I have a lot of Bacillus cereus cultured on nutrient agars, but kept inside the 4°C freezer after 24 hours in 37°C incubator. Then I used the following protocol to do endospore staining:
1. Heat-fix bacteria on glass slide.
2. 5 minutes malachite green (steaming, but not boiling)
3. Wash with tap water.
4. Safranin for 1 minute.
But I couldn't seem to get a perfect endospore staining. So many of the slides are very faintly stained that I couldn't take pictures.
So how should I treat Bacillus cereus so that I could get a better endospore staining?
Dear Mrs. Nurul Adhwa HAR,
below I send you the contact to Prof. Sterflinger who should be able to help you
best regards
Andreas Rohatsch
https://forschung.boku.ac.at/fis/suchen.person_uebersicht?sprache_in=en&ansicht_in=&menue_id_in=101&id_in=5300
Are you sure your bacteria is "Bacillus cereus "? maybe it's a different species!
Yoiur first step should be to ensure that your B. cereus is sporulating. This is unlikely on nutrient agar after even a few days. You would be best growing the bacteria on a suitable nutrient agar and then transfering to a sporulation medium (you can find these in the literature). Once you have sporulating bacteria (you can use phase contrast microscopy to see the phase-bright spores without staining), then you are ready to use the spore stain procedure you mention. However, I would adapt your method by the use of a longer period of Safranin staining (approx. 5 mins). This is because Safranin is a week stain and 1 minute if often insufficient to stain cells sufficiently for visualisation. Good luck.
The endospore staining includes the following steps after heat fixation:
1. The slide is then suspended over a water bath with some sort of porus paper over it, so that the slide is steamed.
2. Malachite green is applied to the slide, which can penetrate the tough walls of the endospores, staining them green. leave for five minutes, do not allow the stain to dry, if needed add few more drops of Malachite Green
3. Leave the slide to cool
4. Rinse the slide with water for thirty seconds. Do not run the water directly on the stained bacteria, but over the side of the slide, or until the green color does not appear in the washed water
5. The slide is then stained with diluted safranin for two minutes, which stains most other microorganic bodies red or pink.
6. Rinse the slide again in water, for 30 sec
7. Blot dry
Examine under oil immersion lens
Hello Andreas, thank you for the contact details. I have contacted Prof Sterflinger, and am currently waiting for a reply from her.
Hello Reza, yes, it is Bacillus cereus. The strain has been Gram-stained and tested biochemically. I so hope it is not a different species!
Hello Hala. My colleagues and I actually performed a series of time duration for the malachite green and safranin. We did 30 second +30 seconds, 1 min + 1 min.... 5 min + 5 min... and all showed similar observations under the microscope. We are all thinking that it is the bacteria that is not sporulating in the first place.
We are wondering how to introduce stress into Bacillus cereus, so that they are sporulating. Seems like all of our plates contains vegetative ones.
0.6 -2 mM Mn (MnSO4, or MnCl2 ) added to the growth medium normally induces sporulation in Bacillus sp.
Did you try stressful conditions like heating at 80o as suggested by Andreas? should be useful to minimize amount of Carbon source
Notice that the cells in the center of the colony which are the old cells may show sporulation cause of depleted nutrients!
Wish you success
if you want Bacillus cereus to sporulate and make spores for your experiment then you can use the experimental conditions mentioned in the manuscript of de Vries et al., 2004. I have given the link below. The sporulation medium is quite a complex one but surely works! Please note that in case of B. cereus it is predicted that the trigger for sporulation is pressed or the decision to sporulate is made only when the cell density is very high. In other words, cell crowding is the cue for B. cereus to sporulate. I hope this helps!
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC383076/
Dear Hala
I work with spores of Bacillus and i alraedy published few articles on this subjet.
I recommande you to read my articles. you can find the good method for spore production
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