I would like to concentrate fungal spores from a bacteria/spore liquid suspension, without having too many bacteria pelleting.
Does anyone know which sucrose concentration would be the best?
At the end I must have a spore suspension in a small volume, with not too many bacteria (doesn't have to be 100% pure).
If you just want to separate fungal spores from bacterial spores you can just culture them on a media containing an antibiotic against that particular bacteria. You will surely get pure fungal colonies.
If you have different fungal spores you can simply use single spore isolation to get pure fungal colonies.
Thank you Haris Ahmed Khan for your answer.
Actually, I would like to count the number of spores per ml and plating is not the best way of detection. I would count them using flow cytometry but I need to concentrate them and I would like to reduce bacterial background.
Ruiz célia I have no experience with flow cytometry but you can count spores using Haemocytometer.
1. Prepare a spore suspension.
2. Prepare a hemacytometer for use.
a. Carefully clean all surfaces of the hemacytometer and cover-slip.
b. Take care to ensure that all surfaces are completely dry using non-linting tissue.
c. Center the coverslip on the hemacytometer.
3. Pipet approximately 9 microliters (this volume will vary slightly with the brand of hemacytometer) of the cell suspension into one of the two counting chambers.
a. Use a clean pipet tip.
b. Be sure that the suspension is thoroughly, but gently, mixed before drawing the samples.
c. Fill the chambers slowly and steadily.
d. Avoid injecting bubbles into the chambers.
e. Do not overfill or underfill the chambers.
4. Count the spores.
b. Count all of the spores in each of the four 0.1 mm³ corner squares.
1) DO count the spores touching the top or left borders.
2) DO NOT count the spores touching the bottom or right borders.
Determine the Spore Count.
a. Calculate the total spores counted in the four corner squares.
b. Calculate the spore count using the equation: spores/ml = (n) x 10^4, where: n = the average cell count per square of the four corner squares counted.
Example: If the calculated average (n) of spores in the four 1 mm corner squares of the hemacytometer is 30:
cells/ml = (n) x 10^4 OR spores /ml = 30 x 10,000 = 300,000 spores /ml.
Yes, it is also a method that we are using. The problem is that we handle spore concentration that are around 100 spores /ml. Thus 9ul is not enough to detect them.
Butu thank you for your contribution Haris Ahmed Khan
- Badges
- Science topic
- Similar topics
- Analytical Chemistry
- Extraction