Hi, I'm testing yeast with gram staining and , as i learned, yeast should be positive but some of them are stained as negative (red sphere shape).
At the first time, I thought it was a mistake of my handling but same results shows up in the other guys hands.
So, is there any possibility that yeast can be stained with safranin ?
I show another possibility.
In the case of gram positive bacteria, cells in old culture may exhibits false negative because the cell wall thickness decreases as it gets old.
On the other hand, cell wall of yeast cells increases its thickness as the culture gets old (Article The possible role of yeast cell walls in modifying cellular ...
). Conversely, cell walls of young yeast cells may be thin and plausibly exhibit false negative. Indeed, I find some budding cells gram negative in your picture (e.g. bottom left).To check this, try staining young and old yeast cells (e.g. logarithmic growth phase and stationary phase).
Hello Dohyeon.
I think these red cells are dead or disrupted yeast cells. These are no longer in the typical physiological state for yeast cells and so may take up the counterstain. This is most likely i think in the areas where the cell wall has been breached.
Dear Dohyeon, while I am thinking similar as Ms/Mrs Amina, I greatly should appreciate your telling more detailed information about the GRAM STAINING proper you used in your staining procedure. There are several if not numerous different recipes for Gram stains/staining mentioned in the literature. Commercial staining kit ?(if yes, please state supplier data), made your own staining components/solutions? Please define either by recipe (ingredients, handling) or by literature reference. Thank you !
Thanks Amina and Wolfgang !
About the Amins's answer, as you said if the cells were disrupted by some reasons, should I check the viability of cells during the cultures? As I know mammalian cells should be checked by days but I didn't hear the same thing on bacterial cells. Is there any protocol about the viability check on bacterial cells?
PS. to Wolfgang, what I used in gram staining is the kit made from the sigma and I followed the protocol of the menufacturer.
Dear Dohyeon, thank you for disclosing the source of your stain.
Regarding your request "viability check available"? I would like to point you to (only two possibilities out of many found by 'googleing'): cf.
https://www.thermofisher.com/at/en/home/life-science/cell-analysis/cell-viability-and-regulation/cell-viability/microbial-viability.html,
cf. onlinelibrary.wiley.com/doi/10.1111/1567-1364.12202/full
M Kwolek‐Mirek & R Zadrag-Tecza - FEMS YEAST RESEARCH 2014: Research Article:
"Comparison of methods used for assessing the viability and vitality of yeast cells" cf.(Open Access): http://onlinelibrary.wiley.com/doi/10.1111/1567-1364.12202/full
or see: http://scholar.google.at/scholar_url?url= https://biblio.ugent.be/publication/395350/file/1130209.doc (found via GOOGLE SCHOLAR: unfortunately no date / year of publication / manuscript writing given: "Comparison of multiple methods for quantification of microbial biofilms grown in microtiter plates" by
Elke Peeters, Hans J. Nelis, Tom Coenye.
(knowing there exist other techniques than staining classically with specific dyes and "differentiating" ("molecular approach" and eg. Fluorescence microscopy
Good luck and best wishes!
I show another possibility.
In the case of gram positive bacteria, cells in old culture may exhibits false negative because the cell wall thickness decreases as it gets old.
On the other hand, cell wall of yeast cells increases its thickness as the culture gets old (Article The possible role of yeast cell walls in modifying cellular ...
). Conversely, cell walls of young yeast cells may be thin and plausibly exhibit false negative. Indeed, I find some budding cells gram negative in your picture (e.g. bottom left).To check this, try staining young and old yeast cells (e.g. logarithmic growth phase and stationary phase).
Thanks all of you. I'll try your suggestions !
One more curious thing is that, if those false negatives is the nature of the gram staining, what is the best way to check the morphology of yeast? Is there other better staining methods?
It depends on what kind of morphology you want to observe.
If you want to ignore the thickness of cell wall, fluorescent dye might be helpful as Dr. Muss suggested, or you might be better to ignore the colorimetry and to focus on the shape.
But, personally, I understand that the differences in cell wall thickness are also important in a sense.
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