Dear Karen van Niekerk ,

Trypan blue is normally used to stain non viable/dead cells and it usually binds with the intracellular proteins. In your case, the observation actually points out to various scenarios which either individually or in combination may be generating the so called "clumps".

Since you have mentioned that the dinoflagellate culture is not axenic, it is safe to assume unintended microbes such as fungi, flagellates, cyanophyceae and other bacteria are presence in abundance due to the dissolved vitamins and nutrients. Since, they can either aid in decomposition and remineralization of the dead dinoflagellate cells to release the intracellular contents or can themselves add to the bio-load itself. Any of them, when dead, can get stained by the Trypan blue.

Secondly, IMK seawater medium comprises of casein hydrolysates. However, enzymatic casein hydrolysates are different from HCl-casein extracts since the latter doesn't fully dissolve in water. And this in itself can lead to the staining of the excess proteins.

Thirdly, there are certain reports available regarding the ability of Trypan blue to actually induce apoptosis in cells during staining. Although this is not globally substantiated but it can certainly not be ignored and if is so, then the dead cells can be easily stained by the Trypan blue.

My fourth explanation is based on the cultured specimens themselves and touches upon the notion stated by you. There are many dinoflagellate species that regularly excrete extracellular metabolites into their surrounding media. Species such as Prorocentrum minimum are known to produce extracellular secondary metabolites (methylated butanediones) in culture Some others ingest food items extracellularly through phagocytotic extension of a feeding veil called the Pallium. These when detached or saturated with food particles may get stained easily and can appear as clumps.

Fifth point ticks off the ability of dinoflagellates to produce pellicle cysts in culture media while under nutrient stress or resting cysts as well. The pellicle cycle wall can be easily stained with Trypan blue and may as well appear clumpy under microscope.

Trypan blue is highly carcinogenic and has a tendency to form precipitates within the medium. But as you have already mentioned that the medium was initially free of clumps, I presume you have had followed the proper steps to ensure minimal ppts and also you should not wait for more than 5 minutes following the application of the stain to the specimen.

I hope that my suggestion be of help to you.

Regards,

Dr. Abhishek Mukherjee