Despite my efforts, I am finding it difficult to label overnight-grown Crypto cells grown in YPD with Aniline Blue. I have tried to alter the pH of the media I washed the cells and resuspended them in (I've tried pH 4, 6, 7, 9, and 10), the composition of the wash buffer (McIvaine's, PBS, and MES), and the concentration of Aniline Blue (0.05%, 0.1%, 0.2%). I have also suspended the stock of Aniline Blue in the variety of buffers and pHs above, to no success.

I thought my struggles might be an issue with Aniline Blue itself so I included cell wall-disrupted mutants of Crypto to see if cells with greater access to beta glucans would allow for labeling. These cells fluoresced, so Aniline Blue itself is not the issue; the issue is with YPD-grown cells.

This would indicate to me that the beta glucans are not possible to label in YPD-grown cells (for example, due to the capsule preventing labeling), but that cannot be the case since other researchers have succeeded in labeling cells with Aniline Blue with YPD-grown cells (1) or even in capsule-inducing media (2). I have been unable to replicate this success.

I am quite confused by this, as my pellets are always very blue during my wash steps. This tells me that the dye is in some way present in the cells, but that does not translate to fluorescence on the microscope.

Frustratingly, there does not seem to be an "established" means of labelling with Aniline Blue, as methods differ from publication to publication. Methods used on other fungi can vary quite a lot, in fact. Is there anyone that can offer me a protocol for this, or even a suggestion about Aniline Blue that I may be missing? Thank you.

Sources:

(1) Puf4 Mediates Post-transcriptional Regulation of Cell Wall Biosynthesis and Caspofungin Resistance in Cryptococcus neoformans | mBio (asm.org)

(2) Cryptococcus neoformans Rim101 Is Associated with Cell Wall Remodeling and Evasion of the Host Immune Responses