in infant milk, or Grade A Milk, before pasteurization. Therefore this milk must remain blue at least 4 hours. It must, however, be remembered that this test gives only approximate results, and is not as yet official.

For Grade B Milk before pasteurization, the Health Department allows not more than 13/2 million bacteria per cc. Therefore this milk must remain blue at least lyo hours.

The strength of the methylene blue solution is of the utmost importance for the correctness of the test. A strong solution certainly requires more time to decolorize than a weak solution. Therfore, it must always have the exact strength, not more and not less. The testing and adjusting of the strength of the solution is to be left to a professional chemist, it being too technical for others. The strength of the solution is so regulated that the results obtained by the color test are about the same as those obtained by plating on agar. If they are not the same, the strength of the solution is corrected until they are the same.