EPEL: One point I don't quite understand 

 is, if these are antibodies against all tliree 

 stages, what is the mechanism of differential 

 staining? 



GREGG: You mean why is there no stain in 

 the prestalk or stalk cells? Apparently the 

 antigens are lost and when you build up anti- 

 bodies, there are apparently no antigens present 

 in here which are specific to build antibodies 

 which would in turn stain these cells. 



EPEL: In other words, these antibodies are 

 differential against the various proteins. 



GREGG: Yes. U you build up antibodies to 

 all three stages, and for some reason these 

 cells lose their antigens, or lose the antigens 

 that they had at an earlier stage, the antibodies 

 would not stain these cells because the antigens 

 are gone. 



KAHN: If they are antibodies against all 

 cell types, why shouldn't the prestalk cells show 

 the same staining response? 



GREGG: They stain on the cell surfaces 

 but not in the cytoplasm. Ifthereareno antigens 

 inside a cell, there is no reason to believe they 

 would be antigenic. 



KAHN: What do you see at a higher mag- 

 nification? 



GREGG: The cell surfaces are stained, but 

 otherwise no essential differences. 



GROSS: Are there vacuoles in the prestalk 

 cells? 



GREGG: They become vacuolated when 

 they differentiate into mature stalk cells. 



GROSS: How much of the area of a section 

 of such a cell would be vacuoles? 



GREGG: What proportion? 



GROSS : Let me phrase it this way. Are the 

 prospore cells and prestalk cells about the same 

 size? 



GREGG: Prestalk cells are inclined to be 

 a little larger in the slug. 



GROSS: Now, is there a difference in the 

 amount of vacuolated space in these two types 

 of cells? 



GREGG: The mature stalk cell is more 

 vacuolated. It's a characteristic of stalk cells. 



GROSS: Then it might simply reflect, not 

 a difference in the number of antigens in the 

 dry weight of the cells, but simply that there' s 

 a lot of empty space. 



GREGG: Do you mean that the antigens are 

 crowded out? 



GROSS: Yes. 



LOVETT: Is that actually true during the 

 migrating stage when you get the same staining? 



GREGG: No, the vacuolation does not occur 



until stalk cell differentiation. 



TS'O: Have you tried mixing different 

 antibodies together? If you have, do you see any 

 different results? 



GREGG: Takeuchi (1) used antisera made 

 only to spores, and he gets exactly the same 

 results as I found with antisera prepared from 

 all three stages. 



TILL: What would happen if you used anti- 

 body only to stalk? 



GREGG: I think you could. In the first 

 place, the stalk cells stain on their surfaces so 

 there are some antigens there. 



LOVETT: The only trouble with that is 

 you'd have to be able to separate just the stalk. 

 There is no stage at where there is only stalk. 



TILL: Well, you could take prestalk cell 

 area that doesn't stain very well. 



GREGG: You can separate the stalks from 

 the spores after fruiting body formation. 



Figure 11 shows a culminating slime mold, 

 D. discoideum. We notice that the intensity of 

 the staining begins to diminish in the prespore 

 area. This probably reflects the fact that the 

 cells are about to form mature spores. I can't 

 account for it otherwise. This is the prestalk 

 area which, of course, remains relatively un- 

 stained. 



Figure 12 shows a section of a mature 

 spore mass of D. discoideum. The trouble with 

 this sort of a preparation (cut at 5 microns) is 

 that it's difficult to determine how many of the 

 cells have been cut. The spores that are cut 

 stain in their cytoplasm to a certain extent. 

 Takeuchi believes they were stained in the cyto- 

 plasm, whereas certain cells appear to be 

 stained only on the outside. He believed these 

 particular cells were not sectioned. In general, 

 however, the cytoplasmic staining seems to be 

 reduced in the spores in my preparations. If 

 you cut through a mass of spores, a consider- 

 able number of them must be cut. So my feeling 

 is that the cytoplasmic antigen is relatively 

 decreased and that, consequently, the staining 

 is reduced in the spore cells. 



SCHRAER: How large are the spores? 



GREGG: They're about 5 microns. They're 

 smaller than the vegetative amoebae or the cells 

 in the later stages. 



SCHRAER: Can you separate the spores 

 and place them on a glass slide without sec- 

 tioning them? 



GREGG: Yes. 



Now, as I mentioned a moment ago, ob- 

 viously development of the slime molds depends 

 on differentiation of these two types of cells 



99 



