Oliver Smithies:
This is an account of VIII Book 1956 starting May 30th, and running through to August 28th, May 30th through August 28, 1956. It’s a fairly straightforward book.
The beginning is just a recording family, and the gels from a family, with hemoglobin added. [name redacted] family and pages three and four similar with — on the left, a girl, [name redacted], and a girl, [name redacted]. These are babies that have had exchange transfusion [00:01:00] because of Rh incompatibility, and they’re noted as first replacement and second replacement, from a girl, [name redacted] gives an image of the difficulty of handling Rh incompatibility in those days, some diagrams of the staining, nothing particularly striking.
A boy, a picture on page five, and the mother, and some other samples from the [name redacted] family.
Seven and eight, more samples being studied. Saturday, June 7th, a typical Saturday, having gone to Norma Ford Walker’s lab, and this [00:02:00] is the rest of the [name redacted] family; it says father, [name redacted], and [name redacted]. So the family being typed.
Fully checked data on page 12 is a summary of what had been going on at various stages earlier; nothing very remarkable.
Mother [name redacted] on page 13, interesting sample taken before the delivery from a sample used for typing her red cells. Previous child had died, probably affected. The father wasn’t available, so that’s [00:03:00] the boy [name redacted] and the mother, [name redacted].
Her more — her two twins on page 15, and her family, the [name redacted] family, on page 18, and so on, more data compiled, page 20. Page 21, 22, another family. So these were just continuing studies increasing the sample size of everything.
If one looks at the gel notation on page 21 as an example, you can see some cut marks, rather obscure cut marks which are translated [00:04:00] on page 200, where there’s the code of the cuts. So, the first gel, because I would stain several at once, the first gel would have two corners off; the second one corner off. The third would be slanted, and the fourth would be straight, and the fifth would be, sort of triangular cut, et cetera, et cetera. So page 200 is the code for the cuts which is shown with the gels, even before page 21. [name redacted] family, and the [name redacted] family, good family on the 25th.
And, on Tuesday, June 12th, page 28, I note that Mother [name redacted], (inaudible) serum for complement fixation, [00:05:00] the results on attached papers. So I got involved in complement fixation work. I think, as I recollect, these were in association with Lois Kitze, Dr. Lois Kitze, with whom I was going out and about, later on to marry, and I was doing some things of her, trying to help her with her work. We’ll see about that in a moment. That was on the same page, page 27, another family talked about.
Normal mothers and babies on page 29. [00:06:00] More families continuing as we move on. And the complement fixation tests are first recorded on page 38 with a very complicated set of attached pages, for the hemolysin titration. I remember, what other people would be able to describe much better than me, that the complement fixation tests were really very difficult, and required a great deal of care, and here was my attempt to help by doing some complement fixation tests, solo, one, two, three, four, five, six, seven, eight, nine [00:07:00] ten — about 13 or 14 pages attached to the notebook of my attempts to help in this hemolysin titration, quite complicated, and not really very relevant to my future work, but just showing my attempts to help get the complement fixation tests working.
Back to more ordinary things, on pages 37 and 38, another family. This is apparently, as far as I can tell here, somebody with a chromosomal deletion, but I’m not quite sure what it is. It just says, [00:08:00] “[name redacted] family with deletion. Relationship to BG, [name redacted], shown.” I was just typing them to see if there was anything unusual about their types, and to continue to document the inheritance of the three haptoglobin types that we understood at that time. [name redacted] family, and the [name redacted] family again, on pages 39 and 40.
And then here, when we get to page 42, entries into the notebooks, that are clearly by Otto Hiller, with a little bit of comment of mine, where he’s writing down various sample, and the gels [00:09:00] run on page 44 were run by him, as can be seen by his writing.
I don’t really understand page 46, a rough test of 1, 2A, 2B interaction. They’re looking for some antibodies and possibility that haptoglobin transfusions that were [00:10:00] not of the same haptoglobin type might need to antibodies, that was what I was trying to consider. Some indication of that line of thought is on page 47 and 48, where I see from the Rh lab, samples with various comments that, [name redacted] for example was A-positive, and subtype DE Rh, and had received A-negative, CDE/CDE, cDE/cDE, Rh- erythrocyte. Some question about parentage in this sample. And whether [00:11:00] antibodies could be detected in the mother.
There’s some more of this thought of looking for interactions, a little bit obscure at this point of my memory, on page 50, where samples were put alongside each other, 1+2a, and 1+2b, to see if I could see any sign of interaction between the types 1a — 2a and 2b, but I didn’t find any.
Continuing with more samples, and I think very straightforward [00:12:00] checks, as usual to run gels which would check previous typings on page 53, and 54, and 55, and 56, with a comment that, “checks are OK; checks are OK,” meaning that they’d been confirmed in the typing.
Continuing in the same general vein, typical received from the Rh labs Saturday June 23rd, my birthday in 1956, I would have been 26 years old on that day, page [00:13:00] 64. And then, 66, no particular comment.
Sunday, Otto must have read these, run these gels, or at least written them in down, because this is his writing. Got some Danish starch on June 26th, page 74, because we’d by then begun to understand the starches, and the Danish starch had been the breast, brought into Canada as a result of a potato blight, which made Canadian potato starch unavailable. [00:14:00] So this was, starch from Denmark was received.
And on page 79 and 80, Otto Hiller’s entries, also 81, 82, more babies. Otto again, 83, 84, 85, 86, all Otto’s writing. Mothers on 30 July 5th, and July 6th, with several others, with Otto, and making the entry [00:15:00] into the book.
Continuing July 10th, more Otto’s entries. Maybe I was away; I don’t know why all of these are by Otto. Trying to make antibodies against human hemoglobin on page 103, July 11, injecting to rabbit hemoglobin from Otto, and preparing — and withdrawing blood out 12 days later. Very faint ring on hemoglobin commented in simple tests. [00:16:00]
Page 108, I begin to try out some agglutination using two — estimated antibodies by preparing a suspension of collodion particle, collodion being the same material that I’d used in my osmotic pressure work, so these would be samples that were made with Parlodion in acetone, similar to the work that I’d used in making gels for osmotic pressure measurement. Collodion particles were prepared approximately according to Cannon and Marshall, with Parlodion in acetone, with the attention of 25% acid-acetone, distilled water, et cetera, et cetera. [00:17:00] And several pages of this work going on into page 114, 116, with a pause to, because some of Otto’s results, going from several pages.
Summary again on page 129, 120, 130 were in Otto’s writing. So, the conclusion of the Parlodion work, I have to see if I can find it. I don’t see a particular conclusion reached in that work. [00:18:00]
In pages 131 and 132, at the beginning of a whole series of tests to try to find a good way of getting photographs of the gels, the gels were, of course, blue on white background that was not completely white, and the blue was not so heavy as to be too dark to reflect light, so were my experience up to that point had been with photographic work in general, I’d enjoyed over the course of many years, as a graduate student [00:19:00] for example, experimenting with photography, and I knew very well, as a result of a good book on developing, I knew well the effect of different developers on the grains — on the gamma, the contrast, that could be obtained with a film, and also the effect of grain size in the emulsion. All of these things where I had a good grasp of, and so, there’s a whole set of maybe 20 pages here devoted to trying to get good photographic attempts, is how it’s labeled on page 132, using a gel [00:20:00] that had been already a good gel as a photographic object.
I’ll pause for a moment; I need to find out what one of the notations is. On page 131 and 132, I’m starting photographic attempts using a film ADOX KB 17. This was a famous film; it was a thin emulsion, but very fine-grained black-and-white negative film, so it was a high-quality film, and exposed it with a K2 yellow film to improve the contrast of blue to various seconds of exposure, et cetera, and printed on white paper [00:21:00], et cetera, and various exposures. And, difference in development time to try to reduce the graininess of the film. Looking at them now, on page 131, the differences are really quite subtle, and it’s always being very fussy about the results. Page 133 is an example of different exposure times of the film. And then of course printed, there’s best from that exposure, so there was a fair latitude, and they have a comment that the contrast is less than or equal to that of Kodachrome, [00:22:00] which had also tested. Comments, or the typical comments, 134, too thin to print well, fairly good, but a bit thin, rather better probably optimal, and still not sufficiently contrasting. A band on this emulsion, when used with this develop — but also try a more contrasted developer, various developers, were available.
Comment on 136, try ADOX KB 14, with Neodyn red developer, Gevapan 27, et cetera, et cetera, different attempts. [00:23:00] Continuing in this vein, page 140 is a typical example with six different exposures of the film, some improvement, but not enough to warrant continuing with KB 14. KB 17, which evidently another film is better, or changed to Gevapan 27, with a contrast in developer, then try microfilm, which I think is what eventually we settled on doing.
On page 141, more tests, and a whole bunch of images that look to have been printed by [00:24:00] somebody else from the same sorts of experiments, having the three types, 1, 2A, and 2B, gels all on the same images, of four unattached ima– photo–prints. Gevapan 27 being tried on page 142, and comments on whereabouts on the exposure gamma curve, the exposure would be best selected.
Photograph continued, I remember this developer on page 144 recording to 1950 photographic almanac, D23, give us [00:25:00] nonshouldering curves at high exposure, and can give a high gamma, so try D23, but result apparently on Thursday, July 26 is, doesn’t appear much improved, nor on the best print, approximately equivalent to Neodyn red.
D11 was another developer which I remember did — trying with microfilm on page 146, (inaudible) try microfilm and reduce the contrast by reduce — by using C5 [d-bill?] filter, so I realized that microfilm was perhaps too contrasted, and needed to have a slightly reduced contrast and picked the development time very — the exposure time very carefully. [00:26:00] So I developed four minutes at 20 degrees with D11 developer, and still not enough contrast visually, and tried a quarter, half, one, two, and four minutes in the developer. Ten’s rather on the long side; 15 seconds is probably the optimal. And of course, whatever papers we used also had a bearing on it. And it’s complicated business.
(laughter) Typical comment on page 148. At this point, I discovered that the generalized fading of the gel, used as an object was giving me a problem. More photographs [00:27:00] on KB 17 with a red filter, which looking at them by now, some are really quite good. Much improved still, may have to develop to a lower gamma as the albumin is still almost clear, and that is, the negative has almost no silver in it.
Photographed on Monday, page 51, and 52, it’s a large frame — number 3 paper, to show the black is not jet black, and white is not jet white, so that I can get a full range of images without saturating, or underexposing [00:28:00] the film, to get off the bottom, as it were. No shading required. Can accept this result. And, that’s the end of the photography for a while.
Back to gels, Otto, page 154, and just another comment on the photography, Monday, July 30th. Exposed KB 17 with some pathological samples, two, three, and four minute, and developed for 14 minute. ADOX E10 developer. Results rather grainy, on the whole, too contrast-y. So, re-exposed, [00:29:00] KB 17, and developed for [Otto sign?], printed two minute exposure for each gel on #3 paper, D-88, with no shading. Backgrounds maxed, just off-white, results perfect. So I’m happy with the photography.
Some examples that Otto ran on August 14, more babies from the Rh lab, 159, 160, and then tried to photograph [00:30:00] a set of gels with hemoglobin patterns, before slicing; I wanted to try to get an image of where the hemoglobin was, unstained, but too faint.
Silk meshing around with photography 163, Kodachromes evaporators, et cetera, exposure roll, on flash, et cetera. And, on page 166, they set up that I used for photographing gels, with a tungsten [00:31:00] 150-watt reflector bulb, and so on.
Comments and on Thursday, August 16th, Kodachrome results. Here’s on [00:32:00] page 174, August 16th, I comment on Kodachrome results. Kodachrome of course was a color-negative, a color-negative film, but I was using it as if it was a black-and-white film by printing it on black-and-white paper, even though it was a color film. There is an example, there’s an image of a Kodachrome image on page 131, just printed and it’s — inverse print; that’s to say that the albumin, for example, looks white, and the other stained bands look white, and the light parts are — the unstained parts of the gel were black. [00:33:00] So it would have been a rather difficult one to use, because it was an inverted image. But anyway, I tried using it again on August 16, rather complicated set of exposures, et cetera, looking at gel.
This was took, showed the procedure. This is — I mean, incorrect about this. Page 174 was just showing various stages of the overall procedure of making gels, using Kodachrome film. It really wasn’t an experiment to determine how to get good gel images; it’s just recording [00:34:00] what a gel preparation looks like, using an electronic flash, that Otto Hiller had. So for example, number seven is the point at which the gel began to — the suspension of starch being heated began to gel, and how it became homogeneous, eight and then degaussing it, was image nine. And pouring the gel, prior to setting it up, and then hemoglobin and sample insertion, et cetera, cutting the slot and starch grain insertion, the whole procedure was recorded in film. I don’t know that it was ever published; it was an attempt to have a record, went all the way through to slicing the gel and [00:35:00] picking it up, et cetera.
And similar experiment with Idaho potato starch, beginning with image number one, boiling gel tray, et cetera. This is on page 178. I’m trying to record it. I don’t know that I ever used the images for any publication.
Page 179, 180 — well 179 really, and some, 180, trying to get some images of two-dimensional results, gels, quite difficult to image because they were rather faint. An attempt with a flash, and again on 180 [00:36:00], photography, flash film, 20x exposure, et cetera, how to do that. The filter paper setup, the filter paper result, the trays, the insertion, and the lighting, how to get — it was not how to photograph the gel, but it was recording the procedure. So this is recording the procedure again on page 180, and 179.
Back to a straightforward family, run by Otto on 181 and 182, and then some beginnings of two-dimensional [00:37:00] electrophoresis, with filter paper strips set up so that I have four narrow strips, and some larger filter paper strips for the first dimension on filter paper, of which the images are there, 6, 1, 2, 3, and 4, but since 3 was used for the two-dimensional apparatus experiment, it’s incomplete, is number 3. Didn’t like the albumin very much, better to have used 0.25 molar [borate?], as albumin back was too sharp.
Another experiment of two-dimension on page 185. [00:38:00] Comment again on page 188, Kodachrome images. Trying to photograph two-dimensional gels before they were stained, on page 187, white background [00:39:00] hemoglobin patterns, various exposures, to see if I could image them, but I was not very successful. Otto running gels 189, 190, 192, and then back to photography again, on the Kodachrome daylight, plus flash results, various images. The copying side, gene nomenclature, trays, et cetera, filter paper results, not clear at this point. [00:40:00] Some two-dimensionals. Missed as either, et cetera, gel one, trying to get images of gels. Two-dimensional gel, the filter paper’s on 195, and the images of the gels are unfortunately not obtainable anymore; they’re buried and lost in some can of microfilm.
The last two entries in this book, page 197, 198, Otto confirming the typing of about 20 different samples, 1, 2A, and 2B, with checkmarks in red crayon. Actually, I mentioned on page 200, the code [00:41:00] of cuts I used for the gels.