Fungi enumeration - (Mar/08/2021 )

Hi there. I am involved in bioburden testing and grow fungi on SDA agar. I am required to count the fungi however sometimes the colonies grow over each other or there is just a white carpet of fungi where I can't pinpoint where the colony started or how many there are. The colonies must be grown for seven days. If any one has any advise I would be grateful. I do have some photos on my phone but can't figure out how to upload them.

You can't upload photos yet because I think the forum has a limit (for spam prevention purposes) on recent members posting photos.

The answer, as you should know if you have any basic microbiological training - is to do a dilution series. Calculate CFU by incorporating the dilution factor into the equation.

I know how to do dilutions. However we are working to a test specification and it says to filter 100 ml of our extraction fluid through a membrane and plate up the membrane. I am relatively new to microbiology so please be understanding.

Can you offer context?  Is this for food, drug, cosmetics?

You might count at earlier time points.  Alternatively, you can forulate with some surfactants to reduce radial expansion of mycelial  colonies. https://sfamjournals.onlinelibrary.wiley.com/doi/abs/10.1111/j.1365-2672.1987.tb02416.x

Thankyou Phil, the research study is very interesting. I am testing fabrics for bioburden. We do add tween to the extraction fluid but I'm assuming that is to aid in the extraction process. I will look at adding a surfactant to the agar. I am trying to count the plates before they overgrow but as it is a Seven day test sometimes they grow lots over the weekend. Seems to me like day four or five is optimum otherwise some overgrow.

Are you using 16404?  Think you can be assured 4-5 is more than enough.

Not sure what the number refers to ?

What fungus(fungi) are you using? A lot of technical/applied protocols specify Aspergillus brasiliensis ATCC 16404.  

https://www.atcc.org/products/all/CRM-16404.aspx

To validate the method yes and yeast.

Yeast Candida albicans 10231?   Assume no issues with that bug.

Folr 16404 - mark the colonies on the reverse of the plate at day ~4 - observe for new foci at 7.