MICROBIOLOGICAL DEVELOPMENTS OLD and NEW E. I?. KOHMAN Research Laboratories, National Canners Association, Washington, D. C.
I t has been said that the most important bacteriological dmelofiments have been made by i.ndividua2s trained i n chemistry. Pasteur i s commonly cited to substantiate such a contention. I n wkw of this, an anonymous article in Lie6ig's Annalen i n 11839,i n which the most a d n a d views i 7 z microbiology are treated with levity' and ridicule, i s a surprise. I t is here brought out that had it been possible to foresee in Liebig's time the developments of today, they would have appeared as fantastic to Liebig's contemporaries as the machinations of a n unbridled imagination.
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O MORE remarkable contribution has ever entered the pages of a serious scientific journal than an article which appeared in Liehig's A n m l e n in 1839. In view of his immortal fame as the father of organic chemistry, it is difficult to realize that Liehig's censorship would permit the appearance of such an article which, as far as I can determine, was written by Wohler, whose renown in the development of organic chemistry is inevitably linked with that of Liebip.* Huxley refers to the article as a "somewhat ~ a b e k s i a nimaginary description of the organization of the 'yeast animals' and of the manner in which their functions are performed, given with circumstantiality worthy of the author of Gulliver's Travels" and brands it as the "most surprising paper that ever made its appearance in a grave scientific journal.. ." It is so pungent and scathing in sarcasm and ridicule, yet so full of humor, that I give here a very literal translation to retain as far as possible the fine shades of meaning conveyed by the German diction: THE SOLVED RIDDLE OF ALCOHOLIC FERMENTATION
(A Preliminary Report by Letter), I am in the act of unraveling a new theory of vinous fermentation. I came upon the trail of this, until now so myherious a change, in the simplest manner in the world and regard it as fully settled. This discovely, too, shows again how simple are the means of which nature makes use, to bring forth the most wonderful phenomenon. I owe thanks t o the use of a remarkable microscope, which, according to the statement of the renowned Ehrenberg, was devised by the distinguished artificer Pistorius. Beer yeast, dispersed with water, is dissolved under this instrument into endless small pellets whose diameter is scarcely I/., that of a line. and into fine threads which are unmistakably. . .-.
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* Edifor's Note:
A communication in a similar vein relating It is to the f, types appeared in A ~ ~33,, 308 , C, H. windier," but was actually the work f, wbhler. A translation by Harold B. Friedman appears in J. CHEM. Eouc., 7,633-6 (Mar., 1930).
a sort of protein material. If one introduces these pellets into sugar water, one observes that they consist of the eggs of a n animal; they swell, burst and tiny animals are evolved from them, which multiply with inconceivable rapidity in a most unparalleled manner. The form of these animals deviates from every one of the 600 hitherto described species; they possess the shape of a tiny "Beindorf" distillation flask (without the cooling apparatus). The nozzle of the helmet is a sort of sucking proboscis which is lined on the inside with bristles '/,rno of a line in length; teeth and eyesare not noticeable; on the other hand one can easily distinguish a stomach, intestinal canal, the anus (as a rose-red colored point) and the urinary secretory organs. From the moment that they have escaped from the eggs, it is observed that these animals gobble u p the sugar out of the solution; very plainly it is seen as i t arrives in the stomach. Momentarily it is digested, and this digestion is simultaneous with and in a most concise mannerdistinguishable from the evacuation of excrements which follows. In a word these infusoria eat sugar, evacuate from the intestinal tract alcohol. and from the urinary organs carbon dioxide. The urinary bladder, in a filled condition, has the form of a champagne bottle; in an empty condition, it is a small button; one notices after a little practice that a gas bubble forms within it which enlarges 10 times; by means of a sort of screw-like rotation which the animal can bring about voluntarily by ring-like muscles surrounding its body, the bladder is emptied. I consider it to be exceedingly probable that herein the same takes place as when galvanic electricity is conducted through a wire; it is known that according t o the opinion of the renowned physicists magnetism is generated under such conditions. This, likewise, instead of following a straight course, in which it is nowise hindered, yet, in accordance with a law of nature, takes the course of a spiral. I cite this only as a proof that in fermentation, as DBbereiner and Schweigger have shown, magnetism and dertricity are simultaneously involved. Our experience here proves the correctness of their observation which was not supported by a powerful microscope. \\'ithour going further into rhis hypothesis, o n < ohrrrvcz t~nintcnuptcdlya sprr~ficlighter liquid rising to ronsidcral.lc hrighr from the anus of these nnimds nnd out of rhrir cnormuus genital organs there is squirted at very short intervals a stream of carbon dioxide. If the liquid is heated to boiling, the fermentation ceases because the animals are killed a t elevated tempefatures. If one adds sulfurous acid, an excess of alcohol, a mineral k i d , or acetic acid, they likewise die and the fermenlation is terminated. If the quantity of water is too small, i. e., that of the sugar too great, the fermentation, as is known, does not begin. This is due to the fact that these little beings, because they cannot change their position or only with great strain in the heavy liquid, die of indigestion as a consequence of a lack of exercise. In order now to give an idea of comparisen of the digestive power of these animals. I submit the estimate of Thenard, according to which 3 parts of beer yeast (in the dry state) are able to transform 200 oarts of suear into alcohol and carbon dioxide. The excrement of these animals, which is discharged in 18 hours, weighs therefore 66 times as much as the animals themselves. Remarkable is the influence of essential mustard oil on these animals; if one introduces a few mustard seeds into the fermenting liquid, i t is noticeable a t once that their activity be-
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comes less, after a few moments they are lifeless, hut after a few hours, especially in contact with air, they revive again and the fermentation begins with its previous activity. This effect is very much like that of anisol, from whose odor, as is known, a flea becomes unconscious and remains so until the oil WaDorates. I have noticed that solanin causes vomitine - in these infusoria: if one adds solanin t o an actively fermenting sugar solution. the animals are observed t o double up and out of their proboscis there streams a Liquid which unquestionably is the fuse1 oil of potatoes, for i t can be identified in the liquid. The fuse1 oil of the grains and of brandy seems to be exuded from the surface of the skin in a sort of perspiration process. There still remain5 . one question t o be answered, namely, the reason for the ravenous. incomparable hunger of there animals for the sugar which they assimilate for their bodies. This, too, is clear now. The fine threads of protein material already alluded to, and which furnish these animals with nitrogen, are consumed and assimilated with a proportionate amount of sugar. The most remarkable thing is the chemical composition of these animals. I subjected t o analysis 6.4376 gram (50,000 million by count) by the usual method with copper oxide and duplicate analyses showed that they contained the four elements in such a combination as thoueh thev consisted of orotein (according to the analysis of h l d d r r ) , ether, and varhon dio\ide This clcars up nor only tlwr whole cligwrive procecq hul d r o the hirherln olscure conjecture :as to a.h:,t might herurnr of the yeast after the fermentation is complete. As soon as these animals find no more sugar a t hand, they mutually consume one another; everything is consumed except the eggs which pass unaltered through the intestinal canal. One has finally yeast capable of fermenting again, namely the eggs which remain. The last corpses of the animals decompose into ammonia and traces of acetic acid. By weight the seeds a t the end of fermentation amount to somewhat less than a t the beginning, which is self-evident. Quantitative data, as well as drawings of the form of thcsc animals are to follow in a more detailed treatise. ~~~
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It is scarcely necessary to'add that the ~romised treatise never followed. Yet Liehig and WGhler would probably admit now that the machination of their unbridled imagination of a century ago scarcely exceeded modem achievements. Some recent developments hid fair to rank with Leeuwenhoek's achievements late in the 17th century, as the father of microscopy, with Spallanazani's efforts in refutation of the theory of "spontaneous generation," or with Pasteur's demonstration of the relationship between bacteria and disease. Work of Ryan and Arnold, of the University of Illinois, now suggests a counterpart to, the old theory of "spontaneous generation." These Illinois investigators plated apparently sterile body fluids by the so-called Hauduroy method. Hauduroy, a Frenchman, proposed' a serial plating procedure in which the material in questign is spread on the surface of nutrient agar or some other appropriate solid culture medium. Although no growth may be in evidence after two or three days of incubation, the surface of the medium is mopped up and replanted on a fresh surface and the operation repeated over and over a considerable number of times, if necessary. Ryan and Arnold started with "apparently" sterile stomach or duodenal smears after feeding various strains of yeasts and Bacillus prodigiosus, a bacterium chiefly characterized by the formation of a red pigment. The first demonstrated growth which they obtained
from B. *rodieiosus ex~erimentswas on the fifth form -spindle.shaped rods with transplant' in very slender tapering ends," not a t all typical of the usual p w t h from this organism. Colonies appeared, with "an internal arranzement suzzestinz a tanzled mass of threads," after ten or twelve transplants. appeared on further transplants, until finally of B. erodigiosus to its weu-known the morphology had been accomplished. In the veast exoeriments the first visible zrowth appeared after two to five transplants, depending upon the particular strain of yeast in question. The first growth was not typical for yeast, however. It sometimes consisted entirely of filamentous forms which, upon transplanting, finally resulted in typical yeast cells. Dr. Arthur I. Kendall, of Northwestern Medical School, has thrown further light on the above discoveries. He has developed a special culture medium, designated as K medium, by which he is able to transform bacteria, visible under an ordinary microscope and non-filterable through a stone filter, into so-called filterable viruses and again hack into the visible non-filterable forms. This has been done with the typhoid bacillus, with a coccus form of bacteria thought to he the cause of influenza, as well as with several other species of bacteria. Dr. Kendall actually observes the bacteria under a microscope change to mere shadows or outlimes actively swimming about, with two or four minute eranules in them. Finally the bacterial outline or wall completely disappears and only the minute granules, which pass through a stone filter and are visible only by special lighting facilities, remain. These now constitute filterable viruses, but by proper culture in his special K medium, Dr. Kendall is able to regenerate from them the original bacteria. Another development along this line is the isolation by Dr. C. G. Vinson and co-workers of the Boyce Thompson Institute for Plant Research, Yonkers, New York, of a filterable virus in -2crystalline form. A very important step has been taken in the analysis and identification of any unknown compound when i t is isolated in a crystalline form. In this instance, it is possible that Vinson's crystals are a foreign substance with the virus in question adhering to them, although this would not detract from the medical significance of the findings. The filterable viruses have generally been considered organisms so minute that they are invisible even with the aid of the most powerful microscope. They are, however, generally regarded as capable of self-propagation. According to Vinson, his isolated product is incapable- of self-propagation, but i t is the infectious principle in the mosaic disease of tobacco. If this is the true state of affairs, it means that a specific, relatively simple substance, placed in juxtaposition or symbiosis with a normal cell may be the cause of disease. The infective principle may thus be an enzyme or other similar product of microorganisms rather than the organism itself. This brings
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into question the microbial causation of all diseases ascribed to ultramicroscopic infections. The basis for interesting theories as to the mode of activity of such phenomenal substances as the hormones and vitamins is also suggested. Quite as remarkable have been the transformations that have been accomplished with pneumococci, the cause of pneumonia. Pnenmococci are classified by means of serum specificity into four (I, I1 111, and IV) types. A serum which reacts with or gives immunity against a certain type is said to be homologous for that type, while it is without effect on the other types. One characteristic of these organisms is that they are encapsulated. Recently i t has been shown that the chemical basis of the capsules is a carbohydrate or polysaccharide of which the chemical composition is specific for each type. In 1927, F. Griffith, a medical officerof the Ministry of Health. England. found that bv culturine " strains of fineumococci in homologous immune serum, attenuated or non-virulent formsmay be obtained. The characteristic smooth colonies of the capsulated organisms give rise to rough colonies lacking the capsules. These new forms were innocuous when injected into mice, and, because of the rough nature of the colonies, have come to be termed R-forms, while the smooth colonies of capsulated virulent organisms are designated S-forms. Griffith later found he could transform one specific type of pneumococcus into other types by injecting subcutaneously into mice small amounts of a culture of living non-virulent R-forms, together with large amounts of beat-killed S-forms of a type other than that from which the R-forms were derived. Living, virulent S-forms of the type of the heat-killed inoculum were recovered from the heart-blood of the culture animals. Later investigators have been able to per-
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form this transformation i n vitro by culturing the Rforms of one type in blood broth containing anti-R serum and the heat-killed S-form organisms of the type desired. Recently J. Lionel Alloway of the Rockefeller Institute made extracts from S-forms of the type into which the R-forms of another type were to be transformed. By culturing the R-forms in brotb-hogserum medium containing some of this extract, Rforms derived from type I1 were converted into types I and I11 by employing the respective extracts. Thesc derived forms were highly virulent. While mice lived when injected intra-peritoneally with 0.75 cc. of the R-form culture, 0.000001 cc. of the derived S-form proved fatal, even upon subcutaneous injection. These pneumococci are thus actually disrobed of their characteristic carbohydrate capsule and mantled with a new cloak. The inner organisms of the various types are therefore apparently identical but the capsular polysaccharide or carbohydrate determines the type specificity. The capsule is thus the allimportant part of the organism from the medical standpoint. Sooner or later, no doubt, the specific substance in the capsule of one type that stimulates the noncapsulated R-forms of another type to envelop itself with a new capsule will be found. So far, it has not been possible to bring about the transformation with pure saccharides isolated from the capsules. These developments may prove of unimagined significance in the future treatment of pneumonia. Were they not proved facts, they would rank well with Wohler's imaginaty yeast cell as "worthy of the author of Gulliver's Travels." Had the writer of the above letter been able to foresee the developments of today, he could have written quite as remarkably by adhering to facts in minutest detail!
