Indications of variability within pure strains of Str. cremoris have been studied.

In one strain changes in acid-producing power were made evident by changes in colony form. A variant which failed to ferment lactose but was still susceptible to the specific race of bacteriophage and exhibited the same morphology (after treatment at 37° C.) as the parent culture is described.

It was found that variation was well marked within some strains, especially in regard to acid production (or vitality), response to high temperature conditions, power to produce ropiness in milk, and degree of resistance to phage attack.

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.

1. Spray-dried (Kestner process), roller-dried and evaporated milks were prepared commercially from one batch of raw milk.

2. The biological value of the proteins (nitrogen), vitamin B1 and the growth-promoting properties of the milks were measured by tests on rats.

3. Vitamin A and carotene, riboflavin and vitamin C were measured by physical and chemical methods at intervals in the course of storage for a year.

4. No significant difference was found between the biological values of the proteins (nitrogen) of the processed milks. The values ranged from 80 for evaporated milk to 84 for spray-dried milk.

5. The proteins (nitrogen) of evaporated milk were significantly less digestible (true digestibility 91) than those of spray-dried milk (true digestibility 94). The difference between either of those and roller-dried milk (true digestibility 93) was not significant.

The destruction of vitamin C in milk under the action of light was studied in different containers. Brown glass bottles showed little destruction, green glass bottles considerably more, and wax impregnated cartons still more. Nevertheless, in the latter, twice as much vitamin C survived as in clear glass bottles.

1. By the use of sodium hexametaphosphate it is possible to eliminate the final filtration in the phosphatase test, and thus appreciably to shorten the time required for the test.

2. It is necessary to avoid direct sunlight during that part of the test in which Folin's reagent is in use. Other minor precautions are mentioned in the paper.

3. The pH. of the milk samples tested was found to vary naturally before pasteurization from pH 6·5 to 6·9 without affecting the validity of the test.

4. The test is also unaffected by the presence in milk of bacteria which produce reducing substances or which hydrolyse the substrate (sodium phenyl phosphate) unless the organisms are present in very large numbers.

1. A rapid method for determining the peroxide value of the fat contained in whole milk powders is described.

2. As estimated by this method the peroxide value is only about 90% of the true value, but in many researches where the method would be applied, it is the relative rather than the absolute peroxide value which is required. Consequently the disadvantage of a slightly low estimation is far outweighed by the rapidity of the method and the ease and reliability with which it can be performed.

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.

By adapting Scharer's “10min. field test” to British conditions of pasteurization, an accurate phosphatase test has been evolved requiring less than 1½ hr. for completion. The method is described in detail.

A small percentage of anomalies was detected when applying the new method to the examination of pasteurized samples bulked from small quantities of milk (less than 100 gallons). This led to a study of several factors thought to be the likely cause of such anomalous results.

No correlation was found between the phosphatase surviving heat treatment and the stage of lactation when milk from individual cows and from small groups of animals was tested, with the possible exception of the early stages of lactation when the enzyme concentration before and after heating was frequently found to be low.

It was observed that pasteurized milk from animals suffering from mastitis tends to give abnormally high results of the phosphatase test. The amount of residual phosphatase is subject to considerable fluctuation.

The oxidation of the fat of butter during cold storage has been studied by measuring the fat-aldehyde value of the fat. A direct relationship was found between this value and the grade after storage.

It was found that acidity, starter organisms, salt and low-pasteurization temperatures each favour the oxidation. Neither diacetyl nor acetoin influence the oxidation.

The results indicate the presence in ripened pasteurized cream and in unripened raw cream of a fat-oxidizing enzyme which is most active at low pH values (about 5) and high salt concentrations.

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.

Simple and rapid methods have been devised which utilize the minimum of apparatus and material for the routine bacteriological testing of butter. These involve the preparation of microplate cultures to obtain counts of total bacteria, “heat-resistant” bacteria, lipolytic organisms, yeasts, moulds; Grimes's method as a measure of the coliform content is also used.

The method for counting colonies of lipolytic bacteria is based upon their ability to produce round each colony a halo of white solid fatty acids in place of the clear glistening fat globules.

The new methods have been exhaustively compared with those hitherto accepted, and have been found to give satisfactory and reliable results.

A variety of comparisons has been carried out to improve still further the methods in use and to provide additional information about the nature of the bacteria contamination present in the butter. The roll-tube method seems preferable to the microplate for mould counts, and is quite suitable for yeast counts.

The results obtained confirm the view expressed by Wood & Thornton(6) that yeast and mould counts provide an inadequate criterion of factory sanitation, and should be supported at least by the total count of bacteria.

The employment of these methods has enabled large numbers of samples to be dealt with. Thus samples from most of the principal New Zealand butter factories have been regularly tested. The information so obtained has been of material value to the Dairy Division's Instructors in drawing attention to factories where sanitary conditions left room for improvement.

1. The effects of alterations in the duration and speed of stirring on the apparent solubility of milk powders have been investigated. It is concluded that 30 sec. shaking of the powder reconstituted in a 10 % mixture is sufficient to dissolve the truly soluble portion of the dried milk, but that the protein which has become denatured during the drying process tends to pass into solution with increasing speed and duration of stirring.

2. A rapid method for estimating the solubility of milk powders on a total solids basis involving only 1 min. shaking is described and discussed.

3. The sediment method frequently used in industry as a guide to the solubility of dried milks is compared with the rapid method described in the present paper. At higher solubilities the sediment volume may serve as a guide to small changes in solubility, but in the lower solubility ranges its usefulness is very limited. It is shown that the washing of the sediment with one or more changes of water merely introduces additional errors into this method of solubility determination.

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.

“Open” cheese were shown to evolve carbon dioxide much more rapidly than did “close” cheese. From open cheese lactobacilli or betacocci capable of producing carbon dioxide relatively rapidly could be isolated. The addition of such organisms to cheese milk resulted in the development of slit openness in the cheese. The most common types causing slit openness appeared to be gas-producing lactobacilli.

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

The reproducibility of Scott Blair's method for measuring curd firmness has been tested and measurements of the “firmness” of cheese curds immediately prior to cutting have been made and related to an expert's opinion. Contrary to her beliefs it was found:

(1) that the mean firmness for normal Cheshire and Double Gloucester curds was the same as that for normal Cheddar curds,

(2) that normal curds, regardless of type, were softer in the summer months than in the winter.

Though confident opinions are often expressed on very small differences in firmness, the smallest difference in modulus which can be correctly appreciated appears to be of the order of 8%.

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.