I was inoculating Trichoderma in broth culture (submerged inoculation) and I want to determine the spore density?
Can somebody help me. Thank you so much!
The spore density of fungus can be determined by using haemocytometer. For details see the page; http://www.microbehunter.com/the-hemocytometer-counting-chamber/. good luck
Thank you Mr. Mathew Baite. Do you know which the method to use for collecting the spores?
It is simple to get spores. Grow the fungal cultures in a particular media. After some days, sporulation occurs . In this stage using a cork borer of any size, cut out the colony (2-3 or more of such). Dissolve in sterilized distill water in a test tube. See for the spores in microscope. If too dense serial dilute it and you will get the spore density measured.
Thanks you so much Mathew Baite.
Have you ever heard about inoculating Trichoderma or other Fungus in the broth culture?
yes. not only heard, i have done it. this is simple way to grow fungi to harvest mycelium.
Are you interested in production of conidia, chlamydospores, or both? The hemocytometer method will work for both, but you may have to macerate the tissue for a good chlamydospore count.
For inoculation in broth culture for spore production, some Trichoderma species (and other fungi) will conidiate reasonably well in liquid culture while others will not if submerged. There have been a number of studies looking at sporulation in broth culture conditions. For example, Lewis and Papavisas (1983) looked at media and growth conditions for Trichoderma chlamydospores and conidia. They showed that factors such as culture medium and growth in submerged versus static culture affect conida production for several species. Chlamydospore production is generally less influenced by whether the growth is submerged or static.
@Mathew Baite: and how do you know how much is Trichoderma biomass? what thing do you base on? spore density or weight of biomass or? Thank you
@Linda E. Hanson: thank you for your answer
I am interested in conidia and maybe also chlamydospores. I inoculated Trichoderma in culture broth and then, I vortex or centrifugate with 6000 rpm the cuture solution (include biomass). I take the vortexed culture (include biomass) to filtrate with the filtration papers and use heamocytometer, or I get the suspension of centrifugated culture to haemocytometer. In the result, I see nothing =.=
Could you tell me what wrong with it or did I make a mistake?
I would suggest you first try taking a sample from your original culture without centrifugation. If you have conidia they will generally be floating in the medium if it is a shake culture or on the surface if a static culture. If you see them at that stage, check after the different steps you are using and see where you might have lost them. Filter paper is one of the most likely. You also may need to have a wetting agent in your final material for suspension. Many species of Trichoderma have hydrophobic conidia so they will not be carried well in pure water. If you do not have conidia in your initial cultures you may want to alter you ingredients or growth conditions.
For chlamydospores, these will usually be intercalary in the hyphae. You will lose most of them if you filter through filter paper. For those, we would macerate the tissue and, if we needed to do any filtration, put it through sterile cheesecloth. A double layer of sterile cheesecloth has generally worked better for us for spores than filter paper..
Thank you, Ms. Linda E. Hanson
After getting your advise, I followed the procedure:
1. Obtain original culture, put in two test tubes (10 ml solution)
2. Add approximately 1 ml Tween 80 to each tube.
3. Centrifugate two samples
4. After that, I get pellets into tube containing distilled water with 1 ml Tween 80
5. Vortex two samples
6. One be filtrated by filter paper (1), one be filtrated by Cheesecloth (double layers) (2)
And (1) maybe, I get chlamydospore and some conidia, (2) I get conidia. Some bad definition pictures below. is it right?
If I want to test Spore density. Can I spread (inoculate) on Petrie dishes, then I use spectrometer to make a standard Curve? Or have to use heamocytometer?
Thank you
- Badges
- Science topic
- Similar topics
- Agriculture
- Soil Science