Harrison Randall - Session III

Dennison:

Well, I thought I would give some of the background first Mr. Randall, and then we’ll…

Randall:

Wait a minute, I’ll have to turn you on more; I don’t follow you.

Dennison:

This is June 21, 1965. I am David Dennison, Professor of Physics, University of Michigan, and I will be one of the two people interviewing Professor Harrison M. Randall, who was for many years the Director of this laboratory. Professor Randall has been interviewed, approximately a year ago, by Dr. James King in connection with the history of physics project of the American Institute of Physics. It was suggested about six months ago that the interview with Dr. King took care of the early part of Professor Randall’s life, but that it stopped at the time of his retirement in 1940. It was suggested that this historical document should be continued and Charles Weiner was very helpful in making the arrangements. So, we will now take on from that point. Later on, we will be joined by Professor Donald W. Smith, who will — let’s see, Smith is from…

Randall:

He’s at the Medical School of the University of Wisconsin.

Dennison:

…the University of Wisconsin, who will then interview Professor Randall, especially on the significance of the recent work. But I thought I would start back at the time of Professor Randall’s retirement and recall at least some of the things as I saw them, with the hope of getting Professor Randall started. Now, my recollection is that at the time of just about his retirement for the next two years, I would think, Professor Randall was very much interested in a recording infrared spectrometer. This recording spectrometer I thought was built about the time when Dr. John Strong was a graduate student and obtaining his Ph.D. here and it was the first recording spectrometer that I know of. Since that time, of course, many recording spectrometers have been developed, but this was the first one. And after Professor Randall’s retirement, I remember his spending a great deal of time in the careful calibration, both with respect to measuring lines and also all the errors of the screw, and so on. Is that right, Professor Randall?

Randall:

Yes, I think that is essentially correct.

Dennison:

It was the first recording spectrometer?

Randall:

As far as I know it was the first one that did that satisfactorily.

Dennison:

It wasn’t a grating spectrometer; it had this salt prism growing in which you and John Strong had done the growing of.

Randall:

When one began to work with compounds, the absorption bands were so broad that it was necessary to precede the grating with a prism of a small refracting angle to prevent overlapping orders. That, the overlapping, did better so long as one was working with emission-line spectra; the higher orders in that type of spectrum could be easily separated. But with molecular spectra that was impossible, and the combination spectrograph of prism-grating became essential.

Dennison:

As I remember, this prism spectrometer had quite high resolving power for those days and it was because it was so carefully built.

Randall:

Yes. The department was fortunate in having a grating loaned to it by the astronomy department, and that had 15,000 lines per inch practically, so presenting those parts of the spectra which were not overlapped was highly resolved. And we were able for the first time to resolve the lines of the spectra of hydrochloric acid vapor, hydro fluoride, hydrobromic acid and hydriotic acid. And this was done by a student, Imes, from Nashville.

Dennison:

That has been in the earlier time. That was in, oh, about 1917 or ‘18.

Randall:

I imagine that was the time.

Dennison:

Now, coming up to the more recent time, though, oh, around 1940, with this recording prism spectrometer, my recollection is that you began getting into taking spectra of more complicated molecules at that time with this recording spectrometer. Is that correct?

Randall:

Well, I had thought of doing that, and the normal way of procedure would have been to make mixtures of known compounds in known proportions for comparison with the spectra of the known compounds. But at that time the Department of Bacteriology of our Medical School was interested obtaining — I can’t think of the word.

Dennison:

Let’s go back just a little bit still. My recollection is that as you began taking spectra, not necessarily of bacterial was things at that time, also one of our students was Fowler and you and he wrote a book which was on these compounds. This must have been written at about this time.

Randall:

Yes, I had agreed with Harper’s Brothers to write a book on infrared radiation; this was before the last World War. But during the World War infrared radiation was used so very extensively and by so many people from all professions that I no longer was an authority on infrared radiation at the end of the World War. I tried to withdraw from this contract, but the company was reluctant to have me do this, and it occurred to me that I might substitute for a general treatise on infrared radiation work that had been done in my laboratory on the infrared spectra of penicillin. During the early part of the war the penicillin available was very impure, and it seemed that it was natural to first obtain the structural penicillin in order to synthesize it. And for this purpose many chemical laboratories, both in England and the United States, were working on this project. It proved to be a very difficult one and so they asked the assistance of infrared spectroscopists. The laboratories involved were Cambridge and Oxford in England, Michigan and a commercial infrared laboratory in San Francisco. These four laboratories worked more or less in conjunction with chemists. To do this the Michigan Laboratory ran the spectra or many compounds which were supposed to be more or less similar in structure to the penicillin. Also, the spectra or many atomic groups, which were supposed to be similar to atomic groups which might be in the complex penicillin molecule. This meant that at Michigan we soon had a group of 10 or 15 people engaged beside myself. There was one full-time chemist, one full-time physicist, and one mathematician. At the end of the… well, by that time, at the end of the war, penicillin had been grown and purified sufficiently so that the synthesis of it was no longer necessary.

Dennison:

That’s Richard Fowler. Was he associated with you at that time?

Randall:

Yes. The physicist that worked with me was Dr. Richard Fowler, at present at Oklahoma University. He had been previously a chemist but had obtained a Doctor’s degree in physics in my laboratory. He was responsible for the chemical work connected with this project. Then, when the Harpers Brothers wished me after the war to write the text on infrared radiation, as I said before so much had been done by so many people of different experiences on the field of infrared radiation that I thought that I was no longer an authority on the whole field and not competent to write a book on that type of radiation. However, they did accept in place of this the work which we had done on penicillin, and a book was published with Randall, Fowler — the fellow at Nashville, what’s his name?

Dennison:

That wasn’t Fuson, was it?

Randall:

Fuson and Donge. This book was about half ready for publication when Harper’s decided that it was proving too expensive and wished to drop it. I assured them that the text was not one that would be ever popular but was one to be used in laboratories and research organizations and that the price they charged for it was immaterial; they could charge any price they wished in order to make it profitable. Under those circumstances the book was published, and there has been ever since about the same demand for it over all the years. Can you turn this off?

Dennison:

Well, it doesn't matter. Why?

Randall:

Well, I'm not sure it was Harpers. What other could it be? [The actual publisher was Van Nostrand (N.Y.: 1949) Randall et al., Infrared Determination of Organic Structure]

Dennison:

Well, it really doesn’t matter because as long as we’ve described the book this much, then it is possible to locate it, so one could look it up. Mr. Weiner, no doubt, in going over this can look up the actual publisher. It had in it, as I remember, the groups of atoms and molecules, and in a way it was identifying what the spectra were of different groups of atoms and molecules.

Randall:

Yes. The value of this book lies in the very large number of wave lengths which were measured in the spectra, so that one can look through the tables of wave lengths and the corresponding computer frequencies of oscillation and find out what kind of compounds might be expected to have produced such a vibration rate. Besides tabulating wave lengths and frequencies examples were given of how to interpret a given spectra-result. This has proved to be a valuable assistance to people beginning to use this kind of spectroscopy.

Dennison:

Well, I think maybe we should just pause here for a minute; I want to talk a little bit. (pause) I notice your hearing aid is playing back a little bit. Maybe it’s a little bit on the strong side.

Randall:

I may have a battery that’s run down. I usually carry…

Dennison:

It’s turned up a little bit too much. Okay, that’s better. Well, I found it very interesting, your work on penicillin, which I’d forgotten and, in a way, this then lead you naturally into working with the bacteriology department. How did that start? Did somebody from bacteriology talk to you about the problems?

Randall:

You see I was working on a spectrograph that didn’t belong to this department; it belonged to the Department of Bacteriology.

Dennison:

Had that been the spectrograph that you did the penicillin work on?

Randall:

Yes. No, no. Let’s see. After I had worked a long time, before retirement, in infrared spectra, it had always been with as pure compounds as possible, whether atomic or molecular. And I was — of course, some or the compounds were quite impure, which meant in a way that I had a mixture with one substance dominating the mixture. Who’s the bacteriologist over in the Medical School?

Dennison:

Is that Nungester?

Randall:

Nungester. He owned this spectrograph, the Medical School owned it, and he had it in his department. He had men trying to use it and getting all kinds or results. I went over — I don’t know whether he called me over; he may have — and found out that solutions they were working with… Well, Nungester was trying to find a cure for tuberculosis, a compound that would cure tuberculosis or help in the cure. What’s the word for it, for tuberculosis…

Dennison:

Vaccine?

Randall:

Yes, a vaccine for tuberculosis. And as I said, he was getting all kinds of results when he was using solvents of the tubercle bacillus. When I first went over there and talked with him — I talked with the men who were doing the work — it turned out right away that a solution to them meant a solution. They were sometimes digesting the organisms overnight, sometimes over a weekend — it didn't make any difference to them and I said right away, “Everything has to be systematized. A method has to be worked out for determining each quantity, and we have to know what the tolerance is going to be for every quantity.” Well, he agreed to that, and we began to work on that basis.

Dennison:

Now, this would have been, oh, 1950, do you suppose?

Randall:

I was never any good at all on dates. I have most all of those papers. I have the very first one I did with them.

Dennison:

I think it must have been because this was a recording spectrometer, and they were commercially made only after the War.

Randall:

It was a very good Perkin-Elmer spectrometer.

Dennison:

Well, then this would have been ‘48 to ‘50 probably?

Randall:

Yes, that’s right. I think ‘49 to ‘50.

Dennison:

But the work that you had done with penicillin must have been done on our own recording spectrometer because there weren’t any commercial ones at that time.

Randall:

It was done with penicillin; I’m sure, on this combination prism-grating spectrograph.

Dennison:

I’m not sure whether it was just a prism one. It’s hard to know.

Randall:

When you work with atoms, atomic spectra, and get lines, then with gratings you have an assortment of first and second and third orders, and so on. Now, the grating we had came from the observatory in 15,000 lines per inch. That meant a whole lot of overlapping. It was all right so long as we worked with metals and we got line spectra. Just as soon as we worked with molecules and began to get molecular spectra and the lines were broad, then it didn’t work.

Dennison:

Now, when you started working with Dr. Nungester, that was before Dr. Donald Smith came into the picture. There was Nungester and some or his people that you were working with.

Randall:

Yes. Well, Smith was one of Nungester’s students.

Dennison:

Oh, he was.

Randall:

And Smith was the first one of his students that worked over there who was willing to do what had to be done in the way of systematic work. He had worked there only a year or so when he had a chance to go to Wisconsin. We kept up the work but Nungester withdrew when Smith went to Wisconsin.

Dennison:

At this time you were beginning to get definite results in that you could reproduce the spectra of these things.

Randall:

Yes. He found that he could grow the bacteria, harvest it, put it in a solution of alcohol and ether, put it through a chromatographic column, and evacuate every 10 cc. that came out of the column. He could fractionate the complicated mixture by putting it through a chromatographic column, and he collected… What do you call that which comes out of the end of a chromatographic column?

Dennison:

Gee, I don’t know; a precipitate?

Randall:

It isn’t a precipitate. He collected the fractions coming out of the — no, he collected the emergent solutions in 10-cc. amounts, dried them, and sent these solid materials to me for infrared examination. And I ran infrared spectra of every eluate. And after a while we got an infrared spectrum which agrees with the infrared spectrum of a known compound. So that it showed that our column was doing a good job; we were getting not simpler mixtures but we were getting compounds, even though they were impure. And in a short time we decided that every time we got an eluate from a compound that a reproduction of what we had already obtained, it meant that we had found a new compound and the more frequently that spectrum was repeated, the surer we were that we had a new compound. And in that way we examined some 140 original growths that he had made.

Dennison:

Now, this work was supported by one of the Federal agencies?

Randall:

We found trouble in getting a publisher. I sent a first report of that work to Science; they turned it down, they wouldn’t publish it.

Dennison:

I wasn’t thinking of the publishing; I was thinking of the financial support. You had several technicians who were working for you there.

Randall:

Oh, yes, I applied every year to one of the governmental agencies that sends out work to be done by universities. I used to get from $15,000 to $20,000 a year. I guess most of it was Public Health; I can’t be altogether sure.

Dennison:

But my recollection is that you had two or three people who were working with you downstairs.

Randall:

Oh, besides myself, there was Fowler, Fuson and Donge as full-time ones, and I had a lot of graduate students.

Dennison:

This was in the earlier time, with penicillin, and I’m thinking later on when you were working with Dr. Smith. Then there was, I remember, a girl downstairs…

Randall:

Oh, yes, I had a girl down there who was working for a degree to be a teacher for second-grade children. She ought to have been working for a Ph.D. degree.

Dennison:

She was very good, as I remember.

Randall:

Oh, she was great. I never knew anybody as good as she was, and she soon had the thing very well organized, and if anything went wrong with the equipment, she was better to fix it up than Paul was. First she went over it, and if she couldn’t do it, we had Paul, but she ordinarily was the one who found out what was wrong.

Dennison:

Well, now, during that period, and that’s really only a few years ago, was the spectrometer that you used here or was it over there?

Randall:

It was over there. We never had one over here. It was all over there.

Dennison:

And it was always the same spectrometer, the Perkin-Elmer. This work stopped about, oh, two or three years ago?

Randall:

Yes, I thought I was getting to be too forgetful.

Dennison:

Well, I remember there was a time when you stopped it, and my recollection that it was about two or three years ago.

Randall:

Yes, it was, just about then. And the department here didn’t seem to think that that work would go on anymore, and everything involved was moved to Wisconsin. All of the records — my records were on ordinary sized sheets, writing paper sheets, and I took them over to one of the offices in the main building and they reproduced them and sent the reproductions to Don in Wisconsin. They were on rather stiff paper. He kept all of those and now he has the original ones, too. I don’t know whether any of the apparatus went or not; there wasn’t very much apparatus here. The University of Wisconsin bought him a spectrograph, so he has his own spectrograph. He’s been made a full professor, by the way.

Dennison:

Oh, fine. Well, I’m trying to think if there is anything else we can do at this moment. When Professor Smith comes, which I hope will be quite soon, we will get him to talk with you about the significance of the work. You were saying that you had some difficulty at first in publishing the work. How did that happen?

Randall:

Well… oh… the author or Science quoted one of the referees to whom he had sent the paper for examination. The referee said that he couldn’t see that we had done more than establish a hope for what we had wanted to accomplish.

Dennison:

I think this is incorrect because, I remember, in looking at these records…

Randall:

Well, what I sent in essentially is what has been published ever since. I came across recently a book in which I had written things at the time we started work, and the publisher who accepted our first paper wrote that he wished we would tone it down some, that we couldn’t really mean to say that by the simple curves and data that we were sending in that we expected to replace the work of so-and-so, who were at that time well-known chemists working on the chemical structure of bacteria. Of course, that’s exactly what we were doing; we intended to do that very same thing.

Dennison:

Well, actually, these were so definite, these curves, and reproducible. This was one of the main features, as I remember, the fact of carrying your process through so carefully and so systematically that the resulting infrared curves were…

Randall:

Yes, you had just to see the curves; as soon as it came off you knew what you had. A curve like that is as definite as a photograph is in many ways.

Dennison:

And what you were doing was to get different curves for the different strains of bacilli, and quite definite ones.

Randall:

Yes.

Dennison:

Okay, we might just stop for a little while. I thought I would just recap what we have to do because I have to go to a class now, and then this will give you the setting to start with, and then if you would like to get the things you can, and we’ll go on from that point. Professor Donald Smith has now joined us and will soon be talking with Professor Randall about the work which they have done together, and its significance. I thought before I go — and I have to go to a class — that I might just recap what we have done so far. In the first place, I want to emphasize the remarkable nature of the work which Professor Randall has done. He retired from the chairmanship, from the active work in the department, in 1940, and at the age of approaching 70, and then began this work at a time when most people stop their research work. This was a new line of work, something that he had not done before, and he is, in that way, I think, very remarkable. He continued with the work until about three years ago and has still done something in the organizing and writing up of the results. This next December he will be 95 years old, and I want to say how very proud we are here at Michigan to have had Professor Randall, not only for all he has done for the department but because of this remarkable thing with respect to the research that’s been done later. Now, just to give Professor Smith a background, what we have essentially covered has been how he got into it with, first or all, working on the penicillin problem during the war, and then later with the work with Professor Nungester — Dr. Smith was one of Dr. Nungester’s students — which then led into the work on the tuberculin bacilli. Now, we’ve covered some of this, but I think the thing we haven’t covered, and since I’ve not been able to do much about it, is the significance of the work and the general results which have been obtained out of it. And so, at this point I will turn it over to Professor Smith, who will continue talking with Professor Randall in bringing out the various points.

Randall:

You might add how much time we have already talked. I don’t know. Don would like to know how much time we have still.

Dennison:

We talked for, oh, I guess, somewhat over an hour, but the thing is just to continue, and you probably are most likely to finish up in the next hour because you can get quite a lot on tape in an hour’s time. On the other hand, if you haven’t finished, I’m sure these people would be glad to come back afterwards, after lunch. (Professor Dennison here introduces Dr. Smith to other persons present.)

Smith:

I’ve brought along with me, Dr. Randall, a number of reports of the work that came out earlier, and I don’t think we need to go through these. I brought them along in case we need a reminder. Could I begin and say what my recollections are of the beginning? The first project in this attempt to use infrared techniques in biological research was the work with Robley Williams and with Bob Bacher, when you were attempting to use the tobacco mosaic virus. And they supplied a certain amount of material, and you were attempting to help them in some of the problems they had in that work. This must have been in 1947 and 1948. Then beginning in 1948 my understanding is that you approached Professor Nungester and asked him if he had any problems in bacteriology that might be assisted by infrared techniques, and you explained that you had some interest in your retirement years in trying to determine if infrared spectroscopy could be applied in biological research in the same way electron microscopy and ultracentrifuge and other physical tools had been applied in biological research.

Randall:

That’s essentially correct.

Smith:

And the problem that Professor Nungester proposed was one in the tuberculosis field, where his laboratory was making efforts to isolate substances from tubercle bacillus which could immunize animals against tuberculosis. They had great difficulty in getting uniform product, so there was a great deal of lack of reproducibility. And in the beginning the people that were involved in this work were Robert Keeber, Robley Williams and Professor Nungester and several students, and the work continued for perhaps a year or so. And my recollection was that they would bring materials to you and ask you to record the spectra and to give them some interpretations from this, but that on occasion you got quite annoyed with it because we found that the materials would vary from one time to another, and when you would inquire about it, you would find that there were perhaps only 15 or 20 different procedures that had been varied and there was every reason to suspect variation in the infrared spectrum. One of the goals in the very beginning of this work was to try and see if there might be some correlation between bands in the infrared and immunizing activity, so that in searching for a fraction of the tubercle bacillus which could immunize against tuberculosis, it would assist materially if there were some way to recognize the chemical nature of this nature, and that this possibly could be accomplished by the infrared spectrum.

Randall:

Yes, that’s about right.

Smith:

Yes, but these were rather high-minded goals, and I think it was through the very great foresight of Professor Randall that we began to recognize that before we could attack such a major goal as this, a great deal more had to be learned about the basic material, and he was insistent that we begin to study every detail of the procedure that could lead to variation in the infrared spectrum. And this led to a period of two or three years of work in which we studied the reproducibility for the growth of the cultures, of micro-bacteria; we studied the effect of medium, the age of the culture, the methods for extracting, and great attention was paid to each step in this procedure in order to determine which ones were significant and could lead to changes in the infrared spectrum. Then, it was during the course of this work that we then studied the reproducibility of the preparation of the extracts and then we wanted to assess the sensitivity of this method, and we were still working with very crude materials. But we then deliberately chose strains of micro-bacteria that we knew to be different biologically and we began to question whether or not we could recognize a difference in the infrared spectrum of these crude materials. And the first two strains that were chosen were a virulent and an attenuated variety of micro-bacterium tuberculosis, H-37-RV, and H-37-RA, and crude extracts were prepared from these strains, and the infrared spectrum was recorded. And we found that although several of the fractions were quite similar, there were several other crude fractions that clearly permitted a distinction between these two strains. And then progressing further, we then selected other strains of micro-bacteria to further test the sensitivity of this method. And the next two groups of organisms chosen had a much closer biological relationship than H-37-RV and H-37-RA, and these strains were human and bovine types of micro-bacteria. And the human and bovine tubercle bacilli are very closely related biologically, but we began preparing tractions of these strains and at this time we had ceased using the original vacuum instrument and were now making our first records on the Perkin-Elmer double-beam spectrometer. They were recorded on the very large-scale paper. This work continued without very much in the way of positive findings for about 12 or 13 months, but it was only after Professor Randall arranged in his laboratory some large sheets of cardboard on which many of these spectra could be displayed at one time and it was possible to see that there was an absorption band characteristically present in extracts of bovine micro-bacteria and absent from the spectra of extracts of human micro-bacteria. And the subsequent study leading to the separation of this material led to the recognition of a substance we called GB at that time. Now it’s called Mycoside B, and this was the first in the series of Mycosides. And the significance of these compounds — eight Mycosides have been recognized — is that they are species specific glycolipides present in micro-bacteria. It was an entirely new series of compounds, and they’ve received rather great recognition in research in experimental tuberculosis.

Randall:

Well, it’s a question of how did we test these various things out. Were you going to give that? You said we determine the various factors, and how did we determine the various factors?

Smith:

Well, we…

Randall:

Let me ask: what were going to say anyhow?

Smith:

I was going to go on and discuss the separation of Mycoside B by chromatography. Just by way of general background, as I mentioned before, about eight Mycosides have now been recognized each one present in only one species of micro-bacteria, and Mycoside B was the first of these recognized. The first Mycoside B purification was by batch chromatography. But then at the insistence of Professor Randall, we began searching for better techniques for making the separations, and we became acquainted with a young man in chemistry who was doing column chromatography. And we then began developing the column chromatographic techniques to our methods, and began recording the infrared structure of column eluates from these various extracts of micro-bacteria. So that the general outcome of this was that by the combination of infrared spectroscopy and chromatography, it was possible for us to survey the lipids of many strange micro-bacteria. The total number now examined exceeds 200, and it was only by examining a very large number of strains that it was possible to recognize which substances were limited in distribution to one particular strain. The recognition of the Mycosides, I think, clearly could only have taken place after a great amount of time was spent on this. But by the combination of the infrared technique and chromatography, it was possible to examine many lipids very quickly and to survey many strains. And the recognition of the Mycoside would only come from a survey of many strains of micro-bacteria. And constantly, throughout this work, Professor Randall continued to stress the need for better techniques for making separations, and there was much searching of the literature and discussion with other laboratories of other techniques that could be used for making separation. On the other hand, there was a continuing search for techniques that could lead to handling smaller samples. It was Professor Randall’s feeling that the materials we could recognize by the infrared spectrum were only those substances present in high enough concentration to make a sample, and usually this requires from one to three milligrams. It was his idea that if we could, by using micro-sampling techniques, get acquainted with techniques that would permit us to use one to three micrograms of material, that it would uncover additional substances that would have been hidden otherwise. And he set people in his laboratory to work on developing micro-sample techniques, and did have, in fact, a successful technique working with solid samples and using as small an amount as five micrograms of material. We will discuss for a moment that at the very beginning of this work there was no published work on the application of infrared spectroscopy in bacteriological research, and after our work had been underway, about a year and a half, we had a visit of two scientists from the Chemical Warfare Center at Camp Detrick in Maryland, and they came for the specific purpose of finding the techniques that we were investigating, the techniques we were using for examination of bacteriological cultures. And their interest — this was near the end of the Second World War — was in using infrared techniques for identifying bacteria that could possibly be used in germ warfare, and they indicated that they received a great deal of encouragement from the consultation with Professor Randall. After this time, this group at Camp Detrick published a number of papers describing their work with infrared spectra of whole bacteria. At the same time, this work was going on in England, in the German Warfare Center, and again the laboratory was particularly interested in infrared spectra of whole bacteria. Professor Randall maintained during this time that the infrared structure of whole bacteria probably would not be quite as useful as the infrared spectra of extracts because of the fact that a very large share of the bacterial cell being protein and carbohydrates, that these materials would be quite common from one bacterial strain to another and that it might be more advantageous to work with extracts of bacteria. And our work was continued, stressing the use of extracts of bacteria. One other interesting aspect of this work: In the continuing search for techniques for improving the sort of separations and for improving techniques of recording spectra, Professor Randall and I journeyed to New York City to visit the Sloan-Kettering Institute, and we were to see their techniques for micro-sampling of urinary ketosteroids. At this time, we made a side visit to the Public Health Research Institute of the City of New York, and met Dr. Hans Noel, who was also preparing extracts of micro-bacteria and subjecting them to column chromatography and recording their infrared spectra, and we found, in fact, as a result of this visit, that the materials that were being separated from our chromatographic column were not always mixtures, but on some occasions were pure compounds. And by comparing our spectra with that of Dr. Noel, we discovered for the first time that we had pure mycolic acid and phthiocerol dimyco-aerosate, two compounds that are present in micro-bacteria. And this was a very great source of encouragement to us in our future work.

Randall:

I remember that trip very well indeed and how I felt when we found that our results agreed with those of Dr. Noel.

Smith:

This next section might be a recapitulation. We’re backtracking a little bit. As I indicated, when I first came into this work, we were still bringing to Professor Randall rather crude extracts of micro-bacteria that had been tested for biological activity in animals, and we would have the infrared spectrum recorded, and Professor Randall would send a report, saying, “Yes, this material is different from something we’ve seen earlier,” and then he would ask, “Well, now were the steps in the preparation of this different from the material you brought us the last time?” Then we would have to point out, well, there were probably 12 or 15 different steps that had been changed between the preparation of this material and the previous one. And, as I indicated earlier, in the beginning he took this rather gracefully, but as it continued, he got rather annoyed, to the point where he insisted then that we must stop and analyze each step which were in the procedure and find out which were critical steps leading to the changes in the infrared spectra of this material. And from that point, we began a very deliberate analysis of every step in the procedure. This was something new to me as a biologist because I had been accustomed to growing bacteria on media and preparing extracts and so on, not being altogether careful. So, this was an altogether new discipline, perhaps in biological research, and it’s something that I think was very important to me in other work. One other point that should be made in terms of the overall significance of this work: The beginning effort was to correlate bands in the infrared with immunizing activity. This was the introduction to the tuberculosis problem, but, as the problem developed, this point actually became lost because we began to see that there was a much greater significance to the technique in analyzing, first, mixtures of lipids extracted from micro-bacteria, and then a detailed analysis of the separated pure compounds from micro-bacteria. So that the introduction to the work was in looking for immunizing activity, but gradually it swung into an analysis s of the lipids of micro-bacteria and became a research project in itself. The overall significance of it was its leading to the recognition of Mycoside, and most recently one of the goals of our research together has been accomplished and this is the first demonstration of lipids of micro-bacteria in bacilli separated from infected tissue. Biologists for some time have maintained that perhaps these compounds that we’ve been demonstrating are artifacts produced by micro-bacteria grown outside of the tissue and bear no relationship to organisms grown in infected tissue or as the organism might grow in man. But recently, using these same techniques, we have been able to demonstrate both mycolic acid and thi in lipids of bacilli separated from infected tissue. Rudolph Anderson attempted to do this in 1940 and was successful, and we feel that our principal advantages over Rudolph Anderson are the addition of techniques of chromatography and infrared spectroscopy, and more particularly infrared using some of the micro-sampling techniques that Professor Randall worked out for us. Now, we would like to begin a general summary of this whole line of investigation, indicating in the beginning that it was the express wish of Professor Randall to see whether infrared techniques could be applied to good use in the analysis of complex mixtures of compounds and to see whether the technique could be useful in biomedical investigation. And we indicated that in the very early phases of this study, very complex mixtures of materials were presented to Dr. Randall for study and that not very much could be made of these because insufficient care was given by the biologists in the preparation of these substances. And it was very early recognized that before very much progress could be made, detailed analysis of each step in the biological laboratory would have to be in preparing these materials. And this led to the phase of the work that we've called the reproducibility phase, in which great attention was given to each step in the preparation of these substances. Then after we determined which steps were vital and which ones had to be carefully controlled, we wished to examine the sensitivity of the method, and in so doing, we selected various known tubercle bacillus combinations where we knew there was a biological difference, and we began to search for some associated difference that could be recognized by infrared techniques. And the sensitivity investigation went on for a period of six to eight months. It led to the recognition of the first of the so-called Mycosides present in mycobacterium bovis. It is of interest to note that the first presence of this material was seen in a mixture where the Mycoside was present in perhaps less than 10 percent of the total mixture, and that by recording the infrared spectrum of this complex mixture, it was possible to see an absorption band that suggested the presence of a substance that was later shown by chromatographic method to be Mycoside B. This was the first of the Mycosides. Then we went on to a study of micro-bacterium avium, another type of tubercle bacillus, looking for a similar compound, and we were able to demonstrate another mycoside in this organism. Then, about this period, in the studies, a group of bacteriologists asked us to take up the study of a new group of mycobacteria that were beginning to be recognized in human disease, and they wanted to know whether the techniques we were using could contribute to any understanding to this new group of organisms. They had not been recognized before. They were essentially unknown in their relationships with other types of mycobacteria. These organisms have been referred to in our papers as atypical mycobacteria, or unclassified mycobacteria, something of that sort. And in the study of the atypical bacteria, using the same techniques, we were able to demonstrate rather clearly several species of mycobacteria and several new mycosides. So, the significance of the mycoside is that it is a substance present in one species of mycobacteria and not in another, and provides a very excellent means of determining species of mycobacteria. Then, if we could go on and speak to the general contributions of this work, we could perhaps say that one of the goals in the beginning was to determine whether infrared techniques could be useful in biomedical investigation, and this seems to have been clearly established in this work. Another point that seems to be established is that contrary to the opinion of a number of chemists that were consulted during this work, who stated that infrared techniques would only be used after you were dealing with pure compounds to identify them, it seems to be clearly established in this work that infrared techniques and chromatography can be very useful as a combination procedure for analyzing complex mixtures.

Randall:

In summation of these methods it has appeared to have a very universal application. One can, in fact, describe different woods by the infrared differences of various samples; similarly for grains or foods. So that while we have developed this method very extensively in the field of physics and bacteriology, it is entirely possible to use the same ideas very universally. I think that sums up this… well… I’m through. I don’t know what to say.