I tried again to use fluorescein 1% dye on a microscopy specimen today. I heat fixed a slide of the culture of Rhys’ mouth and soaked it in fluorescein dye for 10 minutes. I washed off the excess, added coverslip and looked at under the microscope. For this experiment, I used epi-illumination with the LED illuminator a second hand fluorescence filter block I obtained from ebay recently.
Pictures below – not sure what they show though!
Following is a non-stained live image of Rhys’ mouth culture through the Zeiss IM microscope with 63x objective as reminder of what is in the Petri dish culture used for this fluorescence experiment (this is NOT a fluorescence image):
Fluorescence image using fluorescein dye x4 objective (below). I am not sure what the darker blotches represent. This is a low magnification image so they do not represent individual bacteria but more likely large masses from the smear I took off the Petri dish using a needle. I had difficulty attaching the illuminator to the epi-illumination port today and it ended up slightly angled – hence the darker colouration to the left of this picture.
Fluorescence image using fluorescein dye x4 objective (below). The following two images look impressive but I suspect they are only of air bubbles!
Fluorescence image using fluorescein dye x20 objective (below). I have increased the magnification. The MikrOkular was able to show some detail, which is what was used for these photos. I also tried the Mikrocam but it was not sensitive enough. Again, I do not know what the darker patches represent. Far too low magnification for any features within bacterial cells (see size of individual cells in 63x image at start of this post):
Fluorescence image using fluorescein dye x32 objective (below). The highest magnification I could achieve and still see any detail. For this one, I had to stretch the image in GIMP2 and sharpen it. Again – what the features are I do not know. They vary in size from small ones, about the size of a bacterium to larger ones.