American Progress in the Bacteriological Sugars - Industrial

American Progress in the Bacteriological Sugars. Edmund H. Eitel. Ind. Eng. Chem. , 1920, 12 (12), pp 1202–1205. DOI: 10.1021/ie50132a037. Publicati...
0 downloads 0 Views 610KB Size
. I202

T H E J O U R N A L OF I N D U S T R I A L A X D ENGINEERING C H E M I S T R Y

A new t e x t is desirable which will list mineral colors seen u n d e r t h e new illuminator. T h e writer desires t o t h a n k t h e above-mentioned gentlemen a n d m a n y others who h a v e been kind enough t o m a k e suggestions a n d send photomicrographs. A NEW CONDUCTIVITY CELL By Frank E. Rice DEPARTMENT OF CHEMISTRY, CORNELLUNIVERSITY, Received August 6 . 1920

ITHACA,

N. Y .

T h e measurement of electrical conductivity of solutions is being more a n d more employed i n m a n u facturing operations for t h e control of processes. I n such instances a high degree of accuracy is unnecessary, since comparative results are more i m p o r t a n t t h a n absolute. T h e one t h i n g necessary is a n a p p a r a t u s s t u r d y , simple, a n d easy of a d j u s t m e n t , so t h a t a trained physicist need n o t be employed for i t s operation. A t t h e present t i m e t h e r e m a y be obtained o n t h e m a r k e t all t h e electrical p a r t s necessary for t h e installation’of a W h e a t s t o n e bridge of such form t h a t i t m a y be easily manipulated without a n y a d j u s t m e n t or attention. As a source of current, a 60-cycle line m a y be d r a w n u p o n ; alternating current galvanometers were f o u n d sufficiently sensitive for practical work.

A NCW

co,uDucnvirr

CELL.

As an adjunct t o such e q u i p m e n t t h e conductivity cell here described m a y be prepared b y t h e a m a t e u r glass blower in a short time. It will be f o u n d s t r o n g a n d easily cleaned. I n t h e accompanying drawing t h e idea is diagrammatically illustrated, t h o u g h t h e dimensions a n d particular a r r a n g e m e n t of p a r t s m a y be varied a t will.

Vol.

12,

Xo.

12

A glass t u b e about t h e size of a n ordinary b u r e t is used for t h e case. P l a t i n u m wires are blown t h r o u g h t h e sides of t h i s t u b e some distance a p a r t . P l a t i n u m b a n d s m a d e t o fit snugly a r o u n d t h e t u b e are soldered closely t o t h e outside ends of t h e wires. T h e inside ends a r e long enough t o reach t o one end a n d outside t h e t u b e ; t h e inside portion of one wire is covered with small glass t u b i n g for t h e purpose of insulation. Since t h e t e m p e r a t u r e of t h e solution i n which measurement is m a d e is usually i m p o r t a n t , a t h e r mometer passes t h r o u g h t h e t u b e . This t h e r m o m e t e r should be of considerable accuracy a n d one g r a d u a t e d in 0.2’ C. is suggested. This should be planned, however, t o accord with t h e accuracy desired, a n d will d e p e n d also u p o n t h e ther,mal coefficient of t h e solutions in which measurements are made. T h e ends of t h e t u b e a r e finally closed with rubber stoppers. T h e distance between t h e ’band electrodes a n d t h e i r size should be gaged b y t h e conductivity of t h e liquid in which t h e cell is t o be used. T h a t a n idea m a y be obtained of t h e constant t o be expected, t h e following result is reported o n one cell which has been used. ( T h e electrodes are platinized.) Circumference of b a n d . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 mm. 6 mm. Width of b a n d . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 mm. Distance apart (inside to inside) Resistance recorded of 0.01 AT KCl a t 18’ C.. . . . . . . . . . . . . . . . . 261 ohms 0.321 Constant of cell.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

T h e above measurement was made b y holding t h e cell upright in t h e solution, using a 500 cc. wide-mouthed bottle as t h e container. Since t h e p a t h s of current flow are wholly outside t h i s a p p a r a t u s , i t is essential t h a t t h e vessel containing t h e solution be always of t h e s a m e dimensions in order t o insure c o m p a r a t i v e resylts. Furthermore, t h e position of t h e cell i n t h e vessel should always be t h e same. These precautions a p p l y , of course, unless t h e container is of g r e a t size.

ADDRESSES AND CONTRIBUTED ARTICLES AMERICAN PROGRESS I N THE BACTERIOLOGICAL SUGARS

By Edmund H. Eitel SPECIAL CHEMICALS Co., HIGHLAND PARK,ILL

The history of the carbohydrates.covers a brief period of time, during the last ten years of which a large part of the progress in methods of preparation is t o be found in American chemical literature, and practically all of the progress in commercial methods has been due to American ingenuity. The prime investigator in the chemistry of the rare sugars was Emil Fischer, who began his well-known researches in 1883. The first to make a reasonably direct use of the sugars in bacteriology was Escherich, who thirty-four years ago wrote one of the great bacteriological classics, “The Intestinal Bacteria of Nurslings.” By 1902, the possibilities of fermentation reactions with the carbohydrates were recognized by many bacteriologists, and at this time Martini and Lentz introduced the use of mannitol to differentiate the Shiga from the Flexner bacillus. Later intensive studies of bacillary dysentery in America by Park and 1 Presented before the Sugar Section a t the 60th Meeting of the American Chemical Society, Chicago, IIl.,~September6 t o 10, 1920.

Dunham, Hiss and Russell, and by others, differentiated by means of sugar fermentation other strains in the dysentery group, and thus the carbohydrates were established as a necesi sary aid in bacteriology. Germany was the only commercial source of supply for the rare sugars before the war. During the war the stocks of sugars on hand were in most cases soon exhausted, and the U. S. Army Medical School, and other investigators using the sugars in colon-typhoid and other important differentiations, found their work critically handicapped a t the moment it became most vital to the nation. Owing largely, however, to the researches of such men a s Hudson and his co-workers of the Bureau of Chemistry of the U. S. Department of Agriculture, and Levene and his associates in the Rockefeller Institute, methods were developed for preparing the necessary rare sugars in this country. The Carbohydrate Laboratory of the Bureau of Chemistry had been engaged since 1909 in investigating the rare sugars and their derivatives, and this work was continued during the war, and considerable quantities of various sugars were prepared. They were similarly produced a t the Rockefeller Institute, where of special interest were the researches of Levene and Jacobs, who

Dec.,

1920

T H E J O URiVAL OF I N D U S T R I A L A N D ENGINEERING C H E M I S T R Y

succeeded in synthesizing allose and altrose, thus filling in the last gap of the aldohexose series.‘ It is a significant fact that the methods of preparation given in the standard German treatises (Tollens, von Lippman, etc.) are now more or less antiquated, due to the progress made by the Bureau of Chemistry, the Bureau of Standards, Rockefeller Institute, and American industrial concerns. To translate this statement into its practical significance, it may be observed here that the prices a t which the rare sugars, with the exception of levulose, are now available in America, reflect this progress, being in several instances below the pre-war figures, in spite of the far higher price levels of the present day. The work of the Bureau of Chemistry included the production of twenty-one carbohydrates and no less than I j o derivatives. Through the researches of Dr. F. B. LaForge, two new sugars, the only heptoses thus far found in nature, were discovered, manno-keto-heptose in the avocado, and sedoheptose in the stonecrop.* Another work of note was the contribution made by Dr. Hudson and his co-workers toward solving the relation between structure and optical activity, accompbhed mostly through the study and preparation of the various acetyl derivativr.s.3 Mention should also be made of the fact that a more general use of glacial acetic acid, almost essential in many of the sugar methods, was developed in the Bureau of Chemistry. During the war an appeal was made by the Medical Department of the U. S. Army, not only to the public institutions above mentioned, but to individuals, to produce certain sugars urgently required and not then to be had. Largely as a patriotic measure, a few individuals, first in private laboratories, undertook t o prepare certain sugars needed in quantity, and from this beginning their manufacture was undertaken industrially. It should be noted that the industrial stage represents a distinct advance, since the production of gram lots in the laboratory is child’s play compared to manufacture in commercial quantities. The history of the development of cane- and beet-sugar manufacture illustrates the significance of the foregoing observation, which applies not t o one but to a score of sugars obtained from as many different sources. In spite of the research and development expense required to put large-scale manufacture on a satisfactory basis, and in spite of the limited demand for the sugars to date, not all the manufacturers who have attempted their commercial production have abandoned it, but continue in the hope that an increased demand for some purpose will soon be forthcoming. Because of the importance of the sugars to public health and in order to help place the national health beyond the control of foreign countries, the rare sugars were added to the Longworth Dye Bill. The rare sugars now manufactured commercially in this country are the following: I-Arabinose, dulcitol, d-galactose, d-glucose, glycogen, inositol, inulin, invert sugar, lactose, d-levulose from invert sugar, dlevulose from inulin, levulose sirupy, maltose, d-mannitol, d-mannose, melezitose, raffinose, rhamnose, sorbitol, sucrose, trehalose, and 1-xylose. Sugars are being produced in this country in a higher degree of purity than were those formerly imported from abroad. The latest model of Dr. Frederick Bates’ improved sensibility polariscope has contributed to the higher standard of analysis, as has also the increasing knowledge of American bacteriologists of the minute impurities which may give misleading r But above all, the stricter standard is due to a policy of p purity above manufacturer’s costs and profits, because requirements and importance of the science of bacteriology. An instance of the minute quantity of impurity detectable 1

B w . , 43 (1910), 3141.

* J . B i d . Chem., 28 (1917), 61.

*

511; LaForge and Hudson, Zbid., SO (1917),

Hudson, Nichols Medal Address,

THISJOURNAL, 8

(1916), 379.

I 203

bacteriologically is the case of lactose, which is one of the sugars most rarely obtained in a degree of purity suitable for bacteriology. This is used in important differentiations for B. typhosus, which will not ferment lactose; but if minute quantities of glucose or galactose are present, an apparent fermentation of lactose occurs. To illustrate what seemingly insignificant amounts of impurities will give misleading results in bacteriology, the culture media are ordinarily prepared with one per cent of lactose. Yet Dr. Arthur I. Kendall, the bacteriologist to whom the author is indebted for many of the latest facts in bacteriology incorporated in this paper, has made experiments in his laboratory with glucose present in lactose to the extent of only 0.oj per cent, and the bacteria have indicated the impurity visibly. The same thing is notably true of impurities in sucrose. It is no mean achievement of American chemists to have manufactured lactose and sucrose and the other sugars of the required purity, and yet there is no altefnative if accurate results are to be obtained. The rare sugars which are being tried in countless reactions are worse than useless if not absolutely pure, and one of the obstacles to progress in bacteriological research has been the lack, until recently, of uniformly pure sugars, making it possible for one experimenter to check or duplicate another’s work or to repeat his own experiments with uniform results. USES OF THE SUGARS

Before describing the uses of the sugars in bacteriology, where their chief importance now lies, it may be of interest first to indicate their general value and their possibilities in other directions. Dr. E. Frankland Armstrong says in his recent monograph,l “Probably in no other branch of chemistry. . . . . .is so great an opportunity afforded for the study in detail of the influence of structure on the properties of the molecule. Much has already been done in this direction, but we are only upon the threshold of the inquiry.” Raffinose is used in physicochemical measurement of the degree of hydration of ions. Other sugars are used as control reagents in carbohydrate analysis. Mannitol is used in borax determinations in fertilizer analysis. Levulose is valuable for diagnosing certain pathological conditions of the brain. Mannitol has been used in the preparation of the detonator, hexanitromannitol. Glucose, lactose, and maltose are important in experimental nutrition, and have, of course, graduated out of the class of rare sugars and found broad uses in technical grades. Glucose has especial importance in typhoid diet. Thi? sugar, because it is the form in which the carbohydrate is carried in the blood stream, is being injected intravenously in cases of shock, particularly following operations and in cases of exhaustion in pneumonia. Dr. R. T. Woodyatt and Dr. W. D. Sansum, of Chicago, and Dr. Russell M. Wilder, of the Mayo Clinic, have studied and developed this use for glucose as described in the literature.* As illustrative of the wide use of the sugars even in the human body, the following will be suggestive of future fields for the use of the carbohydrates. Practically all of the carbohydrates taken into the body, with the exception of levulose and galactose, must be converted to glucose before being assimilated. Glucose is found in normal blood to the extent of 0.08 to 0.14per cent. So far as is known, the digestive tract is able t o assimilate all of the carbohydrates which are found in nature, with the exception of inulin. Dr. H. B. Lewis, of the University of Illinois, declares inulin is not assimilated. This is due to the fact that there are no specific enzymes in the intestine which convert this carbohydrate to a form which can be utilized by the 1

82

“The Simple Carbohydrates and the Glucosides,” Longmans, Green

co., 1919.

2 J . A m . Med. Assoc., Dec. 11, 1915, 2067; Ibid., Oct. 27, 1917, 1410; June 23, 1917, 1885; Aug. 17, 1918, 71; Jan. 10, 1920, 75; J. Bzol. Chem., March 1917, 355; Ibid., May 1917, 155, March 1920, 315; Ferrar. Am. J . Obstetrics, October 1920.

T H E J O U R N A L OF I N D U S T R I A L A N D ENGINEERING C H E M I S T R Y

I 204

body. The enzyme maltase converts maltose into two molecules of glucose; lactase converts lactose into glucose and galactose; sucrase converts sucrose into glucose and levulose. The normal individual consumes between 500 and 600 g. of carbohydrate daily. The carbohydrate is the most important food because it is the easiest form, of food for the body to oxidize; in other words, the body can get more energy from carbohydrate for the proportion of energy expended than from the proteins or fats. In fact, as is often said, “the fats hum in the fire of the carbohydrates,” or, in other words, carbohydrate must he present in proper proportion to secure the proper utilization of fat.

Val.

iz,

No.

12

cogen is synthesized and found only in the animal body. It acts as a storehouse of energy for the body. In cases of starvation no glycogen is found. The glyeog& content of the body is lowered by severe exercise and almost disappears in cases of severe tetanus, either natural or artificial, such as that produced by strychnine. When a starving dog, whose liver is free of glycogen, is fed on a heavy meat diet free of carbohydrate, the liver and muscles are soon found to contain a high per cent of glycogen, which fact proves that protein may he changed to carbohydrate in the body. When an animal dies, its glycogen is converted very rapidly to glucose because of an enzyme in the tissues. The quantity of glycogen found scattered throughout the animal body in some cases is as high as 15 to 18 per cent (as in the liver), and the ease with which it is converted to glucose indicates that glycogen and glucose are very closely related to the vital processes of life. In this connection there is an interesting fact relating to the nucleus of the living cell, which is the very center of life so far as it can he determined. In the cell is a substance called nucleic acid which is Found to contain B carbohydrate. The identity of this carbohydrate is an important study, and Levene and Jacobs,‘ Neuherg,l and Walter Jonesp have made important investigations to identify it. In the brain of growing infants, galactose is found in large amounts, and is always present even in the mature brain. This, perhaps, explains why nature chose lactose as the carbohydrate in milk, since the disaccharide, lactose, is composed of equal parts of glucose and galactose. This fact may partially explain why any attempt to modify human milk without the use of lactose has not been very successful. The infant seems incapable of synthesizing galactose from any other carbohydrate. APPLICATION IN BACTERIOLOGY

(31 RAE# S o o ~ a a The three test t u b e contain culture media with d-gtucwe (dextrwe). (I) B . oldiscncr leave media unchanged. (2) B . lyphosui famen- glucose.producing acid which tszm indicator red. (31-8. coli not oaly fermenta the sugar but produce%bubble of 8” which _e shown trapped in the media. stiffenedwith agsr. (1)

(2)

D I B W B ~ B N T X ~ ~01 ON BICIBB~A-a



This is proved in cases of severe diabetes, where & high as from 50 to 2w g. of glucose have been excreted daily. The old method of treatment was to remove carbohydrates completely from the patient‘s diet. It was found that the fats were then not completely oxidized and that acetone bodies, namely, 5hydroxybutyric acid, acetoacetic acid, and acetone, appeared in larger amounts, the patient generally going into coma, and death following. The later method is not to remove carbohydrates entirely from the diet, hut to cut down on all the foods in the proper proportion. This would seem to prove that a certain amount of carbohydrate is necessary for a proper utilization of fats. Levulose may he useful as 8 f w d for diabetics, as the levulose is assimilated as such into the blood stream. The body can get its energy from levuloseand in this way levulose can take the place of glucose, which, in diabetics, the body is, for some reason, not able to utilize. In certain diseases the body is unable to utilize the pentoses found in the food and they are excreted as such. This condition is called pentosuria. What would happen if the system were flooded with large amounts of pentoses is a field for interesting future research. Glycogen, sometimes called animal starch, is .qtremely important, and future investigations may develop its uses. Gly-

While the foregoing would indicate that the possibilities of the rare sugars in the future may be great, as a matter of fact their present demand is extremely limited. They have found their chief value in differential media in bacteriology. (And even here they are used in such small quantities that the manufacturers would fall on the neek of any bacteriologist who would train his “hugs” t o use more than one per cent in sugar solutions, so that the sugars might he sold in pounds instead of gramsl) Bacteria which are dBicult or impossible to differentiate by morphology or hy the employment of stains, are differentiated by their reactions upon the rare sugars. Bacteria either do not ferment certain carbohydrates, or else they do ferment them because of their ability to utilize these carbohydrates for their energy requirements. The acid produced in the fermentation reaction is evidenced by a color indicator, such as litmus. Many t+pes produce gas as well. These gas- are carbon dioxide and hydrogen, and the proportions in which they are produced vary more or less with the organism and with the time rhraugh,which the observations are made. Brilliant work upon the gas ratios was done hy Dr. Theobald Smith, of the Roekefeiler Institute. Thb determination of the, gas ratios as a means of identifying the various strains is not now considered so important as it was. In most cases, so far as we know, the cleavage of the molecule is produced by a process of hydrolysis, hut, as a matter of.fact, not very much is yet known about the chemical reactions. The work with the rare sugars is done in racks of test tnhesthe various sugars being arraoged in rows in one direction and the various strains of bacteria being in the rows at right angles to There are many refinements in methods of preparin the the culture media, hut since the value of these refinements is in dispute among the bacteriologists themselves, a simple statement of typical procedure will suffice. The culture media with the sugars are prepared by all bacteriologists with prac1 I. Am. Chcnr. Sw., 8% 231; B e . . 4% 1198.

Bmer.

* Bn.. I

19, 2806. “Nucleic Acids,” Longmaoii, Grcen B Co.. London, 1914.

Dec., 1920

T H E JOURNAL OF I N D U S T R I A L A N D ENGINEERING CHEMISTRY

tically the same formula. The U. S. Army Medical School makes up its media in a typical manner as follows: Sugar-free meat extract, beef or veal infusion Special sugar.. 1 .O per cent Peptone ............................... 1 . 0 per cent End-point neutral, Andrade indicator, autoclaved for 10 min. at 10 lbs. pressure.

.........................

The accompanying chart was compiled by the U. S. Army Medical School, which is doing brilliant work under Col. Frederick F. Russell, and was loaned by Col. M. A. Reasoner, who was one of the first to appreciate the importance of the sugars to American medical research. ’

GROUP I

7 I P H I N T I N G N O C A R B O Y Y O R A I I I WIGYER THAN HEXOSES

GROUPII RRMtNTlNG HLXbSES PYVNANNITi BUT NOT LACTOSC. OTHLR CARBOHYDRATES VARIABLE

NO

GAS.

GROUPIE

pointed out, certain definite differentiations with the sugars have been made, but the work has thus far had the nature of cataloging. Much study is being devoted to the characteristic action of the hexoses and pentoses, etc., to the d- and l-configurations, and various classifications, in the hope of discovering the key to the problem. The problem cannot properly be called the bacteriologist’s problem, nor indeed the chemist’s problem. Whoever solves the riddle will be some rare genius who is both bacteriologist and organic chemist in one. It may be interesting to give certain examples which bring US to the borderland of the known and the unknown. The sugars which are found in nature, whether d or 1 (and practically all are d sugars), are the ones which are fermented. For instance, bacteria which will ferment d-glucose, d-levulose, d-mannose, and d-galactose, will not ferment 1-configurations of these sugars. This brings us to theories of enzymes and enzyme action. To state another problem: Why will a very slight change in the stereoisomeric formula of a carbohydrate, or of a very small change in its terminal groups, determine its fermentability by various organisms? Why, for instance, will B. dysenteriae Flexner ferment d-mannitol and not d-sorbitol? According to Emil Fischer, d-mannitol and d-sorbitol have the following stereoisomeric formulas: H H I

FERHENTING HEXOS€S,MANNITE ARABINOSl.,ANDRII*MNOIE, BUT NOT

H-C-OH

I I

X I L D S C NOR LACTOS+&4SSOHLTIHfS PRODUCED.

H-C--OH

GROUP 14

I

H-C-OH

I I

.

HO-C-H HO-C-H

HO-C-H

rERMENTlNG HUOSES,MlwNlTf,n*LTOS< XYLDID AnD RMNOSC, NOTLACTOSI. bAS USUALLY PRODUCLD.

H-C-OH I

GROUPV ILRMlNTlNG HfXOSES,MLNNITE,~NT~YI f f l D UCTOSE. STWNGAOD R