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The Ginger-Beer Plant, and


Organisms composing

it :

a Contribution

to the

Study of Fermentation- Yeasts and Bacteria.
H. Marshall


Sc.D., F.R.S.,

F.L.S., late Fellow of Christ's



of Botany






Engineering College, Cooper's Hill.

Received January


—Read January

21, 1892.

[Plates 11-16.]

In 1887


attention was directed to a curious substance, or structure, popularly




parts of the country as the Ginger-beer Plant, from

its association

with the domestic manufacture of the well-known summer beverage so often purchased in villages and towns in various parts of the British


it is


put up in brown stone


with tied corks.



specimens of the Ginger-

Kew, who called my attention to its mysterious nature, and from Professor Bayley Balfour, of Edinburgh, then Professor of Botany in the University of Oxford, who exhibited specimens at a
beer plant were obtained from Mr. Thistleton Dyer, of

meeting of the Linnean Society in 1887/


Since then I have obtained specimens

and during the progress of a long series of investigations have elicitated a number of facts as to the constitution and behaviour of this remarkable agent of fermentation, which, whatever their importance,
from various sources in this country and abroad

to be of interest to all biologists.

In addition to the gentlemen referred


kindly provided

me with


obtained from Lincolnshire, Oxfordshire, and even from North America, I have also

thank Dr. Bansome, of Nottingham, and Mr. Adrian Brown, of Burton- on-Trent,
from the towns referred

for specimens

and Messrs. Leete and Appleyard,


further supplies from Coventry
It appears that while,

and elsewhere.

on the one hand, the Ginger-beer Plant has long been known

in the rural districts of this country,

and even abroad, as a mysterious agent which brings about the fermentation of saccharine solutions, to which ginger has been added,

and transforms them into an acid effervescing beverage, usually known



ginger-beer, great or even total ignorance prevails, on the other hand, as to the
# See


Proceedings of the Liimean Society,' July, 1887, pp. 7 and






H. M.



and very little indeed is known as to its real nature. The following studies will at least clear up many of the difficulties on the latter point but I have met with no success in direct answer to enquiries as to when the Gingerbeer Plant was first discovered, and how it was introduced into this country, or what first led people to employ it in making the beverage which gives it its name. Professor Bayley Balfour states :* "it is said the Ginger-beer Plant was introduced into Britain by soldiers from the Crimea, in 1855

original source of the " plant,"

but so

far as I

can discover

was a mere conjecture, and


not to be taken as an accepted piece of history.

was brought from Italy/' but this, again, I have failed to substantiate more definitely. The whole question as to whence it was first derived, in fact, is enshrouded in mystery and it


informs me, in a letter dated April,

891, "

some say




to be

hoped that these studies may so draw attention

to the
it is

matter that some light

will be

thrown upon


at a future time.

All agree that

handed on from family
Passing over

to family


in the

same way

as yeast or "


" is

by brewers and bakers.
Mucor, or


to its nature, the most conflicting views have been put forward.

various mere

conjectures to the effect that it consists of forms of Pemcillium,

other Mould-Fungi, the sequel will show that the nearest approach to the real state of

Balfour's suggestiont that



composed of a Yeast and a Bacterium.


in his

Synopsis of the Bacteria and Yeast Fungi,' J regards

as consisting of

a Yeast, Mycoderma, various species of Bacillus, together with Pasteur's "Mucor-



but he gives no evidence of having examined the matter in

detail, and, as

the sequel will show, he entirely misses the main point.

Notes on the subject have

appeared at various times in the


Gardener s Chronicle '§ and elsewhere, but they

throw no light on the essential nature of the Ginger-beer Pant.
It will be sufficiently evident, therefore, that the

mystery was well worth attacking,
before this investigation

and that practically nothing was known about


was comdifficult

may, however, also be noted, that had

known how long and


task I had set myself, the attempt wou]d possibly have been abandoned at an early

General Description.


seen in the fresh state, as


comes from the ferment

flasks or other vessels,

the Ginger-beer plant presents the appearance of



lumpy masses, not unlike pieces of soaked sago or tapioca these lumps are brittle, like firm jelly, and their size varies from that of a pin's head, or smaller, to that of a large plum, or larger very commonly they are as big as a hazel nut

but, since the pieces often result from the breaking-up of large crusts, or layers,
# Loc.
cit., p. 8.

f Op. cit. p. 8. % See note on p. 67.

1884, pp. 542

and 748


1886, p. 315


1887, p. 148.

in the residue of the fermentations lining the flasks, &e.,



suspended in the liquor of fresh fermentations,
limits of size.



impossible to say

and can grow when freely what are the

and of a pure white, semi-opaque appearance, as said but the opacity and brittleness may both vary, even in the same lump. When



lumps are



touched with needles or forceps, the pieces are very apt to
obtained, to break




a hold is


squeezed between glass plates, they are found to be very
stiff jelly.

pery and


reminding one of some forms of cartilage or



not easy to

out and transfer the floating lumps intact, unless a spoon or

similar instrument





in air or over sulphuric acid

in vacuo, or in absolute alcohol, the
pieces become opaque and

volume diminishes considerably, and the shrunken


and often acquire a yellow tinge


they also

become more
In other

brittle or

more horny, according to circumstances.
in the stages of early



development in the fermenting


the lumps are softer and more viscous than
transitional stage can be
to that of a





and, in fact, every


with from the condition of a slimy, semi-fluid, soft jelly,
like glue or cartilage.

hard horny mass which breaks

These physical characteristics are dependent on the amount of water present
in the lumps.

Obviously the
of water

specific gravity of

the fresh pieces will vary according to the amount

present, also


have not examined

point in



but the

The lumps fall rapidly in alcohol, in distilled water, and even in Pasteur's solution and other media of high specific gravity. Some idea of the amount of water contained in the lumps may be derived from the following On August 27th, I started a culture in a sterilised soda-water flask. observation
following facts

may be

Five grams of the fresh Ginger-beer plant were put in 500


of Pasteur- Asparagin ;*

on September the 11th the carefully collected crop weighed, fresh, after' draining on This, dried at 100° C. till it lost no more water, filter paper, just over 52*5 grms.

Assuming the proportion of water to have been the same in the original 5 grms., its dry weight would have been *66 grm. Although the fresh dried lumps swell rapidly when placed in water, they do not in fact, heating them in water renders them whiter and dissolve in it, even if boiled
weighed a

over 7 grms.


more opaque, much


when they

are placed in absolute alcohol, or dried in vacuo.

In most cases the fresh moist specimens, received by post from a distance, are

though in varying degrees.
but in others (perhaps in

In some specimens the acidity was
the acidity diminishes or even
to be

clearly due, in part at least, to acetic acid,

owing to the presence of impurities to be

explained later on


disappears on boiling in water,
carbonic acid.






to the presence


The evanescence

of the acid reaction to litmus, the effervescence


hot water


poured on the moist lumps, and the precipitation of barium carbonate
* See




H. M.

all afford


when they

are placed in baryta water,

evidence of this

moreover, as will

be seen in the sequel, the existence of dissolved carbon dioxide in the moist lumps

might be inferred with certainty from their behaviour.

The most

striking characteristics of the above-described

lumps of Ginger-beer

Plant, however, only become evident

when they

are placed in saccharine solutions,


they are perhaps best shown roughly as follows.
parts full of Pasteur's fluid, or


soda-water bottle

is filled


any other similar solution of sugar
in a

in water,

and a

lump of ginger added.

Into this mixture are placed a few lumps of the Ginger-beer

the bottle


then well corked, and laid



and observed from

time to time.
the liquor

In from 24 to 48 hours, depending on the season, temperature, &c,

observed to become more and more turbid, and bubbles of gas begin to



the fermentation soon goes on rapidly, and unless the cork

tring or wire,


be blown out.

This primary turbidity


by found to be due
well secured

almost entirely to innumerable yeast-cells, and further examination proves that these
yeast-cells are

shed from the lumps of Ginger-beer Plant (which





varying buoyancy in the

and then multiply

in the

medium, and soon form
of the

a dense greyish deposit at the bottom.

The buoyant dancing



seen to

be determined by the copious evolution of gas-bubbles from their surfaces.

In a few days,


the lumps are vigorous and the conditions favourable, there


serious danger of the bottle bursting,



have had one or two nasty experiences
it is

with such fermentations


consequently, in these rough preliminary experiments,
If this

best to remove the cork for a few minutes every day.

done on, say the

third or fourth day, the cork comes out with the well-known pop of a ginger-beer or



and the

liquid froths over from the copious evolution of gas.

This liquid, on examination,

found to be not only more turbid than before, and

surcharged with carbon dioxide, but

evidently more or less viscous, with a visdirectly imparted to



different from

any property

by the sugar and
and persistent

other materials added

this viscosity

associated with the copious


liquor has of course been


in other respects also

it is sufficient

to say that

it is

now more

or less converted into " ginger-beer."

As time
clearly not

goes on the viscosity increases, and

sometimes happens that the liquid

becomes so thick that the gas -bubbles

comparatively slowly.




due to the mere presence of

yeast-cells, because they fall to the


the lumps of Ginger-beer Plant
to do so daily for

now begin


grow obviously


and may continue

some time.

The microscope shows

that the viscosity

due to the

presence of innumerable swollen or slimy vermiform bodies distributed through the

mass of the


Myriads of rod-shaped bodies {Bacteria), are

also observable.

The increasing deposit below is also found, im the later stages, to consist of Bacteria, swarming amongst the j east-cells. The " ginger-beer " is distinctly acid, as well as

the colour of the liquid


paler than that of the original solution.

As time goes

on, the surface of the liquid usually

becomes covered with a dense

and surcharged with gas Schizomycetes and other organisms are more acid and more and found in the deposit. however. or "lees. Tiresome as some of the failures were. for the trouble and disappointments. and then to determine which essential. and the magnitude of the task my notes refer to very nearly two thousand separate cultures. In all these cases the chief phenomena are the same. found in the fermenting mixture." and allowed to stand and bottled off as before. the villagers usually employ a somewhat different mode of procedure for making " ginger-beer. Meanwhile more sugar solution the original vessel containing the deposit. clearly. in a large . is the yeast which so rapidly spreads in the earlier stages of fermentation ? What What or any. however. and the whole allowed is to stand for a day or two. that interest it it more than repays one B. in a pure state. i. it sheds yeast-cells around. are somewhat as follows. &c. the quantity of gas disengaged having fallen to a : then moulds. with slimy masses in it. and exposed in drunk after two or three days more. and the conversion of the saccharine liquor into " Ginger-beer " is ? Such are the problems of which the solution one's self acquainted attempted in the following studies. putrefaction supervenes/' In practice. each extending over periods of from several days to months. and. or the " ginger-beer : " sets up acetic fermentation in some cases it even " goes bad. or forms. The is pieces of Ginger-beer plant are then placed in the mixture. satisfactory way to accomplish this end was to separate and is cultivate each form by itself. was (if to make with all the various organisms. solution in tap-water. and even in some cases to two years. 129 scum. The bottled liquor simply becomes increase and form a deposit . in part.e. and especially those due to the interruptions inseparable from the conditions under which carried on in a busy teaching laboratory. the excites* MDCCCXCIL — 8 . This entailed very numerous.AND THE ORGANISMS COMPOSING minimum IT. of these organisms to do with the Ginger-beer plant. of various kinds appear. species of Schizomycetes are present ? What does the scum consist of ? finally.. and in the scum at the top. what have all. open vessel. and which any) were mere intruders or foreign organisms having It is equally clear that the only nothing to do with the changes in question." They make a solution of sugar corresponding roughly to a 10-20 per cent. the well-known frothy " ginger-beer " of the country. unless very well corked and protected. The plant grows. there is investigations is must be in so much that fascinating in such work. Then the liquor is poured off into bottles and corked. It will be obvious that the problems which present themselves to the biologist examining the contents of such vessels as the above. a little cream of tartar and a few pieces of ginger are then added some add lemon as well. and these the liquid becomes viscous. wearisome best understood from the fact that attempts. The were first step. and all is alternately buoyed up and falls in : the liquid . are the slimy vermiform bodies in the liquor? What And.

touched with if sterilised forceps only. everything being sterilised as already :• and nothing touched except by means of heated forceps A deep glass ring was placed on a broad glass slide and a drop of previously sterilised olive oil allowed to run between. everything was lifted In special cases the baking for several hours. ." was ensured by check-tubes by the side of the cultures. in the case of test-tubes. necessary. it I have employed the usual methods of culture. or melted paraffin was run in in the same . watch-glass. beaker. Here. they were allowed to two at most) and then either exposed for a couple of hours to 80° C. In all cases were it was desirable to ensure pure to sterilisation. and the baking repeated baked as above at least once. until the paper that enveloped tact with the it began to brown. I employed three kinds of cultures. made in sterilised The latter were described. (1) Large cultures in flasks. may be advisable to give First. then. funnel. not first been thoroughly cleaned. the tube or flask was regarded as " safe.wool plugs for the tubes or the cotton. or kept up Before making the cotton. and in no case was reliance placed on any piece of glass that had Moreover. again. The flasks. treated. or other piece of apparatus was heated in a hot-air chamber to at least 140° C. PLANT. on at least two successive days . tube. all con- hands was avoided by means of After filling series of tubes or flasks with any given solution. but sometimes solid gelatine being employed (2) smaller cultures in tubes and (3) cultures in hanging drops. and thus baked. After plugging the and tubes —already —with such cotton-wool. as some general description of the details. for at least two hours. every flask. sterilised forceps. flasks. WARD OK THE GINGER-BEER Methods. M. During the whole course of the investigation. and when filtering was necessary no filter paper was trusted that had not been baked and treated. and in no case used until they had been again heated to liquids 90° C. in the latter case so that the steam The filling of the culturetubes and flasks was done by means of sterilised funnels. or boiled. or 90° C.wool itself was always baked on flasks several successive days and for several hours at 90° C. cells under the microscope. usually liquids. like the forced itself through the plugs for at least half an hour. glass-cell. ordinarily prepared as follows. . cover-slip. employed as food materials were always prepared in sterilised stocksimilarly plugged. remained clear. under a large clean bell-jar for a day (or . as confidence in the results depends on the accuracy of these cultures. and from what follows. cultures.— 130 PROFESSOR H. by forceps similarly to 150° temperature was raised and even 200°. As will be seen from the above. cotton-wool. or placed on a sandbath and boiled for a quarter of an hour in some cases this was repeated after stand. " safety " If on standing for three days or more I found the This &c. liquid or gelatine. slide. till the plugs began to colour. but. twenty-four to forty-eight hours. or for a shorter period at 120° C. they were again slowly baked.

in hanging drops and in various gases under the microscope. .and water-tight # sterilised chamber. = cover-slip d = hanging drop w = cotton. The much in a laboratory at fairly constant temperature. the this. 131 this cements the ring to the slide. the glass being grou down slide. s 2 . 1. A piece of stout glass tube. Appleyard. especially where it was necessary I to have control over the gases cultures of such composing the atmosphere in the cells.AND THE ORGANISMS COMPOSING way while the slide and ring were hot is . about three is carefully drawn out at both ends. inches long and as thick as possible. In special cases. a drop of is being placed on the upper edge of the glass ring. the tube ready for grinding. L. and the hanging drop quickly put on and oil and then.). A large cover-slip infected. a.. 1. ledging the debt. but the glass should be softened and allowed to contract the opening. devised the following simple form of utility apparatus which has proved to be of the greatest for organisms as the one under consideration. I owe take this opportunity of acknow- Matthews and Mr..chambers. II. IT. chamber ready for use. . until it The narrow tubes must not be drawn thin. side view of the . drop downwards. then placed placed on flat on a support. and the thin glass cover slip experience shows that such cultures will remain for at least a fortnight without danger of infection from without.//s////>///"/ Iff Glass culture-chamber for hanging drops. The hanging drop thus projects from roof into a practically air.1. (X vzzzzzzzzzzzzzzzzzzzZZ I 2ZZZZZZZZZZZZZZ2 r zzzzzzzzzzzzzzzzzz ??§2x ^g2.wool plugs = ordinary glass Passing over the earlier attempts to improve on known forms of gas. fig. The incomplete instrument now looks like 1 (L). and Fig. thanks to Dr. /3. s view of same from above. the one ultimately adopted was the following. /3. III. to the levels c a. In the working out and making of this instrument. looks like a narrow tube with a bulb in the middle of * Practically so because the vapour-tension does not vary its course (fig.

Glass micro-culture chamber in use with gas generators.— 132 PROFESSOR H.e. and the methods employed to obtain pure : cultures. a and is After sterilising.). as parallel. &. b. for of the culture-chamber c. its hanging drop (d) is then fixed by means of freshly boiled to the upper face and the two end tubes are plugged with carefully sterilised cotton. evolving carbon dioxide gas from the marble in d. the lower pierced face (s. with hanging drop ? on the microscope . IL). going into the culture -chamber (see arrows) . PLANT. . It depends on circumstances what further procedure is adopted. but I protect the (sterilised in corrosive clip. upper and lower faces of this " bulb " are perforated. Those most commonly employed have been the following . 1. Obviously any convenient form gas-generator can be attached. the sides are for use. 1. and the cell is ready fixed by means of melted /3. WARD ON THE GINGER-BEER now ground /3. fig.chamber. brass-clips on caoutchouc tubing attached to the plugged tubes vessel containing dilute hydrochloric acid.wool (w. paraffin to a sterilised glass slide A sterilised oil cover-slip (c) with . 2. Fig. M. (See fig. open ends of each glass tube with a piece of caoutchouc tubing sublimate and then washed and boiled) governed by a evaporation the cotton-wool is brass To prevent of moistened. and the accompanying wood-cut shows the apparatus arranged for cultures in carbon dioxide. 2. washing apparatus through which the gas generated in d passes before . in position a = the culture.) Now as to the nutritive media. fig. until shown by the lines a. II.

of asparagin to the above. according to the sugar employed. &c.) "Pasteur's varieties. Pasteur-milk-sugar solutions.. and then water were 50 grms. misconceptions. * Tn boilings. all cases a measured excess of water is allowed.) notes I refer to these Pasteur-glucose." # the made by boiling the best gelatine medium contains from 5 to 20 per cent. present difficulties in sterilising. During one stage of the researches. "because of the loss during the successive " f There is always a certain quantity of starch present in the commercial German yeast. or milk-sugar were used under the headThe constitution of " Pasteur's solution " is well known. pressed yeastt in cold water in a mortar." of which I have employed three (4. when (2. but.or Milk*-) Sugar 150*00 10*00 Ammonium tartrate Acid Potassium phosphate (KH 3 P0 4 ) . 0*20 Magnesium sulphate Calcium phosphate 0*02 0*02 Water In later cultures I found In it 1000*00 advisable to add solutions as 1 grm.— — AND THE ORGANISMS COMPOSING (1. so that of the gelatine. confection is therefore. however. of the syrup or the rhizomes. crushed or whole. 5 Pure starch-paste. to obviate ing sugar. X See the .(Grape. In view of the fact that ordinary decoctions of ginger rhizomes.) " set.. that this ginger " obtained in jars from the grocer s. p. (3. Cane. Latterly. (or Mayer's) solution..) IT. making up solutions containing from 5 to 20 per cent. glucose. I began by using the well-known " preserved It turns out.) made by pounding up sterilising litre. to the : Grms. 5. prepared by slowly boiling from Yeast-water. according as cane-sugar. by successive boilings. in distilled water. 133 Pure gelatine. The proportions of yeast to and reduced by boiling to one-half. my (5. to 20 grms." " Kew Bulletin. I append the form of recipe used filtering." 1891. Ginger solution. I found it was necessary to employ solutions containing ginger. per 100 of water. I made from a species of Alpinia^ and not from made up solutions as follows : Zingiber.

. Glucose Gelatine . the following.) Peter's gelatine : — Grms. gelatine: 414. 1*0 1*0 Asparagin Acid Potassium phosphate . . PLANT. Gelatine 5 Glucose 3 Peptone and extract of meat . ginger.. 5 400*0 Traces. . 0-25 . . . .. 10 to 15 2*5 to 5 Asparagin t 0*25 .. f This consists of * Grams. . ..) Grms. Cane sugar Asparagin t . Traces.'.' 1889. Tap Water 100 50 17 KH PO 3 4 MgS0 4 . especially of pure yeasts. Grms. Water (7.. 0-25 Liebigs extract of meat . ...* — — — —— 134 PROFESSOR H.. « 0*5 MgS0 4 CaCl 3 Tap water . foot note. Haydtjck's mineral solution . . . Peptone In special cases I added from 2*5 to solutions of yeast.. 5 (8. WARD ON THE GINGER-BEER .) The following was employed under the name Haydtjck's ginger # See 'Botanische Zeitung. 10 0*25 5 Mineral solution (9. . Crushed Ginger 10*0 Cane Sugar Tartrate of 40*0 Ammonia . . stiffened with gelatine (2*5 to 5 per cent. or „ 10 per cent. col.. which I solution. sugar..water.) For certain special cultures. term Hayduck's was much used : Grains. 100 : Also a solution of glucose. . and so : and found the following very convenient media for paxticular purposes (6. of gelatine to the above forth...) . M.

or heated to 90° C. . 285-6). and then used. at a suitable temperature. the medium. as : the case might be. and sodic phosphate. placed in like circumstances.. all others. platinum wire.. Arch. I proceeded as : — First. more rapidly than others a small drop of the culture liquid now used for a second infection in a new flask. or both.* Lister ('Pharn Journ. capillary tubes. The clear pale straw-yellow bouillon thus obtained was boiled for twenty minutes.I AND THE ORGANISMS COMPOSING Distilled water IT. and Naegeli Unters. experim. a fair sample of what has gone into the new culture. what may be termed preliminary cultures. Pathol. and at once plug. 135 Grms. was placed in a sterilised flask of the given solution. One pound of lean beef-steak. for at least an hour.) it is possible to obtain at least a flask or tube in which the particular organism favoured abounds almost to the exclusion of follows In order to be sure of what happens at each successive infection. ' niedere Pilze.) this was exactly neutralised with an alkaline mixture of sodic hydrate.100 10 5 Preserved ginger Glucose Asparagin Gelatine 0*25 1 Mineral solution (as before) 5 The following " Bouillon" was much employed in the later researches on the Schizomycetes. # * These are modifications of the " fractionating " methods of Klebs vol. sodic carbonate. 1). and by repeating this a sufficient number of times (depending on the temperature. was soaked over night in 1 litre distilled water. After filtering (10. or other of the organisms By that time (or in some similar period) one would be sure to have commenced budding or dividing. and place in the incubator. none of which were employed again if these are more convenient than used.* 1882. then filtered and boiled for half an hour.' 1877. bouillon-Pasteur. In special cases 5 to 10 per cent. all infections were made with newly drawn glass . on each of four successive days. I should state. &c. A small portion of the presumably mixed mass of Yeasts or Bacteria.. bouillon-sugar. pp. or more of sugar or Pasteur's solution was added bouillon. so on. and equally safe properly made and label. chopped fine. and which of course. touch the liquid to be infected. (' li. Then I examine under the microscope what remains in the capillary. f . now enables us to obtain the given form in yet greater predominance. I simply call these media and It remains to describe the method of infecting and of obtaining pure cultures of the various organisms to be described. . and left for 24 to 48 or 64 hours in the In all cases I started with incubator.. to the above. A drop having been is secured at the drawn end of the tube — the thick part of which not yet cool — is. and again boiled for one hour.

For the study of the fermentations on a large scale. 102. and here the well-known methods of Brefeld.136 PROFESSOR H. p* 191* . all The pure culture else is a mere matter of time. and of discovering the proper environment for the organism.' 1885. I found the following method very convenient. and its cork (a new one) soaked for several days in an alcoholic solution of corrosive sublimate. it is possible to follow the behaviour of the cell under the microscope. Mikroskopie. This method is less although I have succeeded once or twice in isolating a single bacterium in a very small drop. when as possible. for a and then day in absolute alcohol. p. and hi^ for the isolation of the colonies developed ' especial merit consists in bis perfecting from mixed germs. In special cases. M. voh 1. I persevered till the hanging drop contained only one yeast-cell. My procedure was as follows. and always where the final pure culture was to be obtained in the case of the yeasts. t See Hansen in * Zeitschi\ fur wiss. Koch and Hansen # were used. the culture so obtained known to be derived from a single progenitor. and to object in successive stages. A common soda-water bottle is sterilised by heating. by means of gelatine. readily applicable in detail to the study of the Schizomycetes . Die Methoden der Backterien-Forschung. and then boiled for at least one hour on each of two successive days surface of the water this cork is perforated previously. A fair note on the subject is given by Hueppe. I diluted a small quantity of it in a suitable nutritive medium to which enough gelatine was added to just stiffen the mass when cold. were infected by streaking the fine capillary infecting tube over their surface. WARD ON THE GINGER-BEER PLANT. colonies which made their appearance after a culture had been got as pure a few days were isolated by re-infection then. Secondly. the gelatine keeps But although it is not easy to obtain a colony of bacteria it is derived from one thoroughly isolated progenitor. and kept below the by a piece of glass rod. . and specimens from the separate . it was necessary in most cases to work with drops containing several least. The next step —in — De Baky. specimens relying on the fact that. or of nutritive fluids solidified with gelatine. by merely touching it with a capillary pipette and infecting a flask or tube. obtain pure cultures comparatively very easy to by means of the above method. is And. in some cases at the specimens fixed. a culture of guaranteed character and purity can be prepared from the colony which results sooner or later from the budding of the yeast. Klebs. the behaviour of which is definitely known. once obtained. because the gelatine holds it make drawings of the in position all the time. some cases employed forthwith was to obtain pure cultures from these roughly purified ones.\ The advantages same of this method are obvious. that the botanists Brefeld and Klebs devised the * It does not appear to be generally of cultivating known it method on solid media. though Koch improved the method. thirdly. and prepared a hanging drop culture of this.' 1884. tubes of gelatine. In the first place.

arid and peculiar action. that while two specific Cryptoare necessary for its formation gams constitute the ginger-beer plant proper. definite organism. being constant) gives a good approximate measure of the rate and activity of the fermentation.AND THE ORGANISMS COMPOSING The soda-water bottle is IT. pressure. . i Soda-water flask arranged for fermentation experiment. as in the case of a yeast. a. The various Organisms found As will in the Ginger-Beer Plant. the cork pushed well home by means of the glass rod. said. (See is fig. the Ginger-beer Plant is a composite body. plug of cotton-wool . the details of which will be mentioned in Pig. The number of bubbles per I minute (other things. have been gathered from what has been or. that I was for a long time in doubt as to their true relations the MDCCCXCII. after a few hours. 137 then properly charged.) When the fermentation an active one. the rest are merely accessory or to the admixture of spores from outside. and the mercury proximal leg . there rises in the at first considerable absorption of air. — B. the whole column supported in the distal leg of the manometer. the infecting organisms added. the end which pierces the cork being sterilised by heat and plugged with sterilised cotton -wool. doubtless due Of T these there are also two which are so : constantly present. at least yielding more than one Investigation has shown. and presses the mercury forward finally. and A. &c. 3. foreign organisms. this method. mercury in the manometer tube. such as temperature. b. however. have also employed modifications of their proper place. manometer tube is is then added. and the bubbles of gas escape at intervals. 3. . consisting of several organisms. carbonic acid gas is is evolved.

the most important of the forms met with in this investigation. resulting in a copious evolution of carbon dioxide gas. and are certainly intruders. secondly. fungi. and one commonest. any fungus or Schizomycete that will grow in a saccharine solution at ordinary tempera- t. while the other is the vinegar Bacterium Bacterium aceti (Kutz. 138 PROFESSOR H. migW oocu/in to the forms usually the expose/f—tion. # The " Kahmhaut" of the Germans. and by growth on gelatine media from such cultures t . the various fungi of other kinds. I first brought it into pure cultivation in 1889. or glucose. &c. and must be named. but not essential forms. Saccharomyces pyriformis (n. 1-10. WARD GIST THE GINGER-BEER PLANT. (Plate 11. one is a species of Saccharomyces. I have eenfined my attention'^ met with. In fact. Of the two constant. of successive transference from flask to flask. . of Dematium. M. Oidium. sp.). such as might be expected to occur in such a heterogeneous mixture. ellipsoideus). equally well known as the principal constituent .). &c. tubes. and shall prefix a letter to each species in order that reference may be simple and certain.l. both by the dilution method. remainder only occur occasionally. — — of the " Vinegar Plant. Yeast A. which turns out to be a new species. and in the formation at the bottom of the flasks." The foreign intruders most commonly met with are species of Saccharomyces. Pure cultures were readily obtained. syrup. Micrococcus. exposed to air and made with ordinary water. found in all the specimens examined. consisting of characteristic colonies of budding yeast-cells. whether prepared with cane-sugar. the well-known agent of the "mould"* on sour beer. of a voluminous white pasty deposit. one is a yeast-like form. which turns out to be Mycoderma cerevisice (Desm. and lastly. . It induces active fermentation in sugar-solutions.). or two ordinary mould the Saccharomycetes which Penicillium is by far the I shall describe these organisms in the following order :— First. and was at once struck with its general resemblances to the bottom-yeast of some of the Continental wine fermentations (S. the Schizomycetes . Of the two essential forms. Bacillus. Torula. and there can be no doubt that it is the yeast principally concerned in the fermentation of ginger-beer. figs. and is also a new and very remarkable species. and which I shall have to name the other is a Schizomycete. as the usual " brew" of " home-made ginger-beer 57 is. has now been found to occur in every specimen of the Ginger-beer plant that I have examined.) This yeast..

de l'Acad. even at complex to follow in (Plate 11. the cells turn dark sienna red. of gelatine.) and each daughter-cell soon repeats the other daughter -cells from various other low temperatures. the development of ascospores. or to gelatine tubes. at or near the opposite pole. which usually occurs meanwhile. There is no limit to the figs. which I have been distinctness. soon becomes too process. or more commonly ellipsoid. cultures of this yeast. The ordinary behaviour of the cells in hanging drops of ginger-gelatine is well shown in fig. in this and with singular yeast. culture. and the fig. and they often grow to masses as large as a mustard seed. 5. 5 size and shape of these colonies. 6. as the figures (Plate 11. resulting in the formation of remarkably coherent colonies. Its budding. The development of the endogenous ascospores in Saccharomyces has been especially •* Ekeera. or red-brown/* and the colour pales to yellow (or even fades altogether) on warming. detail. or hanging drop. to reappear on able to cooling. opaque. 139 grow in hanging drops of various saccharine solutions. the protoplasm gives a strikingly clear glycogen reaction on adding iodine dissolved in an aqueous solution of potassic iodide. The isolated ellipsoidal cell contains a large vacuole. to 85° C. or ovoid in shape. if or spheroidal clumps at the bottom of the flask.) I have frequently taken advantage of the formation of these colonies in the hanging drops of nutritive solutions stiffened with gelatine. nothing added except the traces of saccharine carried in by the yeast-cells) compressed between a cover-slip and glass slide (all sterilised) also showed that the completely submerged cells can bud at from 0. Plate 11. 5. In very active. in sterilised cells The single cell is globoid. and then infecting the culture is &c. T 2 . though smaller and larger cells are found. of course. though. 11 5 C. kept perfectly (P]ate 11. as shown in Plate 11. fig. employed. One point of physiological importance worth recording. Cultures in pure gelatine (that is. and 6) sufficiently show. even large colonies. vigorous .. were . at or near one end. 6. stiffened under the microscope. to transfer pure cultures — traced from a single cell — to sterilised flasks of Pasteur's or Hayduck's or other fluids. obtained in three or four days. is Another point worth noting observe repeatedly. 'Bull. It buds readily at all temperatures from about 10° C. €. enclosed excentrically in pale finely granular protoplasm. fine glass capillary tube. the process was somewhat slow nevertheless. of several hundred cells. fig. a second may begin in about three hours after the preparation of the . colourless and translucent. apparently. and soon results in the completion of the daughter-cell bud may appear process. and very actively at about 25° C. dome-shaped. and measures from 6 to 7 /x long X 5*5 /x broad.AND THE ORGANISMS COMPOSING I got single cells to IT. visible to the unaided eye as white. &c. and perfectly quiet. also give off points on The mother-cell may its surface. de Bnixelles/ Nov. 1882. to 14° C. This is easily done by touching the colony with a freshly drawn flask. with about 5 per cent.

10). as shown in the figures. and arranged in a tetrad. of Burton-on-Trent. Since it was necessary to have the opinion of practised brewers on the subject of asked it. or the membrane of the mothercell may be burst. fully aerated. formation. protoplasm remains unused. These spores begin to develop in from two to four days at 25° C. his definition of the genus and the distinction of The yeast in question not only developes the spores when spread on moist and sterilised gypsum. this yeast. I my friend. who kindly did this and prepared photographs for me. but the process is slower. 3). Dilute solutions and plenty of oxygen are necessary for the free germination of . samples in the usual manner.140 PROFESSOR PI. consists simply in the swelling up of the endogenous spores to the form of the original yeasfc-cells. Horace Brown. though firmly Each contoured membrane. drying seems to and very little. fig. is no doubt due to the more perfect access also of air and water to the As Hansen showed. The spores are almost invariably in fours. is surrounded by a delicate. is a good temperature. . and I have been able to obtain beautiful preparations by this means. and complete spores were formed in forty-eight This quicker development cells. M. and then sprout. otherwise the spores are not developed. if any. when ripe. which was then folded and wrapped in several successive papers similarly prepared. 25° C. was smeared carefully on to the centre of a thoroughly sterilised filter-paper. '* See Jorgensen. Brown and to Dr. for literature of whole subject. but also very readily on the surface of solid sterilised gelatine. to cultivate it a pure sample of and to have examined from the brewer's point of view. Morris. of a pure culture. and this accords very well with Hansen's results (Plate 11. The germination these sprout-cells. the first indications of spore-formation were evident in thirty to forty hours. I have to express I sent the my sincere thanks. carefully moistened and to fifty hours." 1890. WARD ON THE GINGER-BEER PLANT. in loosely C. Some of the fresh pasty deposit. and I have in vain attempted to follow the details of the spore- cannot decide whether ordinary division or any other method of separation of the protoplasm precedes the complete separation of the spores. so that the membrane of the parent-cell surrounding them is often pulled into a form approaching the tetrahedron. it required four days to obtain the complete spores on . " Die Mikro-organismen der Gahrungsindustrie. Mr. these spores they may be made to germinate in hanging drops stiffened with gelatine. plugged tubes but on sterilised blocks of gypsum. To Mr. which then begin to bud in the ordinary manner of The sprouting may begin in situ. fairly readily if The spores germinate favour the process (fig. &c. the cells employed must be at the height of their vigour.* species chiefly on this peculiarity. which can be stained by hot carbolic-fuchsin solution and mounted in Canada -balsam. white brick. spore. they are completely ripe . who founds I found that at 25° gelatine. and the spores set free. studied by Hansen.

..wort had slightly improved the average size of the cells. fra Carlsb. the fermentation -activity (regarded as a means of obtaining alcohol) is not very great. or some modification of that figure. Morris to return me a similarly prepared sample of his cultures from the above this he was so good as to do. and at the end of forty. new : one. * Hansen. Morris' samples (which had gone through five cultivations in beer. and will be seen to be usually pyriform. (by volume) of alcohol. until a complete skin is formed on the surface./ 1886. and satisfied myself that the form had kept true throughout. number up to the fifth day. and compare them with what is known of other species of Saccharornyces. yeast. The shapes of these aerobian cells (or " involution forms ") are shown in outline in Plate 11. Meddel. These films on the surface of the in contact with air. figs. and I again repeated my observations on Dr. I propose to name it. liquid. at 25° The young were formed cells. in enabling us to recognise the species manner in beer-wort. the yeast under consideration developes the aerobian form or film-growth—in about three weeks this continues during the next two or three weeks.eight hours many spores these increased in out. may be summed up as follows A low. and shows that. or bottom-fermentation. As already pointed still. /x Grown wort of in hopped wort. ellipsoideus* (as Hansen). C. showed distinct traces of ascospores after twenty-four hours. . and this^ came down again in Pasteur's and Hayduck's cells solutions as before. fall to the base of the flask in flake-like clusters. from the characteristic pear-shaped aerobian Its chief characters Shccharomyces pyriformis. and showed no further decrease at the end of fifty-two days. therefore. . This attenuation corresponds to the production of 4-4 per cent." and by us. S. the only point of difference I could detect being that the sojourn in beer. Morris writes " of this form amended by " It was at : thought that it was the same is as one isolated from the air of the film -formation opposed to that conclusion . be regarded as a cells. but the nature It does not agree with any previously described. it If will be evident that my species is one allied to it. . 141 Lad happened. we take into consideration all the facts I have been able to elucidate about this yeast. 8 and 9. lying on earthenware blocks. A specific gravity 1054-0 attenuated in twelve days to specific gravity 1022*7. Labor. ' which inverts and ferments cane sugar. compared with some yeasts. are allowed to ferment malt-worts in contact with certain modified growth-forms arise the whole being kept perfectly of the cells appear as films of great tenuity. and the cells composing them are of considerable importance of Saccharomyces. and patches in this Grown — .." The species must. though not first identical with Dr.wort). the of the yeast range from 9 to 5 in diameter. Hansen's researches # have shown that when yeasts air.— AND THE ORGANISMS COMPOSING To be quite certain that no accident IT. I requested Dr.

as a rule. as sausage-shaped cells. It is. At a separation cultures. though smaller and Ascospores formed in from two to four days. and. the lower surface was thrown into irregular folds. of pyriform. cases. the was covered with a characteristic yellowish. and. f Saccharomyces mycoderma (Reess). figs. wrinkled skin.). this feature gradually intensified. * The cells grown in Pasteur's or Hayduck's solutions are usually smaller than those in wort. reminding one of racemose glands hanging down into the little practice. ranging from 5 to 9 p in diameter. while liquid. WARD ON THE GINGER-BEER PLANT. one of the most variable of the yeast fungi. indeed. and has been the subject of several extraordinary statements as to the vagaries of these fungi. the surface became wrinkled. cerevisice of It is the very polymorphic Mycoderma it DESMAZiERES. M. . and films. looking as if made up of extremely fine sinuous silky fibrillae as its thickness and toughness increased. a dense and. in the narrow sense. in I first many had my was the predominant form from the beginning. might be a matter a form were for surprise that I it should have devoted so much attention to so common its not for the fact that such conflicting statements occur as to it nature and properties. and a . It makes its appearance invariably in the latter stages of all the preliminary cultures. Hormiscium cerevisice (Bonordin). pieces fall of the submerged part of the very thick skin become detached and slowly to the bottom of the flask. These characters are so unmistakable that any one can recognise the skin after a Sooner or later. since this occurred. and. when the air had to filter through sterilised plugs of cotton-wool. On the 30th. Mycoderma cerevisice (Desm." Yeast B. the colour becomes it more and more buff as ages. Aerobian forms. and that was necessary all to be sure of its relation to the ginger-beer plant. This skin began as a very thin iridescent pellicle.) Whenever the fermentations were carried on or wrinkled skin (" mould ") formed at the surface .t It is not a true Saccharomyces. for ascospores. or lower.142 * PROFESSOR H. It and differs in several respects from the true yeasts. more especially if caused to rock or sway as the flask was moved. there can be no doubt as to does not form the origin of the fungus from the infection. finished with access of air. attention attracted by it in the earlier cultures of 1887. at 25° C. It occurs in "home-brewed ginger-beer/' and is the predominant form in the so-called " Ginger-beer Plant. the silky fibrillar character became pronounced. . developed in wort in twenty-one days. at first quite smooth and almost greasy looking it then became thicker and streaky. (Plate 12. later date. On November 16 liquid of that year. Ordinary cells larger ones occur. # ovoid or globoid. 1-6. a flask of Pasteur's solution was prepared for rough lump of Ginger-beer plant put in.

or like that of rancid olive oil is . what what the odour due to. however the separation-cultures . I have nevertheless got it to grow in hanging drops of gelatine—where the supply of free oxygen must. 143 .. changes have gone on to produce to discover 30° C. be very limited. and. . it was often kept in abeyance. cases occur or occur on one side only. and as each succeeding sprout behaves in the same way. In older cultures of the Mycoderma on Pasteur's solution made with glucose. at most. was much more difficult to prevent it gaining the upper hand in glucose solu- tions than in those where cane-sugar was employed. remained without effect for three weeks. 1890. 30° C. it Sooner or later. its Some vitality. of my experiments go to show that Mycoderma does not long preserve Infections made in glucose solutions in May. At higher temperatures. In other cases the lateral all we have curious racemose or dendritic colonies produced.g. even in was not because the Mycoderma will not grow. mass fermentation allowed to go on its open vessels. and so on. This Mycoderma is particularly apt to form in the preliminary cultures made at low temperatures 12° to 15° C. cells. sprouts are alternately suppressed on either side Finally. though (still with refeeuc/to J^&A I cultures) it I . . I have been unable This Mycoderma yeast is is very easily obtained in large quantities. It invariably manifested itself if present. when the dominant fermentation was finished. These facts are quite in accord with Hansen's statements* that Mycoderma about its is unable to invert cane-sugar or to bring fermentation . and therefore infections of glucose solutions. want of oxygen but this is by no means the case with all. — but because it suffered in the struggle with competing forms. 8. and that it is apt to appear on lager beer even in the cold cellars.— AND THE ORGANISMS COMPOSING Many of the cells which fall IT. although Mycoderma is distinctly an aerobian form. 2. this. I have several times noticed a peculiar strong odour.' Paris. apparently from — — — eaulgar. this e. and even in gelatine. then found to be composed of dense colonies of elongated the in budding of which takes place with great regularity. figs. ISTo. and especially when glucose is employed instead of into the liquid die. 1888 . The first cell puts forth a sprout at apex. where the cells are very long and bud so as to produce net-work - * Hansen in ' Atmales de Micrographie. or a may mention that whenever a piece of in was used. easily got pure by repeated successive The skin Plate 12. from flasks kept since the preceding November. this form invariably made appearance at ordinary temperatures.y uo « slow to appear on ordinary Pasteur's solution. and usually in the order its shown 1-3. compressed between a cover-slip and a glass slide where the access of oxygen must have been reduced almost to a minimum. p. In illustration of unsterilised ginger its well-known ubiquity. and then one at each side just below the sprout.

4. Unters. 1869. ' ft Cienkowski in Melanges Biol. ' ** Reess. 67. vol. § and are opposed to those of CiENKOWSKi. and kept them going separately under similar conditions. 2. of Fungi. d. 180. ' Sc. although. 268-9. Peters!}.tt and accepted by De Rary.^ (1) it is probable that all it should be remembered that the earlier observers worked with mixed yeasts. and seems to regard Oidium lactis and Chalara mycoderma as both genetically connected with In this I fail to confirm his statements. its great variability.. u. and offering some difficulties in manipulation under the microscope. St. this Mycoderma seems to be devoid power to form spores.' 1873. Cited by Jorgensen. Bot. which of course affects the question of priority as to the discovery of the ascospores in Saccharomycetes.) Again. Centralbl. 8. WiNOGRADSKYt of the In spite of several statements to the contrary. . 1890. The average different media.' 1872. 747.) || || The small rounded forms are about 2 to Sfi in diamefcer.' 1870.** and In considering this matter. de Bot. in the case of the experienced observers mentioned. (Plate 12. p. describes long branching mycelial forms of Mycoderma. my results confirm those of ZoppJ and Hansen. size of the cells) several peculiarities is 6 to 8 /x long.' 1884. 8. p. because I have separated the Oidium and the Mycoderma from one and the same flask.' vol. These peculiarities had already been noticed by Cienkowsky/* and has also remarked the great variability of this yeast. vol. and their differences remained constant. In this. ' Die Mikro-organismen der Gahrungsindustrie/ Berlin. I have utterly failed to induce the formation of the endospores by any of the received methods. thus giving a peculiar lustre to the films. 20. Cienkowsky that form. see t * In view of footnote. Nat. d. p.H Reess.JI De Seynes. %% 'Morph. and am the more disposed to think he was working with mixed species.' pp. PROFESSOR H. since the . M.|| Engel.' vol. or other yeast. p. 566. much as they vary when cultivated on 1 1| In the first place they are particularly apt to have air entangled among them. p. methods of separating and obtaining pure cultures were entirely modern often easy to mistake oil-globules in the cells error is and (2) it is for spores. 10. §§ If the bodies De Seynes observed in Mycoderma. than the XvJXlilvJX. 154 Ann. so far as Oidium lactis goes (I have not met with Chalara). fig. Akad. of course. they often contain one or more very highly * i Melanges Biologique. vol. St.. I would suggest that this form is worth renewed comparative study from the point of view introduced by Elfying and Laurent. Petersb. as has long been known. the latter less likely.144 like colonies. vol. Botanique/ Tf Les Ferments Alcooliques. de l'Acad. % Schenk's § || * Handbuch der * Botanik. WARD ON THE GINGER-BEER PLANT. 566. by 2 to 4 ^ broad. the credit of discovery belongs to him (' Comptes Rendus/ 1868. Alkoholgahrungspilze.. and there are which characterise them. and Biol. were spores.

the flasks of Pasteur's solution were found to contain rosy-pink specks in the thin buff skin which commonly formed in from two to three days. The pink England flecks soon spread. and also in other cultures derived from {e. Though fairly well characterised by the sum of its properties.g. in specimens from Mr. only it the flasks of Pasteur's solution. at temperatures varying from 56° to 130° F.*) This is ?. Cryptococcus glutinis (Fees. floating loosely in the vacuole (see 4 and 5. came under closer and I have had it separated and in regular laboratory observation for several months. a pink or rosy yeast-like organism. its variability is so remarkable that in regarding the different phases as belonging to one no one would be justified and the same object unless he It traced their connection by continuous cultures.AND THE ORGANISMS COMPOSING IT. and since they original appeared in from 7 or 8 to 20 or 21 days. 145 figs. In some of the preliminary cultures of 1889. 1889.. (in pure cultures) under circumstances where very oxygen must Yeast 0. I have repeatedly failed to find it in cultures of the " plant p obtained from some of the other * I have put a ? to this because it is not certain that the form described by Fresenius is the same as Hansen investigated. figs.. is less translucent than an active Saccharomyces The Mycoderma usually appearing at or near the end of a fermentation. 7-10. while the fact of cell. though. and one of the most beautiful and all interesting of those I have examined. it may be assumed that the germs in were in the specimens from the These pink flecks and patches appeared thus spontaneously. as will be ssen. MDCCOXCIL — B. I suppose this is because the Mycoderrna is aerobian. as already stated. into an atmosphere of hydrogen or carbon-dioxide. and said to have been originally derived from South America. Plate 12. Although there is sufficient evidence to justify the conclusion that this organism was introduced as a constituent of the above-named specimens of the Ginger-beer plant. Leete) first. &c. and partly on the domination of the principal form at the time. but subsequent experience showed that will grow admirably in and on glucose solution. flasks. Plate 12) cell . This was especially the case with a set of preliminary separation cultures of the Ginger-beer plant sent me from Kew. little free can be grown exist. invading the other yeasts of the skin. It was always easy to suppress the Mycoderrna in my cultures by putting the it tubes. probably oily in nature. as I have done. depends partly on the absence of oxygen during the activity of the yeasts in the liquid. so to speak. investigation in a culture of October 21. it is equally certain it is not a necessary part of it : on the one hand. refractive bodies. TJ . as a whole.

sharp spine-like processes. yeast-cell. nor have for its observance. but no motion of translation or pulsation of the vacuoles and the comparison is entirely superficial. while others again were drawn out here and there into thin hypha-like arms. the only feature it has in common with l * It has long been Bacteriology. is As already stated. figs. Indeed. the delicate cells appearing peculiarly hyaline cell. I once obtained under circumstances which strongly air of the laboratory. as 10 or 11 /jl in diameter when the more irregular shapes shown in figs.* p. recorded and described as far as possible. WARD ON THE GINGER-BEER it PLANT. so delicate that they figs. but in no sense indispensable to the mixture. and as regards the mere out- The cells bud like an ordinary cells. : not only the cell-wall extremely thin and transparent. was easily isolated and examined. about 9 //. and watery. and was found to (Plate 12. lines. and very large of this. PROFESSOR on the H. and. 7 and 8. a characteristic feature of this yeast is its extreme delicacy form in . known that a rosy yeast can be obtained from air (see Orookshank. M. be regarded as a foreign or wild form. and favoured clear vacuoles are apt to One consequence observable.. and partly to the large vacuoles the 7 cells are. in fact. by the irregular shapes of the cells is a certain is resemblance to an Amoeba. regard it as not a true yeast at all— the of as not a Saccharomyces. either from some point on the surface of the elliptical regular like protuberances. but the protoplasm is it. 842).146 sources. since every effort to succesfe. and particularly interesting because I have succeeded in eliciting some position. and. and is it would not be impossible for a careless observer to over- look them. 7 and 8 are assumed. but they may be as much size of the cells.) Many of the cells were merely ellipsoidal and regular. The rosy hue I on]y evident when large numbers of the forming a thick mass. in the typical oval yeast-form. and presented a striking similarity to germinating spores.e. I make it develop endospores has been without i. Manual . it is me that this yeast does not induce distinctly aerobian. be a form very variable in shape. and 8). peculiarly hyaline and watery. have not investigated the colouring matter.* indicated its introduction as an adventitious it germ from the All things considered. and other characters. therefore. is I made long measurements as to the thickness of layer necessary The average by 4 ii when broad. simple or branched. and its probable systematic The yeast of the pink patches size.walls. The rosy-pink colour extreme tenuity of the protoplasm (Plate 12. may It is. nevertheless. : is totally invisible in the thin layers used under the micro- scope. sufficiently important to be sometimes occurring as an impurity in the lumps of Ginger-beer plant. new discoveries concerning it. but others were provided with short. partly owing to the and very watery seem almost transparent cells are together. or from the tips of the sharp spine-like or the longer arm- Cultures in various media have convinced alcoholic fermentation. other.

arranged in large cells and with abundance of fresh obtained excellent results. and leave the question of name and synonymy New was thrown on the question by cultures air. This was at 10. the only distinct references to pink yeasts.30. zur Mykologie. iii. name mination. Before doing this. cell. but it is. this was sufficiently distinct to be shown in a drawing (6). 339. Heft 2. unfortunately. 10. I cannot get at oval regular form of Hansen's and my rosy yeast. at least.m. Fbesemus§ a note on a red yeast. 1876.. When I (fig. 5. 147 its cells as habit of budding. U 2 .. I am.AND THE ORGANISMS COMPOSING true yeasts is its IT. to make and it very probable. I in sterilised extract of beet-root. that his " Bodtfarvede gjcersvampe " my rosy yeast are one and the same form. and Cohn's form the same. gives As regards the or other references. ' Landwirthsch. 187. 43-46. Hansen's opinion on this point. § 'Beitr.' Copenhagen. Jahrb. indeed. t % * Beitrage zur Biologie der Pflatizen. ' : So far as 1 have been able to discover. however. 77. unless we regard the forms of com- palpable to the aerobian forms of some species of Saccharomyces* However. though the latter altered the The question then arises whether this form is the Unhappily. I have. ii. containing only one cell. 1879. 10. In the hanging drops. * it had become as large as the parent vol. oL-q). a few from a pure culture of the rosy yeast. %. viii. At noon of the same day. This matter of species deterto Saccharornyces glutinus. which will be best illustrated by a concrete case cells (see Plate 12. it was fixed under the microscope and then sketched drop culture was prepared from this.t and Hansen. were trans- ferred to a large drop of the beet-gelatine on a sterilised glass plate. my light rosy yeast. stiffened with 5 per cent. figs. and a small hanging had succeeded in obtaining a satisfactory drop. are in Bbefeld.30 a. latter is in Danish.. fortunately. and by 12. the full detail. p. Taf. and are useless all in this connection. at 4 p. fig. it will be well to state what I have been able to find out about CD the " rosy " or " pink " yeasts in the literature this is not very much. however. 110 and p. contented myself with recording its I could find out about to others. ' Organismer i 01 og Olurt. the ordinary descriptive fungus floras abound with mere names of yeasts and bacteria. and not even the aerobian form of one. almost certain. 6. a). this cell had commenced to put forth a protuberance at the lower rounded end. the thermometer stood at 12° C. while. in a position to give much is better evidence than the above in support of my contention that this pink yeast no Saccharomyoes..' vol.' Taf. On the 15th March. 10. a conclusion that is fully borne out by the glimpses into the meaning of the text which I have been able to get with the aid of German. 1. . p.m.' 1850-63. fig. which he is named Cryptococcits glutinus. they are so incomplete that they afford very little no help to us. 1872. is hardly worth following out in the present state of our knowledge . likely that what Fresenius and Cohn described is simply the young stage of our rosy yeast. and I cannot read it in Some of Hansen's figures are sufficiently characteristic. Taf.. of gelatine. J and. therefore.* CoHN. p.

Mykol. (e). on the 15th separated. 10. conidiophores are at shown p and Obviously. 138.m. continued up to 4 P. the cells of which were very nearly in : spherical. known to us. however. (Plate 15. the general resemblance between my m-q. which one of the bud-conidia detached and germinating. the cell budded off. fig. while at m is a characteristic group of these conidia-bearing hyphae. budding conidia. During the night. is That they are to be regarded as conidiophores is evident from their further behaviour. as shown in fig.148 (c) PROFESSOR H. a change was noticed in cells budding the behaviour of some of the which were beginning to elongate. this was formed (f) and this time. From shown the colony (shown in h) of o. as on the preceding day. are shown the stages of further development of the hypha marked X in h . on the 17th. The further progress of events is evident from the succeeding figures. as stated on p. to the slight rise of temperature which occurred in the laboratory. the well -nourished "rosy yeast" has developed into a true hyphal fungus. and I. M. Gebiete d. At i. and we must look for its allies For my own part. and at 9 A.M. the temperature being the like cells 15° 0. .88-89. and appeared culture remarkably dense. and considerable progress was made during the ensuing night. In the dense centre of the radiating mycelial mass the pink hue is now quite evident. 10. because.M. WARD ON THE GINGER-BEER PLANT. and from n. had put out long hypha-like arms. quite a colony of (g). and those may be put forward in the hopes that further investigations may * ' Fnters. . probably. By cells." it have made no very extended cultures of it found that was not a normal constituent of the Ginger-beer plant but one or two interesting facts about it came to light during the examination of the pure cultures. and some of the conidial forms of the Basidiomycetes lately described by Brefeld*. 10. I frequently met with a small yeast./ 7 and 8. 18. Yeast D. it seems probable that the common "rosy yeast" is a mere growth form of some member of that group. and two of the q on a larger scale. aus d.m. mycelium in which represents the state of on March 21st the small dots shown in this figure (drawn to smaller scale) are the above-named conidia. on the 16th. I cannot help being struck with figs. March 18th affairs arose the dense radiating . referred to this form in I I my notes as the " floury yeast. d) to this a third was added by 6 p. owing. by 10 a. some of which produced terminal cells. still On the morning of the 18th. . with septate filaments and conidia. and although several links are required before this form can be definitely allied with the Basidiomycetes. white. Gesammt. h. dusty looking films on the top of the these films liquids were so like flour in appearance that I habitually in detail. and averaged about 2*3 to S'7 /x in diameter. and the next step like : is to see what it is most clearly it is not like any yeast among some of the higher forms. Jc. In 9 fact.) In some of the earlier separation cultures. a new one was being formed near its point of origin (fig.

how- certainty. Of course my . it was relatively abundant.AND THE ORGANISMS COMPOSING be undertaken to clear up its life -history. is In the film on. and in sugar solution I to which ginger had been added.g. (Plates 13 and 14. but it seemed to grow best in glucose solutions.exist. first place. but the results do not require detailed description. swollen. I. Sehizomycete No. I kept them in cultivation for a few months. as shown in it and if reliance may be placed on these morphological characters. and even made a few cultures of them in company with Bacterium vermiforme. Other Yeasts. and everything points to it being capable of inducing alcoholic fermen- tation. enclosed in hyaline. it neither liquefied the medium nor and the In some grew I to any marked extent on it. have already referred to its association with the acetic Bacterium. &c. and were obviously not concerned in the formation of the Ginger-beer plant. in the brain-like masses formed by its convolutions. as to whether it forms spores — seems unadvisable to give any specific name. home-made ginger-beer" in 1890. production of acetic ether in those flasks where both organisms go. the same as a form isolated from a bottle of " where. cases I noticed a pressure of carbon dioxide sufficient to blow the corks from the culture-flasks. &c. Bacterium vermiforme (n. it IT. In addition to the foregoing. I did not follow out the details of their life-history. colonies.) of this investigation the Of the several Schizomycetes met with during the course one constant and essential form as such without — essential because the Ginger -beer plant cannot exist It is it—is a peculiarly vermiform organism. gelatinous sheaths. as It developed if the liquid did not wet an evident fermentation in Hayduck's solution. sp. ever. and imprisoning the yeast-cells of Saccharomyces pyriforme. in question develops characteristic 10. the swollen sheaths of this 5 ' organism which constitute the jelly-like matrix of the " plant. is fig. and has a dull dusty appearance.). medium. 149 Owing to the gaps in it my knowledge of its characters —e. I occasionally found the ordinary beer-yeast (Saccharo- myces cerevisice) and two other forms which is I have been unable to identify with Since these were rare. though one of them probably S. in chains. forming a pure white floury the surface of the culture-liquids this film creeps up the sides of the tubes several millimetres beyond the general level of the it. could not get any material results on gelatine .. The yeast Plate 15. it aerobic in a marked : degree. mixed with several forms of other yeasts and Schizomycetes. apiculatus.

When the contents of a suitably prepared fermentation-flask. They have a field. am here assuming that the culture pure. curved. have attention was particularly directed to from the first. and obtainable in any quantity at short notice. may be excentric (Plate 1 3. 3. rods. rodlets. the observer can assure himself that they are all modifications of a sausage-shaped or cylindrical form. turns. several is objects are noticed besides the yeast-cells. singularly respon- sive to changes in the environment —nutritive materials. twists. but in a third day fermen tation of Pasteur's solution (with asparagin pale. or filaments). &c. and following (Plate 1 the coils. M. on the other hand. 6. egg-shaped. o. and its vagaries. but contain specks. and are easily overlooked in some lights. in which the Ginger-beer plant has been acting for about three days. or twisted in kinds of ways. are I examined. it is been the chief puzzle throughout atmosphere. involving numerous failures before was attained. These contents (whether specks. though. on the one hand. is. coiled. The commonest types of these objects are shown in figs. just described. . or the specks. it is by no means a sensitive or delicate organism in is other senses . and ginger) large numbers of (figs. which envelopes the organism. WARD ON THE GINGER-BEER it PLANT. are not necessarily symmetrically disposed in their investing substance . b). 3-6 of Plate 13. the gases composing the it &c— it. 6. figs. these vermiform bodies present two modifications. these may be long or short. or variously twisted bodies will be seen floating in the liquid . refringent substance in 6-8. rodlets. as the much more 3-5). 150 PROFESSOR H. (figs. of the investment 3. Fortunately. and since these can only be carried out with the expenditure of success much time and trouble. straight. figs. Careful observation shows that others of these bodies are not homogeneous. indeed. curved.. and equidistant from the all contour throughout. that had to be tested in the actual investigation. can be dried and kept for long periods. of course. where the intervals necessary for continuous observation cannot be arranged during term. figs. they may and 7) be central in the latter. so remarkably polymorphic that was impossible to trust the mere tube. and 8) or partly exserted if I or absent from one part or the other. Some of them are perfectly homogeneous throughout all 4 and 5) . it will readily be understood that results could only be got slowly in a busy teaching laboratory. although this Bacterium conditions of its it is so variable in its behaviour according to the environment. curious lustre. It will simplify matters say at once tha"B the brilliant refractive contents. rod-like. 6) It depends on the stage of the fermentation which one predominates.and flask-cultures without full confirmation from cultures in hanging-drops under the microscope. for while. or filamentous. glassy- looking spheroidal. and Plate 14. in various stages of development while the paler investment is a peculiar swollen gelatinous sheath. or filaments (fig. 6 . 4. . though the brilliance due to their moderately high refractive index usually picks them out in some part of the above. are the Bacterium we are concerned with. figs. or axile. and as they roll over. or filaments of their interior (Plate 13.

as it is it were.. in many cases. rodlets. are Schizomycetes at all—nay. which rapidly All I could when dried.prepared ammoniacal cupric sulphate.AND THE ORGANISMS COMPOSING IT. and I have kept them several times for periods ranging from five to seventeen days in a hanging drop. and perhaps (but not always) some longer filaments fact. and from their behaviour in cultivations. not even obvious that they are organisms of any kind. are all these variously-shaped naked bacteria conclusion could not be taken for granted. . —in and leptothrix-forms. in the natural state of affairs. unassociated than some substance resembling a gum-like or living being at all. the case described. that the addition of alcohol merely causes the convolutions or the separate vermiform bodies to shrivel up and become very granular. merely the empty As a matter of fact this is . above referred Are they.wall. or not ? It should also be said that it was a long time before the question of their identity assumed a definite form. . to use the current phraseology. there are several Schizomycetes to be looked for in the fermentations. sheaths ? and are the homogeneous structures. bacteria. and the first and most obvious of such questions was. freshly. of course each has its proper cell. it by no means obvious that the convoluted masses which it form the gelatinous matrix.. these somewhat violent procedures enable one to judge whether the * 1 use the term simply in contra-distinction to sheathed. as seen in Plate 13. cocci. 13. 3 These are unquestionably naked * Schizomycetes (Plate and 8) from their form. &c. Isolated specimens remain for days unaltered in the nutritive solutions. and rods of various lengths. merely the Schizomycetes escaped from their investing first sheaths. of the investigation I assumed that they probably were partly on more general grounds. with a When this is the condition of affairs. Hence. figs. make Of out was that strong sulphuric acid dissolved the masses in question. B. It is now easy to see how natural were the questions — Have these naked forms anything in to ? common with the sheathed in fact. water or glycerine. and instances occur where difficult to would be decide directly that the convolutions consist of anything more cellulose-like body.wool in contact all night . cocci. At the commencement distinct. for example. which proved to be quite distinct organisms. bacilli. When examining a specimen of the Ginger-beer plant is directly. but it will readily be understood that such a and several other questions had to be propounded and answered before these facts were demonstrated. Water causes no apparent change in them dissolved cotton. A. &e. 5. filaments. the stages of division which they present. and partly because at an early stage I found several Bacilli and Bacteria in the separation cultures. at any in rate. it may happen fig. did no more than faintly tinge them blue after being and the other cellulose reactions failed similarly. course.. 151 Mingled with the objects referred to are large numbers of minute spheroidal specks. different species. reactions.

curved. the question and would not do to conclude off-hand that the free bacilli among the yeasts were simply the Schizomycete in question. escape from the sheaths. it Over and over again plant. unless certain ordinary. only the task was. so far. The methods employed for separation were the same as those already described as being used to obtain the pure yeast-cultures. M. looking free bacilli could be taken for it it This was. they are masses of dead substance. —During — the — the the course of events first was almost invariably the same. and none of these obviously resembled the sheathed and convoluted form commenced with. masses in question enclose any other bodies. I was led to the second conclusion. The only fact really established. or in cells kinds of media. as follows rapidly. I have found no dye stain them satisfactorily. the convoluted masses contain nothing in the nature of a filament. and the above-named reagents place beyond all doubt the fact that the convoluted or vermiform masses of gelatinous matrix are to. and the iodine test particularly convinces the observer that. and attempts were made to bring the gelatine media more into use. forty-eight hours or so the yeast multiplied and soon rendered the liquid media turbid yeasts falling to head. coccus. As a matter of fact. because. rodlet. the swollen sheaths of the long or short rodlets and filaments just referred This point established of course led to the conclusion that we have to do with a form of Schizomycete with remarkably pronounced sheaths. or.152 PROFESSOR H. quite naturally. liquid. turned out that three forms at least were obtainable. cases. it On looking for the sheathed Schizomycete in such experience soon showed that was rarely to be found. in the state I am referring to. in cases where the . or spore of any kind . and soon ceased and then fermentation came to a the bottom. WARD ON" THE GINGER-BEER PLANT. in tubes. however. flasks. the sheaths may be found devoid of the Schizomycete. forming a thick film on the top. the microscope at once shows the presence of the brilliant rodlets or filaments. as a whole. based on the observation that when the : . or even coiled into corkscrew-like forms of numerous turns. in tures. I also employed the following method. at high or low temperait under the microscope. In the other condition of the specimens. of course. on separating all the latter and cultivating them apart. from the results of the cultures which established the existence of the dead sheaths. and in some cases more. straight. Mycoderma was favoured. or under certain conditions. or —an idea that arises subsequently—because the living schizomycetous rods. either owing to the death and disintegration of the proto- plasm and cells proper. more difficult. &c. and the facts observed suggest that at certain stages. of course. was observed that. when minute pieces of the Ginger-beer were employed for infecting the sterilised media—whether solid or in tubes or flasks : namely. was that the vermiform sheathed bacillus was not easily capable of direct cultivation in the form met with in the Ginger-beer a fair inference was that it probably escaped from its sheaths in the cultures plant and then carried on its life as a non-sheathed Schizomycete. and that some one or other of the various forms found in the yeast deposits was the one in question.

it was found that short exposures (five to ten minutes) at the latter temperature killed neither yeast nor Schizomyeete. and very few Schizomycetes could be found among these those that were found had . however. : The question. which aggregated at the surface and produced spores in their interior. and the yeast entirely suppressed by a bacillus which rapidly passed through all its phases of long filaments. also with negative results. more than the I therefore raised the walls of the yeast-cells can protect their contents. of the " Ginger-beer Plant/' to 100° C. as subsequent infections in 30° kinds of media at 15° C. I placed a minute piece of the "plant" in each of twelve thoroughly sterilised gelatine tubes. almost amounting to conviction. On with December 19. 25° C. and further experiments proved that the second conclusion was correct. for periods varying from a week to a year sufficiently demonstrated. also give no satisfactory results . that these free forms " belong to the life-cycle of the Schizomyeete in question. but the puzzling b. — among the sedimentary cells . containing several lumps for one minute. all This killed both the yeasts and the Schizomyeete. I inferred that either the latter were sup- pressed by the copious development of the yeast..AND THE ORGANISMS COMPOSING « IT. The mere culture most of small quantities of the Ginger-beer plant itself in gelatine tubes gave the which I may quote the following as an example. Eight of the tubes developed yeasts in and on the gelatine which was not liquefied. and C. The next method employed was that distracting results. the sheaths are so thick and look so dense that they may possibly protect the protocell- plasm of the cells proper against the action of high temperatures. bore in which did not more especially favour the the suspicion. in the incubator. I then exposed similarly washed specimens in water to 80° C. under Culture after culture proved that ordinary bacilli conditions. are they the free forms. and so I abandoned this mode of attempt to separate the organisms. The other four tubes. even in those where the yeasts gained the upper hand. exactly like those referred to mdcccxcii. x . Exposures to 70° C and 60° C. to illustrate of isolation on gelatine. Schizomyeete In other words. which have ? escaped from the sheaths. temperature of a flask of water. breaking up into typical swarming bacilli. the following results. the older cultures showed swarms of bacilli. 153 sheathed Schizomyeete in question is at its fullest activity in the ginger-beer bottles. and kept the cultures at 25° to 28° C. had their gelatine liquefied. and thus behave so differently it was no use looking for the convoluted sheathed forms to multiply as such on or in the usual gelatine media in such tubes. or they are of a kind which does not flourish in gelatine. for ten minutes. broken up into minute cocci or rodlets. arose ? Are these bacilli and spores those of the Ginger- beer plant. cases Moreover. Evidently the death temperatures do not differ sufficiently for success. of course. and then allowed all to cool. the repeated observation that just such free spore-forming in those tubes commonly appeared yeasts.

I infected a tube of ginger-gelatine. M.154 PROFESSOR H.e. 28th February the tube contained abundance of the typical Ginger-beer plant. course this did not prove that the free rodlets gave rise to the sheathed it fila- ments. and generally two. and control experiments. I again infected a tube of equal parts Hayduck's gelatine- ginger and Peter's gelatine with the rodlets and yeasts from the last culture. how is ifc these free forms do not eventually become sheathed. rod-like Schizomycete became a dominant form. Ginger-beer plant from one the above flasks. beer plant" was developed. . and up to February 28. equal quantity of Peter's gelatine. Up any February 9 things went on as before. My methods. 1891. on January 26. evidence was gradually forthcoming. On December 1890. Copious fermentation and multiplication of the yeasts ensued. But on the i. and a thick deposit was formed. and by comparing their behaviour. whereas under other conditions I found the sheathed and gelatinous forms. free rodlets It should be noted that throughout these cultivations the species. with the mixed yeasts and rodlets of the last Things went on much as before. On December 19. though the yeasts and rodlets were very abundant and healthy in appearance. small pieces of the Ginger-beer plant were placed in flasks of glucose-Pasteur. convoluted sheathed rods and filaments entangling the yeast-cells in the gelatinous Of that. but no typical Ginger-beer plant had developed in these flasks up to the end of January. I employed at least one. and more. the deposit consisted. Either some free and one of two explanations only could be entertained. 28. and grow into the convoluted gelatinous masses of the Ginger-beer plant? this And since they refused to do even in cultures three and four months all old. as I carefully convinced were of one predominent myself after each transfer. question arose. is On the other hand. entirely of yeasts and free bacillus-like rods. 1891 . and suppressed side * In all such cases as this. into a tube* free bacillus-like rods of ginger-gelatine and here again and yeasts alone were recognisable after a few days. mixed with an gelatine. On February to 1891. check-tubes. and up to February 28. aud . no " Gingerculture. 1. convinced me that there was here no case of casual infection from outside. but. I placed a drop of the still recognisable . I was again inclined to suspect that after they had nothing to do with the Schizo- mycete I was hunting for. this culture showed no typical " Ginger-Beer Plant . as I have said. 1890." simply free bacillus-like rods among the thickly deposited yeast-cells at the bottom of the liquefied Meanwhile. I had no reason to anticipate greater success than in previous experiments the copious deposit at the bottom of the tube consisted of yeast-cells with crowds of intermingled rodlets. mass. of which the following one sample selected from many others. all by side. 1891. was only one case out of many others which went to show I could under certain conditions only find free rodlets among the yeast-cells. WARD ON THE GINGER-BEER PLANT..

however. of the free rodlets during the early stages it would seem to disprove the former stress on such evidence nevertheless.m. namely. After two days. was made and the specimen drawn at 3 . or. 1890. and so became evident that the rodlets among the yeasts watched a slightly coiled sheathed one marked a in fig. behaviour of the Ginger-beer plant in the fermentations in soda-water bottles for.m. as pointed out previously. I were changing.investigation of the particular A considerable step forward fig. " IT. that the growth of these filaments only begins after the yeastcells have exhausted the oxygen of the culture-drop and revision of all it its surrounding atmosphere. the first thing observed . the latter organism does really lose and live and multiply as a it changes in the environment compel moving bacillus-like form. for the curious stage h was little not reached until 11 a. was made December. 7. A careful my previous notes and tube. 7. &.m.AND THE ORGANISMS COMPOSING the true " Ginger-beer sheaths.and flask-cultures bore out this suggestion. on December 31. moreover.m. 1891) at 11 o'clock 2) . seen at e. of course does not do to lay too much where bacteria are concerned. No more 9. together with investing envelope or sheath. At p. free fact of the suc- The latter seemed the more likely alternative. the increase of the plant as a whole ceils will not observed Obviously these yeast have used up all the free oxygen by that time. which.m. The growth was yet more pronounced at on that day. though intercalary elongation also occurred. a copious shedding and is multiplication of the yeast-cells until later. affairs at Such was the state of form referred to. and f at 9 p. but the growth now slowed had off considerably. d exhibits the state of affairs next morning (January 1. the stage g was reached at 10. 155 its Schizomycete . the first note on the same date. explained some previous imperfect observations in trapped gelatine cultures. because the very cessful cultures containing so many . Specimens of both yeasts and rodlets of a clean culture of the Ginger-beer plant gelatine. the filament had its evidently elongated considerably. for in the fermentation-cultures in soda-water X Zt . and was really only a confirmation of what is I had suspected from the . it The results are evident from the drawings. until certain once more to assume the sheathed state. Indeed this latter statement does not depend on mere inference from what we know of the general life-history of the yeasts. an obvious inference was drawn. 9 a. as 9 p. were growing together a hanging-drop of ginger- My attention at the time was being more particularly directed to the yeast. on January contortion or screwing up of the coils and it is evident that more than a it occurred. on the same date.. e was drawn at 3 p. as shown at and a comparison of the drawings shows that the growth was chiefly at the righthand end of the filament. when I found the case in recorded in Plate 13. I although the specimen was carefully watched till January and thought probable that the low temperature accounted for the cessation of activity. which I took up the in re.. Next day (January 3.m. changes were observed.M. Having once satisfied myself of the above phenomena.30 A.

coils This cushion proved to be composed of dense I and masses of the typical sheathed Schizomycete was seeking washed for.steur s solution. As a matter of fact. but a sort of cushion of jelly-like substance had formed at the bottom of the tube. consisted solely of the form in question. obviously due to the vigorous absorption of the oxygen of the air by the yeasts. although it is easy enough to separate the Schizo- mycete now. the difficulties were natural so long as I looked only for the sheathed and gelatinous form. A It should be borne in mind that I did not certainly know that this cushion first. to was then regarded at any rate be worked out as time and opportunity — admitted. and on February 27. that . M. carefully pure water. satisfactory pure cultures but. the same medium and one of these tubes. Be this as it may. I had meanwhile attempted over and over again to isolate the Schizomycete..e. in plant. afterwards became very useful. into tubes of gelatinere-infections were made from these tubes into new tubes of after incubation for three days. but it provisionally —as a separate species. and. as small as possible. as said. in the first place. WARD ON THE GTISTGEII-BEER PLANT. there was a cushion-like. and on February 14a tube of H ayduck's solution was infected from one of the above tubes* Further cultivation showed that the yeast had now become eliminated. and examine its properties in pure cultures." infections.or dilution-cultures. On the 9th January. Two days later . because. gelatinous This tube deposit. p. or suppressed entirely. of the sediment containing the "bacilli. had placed a lump of the Ginger -beer of separation cultures were On the 9th of the previous December. into a flask of glucose-Pa. the volume of gas absorbed was in agreement with our knowledge that nearly one-fifth of the air. composed of the sheathed rodlets and filaments in question. the first time I thoroughly satisfied myself that my tubes con- tained the Schizomycete was on February 27. was used for further infections. and a number glucose. leading to the absolutely pure cultures obtained subsequently.— 156 PROFESSOR H. I The history of this culture was as follows. by volume. was found which to be a bacillus-like body. and at last succeeded in doing this. The liquid in the upper two-thirds of the tube was clear. and supplied me with material for successive redrop. occurred when the culture — It seems now almost ridiculous that I should have failed so long in obtaining . made on the third day. when I know something of its vagaries. the only conclusions I drew were. I had isolated the organism long before this. the predominating organism in a selected tube of this series of transfer. flourishing also occurred in the tube. consists of oxygen. when I found that a culture in Hayduck's solution presented the following appearances. among the few suppressed yeast-cells was now (January 9) transferred to tubes and flasks of Hayduck's gelatine-glucose. it only was made in air. in the second place the diminution of volume i. 1890. The usual fermentation followed. bottles connected with manometer tubes (described on 137) the first thing was always a rise of the mercury in the proximal leg of the U-tube.

the rod-like Schizomycete either escapes altogether from (fig. and 4* The rodlet travels forward (fig. 1 only. but it it is connected so closely with the conditions of the environment. merely escape from the sheaths. from gelatinous investment. and use rodlets. After numerous failures. Plate 14. or sideways (fig. but figs. Plate 14). it it — the sheathed Schizomycete— promised to be a comparatively easy task to cultivate . I used about 1-2 per cent. and in part to the sensitiveness of the organism to transference from tube-cultures to the hanging drops. whereas any interference with the medium induces the to. it as a hanging-drop. gelatinous investment Plate 1 4) . At length I obtained some very puzzling results. according to their length at the time. it does this very generally in the fermentations carried on in soda-water flasks.. secondly. 1-4. naked filaments.And the organisms composing it. the sheathed organisms behave as above described— the rodlets. according to the temperature. it could be developed apart from the Having now obtained the long-sought organism separate from other forms. some of the specimens at least do not thus cast their sheaths forthwith. due in part to the unsuitable media. as in the one figured at Plate 14. however. the organism remains in the sheath. I transfer such a drop carefully into a hanging-drop of suitable food materials. in the stiffened media. &c. 1). This may be fairly concluded from the observations that so long as the conditions in the hanging-drop are favourable. Difficulties by the use of beet-gelatine and of bouillon-Pasteur- arose from the facts that the sheathed organism will not was necessary to employ as little as possible of this medium only just as much as would suffice to slightly fix the specimens. unequal growth just referred I found that if I simply transfer a drop from such a fermentation to a cover-slip. and goes on adding to the sheath as forward and 4. and. and goes on continually . in its sheathed readily as such in gelatine. that my explanation of may probably be accepted as the true one. If. &c. and grows symmetrically in it . or. and become isolated. In the cases referred its to. travels it does so partially (see figs. that yeast. it more frequently. shown in first figs. I arrived at results gelatine. . grow and that it — form. that may be taken as an indication on the part of the organism that some change has occurred in the surroundings. 4). The next obstacle was the frequent refusal of the Schizomycete to grow at all. 1 they behave in most cases. 2. I supposed that this was a pathological phenomenon it but it- now been followed so often and so closely. i the vermiform sheathed organism sought for could be grown in the medium (Hay- duck's solution) mentioned. is its not only a curious fact in itself. moreover. pure in hanging drops under the microscope but this proved to be by no means so simple as I supposed. When has observed. This process of partial or total emergence of the Schizomycete proper.

forming more and more of the sheath substance it wake. rodlets. so long as any free oxygen if present whereas developes rapidly enough (in suitable nutritive solution) a film of Myco- derma or mould forms at the top. became intelligible at once in the Tt now remains to see what information is forthcoming as to the conditions which gelatinous investment. all I This quite in accordance with have been able to find out as to the developIt is ment of the Ginger-beer plant as a whole. presumably because the Saccharomycete rapidly uses up all is the oxygen. &c. however. If there are yeast-cells present in the drop. (fig. — as it were. as recorded The most curious paths the curves. of the sheath substance 1.e. 3 to 6. coils. air containing that gas seems to cause the phenomenon at once. WARD ON THE GINGER-BEER in its PLANT. M. show that the nutritive medium must contain carboThe Schizomycete lives and grows an bouillon alone. or when the yeast has used up all the oxygen.— — . figs. not found in open vessels. as shown Plate 13). but it does not develop the sheathing substance in them. only it is thin at the forward end. were .. ordinary cane-sugar seems to be more suit- . devoid of carbo-hydrates. but the most remarkable thing about the whole phenomenon end on or side on that in : many cases the rodlet does not perceptibly elongate or enlarge during the process it simply goes forward it. and the sheaths with eccentric 8). provided the nutritive medium is suitable in other respects. and I shall have something to say of its behaviour in such Similar comparative cultures solutions presently . the sheaths are soon developed again. 131. are described by the onward growing organism. and the air thoroughly replaced by carbon dioxide. and even appears to be projecting free from this sheath substance. light of the above cultures. like an erratic rocket leaving a tail behind The above phenomena amply explain the meaning of my some of the forms found in the fermentations. sheathing jelly in an atmosphere containing it oxygen but in a constant atmosphere of carbon dioxide does this readily. is by &c. as it and although keeps ahead. and 8 The empty sheaths (figs. (see Plate 14. in the gas-chamber described on its In no case have I been able to make the organism develop . or if the cultures are placed in the receiver of the air-pump. or even is in tubes and it flasks plugged with cotton wool. earlier observations of in figs. bends. determine the escape of the organism from its The presence of oxygen i. 158 PROFESSOR H. or apparently protruding filaments. &-g). Moreover. Of the carbo-hydrates I have employed. one it all can see that the latter really invests round. I have satisfied myself of this by means of duplicate cultures in hanging drops of the same nutritive side with one in air medium — — one in carbon dioxide being carried on side by p. and in other media hydrates. the rodlets at once proceed to free themselves of the sheathing substance. for I have observed over and over again that when the sheathed organisms are transferred from a strong fermentation (in which it — may be assumed that no trace of free oxygen exists) to a newly-made hanging drop of the same as that medium employed for the fermentation. 3-5).

and when the yeast is present the carbonic acid aids I have not made any special experiments as to what acids will or will not matters. so that the strong perforated glass plate (c) may be fitted to it. Although this is certainly not the case (because the pure cultures disprove better the organism developes. and one is impelled to the suspicion that some product of the yeast's action on the saccharine solution I tried to test this made use of by the Schizomycete. and my usual method to expose crushed ginger. . glucose. to 90° C.— — AKD THE ORGANISMS COMPOSING able than glucose. and second. if Experiments showed is that the ginger might be sterilised.' vol. it is. for I seems to be the have obtained very good results by substituting sterilised ground rice for the ginger. but no doubt tartaric. for the development of the gelatinous sheaths is that the is medium effected In such solutions as those of Hayduck. J* it presents to the " Kephir-grains " of the Caucasian milk-ferment See Husemann. is in presence of the yeast apart from it. the growth is very unsatisfactory unless either solutions as Pasteur-bouillon asparagin bouillon added. p. 159 Milk-sugar ' is of no use. although developis does occur in the first or.. and other vegetable acids would be found to be suitable. in nevertheless.t oft*. e. 4) A tall museum jar of glass (a). But even such are rendered much more suitable for the development of the Schizomycete if a little ginger is added. For a long time for the I was driven to believe that the presence of the yeast was essential growth of the sheathed Schizomycete. Pasteur-bouillon. ' Die Pnanzenstoffe. and investigation showed that the this in the ginger rhizome are untouched by the organism. &c. but does not destroy oil is several successive days. and rendered gas-tight by the compressed caoutchouc ring (6). is Into this plate is fitted the manometer tube dd\ with mercury. : by means of the following apparatus (fig. finely-sifted care is used. better. for believing that the is Ginger-beer plant would possibly do such not the case. The glass jar (a) about two-thirds filled with the solution to be employed. Another condition shall be acid. was difficult to believe that the essential a hydro-carbon. and alternative was to suppose that the case. 423. This was for a long time a puzzle to me. 1. very remarkable how much than some of the media. this by the di-hydric phosphate. though some of the It probably driven oil.g. do. because there were certain grounds 4 well in milk-sugar . footnote). IT. Pasteur. citric. could be utilised reservoirs of oil by the Schizomycete. malic. however. On ment comparing the cultures in Pasteur's solution made with the various sugars cane-sugar. its pungency. (see p. This is a point of interest. 187. Easily standing inside the jar a is a Chamberland filter-tube (e) of unglazed porce- * Based on the resemblances which. for a couple of hours on This slightly browns the ginger. it). in a stoppered bottle. and milk-sugar — it comes out very clearly that. starch in the cells was the useful The adjunct. has its edge ground flat.

PROFESSOR H. Apparatus for the simultaneous growth. but separated by a porcelain film . WARD ON THE GINGER-BEER is PLANT. manometer-tube plugged with cotton wool where the proximal g. and finally washed and boiled in sterilised water for an hour. All the glass parts are sterilised at 150° to 180° C. glass-plate . Chamber- land filter of unglazed porcelain. end jammed . This is quite effective. a. and outside . 160 lain. glass cylinder partly filled with the culture-medium . After charging . e. into the plate /.. continuous through the porous porcelain. All sterilised before infection in semi-diagrammatic section and reduced. of the yeast and the Schizomycete. e is and dd' also filled to the same level as a with the same medium. in the same medium. The liquid in b. <*-» closed by a piece of glass rod (/) held Pig. 4. The glass plate (c) is held down by double cords passing over the plate and below the base of the jar. by well- the glazed nipple (g) of which fitted caoutchouc-tubing. a stopper fitted into a piece of caoutchouc tubing. as proved by the apparatus being gas-tight even under high pressure. for several hours. and the caoutchouc parts by steeping in corrosive sublimate for several days. M. but the organisms cannot pass . and twisted (after the fashion of a tourniquet) by means of wooden pegs. then in absolute alcohol. caoutchouc ring is c.


the jar with the previously sterilised nutritive




the Chamberland tube


two successive days. and ready, a few drops of the yeast (Saccharomt/ces pyriformis) from a pure culture were put into the Chamberland tube, into which a certain portion of the nutritive fluid had diffused through the porous porcelain sides this tube was


the whole sterilised at 80° C. for an hour on


finally cooled


then stopped, by the glass rod
exterior through the nipple g.

f in

order to prevent any yeast from escaping to the

I proved clearly,


several experiments, that neither

the, Schizomycete nor the yeast-cells can pass through the pores of this tube.

Then a

trace of the Schizomycete (Bacterium vermiforme)

was put into the liquor

in the outer tube,

and the whole apparatus closed by the glass plate



attached manometer, dd', the short arm of which was plugged with sterile cotton-wool.


course of events in this apparatus


was a suitable




was always the same, provided the nutritive a distinct absorption of gas was noticeable, as
This usually

indicated by the rise of mercury in the proximal leg of the manometer.
lasted for about twelve to twenty-four hours,
of the

and was obviously due

to the absorption

oxygen by the yeast in the porcelain tube.
sets in a period during

this is

which the level of the liquid

in the glass jar (a) rises

due to the pressure of the carbon dioxide, now being evolved by the ferment

activity of the yeast increasing in the inner tube

and driving the liquid through

the pores out into the jar


At the same time

the mercury in the distal leg of the

manometer begins
gas increases.

to rise,

and continues to do

so in proportion as the pressure of the


the pressure of carbon dioxide increases, the level of the liquid becomes gradually

equalised in both tubes, and

may be assumed,

that while neither the Schizomycete

nor the yeast-cells can eome in actual contact (because they cannot traverse the porous porcelain), any soluble ingredients due to the fermentative action of either

can pass and mingle


both tubes.




I first

proved conclusively that the Schizomycete can carry on from the yeast,



life-actions separately

arid in the course of a

few weeks the

whole of the liquid in the tube

which was outside the porous tube



with cloudy clots of gelatinous masses, which were simply aggregations of the sheathed
bacterium already described, and named Bacterium vermiforme.

Then the whole

mass becomes a semi-solid

jelly, consisting

almost entirely of the coiled, sheathed


next departure dates from a discovery made when examining a tube of bouillon
This particular tube* had been in an atmosphere of hydrogen for
liquid (bouillon)

which had been carefully prepared, and belonged to a very successful and satisfactory
series of cultures.

seven days.


was found that the turbid

was teeming with


* It should be noted here, again, that I select one tube only for definite descriptions, though, as in all


conclusions are derived from the results of several parallel cultures which agree.






H. M.





so short as to be almost cocci, others several times longer

broad, others again united into long filaments.


already said, the tube was one of a series that had been very carefully prepared,




had great confidence, and examination showed that none of
others in carbon dioxide



fected fellows were turbid, while all the rest of the series

others at 25°




in air at 15°


similarly teeming with the


bacteria, after different periods in each case.


certain, therefore, that the

swarmers had developed from the organisms with which the tubes were infected, and
not from bad sterilisation or from the outside.

The following


the history of the tube in question.


had been



discontinuous heating on four successive days, and was perfectly clear

when put with

under the air-pump, after being infected.


receiver (see




of apparatus for

growing cultures in hydrogen (or carbon dioxide





cock of air-pump, under the receiver of which are the plugged tube-cultures,



U-tube with AglTO 3



pumice saturated with




pyrogallic acid solution to

rid of traces of

pipe conveying dilute sulphuric acid

(of, fig. 2, p.


matic section, reduced.

then exhausted as thoroughly as possible, and hydrogen passed in
hours the processes of exhaustion and


after twenty-four

were repeated, and the receiver tied

down with a
explained by


and the gas forced
for this

under a pressure equal to about
similar gas culture

3 inches of mercury.
fig. 5.

The apparatus
All was



then left at 15° to 16° C. for seven days.
in all the tubes referred to,


infecting material

was the same

and had come from
as follows

a source whose rather curious history was duly chronicled, and
of Ginger- beer plant, thoroughly

flask of


in distilled water,

were put into a

Pasteu&'s solution, made up with glucose.

In three days the turbid liquid contained

abundance of mixed yeasts and bacteria, and tube-cultures were prepared. Next day re-infections were made into fresh tubes, and so on till the yeast was gradually


these final separation cultures, which showed only the bacterial rodlets,


infections were





Haydtjck's solution, and in fourteen days the bottom of

these cultures contained the clots of gelatinous sheathed bacteria


so well


From one

of these last tubes I

made a


number of comparative



there was no question as to the isolated bacterium being the right one.
these cultures was


now employed.

was a tube of gelatine-ginger

solution, infected

on February 28th with a minute trace of the gelatinous cushion at the base of the
tubes referred


first clear,

the liquid went through various stages of turbidity,

and ended by


forming a similar gelatinous cushion at the base of the tube, and

on March 30th this cushion was quite normal, as in

the other tubes of the


On March

8th, a culture in


solution of the above

w as made


this pro-

gressed normally and yielded the gelatinous bacterial masses in such abundance that

the tube could be upturned

— the

whole of the contents were transformed to a


jelly of sheaths enclosing the bacteria.


this tube, of the
fig. 6,

pure sheathed form, cultures were made in vacuo





described on pp. 170 and 171), in which no gelatinous form appeared

free filaments, rodlets,


cocci (see Plate 14, figs. 6




of these free forms, after one

employed to infect a series of form was eventually obtained.

month in the vacuum apparatus, were then tubes of Hayduck's gelatine, and again the gelatinous

impossible to give even a resume of

the series between this last one and

the one started with.


suffice to

say that I satisfied myself at every stage

that the cultures were true throughout, and that, in


where the conditions

given on pp. 158 and 159 were fulfilled, the gelatinous sheathed bacteria were at length developed but where, as in the vacuum experiments referred to, the condi;

tions were altered in certain directions, the sheaths were not formed,
filaments, rodlets,

and only the
162. It


and cocci were developed.


return to the tube of bouillon, in hydrogen, referred to on


been infected, as one of a large
certain tube

with the gelatinous sheathed bacteria of a
said, after

whose history has been described, and, as

seven days in the

hydrogen, the whole of the bouillon was uniformly turbid with swarmers




to 5

filaments measured from 10

to 50


and more in length


the rodlets from

and the


were about 0'5

in diameter,

also the average diameter

of the rodlets and filaments.

Microscopic cultures in hanging drops showed that the rodlets and the filaments
alike break up.


rodlets into smaller rodlets,

and these into



the filaments

into shorter

and shorter rodlets and
all in

cocci (see Plate 14, figs. 6

the final fate of them

the bouillon, there

But while an intermediate stage when

the shorter rodlets are actively motile, and dividing with greater or less rapidity.

In drops of bouillon-gelatine with a


sugar added I was able to follow the

(retarded) division of these motile rodlets (Plate


Each rodlet simply





H. M.

in the middle,

the process.

elongates, constricts,

and divides


each, half repeats

This goes on several times until at last the short rodlets halve into two cocci (Plate 14,

6,f), which remain unaltered in the medium. I made numerous attempts to demonstrate the existence of flagella on these motile

by staining with hematoxylin, but also by the following more complex method, which is said to be very good in many cases. # The culture in bouillon is then a drop diluted with pure water, and a drop of the fluid placed on a cover-slip of 10 per cent, alcohol is added, and the whole placed to dry at 40° C.
forms, not only


dry the residue


stained with the following mixture


— 20 parts of a 10 per
made up
to 100

cent, tannin solution, 5 parts ferrous sulphate, 1 part methyl-violet,

parts with water, and used either just acid or alkaline.

or the cocci.


no case could I determine the presence of

flagella, either

on the rodlets

This leaves us in the dark as to whether such motile organs exist




true for other active forms, in which, as here, the
natural to look for


are so vigorous

it is

as producing them.



the media

which, like bouillon, induce the

development of these motile forms

only, the succession of events seems to be the same.


into shorter

and shorter dividing


The long naked filaments break which eventually break up into cocci

(Plate 14,

figs. 6, 7,



and although


have cultivated these forms



in hanging-drops,

no other result has been obtained.

Having got thus
phases of this

and having satisfied myself of the existence of two distinct organism the vermiform sheathed stage found in acid saccharine media

saturated with carbon dioxide, as contrasted with the motile naked filaments, rodlets



met with

in neutral bouillon

and other incomplete nutritive media—it was

necessary to take the precaution of cultivating both forms side by side, under exactly
similar conditions, varied one

by one

similarly for each.
it is

Before describing the experiments to this end, however,

necessary to say a
this Schi-

more about the macroscopic appearances presented in tube-cultureS of
a small piece of the gelatinous formthe compacted




of sheathed

filaments, rodlets,
(e. g. y


—of B. vermiforme


put into a test-tube of suitable nutritive

Pasteur-bouillon, beet solution, bouillon


5 per cent, of sugar, &c.)


kept at 15° to 18° C, the usual course of events

as follows




becomes more and more turbid after 48 hours or so

then a whitish film
In from "%

begins to form above, and a deposit at the edges of the level of the liquid, while a
similarly whitish, granular or cloudy looking deposit falls to the bottom.

to 14 days the rapidly increasing deposit becomes more and more gelatinous, and at

length assumes the consistency of a sort of jelly.
consists of the sheathed coils so often referred to

This gelatinous cushion at the base

the film and ring at the level 6f the

* E.g., by Woqdhead,


Bacteria and their Products,' 1891, p. 413.

is Over and over for again— that to say. 157 and 158) that the rodlets certainly do escape from their sheaths when put into a fresh supply of nutritive liquid containing oxygen It . and the turbidity. &c. probably motile. and I shall put them in order here to enable the reader to how the matter stood up to this stage in the investigation. as well as by the apparent identity in size. In the early is stages of the enquiry I missed the point that the preliminary turbidity of the liquid due to the motile forms of these filaments and rodlets. shape. from which I started in the to separate the Schizomycete moreover. the formation of the gelatinous cushion. it was not unnatural that form to be a sort of so-called zoogloea stage of my Schizomycete. and these free filaments which break liquid . members of forms then grow out into of up into motile rodlets. was further strengthened by the future behaviour of the tubes preliminary turbidity. and the film of filaments and rodlets at the top. the preliminary turbidity of the liquid by motile rodlets. was strengthened by the constant recurrence of the phases (preliminary turbidity. vermiforme. 165 chiefly and the turbidity throughout the body of the same. &c. Only two explanations of this seem namely. the other hand. and the temperature. are due to the rapid spread and divisions of its members. Ginger-beer plant. is and so occupy the is upper parts of the or (2) the only alternative that some foreign form mingled with the gela- tinous sheathed organism. are due to the free filaments and rodlets already described as escaping from the sheaths. . but on further looking matter such turns out to be the case. composed of the typical coiled and sheathed forms. was completed at the base of the tubes. are caused by the escape Bacterium vermiforme from their sheaths. by the methods of dilution. the nutritive will medium. and. because it will not flourish and hence Koch's method was inadmissible. IT. film. My suspicion that the above-described free and motile form really belongs to B. and flocculent deposit) above referred to in all the cultures.. in view of the admitted impurity of the usual supplies of first . since I had on gelatine. in that case. and other characters of the in which. and of which more # be said The actual number of such in dividual cultures amounted to over three hundred. scores of times— I found that if these tubes were kept periods varying from about tlrree to six weeks (depending on. was to be expected from what we know of other forms. film.AND THE ORGANISMS COMPOSING liquid. the atmosphere. however carefully instance my cultures were made. in favour of the first of these alternatives are so far conclusive The arguments see clearly against the second. there was always present in doubt lest I had carried over with the I should suspect the gelatinous my mind On in the early period of investigation the desired organisms traces of some form as yet unknown. the possibility of the admixture of two organisms could not be denied. Of course. the existence of a free stage. after the free forms in both cases. either (1) into the possible.^ It was further strengthened by the discovery (see pp.

L.. II.X. CO.XJL.. . the contents became uniformly throughout. G. For the sake of may 1. XVII. and XXVI.. WARD ON THE GINGER-BEER stiff PLANT. I.. XVI. XXII.. now remains media to describe a comparative series of tube cultures made during the long vacation to decide the above question certain (e.. BB. — Are the motile rodlets..X. carbon dioxide was passed in to saturation the exhaustion was then repeated.. H.X. stiff jelly.. and tubes I. K. XVIII. XXL. . F. translucent. R. and then the vessel was finally closed and put siside at . XIII. which was first securely closed. 0. The exhaustion and refilling with pure carbon dioxide gas were repeated next day.— — 166 PROFESSOR H.. p. B. of sheaths in which the bacteria were evenly distributed It and firmly embedded. Thirty tubes. M. M... IX.. tubes were prepared... Five tubes charged with bouillon-sugar and labelled K. XIV. IV. E. C. XXVIL. VII. The first tube of each group of five (i. W.. XXV. 5. XV../UL. Q. . &c. AA. DD. and fitted to a good air-pump. Y. The whole were transformed into a yellowish.. infected with the motile form. T. V.. 162. alone found in Sixty bouillon). and again pure carbon dioxide passed in till a slight pressure was obtained. comprising I. of the contents presently). Five tubes charged with bouillon-glucose and labelled VI.. Five tubes charged with dilute ginger-solution and labelled J\_. Thirty tubes. infected with gelatinous form. P. . Five tubes charged with dilute ginger-solution and labelled AA. * Apparatus described and figured at fig. X. Five tubes charged with bouillon-sugar and labelled XI. # Having exhausted it of air as thoroughly as possible. XXIV.. VI..e.2SLIA. with the typical sheathed bacterium met with ? in the saccharine media bouillon-Pasteur) saturated with carbon dioxide .) was then put into a strong glass vessel. Five tubes charged with bouillon-Pasteur and labelled XVI.. classify them as follows : Series No. identical (e. VIII. Five tubes charged with plum-decoction and labelled XXI.. XXIII.g. J.. S.. -A.... Five tubes charged with bouillon-glucose and labelled F.. .X. 2. U. In each series of thirty. comprising Five tubes charged with bouillon and labelled A. tubes A. there were six groups of clearness I five tubes each. . the other was infected with the free motile rodlets obtained in a bouillon culture. V. Five tubes charged with bouillon and labelled III. N.. Five tubes charged with plum-decoction and labelled U. and divided into two series of thirty each infected one series was from the stiff gelatinous mass composed of the sheathed form of our bacterium. XXVI...X. Five tubes charged with bouillon-Pasteur and labelled P. X. 15° 0. XII. EE.g. D. Series No. XL.VIII.

but was kept in the receiver over the air-pump and filled under slight pressure with hydrogen. V. these conclusions were strengthened by the after events. very faint films. All such events are noted. by weight. of Series for three No. (Fig. maintained - at 25° C. to examine samples of the contents of . decoction remained perfectly clear throughout the whole period of experiment. I do not know .. to XXV. AND THE ORGANISMS COMPOSING IT. behaved remained perfectly clear in air. In the ginger solution— fifteen parts. months... L. And the fourth and fifth tubes of each group of five were kept in air at 15° C. there is something to be said for the latter view. 1. worth notice that the tubes put into C0 2 and then in hydrogen also started to form these rudimentary films after being a week * These tubes were kept for several months longer and nothing occurred to alter the conclusions drawn at the time I regarded the experiments as ended.. or It should also be noted that both series and at 25° C. and The second tube of each group of five (i. at 15° C.. 167 XII. it is my practice to infect a duplicate tube at once. In some cases. .) The third tube of each group of five was kept in air in the incubator. and II. and and then the tubes in carbon dioxide and those in hydrogen had air would generally find access to them. G. tubes U to Y inclusive. as in those prepared with saccharose. to eighty-five of water —there was very fectly clear for three weeks. Q. inclusive. The tubes in air developed little growth. the and the tubes XXI. only. XVII. VII.) was treated in exactly the same way. but no further growths occurred even after three months. to be removed. in hydrogen. and the following It some time. more than one duplicate has to be made. more pronounced at 25° It is C than at 15° C. whether in carbon dioxide. on the contrary. accounts for this. those in The tubes in C0 2 remained perhydrogen the same. conclusions were drawn from comparison of the original tubes eliminate the tubes which yielded no results. tubes B. does not do so well in glucose-solution. .. in case anything goes wrong with the original one in the act of rapidly removing and replacing the cotton-plug. and some tubes with the microscope when this is done. XXII. of preserved ginger syrup. curiously enough. or an insufficiency of nitrogenous materials. or the kind of sugar. XXVII.e. throughout. Whether the want of acidity. We may first All the tubes of plum i. and then examined they were then replaced and not re-examined until fourteen days had passed again at the end of . 162. The cultures were left undisturbed for a week. exactly alike. # detailed. Be this as it may.e. the third week . three months. and pyrogallic acid. purified by passing through silver nitrate. as all the information I It will be understood that the detailed examination series occupied and reporting upon such a was necessary to compare all the duplicate sets. The further examinations wanted was forthcoming before they need not be occurred. but. and taking a sample on the end of a freshly drawn glass capillary. 5. since the bacterium. . so far as exhausting went.. p. potassium hydrate.

) VIII. In hydrogen the tubes Nothing more happened. and although the gelatinous cushion was not so large. and contained more of the swarming rodlets on the seventh day. in three days at this consisted of 15° C. resulting in the whole mass being converted into a stiff jelly . p. and then began to develop thin films. These again behaved similarly. even after three months. In first L and XII. cases —except that the tube more advanced during the first fortnight it was impossible to distinguish between the tubes infected with the motile rodlets and the corresponding tubes infected with the gelatinous coils. but the intervening liquid was not quite gelatinised throughout. free (in hydrogen) also the gelatinous cushion was developed during the week. at the same date. I. but more slowly. . but In the tube H (at 25° C. even after three months. comparing tube with tube of each those tubes infected with the gelatinous form behaved exactly like those infected with the free motile rodlets. all the other tubes became turbid. M. I confess. in advance of XII. but typical growths. 163. Nothing further occurred. After fourteen days a considerable was formed. In C0 3 the tubes A and remained clear at first. and formed the precipitate during the first week. to V. In the tubes of bouillon-sugar (5 per cent. In the tubes of bouillon -glucose — In 5 per cent. VI. .. the turbidity was due the minute swarming rodlets. which precipitated white flocks to the bottom of the tubes. B and II. striking fact is in air hut not so long as they were in hydrogen. Now let us turn to the two groups. H was So — far. rodlets and In hydrogen similarly. of brown sugar) in C0 3 the tube K became very turbid in two days. Nothing further occurred. the course of events in the corresponding tube XL was just the same.sheathed forms below its further behaviour was as described on . but.) advanced more rapidly than the tube (also at 25° C. and then formed a whitish deposit of to a less extent in both tubes. C0 2 air the tubes F and cocci. and I. in two days at 25° deposit to C. comparing tube with tube. and in five days deposit and film smaller ones were found in had formed a large flocculent The other VIII. that. . 168 PROFESSOR : H. and again L was below. it the bacteria to good growth at. A to E inclusive. by a day or two. In air. began by being turbid. short rodlets and cocci. first. turned slightly turbid. because this medium is an admirable one for most ordinary fungi) proved the worst. PLANT. In all tubes behaved quite like these. be it noted. the slight development of the organism was evidently due to the nutritive fluids being incomplete. the tubes of bouillon. and within the week had formed a large brain-like mass of the coiled. of glucose—the growth was distinctly more pronounced in both series. also The bouillon-glucose stimulated was incapable of supporting large We now come to very different phenomena. even after three months. The plum decoction (somewhat to my surprise. it was quite typical. After six weeks both tubes had a large normal cushion and a film above. and the dilute ginger-solution w as but T little better. inclusive. WARD ON THE GINGER-BEER But the most series.

I also owe much to bis making and use of the vacuum apparatus Z to be described below. preserved ginger solution. occupying about a third of the . The tubes behaved exactly like M. that gas.) were very interesting. accordingly. and I would call attention to the fact that the cushion is the gelatinous — do not form until there a large quantity of carbon dioxide in the liquids. about half an inch internal dianjeter. in bouillon -sugar and bouillon-Pasteur. have already stated that this Schizomycete can be cultivated in vacuum tubes. to XX. however. but more slowly. an atmosphere of hydrogen.. MDCCCXCII. throughout in (in air at weeks. not only that the above conclusions are correct. and beet decoction. in every respect. was drawn carefully at one end and sealed at the other. in tubes of Haydtjck's exhausted as perfectly as possible by means of a very good mercury pump. behaved very similarly to the last series. both formed so cushions. and XV. did not solidify it was similar to in all other respects. . only they did not stiffen inclusive. a fact explicable by the enhanced activity of the organism in its early stages. several points of interest are noticeable. it is In the degree coils . I have other evidence. in its free stages. Professor M'Cleod. though the cultures in carbon dioxide prove that oxygen is not necessary. but that the presence of the carbon dioxide is necessary for the proper development of the sheathed. Tubes XIX. designed to go .e. and were quite comparable with N to The tubes P T and 0. the organism can be aerobic. and XX. like that of the its behaviour was. charged with bouillonis inclusive. of greater nutritive value of these media. Pasteur. indicating the source and it is noticeable how much sooner the cushions were developed in the tubes kept (in air) at 25° C. and kept in an atmosphere of the medium gradually became saturated with carbon dioxide in those cases fact.. than in those at 15° C. These tubes in air also show that. coiled forms I i. liquid. which goes to show. — B. was inadvertently placed in air at 15° C.— — AND THE ORGANISMS COMPOSING The tubes day. prepared as follows. M . IT. evident once more that the organism is anaerobic in a high i. in addition to the proof On much looking over the records of these two double series of cultures. only. first place. Both became very turbid on the third day.* During the year 1890 I made a large number of cultures solution.. and infected partly with the Ginger-beer plant. though 15°) N five and O was a stiff jelly throughout XIII. All that concerns us here that the whole of the five tubes infected with the swarming rodlets developed the gelati- nous cushions within the fortnight. It remains to stiff throughout in from three to into five add that Tube XVII.e. the gelatinous cushion. (in air at 25° C. 169 M and XIII. and had begun to form their gelatinous cushions on the fourth In a fortnight the cushions were very In three weeks five large. and were solid M throughout in weeks XIV. Strong glass tubing. Even in the case of the tubes charged with hydrogen. and partly with the Schizomycete possible. and then exhausted as thoroughly as * Kindly placed at thetic aid in the and the sympa- my disposal by my colleague.. as where a carbo-hydrate was present—& significant of the carbon dioxide . I think. and were weeks. and XVI. much.

When finally cool. and these are sterilised is by heat carefully the glass tube m. The tube vapour. to the sterilised tube a. 170 PROFESSOR sealed. at least three successive days.m. by When the click of the mercury has reached is and remained at it for some time. and being still almost boiling. M. drawn end the large quantities of carbonic acid gas formed. then connected to the mercury pump by x. and at one time I proposed to use this method to separate it from the yeast but the internal pressures became so great. = 20° C. and the mercury contains. that several of the tubes burst spon- taneously — one its as I held it in my hand — and I had to abandon the method on its use. are also heated. absolute is and The tube/* then charged with the nutritive liquid to be used. and a vacuum produced. and plugged with sterilised cotton. they were then sealed again. a. The drawn ends were then broken by means of forceps in the nutritive fluid employed. the latter having been freshly boiled for some time. and heated for several hours on two or three successive days. the click again indicated much vapour. a\ it &. the pump obtained the gas is stopped. It will be best to follow the details in a concrete case. iv) sterilised by corrosive sublimate. The sterilised tube.30 p. the pump-apparatus was again set going the vacuum was . is After a few hours the exhaustion . the apparatus shown in fig. H.wool (w'). was infected on September 11th with a trace of the white gelatinous cushion. The joint completed by filling m with previously heated a is mercury. very good. The Schizomycete increased considerably in those tubes infected with the Ginger-beer plant. On September 1 2. and the whole kept at 80° to 90° C. It was then left until September I On starting the pump. 3. (w/). The liquid in^is then and the tube plugged is and joined a. while vapour was being driven through the drawn necks. nothing but vapour being obtained: Repeated the exhaustion 14. repeated. These successive exhaustions are continued at intervals of six to twelve hours for three days. but another plan was finally adopted instead. filled with equal volumes of Pasteur's solution and bouillon. WARD ON THE GINGER-BEER PLANT. account of danger. they were infected. being finally warmed to drive out the last traces of air its best. or longer. they were finally sealed. in the evening — same of mercury placed over the escape-tube of the pump found two divisions of gas in the . &c. on infected. The tube collar f is The detachable from the piece a. at the bottom of a pure culture of Bacterium vermiforme. to the tubes. as follows : With Professor M'Cleod's assistance. for several hours. two tubes (near boiling. the tube a. vapour and a trace of gas being totally absorbed at once by potassic hydrate.— . as also the cork is h piece of caoutchouc which joins the alcohol. and then the culture is left to itself for periods of twenty-four to forty-eight hours. but on examining the graduated catch. and exhausted very thoroughly at Temp. 6 was made. and again attached to the pump and exhausted. owing to . a\ also plugged at w. It is true I have occasionally continued with such precautions as wire-gauze jackets. then.

a'. lessened the loss somewhat. a. and m. 6. caoutchouc-tubing connecting the tubes a and/. so I placed a small flame near the tubes. was all absorbed by potassic hydrate. a loud sharp click being left each time. glass tube with mercury and making a gas-tight junction over beaker containing water Gr. The exhaustion was repeated in the afternoon. glass tubing attached to mercury pump beyond x . and more vapour obtained. a. a bulb condensed vapour /. thermometer. v 9 glass h cork-ring.AND THE ORGANISMS COMPOSING eudiometer tube employed to collect it. IT. w. similar plug in the sterilised s . T. 171 This gas apart from the condensed vapour. . but distillation into trifle f by keeping the temThis perature of these distal parts of the apparatus a it higher than that of f. On September 1 5. indicating a good vacuum. and caused a slow backward device only partially succeeded. The loss of vapour was now telling considerably on the quantity of liquid in the culture-tube. and a Apparatus for cultures for in vacuo 9 : a. this is placed over a small burner connected by tubing with the gas-regulator thermometer. 6). and b (fig. Similarly on the 15th. c. . w cotton-wool plug in the sterilised tube a. tube 9 which contains the culture growing at filled c. I could distinctly see that the mass of organism was increasing .

On October . 3. temp. I stopped the experiment at the point referred to. It will be noticed that the total volume of carbon dioxide obtained amounted to I carried more than sixty -nine divisions. = 10° C. &c of the bacterium — an On assumption which the sequel shows was impossible. On the 17th. The apparatus was again left quiet until November 3. # The temperature of the tube / was allowed to fall to 10°-12° 0. five more 5.* The gas was allowed to accumulate in the catch-tube from the 5th October to the 20 th the total volume was just over twenty divisions. on October five divisions on the 6th. gas was present. divisions of . the pump again brought off carbon dioxide. that although the organism had been in the apparatus. M. off. Temp. still = 10° C. because I wanted to answer two it questions with regard to which doubts might possibly arise. and that the carbon dioxide . now or at any other time. C0 2 were pumped on October . and the apparatus alone until November 14. and that more would have been obtained had the experiment further. however. thers was no other kind of gas evolved. coming off was due to decomposition of the proteids. fact that I might possibly be owing to the it the carbon dioxide. During in all this period a slow but quite perceptible growth had been going on daily in the tube the culture medium f 9 and there was now a flocculent cloud extending half-way up the tube: this evidently corresponded to a material increase of the organism. and on again exhausting . I collected eighteen divisions of carbon dioxide. and evidently would it have yielded much more. I pumped off five divisions of carbon dioxide. I obtained a few divisions more. I obtained eleven divisions of carbon dioxide. should be stated. for more than six weeks (September 11 to had enormously increased I argued that this off in volume. four divisions on the 7th. . eight divisions divisions. WARD ON THE GINGER-BEER PLANT. On November the 18th I 14. between the 8th and the 20th.172 inf. it was noteworthy had been drawing that no characteristic gelatinous cushion had made its appearance. only I stopped after a few divisions were drawn off. owing to my I finger slipping and letting air into the measuring tube. PROFESSOR H. almost as fast as was formed. and weight. on that day. pumped out thirteen divisions of carbon dioxide. and that probably from what occurred in similar cultures in an atmosphere of carbon cushion would form if I let —judging dioxide — the that gas accumulate. at the expense of the nutritive materials in the medium — unless we assume that the increase was a mere volumetric one. On November 9. two and so on. and presumably in November 18). and deprived of atmospheric oxygen. On September and 30 —the apparatus and contents having remained untouched during pump at once the interval— the starting of the showed that much. left now (November 9th) again raised the temperature of the culture to 25° C. First. but did not measure them. 1.

I sealed off the it tube f with a blow- pipe flame # just below the cotton. I wrapped its this one in wire-gauze. the loose. does not kill the bacterium in the vacuum or other cultures. 6. 170). and put them all C. 5th I held the point of the sealed tube in the flame of a Bunsen's burner until and noticed a sharp puff which blew outwards a minute hole I in the side of the tube. 173 Since it would not do to leave the apparatus as was until the pressure of gas dislocated the mercury junction with the 9 pump. and on December soft. with sterile check- tubes by their sides succeeded. Prompted by previous and experience with sealed tubes (see p. placed it in the incubator at 25° as said. possible that light has some effect on the cultures under in the point deserves special investigation.— AJSTD THE ORGANISMS COMPOSING it IT. In a few days the typical gelatinous cushion made appearance. lower part of the liquid. be concluded that this remarkable Schizomycete is to. The two questions were: tube referred to above could now be answered. able to live and grow in is an acid saccharine in solution. tained the idea of using the method separating the bacterium from foreign organisms for pure cultures. cloudy. not only so an atmosphere totally deprived of oxygen. and that no burnt cotton-wool interfered with this operation. sowings from this tube into ordinary tubes of bouilloninto the incubator at 25° Pasteur (twelve in pp. and in no case found the Schizomycete killed by even more continuous exhaustion than these. the tube also C0 2 C. but was capable of forming the typical sheathed form. but in one of vapour. and followed the normal course described on Clearly the organism was not only alive. . f I say no more on this subject. at least. with suitable minerals and nitrogenous materials. and I have experiments hand. On November the 18th. 166-169. and vapour of water. all this — (1) Had (2) the organism really lived and grown The questions time in the vacuum% f% and Would it develop into the typical sheathed form of the Schizo- mycete if left in the atmosphere of C0 3 produced by its own metabolic activity Both questions received their definite and satisfactory answer. It must. slightly flocculent mass extended about half-way through the the tube . . because certain conditions .! It ought to be added that I have confirmed these results by several experiments So successful were such cultures that I enterfor with the above apparatus. interesting also to note that the exposure to ordinary daylight. and thus were answered the two questions referred therefore. which half contained a partial pressure of filled as we have seen.wool v/ and put aside at 25° C. then made a series of all). and which it is attenuated that practically a its vacuum — so far as if permanent gases are concerned is but that it only forms It is gelatinous sheaths carbon dioxide present. # It should be noted that there was more room below it si w' than appears in fig.

C). in it would be premature to propose a sterile name for it as a new it species. were sown on January 10. rapidly passes through the filamentous and bacillar stages (which may be motile) to the formation of it the endogenous spores.) . At 9 p. with It grows readily on nutrient gelatine. Recorded in my notes as the Ginger-bacillus. measuring about 2*5 /x. prepared so that in Pasteur. many of the rodlets were swelling up. 5. In such cultures it formed a dense whitish membrane on the surface. 5. D).m. (Plate 15. and passing through fall all its stages to the development of which to the bottom of the cultures. Cultures of these filaments on gelatine showed the breaking up still more clearly (fig. forming the oval spores 5.. The germination of the spores was glucose. M. and broke up into zigzag jointed rodlets. or blunt spindle. 1. figs. to which lumps of unsterilised ginger were added. WARD ON THE GINGER-BEER Schizomycete No. they had germinated and given rise to rodlets and filaments. the gelatine. 3). but desire to put forward this explanation all reserve. as traced in a hanging drop of Peter's gelatine. of glucose. 2. I attributed the difference to its inability to invert cane-sugar. in some of the separation and suspicions aroused by the frequency of this form in fermentations.asparagin no inversion of the sugar occurred. breaking up into bacilli (fig. about 1 to 1*5 [i broad. and forty-hours later these bacilli were replaced by definite spores frequently in twenty-four to forty-eight hours. (fig. B). 2) into bacilli. PLANT.m.174 PROFESSOR H. 6. composed of closely woven and often parallel filaments. and well-marked Schizomycete. especially with 5 per cent. have not been able to definitely identify this form with any known species of its life-history Schizomycete. rapidly liquefying spores. fig. on the next day. These filaments were septate (B). I was led to look for the bacillus on ginger- From rhizomes. before developing the oval spore. Grown Pasteur's solution with ginger and asparagin. . it appeared Hence the name Ginger-bacillus. 1-6. X 1 to 1"5 //. and found that on putting these into sterile glucose solutions. and the culture placed in the incubator at 27° C. others were in the stage of filaments. fig. The spores (fig.) In the early stages of the investigation I was much concerned with a very pretty cultures. on January (fig. still and. and showed that the filaments swell to an elongated oval. A). as described. the latter disjointing into rodlets (fig. but I failed to grow alone. I also observed in a hanging-drop of gelatine- (Plate 15. and of various lengths (Plate 15. but as I regard the investigation of still and properties as incomplete. 5 I have recorded the chief stages in the life-history. In fig. These spores were easily obtained after bacillar rods or five days' culture on gelatine-glucose (fig. 5.. met with easily cultivated on gelatine. at 4 p. 4). 10-12 p or more in length (A).m. A and B). At 10 p. 12.

The latter recall .). Soc. ginger-bacillus some resemblances to Bacillus sizes of the subtilis. it 138) and so I abandoned the was necessary to concentrate presents my energies on the essential The above seems to different.. in 'Journ. that the bacterium of the acetic ferment forms a well developed in and even massive zooglcea-like skin # See Hansen. pp. which only differs in that it turns blue in iodine its owing to the presence of a starch-like substance in the matrix surrounding ' protoplasm. Beown. 7-9. looking like miniature honey-combs.). pasteurianum. vols. however to say nothing of the recent discovery that there are at least two physiological forms (races or species) of the organism. 45 These facts are. That in it is an aerobic form was abundantly proved by the failure of all cultures C0 2 . will be readily understood it soon became quite certain that they had nothing to do p. The acid smell and this reaction referred to on p. and the shapes and sporogenous rods and spores are The is sizes accord better with Cohn's Bacillus ulna. but produces a greenish shimmer in the My reason for not attempting to thoroughly unravel the life-history of these forms . unless glucose was added. On Peter's gelatine it did remarkably well. leaving questions as to its fully name and position until its characters have been worked out. but the oval sporo- genous cells are different. and Hansen's B. and eventually developing spores as described. 3. of Chem.. I confounded this During a certain period of the preliminary cultures. Also A. figs. the specimens of Ginger-beer plants sent to me. and the curious spores and germination seem to accord. Die Mikro-organismen der Gabrungsindustrie/ 2 Aufl. the ordinary Bacterium aceti (KtiTZ. forming thick grey. of a small bacterium (about 1 to 1*5 long. Schizomycete No. somewhat larger. 49 and 51. but me larger. It also failed in vacuum tubes. form with another which also liquefies the gelatine.white crumpled films. 127 are principally due to this intruder. Bacterium In all aceti (KtiTZ. Two facts made it . 175 grow it on yeast-water gela/fcine. necessary to undertake the further investigation of Schizomycete. it seems wiser to be content with merely recording the presence of this form. and 0*3 to 0*5 [i broad) which turned out to be the well-known bacterium of acetic acid fermentations. ' which yeast-cells and 2. te 1879 (quoted by Jorgensen. matrix. 43-47). viz. I found varying quantities ju. Prazmowsky's Clostridium butyricum.' 1886 and 1887. 1890. all kinds of organisms may * Medelelser fra Carlsberg Laboratories' H. as forms. though the sizes of mine are On more the whole. but that markedly anaerobian the habitat would do very well. (Plate 15. all with the formation of the Ginger-beer plant (see further pursuit..) AND THE ORGANISMS COMPOSING I failed to IT. first. .

. the bacterium was comparatively easy to isolate the Schizomycete by placing it it in solutions too all acid to permit the development of the yeasts. in the second.and flask-cultures. but I Mr. so strong that it pervaded the whole laboratory. and cases of the latter kind of impurity rarely occurred. or of preparing food-materials. not only that bacterium it not a normal or necessary constituent of the Ginger-beer plant. nearly the preliminary cultures and the samples of ginger-beer I examined went "sour" after gained access to them. it was. . very easy to compound mentioned when repeat the phenomenon by adding life-history of the two organisms to a saccharine solution. and I found to agree in respects with the well-known descriptions given by the chief authorities. milk.g. was to be expected that various casual bacteria would occur as intruders in the very numerous tube. with the properties and itself. compared it with pure cultures obtained by the kindness of Not only so. In some cultures of the 1889 and 1890 series especially. Other Schizomycetes. neither the bacterium nor the yeast was able to produce the working alone. M. and the bacterium in question. secondly. indeed. and such was the case. but I. in the first place. These were not numerous. established that while. however. and the sourness was found to be accompanied by this bacterium. all become imbeddeda time if air —the so-called "mother all —and. which. like others which turned up during the investigation under consideration. seems well worth the attention of the biological chemist . It was impossible for me to go further into this phenomenon. and due to acetic acid. WARD ON THE GINGER-BEER of vinegar" PLANT. I observed a very curious and penetrating odour of acetic ether (ethyl beneath the bell-jars covering them. acetate). as could be detected. and from This was found to be due to the co-operation of p. Some of these were obviously forms introduced as impurities in the process of opening and examining the It tubes. except with certain media. We It are more particularly concerned. who this has devoted much attention to this It suffices to is species. but that cannot be induced to form a submerged commensal growth with any of the yeasts. and so forth. plums. at least. It it is so markedly aerobic that no wonder can now be entertained at point. Adrian Brown. however. consequently the mixed vapours of alcohol and and combined to form the very fragrant ethyl acetate. " was necessary to test the mother of vinegar present considerable likeness to the gelatinous investment of the Ginger-beer plant. and ginger.. and was explained as due to the yeast producing more alcohol than the bacterium could completely oxidise to acetic acid acetic acid escaped together . add that subsequent cultures demonstrated. because the dense skins of the " this . very difficult to sterilise in the summer e. escaping through the cotton-wool plugs of the the very small and white top-yeast (see yeast. as already quoted. flasks.— 176 PROFESSOR H. by the smell in old cultures. decoctions of beet. 149).

seemed to do best beyond observing that It was always closely assoit. and appeared I to constantly reproduce in the micrococcus mode. One of the commonest of these. nothing definite came of it. although I tested the idea to some extent. which were very I have only mentioned the possibility that these gelatine. and figured on Plate (%. traced to the Coventry specimens. which grew rapidly on causing a putrid smell. bodies. quite independently and on other grounds altogether. like that of burnt or smoked some attention in 1889. with the remark. and confine my remarks entirely to forms really carried in with the specimens of Ginger- beer plant. forms. A second Schizomycete. though by no means a constant accompanimentj of 1 was a very minute micrococcus. In some of the earlier cultures in ginger solution. but a regular mycelium. liquefying it. nor would it grow oh that substratum to any observable extent. it was unable to render any account of in the cultures in beet -gelatine. and even suggested parasitism. and was much contaminated. though they only occurred once — each in a set of cultures which gave stituents. forming Ascococcus-like colonies in the matrix some of the hanging-drop cultures of Bacteriumm veriforme. formed the opinion that the two are quite different. was a slender fila- The coccus itself mentous form. I paid It was associated with a fragrant to it smell. — b. and It I dismissed it after identification. Coventry. which formed yellow patches on yeastIt water gelatine. It did not liquefy gelatine." mdcccxcii. was less than 1 /x in diameter. was an extremely minute form. who sent me specimens of the Ginger- beer plant. of course. was not unlike that observed in some specimens of ginger-beer it was so assuredly not conits cerned in the building up of the Ginger-beer plant. 2 A . ciated with the matrix of B. and lost before it could be separated. however. the colonies white. blance to that of the Actinomyces. including even pathogenic The same idea occurred. aurantiacus (Schrot. "I was rather surprised to find not only torula-like which in cover-glass preparations bore some resemI have. a beautiful micrococcus in long was met with. me trouble in connection with the isolation of the con- One of these was a rather large micrococcus. Two other forms may be just referred to. vermiforme. came in specimens of the Ginger-beer plant obtained from The other was a small micrococcus. handed from family to family as them all kinds of microbes. was almost certainly M. 177 any further account of such forms as were traced to these sources. only found chaplets in the earlier cultures.). lumps of Ginger-beer plant. and that the fungus has no connection with this disease. sugar. because the fragrance .5 AND THE ORGANISMS COMPOSING I neglect IT. however. and also It only occurred in one set of cultures in 1889. but. may carry in to a medical man in the midland counties. two last forms because they impressed me with the they are. that behaviour was not followed further. It ii). also of comparatively rare occurrence.

Proof of this may be obtained by anyone who places a solution of ordinary sugar and water. of course. figs. investigation. pasty masses of cylindroid-oval cells. when my mind was clear of any preconceived ideas. which.white. happily. common sugar and ginger direct from the grocer's. pieces of paper or cloth.) To the ingredients employed ordinary unboiled spring-. and. of a household — the walls of air. Higher Fungi occurring as Impurities in I have now concluded the biological analysis of the Ginger-beer plant. As matter of fact. and remembering the tions are carried on in country houses. well-." that is Looking at all known of the lumps of Ginger-beer plant. I never get into the mixtures and contaminate had a specimen of the Ginger-beer plant sent to I attribute the me that did not contain several species of these mould-fungi. and the commonest foreign yeasts and Schizomycetes are concerned. I will first take a large yeast-like form. (2. I have if by no means attempted to exhaust the list of forms that might be observed but since one or two one made a systematic search through the specimens . 12 and 13. which (3. different as examined of a number of higher mycelial forms of in detail.-M. Plate 15. for fortyeight hours or so the brew swarms with yeasts. and exposure to as the specimens are passed from one person to another. it way the ordinary fermenta- might be predicted off-hand that kinds of spores of common mould-fungi would them. with delicate walls and vacuolated protoplasm. pipkins. bacteria.— 178 PROFESSOR H. WARD ON THE GINGER-BEER PLANT. think the suggestion was decidedly worth the turned out to be wrong in the present instance. however. so far as the essential constituents. It remains to place on record. interesting species turned up during the earlier stages of the enquiry. it may be worth while to record what was made out about them. which was particularly abundant in the It is figured on separation cultures of one set of specimens examined in 1888. with a lump of ginger in it. were either isolated or united in . Contact with the hands. — kinds. The last sentence refers to my correspondent's suggestion (in a previous letter) that his a patient of his who gave him the specimens might have contracted I it malady (which turned out to be due to Actinomyces) from partaking of ginger-beer made with the Ginger-beer plant. they are may all be regarded as ef the nature ef « naeulde. the Ginger-beer plant. unsterilised. of course. the existence in the specimens fungi. and how they all are handed about in the fresh state.) To the vessels —ordinary jugs. It occurred as yellowish. and mould-fungi of various . &e. or tap-water. though.. jars. and ready to receive any form as possibly concerned in the constitution of the Ginger-beer plant. or thick deposits at the bottom. never biologically clean without special treatment. &c. and in a rather warm room.) are. forming dense These cells films on the surface of the cultures. in an open vessel. : presence of these intruders to three chief sources (1.

2 A 2 . lactis. were umber-brown in colour. On and a It sowing these cells again in Pasteur's solution. One of these cells. . germinated in a rise to a few hours. isolated in a hanging-drop of peptone-gelatine. feeble alcoholic fermentation was induced. fig. Gesammt-Grebiete der Mykologie. which. I have not obtained it is development of that Oidium mycete # 9 fungus than the forms described. '"[Inters. aus d. possibly of a Hymeno- In form. fig. and in ginger- Pasteur. 1889. is from odd specimens of the of the form long known under the name Dematium pullulans (De Bary). had attained the relatively large dimensions y. 5. or had protuberances budding from the sides of their ends. descriptions of and general behaviour. giving mycelium. Pasteur-glucose. 144) to have already referred (p. that I have no hesitation in referring it to that is all the species. heated with hot water to 20°-25° C. by abscission of its joints. three. it stood fully exposed to light. my failures to confirm Cienkowsky's suggestion Oidium and Mycoderma are identical. fig. which on examination was found to consist of countless numbers of cells like those represented in fig. into the cylindroid yeast-like cells started with. viii. they again behaved like a yeast.. especially 2. It seems likely that a thorough investigation of Oidium lactis would repay any competent worker. the hyphal form was interesting to note that this penetrated into and partly liquefied the gelatine. others. and in forty-eight hours was breaking up. fig. 179 imperfect chains of two.' 6. 4. growing on gelatine. in twenty-three hours from the moment of sowing. In about a fortnight the brown colour was when the much deeper. They * See Plate Brefeld. size. It would be that especially worth w hile to see T if 0. and within the month the whole mass was covered with a dull. H. and placed as said to see if the Schizomycete could be induced to form spores in the interior of the lumps. as seems to be the case. in tubes of nutrient gelatine. It first turned up on some lumps of Ginger-beer plant which had been plunged into boiling water and put aside in a dry sterilised flask. the buds breaking off as soon as formed. 14. plant had been washed in boiling distilled water. 22 . A.AND THE ORGANISMS COMPOSING IT. 13. Another of the queer mould-fungi which Ginger-beer plant. As there seen. The pieces of Ginger-beer cotton.wool. plugged with sterile This flask was placed in a glass cultivatingrchamber. some of the cells were almost colourless. like the budding of a yeast (Plate 15. surrounded by a firmer wall. but any higher by no means improbable is merely a growth-form of some higher fungus. or more. shown at Plate 15. unsatisfactory as the form I from the points of view of modern biology. this fungus agrees so well with Oidium lactis. Plate 15. fig. In two or three days a brownish-yellow hue appeared plant is — as is usual allowed to dry. Plate 25 j Plate &c. produces I isolated lactic acid. 12). dark brown film.

as The arrangement was at b and c. ellipsoidal or sausage- shaped some were hyphee. and showing that the specimens so produced act like the original specimens. had to abandon and the GingerIn addition after satisfying myself of the want of genetic relations between its life-history it beer plant. in a hanging-drop of Hayduck's glucose-gelatine. I For the sake of completeness. and so * The brown cells average 8-10 jm long by 6-7 /t broad. This I have done. in the same figure. in a hanging-drop under On the microscope. in connection with them. 558 and 581). others some //. septate. septate. on February 14. I have depicted the behaviour of a more complete culture obtained by These cells grew readily in ginger-gelatine. as described on p. and others again had All colourless about 3-4 diameter.m. pp. . Interestingt as the further pursuit of this form would have been. sowing the segment a at 4 p. The most conclusive proof of the accuracy of the foregoing studies. Similarly with the series d to g and x. WARD OK THE GINGER -BEER in chains of three or more. colourless buds in gelatine (which they /a long by 1-3 /* broad. rapidly. The details of the development of these buds were also traced (g to I. and in a little more than two days had formed a copious mycelium (/) throwing out buds from numerous points of the older segments of its hyphse. de Hnstitut Laurent See f Pasteur/ vol. in PLANT. where he claims Bematium as a mere growth form of this fungus. is Beyond having proved that it is capable of life-history developing a mycelium. Synthesis of the Ginger-Beer Plant from Pure Cultures. It germinated shown at about 17° C. that Penicillium glaucum was a frequent and that a Mucor (apparently M.) Elfving asserts that several races of growth-forms can be got from this fungus by altering the conditions. in size* and shape. 1. much . M. and the further development of the specimen fig. 1888. consisting of isolated spheroidal cells varying in colour from brown to green and greenish -blue. " Becherches sur le Polymorphisme du Cladosporium Herbarum " (* Ann. and only occurred occasionally. I it. 2. must evidently be afforded by my being able to re-constitute the Ginger-beer plant.) and a full description of the figures is given with the plates. oil the darker ones and most of the others contained much in the form of drops scattered through the protoplasmic contents. by bringing together pure cultures of the organisms composing it. 131. racemosus) made its appearance once. which of these. impurity may add in the Ginger-beer plant. some being spheroidal. Plate 16. 1890. to the above. I have not gone further into the They were obviously intruders.180 varied PROFESSOR H. Plate 16. as such. 1 hope to follow out at a future opportunity. a (Plate 15) was traced in this medium. I have at various times isolated a curious form — or perhaps series of forms— of Torula. Laurent's results are particularly interesting in view of the similar results with a totally liquefy) average 2-8 different mould-fungus (Eurotium Aspergillus glaucus) by Elfving (' Studien ilber die Uinwirhing des Lichtes auf die Pike? Helsingfors.

cells. which lives on the debris of any haphazard yeast-like form in the It medium. I determined to attempt to build up the is the essential yeast compound organism again. In the more suitable saccharine media. or abeyance of the latter. with each of the bacteria or other Schizomycetes itself into which forced the foreground of the experimental cultures. I infected them in the ginger-bacillus. \ but in no case with success. With these ideas in view I started with Mycoderma cerevisice. e. in carbon dioxide. it (and dissolved in the liquid) In neutral gases. and (3) Bacterium vermiforme. I then tried cultures of Oidium together with the above forms. 181 completely that no further doubt need be entertained as to the biological nature of the phenomena concerned. the Mycoderma rapidly succumbed. to pure cultures of this Saccharomyces. moreover. but soon became conto the bottom of the cultures. that Mycoderma is incapable of inverting cane-sugar but must not be overlooked that such inversion might be brought about by the was pretty clear. in Haydtjck's solution. in order that there should be no stone left unturned in the research. by adding pure cultures of the different Schizomycetes.AM) THE ORGANISMS COMPOSING IT. is Mycoderma The at. a conclusion fully borne out by the sequeL lactis. each of the yeasts or yeast-like forms which occurred commonly in the investigation.g. from these experiments. that there might be more than one combination capable of existence as a dual or symbiotic form. that the Bacterium vermiforme is not a mere saprophyte.. but with negative results in all cases. I thought it not impossible. At the same time. and remained dormant variously. The verted into spores. (2) Bacterium aceti. After satisfying myself that Saccharomyces pyriformis concerned. I also brought together. in turn. some growth of the Mycoderma was observed. in turn. it often commenced cells. and in vacuo. it perhaps not to be wondered . form the sheaths. and in Pasteur-bouillon— but no symbiotic relations were established between these and the death. But. The strongly marked aerobian character of the Mycoderma was itself sufficient to make the attempt almost hopeless and I found that in no case would the cultures endure being bottled up away from the air. when we remember Schizomycete. in succession. in such solutions. turn with (1) Having prepared pure cultures of this. but only at the top of the liquids. evidently only so long as the air in contact with lasted. which fell The acetic bacterium died rapidly when ginger-bacillus grew at first. among the dead and dying Mycoderma The Bacterium vermiforme did not so rapidly succumb. . but generally broke up to eventually into short rodlets and cocci. and never formed the typical cushion-like gelatinous masses of successful cultures. The Schizomycetes behaved continuously submerged. and .

9) which seemed to promise more success* Nevertheless I was unable to produce satisfactory clumps of the Ginger-beer plant . was interesting to note. able light on this most remarkable dual organism—the Ginger-beer Never- theless. however. mixed with bacillar rodlets. on the one hand." which. T have named Saccharomyces company with the ginger-bacillus. that and it when this form was sown with Bacterium access. In some cases the latter seemed to be suppressed altogether. though in others the Schizomycete formed numerous spores. and died. that a sort of symbiotic . in the The results obtained by cultivating together. however. in virtue of its fermentation powers. WARD ON THE GINGER-BEER air. developed no gelatinous sheaths. the latter by the rapidly dividing bacteria (Plate 14. No trace of anything like the Ginger-beer plant could be detected in any of the cultures. and the bacterium grew ou' long rods and filaments (Plate 14. side by side. moreover. fig. same medium. M. on the top of the aerated liquid which the yeast produces alcohol. the two forms. 148 and 149. because. turned yellowish. one of the products being the fragrant acetic ether referred The only other yeast of importance was the one piriformis. PLANT. the bacterium oxidises more or less to. In cases where I employed bouillon as the medium no appreciable success was obtained. and consequently no gelatinous lumps *were made. which could be detected. seemed to rapidly succumb to every attempt to confine it from the free access of One series of attempts was also made with the small white aerobian yeast. but throw considerplant. the skin formed by Bacterium aceti is strongly aerobic whereas the gelatinous cushions of B. aceti. life is led by the two organisms. 176). The yeast-cells budded very slowly. again without success.brown. and although they became enveloped 10).182 PROFESSOR H. they formed the acetic ether referred to viz. on the failed : other. In bouillon-glucose the yeast did much better. referred It to on pp. This yielded no results in when sown at the end of the fermentation (due to the yeast alone) in the deposit of yeast-cells. have said that I was attracted at an early by the notion that the dense gelatinous skin (zoogloea) which the acetic bacillus forms on the top of vinous media presents some resemblances to the gelatinous masses due to the coiled sheaths of the Bacterium vermiforme. fig.. to which the air had (p. vermiperiod in the investigation forme are formed best in acetic bacterium submerged with yeast simply fell — carbon dioxide —and. Complete the failure also attended every attempt to combine the acetic bacillus with I Saccharomyces in question. on saccharine media. Similarly with the "rosy yeast. were eventually so successful as not only to exceed all my most sanguine expectations. completely. also to Saccharomyces pyriformis and Bacterium vermiforme. was this which led me to the conclusions already stated. it is necessary to fulfil certain conditions in order that the synthesis referred to may be established. all attempts to grow the the rodlets of the acetic Schizomycete down in the closed tubes. This notion had to be abandoned.

This enhanced activity was not shown merely by the more active budding of the yeast-cells and the growth of it also manifested itself in the steady and continued evolution of the bacterial coils : large quantities of carbon dioxide. the yeast had already finished its fermentation of its own culture-medium the primary fermentation was over. though temperature. led to some observations which seemed instructive. affects the question. p. was that both yeast and bacterium seemed to gain in activity as soon as they came into direct contact in the embraces of the latter. 11). Things were very different in those cases where bouillon-Pasteur was employed. as also do the media. of course. and the medium exhausted. I found. typical lumps of the Ginger-beer plant were developed in a few days. differences in the mode of culture of the two samples. and then added a few drops of a pure culture of Bacterium vermiforme. 160) how successful were Bacterium vermiforme in tall glass cylinders. but not for soluble matters in the liquid) impenetrable porous porcelain wall of the I found. In the first place. Now we are justified in inferring medium was yeast-sick from all that is known of such fermentations— the ! . — If the reader chooses so to express it. will. in fact. that if I prepared pure tube-cultures of the yeast (S. The experiments just described. and often buoyed them up to the top. 4. bubbles of which rose from the lumps of Ginger- beer plant. even after several weeks. when compared with some its my previous attempts to re-construct the Ginger-beer plant from constituent organisms. coils of the sheathed bacterium entangling the yeast-cells (Plate 14. but the most extraordinary result.AND THE ORGANISMS COMPOSING as such. Chamberland tube. if I now. and I pointed out then that the successful development of the bacterium was probably due to the culture-medium being simultaneously affected by the action of the Saccharomyces on the other side of the (for the organisms. These lumps were formed by the fig. on a method which seems as if it would enable one to produce almost the cultures of the filters any desired weight of the Ginger-beer plant at have already described (on p. it only required a few days in these cases to make the yeast and establish the the bacterium effect their symbiotic union. except in one or two cases. it at last became clear that some of these partial or temporary failures were due to the fact that at the time I added the bacterium. in this IT. the union Putting aside all cases obviously depending on of the two was often delayed. the Chamberland tube (see 160) with a little of the yeast grown apart inside the Chamberland tube. that infected the liquid containing the bacteria on the outside of fig. whence they then descended as of the gas escaped from their surfaces. pyriformis). which had then ceased and at I last I hit to be interesting on account of success in other directions. 183 medium. to my mind. containing Chamberland charged with the Saccharomyces piriformis. whereas I had found that in cultures in ordinary test-tubes union. it often needed several weeks —three to six— to This was not due to difference of temperature.

and the numbers of Schizomycetes added. as they accumulate undergo further alterations which entirely alter its nature. has been shown that S. in the altered seems impossible to avoid the conclusion that the delay in forming the symbiotic union. in direct contact with the yeast-cells. and at the end of the fermentation . Everything points to the view that the relations between the yeast and the bacterium are those of true symbiosis. unstable bodies w hen they T first leave the yeast-cells. it time is needed might be simply due to the quantity of bacteria being so small that for their multiplication in the medium. culture of the yeast. seem at unfit for the first sight to preclude the idea that the medium is rendered growth of the Schizomycete by the soluble products of the fermentation due to the Saccharomycete. It is significant that the synthesis of this dual like a organism —which is so strikingly Lichen that we easily may compare it forthwith to one of the gelatinous forms was most brought about by adding the yeast-cells to already advanced cultures . and for the development of sufficient of the sheathed coils to fully entangle of yeast. is when the bacterium difficulty is added to an already advanced due to some which the Schizomycete meets with :- medium.cells. WARD ON THE GINGER-BEER PLANT. and embrace the large supply Secondly. But it must not be overlooked that these products may be very different at the beginning be.— — 184 it PROFESSOR H. it might be due to some inability on the part of the Schizomycete yeast-cells. can take them at first-hand. effecting and I could detect no correspondence between' the time occupied the symbiotic union. pyriformis induces an alcoholic fermentation to —that they fermented come an end owing to the inhibitory action. on the .g. or decoctions of such. since these products must have diffused through the porous porcelain. of the products of fermentation liquid. because every attempt to feed the Schizomycete with dead yeast-cells. This difficulty might be one of the three following Firstly. Thirdly. because similar delays were met with if I added larger quantities of the bacteria to such in yeast-cultures. or to detect it embracing such cells in a dead or feeble condition has failed. and that these products. whereas the Schizomycete only enters into symbiotic relations with active yeast-cells. In the latter event they might e. become the bacterium growing at a distance from the yeast on the distal side of the porous septum —than they would be when newly formed. M. containing the yeast. It is less The successful cultures of the bacterium on the outside of the porcelain tube. and when the Schizomycete. is to assimilate substances left in the I medium by the first came to the conclusion that the explanation not the right one. it in the In view of the above. which alter as they pass out into the surrounding less useful to liquid. it might be due to the fact that the yeast-cells are now in an exhausted condition. yeast-cells. and that they may and probably are. easy to decide with respect to the second and third possibilities.

separately. they leave the sphere of metabolic activity of the yeast-cells it can benefit by the presence of these substances even apart from the living yeast. In addition to yeasts of various kinds and several species of bacteria. I have proceeded some distance in this direction. which have not as yet been undertaken systematically. almost entirely to elucidating the morphology and physiology the dual organism the biology is — of known as the Ginger-beer plant. and under and the previous considerations. —the yeast and the bacterium—by to the Lichen-like but still more the symbiotic fermentation due little compound organism. though to a less extent. led me : to the conclusion that we must look on the symbiosis somewhat as follows The Schizomycete is favoured by obtaining some substance or substances directly . IT* 185 like grown in the same medium. but only sufficiently far to show that in addition to the large quantities of carbon dioxide evolved. The yeast. until we obtain some know- ledge of the products of the several fermentations. that I have inverted the order of events might be objected it — that. and compare those formed by the two organisms as a whole. it may be pointed out that my researches have so far been directed i. with those developed by the symbiotic Ginger-beer plant Conclusion. both having been conditions. may be its that its powers are enhanced by the yeast-removing inhibiting substances of activity. the ginger- beer contains traces of alcohol and acetic acid during early stages of fermentation. the organism excretes such inhibiting substances All these questions ? must wait for answers. Undoubtedly there a pro- mising field for investigation in connection wdth the chemistry of the fermentations not only those due to the action of each organism itself. for instance. hence renewed and continued activity — as evinced by the It steady and copious evolution of carbon dioxide for weeks. and the corresponding increase of the yeast-cells by budding —when the symbiosis is established.— — . and that relatively large quantities of some body resembling lactic acid are formed. benefits by these substances being removed and its destroyed. In conclusion. (if not identical with) The details of this question can only be determined by quantitative methods and combustions. however. This.. — B. on the other hand. For the present this can only be regarded as a hypothesis. but I think the former hypothesis explains most if facts how. since the Schizomycete is able to evolve small quantities of carbon dioxide daily. from saccharine solutions. The : objection is possibly valid. I have also made some biological examinations of bottled ginger-beer (so-called home-made).e. MDCCCXCII. is it to be explained that the Schizomycete slowly and steadily converts the whole of the liquid sugar-solution into a solid gelatinous mass. 2 B . AND THE ORGANISMS COMPOSING of the bacterium.

Other series with the latter gave negative results. in some parts of the Ehine-lands. 17. WARD OIST THE GINGER-BEER PLANT. suggesting that both yeast and bacterium were introduced with the sugar. is in after-fermentations.: 186 I PBOFESSOR H. and was decidedly an oversight not to weigh the quantities of ginger and sugar employed and still more so not to employ non-sterilised sugar and sterilised ginger in the same series. little doubt that the idea here introduced likely to bear fruit if It has long been known that certain micro-organisms so act on particular nutritive substrata. except that referred to previously but it may be worth while to say something on the general subject of what I would call " symbiotic fermentation. . PERDBixt has recently isolated from water an interesting anaerobian bacillus which ferments starch into a glucose-like body and finds that if a yeast the cultures it completes the fermentation as an alcoholic fermentation. involved in obscurity. &c. A number of liquid. case. Be this as it may. for other organisms. The origin of the Ginger-beer plant is as has been stated. s is added to In these and similar cases. while the bacterium vermiforrne) introduced with the ginger." is There can be pursued further. regard the fermentations as not 1888. 286-311. sugar. Again. we * Thiel's ' may 5. excepting that I found the yeast on it. Landwirtksch. 83-160. so to speak. have isolated from two different brands certain minute micrococci which appear to be fig. As Muller-Thurgau by a tion of acids. The negative results with the ginger. sterilised filled with the saccharine sterilised and charged with rest Some had sugar and a lump of unsterilised . An excellent example afforded by the grapes affected with JSdelfaule. sterilised. ginger . none.' vol. which so alters the constitution of the grapes that the propor- and nitrogenous matters are altered before they go into the must " such grapes yield wines of higher quality and finer " bouquet than merely ripe healthy ones similarly fermented. . suggested that the bacterium was introduced with the sugar . properly speaking there . is As to the literature of the subject. in two of the flasks with both ginger and sugar not sterilised the Ginger-beer plant made its appearance after 10 weeks. identical with those on Plate 14. Jalirb. but I accept this suggestion cautiously. introin duced from the and brown sugar employed is ordinary practice. pp. is. or both. or the ginger. M. of yeast and mould. however. are well-known cases in point.' vol. 6. that they prepare the latter. " has shown. others had both ginger and sugar non-sterilised and the had both it .* these grapes are rendered mouldy and " rotten species of Botrytis. t ' Annales de Tlnstitut Pasteur. Unfortunately I have not had time to continue these experiments far enough. In one series of experiments this was very clearly the soda-water bottles were three-parts ginger and sugar as follows. and hope to follow it out more carefully another summer. pp. is but there evidence to show that the yeast (Saccharomyces pyriformis) grocers' shops attached to the ginger (J9.. 1891. The successive crops of bacteria in putrefaction.

of December 19. Zeitung.). investigating certain phenomena of antagonism between bacteria. also induces symbiotic fermentations. and no line of biological research more likely to yield results than this one. &c. (n. t It has long been recognised that many of the " diseases " of beer. It seems extremely likely that the Kephir-ferment. Zeiss. 102-114). Schweizer. Nat..g. IT. are due life- to mixed ferments. Zeitg. 1-10.e. Med. 1. 187 the action of the one organism follows on that of the other. Acad. Description of Plates 11-16. and it is almost certain that some of these carry on symbiotic actions. 1887. " Ueber ein neues Milch.' Jahrg. p. 184). of Arts and Science/ vol. e.^ found that two forms may grow side by side on the . in other cases. the first paper to consult is Ketw. the one form ousts the other by poisoning the medium for it (antibiosis). same medium. " On a Kephirfound in the United States " (' Proc. ginger solution (old) from Flask (4). that many of the best brews are sense. 1891. but I think we must uphold it any rate compound fermentations where the actions go on more simultaneously. Yeast a.' 1886. viz. obtained from a nine days' culture in 5 per cent.AND THE ORGANISMS COMPOSING symbiotic.. L/4. and antibiotic fermentations to yeasts but I would draw the attention of brewer s technologists to be to another side of the question. Garre. 17. p. It should be noted that Lett (' Deutsch. it is strain. Imp. and carry on a symbiotic existence whereas. Ed. Probably the distinction for the present at is . Lectures on Bacteria' (Engl. de Moscou... pp. it renders the medium more favourable for a second form. though many of the best-known "diseases" of beer are due to rnetabiotic . able that a better brew can be obtained by symbiotic ferments than by a pure In any a logical consequence of the study of the separate fermentations that the behaviour side-by-side of those which live symbiotically shall also be tested.' 1882). Further ' literature in like Yeast De Bary. " dem Kaukasus (' Bullet. 1887). Fig. d. de la Soc.. wine. known due which are not of a pure case.ferment aus f With regard to Kephir. 783) says the ferment * is not necessary. Aerzte. not so real as for there are it appears. i. of the Amer. For instance. sp. 1890. or. in Hansen's and it is not at all improbone. Characteristic groups of the yeast. which presents so I can conceive of many morpho- logical resemblances to the Ginger-beer plant. 2 B 2 . and Bot. and in Mix. figs. but rather rnetabiotic. nutritive gelatine. * " Ueber Antagonisten unter den Bakterien " (' Correspondenzblatt f. —Saccharomyces pyriformis PLATE 11. 26.' 1881. in yet others.

29. at 3 p. three. . . Fig. and drawn to cell Similar culture from an isolated glucose. = the cell at 1 p. on January (temp. Zeiss. 4.m. or four spores. 6. € = 5.m. S. but in 3. above. but returns on cooling Zeiss. y. E/4. D/4.— 188 Fig. a vigorous bud was developing as seen at At y we have the (temp. WARD ON THE GINGER-BEER PLANT. two new buds were formed (temp. From Flask 15° 4a. in in a drop of Hayduck's gelatine- which it was completely submerged. y. (temp. . e. = 19° C). = 12° C) .) . drawn to a large scale.m.m. E/4. From Flask 19° C. A continuous culture of one yeast-cell in a hanging-drop of old ginger gelatine. 1891. on January 5. at 9 p. in diameter. The sienna colour pales to straw-yellow.m. am. the was easily visible to the unaided eye. 2. Zeiss. on January 2 same day (temp.m. was Jly visible as I y*. Two groups of old yeast-cells of this species. colony = 13° C).m. the colony was too complex to draw.30 p. = ll°*l). = 14° C). on January 10 A. 5. 14°*5 C). Pig. after four days' culture on pure gelatine at a temperature of 22° C. 6. Two groups of the same yeast. A group of the above after treatment with a dilute solution of iodine in potassium iodide. those of the lower group (B) are cell. 17° C. bottom of the to spores. seen as an opaque object on the surface of the gelatine drop perfectly visible to the unaided eye. tion of pure cultures. as the thinner Zeiss. at 11 a. the temperature /3. They are not dead.. (temp. (temp.) . 3. (temp. gelatine only (with traces of the saccharine solution). such colonies are for the prepara- and can readily be used scale. on January it = S. but Fig. being about 1 mm. December 30. and. condition of affairs next morning.) as seen at S same day. The spores in the upper group (A) are quite ripe not. At 11 a. = 12° C. on January at 10 a. on January 4. but the protoplasm undergoes a kind of fatty degeneration and the fat globules may present a resemblance Many of these cells germinate readily in fresh media.) 16° C. (temp. On December 15° C). indicate. Fig.. 2 „ dia-nete. white Jt. /3.m. (temp.) 4. the oil-drops running together and disappearing before budding recommences. .. = = o1 18° C. or even dis- appears on warming. a .30 a. 4.M. C.m. and e shows the rapidly developing colony at 10. = ft. Errera's glycogen reaction. M.walls having but just completed their development. a similar colony to .m* on December 31 (temp. risen to = At 9 p. at 11 S. Twenty-four hours later. 1. Fig. at 3 p. A similar culture to the a.m. 1890. at 11 a. 1891. 7. at 11 a.m. L/4. about Zeiss. the cell was as shown having at a at 4. the contents of which have become converted into two. L/4. On January (temp. PEOFESSOR H. and Led for the preparation of flask cultures. 1890. at 9 p. after lying for some months at the flask.. From tube 3c of December 19.m.

occ. either escaping from the mother-cell wall. which is and vacuoles. J. 4. as it hangs from the base of the thicker. a was taken from a three weeks' culture in yeast-water. as found growing on the top air. D/4. D/4. 1-6. From surface of sugar solution in soda-flask with G. on the surface of which with air entangled. 3. of the saccharine liquids in contact with plenty of well developed. Zeiss. minute this dot is very characteristic of the fungus. and looks brilliant dot. Zeiss. 4. The In forty-eight hours (6).B. As when black under the microscope. dry ripe spores are very brilliant and contracted (a). Germination of the yeast-spores in dilute Pasteur-asparagin. as a thin. figs. Similar preparations are obtained in old hanging-drops of gelatine. PLATE Fig. from specimens which had developed the spores on gelatine eleven months previously. and number of the branch determine the forms of the Fig. 8. December 11. imm. 10. D/4. (c) or protruding the new bud through Zeiss. b was from a twenty-two days' culture on yeast. J. greasy film. Morris. and are less highly refractive and during its cell- the next twenty-four hours begin to bud like the ordinary yeast-cells. shown mature at a. H. fall from them into the Fig. The pyriform shape is very pronounced. Zeiss. 2.AND THE ORGANISMS COMPOSING IT. occ. Zeiss. the cells each contain a . D. G. rapidly growing. on the surface of which it flat. thin. Characteristic groups of Mycoderma cerevisice. Fig. in. 189 Zeiss.P. twenty-four hours old. Zeiss.) c shows a form also often met with in the solutions. 12. form"). It will be noticed that the direction colonies. and cells growing rapidly. shown at b. medium. Mycoderma cerevisice (Desm. A characteristic group (so-called of the yeast cells from the aerobian films of old cultures " involution 4. Zeiss. from a fifty-two days culture in beer-wort. kindly photographed for me by Dr. A group of the aerobian forms.). dull-grey.. The dull appearance entangled among the cells. but with a limited Specimens taken from a tube. is of the films due to the air. (From Flask 33. growing slowly on or in bad nutritive media. floating membranes. Fig.water gelatine. J/4. Groups characteristic of starved Mycoderma. as Fig. 9. of the submerged Mycoderma. E/4. they have swollen considerably. Fig. 1. of Burton-on-Trent. Groups characteristic liquid. it floats in dull grey. feebly growing istands. Characteristic groups as found growing on a good supply of floats air. .

= 18° C). Successive stages in the development of a (of Flask tions mycelium from a (fig. to March 14. = 12° C. growth began at once e b = 17. o. 12. but no growth occurred till the 23rd. January 24 (temp. obtained by infecting beet solutions from Flask 3. 1891. mycelium (March 21). on March 15 ..m. f= March off 10 a. Fig. = 4 p. 26. same day . Fig. on January 25 (temp. PLATE Fig. A. . ment of the hypha. the same at 5 p.m. obtained from the pink film on 28. = 12° C. from .m. i = at 12 noon on March 18 k. 1890. 9.) December. y the colony at 9 a. March 14. 1891 (temp. septate). The observa- were started at 10.m. 1890.) B.m. Three specimens of the above yeast. as seen at h. on the 26th Next day the colony was too large to draw. D/4. =6 p. = 12° C). M.m. due (cf.m. 5. transferred from beet solution to a hanging-drop of beet gelatine. g = at 4 p. as solution. Fig. m. PROFESSOR H. at 10 a. (temp. marked X in h. d = March 16.30. At 9 a. C. Zeiss. but the cell completely immersed in glucose-ginger-gelatine. when the cell began to bud at one end very slowly (temp. December 21 at 4 p. the three specimens at 3. 10 (a-q). and d on the 27th. The experiment was commenced on between a cover-slip and slide. and 12. A characteristic group of the yeast. a).M. the temperature had c : . . 6. /3 the same at 11 a. = 16° C. p. Zeiss. at I. .m. . Culture from a single hanging-drop of Haydtjck's gelatine-glucose. Similar culture. a the cell at 3 p.m. Pasteur's Zeiss. = 14° C) = 14*5° C). . The film was four days old from Fig. 8.30 . (temp. Rosy Yeast. which bud i to I = stages in further develop. now risen to 15° C. one of the conidia which had fallen large and germinated in the beet gelatine h.) . It should be noticed that the shrinkage and cracking of the gelatine let m air more and more as the culture progressed. 7. single cell B 4). (Flasks 24.m. Groups of same. next day. WARD ON THE GINGER-BEER cell in PLANT. temp. and . = 15°-16° C).m.30 a.190 Fig. the enlarging mycelium (March 20) . and on 25th the drawing b was made. at 9. Zeiss.30 on March 19 4 p. (temp.m. a group of conidia-bearing hyphae. at 9 a. . n. D/4. 12. on March 18 the mycelium was beginning to by the outgrowth of the cells into hyphse (eventually terminal conidia .m. C/4.m. cultivated in a hanging drop of beet gelatine. c represents the state of affairs on the 26th at 10. .. to the further development of The radiating hyphae bear conidia m). figs 7-10.30 A. . the original infecting material had remained in its flask May 20. form. D/4.m.

Fig. Treatment with iodine . enveloped by a thick : sheath. taken from a very active fermentation. AND THE ORGANISMS COMPOSING IT. 5. —Bacterium vermiforme. and two of these conidiophores are drawn at p and . many rodlets are free. perhaps more. KHO for They remain days and weeks intact in water. Fig. but the lustrous sheath prevents their being easily seen in the fresh state some are devoid of contents. bodies is now seen to be a filament.." is also seen in the centre. do not dissolve in freshly prepared . n. sizes — rodlets. as also the sheaths in The masses become 3 shrivelled and granular. One of the very small opaque object. Zeiss. they do not and 4. Zeiss. as the In 6. Specimens of loose filaments and found floating about in the earlier of the bright vermiform bodies stages of growth of the organism. but they dissolve Zeiss. A similar group of empty sheaths figs. and Fig. filaments. 3. characteristic of the masses of " yeast. in beet solution or in Fig. though they swell in it. 13 and 14. this is by no means always the case. Zeiss. the sheath. sizes are length. PLATES (Average Fig. bacilli. in the fresh state B. D/4. 6. 0*5 fi diam. . These. have bacillar rods or filaments in them. ja breadth of enclosed rodlets. ammoniacal cupric oxide. 5 p to over 100 /x breadth of sheath. Magnified about 5 diameters. or Each of the vermiform row of rods. 1. 1-5 X 0*5 /x . nor in iodine and sulphuric acid. The average to 5*5 fi . however. Four selected specimens of the above. D/4. 191 q. often corrugated. and more highly magnified with strong transmitted light. even when boiled in in strong sulphuric acid. 1 would present the same structure. D/4. 1. Specimens of the filaments. in the axis of Though the Schizomycete is often. sheath . : A. and p and q = Zeiss. Such specimens are very common during of the filaments round themselves. specimens a and c show. after treatment with alcoholic iodine solution. 4. D/4. the filament is escaping terminally from its in c it is throwing off the sheath laterally. or a little mostly. sp. D/4 o = hyphse are most densely packed. /x. Hayduck's solution. Zeiss. &c. the active early stages of the fermentation. cocci. brain-like colonies in young cultures. zinc iodide. which at once dissolves cotton wool turn blue in chlor.) Two specimens of the colonies obtained in pure cultures. The pink hue. where the a to n. &c. it . B/4. Schizomycete No. brilliant lustre. to show the extraordinary coiling. 2. Many . 4*5 about 0*5 /x. does not obviously dissolve them. Fig. with a Any of the smaller knobs on the masses in fig. magnified as an It consists of closely convoluted sheathed filaments.

facts solution or with picric aniline blue brings out the Zeiss. Time records of the growth of one of the filaments. December 31 (temp. the one markedy (temp. 10 A. has occurred moreover. considerable intercalary growth. for the stage as seen at = till 16° C).. noticed at stage /. as well as terminal. no further changes were observable. &. c. 15° d shows the it on January 1891..m.M. made (temp. its of old plant of the found that several of the Schizomycete filaments were also growing. and at I attributed the cessation to these causes. the temperature each morning as usual when observing the : and append the numbers for the period in question °C. the same at 3 p. I recorded cultures. On that date I specimen was marked and a. at 11 a. (yeasts and bacteria) on December 29. = 19° C). increased daily in the older portions of the filament. diminished growth and cessation at stage colonies In the first place. first but this is irreconcilable with the behaviour of the Ginger-beer plant in corked bottles and in sealed tubes under pressure and in vacuo. and although watched the specimem January 9. January 2. the growth throughout filament. C. = 15° C). It is uncertain which of the following phenomena accounts for the h.m. on the 1. . = 14'5). (temp. 1890. M. and the behaviour of one yeast-cells was traced up to the 31st of December. WARD ON THE GINGER-BEER PLANT. Next morning. 7. At 3 p.m. Fig. and eventually (January 9) were . and is noticeable that a . same very clearly.— — 192 PROFESSOR H.. off. first It will be seen that the corrugations in the sheath. 10 10 „ * >.M.30.M. and diminution of the oxygen. is confined to the right-handed portion of the coiled 1. the coils had increased considerably. in a hanging drop In this was placed traces of the Ginger-beer ginger-gelatine. g (temp. It is much more 9 probable that the diminution in temperature from for the January onwards was responsible stoppage of growth. but the growth now slowed h was not reached I 11 A.? „ } ? 12 noon 1 s = 6 = 6 = 7 = 8 = 9 = 10 = 11-5 8-5 11-5 9-0 11-0 12-0 lO'O . J anua. 5» 10 A. the yeast were increasing all the time.m. = 17° C).m. L/4.. state of affairs and at 9 p. same date (temp. and an isolated behaviour traced.ry 5 10 „ 5? 3 P. till on January 3 (temp.M. at 10.m.) . with the following results the filament at 9 a. invading the neighbourhood of the filament this must have entailed an accumulation of carbon dioxide. 17° C). on January the drawing e was and at 9 p.

at a = a coiled sheath 10 a. August 16 is . Changes observed in Pasteur-bouillon (equal volumes) stiffened with gelatine. more voluminous) Zeiss. Various stages in the development of the gelatinous sheathing substance by a rodlet observed in a hanging-drop of bouillon-Pasteur.. 18).m. 18° C). Types of the Schizomycete from a pure culture in Hayduck's solution. 2 The preparation was made from a — B.m.. 3. and 9 e = 7 p. . same day — (August 15) b the condition of the rodlet has advanced considerably to the left. are free and unsheathed rodlets. on May At at &. again. = = .m.m. of same date. Fig.M. 14. and loops are completed (g = 9. but does (d = 1 p. = 13° C. one had added considerably to sheath. the rodlet lying at one at 4 p. Stages in the behaviour of the small sheathed rodlets. the Next morning. and the its (also slightly second rodlet had escaped bodily from sheath. occ. the free rodlet remained unaltered (temp. formed (f= a. the first sheath had grown yet larger. D. May. 2.m.. its at 10 A. the temperature having again fallen to 17° C.m. Others. and that the products of action of the yeasts and Schizomycete were accumulating.m. c shows the further growth of the sheath of the former slight sheath at 4 p. the latter being indicated c tinous substance found. the two rodlets shown at a were fixed and drawn. = 7 a. 20° CL). August 16). 8. (temp. with the rodlet at the affairs at 9 p. observed in a hanging- drop of beet-gelatine. on the 15th the development had begun. The drop was infected with organisms from a marked tube. PLATE Fig. as shown and had added to its sheath . Zeiss E.30 a. and during the next twenty-four hours a series of August watched Fig. and begun to return across its path. but others are short rodalso be lets must symmetrically or excentrically immersed in the gelatinous sheaths.. temp.AND THE ORGANISMS COMPOSING Of course it IT. and MDCCCXCTL in an atmosphere of carbon dioxide. 193 Fig. occ. . and a first now invests the other rodlet.. 1. though the specimen was coils 4. stiffened with gelatine. At 10 a.. 1891 (from a marked tube). changes occurred. 4.m. at 4 p. No further changes could be observed..m.m. 4.m. 29. and the free rodlet end (d) had now a very evident swollen sheath. remembered that the nutritive materials in the hanging-drop were being exhausted. it 12 noon of same date the sheathed one had changed position. August 14. occ. and the erratic rodlet not complete another loop it is forming another. by the gelaa new loop had been formed during the night. same date. D. Temp. = 17° 0. Zeiss.. left end. . till No August further 27. some so short as to be mere cocci. March 29. Some of the forms are coiled as before. On August 17 August 17).

E. c — 9 a. so.m. . the swelling has increased. fourth day's culture of the Ginger-beer plant. and on September 9 A. 4. in bouillon-glucose. Zeiss. Typical group of the swarming filaments and rodlets. and breaking up into segments. Changes induced another.. the growth of which is scarcely noticeable. 4. Type of a fifteen days' synthetic culture of the jS. and finally cocci. occ. shows the condition of Zeiss. Two of the filamentous forms of Bacterium vermiforme. . on September 2 6.m. 1 the yeast-cell and a 3. in a hanging-drop of bouillon-gelatine-sugar. 8. specimen a was fixed under the microscope at 10 a. occ. o'clock. apparently owing result was the formation of cocci.m.m. in Pasteur's solution. and produced contortions of the filament. d at 2 p. Nothing Pasteur's solution and ginger. 6. a culture ginger. as seen under Swift's y^— in. the right-hand longer rodlet had begun to break up into bacteria cocci. drawn after staining with methyl-violet. September 2. in a soda-water flask (B). in sheath. a at 10 a. at 4 p. D. d = 9.30 p.. (c). and advanced they did affairs laterally to the left. Fig.30 a.m.m. . neighbouring row of rodlets.m. which remained Zeiss. vermiforme together with Saccharomyces pyriformis.m. WARD ON THE GINGER-BEER At 4. 7. occ. as it were. September 4. breaking up into cocci.30 a.30 p. 4. (b) . occurred further except a slight increase of the swellings on the right hand. the swelling and wrinkling of the sheath have increased. and e at f on the third day. . which had next day divided again into Zeiss.m. to the gelatine. apochromatic oil-immersion. . The yeast-cell remained quiescent throughout. and XXX c had divided. in a sheathed filament when transferred from one medium to The specimen was from a four days' fermentation in normal On September 1 it was transferred to a drop of Pasteur's solution. similarly with those on the right.. 4. . At 10 a. (b). a was sketched at 9. 5. shorter rodlets. form in a drop of bouillon-gelatine-sugar. From an was made eight days in a drop of several yeast-cells were present in the .194 PROFESSOR H.m. 5 p. the rodlets were growing and dividing in the sheath 3. Fig. adding to the gelatinous matrix as further changes were traced. although the specimen was watched Fig. The. E. Division of the motile bacterial till September next day 7. XX. next morning at 10 No D. the rodlets marked X. to which Pasteur-bouillon-gelatine was added. a). (3 at 4 p. then shorter and Zeiss. c at 7 a. occ.. The final The division was very slow. 9. M. from a culture in bouillon Fig.m. on September PLANT. its sheath is swelling and peeling off.m. of the peripherally Fig. in the matrix. separated.M.gelatine on September 9 drop. unchanged Fig. 4. This went on.m.. E. 4. were fixed (fig. Fig. at 2. . Curious branching of the sheathing matrix by the repeated growth and division situated Schizomycete. The yeast is budding . same date. September 3. occ. fermentation in Pasteur-bouillon-gelatine.

short rodlets (bacteria) and cocci. E/4. 1. Zeiss. E/4. A mass of spores obtained from the above culture forty hours later than last All the filaments and rods at the top of the liquefied gelatine have : phase. at 10 p. E. which rapidly break up into very Zeiss. Zeiss. occ. E/4. up into the swarming rods of various lengths. and entangle the well-developed yeast-cells in the coils of the gelatinous matrix. . and fig. figs. The Schizomycete grows out into long filaments. next day. Schizomycete No. Zeiss. by twenty-four hours' culture of above filaments on disjoint The filaments simply still into rods. 1. 195 Fig. 5. and at last forms the hard brain-like so like those figured at Plate 13. below the surface they are still breaking up.m. Fig. on the fig. in same culture. 4. fig.. some are breaking up. formed spores thus fig. The mass becomes denser and it denser. These spores are embedded in an indistinguishable matrix. many of the filaments run parallel. The yeast buds slowly and for a short time only. 1-6. Type of a fifteen days' synthetic culture of the yeast and bacterium in bouillon only. 3 filaments which are segmenting. January 5. C Bacilli and figs. of the longer rods are disjointing. Type of a fifteen days' synthetic culture of the yeast and bacterium together in a suitable saccharine medium (Pasteur-bouillon). in In B. Fig. which then swarm. A. 2. A. B Fig. January 11.. 6. Zeiss. which then break freely. Zeiss. glucose a =a Zi sowing of the ripe spores in gelatine- made on March 30. as in 1. and kept at 28°-3Q° O Lt C f On the 31st. Two groups of spores and spore-forming segments. 3. occ. E. 4. Bacilli developed from the spores at 9 p. A from the original culture in glucose. that was unnecessary to draw special figures of them. 10. Two characteristic groups of the bacillus in the filamentous condition. and show septa A.m. Spore-forming segments Fig. All Zeiss. at 10 a. E/4. D. and placed in the incubator at 27° C. on January 10. The filaments and rodlets ensheath themselves as soon as the carbon dioxide is in excess. 11. from the group shown in 3 day. PLATE Fig. Bacillar rods obtained gelatine. 2. Germination of the spores. 4. (under a lower power) and stained with fifth iodine.— AND THE ORGANISMS COMPOSING IT. Fig. Ginger-bacillus.m. more highly magnified (Zeiss. 15. The Schizomycete grows out into filaments. 2. as found on the surface of glucose solution forty hours after immersion of a piece of ginger. B. Group of the spores of and 4 sown in a hanging-drop of Peter's gelatine at 4 p. L/4). occ. 4. 1891. lumps of the Gingerrbeer plant. E. .m. Some Fig.

). 9. occ. and PLATE 16. PLATE 15.. y. J. figs. the above segment at 10 a. Zeiss. (fig. obtained from a strong " Vinegar- plant/' Fig. Dematium pullulans (De Baky). of " involution forms " of Bacterium aceti.) . next day c. 4.m. the same at 10 a. 1. 14. 8. 196 PBGFESSOU H. D/4. figs. WARD ON THE GINGER-BEER oil PLANT. November 7th. .m.m. a characteristic group pf the cells Ginger-beer plant. E. obtained from the film of an old culture of the Ginger-beer plant. b. 6 3 (3) into (Swift i\ homogeneous immersion. 15. Zeiss. 4. from the top of a tube-culture of the " Ginger- beer plant " with free access of atmospheric oxygen. 7.) and watched. Fig. A. PLATE 15. 9 series traced for other segments. grown on acidified claret groups of Bacterium aceti and water. M. 14°-15° 0. 12 and 13.) Zeiss. 4 t Oidium lactis (Fresen. A group Zeiss. on the 16th February 14. next morning S. December a. 9 p. imm. part of the mycelium twenty-four hours later it is becoming disjointed into the yeast-like segments of (Temp.m. Culture from a single cell isolated in a hanging-drop of peptone-jgelatme. they had swollen. and placed in and mycelium as found on the boiled ginger-gelatine in a hanging-drop on The segment a was marked (2 p. 1889. 7-9. D/2 and D/4. similar Zeiss. 14.m. 1889 : /3. 14°-15° C. same day fig. Zeiss. . Fig. the cell at 10 a.). 9 a.— . Bacterium aceti (Kutz. 13.m. fig. 13.) Schizomycete No. PLATE Fig. Fig. (Temp. 12. J. on . Fig. Two Two groups of the normal Bacterium aceti. occ. 'd-f and at x. E/4. 3. # . Characteristic group of the yeast-like form as found on the surface of flask 18. and several of them were germinating bacillar rods. occ. throughout.

15th (temp. 1. at 11. and much less highly magnified (Zeiss. showing the development of one of the buds i.m. Zeiss. when fully formed. unless transferred to fresh pabulum. 197 16. . on February 17th.m. at 4 p.30 a. J/4. at 10 a.30. and I.AND THE ORGANISMS COMPOSING JlIjA.30 a. The brown colour pales somewhat. Culture from a single segment. Fig.m. isolated in a hanging-drop 6.50 a. 'D/4. and e.15 p. h at 12. of Hay- duck's glucose-gelatine. at 8 p. c.m. h at 10. : h-l. fall into the drop and remain unaltered. d. at 2 p. 10. B/4). The buds. Zeiss.m. = 17° C).. f y the mycelium at 10. All the above. on February 14. time drawings. g a y small piece of a pullulating hypha. the oil-drops gradually enlarge and run together as growth commences. to the same scale as before (D/4) .30. same day. on February 16th. at 1.IJlL IT.m. on same day.. showing the copious development of buds now going on.m.m. a.

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walls having but just completed their development. gelatine only (with traces of the saccharine solution). A similar culture to the a. = 17° C. E/4. G. in indicate. 1-10. imm. (temp. Similar culture glucose. 6. = 12° C.) = 16° C. (temp.m. Characteristic groups of the yeast. A continuous culture of one yeast-cell a hanging-drop of old ginger 29. Fig. a drop of Hayddck's gelatine- which 1891. = ll°'l). they have swollen considerably. A group of the above potassium iodide. a . . being about 1 mm. colony was easily visible to the unaided eye. 1890. of December 19. On December 15° C). figs.m.) 4. on December 31 (temp.. at 11 a. after treatment with a dilute solution of iodine in to straw-yellow. On January (temp. 10. at 11 a. either escaping from the mother-cell wall.m. = 12° C. form "). or The sienna colour pales appears on warming. was as shown at a at 4.. on January was clearly visible as a pure white spot. L/4. Fig.) at 9 P. on January at 10 a. in from an isolated it a. y.m.M. 14°'5 C). Two groups of the same yeast. Zeiss. Similar preparations are obtained in old hanging-drops of gelatine. . about 2 mm. H. Fig. at 3 p. 3.m. dry ripe spores are very brilliant and contracted (a). E/4. above. was completely submerged. Fig. L/4. 1891. on January 10 a. occ. the cell gelatine. Fig.30 a. 4. 5. and cell drawn in to scale. = e. am.M.. =s 19° C. At y we have the condition of affairs next morning. the cell at 1 p. same day. Two groups of old yeast-cells of this species. risen to = At 9 p. J. but drawn to a large scale. 7. /3.m. occ. . 6. in diameter. even disErrera's glycogen reaction. Fig. (temp. or four spores. after four days' culture on pure gelatine at a temperature of 22° C. a similar colony to . Morris. at 9 p. From Flask . the 10. on January it the colony was too complex to draw. The spores in the upper group (A) are quite ripe not. of Burton-on-Trent.M.m. and are less highly refractive and during its cell- the next twenty-four hours begin to bud like the ordinary yeast-cells. y. E/4. The In forty-eight hours (6). From tube 3c of December 19. and. L/4.) . The pyriform shape is very pronounced. those of the lower group (B) are cell. Fig.) as seen at 8 and e shows the rapidly developing colony at Twenty-four hours later. = 18° C.. Zeiss. the oil-drops running together and disappearing before budding recommences. They are not dead. but the protoplasm undergoes a kind of fatty degeneration and the fat globules may present a resemblance in Many of these cells germinate readily fresh media. obtained from a nine days' culture in 5 per cent. on January 2 at 3 P. At 11 a. Zeiss. as the thinner Zeiss. Fig. but returns on cooling Zeiss. 1890. on January (temp. D/4. from specimens which had developed the spores on gelatine eleven months previously. Zeiss. the contents of which have become converted into two. (temp. C. 8. 1. such colonies are for the prepara- and can readily be used tion of pure cultures. diameter. (temp. 8.m. but in 3. from a fifty-two days' culture in beer-wort.30 p.m.. Fig. at 11 A. on January 4. 1. 1890.m. Zeiss. j3. 2. seen as an opaque object on the surface of the gelatine drop perfectly visible to the unaided eye. after lying for some months at the flask. three. vigorous . 5. kindly photographed for me by Dr. Zeiss. = 14° C). at 11 8. same day (temp. ginger solution (old) from Flask (4). the temperature having bud was developing as seen at y8. = 13° C). (temp. bottom of the to spores. = 19° C). 4. and used for the preparation of flask cultures. December 30.c9 © o 6 B ^ 7 A 7 i — \bi o 9 10 c^o PLATE 11. 4. A characteristic group (so-called of the yeast cells from the aerobian films of old cultures " involution 4. Germination of the yeast-spores in dilute Pasteur-asparagin. J. Fig. (temp. A group of the aerobian forms. From Flask 15° 4a. 9. two new buds were formed (temp. e = 5. .m.) 8. (c) or protruding the new bud through Zeiss.

and cells growing rapidly. marked X in h. . Zeiss. Groups characteristic of starved Mycoderma. I.. b = 15°— 16°G). Successive stages in the (of Flask tions development of a mycelium from a single a). 6 was from a twenty-two days' culture on yeast-water gelatine.M. greasy film. . Mycoderma cerevisice (Desm. a. 9. but no growth occurred the when the began to bud at one end very slowdy (temp.B.M. on March 15 growth began at once b 12..m. Specimens taken from a tube. i = at 12 noon on March 18 k. from . form. which is entangled The among the as it dull appearance cells. temp. where the. 26. Fig.30 on March 19 m. with air entangled. 4. January 24 (temp. (Flasks 24. of the thicker. The observa- were started at 10. Characteristic groups as found growing on a good medium. D/4. December 11.M. From surface of sugar solution in soda-flask witli G. of the submerged Mycoderma. at 9.m. . A characteristic group of the yeast. A. I). A. Zeiss. wi). . h. Fig. Next day the colony was too large to draw. the cell at 3 p. now risen to 15° C. = 16° C. as a thin. feebly growing islands. and c 4 p.30 a. n. Pasteuk's Zeiss.) . J/4. Groups characteristic liquid. .m. hangs from the base Fig. Rosy Yeast.) . Zeiss. and 12. same day . D/4. and vacuoles. Three specimens of the above yeast. e = 6 P.30. D/4 . the further development of o. /'= March 17. (fig. C/4. . Fig.M. growing slowly on or in bad nutritive media. but with a limited it supply of floats air. Zeiss. and two of these conidiophores are drawn at p and characteristic of the masses of " yeast. 1-6. hypha? are most densely packed. to n.M. between a cover-slip and The experiment was commenced on till December 21 23rd. 2. is of the films due to the air. and p and q = Zeiss. at 4 p. It will be noticed that the direction colonies. 1890. cell Culture from a single a. as seen at h. transferred from beet solution to a hanging-drop of beet gelatine. Zeiss. Zeiss.m. y the colony at 9 on the 26th Zeiss. 6. The pink hue. shown at 6.. As when this dot is very characteristic of the fungus. figs 7-10. also seen in the centre. the enlarging mycelium (March 20) . 1891 (temp. 10 A. as solution. due to The radiating hyphae bear conidia (ef. Similar culture. on It should be and on 25th the drawing was made. /? the same at 11 A." is q. 1890.m. at 10 a. 1. Fig. noticed that the shrinkage and cracking of the gelatine let in air more and more as the culture progressed. 10 (a-q). the three specimens at 3. fall from them into the (From Flask 33.) December.M. and d on the 27th.M. = 12" C. the cells each contain a minute brilliant dot. cell B 4). cell = = 12° C). a group of conidia-bearing hyphse. o = . = 12° C. (temp.30 A. c represents the state of affairs the 26th at 10. at 4 P. which bud off terminal conidia i to I = stages in further development of the hypha. (temp. and looks black under the microscope.). shown mature at a. slide.30 . Groups of same. rapidly growing. (temp. one of the conidia which had fallen and germinated in the beet gelatine.m. PLATE Fig. . P. 1891. PLATE Fig. 8.M. obtained from the pink film on 28.) B. g = at 4 P. 18° G). from Fig. at 9 A. as Fig. large mycelium (March 21). May 20. C. 12. = cell 14° C. of the saccharine liquids in contact with plenty of well developed. March 14. = = d = March 16. to March 14. but the completely immersed in glucose-ginger-gelatine. : . by the outgrowth of the cells into hyphae (eventually septate). in. floating membranes. Zeiss. D/4. Zeiss. next day. D/4. on March 18 the mycelium was beginning to .M. figs. Characteristic groups of Mycoderma cerevisice. D/4. and number of the branch determine the forms of the Fig. obtained by infecting beet solutions from Flask 3. on the surface of which it floats in dull grey. the same at 5 p. B/4.M.P. 7. D/4. in hanging-drop of Hayduck's gelatine-glucose. as found growing on the top air. on January 25 (temp. = 14'5° C). cultivated in a hanging drop of beet gelatine. twenty-four hours old. The film was four days old the original infecting material had remained in its flask . thin. the temperature had At 9 A.) c shows a form also often met with in the solutions. 12. 3. a was taken from a three weeks' culture in yeast-water. on the surface of which flat. dull-grey. 5.

Zeiss E. 17° G).. is confined to the right-handed portion of the coiled 1. in the fresh state B. with a fig. Time records of the growth ginger-gelatine. on January the drawing e was and at 9 p. 1 Any of the smaller knobs on. and diminution of the oxygen. On that date I found that several of the Schizomycete filaments were also growing.) . as the its specimens a and sheath . many rodlets are free. and an isolated specimen was marked and a. Zeiss. 4.m. had increased considerably. Specimens of the filaments. = 19° G). to show the extraordinary coiling. for the stage h was not i-eached till 1 1 a. Zeiss. in a hanging drop of old In this was placed traces of the Ginger-beer plant and bacteria) on December 29. taken from a very active fermentation. It is much more probable that the diminution in temperature from for the January 9 onwards was responsible stoppage of growth. &c. ammoniacal cupric oxide. are free and unsheathed rodlets. occ.M.M. some so short as to be mere cocci. Next morning. Types of the Schizomycete from a pure culture in Hayduck's solution.. the filament escaping terminally from in c it is throwing off the sheath laterally. 8. at 10. 15° d shows the and it on January 1891. and Fig. bodies is Each of the vermiform length. nor in iodine and sulphuric acid. L/4.. 4. These.m. Others. Fig. has occurred moreover.. the yeast were increasing all the time. magnified as an It consists of closely convoluted sheathed filaments. its behaviour traced. as seen at g (temp. In b. D/4. The masses become 3 shrivelled and granular. at 11 a. and although I watched the specimem till = = January 9. sizes are : ft 100 /a. Two specimens of the colonies obtained in pure cultures. off.m. Others. &c. they do not and 4.m. The average to 5'5 . but they dissolve strong sulphuric acid. 4. same date (temp. C. It is uncertain which of the following phenomena accounts for the h. of the filaments round themselves. breadth of sheath. December 31 (temp. considerable intercalary growth. first It will be seen that the corrugations in the sheath. Zeiss.m. noticed at stage /. Fig. 3. but others are short rod- symmetrically or excentrically immersed in the gelatinous sheaths.M. filaments. the same at 3 P. which at once dissolves cotton wool does not obviously dissolve them. 1. about - 5/i. occ. as well as terminal. as also the sheaths in figs. 14'5). and at I attributed the cessation to these causes.30. 2. or row of rods. bacilli. 7. Fig. and the behaviour of one of the yeast-cells was traced up to the 31st of December. zinc iodide. do not dissolve in freshly prepared . Hayduck's solution. on the 1. .m. the growth throughout filament. in beet solution or in Magnified about 5 diameters. Zeiss E. often corrugated. c show. no further changes were observable. 10 a. in the axis of the sheath. the one marked/ (temp. state of affairs and at 9 p. 4'5 p. 1890. Many . it. 6. but the lustrous sheath prevents their being easily seen in the fresh state some are devoid of contents. Zeiss. again. have bacillar rods or filaments in them. A similar group of empty sheaths : A. or a little more. I recorded the temperature each morning as usual when observing the : cultures. p Though the Schizomycete is breadth of enclosed rodlets. D/4. . Specimens of loose filaments and found floating about in the earlier of the bright vermiform bodies stages of growth of the organism. again. perhaps mostly. is noticeable that a . are free and unsheathed rodlets. but this is irreconcilable with the behaviour of the Ginger-beer plant in corked bottles and in sealed tubes under pressure and in vacuo. and append the numbers for the period in question °C. Treatment with iodine solution or with picric aniline blue brings out the same facts very clearly. D/4.— « — On t B ft Jt Fig. Four selected specimens of the above. brilliant lustre. after treatment with alcoholic iodine solution. One of the very small opaque object. 10 A. (temp. made (temp. = 17° G). Such specimens are very common during the active early stages of the fermentation. Zeiss.m. Fig. Fig. 16° G). = 15° O). c. At 3 p. January 2. 10 „ „ „ „ 10 „ „ 12 noon = 11-5 = 8-5 = 11-5 = 9'0 8 = 11-0 9 = 12-0 10 = 10'0 6 6 7 in Of course it must also be remembered that the nutritive materials the hanging-drop were being exhausted. though they swell in turn blue in chlor. enveloped by a thick sheath. diminished growth and cessation at stage colonies In the first place. Some of the forms are coiled as before. the coils but the growth now slowed on January 3 (temp. even when boiled in in it . D/4. some so short as to be mere cocci. increased daily in the older portions of the filament. KHO for They remain days and weeks intact in water. is often. 6. Fig. 5 to over now seen to be a filament. (yeasts of one of the filaments. with the following results the filament at 9 a. however.the masses in would present the same structure. brain-like colonies in young cultures. eventually (January 9) were this invading the neighbourhood of the filament must have entailed an first accumulation of carbon dioxide. this by no means always the is case. January 5 10 „ „ „ 3 P. and . March lets 29. a. and that the products of action of the yeasts and Schizomycete were accumulating. and more highly magnified with strong transmitted light.

at 4 P.. (6).M. lumps of the Ginger-beer plant. and in an atmosphere of carbon dioxide.m. a). a new loop had been formed during the night. . which rapidly break up into very short rodlets (bacteria) and cocci. The yeast-cell remained quiescent throughout. Typical group of the swarming filaments and rodlets. up into Type of a The Schizomycete grows out into long filaments. of same date. to which Pasteur-bouillon-gelatine was added. . Zeiss.. occ. August 16 is . adding to the gelatinous matrix as c so. the temperature having again (temp.. Zeiss. At 10 a. D. September 2. till September 7. in Pasteur's solution. were fixed (fig. XXX separated. but does another loop twenty-four hours a series of = 7 p. . vermiforme together with Saccliaromyces pyriformis.M. 13° C). 1891 (from a marked tube). the latter being indicated by the gela- tinous substance found.PLATE Fig. May.m. from a culture in bouillon Fig.M. — 7 a. and on September had begun to break up into bacteria (c). August a. the free rodlet fallen to 17° C. a at 10 a. its sheath swelling and peeling off. 4. . Zeiss. 5. fifteen days' synthetic culture of the yeast for bouillon only. occ. . XX. c at 7 A.m. Fig. E. breaking up into cocci. 8. and the erratic rodlet not complete it forming another. occ. Sep- the swelling and wrinkling of the sheath have increased. E.. The drop was 10 a. its (also slightly larger. The final result was the formation of cocci. as seen under Swift's -fg-m. Fig. as it were. which had next day divided again into cocci. though the specimen was August 18). the two rodlets shown at a were fixed and drawn. apparently owing to the gelatine. watched till August 27. Fig.m.30 p. 4. .m. 14. Nothing occurred further except a slight increase of the swellings on the right hand. temp.M.M. c drop of Pasteur's solution. =a coiled sheath with the rodlet at the affairs at 9 p. freely. shorter rodlets. d = 9. The mass becomes denser and it denser. Zeiss. occ. 4.M. the The specimen a was fixed under the microscope at 10 A. then shorter and Zeiss... and the had now a very evident swollen sheath. D. 14. The division was very slow. in sheath. At 10 a. and had divided.m. Changes induced another. 4. 9. On August 17 August 17). on May at b. a culture was ginger. same day — c (August 15) 6 the condition of the rodlet has advanced considerably to the left. at 10 A. Fig. stiffened with gelatine.m. D.m. in the matrix. Type of a fifteen days' synthetic culture of the S. the 4. the sheathed one had changed . 1 . 29. Zeiss.m... 2. 10. 1. The preparation was made from a the yeast-cell and a 3. infected with organisms from a marked tube. D. b.30 a. 3. unchanged Fig.. in a soda-water flask (B). 11. which remained 4. it position.m. 4. and at last forms the hard brain-like 1 3. The yeast buds slowly and a short time only. August 16).m. the rodlets were growing and dividing in the sheath 3. in a hanging-drop of bouillon-gelatine-sugar. Fig. .M. and e is formed (/= 9 A. on September 2 is . by the repeated growth and diviFrom an eight days' fermentation in Pasteur-bouillon-gelatine. and bacterium in The Schizomycete grows out into filaments.m. and advanced they did affairs laterally to the left. Fig. same date. 7. made in a drop of several yeast-cells were present in the .30 p. in bouillon-glucose. E. No further changes could be observed. coils Fig. and during the next and loops are completed (</ = 9. 4. next day. 4. 4. Various stages in the development of the gelatinous sheathing substance by a rodlet observed in a hanging-drop of bouillon-Pasteur. On September it was transferred to a at 2. and and rodlets ensheath themselves as soon as the carbon dioxide entangle the well-developed yeast-cells in the coils of the gelatinous matrix. and a now invests the other rodlet. E. a was sketched at 9. in a sheathed filament when transferred from one medium to The specimen was from a four days' fermentation in normal 1 Pasteur's solution and ginger. = 9 A. and begun to return across its path.m. Division of the motile bacterial form in a drop of bouillon-gelatine-sugar. Two of the filamentous forms of Bacterium vermifurrne. Next morning. fig. d at 2 p. and produced contortions of the filament. Stages in the behaviour of the small sheathed rodlets. observed in a hanging- drop of beet-gelatine. occ. Zeiss. E.gelatine on September 9 drop. on September 1 neighbouring row of rodlets.. The yeast is budding Fig. 4. same date. so like those figured at Plate that was unnecessary to draw special figures of them.m. o'clock. although the specimen was watched Fig. occ. Zeiss. shows the condition of Zeiss. Sep- tember tember 3. At 12 noon of same date and had added (temp. the swelling has increased. the right-hand longer rodlet This went on. Changes observed in Pasteur-bouillon (equal volumes) stiffened with gelatine. 9 A. and finally cocci. (b) . Type of a in fifteen days' synthetic culture of the yeast and bacterium together a suitable saccharine medium (Pasteur-bouillon). the first sheath had grown yet second rodlet had escaped bodily from sheath. = 17° C. growth of which is scarcely noticeable. which then break the swarming rods of various lengths. (d 1 = p.30 a. drawn after staining with methyl-violet. apoehromatic oil-immersion. at 4 p. = 13° C. 4. fourth day's culture of the Ginger-beer plant.m. left end.M. 5 p.. and breaking up into segments. and e at f on the third day. and the more voluminous) Temp. The is filaments in excess. similarly with those on the right. = . next morning at 10 No further changes were traced. Zeiss. occ. first one had added considerably to its sheath. (8 at 4 p. Curious branching of the sheathing matrix sion of the peripherally situated Schizomycete. c shows the further growth of the sheath of the former slight sheath at 4 P.30 A.M. At 4. No further changes occurred. as shown to its sheath the free rodlet remained unaltered = 20° C). E.m. occ. . occ. occ.. rodlets marked X. the rodlet lying at one end (d) at 4 p.m. at on the 15th the development had begun. G.

2. L . 9 A. next morning S. Fig. 2." Fi igFig. 2. the same at 10 A. A. L/4). 13. Two groups of spores and spore-forming segments. Two Two groups of the normal Bacterium aceti. 3. Ginger -bacillus. obtained from a strong " Vinegar- plant. 7. 1889 : ft. 1889. Dematium pullulans (De Bary). next day. E/4. next day cl-f.m. 3. Zkiss. y. and fig. A. as found on the surface of glucose solution forty hours after immersion of a piece of ginger. .) February 14. Spore-forming segments Fig. f : ' ^ V < ' • Schizomycete No. . occ.) Schizomycete No. Fig. January 11. 1891. groups of Bacterium aceti from the top of a tube-culture of the " Ginger- 9. Zeiss. 14. more highly magnified (Zeiss. /8) into (Swift ^j homogeneous oil immersion.M.M. 9 P. 6. and PLATE 16. E/4. and 4 sown in and placed a.M.m.m. on the 16th (Temp. B Fig. The segment as was marked . Bacillar rods obtained gelatine. 7-9. on January 10. December a.) Zeiss. of the longer rods are disjointing. . J. still Some Fig. 8.M. a characteristic group of the cells Ginger-beer plant.. J. figs. occ. These spores are embedded in an indistinguishable matrix.M. E/4. occ. 6. D. Fig. on . (fig. and placed in and mycelium as found on the boiled ginger-gelatine in a hanging-drop on (2 p. Zeiss. 14°-15° C. the cell at 10 A. Zeiss. E/4. A from the original culture in glucose. E/4. 1. Zeiss. 4. 4.. Bacterium aceti (KtiTZ. PLATE Fig. PLATE 15. some are breaking up. Two characteristic groups of the bacillus in the filamentous condition. January 5. many of the filaments run parallel.— \-*L. Germination of the spores. = they had swollen. 15. — ^ . PLATE 15. 4. . at 1 a. and several of them were germinating bacillar rods. throughout. of spores obtained from the above culture forty hours later than last All the filaments and rods at the top of the liquefied gelatine have : formed spores thus fig. of " involution forms " of Bacterium aceti. glucose made on March 30. imm. 14. A. in In B. a a sowing of the ripe spores in gelatineand kept at 28°-30° C. 3 gelatine at 4 P. by twenty-four hours' culture of above filaments on The filaments simply disjoint into rods. same day fig. D/2 and D/4. in same culture.. part of the mycelium twenty-four hours later it is becoming disjointed into the yeast-like segments of (Temp. A mass phase. Bacilli in the incubator at 27° C.. Group of the spores of figs. Bacilli developed from the spores at 9 P.) Zeiss. 13. 12. D/4.). c. Characteristic group of the yeast-like form as found on the surface of flask 18. hanging-drop of Peter's C. figs. figs. below the surface they are still breaking up. B. from the group shown in 3 (under a lower power) fifth day and stained with iodine. I . . fig. b. 1. On the 31st. the above segment at 10 a. PLATE Fig. . Culture from a single cell isolated in a hanging-drop of peptone-gelatine. A group beer plant " with free access of atmospheric oxygen. which then swarm. Fig. on the fig. and filaments which are segmenting. grown on acidified claret and water. All Zeiss. E. similar series traced for other segments. Oidium lactis (Fresen.m.M. 15. 12 and 13. at 10 p. November 7th. as in 1. Zeiss. show septa A. 5. obtained from the film of an old culture of the Ginger-beer plant. 1-6. and Fig. and watched . and at x. 14°-15° C.). 4. Zeiss.

PLATE Fig. on February 16th. at 1. to the same scale as before (D/4) h-l. mycelium at 10. on February 17th.m. i. Zeiss. g.50 A. fall into the drop and remain unaltered. 16. showing the development of one of the buds h at 10.30. Culture from a single segment. d.. showing the copious development of buds now going on. at 4 p. a small piece of a pullulating hypha. the unless transferred to fresh pabulum. of Hay- duck's glucose-gelatine. The brown colour pales somewhat.M.m. at 11.. and much less highly magnified (Zeiss. c.m. All the above.15 p. when fully formed. f. and at 10 A.M. on February 14. same day. on = 17° C). The buds. at 8 p. Zeiss. at 2 p. : . h at 12.30. isolated in a hanging-drop b. time drawings. 15th (temp. the oil-drops gradually enlarge and run together as growth commences. and I.m. e. J/4. 10.30 A. a.M. D/4. same day.m.30 a. B/4). . 1.