Part 26 (1/2)

V--ANOTHER EXAMPLE OF LIFE WITHOUT AIR--FERMENTATION OF LACTATE OF LIME

As another example of life without air, accompanied by fermentation properly so called, we may lastly cite the fermentation of lactate of lime in a mineral raph, it will be reernation cah only for a very brief ti that we possess exact observations which prove that the diffusion of oxygen and nitrogen in a liquid absolutely deprived of air, so far fro place rapidly, is, on the contrary, a very slow process indeed; yet ere anxious to guard the experihtest possible trace of oxygen at the nation

We employed a liquid prepared as follows: Into froallons) of pure water the following salts [Footnote: Should the solution of lactate of lime be turbid, ita small quantity of phosphate of ammonia, which thron phosphate of lime It is only after this process of clarification and filtration that the phosphates of the formula are added The solution soon becomes turbid if left in contact with air, in consequence of the spontaneous formation of bacteria] were introduced successively, viz:

Pure lactate of lirarararara 11]

On March 23rd, 1875, we filled a 6 litre (about 11 pints) flask, of the shape represented in FIG 11, and placed it over a heater

Another fla the saed The liquids in the flask and in the basin were raised to boiling together, and kept in this condition for more than half-an-hour, so as to expel all the air held in solution The liquid was several tiain; but the portion which re-entered the flask was always boiling On the following day when the flask had cooled, we transferred the end of the delivery tube to a vessel full of mercury and placed the whole apparatus in an oven at a terees C (77 degrees F and 86 degrees F) then, after having refilled the small cylindrical tap-funnel with carbonic acid, we passed into it with all necessary precautions 10 cc (035 fl oz) of a liquid similar to that described, which had been already in active fermentation for several days out of contact with air and noarmed with vibrios We then turned the tap of the funnel, until only a sh to prevent the access of air In this way the ination was accoerht in contact, even for the shortest space, with the external air The fermentation, the occurrence of which at an earlier or later period depends for the eran to manifest itself by the appearance of minute bubbles from March 29th But not until April 9th did we observe bubbles of larger size rise to the surface Fro number, from certain points at the bottom of the flask, where a deposit of earthy phosphates existed; and at the same time the liquid, which for the first few days rerow turbid in consequence of the development of vibrios It was on the same day that we first observed a deposit on the sides of carbonate of lime in crystals

It is a matter of some interest to notice here that, in thecombined to prevent the interference of air A portion of the liquid expelled at the beginning of the experiment, partly because of the increased teas, as it began to be evolved from the fermentative action, reached the surface of thethe rowth of bacteria, it speedily swaranisms [Footnote: The naturalist Cohn, of Breslau, who published an excellent work on bacteria in 1872, described, after Mayer, the coation of these organisms, which it would be well to compare for its utility in studies of this kind with our solution of lactate and phosphates The following is Cohn's formula:

Distilled water 20 cc (07 fl oz) Phosphate of potassiunesiurains) Tribasic phosphate of lirain) Tartrate of arains)

This liquid, the author says, has a feeble acid reaction and fore of air, if such a thing were possible, between the ether prevented, since the bacteria would consuht be dissolved in the liquid lying on the surface of the htest trace of oxygen could have got into the liquid in the flask

Before passing on we en by bacteria we have aferas with a facility and success equal or even greater than by the preli

Such a solution as we have described, if kept at su, becomes turbid in the course of twenty-four hours from a SPONTANEOUS development of bacteria; and it is easy to prove that they absorb all the oxygen held in solution [Footnote: On the rapid absorption of oxygen by bacteria, see also our Memoire of 1872, sur les Generations dites Spontanees, especially the note on page 78] If we coallon) (Fig 9) with the liquid described, taking care to have the delivery-tube also filled, and its opening plunged under ht hours afterwards by means of a chloride of calcium bath, expel froas which it holds in solution, this gas, when analyzed, will be found to be coas, WITHOUT THE LEAST TRACE OF OXYGEN Here, then, we have an excellentthe fermentable liquid of air; we simply have completely to fill a flask with the liquid, and place it in the oven,any addition of butyric vibrios, before the lapse of two or three days We er; and then, if the liquid does becoerms, the liquid, which at first was turbid froain, since the bacteria, when deprived of life, or, at least, of the power of en in solution, will fall inert to the bottom of the vessel On several occasions we have deter fact, which tends to prove that the butyric vibrios cannot be regarded as another forinal relation between the two productions, butyric ferrowth of bacteria

Weexperiment, well suited to show the effect of differences in the coation of s The fermentation which we last described commenced on March 27th and continued until May 10th; that to which we are now to refer, however, was co similar in composition and quantity to that employed in the former experiment On April 23, 1875, we filled a flask of the sa 11, and of similar capacity, viz, 6 litres, with a liquid coe 69 This liquid had been previously left to itself for five days in large open flasks, in consequence of which it had developed an abundant growth of bacteria On the fifth day a few bubbles, rising fro intervals, betokened the commencement of butyric fermentation, a fact, moreover, confirmed by the microscope, in the appearance of the vibrios of this fermentation in specimens of the liquid taken from the bottom of the vessels, the middle of itswith bacteria We transferred the liquid so prepared to the 6 litre flask arranged over the un towith astonishi+ng rapidity, which continued during the 25th and 26th During the evening of the 26th it slackened, and on the 27th all signs of ferht be supposed, a sudden stoppage due to some unknown cause; the fermentation was actually completed, for e examined the fermented liquid on the 28th we could not find the smallest quantity of lactate of lime If the needs of industry should ever require the production of large quantities of butyric acid, there would, beyond doubt, be found in the preceding fact valuable infor that product in abundance [Footnote: In ay are we to account for so great a difference between the two fer to some modification effected in the medium by the previous life of the bacteria, or to the special character of the vibrios used in iht have been due to the action of the air, which, under the conditions of our second experiment, was not absolutely eliainst its introduction at theour flask, and this would tend to facilitate the multiplication of anaerobian vibrios, just as, under similar conditions, would have been the case if we had been dealing with a fero any further, let us devote so fermentations

On May 27th, 1862, we co 2780 litres (about five pints) with the solution of lactate and phosphates [Footnote: In this case the liquid was composed as follows: A saturated solution of lactate of lirees F), was prepared, containing for every 1OO cc (3 1/2 fl oz) 2565 grarains) of the lactate, C6 H5 O5 Ca O (NEW NOTATION, C6 H10 Ca O6) This solution was rendered very clear by the addition of 1 gramme of phosphate of ammonia and subsequent filtration For a volume of 8 litres (14 pints) of this clear saturated solution we used (1 grarains):

Phosphate of ararararaerms The liquid became turbid from a development of bacteria and then underwent butyric fermentation By June 9th the fermentation had become sufficiently active to enable us to collect in the course of twenty-four hours, over mercury, as in all our experias By June 11th, judging froas liberated in the course of twenty-four hours, the activity of the fermentation had doubled We examined a drop of the turbid liquid Here are the notes acco 12) as they stand in our note-book: ”A swarreat difficulty in following thehout the field, apparentlyefforts to separate from each other The connection would seeelatinous thread, which yields so far to their efforts that they succeed in breaking away from actual contact, but yet are, for a while, so far restrained that the movements of one have a visible effect on those of the other By and by, however, we see a complete separation effected, and each reater than it ever had before”

[Illustration with caption: Fig 12]

One of the best methods that can be employed for the

microscopical examination of these vibrios, quite out of contact with air, is the following After butyric fer 13), we connect this flask by an india-rubber tube with one of the flattened bulbs previously described, which we then place on the stage of the13) When ish to make an observation we close, under the mercury, at the point B, the end of the drawn- out and bent delivery-tube The continued evolution of gas soon exerts such a pressure within the flask, that e open the tap R, the liquid is driven into the bulb LL, until it becolass V In thisthe vibrios under observation without their co into contact with the least trace of air, and with as much success as if the bulb, which takes the place of an object glass, had been plunged into the very centre of the flask The movements and fissiparous multiplication of the vibrios may thus be seen in all their beauty, and it is indeed a ht The movements do not immediately cease when the temperature is suddenly lowered, even to a considerable extent, 15 degrees C

(59 degrees F) for example; they are only slackened

Nevertheless, it is better to observe them at the temperatures most favourable to fermentation, even in the oven where the vessels employed in the experirees C and 30 degrees C (77 degrees F and 86 degrees F)

[Illustration: Fig 13]

We may now continue our account of the ferression On June 17th that feras as it did on June 11th, when the residue of hydrogen, after absorption by potash, was 726 per cent; whilst on the 17th it was only 492 per cent

Let us again discuss the e Appended is the sketch we14) and our notes on it: ”Aor undulating They have grown considerably in bulk and length since the 11th;sinuous chains, very mobile at the articulations, visibly less active and o to forth of the individuals” This description is applicable to the majority of the vibrios which occur in cylindrical rods and are hoeneous in aspect There are others, of rare occurrence in chains, which have a clear corpuscle, that is to say, a portion ment has the corpuscle at one end, soth of from 10 to 30 and even 45 thousandths of a millimetre Their diameter is from 1 1/2 to 2, very rarely 3, thousandths of a millimetre [Footnote: 1 millimetre = 0039 inch: hence the dith, from 000039 to 000117, or even 000176 in; diameter, from 0000058 to 0000078, rarely 0000117 in--D C R]

[Illustration: Figure 14]

On June 28th, ferer any trace of gas, nor any lactate in solution All the infusoria were lying motionless at the bottorees, and in the course of a few days becaht Here we may inquire, were these motionless infusoria, which from complete exhaustion of the lactate, the source of the carbonaceous part of their food, were now lying inert at the botto vessel--were they dead beyond the power of revival? [Footnote: The carbonaceous supply, as we remarked, had failed them, and to this failure the absence of vital action, nutrition, and multiplication was attributable

The liquid, however, contained butyrate of li properties similar to those of the lactate Why could not this salt equally well support the life of the vibrios? The explanation of the difficulty seems to us to lie simply in the fact that lactic acid produces heat by its decomposition, whilst butyric acid does not, and the vibrios see the che experiment leads us to believe that they were not perfectly lifeless, and that they ht behave in the same manner as the yeast of beer, which, after it has decoar in a fermentable liquid, is ready to revive and multiply in a fresh saccharine medium On April 22nd, 1875, we left in the oven at a terees F) a fermentation of lactate of lime that had been co 15), in which it had taken place, had never been withdrawn from under the mercury We kept the liquid under observation daily, and saw it gradually becohter; this went on for fifteen days We then filled a similar flask, B, with the solution of lactate, which we boiled, not only to kill the gerht contain, but also to expel the air that it held in solution When the flask, B, had cooled, we connected the two flasks, avoiding the introduction of air, [Footnote: To do this it is sufficient, first, to fill the curved ends of the stop-cocked tubes of the flasks, as well as the india-rubber tube C C which connects thehtly shaken the flask, A, to stir up the deposit at the bottom There was then a pressure due to carbonic acid at the end of the delivery tube of this latter flask, at the point A, so that on opening the taps R and S, the deposit at the bottom of flask A was driven over into flask B, which in consequence was inated with the deposit of a fermentation that had been conation the flask B began to show signs of fermentation It follows that the deposit of vibrios of a completed butyric fermentationthe power of causing fementation It furnishes a butyric ferment, capable of revival and action in a suitable fresh fer 15]