cause materials are only fermented after they are taken into the bacterial cell. Fermentation products are metabolic products produced under the influence of special nutrition. (Compare p. 64.)

I. The Bacterial Ferments and the Changes Produced by Them.

Under ferments in the restricted sense-enzymes(the custom of calling micro-organisms "living ferments is passing into disuse) one understands certain chemical bodies, which, in a minimal quantity and without being thereby destroyed, are able to split up large quantities of definite elaborate organic molecules into smaller, simpler, more soluble, and more diffusible ones.1

We can only properly speak of chemical ferments after the following properties have been demonstrated:

1. Fermentation continues in the presence of materials which are surely bactericidal, but do not injure ferments; for example, phenol, 3%; thymol, 0.1%; chloroform, ether; or

2. The power of producing fermentation is possessed by the germ-free filtrate, obtained by passing cultures of the bacteria through clay or porcelain cylinders; or

3. This activity is possessed by the pulverized and sterile ferment-preparation obtained from cultures.

Of the extraordinarily numerous details which have been taught by Fermi's 2 methodical and exhaustive studies, only the most important can here be given. All ferments dialyze a little, like ordinary albuminous bodies, through good parchment paper.

Proteolytic or albumin-dissolving enzymes are widely distributed. The liquefaction of the glue in gelatin (closely related to albumin chemically) is a sure indication of the presence of a proteolytic ferment. Since the reaction of the gelatin when dissolved is always or may be alkaline, the liquefaction is not due to pepsin (which is active

1 This definition evidently does not apply to rennet ferment, which coagulates milk.

2 A. H. X, 1; XII, 240; C. B. XII, 713; C. B. L. 1, 482.

only with an acid reaction), but to a trypsin. The individual bacterial trypsins differ very much as regards resistance to heat (they withstand 55° to 70° moist heat for one hour), susceptibility to injury by various acids, etc. Some are active with a suitable acid reaction, but never more so than with an alkaline reaction.

Much more feeble than the action upon glue, is that upon fibrin. 1 Fermi has employed the following method as the easiest and surest way of proving the presence of even traces of proteolytic ferments: Tubes of the same size are filled to an equal height with an unneutralized 7% solution of gelatin in 1% aqueous solution of carbolic acid. The solution to be tested for proteolytic ferment has 2% carbolic acid added to it, and is placed in layers upon the solidified gelatin. The tubes are kept at room temperature and observations are made by means of a millimeter scale, as to how much the liquefaction of the gelatin extends in the course of days and weeks. For qualitative examination, the upper layer may consist of 1 c.c. of a liquefied gelatin culture, sterilized with carbolic acid. This material also suffices if one wishes to test the influence of the nutrient medium on ferment formation. One may also, by this method, compare the action of various concentrations of different purely prepared bacterio-trypsins. The lower the percentage of gelatin and the nearer the temperature approaches that of the incubator, the more certainly will one observe effects from traces of ferment. In such critical cases the examination is continued fourteen days, and it is determined whether the gelatin remains fluid in the ice-box, while that in the control tube solidifies. To demonstrate the formation of true peptone from the albuminous bodies one proceeds as follows:

The variety of bacterium to be tested is grown upon a fluid nutrient medium, rich in albumin, but containing no peptone (blood-serum, milk-serum, milk). After the culture is grown, all albuminous bodies except peptone are precipitated by the addition of strong ammonium sulphate (about 30 gm. to 20 c.c.). Milk and milk-serum may be warmed to 60° to 80° and blood-serum to about 40°. The precipitate is filtered off, and the filtrate cooled. A sample is made strongly alkaline with potassium hydroxid, and 1% solution of copper sulphate added drop by drop. A rose-red color indicates the presence of peptone.3 Fermi has shown, by similar methods, that no variety of bacterium produces true peptone.

1 Fermi found only a few bacterio-trypsins acting upon fibrin, and none upon egg-albumin.

2 Naturally, a control test with 2% carbolic-water (ferment-free) must never be omitted.

Through more recent investigations it is certainly known that some albumoses besides peptone remain partly unprecipitated by ammonium sulphate.

The production of proteolytic ferments fluctuates with many, perhaps with all, species in a greater degree than one would be led to suppose from the ordinary descriptions. Beijerinck found that one of two photogenic vibriones at first liquefied slowly, but that after longer culture gelatin was always liquefied more rapidly; the other showed exactly the opposite. The same was observed by Katz in the Australian photogenic bacterium. Max Gruber and Firtsch (A. H. VIII, 369) have studied particularly closely liquefying cultures of Vibrio proteus, but they have also reported similar experiences with the cholera vibrio, Bact. vulgare, the Micrococcus pyogenes; indeed, many observers have even seen liquefying Streptococci pyogenes.

We have observed, also, in many varieties, that upon thin plates single, distinctly visible, superficial colonies of the same bacterium present such varying degrees of liquefaction that a beginner could scarcely be convinced that several varieties were not present.

It is very unfortunate that, through these observations, one of the readiest applied diagnostic aids, the liquefaction of gelatin, has lost not a little in value.

The causes of the decrease and increase of liquefaction under prolonged cultivation we ascribe to our artificial nutrient media or to the influence of the metabolic products of the micro-organisms, but without being able to give anything more decisive.

Regarding the influence of nutrient media upon the formation of trypsin in a culture and the liquefaction of gelatin, the following facts are known:

1. Most circumstances which interfere with the growth of a variety of bacterium on a nutrient medium also interfere with the liquefaction of gelatin; for example, the addition of phenol and a large amount of glycerin. Wood has observed that the lessened power of liquefying gelatin produced by phenol may be propagated for several generations upon favorable nutrient media (C. B. VIII, 266).

2. In hydrogen and nitrogen the liquefying facultative anaerobes do not liquefy gelatin, while in CO2, if they

1 A single exception is the B. prodigiosum; but if grape-sugar be added to the gelatin, it also ceases to liquefy.

can thrive in it, the contrary is true. 1 (Compare Table I.) Since the gases, according to Fermi, are without influence on the activity of the ferment, they must influence the formation of ferment. On the contrary, obligate anaerobes preponderately present most beautiful liquefaction of gelatin.

3. The addition of sugar does not interfere with the growth, but does with the liquefaction of gelatin in the case of many bacteria; for example, Bact. vulgare (Proteus vulgaris) (Kuhn, A. H. xIII, 40).

Auerbach has shown in my Institute (A. H. XXXI, 311) that sugar influences, in varying degree, the liquefaction of gelatin by various bacteria. In the instances examined, this checking was dependent upon the fact that no proteolytic ferment was formed in media containing sugar, and not that the sugar or acids formed from it interfered with the action of the ferment.

4. In fluid non-albuminous nutrient media, containing glycerin but no sugar, only a few bacteria produce proteolytic ferment; for example, B. prodigiosum and B. pyocyaneum. Also in peptone bouillon the ferment formation appears less than in peptone bouillon gelatin (Fermi). Upon albuminous nutrient media the liquefying bacteria produce bitter-tasting metabolic products; for example, from milk in the case of many varieties (Hüppe).

An enumeration of the varieties forming trypsin may be omitted, since they are characterized by their liquefaction of gelatin. The other bacterial ferments have been less thoroughly studied.

Diastatic ferments change starch into sugar. They are recognized in this way: To a thin starch paste containing about 1% thymol, a culture containing 1% to 2% thymol is added. After keeping it in the incubator from six to eight hours, it is tested for sugar with Fehling's solution, when it is recognized by the reduction of the copper salt (reddish-yellow precipitate). One may also examine directly potato infusion cultures of bacteria for sugar, in which case the sugar is extracted by boiling with alcohol and the extract evaporated to a syrup. It is then dissolved in water and the reaction carried out.

It is questionable whether, in these experiments, equal care was always taken to absolutely exel

According to Fermi, about one-third of the varieties investigated possess the ability to form such ferment, but only on albuminous nutrient media (A. H. XI, 1, and C. B. XII, 713). It is produced by bacilli of the subtilis group (anthrax, megatherium, Fitzianus, etc.), the vibriones related to the cholera vibrio; besides, Micrococcus tetragenus, Micrococcus mastitidis, Bact. janthinum, Bact. mallei, Bact. pyogenes foetidum, Bact. phosphorescens, Bact. pneumoniæ, Bact. synxanthum, Bact. aceticum. The remainder do not elaborate it or it is doubtful whether they do. Besides, all actinomyces and Oosporeæ (with the exception of the Oo. carnea) form such a ferment. Most of these varieties afterward utilize the sugar further to form acid, while others do not; for example, Bacillus subtilis.

Inverting ferments—i. e., such as convert cane-sugar into grape-sugar-are rare, according to Fermi and Montesano (C. B. L. 1, 482). Their presence is easily demonstrated by mixing a 1% to 2% solution of cane-sugar containing carbolic acid and the culture treated with 1% carbolic acid, and after a few hours testing with Fehling's solution, and learning if it reduces the solution after standing, which cane-sugar admittedly does not. Control experiments with a solution of cane-sugar alone are always necessary. Fission fungi invertin can (always?) stand 100° for over an hour; it is also produced in non-albuminous nutrient media, if glycerin is added. As producers of inverting ferments the above authors mention only: Bacillus megatherium, B. kiliense, B. fluorescens liquefaciens, B. vulgare, and Vibrio cholera and Metschnikovii.

Efforts to find a ferment resembling emulsin have been frustrated. The "Micrococcus pyogenes tenuis" splits off benzaldehyd from amygdalin, but without the function being separated from cell-life.

Rennet ferments—i. e., bodies which coagulate milk of neutral or amphoteric reaction, unconnected with the action of acid—are not lacking among the products of bacteria. For example, cultures of the Bact. prodigiosum, if not too old, sterilized by heat at 55° to 60°, cause a solid coagulation of sterile milk in one or a few days (Gorini, C. B. XII, 666).

Thorough investigations regarding the distribution of this ferment are unknown to me. We may suspect it in all varieties which coagulate milk without being able to form lactic acid from milk-sugar.