Factors that affect zones of inhibition and zone of inhibition analysis (last part)
Factors that affect zones of inhibition:
1. Pathogen susceptibility
Selection of the antibiotic is based on the type of organism being tested. If the organism is susceptible to the antibiotic, they will not grow near the disk. However, if they are resistant, they will grow right up to the disk.
2. Antibiotic diffusion effects
The rate of diffusion of an antibiotic through the agar is not always same. The rate of diffusion of the antimicrobial through the agar is dependent on the Concentration of antibiotic, Molecular weight of antibiotic, solubility properties of antibiotic, pH and ionization, binding upon agar. Larger molecules will diffuse at a slower rate than lower molecular weight compounds. These factors, in combination, result in each antimicrobial having a unique breakpoint zone size indicating susceptibility to that antimicrobial compound.
3. Agar depth
If the nutrient agar plates are prepared from dehydrated media, the plates must be poured to a depth of four mm. Plates that are too shallow will produce false susceptible results as the antimicrobial compound will diffuse further than it should, creating larger zones of inhibition. Conversely, plates poured to a depth below four mm will result in false resistant results.
The nutrient agar medium should be checked through the test. The agar medium should have a pH between 7.2 and 7.4 at room temperature because optimum pH range of most bacteria to grow is pH 6.5- 7.5. Exception Vibrio cholerae is the only human pathogen that grows well above pH 8. If the pH is too acidic, certain drugs (e.g. amino glycosides, quinolone, and macrolides) will appear to loss potency, while other drugs may have excessive activity (tetracyclines, novobiocin, methioillin) and thus result in a smaller or larger zone of inhibition. If the pH is besides high, the opposite effects can be expected.
5. Size of the inoculated organism:
The size of the inoculated organism must also be standardized. The reasons are because if the size of the inoculum is too small, the zone of inhibition will be larger than what it is supposed to be and if the inoculum are also large, the zone of inhibition will be smaller.
6. Presence of other metals:
Excessive thymidine or thymine can reverse the inhibitory effects of sulfonamides and trimethoprim resulting in smaller and fewer distinct zones of inhibition, or no zones at all. The incorrect concentration of divalent cations (calcium and magnesium) will affect the results of aminoglycoside and tetracycline tests against Pseudomonas aeruginosa. Excess cation concentration will result in reduced zone sizes, and low concentration will increase zone sizes. Excess calcium will increase the zone size of P. aeruginosa against daptomycin. Excess zinc ions may reduce the zone size of carbapenems against P. aeruginosa. Zone of inhibition also 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, etc.
Zone of Inhibition Testing
Strengths of Zone of Inhibition Testing:
1. Zone of inhibition testing is fast and inexpensive relative to other laboratory tests for antimicrobial activity. In addition, it requires media, reagents, equipment and supplies that are readily accessible to most clinical laboratories.
2. Zone of inhibition testing is especially well suited for determining the ability of water-soluble antimicrobials to inhibit the growth of microorganisms.
3. A number of samples can be screened for antimicrobial properties quickly using this test method.
4. A variety of antimicrobial product types can be tested using this method. Liquids, coated antimicrobial surfaces, and antimicrobial-impregnated solid products can all be tested for their ability to produce a "zone of inhibition.
Weaknesses of Zone of Inhibition Testing:
1. Zone of Inhibition tests does not necessarily indicate that microorganisms have been killed by an antimicrobial product - just that they have been prevented from growing.
2. Microbial growth agars themselves may interfere with the function of some antimicrobial agents.
3. The method cannot be used to test the activity of antimicrobial agents against viruses, since viruses don't "grow" on agar plates like bacteria.
4. The method has some natural variability, and zones of microbial inhibition do not always have clear or regular boundaries.
5. The method is not classically quantitative (though sometimes the diameter of the zones of inhibition are measured and recorded).
Modification and Previous research work on zone of inhibition:
1. Determination of methicillin-resistant stains of Staphylococcus aureus. The resistance of Staphylococcus aureus to methicillin and related drugs can be reliably determined by using the Kirby-Bauer method of susceptibility testing if the incubation temperature is 35 °C or below, but resistance may be missed at 37 °C. The 1-aug discs of oxacillin and nafcillin or the 5-gg discs of methicillin may be used for this purpose but not the 1-, gg discs of cloxacillin.
2. Rapid, Modified Kirby-Bauer Susceptibility Test with Single, High-Concentration Antimicrobial Disks.
A rapid (6-7 hours), modified Kirby-Bauer disk susceptibility method is developed by Sterling-Winthrop research institute in which derivatives of tetrazolium dyes are used for the examined the susceptibility of a number of grams negative bacteria and Staphylococci.  The organism employed for the test was Escherichia coli and Serratia marcescens strains.
3. A Quantitative Comparison of the Zone of Inhibition against Streptococcus mutans and Oral Bacterial Growth using Crest®, Colgate®, and Aquafresh® Toothpastes.
4. A simple, rapid test to different Penicillin-susceptible from penicillin- resistant Staphylococcus aureus.
The purpose of the work was to determine the efficacy and low cost benzylpenicillin to the different Penicillin- susceptible to penicillin- resistant Staphylococcus aureus.
5. Development of a Disk Diffusion Method for Testing Moraxella catarrhalis Susceptibility.
An examined on 318 clinical strains of M. catarrhalis obtained as part of the SENTRY (Asia-Pacific) Antimicrobial Surveillance Program, plus two ATCC strains to determine a standard method for testing Moraxella catarrhalis susceptibility.
Zone diameter standards for varies bacterial species to vary antibiotics are given below:
Zone of inhibition has potential application not only the clinical sectors but also the industrial sectors when performed and evaluate carefully.The scope and future of the zone of inhibitions. However, works on it is not sufficient until today. So we should work on it and think about it as how we make it more useful.
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