I have done it using Haemocytometer but there are some problems with the fungal spores i.e. some fungi are not sporulated in the broth and some fungal spores are too big in size so hardly one or two fungal spores are present on the whole slide of haemocytometer and sometimes not a single spore is observed on the slide. Any suggestions?
I'm a bit confused here. A typical Neubauer haemocytometer has a depth of 0.1mm (100 micron) and that should not be a problem with most fungal spores. Can you please explain more on the kind of fungus you are using?
Excellent method for determining numbers of P. infestans sporangia, zoospores or spore number of other fungi.
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.
the explanation of Julius Sserumaga is the most common standard method available so far. It works well
Serial dilution is used to estimate spore concentration/ml. However, for accurate spore count, hemacytometer is generally the standard method used. Follow the procedure outlined by Julius Sserumaga above. Goodluck!
I agree with Julius Sserumaga!!!!And I only add one information, you can prepare your suspension in a detergent solution (use triton, for exemple), this step will help you with glued spores!!!!Goodluck
The only thing I would like to add to Julius Sserumaga method is that in many fungal species spores are hydrophobic so it is better to make spore suspension in 0.01-0.1% Tween 80 solution. Best wishes
The method outlined by Julius works well, It is important that the saline isotonic solutions do contain tween 80 (0.05% is often enough).
Prepare the dilution spore suspension as suggested by Sserumaga spread 1-2 ml on agar plate and count the spores under high power steriobinocular for larger spores. While smaller can be observed next day after their germination or by myelial growth again under sterio biocular microscope- P.N.Chowdhry
Use serial dilution for both culturable and non culturable fungi. For clturable after dilution put in medium sread evenly and count. for non culturable count the spores direclty mounting on slide and calculate for the remainind drops.
Swapna Kalbende,
It sounds like your problem is the size of your spore and development of the spores in your culture media. What is the organism? As mentioned the Hemocytometer is an excellent tool, but it would exclude anything larger than 100um in diameter.
If you don't have access to a microscope you can perform serial dilutions of your culture and spread these on plates. This is very common for soil plating for example. Take 100ul of your liquid culture and dilute it in 1ml. This will be a 1:10 dilution, then take 100ul of this 1:10 and dilute again, this is now a 1:100 dilution, etc etc. Take each dilution and plate 100ul on a plate of growth media (potato dextrose agar plates). The 100ul of each dilution you put on your plates is a 1/10 fraction of said dilution. Allow the plates to incubate for some time so you can visualize and count your colonies. This technique will give you an estimate of "viable" colonies in your original culture.
If you are still having problems with getting sufficient numbers of spores you should then trouble shoot why. Many fungi do not produce spores and even if the species is known to, there are some isolates which might be exceptions to the norm. It may also be your culture conditions preventing spore production, temperature, media composition, and light are all variables here and could be very species specific. Again, what organism?
Good luck
Microbiology of food and animal feeding stuffs — Horizontal method for the, ISO 21527-2 First edition, 2008-07-01
enumeration of yeasts and moulds — Part 2:, Colony count technique in products with,water activity less than or equal to 0,95
Check out the following for some things to consider. Smith et al. (1988) Sources of Variability in the Measurement of Fungal Spore Yields. Applied and Environmental Microbiology. P. 1430-1435.
If fungus do not produce spores you can try to break the hyphae with glass beads and vortex then you count with the hyphal fragments as hyphal units.
Prepare spore suspension with tween 20 count using haemocytometer or do slide culture and count
I also use the haemocytometer. I grow the organism on solid media and after sporulation, add a known quantity of water (in little aliquots) and scrape all the spores and suspend in sterile water. The this is filtered through a double layer of muslin cloth to remove mycelial fragments as much as possible. Make sure that you take almost all spores into the measured quantity of water. Then transfer into the haemocytometer. After counting results are expressed as spores/ml of suspension. It doesn't matter the number you get in the counter.
By making a serial dilution you will get the number of colony forming units. The colonies may arise from spores or mycelial fragments. For a spore count this is not a good method.
Sporulation in liquid media is difficult with some fungi. If your purpose is use of liquid media, then you need to find out the reasons for not sporulating. Some fungi need total darkness, some need exposure to IR shortly, etc. Need to do a good lit survey on your fungus.
Have you any data on spore counts in the UAE? We are very interested in determining if high fungal couts are responsible for the high incidence of chronic sinus disease seen in the UAE and Gulf area. Please email directly to [email protected]. If required, I have a colleague on the ground in Al Ain who trained with me who could research this topic with you.
Regards MD
Haemocytometers procedure
(modified from Cell counting using a hemacytomer Bio Whittaker)
brilliant method for determining numbers of P. infestans sporangia, zoospores or spore number of other fungi.
1. organize a spore suspension.
2. organize a hemacytometer for use.
a. cautiously 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 labeled A thru D in Figure 1 on the next page.
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.
As I mentioned earlier , the best method is: Haemocytometers procedure
I am facing problem with counting the Alternaria fugal spore suspension using Haemocytometer. The spores remained clumped and i am able to count only smaller spores.
I think the best method for small fungal spore counting is using Haemocytometer. In spore suspension add little Tween to get spores free.
Sir i did tried adding it but it does not work with Alternaria.. What i suspect is this issue might be due to large spore size
Vortex your sample tube first, and then count in hematocytometer. It helps a lot.
I think you should go for direct counting under microscope as Alternaria spores and larger in size and shape also different. You make the dilutions and go for direct counting under light microscope
The conidial concentration of the suspensions determine with a haemocytometer
How to adjusted to 2x104 zoospores per ml of Phytophthora capsici zoospore?
Yes haemocytometer can be used for the spore counting of Phytophthora capisici zoospores. Dilute the spore suspension appropriately and then count in WBC or RBC square and calculate the required concentration from the formula.
Hi ,
You can use flow cytometry , more information are available here:
https://www.sciencedirect.com/science/article/abs/pii/S0953756209801093
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
Hemocytometer is an excellent tool for the estimation of fungal spores but it is good to note that you have homogenize your suspension very well so that you can get the good representation of the calculated averages. if the fungi concentration in the suspension is very high, it is recommended that you do serial dilution so that you will be able to count the spores easily
The use of hemocytometer is an excellent method, although, it should be used with caution. One may count artifacts as spores if not properly observed.
What's the estimated weight of a million spores of fungi for e.g Metarrhizium anisoplae
Serial dilution is used to estimate spore concentration/ml. However, for accurate spore count, hemacytometer is generally the standard method .
Hemocytometer is best for estimating the concentration of fungal spores for inoculation study or spores counting.
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