The CHEMIST at WORK ROY I. GRADY
AND
JOHN W. CHITTUM
The College of Wooster, Wooster, Ohio
XXXIV. CHEMISTRY I N A TUBERCULOSIS SANATORIUM GEORGINE A. MOERKE
Dr. Moerke is biochemist i n the Detroit Department of and during the early part of her graduate work she served Health. She is assigned to the W m . H. Maybury Sana- as a chemist at St. Luke's Hospital, Chicago. Dr. Moerke is a member of Phi Beta Kappa, Kafipa torium (Detroit Municipal Tuberculosis Sanatorium) at Mu Sigma, Sigma X i , The American Chemical Society, Northwille, Michigan. She received the S.B., S.M., and Ph.D. degrees i n or- Biological Photographic Association, Biochemical Soganic chemistry from the University of Chicago. Before ciety (England), and the American Institute of Chemists.
Our institution is located thirty miles from the city which owns it, and is really in the country. To say that it is an eight hundred forty bed hospital does not describe it, for the sanatorium comprises a vast array of buildings scattered over twelve hundred acres. There has always been and probably always will be considerable maintenance work for the chemist, inasmuch as the water supply is chlorinated, the hot water undergoes a softening process, there is a brine refrigeration system in the main chain of buildings, there are the laundry, painters', carpenters', electricians' and blacksmith's shops, the kitchens and the bakery, the pasteurizing ~ l a n tthe , garage, the farm, the sewage disposal system, the experimental animal quarters, and so on. In such an isolated community many emergencies arise in which the laboratory is called on for help. To make it harder, suppljes can only be ordered four times a year. The jobs the author has worked on make a ludicrous list, from analyzing axle grease to refilling steam radiator diaphragms (I hope that was not illegal-but the city was broke a t the time and could not afford new ones). The laundry may ask what to do when a whole batch has come out pink because a nurse left her lipstick in her uniform pocket. The painter has no mahogany stain; what can we fix up that he can use for repairs to a desk? The rats are eating up all the guinea-pigs' oats; will I fix up some of that good cheese rat poison? Will I bring some large sterile bottles and go on a tour with the engineer to collect samples of raw sewage and filter bed effluent from two other municipal institutions in the vicinity? (They laid tar paper down first in a certain filter bed and-not very surprisingly-the efficiency of that bed is variable.) You may be wondering when we will get around to work on tuberculosis. This is a question that has frequently puzzled the author. Well, with eight hundred forty beds, all full, with a waiting list, there is a large amount of biochemical routine work to be done. The usual clinical, and some surgical, facilities are provided,
and since the institution has no pharmacist, the preparation of much material falls on the laboratory, and that of special solutions on the chemist. In general, the biochemical tests run on tuberculosis patients deal with other matters than tuberculosis and cover the same aspects of disease as biochemical work in a general hospital, even to following the effect of sulfanilamide therapy in streptococcus and pneumococcus infections. Until my camera wore out, I was called on for photography of the most varied subjects-clinically interesting aspects of patients, pathological specimens, experimental animals, charts, bacterial cultures, apparatus, and so forth. At present our equipment permits only photomicrography. One of the most time-consuming assignments, although interesting for the photographic problems it presented, was a series of several dozens of pictures of interiors and exteriors of buildings and of equipment in many parts of the institution, showing things badly in need of repair or replacement. These pictures were used to illustrate a plea for additional funds from the city fathers, and the plea was partially successful; one favorable result was the repainting of the chemistry laboratory itself, covering the old dark mahogany-stained cupboards and woodwork with glistening white, and thus improving the illumination very agreeably. Until very recently, the chemist was expected to handle all the foregoing work alone and, moreover, carry out laudable biochemical research in tuberculosis. Lately, however, the laboratory staff has been somewhat reorganized and certain routine jobs assigned to others. Tuberculosis occupies a unique position, in the very wide study it has been given for many years. Since the bacillus or mycobacterium causing the infection was discovered by Koch in 1882, many and varied chemical investigations have been carried out along whatever line, in the light of current knowledge, seemed likely to prove fruitful. Much was known of the
disease before the causative organism was discovered, and the literature of a century ago contains some strikingly interesting observations. A great deal of the chemistry of tuberculosis has been learned, but unfortunately very little of that is of any direct use to the infected individual. One outstanding annoyance to biochemists occupied with research in tuberculosis arises from the painfully slow growth of the cultures one must work o n s o that one has to wait two months to get the amount of material which many other kinds of bacteria produce overnight. The progress one makes is thus necessarily of slow-motion variety. Because so many extensive chemical studies have already been made, and all aspects of the disease have received a t least some attention, the development of an original research program in such a field is not simple. On the other hand, in spite of what has so far been done, there is the challenging feeling that the really vital facts are still to be dug out. We are fortunate in having as director of our laboratory a pathologist whose observations in tuberculosis are made with a care which should delight the sages of old. As a happy result of his curiosity, we are provided with really exciting questions to answer, many of which lead to fascinating chemical research. For example, in order to explain a pigmentation observed under several conditions, it was necessary to consider the types of pigment which might be involved, decide on a likely one, and then cast about for the possible mechanism of its formation under the circumstances permissible. This led to a consideration of a certain group of enzymes, and then to the actual search for a particular member of the group-and the author found that the tubercle bacillus produces a polyphenolase of the catechol oxidase type. (Until a few years ago, I was unwilling to believe that there really were enzymes, and felt that physical chemistry could explain i t all. Retribution was swift! I now concede that the enzyme is mightier than the chemist; but the chemist is somewhat more durable, if less powerful.) Although enzymes of this type have been reported in tissues of vegetable and lower animal orders for more than forty years, the actual significance of their presence is only now receiving belated attention. It appears increasingly probable that they are important factors in a variety of biochemical transformations. One aspect of this kind of work which rarely receives mention, but which requires much extra time and care, is the need for using sterile apparatus from start to finish. When you consider how hard i t is in regular chemical work to foresee all the kinds of glassware and how much of each kind you are going to need, when you don't know what chemical action is going to occur or how much unintended experimentation will e n s u e you can imagine how difficult i t is to provide everything you can possibly need in sterile form and keep i t uncontaminated. There is no awful moment in ordinary chemistry like the sudden desperate need of a sterile funnel and sterile filter paper.
Frequently i t is just as important not to contaminate your person with your working materials as it is to keep the materials themselves sterile. Besides the danger of direct infection there is the occurrence of sensitization to bacterial components, so that manipulation of materials in themselves quite non-infectious may still be vastly uncomfortable and even put one to bed. You may smell, see, taste, or touch no material outside the container yet find yourself the victim of a violent reaction! Another obstacle to work in a laboratory situated in the woods, as ours is, is the contamination of material by mold growths. Molds attack not only the culture media and a surprising number of pure chemical solutions, but construction materials of cloth and leather, such as those in book bindings, framed charts, typewriter cases, camera bellows, even moist spots on work benches. The author covered everything with a coating of shellac saturated with thymol, so that perus'al of the reference books is now accompanied by a wholesome emanation, if not inspiration. Very few buffer solutions can be kept long after preparation, although the component solutions can be kept and mixed as needed. Since various simple phenolic substrates for the type of enzyme whose presence was suspected in the tubercle bacillus, were already on hand in the laboratory, it did not require a great deal of time to establish the existence of the enzyme by qualitative tests, yielding colored oxidation products. Quantitative studies, however, required the measurement of oxygen absorption during the enzyme-catalyzed oxidation of the phenols, and since the institution's funds did not permit any special expenditures for such a necessary luxury as a Warburg-Barcroft apparatus, the author had to design and make the equipment herself. An ancient buret of a type no longer on the market served as a starter and a trial hookup with mercury reservoir and reaction flask showed that the idea was practical. After that a set of ten outfits were made up on as similar lines as supplies permitted. This involved a good deal of glass working. (I have never regretted the time spent or the skin lost in a course of glassblowing taken after hours during a very hot spring quarter.) There is really no thrill quite like that of setting up wholly new experiments, in apparatus of one's own design and manufacture, and collecting extensive data therefrom which can go into the always-satisfyinggraph. In studying the action of a particular and substratespecific enzyme, it is not always necessary or even desirable to work with the isolated enzyme in pure form. An enzyme may be more stable in the presence of other proteins. It may perform its function more efficiently in the presence of added protein. At present it is important to know the significance of this particular enzyme in the development of the tubercle bacillus and in the infected body. Since it is diffusible, studies have been made not only on masses of bacteria themselves, but also on bacterial culture filtrates, and on extracts from crumbs of bacteria. Thermostability
in the presence of other tuberculo-proteins, and precipitability by protein reagents were established. Methylene blue was found not to serve as hydrogen acceptor in this t p e of enzyme-catalyzed oxidation, free oxygen apparently being needed for the occurrence of the reaction. During some of the quantitative studies of oxygen absorption it was discovered that a
certain popular buffer, much used in this type of work, itself undergoes photodecomposition. Thus while working on a complex problem in biochemistry one may be thrown right back into "pure" chemistry. There is much more than etymology involved in the fact that the farther one delves into "pathological chemistry" the more organic chemistry is involved.
