Isolation of Bacillus megaterium from a soil-sample
To isolate any Bacillus sp. from a soil-sample, you can follow two strategies: either you boil your probe solved in water for 10 minutes by 100°C or you add ethanol (50% to 70%) to your probe and shake this solution for 5 to 10 minutes. If you boil your probe and you want to isolate Bacillus megaterium, be careful not to overheat the solution, because the spores of this special Bacillus are not so heatresistant like all other. 80°C for 10 minutes are the maximum, therefore it is easier to add ethanol to the soil. The ethanol kills all living cells and just spores of sporeforming bacteria (like Bacillus sp. and Clostridium sp.) remain “a life”. After that, dilute your probe 10fold to 1000fold (with water) and plate it out. You can use a special screening-agar to identify Bacillus megaterium, called “Claus-mineralsalt-agar”.
- 0,8 g K2HPO4
- 0,2 g KH2PO4
- 0,05 g CaSO4
- 0,01 g FeSO4
- 0,5 g MgSO4
- 1,0 g (NH4)2SO4
- 10,0 g sucrose
- 15,0 g agar
- fill up to 1000 ml with H2O (dest.) and autoclave
After two days of incubation by 30°C and with access to oxygen the colonies of Bacillus megaterium are round formed, up to 4 mm in diameter and with a white to yellow or light brown color. Take some of these colonies and smooth it out for dilution on Nutrient-Broth-agar or the Claus-Agar. Over night by 30°C you can take samples and screen under a microscope for the size of the cells. Bacillus megaterium are very well visible with his size up to 4 µm. You can also test for Gram-staining and starch-hydrolysis, both should be positive.
Oh woher wusstest du, dass ich genau in dieser Minute genau diese Informationen gesucht habe?!?!
Eingebung.
Ich hab erst überlegt, ob ich mein Protokoll darüber schreiben soll, oder ob ich die Welt daran teilhaben lassen soll. Nachdem ich so ewig gesucht hab, wie man dieses Vieh isolieren kann, hab ich mich dann für die zweite Variante entschieden.
hi~
I see that you worte protocol.
I will isolate Bacillus megaterium from soil. so..
Can I ask you detailed protocol?
Thank you *^^*
More detailed…
I can just provide you with same more hints: I used about 1/4 vol. of an eppi ful of soil from my compost and added the double vol. of 70% ethanol, instead of boiling the sample. After 10 min of shaking I made two dilutions, 1:10 and 1:100, out of the ethanol, just with normal water. It gets a litte bit dirty, but this is ok. Afterwards I plated it on the agar described above and after two days I had different colonies. Besides bacillus megaterium there were also bacillus cereus and bacillus subtilis. But you can distinguish the colonies very easyly: b. megaterium has bigger colonies than the others, b. cereus looks more like a yeast colony and b. subtilis has small colonies.
I hope that helps you a little bit.
Thank you.
Ah! I have a question.
you worte that if you want to isolate bacillus megaterium, you don’t overheat the solution.
That case,,
What will the temperature boil solution?
80°C or 100°C?
“Boiling” normally is at 100°C but at this temperature the spores of b. megaterium will die. So it is better to heat the solution only at 80°C for 10 minutes.
But as I said before, the easier way is to add 70% ethanol, because in this case you can be sure that the spores of b. megaterium are still alive and you don’t need to be careful with the temperature.
The best is, as every time in science, try it yourself. Good luck! (but if you run into problems, just ask)
HI~
You wrote that….
but… I didn’t understand. I’m sorry. ^^:::
so….
Can you explain it one more time?
Ah! you worte that I can test for isolation of B. megaterium as Gram-staining and starch-hydrolysis.
If it is positive, don’t I need any more test?
Thank you. *^^*
I used about 1/4 vol. of an eppi ful of soil from my compost and added the double vol. of 70% ethanol, instead of boiling the sample.
<= I can’t understand this part.
It means, I took about 250 “µL” of a soil sample (about 1/4 volume of an eppendorf reaction tube) and added about 500 µL of 70% Ethanol. This solution was not boiled, just simply shaken for 10 minutes.
For identification of b. megaterium gram staining is good, but the easiest way is by looking at the bacteria through a microscope, because the cells are very big and simple to see. Also all other species of bacillus have a negative gram staining and can hydrolyse starch.
Thank you very much. *^^*
Your advices make the experiment successful.
and..
What’s your job? I think that you are a student or researcher. That’s right?
I’m a student of national university. Nice to meet you. ^^
I’m PhD student at a small university in Germany and my work focuses on cell division of the african clawed frog Xenopus laevis.
Glad to help you.
Ah!
I find that gram-positive bacteria include Bacillus in http://en.wikipedia.org/wiki/Gram-positive_bacteria.
and,,,
B. macerans is positive in hydrolysis of starch. ^^
wow.. PhD student? in Germany?
I’m master student in Korea.
Thank you for your advice and replies.
maybe.. I’ll come to you for something.* ^^*
Auf wiedersehen.
Entschuldigen Sie bitte.
Lately, I did test about starch hydrolysis.
but.. it wasn’t stained well by Gram’s iodine.
so I want to hear your advices about it.
How do I stain it?
Sorry, but I can not help you with this issue.
At my time, I didn’t stain it so I don’t know a working protocol. And now I’m not any longer in to this stuff.
Danke für die Tipps.
Auf wiedersehen.