Gas production in dairy products is a subject of considerable importance, since it is responsible for certain defects in milk, cream, cheese and occasionally condensed milk. Four groups of micro-organisms are concerned, namely, yeasts, lactic acid bacteria, coliform bacteria and spore-forming anaerobes, and of these the fixs two produce carbon dioxide only while the last two produce a mixture of carbon dioxide and hydrogen (with some anaerobes there may be methane as well). Where both gases are evolved the effectiveness of the two as regards ability to produce effervescence or to develop pressure in a closed container or in a hard-pressed substance like cheese differs by virtue of the fact that carbon dioxide is soluble in water (its solubility depending on the pressure and pH of the medium) while hydrogen is insoluble. Until the medium in which such micro-organisms are growing becomes saturated with carbon dioxide the effective gas production is therefore due to hydrogen. On the other hand, in some media the hydrogen may be involved in secondary reactions with some of the constituents while in the nascent condition and will therefore not form gas.

In a previous paper (Allen (1)) some methods have been described for measuring gas production by micro-organisms. The objects of the work described here were (1) to measure the gas production (if any) by pure strains of lactic acid streptococci and by starters growing in milk; (2) to measure the total gas evolved by Bact. coli in the early stages of growth in different media, particularly milk; (3) to find the effect on gas production by Bact. coli of growing Str. lactis in the same medium with it and to measure the proportion of Str. lactis relative to Bact. coli required to inhibit gas production completely—information which has a bearing on the defects due to coliforms growing in the early stages of cheese making; (4) to find the influence on gas production by Bact. coli, with and without the concomitant growth of Str. lactis, of subjecting it to anaerobic conditions from the outset; and (5) to measure the effect due to the hydrogen alone (by absorbing the carbon dioxide) on the gas produced by Bact. coli under different conditions.

Though the lactic acid bacteria are recognized primarily as saccharolytic, several workers have recorded observations on their slow proteolytic activity. Von Freudenreich(1) was the first to record the fact that cultures of these organisms in milk, to which chalk had been added to neutralize the acidity, formed appreciable amounts of soluble nitrogen, and these findings were confirmed by Orla-Jensen (2), Barthel(3), and Barthel & Sandberg(4). Anderegg & Hammer (5), in a study of a large number of strains of Str. Lactis, found an increase in soluble nitrogen in some cases and a decrease in others, while occasionally the same strain differed in different tests. In general, cultures which clotted rapidly were more inclined to proteolysis than those which were slower in forming acid. Str. citrovorus and Str. paracitrovorus did not cause protein breakdown. Addition of 0·3% peptone to the milk tended to retard proteolysis or to increase negative values while addition of chalk resulted in more extensive proteolysis. Barthel & Sadler (6) found that starters consisting of mixed cultures of streptococci produced more extensive proteolysis than single species, indicating a symbiotic effect. Sherwood & Whitehead (7) tested the proteolytic powers of several strains of Str. cremoris in chalk milk cultures and found some active and some comparatively inactive. Two strains appear to have formed surprisingly large amounts of non-protein nitrogen. In general they found that acid-producing power was linked with proteolytic power.

1. In good quality raw milk reducing methylene blue at 37–38° C. members of the coliform group and Staph. aureus were among the commonest organisms present at the time of reduction. Other types of staphylococci, micrococci, and certain streptococci were not infrequently present in smaller numbers. In any given milk one type of organism tended to assume predominance.

2. Using the modified test described by Wilson et al. (1), various organisms isolated from milk at the time of reduction were examined for their rate of decolorization of methylene blue when growing in pure culture in raw “sterile” milk (milk freshly drawn from the healthy udder under aseptic conditions and having a plate count on milk agar at 37° C. of 200–300 per ml.). The most rapidly reducing organisms belonged to the coliform group. Following them, in decreasing order, came Str. lactis and some of the faecal streptococci, Staph. aureus, Staph. albus, Staph. citreus, some micrococci, group C haemolytic streptococci and some strains of Str. agalactiae, and aerobic spore-bearers

In recent years the attention of those interested in all aspects of the dairy industry has been focused upon the subject of mastitis, particularly the chronic streptococcal type. On account of the wide distribution of this disease and its adverse effect upon milk and milk products, it has become urgently necessary for the producer, distributor and manufacturer to be able to recognize mastitis in the cow and its effect upon the milk. It is, therefore, desirable that all those interested in dairying should be as fully informed upon the subject as possible. Many simple tests for the detection of mastitis have been described and are in common use, although so many papers have been published showing their limitations that it is surprising that workers should continue to use them(1, 2, 3, 9). Opportunity has been afforded to the writers to make an extensive examination and comparison of such, tests, with the main object of finding out if any simple test, or combination of simple tests, is really satisfactory for the diagnosis of chronic streptococcal mastitis. The enormous literature upon mastitis (much of it uncritical and, therefore, of little use) makes an adequate summary impossible, even if it were desirable in a paper of this nature, but reference may be made to a useful summary by Munch-Petersen (1).

Many tests have been used for the detection of subclinical mastitis and these have been enumerated by Munch-Petersen(1) and Rosell(2). In view of the continued recommendation and use of these tests in many countries it was felt desirable to make a careful assessment of their value.

By the term “udder count” in this series of papers is understood the colony count obtained when samples of early mid-milk (taken after about ½ pint has been removed from each quarter) are plated in milk agar and incubated for 2 days at 37°C. Dairy bacteriologists have long recognized the fact that cows with udder disease give high-count milk. In fact, some of those engaged in control work have made a practice of advising producers to search for mastitis when, in the absence of other causes, unusually high counts have been obtained in a series of samples. Therefore in low-count milk, at least, the milk agar plate gives some idea of infection in herds. The early observations, however, were not as a rule confirmed by satisfactory evidence of mastitis, i.e. by plating on blood agar or by cultural or serological typing of colonies picked off plates. This criticism also applies to many papers and statements in articles on mastitis as it affects other aspects of the dairy industry, e.g. off-taints, “slow starter” in cheese-making, and butter-making generally. Unless satisfactory evidence of mastitis is produced such work is valueless. Conclusions based on the results of the indirect tests are unreliable.

The adoption of the methylene blue test as an official method of grading milk in England makes this subject one of considerable importance. The predominating factor in this, as in other tests, is the condition of storage (time and temperature) of the sample. If mastitis affects the methylene blue reduction time of milk the factors responsible may be any or all of the following:

(1) The mastitis organisms themselves.

(2) Other organisms present (secondary invaders).

(3) Body cells, e.g. the so-called leucocytes of milk and also blood cells.

(4) Chemical changes in the milk as the result of the mastitis.

A related aspect is the value of the methylene blue test on single cow or quarter samples as a test for mastitis.

Instances of the deleterious effect of “mastitis” on milk for cheese-making have been recorded over a considerable period. Until recently it was neither possible to define what was meant by mastitis nor to consider it in its chemical and bacteriological aspects. Many of the early reports must therefore be accepted with caution, a point emphasized in a review by Davis & Mattick(1).

I. Methods of taking and examining samples of milk for Str. agalactiae are described.

Where delay in examination is unavoidable Edwards's (1) medium is recommended. This selective medium, however, does not permit the growth of some types of organisms which may cause a subclinical mastitis. It is therefore recommended that, where possible, samples be examined immediately after taking and a non-selective medium used. In this way a measure of the udder count is obtained which is of great value. Where samples can be examined within a few hours plain blood agar is preferred. If blood is unobtainable ordinary milk agar may be used. Non-blood media have certain advantages over blood media, which compensate for the loss of ability to detect haemolysis when it occurs.

A simple confirmatory test for Str. agalactiae is described. Confirmation of type is regarded as essential in all but the obvious cases.

II. The results of a number of simple tests for mastitis have been compared with those from examination in blood agar. These all have roughly the same order of efficiency in that they detect about half the positive cases and give 10–30 % false positives. It is suggested, therefore, that with two exceptions (the milk agar count and the rennet test, which may be used for special purposes) they are so unreliable as not to be worth doing in any laboratory examination.

The brom-cresol-purple paper, strip cup and induration tests, which are very simple and can be carried out by the milker, are worth doing in order to get a general idea of the incidence of mastitis in the herd. Their limitations should be realized, however, for no simple test, or combination of simple tests, is really satisfactory, especially if the owner wishes to secure eradication of the disease from the herd. Our results thus confirm those of other workers. III. Mastitis has a marked effect on the “udder count” as given by total colony count on milk agar. No samples from mastitis-free cows were found to have a count of over 2000 per ml. and very few over 500. About 50% of infected cows yielded milk with a count of over 1000.

The milk agar count is perhaps the best of the indirect tests as although it detects no more than other tests (about 50 %) it gives very few false positives. A count of under 100 per ml. may be taken to indicate freedom from infection and a count of over 1000 to indicate mastitis.

IV. The methylene blue reduction test is not capable of detecting mastitis. Reduction time appears to be more closely related to the cell content of infected milks than to the total count. Storage of the samples for 16 hr., either at 4 or 15·5° C, did not materially improve the efficiency of the test.

V. The possible causes of the unsuitability of mastitis milk for cheesemaking are classified and discussed. The brom-cresol-purple-rennet test is described and recommended as a general test for mastitis and suitability of the milk for cheese-making. The effect of mastitis on the chemical composition and enzyme content of the milk is discussed. Mastitis adversely affects the “body” of cheese and is an important factor in the fault known as “red spot”. Many samples of mastitis milk give slow growth of starter, and these are usually detected by the rennet test. Deficiency in bacterial growth factors may be one factor in causing slow starter. This phenomenon is also correlated with rapid reduction of methylene blue and high total count, but the real factor responsible is probably abnormal chemical composition.

VI. The incidence of mastitis increases steadily with age of the animal. Older cows do not give milk of higher “udder count” than younger cows if infection is absent. The increased count is entirely due to the increased incidence of mastitis. There appears to be a somewhat lower udder count in late lactation in infected cows, but a slightly higher one if mastitis is absent.

Finally we may emphasize the fact that for eradication purposes there are only two methods of diagnosis worth using—simple tests on the farm and carefully controlled bacteriological examination in the laboratory.

The following indirect tests for the detection of subclinical mastitis were applied, under bacteriological control, to a series of milk samples from the individual quarters of cows in full milk: presence of clots, reaction to bromcresol-purple, reaction to brom-thymol-blue, amount and character of centrifuge deposit, catalase content, chloride content, casein number and solids-not-fat content.

These tests detected 12, 61, 14, 55, 49, 77, 91, and 63% respectively of the infected quarters, and diagnosed 0, 16, 0, 3, 34, 0, 8, and 9% of the uninfected quarters as infected.

For each indirect test the mean square contingency was calculated as a measure of agreement between it and the direct bacteriological method. The tests were placed in the descending order of their value for diagnosis thus: casein number, chloride content, centrifuge deposit, solids-not-fat content, brom-cresol-purple reaction, brom-thymol-blue reaction, presence of clots, catalase content.

The vitamin A and carotene content of 14 samples of butter fat churned from milk in the usual way has been compared in spectrophotometric and Lovibond tintometer tests with figures similarly obtained for the ether-extracted fat from the same milks. On the assumption that there is no loss of either carotene or vitamin A in the course of the ether extraction, it has been found that some carotene is lost in churning but that vitamin A is not appreciably affected.

The white specks appearing in mature Cheddar cheese have been shown by analysis to be calcium lactate.

Single-strain cultures of streptococci used as “starters” in Cheddar cheese manufacture in New Zealand frequently suffered failure due to the apparently spontaneous appearance of bacteriophage. The trouble was cured at the Institute experimental factory by increasing the inoculum used in the starter culture from about 0·2% to 1·0–1·6%. The culture then showed a regular activity in the cheese vat from day to day for long periods of time. Field trials during the past two years have indicated that single strain cultures can be maintained for long periods also in commercial factories if the correct technique is practised.

The theory is postulated that the effect of heavy inoculation in eliminating the spontaneous appearance of bacteriophage is due to the more rapid growth of the streptococci when they are transferred to each new batch of milk, i.e. to the shortening of the lag period. Bacteriophage tends to appear whenever the lag period is prolonged as a result of light inoculation, low incubation temperature, and aeration or ageing of the milk medium. Until more is known of the nature of phage it is not possible to suggest why a prolonged lag period in the initiation of growth should favour the appearance of phage.

Under commercial conditions failures of starters occur from time to time due to faulty technique which permits the entry into cultures of extraneous phage. The source of the contamination is most probably the whey from cheese vats, since it has been shown that phage commonly occurs in the whey even in vats where acid develops quite normally within the the cheese curd. A recent case of failure, which appears to be due to some milk abnormality, indicates that there are some milk supplies in which the heavy inoculation technique described in this paper is not completely effective in preventing the appearance of bacteriophage.