Bacteria / Especially as they are related to the economy of nature, to industrial processes, and to the public health

by Sir George Newman

BACTERIA IN MILK PRODUCTS

Cream is generally richer in bacteria than milk. Set cream contains more bacteria than separated cream, but germs are abundant in both. Yet whilst it is true that cream contains a large number of bacteria, it must be pointed out that the butter fat in cream is a less suitable food for organisms than is the case with milk. Hence the fermentative changes set up in cream are of less degree than in milk, particularly so if separated from the milk. Butter-milk and whey vary much in their bacterial content. Butter necessarily follows the standard of the cream. But as the butter fat is not well adapted for bacterial food, the number of bacteria in butter is usually less than in cream.[65] Moreover, they are soon reduced both in quality and quantity. Butter examined after it is several months old is often found to be almost free from germs; yet in the intervening period a variety of conditions are set up directly or indirectly through bacterial action.

Rancid butter is partly due to organisms. Putrid butter is caused, according to Jensen, by various putrefactive bacteria, one form of which is named Bacillus fœtidus lactis. This organism is killed at a comparatively low temperature, and is therefore completely removed by pasteurisation. Ill-flavoured butter may be due to germs or an unsuitable diet of the cow and a retention of the bad quality of the resulting milk. Lardy and oily butters have been investigated by Storch and Jensen and traced to bacteria. Lastly, bitter butter occasionally occurs, and is due to fermentative changes in the milk. Butter may also contain pathogenic bacteria, like tubercle. The B. coli can live for one month in butter.

Cheese suffers from very much the same kind of "diseases" as butter, except that chromogenic conditions occur more frequently. The latter are, under certain circumstances, more the result of chemical than bacterial action. Most of the troubles in cheese originate in the milk.

Method of Examination of Butter. Several grams of the butter should be placed in a large test-tube, which is then two-thirds filled with sterilised water and placed in a water-bath at about 45° C. until the butter is completely melted. A small quantity may then be added to gelatine or agar and plated out on Petri dishes or in flat-bottomed flasks in the usual way. After which the tube may be well shaken and returned to the bath inverted. In the space of twenty or thirty minutes the butter has separated from the water with which it has been emulsified. It is then placed in the cold to set. The water may be now either centrifugalised or placed in sedimentation flasks, and the deposit examined for bacteria.

The Uses of Bacteria in Dairy Produce. In considering the relation of bacteria to milk we found that many of the species present were injurious rather than otherwise, and when we come to consider bacteria in dairy products, like butter and cheese, we find that the dairyman possesses in them very powerful allies. Within recent years almost a new industry has arisen owing to the scientific application of bacteriology to dairy work.

As a preliminary to butter-making the general custom in most countries is to subject the cream to a process of "ripening." As we have seen, cream in ordinary dairies and creameries invariably contains some bacteria, a large number of which are in no sense injurious. Indeed, it is to these bacteria that the ripening and flavouring processes are due. They are perfectly consistent with the production of the best quality of butter. The aroma of butter, as we know, controls in a large measure its price in the market. This aroma is due to the decomposing effect upon the constituents of the butter of the bacteria contained in the cream. In the months of May and June the variety and number of these types of bacteria are decidedly greater than in the winter months, and this explains in part the better quality of the butter at these seasons. As a result of these ripening bacteria the milk becomes changed and soured, and slightly curdled. Thus it is rendered more fit for butter-making, and acquires its pleasant taste and aroma. It is then churned, after which bacterial action is reduced to a minimum or is absent altogether. Sweet-cream butter lacks the flavour of ripened or sour-cream butter. The process is really a fermentation, the ripening bacteria acting on each and all of the constituents of the milk, resulting in the production of various bye-products. This fermentation is a decomposition, and just as we found when discussing fermentation, so here also the action is beneficial only if it is stopped at the right moment. If, for example, instead of being stopped on the second day, it is allowed to continue for a week, the cream will degenerate and become offensive, and the pleasant ripening aroma will be changed to the contrary.

Bacteriologists have demonstrated that butters possessing different flavours have been ripened by different species of bacteria. Occasionally one comes across a dairy which seems to be impregnated with bacteria that improve cream and flavour well. In other cases the contrary happens, and a dairy becomes impregnated with a species having deleterious effects upon its butter. This species may arise from unclean utensils and dairying, from disease of the cow, or from a change in the cow's diet. Thus it comes about that the butter-maker is not always able to depend upon good ripening for his cream. At other times he gets ripening to occur, but the flavour is an evil one, and the results correspond. It may be bitter or tainted, and just as certainly as these flavours develop in the cream, so is it certain that the butter will suffer. Fortunately the bacterial content of the cream is generally either favourable or indifferent in its action. Thus it comes about that the custom is to allow the cream simply to ripen, so to speak, of its own accord, in a vat exposed to the influence of any bacteria which may happen to be around. This generally proves satisfactory, but it has the great disadvantage of being indefinite and uncertain. Occasionally it turns out wholly unsatisfactory, and results in financial loss.

There are various means at our command for improving the ripening process. Perfect cleanliness in the entire manipulation necessary in milking and dairying, combined with freedom from disease in the milch cows, will carry us a long way on the road towards a good cream-ripening. Recently, however, a new method has been introduced, largely through the work and influence of Professor Storch in Denmark, which is based upon our new knowledge respecting bacterial action in cream-ripening. We refer to the artificial processes of ripening set up by the addition of pure cultures of favourable germs.[66] If a culture of organisms possessing the faculty of producing in cream a good flavour be added to the sweet cream, it is clear that advantage will accrue. This simple plan of starting any special or desired flavour by introducing the specific micro-organism of that flavour may be adopted in two or three different ways. If cream be inoculated with a large, pure culture of some particular kind of bacteria, this species will frequently grow so well and so rapidly that it will check the growth of the other bacteria which were present in the cream at the commencement and before the starter was added. That is, perhaps, the simplest method of adding an artificial culture. But secondly, it will be apparent to those who have followed us thus far, that if the cream is previously pasteurised at 70° C. these competing bacteria will have been mostly or entirely destroyed, and the pure culture, or starter, will have the field to itself. There is a third modification, which is sometimes termed ripening by natural starters. A natural starter is a certain small quantity of cream taken from a favourable ripening—from a clean dairy or a good herd—and placed aside to sour for two days until it is heavily impregnated with the specific organism which was present in the whole favourable stock of which the natural starter is but a part. It is then added to the new cream the favourable ripening of which is desired. Of the species which produce good flavours in butter the majority are found to be members of the acid-producing class; but probably the flavour is not dependent upon the acid. Moreover, the aroma of good ripening is also probably independent of the acid production.

Of all the methods of ripening—natural ripening, the addition of natural starters, the addition of pure cultures with or without pasteurisation—there can be no doubt that pure culture after pasteurisation is the most accurate and dependable. The use of natural starters is a method in the right direction; yet it is, after all, a mixed culture, and therefore not uniform in action. In order to obtain the best results with the addition of pure cultures, Professor Russell has made the following recommendations:

1. The dry powder of the pure culture must be added to a small amount of milk that has been first pasteurised, in order to develop an active growth from the dried material.

2. The cream to be ripened must first be pasteurised, in order to destroy the developing organisms already in it, and thus be prepared for the addition of the pure culture.

3. The addition of the developing starter to the pasteurised cream and the holding of the cream at such a temperature as will readily induce the best development of flavour.

4. The propagation of the starter from day to day. A fresh lot of pasteurised milk should be inoculated daily with some of the pure culture of the previous day, not the ripening cream containing the culture. In this way the purity of the starter is maintained for a considerable length of time. Those starters are best which grow rapidly at a comparatively low temperature (60–75° F.), which produce a good flavour, and which increase the keeping qualities of the butter. Now, whilst it is true that the practice of using pure cultures in this way is becoming more general, very few species have been isolated which fulfil all the desirable qualities above mentioned. In America starters are preferred which yield a "high" flavour, whereas in Danish butter a mild aroma is commoner. In England as yet very little has been done, and that on an experimental scale rather than a commercial one.[67] In 1891 it appears that only 4 per cent. of the butter exhibited at the Danish butter exhibitions was made from pasteurised cream plus a culture starter; but in 1895, 86 per cent. of the butter was so made. Moreover, such butter obtained the prizes awarded for first-class butter with preferable flavour. Different cultures will, of course, yield different flavoured butter. If we desire, say, a Danish butter, then some species like "Hansen's Danish Starter" would be added; if we desire an American butter, we should use a species like that known as "Conn's Bacillus, No. 41." But whilst these are two common types, they are not the only suitable and effective starters. On certain farms in England there are equally good cultures, which, placed under favourable temperatures in new cream, would immediately commence active ripening.

Professor H. W. Conn, who, with Professor Russell, has done so much in America for the advancement of dairy bacteriology, reports[68] a year's experience with the bacillus to which reference has been made, and which is termed No. 41. It was originally obtained from a specimen of milk from Uruguay, South America, which was exhibited at the World's Fair in Chicago, and proved the most successful flavouring and ripening agent among a number of cultures that were tried. The conclusions arrived at after a considerable period of testing and experimentation appear to be on the whole satisfactory. A frequent method of testing has been to divide a certain quantity of cream into two parts, one part inoculated with the culture and the other part left uninoculated. Both have then been ripened under similar conditions, and churned in the same way; the differences have then been noted. It is interesting to know that, as a result of the year's experience, creameries have been able to command a price varying from half a cent to two cents a pound more for the "culture" butters than for the uninoculated butters. The method advised in using this pure culture is to pasteurise (by heating at 155° F.) six quarts of cream, and after cooling to dissolve in this cream the pellet containing bacillus No. 41. The cream is then set in a warm place (70° F.), and the bacillus is allowed to grow for two days, and is then inoculated into twenty-five gallons of ordinary cream. This is allowed to ripen as usual, and is then used as an infecting culture, or "starter," in the large cream vats in the proportion of one gallon of infecting culture to twenty-five gallons of cream, and the whole is ripened at a temperature of about 68° F. for one day. The cream ripened by this organism needs to be churned at a little lower temperature (say 52°-54° F.) but to be ripened at a little higher temperature than ordinary cream to produce the best results. Cream ripened with No. 41 has its keeping power much increased, and the body or grain of the butter is not affected. More than two hundred creameries in America used this culture during 1895, and Professor Conn reports that this has proved that its use for the production of flavour in butter is feasible in ordinary creameries and in the hands of ordinary butter-makers provided they will use proper methods and proper discretion.

Bacteria in Cheese-making. The cases where it has been possible to trace bacterial disease to the consumption of butter and cheese have been rare. Notwithstanding this fact, it must not be supposed that therefore cheese contains few or no bacteria. On the contrary, for the making of cheese bacteria are not only favourable, but actually essential, for in its manufacture the casein of the milk has to be separated from the other products by the use of rennet, and is then collected in large masses and pressed, forming the fresh cheese. In the course of time this undergoes ripening, which develops the peculiar flavours characteristic of cheese, and upon which its whole value depends.

We have said that the casein is separated by the addition of rennet, which has the power of coagulating the casein. But this precipitation may also be accomplished by allowing acid to develop in the milk until the casein is precipitated, as in some sour-milk or cottage cheeses. The former method is of course the usual one in practice. It has been suggested that the bacteria contained in the rennet exert a considerable influence on the cheese, but this, although rennet contains bacteria, is hardly established. It is not here, however, that bacteria really play their rôle. After this physical separation, when the cheese is pressed and set aside, is the period for the commencement of the ripening process.

That bacteria perform the major part of this ripening process, and are essential to it, is proved by the fact that when they are either removed or opposed the curing changes immediately cease. If the milk be first sterilised, or if antiseptics, like thymol, be added, the results are negative. It is not yet known whether this peptonising process is due to the influence of a single organism or not. The probability, however, is that it is to be ascribed to the action of that group of bacteria known as the lactic-acid organisms. Nor is it yet known whether the peptonisation of the casein and the production of the flavour are the results of one or more species. Freudenreich believes them to be due to two different forms.

However that may be, we meet with at least four common groups of bacteria more or less constantly present in cheese-ripening, either in the early or late stages. First, there are the lactic-acid bacteria, by far the largest group, and the one common feature of which is the production by fermentation of lactic acid; secondly, there are the casein-digesting bacteria, present in relatively small numbers; thirdly, the gas-producing bacteria, which give to cheese its honeycombed appearance; lastly, an indifferent or miscellaneous group of extraneous bacteria, which were in the milk at the outset of cheese-making, or are intruders from the air or rennet. All these four groups may bring about a variety of changes, beneficial and otherwise, in the cheese-making.

In order that the relation of bacteria to cheese may be more fully understood, we may draw attention to some experiments conducted by Professor H. L. Russell as to the numbers of bacteria present during different stages of the ripening, excluding those already referred to as present in the rennet. It appears that there is always at first a marked increase in the number of micro-organisms, which is soon followed by a more gradual decline. While the casein-digesting and gas-producing classes suffer a general and more or less rapid decline, the lactic-acid bacteria develop to an enormous extent, from which fact it would appear that cheese offers ideal conditions for the development of the latter. In some most interesting records Professor Russell has divided the ripening process into three divisions:

1. Period of Initial Bacterial Decline in Cheese. Where the green cheeses were examined immediately after removing from the press, it was usually found that a diminution in numbers of bacteria had taken place. This period of decline lasts but a short time, not beyond the second day. Lower temperature and expulsion of the whey would account for this general decline in all species of bacteria.

2. Period of Bacterial Increase. Soon after the cheese is removed from the press a most noteworthy change takes place in green cheese. A very rapid increase of bacteria occurs, confined almost exclusively to the lactic-acid group. This commences in green cheese about the eighth day, and continues more or less for twenty days. In Cheddar cheese it commences about the fifth day, reaches its maximum about the twentieth day, declines rapidly to the thirtieth day, and gradually for a hundred following days. During the first forty days of this period the casein-digesting and gas-producing organisms are present, and at first increasing, but relatively to only a very slight degree. With this rapid increase in organisms the curd begins to lose its elastic texture, and before the maximum number of bacteria is reached the curing is far advanced. Freudenreich has shown that acid inhibits the growth of the casein-digesting microbes and vice versâ.

3. Period of Final Bacterial Decline. The cause of this decline can only be conjectured, but it is highly probable that it is due to a general principle to which reference has frequently been made, viz., that after a certain time the further growth of any species of bacteria is prevented by its own products. We may observe that the gas-producing bacteria in Cheddar cheese last much longer than the peptonising organisms, for they are still present up to eighty days. Professor Russell aptly compares the bacterial vegetation of cheese with its analogue in a freshly seeded field. "At first multitudes of weeds appear with the grass. These are the casein-digesting organisms, while the grass is comparable to the more native lactic-acid flora. In course of time, however, grass, which is the natural covering of soil, 'drives out' the weeds, and in cheese a similar condition occurs." In milk the lactic-acid bacteria and peptonising organisms grow together; in ripening cheese the former eliminate the latter.

We have seen that the conclusion generally held respecting these lactic-acid bacteria is that they are the main agents in curing the cheese. Upon this basis a system of pure starters has been adopted, the characteristics of which must be as follows: (a) The organism shall be a pure lactic-acid-producing germ, incapable of producing gaseous products; (b) it should be free from any undesirable aroma; (c) it should be especially adapted for vigorous development in milk. The starter may be propagated in pasteurised or sterilised milk from a pure culture from the laboratory. The advantages accruing from the uses of this lactic-acid culture, as compared with cheese made without a culture, are that with sweet milk it saves time in the process of manufacture; that with tainted milk, in which acid develops imperfectly, it is an aid to the development of a proper amount of acid for a typical Cheddar cheese; and that the flavour and quality of such cheese is preferable to cheese which has not been thus produced. Professor Russell is of opinion that the lactic-acid organisms are to be credited with greater ripening powers than the casein-digesting organisms, but it must not be forgotten that these two great families of bacteria are still more or less on trial, and it is not yet possible finally to dispose of either of them. Mr. F. J. Lloyd holds that though "the greater the number of lactic-acid bacilli in the milk the greater the chance of a good curd," still "this organism alone will not produce that nutty flavour which is so sought after as being the essential characteristic of an excellent Cheddar cheese."[69]

There are several difficulties to be encountered by dairymen starting a ripening by the addition of a pure culture. To begin with, there is the initial difficulty of not being able to pasteurise milk intended for cheese, as rennet will not coagulate pasteurised milk (Lloyd). Hence it is impossible to avoid some contamination of the milk previous to the addition of the culture. The continual uncontaminated supply of pure culture is by no means an easy matter. The maintenance of a low temperature to prevent the rapid multiplication of extraneous bacteria will, in some localities, be a serious difficulty. These difficulties have, however, not proved insurmountable, and by various workers in various localities and countries culture-ripening is being carried on.

Abnormal Ripening. Unfortunately, from one cause or another, faulty fermentations and changes are not infrequently set up. Many of these may be prevented, being due to lack of cleanliness in the process or in the milking; others are due to the gas-producing bacteria being present in abnormally large numbers. When this occurs we obtain what is known as "gassy" cheese, on account of its substance being split up by innumerable cavities and holes containing carbonic acid gas, or sometimes ammonia or free nitrogen. Some twenty-five species of micro-organisms have been shown by Adamety to cause this abnormal swelling. In severe cases of this gaseous fermentation the product is rendered worthless, and even when less marked the flavour and value are much impaired. Winter cheese contains more of this species of bacteria than summer. Acid and salt are both used to inhibit the action of these gas-producing bacteria and yeasts, and with excellent results.

We may remark that the character of the gas holes in cheese is not of import in the differentiation of species. If a few gas bacteria are present, the holes will be large and less frequent; if many, the holes will be small, but numerous. (Swiss cheese having this characteristic is known as Nissler cheese.)

Many of these gas germs belong to the lactic-acid group, and are susceptible to heat. A temperature of 140° F. maintained for fifteen minutes is fatal to most of them, largely because they do not form spores. The sources of the extensive list of bacteria found in cheese are of course varied, more varied indeed than is the case with milk. For there are, in addition to the organisms contained in the milk brought to the cheese factory, the following prolific sources, viz., the vats and additional apparatus; the rennet (which itself contains a great number); the water that is used in the manufacture.

In addition to the abnormalities due to gas, there are also other faulty types. The following chromogenic conditions occur: red cheese, due to a micrococcus; blue cheese, produced, according to Vries, by a bacillus; and black cheese, caused by a copious growth of low fungi. Bitter cheese is the result of the Micrococcus casei amari of Freudenreich, a closely allied form of Conn's micrococcus of bitter milk. Sometimes cheese undergoes a putrefactive decomposition, and becomes more or less putrid. These latter conditions, like the gassy cheeses, are due to the intrusion of bacteria from without, or from udder disease of the cow. Healthy cows, clean milking, and the introduction of pure cultures are the methods to be adopted for avoiding "diseases" of cheese and obtaining a well-flavoured article which will keep.

Finally, we may quote five conclusions from the prolonged researches of Mr. Lloyd[70] which cannot but prove helpful to the Cheddar cheese industry in England:

1. To make Cheddar cheese of excellent quality, the Bacillus acidi lactici alone is necessary; other germs will tend to make the work more rather than less difficult. Hence scrupulous cleanliness should be a primary consideration of the cheese-maker.

2. No matter what system of manufacture be adopted, two things are necessary. One is that the whey be separated from the curd, so that when the curd is ground it shall contain not less than 40 per cent. of water, and not more than 43 per cent.; the other point is that the whey left in the curd shall contain, developed in it before the curd is put in the press, at least 1 per cent. of lactic acid if the cheese is required for sale within four months, and not less than 8 per cent. of lactic acid if the cheese is to be kept ripening for a longer period.

3. The quality of the cheeses will vary with the quality of the milk from which they have been made, and proportionately to the amount of fat present in that milk.

4. "Spongy curd" is produced by at least five organisms, and one of these is responsible for a disagreeable taint found in curd. They occur in water. Hence the desirability of securing clean water for all manipulative purposes, and also for the drinking purposes of the milch cow.

5. The fact that certain bacteria are found in certain localities and dairies is due more to local conditions than to climatic causes.

It is needless to remark that these conclusions once more emphasise the fact that strict and continual cleanliness is the one desideratum for bacteriologically good dairying. That being secured in the cow at the milking, in the transit, and at the dairy, it is a comparatively simple step, by means of pasteurisation and the use of good pure cultures of flavouring bacteria, to the successful application of bacteriology to dairy produce.

Methods of Examination of Milk:

1. Preparation of Microscopic Slides. This course might at once occur to the mind as the first to adopt in searching for bacteria in milk. Devices have accordingly been proposed for saponification previous to staining. Some recommend the addition of a few drops of a solution of sodium carbonate; others use methylene blue and chloroform. But, whatever plan of staining is adopted, this method of examination in its simplest form is in no degree a criterion of the bacterial content of a large quantity of milk.

Hence it has come to be recognised that one of two manipulations must precede such microscopic examination. These simple processes are known by the terms of sedimentation and centrifugalisation. Sedimentation means merely

A Centrifuge
Used in the Examination of Milk placing the milk in conical glasses in a cool place for twenty-four hours. The introduction of improved forms of the centrifuge has brought the second method of securing a sediment into preference. Five cubic centimetres of the milk are introduced into the graduated bottle, which is then placed in the centrifuge, and whirled for one or two minutes. Thus a deposit of particulate matter is ensured. Cover-glass specimens of the sediment or deposit are then prepared and stained in the ordinary way.

In testing for tubercle something more is generally necessary. To the 50 cc. of the milk set aside for sedimentation 10 cc. of liquefied, colourless carbolic acid are added. The mixture is shaken and poured into the conical glass. After standing for twenty-four hours a little of the sediment is taken by means of a pipette and examined by ordinary methods, though after "fixing" the films with heat they are some times passed through equal parts of alcohol and ether. The stain is of course that usually adopted in tubercle, namely, the Ziehl-Neelsen. Scheurlen suggested a method for demonstrating the tubercle bacillus in milk by steeping the cover glasses first in alcohol and then ether, after which they were stained with Ziehl-Neelsen.

2. Plate Culture. The milk is to be diluted a thousand or more times with sterile water, and ordinary plate cultures made in Petri dishes or flat-bottomed conical flasks. The colonies should be counted as late as possible; but even then the isolation of pathogenic germs is uncertain. As regards further procedure, the ordinary methods of sub-culturing adopted in water examination must be strictly followed, and the special tests for Bacillus typhosus and B. coli applied. As we have already seen, the quantitative estimation of organisms in milk is not of the same value as in water.

3. Inoculation. To test the capacity of the milk for causing disease, before or after centrifugalisation, preferably the latter, a certain quantity of the sediment may be inoculated into guinea-pigs. In suspected tubercle 2 cc. may be taken; in diphtheria a little less will suffice. The inoculation should be either intraperitoneal or subcutaneous. Many authorities hold that this test is the only safe one to protect the public from milk containing germs of disease.