cells absolute or do you have a graded series? 



GREGG: In my opinion it is a graded 

 series. The ones I pointed out were the ex- 

 tremes. Although photographs may be mislead- 

 ing I noticed in Takeuchi's black and white 

 photos that there appear to be more darker- 

 staining cells than bright cells. This is strange 

 if these cells are going to sort out to form a 

 slug with certain proportions. 



KAHN: If you were to establish criteria for 

 classifying light and dark cells and were to 

 score the cells found in the aggregate do you 

 think you would find the 30 (prestalk):70 (pre- 

 spore) ratio? 



GREGG: No, I don't think so. As a matter 

 of fact, you might find various transitions and 

 not necessarily this final proportion of very 

 bright and very dark cells. You may find all 

 intermediates in about equal proportions. Ta- 

 keuchi has also referred to the various grades 

 of staining caused by the granules in the cells. 



KAHN: One final question. What was your 

 antigen? 



GREGG: All three stages injected into a 

 rabbit. In theory we had antibodies to vegetative 

 amoebae, slugs and mature stalks and spores. 

 They were homogenized before being injected. 



B. WRIGHT: How do you prepare these 

 sections initially? 



GREGG: They are fixed in Carney's and 

 run through an alcohol series. 



B. WRIGHT: Yes, but how do you kill them? 

 What's the initial step? Do you freeze them? 



GREGG: No, the fixation kills them. Car- 

 ney's is essentially acetic acid alcohol and 

 chloroform. These are paraffin sections. 



B. WRIGHT: I see. Could this treatment 

 itself differentially leach the two cell types? 



GREGG: That's a possibility. However, 

 Takeuchi used methanol and we have obtained 

 identical results with these two methods. 



GROSS: You're presumably looking at pro- 

 teins with the fluorescence. Carnoy's fixer is 

 3:1 acetic acid and chloroform. It's a very 

 effective protein fixer. It's unlikely that it would 

 wash out antigens. 



GREGG: This is more or less a conven- 

 tional histological technique when using fluo- 

 rescent antisera. 



PAPACONSTANTINOU: Well, there's one 

 thing that bothers me. Although it may be a 

 little trivial here, I'd like to find it out. Dr. 

 Deering asked you what was the staining mate- 

 rial along the outside and you said it was slime. 

 Is there any protein in that? 



GREGG: The slime sheath could be poly- 



saccharide. 



PAPACONSTANTINOU: Well, would that 

 stain? 



GREGG: Perhaps the polysaccharides are 

 antigenic. 



PAPACONSTANTINOU: Oh, I see; you've 

 got polysaccharide as well as protein antigens. 



GREGG: Oh yes, that's very likely. 



PAPACONSTANTINOU: You don't know 

 whether that difference in staining is due to 

 polysaccharide or to something else? 



GREGG: No. That cannot be determined 

 yet. I'm not sure exactly what the composition 

 of the slime sheath is, but I would say it's 

 probably polysaccharide. 



ZIMMERMAN: Could you tell us once more 

 how long it takes this thing to flop over? 



GREGG: It's a matter of a few minutes. 

 I'm quoting Dr. Kahn on this. At any rate, by 

 the time the migrating pseudoplasmodium has 

 formed, prestalk cells have developed such that 

 this will result in the development of propor- 

 tional fruiting bodies. Again you can see that 

 one of the characteristics of prestalk cells, in 

 both D. discoideum and D. mucoroides , is that 

 they tend to lose their cytoplasmic stain. Con- 

 sequently, the cytoplasmic antigen must be lost 

 at the anterior end. Obviously development of 

 the slime mold depends upon differentiation of 

 these two types of cells, prespore and prestalk 

 cells. How do we account for the loss of cyto- 

 plasmic antigens in these prestalk cells? 



DEERING: I have one more point I'd like 

 clarified. Is this a mixture of the antibodies to 

 all three stages injected at the same time? 



GREGG: Yes. 



Figure 10 shows a preculmination stage. 

 There is no drastic change in prespore staining 

 in the preculmination stage. The stalk has begun 

 to develop; the lack of cjrtoplasmic antigens is 

 seen to continue in the prestalk cells. 



DEERING: Is that the disc at the bottom? 



GREGG: Yes, the basal disc has begun to 

 form here, and it also loses its cytoplasmic 

 antigens, and consequently loses its staining 

 capacity. 



KOHNE: Are the cells rapidly dividing as 

 it's falling over? 



GREGG: No, there is very little, if any, 

 cell division once the aggregate is formed. 



For those of you who are not familiar with 

 the way the slime mold develops, the prestalk 

 cells in this area move up and flow down into a 

 funnel-shaped area formed by the stalk. The 

 cells pile up on top of one another in the same 

 process by which a chimney is formed, and this 

 results in the raising of the spore mass. 



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