from the source of heat and cooled to about 50° C., when a small amount of fresh albumen 1 is added; the latter is first beaten up with a little water, this being most readily done by shaking up violently in an ordinary medicine bottle. The white from one egg is sufficient for two litres of liquid. The gelatine solution is thereupon mixed well with the white of egg solution, the whole then put on the sand bath and cautiously heated to boiling without stirring. This coagulates the white of egg in a few minutes, and it separates out in large flocks which aggregate all the suspended matter and impurities present in the gelatine. The whole is now weighed to ascertain if the weight has remained the same; if the weight is too small, sterile water is added; otherwise the liquid must be carefully evaporated at a gentle heat until the proper weight is arrived at. The gelatine solution is strained through a flannel while it is still warm, the flannel being stretched on a wooden frame. In many cases a perfectly clear gelatine is not required, and it is then unnecessary to filter it. If on the contrary perfectly clear gelatine is required, it is filtered boiling hot through a paper filter provided with a toughened point. This filter is placed in a glass funnel fitted in a copper funnel (see Fig. 44). The latter is double walled and provided with a closed side tube. There is an opening on the upper edge of the funnel which allows of the space between the walls being filled with warm water. Under the side tube a gas flame is placed which keeps the water boiling during filtration; the gelatine is thus prevented from setting.

Smaller quantities of nutrient gelatine may be prepared with least trouble on the water bath.

1 According to Rich. Meissner the use of dry albumen is not to be ecommended, as it has been shown that when it is used for clearing the gelatine, organisms sown on the latter are hindered in their development. This probably arises from the formation of secondary products (ptomaines?) during the drying.

Pasteur flasks are best for preserving nutrient gelatine as they do not allow any drying up. The strained or filtered gelatine while still hot is poured into a flask, previously sterilised, and boiled for five minutes on the sand bath. A smaller amount may be stored in different small flasks according to the purpose for which it is intended. As regards preservation of nutrient gelatine to be used for ordinary plate culture, it is advisable to use the globular flask represented in Fig. 36, which is charged with about

15 c.c. of gelatine. The gelatine is here also boiled for five minutes on the sand bath, after the flask has been plugged with cotton wool and covered with a double layer of filter paper. In case the flask is to remain for a long time, it is of advantage to use a rubber cap in place of the filter paper These flasks are to be used in preference to test tubes or Freudenreich flasks, because the liquefied gelatine seeded with a culture can be better shaken up in the former than in the latter, it being above all desirable when making

ordinary plate cultures to distribute the germs as much as possible. If the gelatine is to be applied for surface plate cultures, test tubes or Freudenreich flasks may be used in which the culture gelatine is only liquefied without being mixed with the germs; but globular flasks are also preferable in this case.

In Freudenreich flasks, gelatine is sterilised for a quarter of an hour in steam. After the sterilisation is finished, the flasks are placed in an oblique position until the gelatine has set. When these flasks are to contain their gelatine for a long time, the cap tube ought to be closed with wax or with the S-shaped tube mentioned previously (Fig. 33), to prevent the gelatine drying up. Gelatine ought in

all cases to stand for some time before use, partly to see if it is sterile, and partly because it can then resist higher temperatures better without melting. With regard to the latter property, different kinds of gelatine behave differently.

Hueppe recommends the discontinuous method of sterilising gelatine, this consisting of subjecting it daily, for four to five days, to a boiling heat for five minutes.

Wort gelatine, yeast water gelatine, fruit syrup gelatine, etc., are prepared with a content of 7 to 10 per cent. of gelatine, that is, as much as will enable them to stand 25° C. without melting. Meat extract peptone gelatine is, on the other hand, always prepared with at least 10 per cent. of gelatine. Ten grams of gelatine are dissolved in 100 grams of meat extract in the usual way; sometimes it will be necessary after adding the gelatine to neutralise with sodium carbonate, for the gelatine, as is known, gives an acid reaction, and many bacteria do not thrive even on a feebly acid medium. Meat extract peptone gelatine is always sterilised by the discontinuous process.

Nutrient

agar-agar is prepared in a similar way to the gelatine; but it must be cut into very small pieces before

being put into the boiling liquid, and has to be boiled for some time before it dissolves. The proportions in this case are 100 grams of liquid to 2 grams of agar-agar Filtering is, as a rule, avoided, as it is only accomplished with difficulty, agar-agar requiring a higher temperature to keep it liquid. Hence, white of egg is not used for clearing. If it is desired to remove coarser suspended matter, the solution can be strained through linen.

If, however, the agar-agar has to be filtered, Giesenhagen recommends the following method. To accelerate the agar filtration he employs filtration in steam, and distributes the substance among several filters working simultaneously. Small tin funnels with turned down edges are used for filtering, and these are provided with flat enamelled covers with projecting edges. Three funnels are placed in rings round the stem of a special stand arranged over Erlenmeyer flasks of appropriate size (each of the three filter stands is 165 cm. high and 8 cm. wide). The wadding plug for each flask is fixed in the meshes of the wire stand. Two, or in high steam chambers even three, such sets of filters (ie., 6 to 9 filters) can be arranged for steaming. The filtering is done through two folded filters. Nutrient agaragar is employed for cultures at high temperatures, gelatine being unsuitable as it becomes liquid.

Mixtures of agar-agar with gelatine are prepared in the proportions of 100 grams of culture medium, 1 or 2 grams agar-agar, and 4 or 3 grams of gelatine. The addition of gelatine prevents the separation of water which always takes place when pure agar-agar is used.

Litmus gelatine is sometimes used as a reagent for detecting the formation of acid during the growth of a micro-organism. It is prepared according to Hueppe in the following way: An aqueous solution of the colouring matter is made up, sterilised and allowed to cool. The

gelatine is liquefied at 30° C. (the agar-agar at 40°), and is then mixed with the equally hot litmus solution. The latter should only be so strong that its action as a reagent is just distinguishable.

Other Solid Media.-Bread might be mentioned as one of those solid media which are sometimes employed. It is made into a paste with a little water and sterilised by steam; rice may be sterilised in the same way. Manure (with or without addition of water) is sterilised several times with an interval of one or two days. These substrata are employed, e.g., in cultivating moulds. Potatoes are frequently used for the culture of bacteria. The potatoes are cleaned well with a brush and laid for some minutes in a 10 per cent. solution of sublimate and afterwards in a 01 per cent. sublimate solution for half an hour to fifteen hours. Finally they are well washed with water and then sterilised for two hours in steam and cut up.

1.-Microscopical Investigation of Micro-Organisms. Preparation Making.-A preparation of a micro-organism is made as a rule by putting it in a drop of liquid or in Canada balsam, etc., on a glass slip and laying a cover glass on it. Water is often used (best when distilled and sterile so as to exclude outside organisms from the preparation); a little of the growth to be investigated is taken out with a platinum wire or similar instrument and stirred in the water. If it is in a culture liquid, a sample can be taken out with a small glass rod and placed direct on the glass slip without addition of water. The needles and rods used for taking out samples must of course be sterilised beforehand, when the cultures are to be preserved pure; all the usual precautions must be observed. When the cover glass is laid on the drop the enclosing of air bubbles in the liquid is to