Hi Davide , you can add ZnO NPs in Potato Dextrose Broth  or Mueller Hinton Broth befor solidify in different concentration  then you can Measuring the diameter of colonies and comparing them with free of added colony . ...this method its useful I have used in my research..please feel free to contact me for any help......good luck

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It seems streaking the conidia did not work well. I recommend another way to test with disk.

I usually prepare the agar medium containing live conidia that are the object in the test. Before solidifying the PDA plates (about 45C), conidia suspension is added (final concentration: 104/mL). Then, put disk on the plates and test the compound. You can see a halo around the disc if the compound has antifungal activity.

Good luck. 

Hi Davide

You can use a lower load of spore suspension and consider using 0.05% tween 80 and 0.2% agar for preparing spore suspension.

Good Luck

instead of using disc diffusion test you can use agar-well diffusion assay 

Hi Davide

Disc diffusion test is the best method to detect the effect nanoparticles on fungi

I agree with Shafeeq Hassan A..

Dear Davide, you did not make any mistake. The type of method used gives you specific results. I would recommend something similar as Daisuke Hagiwara: After sterilizing the PDA, cooled it down until 55 Celsius degrees. Then, into to medium pour the fungal cell suspension, mix it and then pour it into Petri dishes. After the agar is solidified put your samples imersed previously into the ZnO Nps solution on the agar.

Indeed streaking its not working to good with microorganism like fungi because of their growth characteristics. It is going better with bacteria or yeast. Good luck!

I agree will Alaa Jabbar. But be aware that fungal inoculation must be done by three-point inoculation or single-point inoculation, not by surface distribution of spore suspension.

In alternative, you can also try inoculating the fungus in a position opposite to the disk (similar to dual cultures): on one side of the plate you put the disk, and on the other side inoculate the fungus using the single-point inoculation method. The compound will diffuse towards the fungus, and the colony will only grow in the opposite direction if some effect is exherted by the compound. Check the attached figures.

I agree will Daisuke Hagiwara and with  another alternative of P. Rodrigues. 

Seems like the way you did is completely fine. However, you have not mentioned about the spore concentration that you used to make a fungal lawn. If you have lots of spores they will tend to grow faster and vigorously masking the activity of your compounds. Use 10^5-10^6 spores/mL to make the lawn. It is also possible that your nano particles are not effective against this fungus. It is better to use one of the control that works best against this fungus (eg posaconazole). That will give you the clear picture of whether your technique is working or not for sure. OR do microbroth  dilution assay to score MIC. All the best.

Really good suggestions here, except that of using a lower spore concentration. For either spreading the spores on the surface or embedding them in the agar, the aim should be to get a uniform lawn of mycelial growth, not individual colonies, as shown in the photo. As someone suggested, a bit of Tween might also help the spore distribution. But you need enough spores for the colonies to merge at a young age.

You might also want to consider using a medium on which there is less sporulation, unless you are specifically looking for compounds which inhibit sporulation. You would need to test some different media for your specific strains, since sporulation can be quite variable. A rich medium like YPD (yeast-peptone-glucose) can be good, since many Aspergilli won't sporulate until they run out of nitrogen. But also sporulation is sometimes suppressed on weak bacterial media like Plate Count Agar. Similarly a defined medium with ammonium as N-source may be suitable.

It is also worth keeping in mind that one reason for not seeing clear inhibition zones is that your zinc oxide nanoparticles aren't inhibory for these 2 fungi. At least at the concentration applied in the photo above.

Hi Davide,

Whilst a couple of suggestions have been made regarding the method of investigating the antifungal activity of ZnONP, I suggest you look at your ZnO suspension because you need to consider factors like dispersion, aggregation/agglomeration and most importantly dissolution (if you intend to use your initial disc diffusion method). You should think about 3 questions:

1) The Zn ion effect

2) The particle size effect.

3) Concentration effect ( because I imagine the ZnONP would not dissolve even in proprietary solutions from commercial sources).

 Summarily, you need to characterize your ZnONP in solution; find your ion concentration, and the dispersity. If there is monodispersion of your ZnONP, I believe your result will be different with the same technique.

PS: Accept my apologies for any typo (using a phone).

I agree with the answers above: why are you not getting a continuous lane of mycelia?

You need an independent control, through serial dilutions, of the concentration of viable spores.The fact that you see individual colonies suggest a very low number of viable spores. And yes, suspend your spores in something like 1% tween, and vortex them for a minute or so.

Then before testing nano-particles you should test whether ZnO is toxic. But it is virtually insoluble! And I bet that fungi don't take up nanoparticles. Is there any evidence that they can? Inhibition of course may result from adsorption of the particles to the membrane and either a direct effect on the membrane or release of the toxic compound by the nanoparticle.

Dear  Davide

I suggest  that the actual problem in the used concentrations of zn-nps , where you must used gradual concentrations till reach the effective inhibitory conc. 

.In addition to the used NPS  must be characterized and identified by electron microscope to insure that the used Zn metal  reached to nanosized .

Therefore, increase the used concentrations to obtain the inhibitory doses . As the concentrations increases , the zone of fungal inhibition become clear an measurable as you want .

 I work with my team over 5 years in this subject   .  see below and attached files

 Disc diffusion technique :

i- Preparation of paper discs of commercial antifungal feed additives: Filter paper

discs Whatman of 5 mm Φ were impregnated for 10 minutes with different

concentration of (20, 25, 50, 100, 150, 200, 250 ug/ ml) of metal nanoparticles (ZnONPs& Fe2O3 -NPs),  The prepared discs were dried by heating at 40- 50°C for one hour.

ii- Placement the discs of tested :

One ml of 105 spore suspensions was added to sterile plates and over layered with SDA. The plates were rotated to mix the content and allowed to solidify at room temperature. On the surface of plates the paper discs of the drug were pressed firmly for complete contact with the agar. The discs were distributed evenly in a manner such as to be not closer to each other, 15 mm from edges of dishes, 20 mm between each 2 discs and 24 mm from center of plates. The plates were incubated at 25°C for 2-5 days.

iii-Reading the results (inhibitory zone):After the end of incubation period, the

sensitivity of fungi to the tested against each drug was determined by measuring the diameter of the growth inhibition zone in mm.

I am giving below the extract ot needed write-up of the standard method for you to adapt with innovation or any required modification. IF your require the full PDF of the article , tell me your e-mail ID

Yours sincerely

Harish C. Gugnai, PhD., FRC. Path.e-mail: [email protected]

Professor of Microbiology and Epidemiology & Biostatistics,

Saint James School of Medicine, Anguilla (BWII

. Lo´pez-Oviedo,1 A. I. Aller,1 C. Martı´n,1 C. Castro,1 M. Ramirez,1 J Evaluation of Disk Diffusion Method for Determining Posaconazole Susceptibility of Filamentous Fungi: Comparison with CLSI Broth Microdilution Method. ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 2006, p. 1108–1111 Vol. 50, No. 3 0066-4804/06/$08.000 doi:10.1128/AAC.50.3.1108–1111.2006

The procedure for the disk diffusion method as mainly that of CLSI document M44-A (10). Posaconazole disks (5

g, Oxoid) were used. The following culture media were tested: RPMI agar with 2% glucose (RPMI-G; Izasa, Spain) and Mueller-Hinton agar (Difco) supplemented with 2% glucose and methylene blue (0.5 mg/ml) (MHB) (10). The plates were incubatedat35°C and read at24 and 48h.The inhibition zones (IZs, inmillimeters) were read at the point of marked decrease in fungal density (IZ-2) and the point of complete inhibition of growth (IZ-0). A. fumigatus ATCC 204305, A. flavus ATCC 204304, Candida parapsilosis 22019, and Candida krusei 6258 were included in each susceptibility test for quality control and assessment of reproducibility. For analysis of the results, geometric means (GM) and ranges of MICs and arithmetic means and ranges of IZ were calculated. To determine the correlation between the disk diffusion method and the microdilution method, the IZs were plotted against their respective MICs. Pearson’s correlation coefficient was used to calculate a regression line for each comparison.

I agree with the answers above.. you can try antifungi in broth media ...good luck.