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Proper staining method?
Viktorie Halasu,
16-11-2018 19:57
Hello forum,I'm looking for some protocol how to stain fungal tissues, specifically lipids with Sudan III. The biggest problem I have is how to remove excessive lipids from damaged spores and other structures that got spilled into the preparation, and yet preserve undamaged the lipids inside cells (spores and other). I tried rinsing it with 60% ethanol, but the lipids merely coagulated into bigger drops and stayed attached to the surface of the section. Using a kitchen detergent helped more but that one doesn't exactly look like a publishable method. : )
Thank you in advance.
Viktorie
Hans-Otto Baral,
16-11-2018 22:06
Re : Proper staining method?
Not sure if I understand you well. I assume you mean that staining lipids requires damaged cell walls?
I can actually confirm this for spores with thick walls. Only a few times I tried Sudan and also found alkalinized Cresyl Blue (CRBA) useful. The following text I wrote in my VT paper in 1992:
Lipophile dyes are commonly used to give more or less specific stains for fatty matters (see KIRK, 1966: 87ff.). Most of these tests are lethal to the cells. I have tested Sudan III in lactic acid in se¬veral species: a distinct reddish stain of the LBs was rapidly obtained especially after heating the slide, while CRBA gave the characteristic amber stain. KIRK (l.c.) recommended two tests (using benzapyrene-caffeine, and neutral red) for staining LBs in statu vivo by fluores¬cence.
KORF & ERB (1972) and KORF (1977) found Trichophaeopsis bicuspis to differ from Trichophaea by ascospores with "non-oleaginous, somewhat resinous inclusions" instead of LBs since the inclusions failed to absorb oil stains such as Sudan IV. My material of T. bicuspis showed two polar LBs 3.5-4 µm in size. Indeed, the whole spore content remained unstained by CRBA or Sudan III for hours, even after boiling, except for immature, and ruptured mature spores, where the LBs stained amber in CRBA and red in Sudan III. Thus, the negati¬ve result is clearly due to the impermeability of the thick wall of the mature spore to these dyes.
Zotto
I can actually confirm this for spores with thick walls. Only a few times I tried Sudan and also found alkalinized Cresyl Blue (CRBA) useful. The following text I wrote in my VT paper in 1992:
Lipophile dyes are commonly used to give more or less specific stains for fatty matters (see KIRK, 1966: 87ff.). Most of these tests are lethal to the cells. I have tested Sudan III in lactic acid in se¬veral species: a distinct reddish stain of the LBs was rapidly obtained especially after heating the slide, while CRBA gave the characteristic amber stain. KIRK (l.c.) recommended two tests (using benzapyrene-caffeine, and neutral red) for staining LBs in statu vivo by fluores¬cence.
KORF & ERB (1972) and KORF (1977) found Trichophaeopsis bicuspis to differ from Trichophaea by ascospores with "non-oleaginous, somewhat resinous inclusions" instead of LBs since the inclusions failed to absorb oil stains such as Sudan IV. My material of T. bicuspis showed two polar LBs 3.5-4 µm in size. Indeed, the whole spore content remained unstained by CRBA or Sudan III for hours, even after boiling, except for immature, and ruptured mature spores, where the LBs stained amber in CRBA and red in Sudan III. Thus, the negati¬ve result is clearly due to the impermeability of the thick wall of the mature spore to these dyes.
Zotto
Viktorie Halasu,
16-11-2018 22:29
Re : Proper staining method?
Hello Zotto,
no, I meant that after some spores are accidently badly damaged (with the razorblade or by whatever cause), their contents will escape and float in the preparate. Now I need to observe some small lipid bodies (?) in cells of outer excipulum (it's dead herbarium material). But because of those cracked spores etc., I cannot be sure whether the guttule I'm observing is actually inside of the cell (= a morpholog. character), or if it is one of these freely floating guttules that's just sticking to the cell surface from outside (= an artefact). And these floating lipid particles I want to remove. I appologize I don't have an image at hand.
Viktorie
no, I meant that after some spores are accidently badly damaged (with the razorblade or by whatever cause), their contents will escape and float in the preparate. Now I need to observe some small lipid bodies (?) in cells of outer excipulum (it's dead herbarium material). But because of those cracked spores etc., I cannot be sure whether the guttule I'm observing is actually inside of the cell (= a morpholog. character), or if it is one of these freely floating guttules that's just sticking to the cell surface from outside (= an artefact). And these floating lipid particles I want to remove. I appologize I don't have an image at hand.
Viktorie
Hans-Otto Baral,
17-11-2018 08:09
Re : Proper staining method?
Yes, now I understand. The same problem I know personally. It is usually a matter of the living vs. dead state, that one cannot be sure if the guttules are cell contents or have a secondary, alien origin. Are the excipular cell walls impermeable for Sudan? I have little experience with that stain. With CRB it works, it esily penetrates the cell wall and it stains oil in a rose colour while vacuoles in blue.