Dear

What is extraction or from any thing?

Generally , found more factors was effected on inhibition  zone. such as method of extraction , solvent use in extract method ......etc.

I best you, to use  inhibition method in broth media and determination of inhibition percentage. 

or use new method,  ATP product determination from tested bacteria 

Maybe you must look at which bacterial or fungal species you chose. Sometimes intrinsic resistance occurs, or even strain-related resistance. Other point is the amount of antimicrobial you are pouring into each disk. Maybe those insight help you. 

One possibility is that dragon fruit does not contain anything with antimicrobial activity, and the first report was wrong.

Another possibility is that you failed to extract the antimicrobial activity, and a different extraction procedure/solvent is needed.

A third possibility is that the antimicrobial activity is unstable, and degraded before the susceptibility test.

A fourth possibility is that the antimicrobial substance is not diffusible and therefore does not enter the agar. This could be due to a high molecular weight, chemical reactivity, or lack of solubility.

There are other methods of measuring antibacterial activity that might be better in this case, such as broth microdilution.

How you prepare the extraction?

What is the concentration of the active material required and whether you have achieved the harmful concentration of extract for bacteria?

Is this substance had been used on other bacterial species and what are the results?

Is it possible to increase the concentration of the active substance in the extraction (What was the dilution of the extract in your study) ?

Did you prepare the agar correctly?

What is the intensity of microbial growth of the broth cultured on agar  ?

How many hours have been incubated  cultured media, and the conditions of incubation ?

Is there a possibility  of developing resistance against your extract (Is this material had a common wide use so that  resistance may be has evolved)?

You should check these notes to find a solution in order to complete your study correctly .

Regards

 Hi dear

Zone of inhibition may be  affected by the Concentration of bacteria spread onto agar plate, Drug antagonists, incubation temperature, incubation time, size of the plates, proper spacing of the disks, reading of the zone, Agar depth ,Size of the inoculated organism,pH etc.

I agree with Alaa Abed and Adam B Shapiro.

Regards

Either the compound you are testing has no antibacterial activity or you used small concentration that does not cause inhibition. Another possibility is that  you forgot to add the bacteria. Lastly the bacteria is highly resistant. In viruses the situation is different and the inoculum used may be toxic to the cells. So when you want to plaque the virus, the inoculum should be washed out with PBS before adding the agar. i

Three main factors:

-Molecular weigh of agar material that would impact on the diffusion of the tested antibiotic.

-Thickness of agar in the plate, also dimension of the wells if the wells technique was used.

-Concentration of the extract used. Basically higher concentration (very thick) of the extract makes it difficult to diffuse into agar particles.

also double check the effectiveness of the extract, it may need to be discarded.

Hi:

First of all, don't be frustrated sir, Negative results, are still results.

Your test organisms might be resistant to your extract,

also Have you applied Control positive and Control negative organism?, its very important to find out what happened.

regards

Hi:

First of all, don't be frustrated sir, Negative results, are still results.

Your test organisms might be resistant to your extract,

also Have you applied Control positive and Control negative organism?, its very important to find out what happened.

regards.

Hi:

First of all, don't be frustrated sir, Negative results, are still results.

Your test organisms might be resistant to your extract,

also Have you applied Control positive and Control negative organism?, its very important to find out what happened.

regards

It is evident that the extract has no antibacterial activity against the tested strains. You can try first with the juice of this fruit and with a concentrate (prepared at low temperature). But I think the antimicrobial activity is very low or absent.

Kirby Bauer test is not broad suitable for antimicrobial drug discovery due to non polar compounds with antimicrobial activity does not diffuse in agar, you can repeat experiment using broth dilution test recommended by CLSI. With best regards

http://ac.els-cdn.com/S2095177915300150/1-s2.0-S2095177915300150-main.pdf?_tid=468be88e-6716-11e7-81fb-00000aab0f02&acdnat=1499873282_53c4649870e43ac9904cc4d3b42c8a7d

Factors that can reduce the ZOI:

1. Method of MHA agar preparation is wrong.

2. The suspension of bacteria that you used is more than 0.5 McFarland' s unit.

3. The discs used are impotent.

4. The specific drug does not have any antimicrobial activity.

5. The bacteria is resistant to the specific agent.

However, to test the antimicrobial activity of a new extract, I would recommend you to use broth dilution method. It will also help you quantify the degree of susceptibility in form of minimum inhibitory concentration. Or else you can try with some other test organism. At first, you can test it with E. coli ATCC 25922 (If you think your extract is active against gram negative organisms) or Staph. aures ATCC 25923 (if you think your extract is active against gram positive organisms) as positive control and then test it against clinical strains. 

Dear, you could be give attention to some factors like the zone of inhibition may be  affected by the Concentration of bacteria spread onto agar plate, Drug antagonists, incubation temperature, incubation time, size of the plates, proper spacing of the disks, reading of the zone,pH, Agar depth ,Size of the inoculated organism, so on .

Thanks

Hello,

All of the above answers are valid reasons why you failed to have a zone of inhibition. in your question, you did not say what your test organism was and you did not mention if you used a pure culture or not. In a situation of mixed organism you can fail to have a zone of inhibition if a member of the mixed population is resistant . Checking the purity of your test organism can help.

May be your extract does not contain anything with antimicrobial activity, and you bacterial inoculum is very high.

Within 12-14hr, I can see the zone of inhibition very clearly, but after that it stars to go away, simply the test bacteria are growing and thus zone of inhibition starts disappearing. what does it suggest?

how to interpret mic measurement ?if the values are high

The minimal inhibitory concentration (MIC) is the lowest concentration in a set of (usually) 2-fold serial dilutions that prevent the growth of a bacterial culture, under a standardized set of conditions. For a specific strain of bacteria used in the test, differences in the MIC measurements for various test substances reflect differences in the ability of those substances to inhibit bacterial growth under the specific set of conditions, with higher values representing weaker inhibitory activity.

Please can you explain this data in given table ??G is Garlic extract

Table

I suppose that NC means negative control, meaning no growth (possibly no bacteria added), and PC means positive control, meaning no inhibitor. It looks like the positive control didn't grow as well as it should have, since higher optical densities were observed in the experiment. Nevertheless, the result is that the bacteria were resistant to ampicillin up to at least 50 ng/mL, but growth was inhibited by all concentrations of garlic extract tested.

Yes thanks 🙂

How we can get more accurate result ??

An MIC determination is basically an all-or-none measurement. Either the culture grows to maximum density overnight, or it doesn't grow at all. Since it is typical to test 2-fold serial dilutions of the antibacterial substance, the precision of the measurement is limited to 2-fold. In fact, we generally think of an MIC as being precise to +/- one 2-fold dilution. If multiple repeated measurements are made, it is usual to see some experimental variation in a range of 2-4 fold. However, if you want to get a more precise measurement, you can use smaller dilution steps.

It is important to recognize the distinction between accuracy and precision. By using smaller dilution steps, you can increase the precision of the measurement, but this may not result in an increase in accuracy. Accuracy measures how close the measurement gets to the true value. Since the true value is unknown, you can't tell how accurate the measurement is. One thing you can do is make the measurement multiple times and report a measure of the spread of the values as the average +/- the standard deviation, the mode value, or the range of values. These are measures of the reproducibility of the measurement.

Thanks

Hi good evening. Me and my team are actually currently conducting a research on the antibacterial potential of citrus fruits (calamansi, lemon, mandarin) against Pectorbacterium caratovorum, to prevent soft rot on tomatoes. So we decided to find the ZOI of our treatments first. We made use of a 20% pure extract which we diluted in 80% water. We have 5 treatments in total, 3 citrus fruits, and one positive and negative control. But the problem is literally mo ZOI appeared. Not even on the positive control. What do you think is wrong? We're currently at a lost on what to do next

What was the positive control, and how was it prepared and tested?