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Team:Groningen/Template/MODULE/Notebook/bandage/week15
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October 13 - 19 October
The freeze dried cells made last week were used to prepare several gels for microscopy. From literature we found out that the pore size of a polyacrylamide gel of 2.5 % is approximately 200 nm and the pore size of a 10,5% gel is approximately 20 nm1
Also we found that the pore size of a 1.5% low melting point (LMP)agarose corresponds to a pore size of around 150 nm2 as can be seen in figure 6. A L. lactis cell varies between sizes of 500nm up to 1500nm3
Figure 6:
Pore sizes of LMP agarose gels in relation to the concentration of agarose added to the final volume2
For these reasons we attempted to grow the L. lactis cells in a 2.5% polyacrylamide gel, a 5% acrylamide gel, a 1.5% LMP agarose gel and on the surface of a 5% polyacrylamide gel.
Half an hour in advance of making the gel the cells were induced with nisin to start the GFP production. In this way we could find out whether the cells were still viable after polymerization of the gel they were poured in.
The gels were observed and followed using a widefield (confocal) microscope. So far we can prove the cells are able to grow on top of a 1.5% LMP agarose gel as well as inside a 1.5% LMP agarose gel, results are shown in figure 7
Figure 7:
Cel growth inside the gel. A. After 30 minutes incubation B. After 60 minutes incubation and C. After 90 minutes incubation. And cel growth on top of the gel. D. After 30 minutes incubation E. After 60 minutes incubation and F. After 90 minutes incubation
The slides were then incubated overnigtht at 30 C, to check for significant differences in growth see figure 8
Figure 8:
Growth after overnight incubation at 30 C. A,B. Cells poured inside the gel. C,D. Cells on top of the gel.
Ultimately it would be the best to prove the ability of L. lactis to grow inside a gel, by making a time lapse of the growth inside a polyacrylamide gel, but for now this will remain a future prospect.
