I would like to test certain bacteria, yeast and mould against various liquid anti-microbials using the disk diffusion test. The anti-microbials are made in-house and are thus not available in commercial impregnated disks. The current way of testing is just to place the blank sterile disk in the middle of an inoculated agar plate and then apply the anti-microbial liquid to the disk using a pipette. The problem with this method is that only 10 ul of solution can be added to the disk before it floods (I think this is because the disk starts to absorb moisture from the agar as soon as it makes contact thus reducing how much liquid it can hold). In some cases using only 10 ul results in very small zones of inhibition. Can I maybe place the disks in a empty sterile petri dish or such and then apply the antimicrobial solution to the disk and allow it to absorb before placing on the plate? Also if I do it that way should I allow the disk to dry first or apply it to the plate while still moist?
Hello Nivana, antibiotic susceptibility testing is a very serious microbiological assay adopted frequently in clinical, industrial and environmental microbiology experiments. There are standard ways of preparing discs and impregnating them too (NCCLS) in a disc diffusion assay. This method of first dropping the disc on pre-inoculated agar medium before adding your antimicrobial solution is strange to me. The proper thing to do is to prepare your discs with appropriate concentrations using standard methods which will always require sufficient time for equilibration (1-2 h) before you use them. Please consult the Manual for Antimicrobial Susceptibility Testing, chapter 4, pages 42 and 43.
Hello, Nirvana! I also did the same tests with different chemicals. I used disc-diffusion method as skrining-test. To confirm obtained results serial dilution method is usefull.
You have too prepare your disc separately and before your test.
Ekpenyoug is right .
Most important things is that you determine of concentration of your agent neither in disc or in serial dilution.
8/23/18
Dear Nirvana,
I agree with others who advise adding the antimicrobial to the discs prior to putting them on the agar plate. Allow sufficient time for the antimicrobial solution to dry before use.
If a 10 uL aliquot is too large for the disc to absorb, there are a couple of things you can try. One approach would be to apply the desired volume of antimicrobial in stages, e.g., 5 uL at a time, allowing it to dry, then add another 5 uL. Another way that might work would be to prepare a more concentrated solution of the antimicrobial, in which case you could apply a smaller volume to the disc.
I hope this information helps you.
Bill Colonna Center for Crops Utilization Research, Iowa State University, Ames, IA , USA [email protected]
Hi Nivana,
I am fully agree with others who advise to add antimicrobial to the discs prior to putting them on the agar plate and allow sufficient time for the antimicrobial solution to dry before use. If you require to test higher volume of antimicrobial, use double layer agar assay with Assay Cylinders or cut wells. Under this assay double layer of agar is used, first pour hard agar layer and onto this add the soft agar (alongwith properly mixed test pathogen) and let it solidify. After that place the asssay cylinders and press down upto the hard agar layer and load the desired amonut of antimicrobial compounds in it or cut small well in the soft agar layer with the help of sterile cork borer and remove the cut agar portion (a well will be made) and fill this well with desired amonut of antimicrobial compounds in it.
Hello Nirvana,
The discs should be loaded with the antibiotic solution before you put the disc on the agar surface. Try to use sample disc to knw how many microliters will take first without leaking the extra liquid outside. Then you can add the exact amount on the disc and then move it under aseptic conditions to the agar plate seeded with the test organism. Keep the plate for 2 hours in the refrigerator and then incubate it inverted.
Sometimes it is just as cheap to buy standard antimicrobial discs from oxoid or similar. If you want to make your own you can pipette 10 or 20 ul of antibiotic onto the disc and let it soak in . Perhaps compare to a control or a standard Type organism. If you are testing an unknown substance then test in parallel with a known positive antibiotic and a negative control. Alternatively you can use the agar well method, an ultrasesitive version of which is detailed in a paper by lehrer et al i think Article Ultrasensitive assays for endogenous polypeptides
I usually start with a rough assay on a very sensitive organism and see what concentrations are effective. Then I know what organisms and concentrations will work for a good image ffor publication.
Moustafa Y. El-Naggar What would be the reason for refrigerating the plates before incubating?
You have to immerse filter paper discs in the liquid antimicrobial agents, then take the filter paper discs and make the test.
Hello Nirvana Jaganath
Just like what others have said, you have to load antibiotic solution onto the disc and let it dry first before you place it on agar. As for the volume to be added to the disc, it depends on the disc concentration you desire. For example, you have antibiotic stock solution of concentration 10 ug/ul. If you want to prepare prepare disc concentration of for example (30 ug/disc) then you need to add 3 ul of the stock solution per disc.
10 ug/ul x 3 ul/disc = 30 ug/disc
Hope it helps.
i will be grateful if anyone can share a research article in which the advice given on this forum was used.
Ibanda Hood Hi. Unfortunately the paper I used the info for contains proprietary information and cannot be shared. Do you need the paper as a reference or do you want to see the method itself?
You can give me both, the paper as a reference, and a description of how you did it.
Thank you so much.
To impregnate a disk for antimicrobial susceptibility testing, you have to saturate a sterile filter paper with a specific concentration of the antimicrobial agent that depends within the range of your testing requirements. In handling or preparing the filter paper disks, make sure to use forceps to avoid contamination. After dipping the filter paper into the concentration/stock solution, make sure that it is fully saturated, but not excessively. Let the impregnated disks to air dry on a sterile surface, then to a sterile container. Label it afterwards.
- Badges
- Science method