Antifungal Assay anyone?

[extrachride]

Hi, I'm Lorenzo
and i would like to ask if someone could introduce me to an assay/method that would measure the antifungal activity of mango leaf extracts. I know there is a lot available, but i am looking for a method that would be inexpensive ,understandable, feasible in a highschool laboratory and still make exact and accurate results on the antifungal activty of an extract. If there is one, i'd like to ask if a soft copy or a .pdf file of the method will be available. thanks! and i'm hoping for your reply!

[donnahardy2]

Hi Lorenzo,

Here is a website that includes a protocol for an antifungal assay. This website describes a disc diffusion method where the fungi are grown on an agar plate, the antifungal agent is added to a disc of filter paper, and the zone of inhibition of growth around the disc is measured in millimeters after incubation.

http://www.mycology.adelaide.edu.au/Lab ... thods.html

There are other methods, but this one would probably be easiest to adapt to a high school laboratory. It would require agar, Petri dishes, and cultures of fungi.

Before you start on this project, I recommend that you review the safety information for working with microorganisms from the science buddies website:

https://www.sciencebuddies.org/science- ... fety.shtml

And, you should also find the information on general microbiological technique helpful:

https://www.sciencebuddies.org/science- ... ques.shtml


You will probably need to adapt the method to the resources that you have available. Please do let us know if you need any additional explanation about this subject.

What type of fungi are you planning to work with? What types of antifungal compounds are you expecting to find in mango leaves? Have you done background reading about fungi and antifungal compounds? When is your project due?

Donna Hardy

[extrachride]

hi, thanks for giving me that method. it's really great that you took your time to give aid to me. I still have some questions, since i can't quite understand some parts of the method. Could you please explain to me how the whole method is done, especially the part where the fungi are cultured.(a) how do you get the inoculum, i mean how do i get the single isolated colony (will i just swab the culture with wet cotton, will I cut the agar with the colony?? i don't know). (b) at what speed will i set the vortex mixer so as to achieve a smooth suspension from the inoculum? (c) I would also like to ask if there is another way of approximately adjusting the density of the suspension to a #0.5 McFarland Standard other than using a densitometer? (d) and where could i store the agar plates at 35C?
thanks again! and i am hoping for your replY!

[donnahardy2]

Hi Lorenzo,

You have very good questions. What kind of fungi are you planning to work with? The method given is for a rapidly growing yeast, Candida, which causes human infections. There are many other types of fungi, some that cause human infections and some that grow on food, plants, textiles or buildings. You need to pick a specific topic for your project and it would be very helpful to know what your subject is.

You will need a pure culture of a test organism growing on an agar plate. Where are you going to get your test organism. Before you set up your method you will grow up the test organism on a suitable agar plate. Then you can use a sterile swab to transfer one whole colony to a test tube containing sterile water. Vortexing is done at a fast speed until the sample makes a homogeneous suspension. Then, the sample is diluted so that the turbidity matches the McFarland standard. McFarland standards are traditionally made with barium sulfate and the idea is to match the turbidity, or optical density of the fungal culture to a standard to ensure that you inoculate a certain number of organisms on the agar plate for the susceptibility testing. With this type of testing, it’s very important to set up the method exactly the same way every time to ensure consistent results. Here is information for making McFarland standards:

http://en.wikipedia.org/wiki/McFarland_standard

You don’t need a densitometer; you will be matching the standard to your culture suspension using your eye to compare results.

35 degrees Centigrade is the incubation temperature recommended for human fungal pathogens like Candida, and this temperature is maintained in an incubator. Fungi also grow very well at a lower temperature, so you can incubate them at room temperature of 20-22 degrees. So don’t worry if you don’t have an incubator available.

What are you going to use to extract potential antifungal compounds from the mango leaves? Are you familiar with the chemistry of other antifungal compounds? If not, you should look for more information on this topic so you will understand the science behind your project. Do you know what types of solvents other antifungal compounds are soluble in?

Also, you need to learn more about the biology of the fungi you will be working with. What type of an organism is a fungus? Is it a plant, an animal, or something else?

Donna Hardy

[extrachride]

Hi!
Well, as of now, the fungi that I would be planning to do the assay unto would be Aspergillus niger, but it isn't final yet. A lot of things will probably be changed as I probe further into my research.
In getting the crude extract from the mango leaves, i will use methanol. after that it will then be fractionated, attached is a concept map of the process i will be doing, it was suggested to me by an author of a study that used fractionation.
Thank you for helping me!
I'll be glad to ask again if there are some gaps in my study. Thanks again!

[extrachride]

The potential antifungal compound that i hope will be the reason for the mango leaf extract's antifungal ability would be mangiferin. And I think it would be best if i use it as a positive control. It was reported that mangiferin protected safflower seeds from the invasion of Fusarium oxysporum f. carthami but it was from the extract Canscora decussata.(http://www.apsnet.org/phyto/SEARCH/1977/Phyto67_548.asp)So i am trying to find out if the extract from mango "Mangifera indica" would be any better as a fungal growth inhibitor (since mangiferin, is also found in mangos, hence the name mangiferin). And then i will also be fractionating the extracts, with dichloromethane and butanol so as to possibly further isolate the mangiferin and find out if the fractionation made the extract more efficient in inhibiting fungal growth.
I'd like to ask if what concentration of mangiferin would be best so that it can be used as a significant control in my study?
Thanks! i'll be hoping for your reply!

[donnahardy2]

Hi Lorenzo,

I can see that you have done a lot of work in researching your topic, and I think this has the potential to be an excellent project. However, before you get started, I have some comments that you should consider. I have also included some links with additional background information that should be useful to you.

First, in searching on your topic, I found the following article, which describes the results of testing mangiferin, the yellow color from mangoes and mango leaves, for antifungal activity against a number of organisms, including Aspergillus niger. The authors found no antifungal activity against this organism.

http://www.iripz.pl/ftp/51_1_2_05_6.pdf

Do let me know if you are not able to open this link. One basic principle of doing scientific research is to do a background literature search and design an experiment that will add to existing knowledge on a topic. Therefore, since someone has already worked on your project already, I think you should do more background reading and perhaps consider changing your topic slightly.

Your basic project is good, but perhaps you could try a slightly different approach. For example, perhaps instead of doing a methanol extraction of the mango leaves, you could also extract directly with n-butanol and chloroform to extract a variety possible compounds to test. If you start your extraction with methanol, you will extract only compounds that are soluble in this polar solvent.

Here is another research paper that reports anti-viral and anti-oxidant activity of mangiferin.

http://www.ncbi.nlm.nih.gov/pubmed/11165750

Here is a study that reports the antihyperglycemic effects of mangiferin.

http://jhs.pharm.or.jp/data/55(2)/55_206.pdf

Here is an article that describes the structure and chemistry of mangiferin.

http://www.journalarchive.jst.go.jp/jnl ... nlabstract

Here is an article that describes a procedure for evaluating the antifungal activity of extracts from a fungus:

http://www.riceblast.snu.ac.kr/file/44/ ... (5)-08.PDF

Here is a subscription website that includes an abstract describing analysis of naturally occurring antimicrobial compounds from mangoes. Please not the extraction was done in dichloromethane.

http://www.actahort.org/members/showpdf ... nr=682_279

Here is a paper that describes the details of a study designed to evaluate antifungal compounds from a plant. The details of the experimental protocols are very good in this paper.

http://www.academicjournals.org/AJB/PDF ... 0Eloff.pdf

Please read through these sources and decide if you would like to incorporate any ideas from these papers into your topic. Please let me know if you have any questions about any of the details in the papers. I would be happy to explain anything that you don’t understand, as I do want you to have a very successful project.

Donna Hardy

[extrachride]

Hi! This is what i meant about the changes that would happen. Thanks! you have warned me about that!. It was great of you to do so.

Well, Is it advisable that i only change the test fungi that i will be working on? Because that's what i'm planning to do. And I'm still in the search for a new test fungi.
If it isn't then what logical and scientific thing should i do with this problem?
And I have a problem concerning the positive control in my study, i don't know what concentration of mangiferin i will use. because in the article, wherein it was reported that mangiferin isn't good enough for Asperigillus niger but is effective against other fungi. they used different concentrations of the solution with polyethyleneglycol and mangiferin. the concentration of the solution wherein they found it's antifungal ability was at 35%(w/v).
on the other hand, in the study about mangiferin's ability to protect safflower seeds from a fungal infection, it was shown that at a concentration of 1x10^-3M or 0.001M of mangiferin solution. (i'm not sure if this is a solution with water though, i still can't understand the first sentence in the materials and method chapter, where the concentrations of mangiferin were introduced, i was hoping if you can help me understand this business about the Aqueous sodium carbonate solutions and mangiferin)
here's the link to the pdf file so that you could see what i'm talking about.
(http://www.google.co.uk/search?hl=en&q= ... arch&meta=)
I don't know which concentration i would use as a positive control. whether it's 35%(w/v) solution with polyethylene glycol or 0.001 M of mangiferin solution.

Thanks again! you literally saved me. I am grateful and will be waiting for your reply!

[extrachride]

This concerns the method for antifungal activity.
how do i transfer one whole colony to the test tube using a sterile swab?
will i just let the cotton tip touch the fungi or will i have to get (scrape) the fungi off the agar and put in the test tube?

[donnahardy2]

Hi Lorenzo,

Here are answers to your questions.

In the link from your last message, the mangiferin was 1 x 10 -4 M dissolved in a 1% sodium carbonate solution. To reproduce this solution, you would weigh out 1 gram of sodium carbonate (Na(CO3)2. To make a 0.0001 M, or 0.10 mM solution of mangiferin, which has a molecular weight of 422 Daltons, you will need the following:

mM x 422 mg/mM = 42.2 mg per one liter of 1% sodium carbonate

I don't know if there is any difference in the activity mangiferin preparations, so perhaps you could try a range of concentrations. The authors in the paper used a concentration ranging from 4.2 to 422 mg/L for their experiments.

What are you going to use for controls? You should use a negative control of a filter disc that has been soaked in the sodium carbonate without mangiferin. A positive control could be an antibiotic disc of a known antifungal agent with known activity against your organisms. The controls will ensure that your assay is working properly.

To scrape off a colony, you would use a sterile cotton swab and rub it on the colony, and transfer as much as possible to the sterile water blank to make your suspension. It is important to use a single colony to make sure you are not transferring a contaminated culture to the dilution tube.

Here is an article that provides good background information for your topic, which is information on the mechanism of various antifungal agents. The key to understanding this article is to focus on the chemistry of the antifungal compounds and the fungal cell to understand how the molecules interact.

http://cmr.asm.org/cgi/content/full/12/4/501

After you read this article, look at the structure of mangiferin and try to decide how it might interact with fungal cells.

Good luck!

Donna Hardy