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once and is connected with the Miquel flask, B. The latter is shaped somewhat like a Chamberland flask and is provided with two side tubes, c and d, but the neck is fitted with a glass tube, b, which reaches almost to the bottom.

During sterilisation, B carries a glass cap with a little tube filled with cotton wool (as in the Chamberland and Freudenreich flasks); the one side tube is likewise closed at c by a wool plug. At e a very loose wool plug is fitted which can be easily blown into the water in B at the end of the experiment. At d there is a rubber tube which is closed. by means of a glass tube drawn out to a fine point and sealed. The flask further contains a measured quantity of water in such amount as to immerse the opening of the tube, b. In this condition the flask, previously sterilised in the dry state, is again sterilised. The whole apparatus is then set up at the place where it is desired to examine the air, the tube, c, being connected with A by a rubber tube.

The experiment now consists in sucking the air to be examined through the water in B, so that the germs are retained by the latter; this is done by opening the pinchcock, a, so that the water in A runs out. The air must not be drawn too quickly through the water; the water in A is therefore allowed to pass out at a drop by drop. As the experiment proceeds the pinchcock at a may be opened more and more as the flow of water gradually lessens. The wool plug, e, retains those germs which may be carried off by the air without being taken up by the water in B. It is obvious that the volume of air sucked through the water in B is the same as the volume of water which runs out at a. The quantity of water in A must therefore be known. This bottle, however, cannot be completely emptied through a while in an upright position, and this should be kept in mind when the quantity is determined. For this reason it

is advisable to first pour as much water into A as will fill

it over the inner opening of the tube a, and then to mark the level of the liquid in the flask; a volume of water equal to that of the air to be analysed is then added.

As soon as the desired quantity of air is passed through the water in B, the pinchcock, a, is closed and the cap placed on B; the tube, c, is then disconnected from the rubber. There are still some germs in the tube, b, which have not reached the water; the tube, c, is therefore blown through until the water rises in b. This is repeated several times until b has been washed out. Finally the wool plug, e, is blown or pushed into the water. B is now well shaken up, partly in order to distribute in the water the germs which are on e, and partly to effect a thorough and uniform distribution of the germs throughout the water. The procedure is then exactly as in water analysis, the water being dropped through d into the flasks containing culture liquid. This is done by cautiously breaking off the point of the thin glass tube which is fitted into the rubber tubing. The dropping can be regulated by holding the finger over the opening of the tube, c.

If there is no more water in B than is used for the inoculating, it is not necessary to know the amount of this water. If, however, only a part of this is used, which will probably be the most frequent occurrence, it is necessary to know how much of the total amount of water has been used. Suppose for example that 6 litres of air have been passed through the water in B, and that B was charged with 10 c.c. of water, and assuming that one drop 0.05 c.c., it follows that if 100 flasks are inoculated with one drop each, 5 c.c. of water in all, or, in other words, the half of the water has been used, and therefore the number of germs in half the quantity of air, i.e., in 3 litres, is determined. Sometimes it happens that the air is so rich in germs that it is necessary, as in water analysis, to dilute the water.

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If it is required to quickly obtain some idea as to the purity of the air with regard to germs, Petri dishes with wort gelatine may be left open for fifteen minutes. The covers are then replaced and the plates put into a thermostat at 25° C. The germs then develop.

Hansen's Results. In his researches on the microorganisms of the air, Hansen had partly in view theoretical considerations, such as the circulation of species in nature, especially that of the saccharomycetes, and he partly followed out purely practical problems concerned with brewing. As regards the solution of the latter the air was examined in different parts of the brewery (fermenting cellar, cooling vessels, etc.). In connection with this, experiments were carried out to discover whether the vapours from the grains carried infection by means of the numerous bacteria they contain. Hansen arrived at the result that this does not take place; on the other hand, dried grains become dangerous in a high degree as soon as they are carried by the wind as dust in the air. Therefore it is not advisable to employ any apparatus for drying the grains in the neighbourhood of the brewery; for, according to Hansen, the bacteria are not killed by this drying; if, therefore, drying apparatus is set up in such a position that the dry particles with numerous bacteria can find their way on to the cooling vessels and into the fermenting cellars, much harm may be caused by this means.

Hansen further found that the purest air in the Old Carlsberg Brewery was in the fermenting cellars, this being due to the fact that the cellars are provided with cold air which has been previously purified. In analyses of the air in the fermenting cellars of other breweries which were without purified air bacteria were observed, among which were Sarcina and various species of wild yeasts, disease forms also being found. The cooling vessels are exposed

to infection from the air, especially at the time when sweet, juicy fruits are ripe, when the dust from the ground is very rich not only in yeasts but also in bacteria.

Analyses of air should be performed in breweries from time to time; a clear idea is thus obtained of the progress of the different processes, and will in many cases avert mishap. It is the air of fermenting cellars and coolers in particular to which special attention must be paid.

Soil Analyses. In soil analyses a small sample is placed in an Erlenmeyer flask containing a culture liquid chosen with regard to the organisms to be sought. The flora of the soil is a very rich one, especially as regards bacteria and mould fungi. If saccharomycetes are to be looked for, it is advisable to seed the soil sample in wort to which tartaric acid has been added, since this prevents to a great extent the development of most bacteria. In this case also it is desirable to allow the cultures to remain for a considerable time (about fourteen days) at 25° C., and then to prepare a second culture in wort, as saccharomycetes are generally tardy in their development.

It has also been proposed to mix a small quantity of the sample of soil with liquefied nutrient gelatine and then to prepare plate cultures. However, the result is in most cases bad, the number of germs in the soil being too large. Others prefer making a paste with the soil in sterile. water; plate cultures are then prepared from an average sample.

These analyses, in common with all the foregoing, have the object of discovering the source of the infection which may take place in a brewery. The groundwork for this was furnished by Pasteur's and Hansen's investigations.

11.-Hansen's Pure Culture System in Fermentation Industries.

The Pure Culture System in Bottom Fermentation Breweries. The following information on the introduction of the systematically selected yeast race into the bottom fermentation brewery is extracted from Hansen's Practical Studies in Fermentation. The starting point must be the yeast which has proved its superiority in working in practice, and has yielded that product which it is wished should be the future normal production of the brewery. Since the species which lends its character in great measure to the product will be present in superior numbers, it will also be the most easily isolated.

If there is wild yeast present in the stock yeast employed, it will, according to Hansen, be present only in small amount in the surface beer at the beginning of the primary fermentation, or it will be totally absent, while at the finish the reverse is the case. A sample of the surface beer is therefore taken just at the time when a frothy head has formed in the fermenting vessel; we are then certain that the race or species to be isolated is in preponderance, and this sample is used for the preparation of absolutely pure cultures, the starting point being made as usual from a single cell (see page 106). Preliminary fermentations are now made with these pure cultures in flasks in the laboratory. It is advisable to use the same wort as that employed in the brewery, and this should not be re-sterilised in the laboratory. The method of procuring this is described on page 76. While fermentation proceeds in the flasks, certain preliminary observations are made which will be of use later on. It is observed to what extent the wort remains clear, whether the yeast lies compactly on the bottom, and whether the beer has any peculiar smell and taste, etc. A microscopic examination and also a spore

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