We use to prepare water agar, to avoid a massive fungal growth. We inoculate a diluted mixture of spores, and collect the germinated conidia looking under microscope by cutting the piece of agar that is moved to rich media PDA, malt, etc.

1. Prepare a dilute spore suspension in 1% aqueous glycerine or in water to which a small amount of detergent has been added.

2. Using a sterile loop, remove a loopful of the spore suspension and place it on a slide. Examine the slide to determine relative concentration and appearance of the spores.

3. Using a sterile loop, streak another loopful of the spore suspension over the surface of agar in Petri dish (both agar and Petri dish should be sterile). Sterilize the loop and re‐streak (without additional inoculum) in the opposite direction.

4. Place the Petri dish on stage of the microscope and, using low power, locate the spores, or allow the sample to germinate overnight, and then locate them.

5. After single spores have been located, use a sharp sterile needle to cut out a square of agar containing the spore and transfer it (agar and spore) to a fresh sterile agar plate or test tube slant.

Good luck in your research..

Best regards

Simon Francis Shamoun, Ph.D.

Research Scientist

If you know the structure and spore morphology and have access to a basic stereo microscope, I found the method described by - Choi, Y.W., Hyde, K.D. and Ho, W.H. (1999). Single spore isolation of fungi. Fungal Diversity 3: 29-38 - is very useful, as well as you steady hand. Good luck.

I will do a dilution of spore and later transfer to minimal media plates and follow their germination and isolate them to a new plate. It might take for germination tube form between 12-24 hours. Do it according to your works hours, because if you pass that time they will overgrown and you will need to start again.

I will do a serial dilution to get the right one.

Hope it is helpful to you.

Carmen Rodriguez

Dr Simon gave the answer and that is good enough[;clear and simple

Already many good answers. Beauveria conidia are very small (

1. Three ml of 2% water agar Poured into unscratched Petri dish and allowed agar to solidify.

2. A suspension of conidia either from a sporpdochium or from aerial mycelium was prepared in 5 ml sterile water in a sterile vial.

3. A micro pipette was used to transfer 5 μl of conidial suspension aseptically on a drawn circle with a permanent marker on the back side of a Petri dish containing water agar medium..

4. An L-shape inoculum needle touched the conidial suspension located on the drawn circle several times and streaked across the water agar plate.

5. The plate was incubated for 12 to 16 hrs at 25 °C.

6. The Petri dish was examined under a dissecting microscope. A lot of germinating conidia appeared on the site of inoculation and by following the streaked lines using the low power of the microscope, single germinating conidia could be observed [Fig., 1]

7. Suitably positioned germinated conidia [Fig., 2] were removed using prestrelized surgical blade [Wuxi Xinda Medical Device co., ltd. Sterilized by GAMMA radiation 25 KGY, STERILE].

8. Single spores were cultured on potato sucrose agar medium [PSA] [Booth, 1971, Booth, 1977].

9. Finally, identification of F. verticilloides and F. subglutinans was accomplished according to: [Booth, 1977, Nelson et al., 1993, Leslie and Summerell, 2006]. [Fig., 3, 4, 5 & 6].

A simple method is making serial dilutions (1:10) of the spore suspension . and take 0.1 ml of the different dilutions and plate on the agar (one plate per dilution). Afte incubation look for the plates with monosporic growth. It is very easy and it can be use with bacteria also

Dear Farzaneh

Although there are similar methods introduced, but reviewing them it seems the most complete approach is that kindly presented by dear Shawn. Best Luck,

Firstly, you should isolate the single spore from B bassiana under sterilized conditions> Take carfuly and used steril needle to pick up the single spore on water agar medium and incubate at 27 C for one week> Several subculture should be needed to purifie the isolated spore.

1) make a suspension of fungal cells

2) using a cell strainer (100microm) separate spore from hyphae

3) count spore supsension in Burker chamber

4) make a limiting diluiton in RPMI+MOPS and seed in 96 well microtiter plates. Incubate at at 25-30°C. Perform microscobic observation under an inverted microscope at 4-6-8 h to detect single-spore germination.

I agree that a simple method is : Prepare: a pure culture(s) on whatever choice of growth medium (e.g., PDA) and them making serial dilutions (1:10) of the spore suspension .The protocol proposed by Shawn Kenaley is Ok.