Microbiological Antibiotic Assay

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MICROBIOLOGICAL ASSAY OF ANTIMICROBIAL AGENT CONCENTRATIONS

Introduction        

Testing for antimicrobial susceptibility to significant bacterial isolates is an important task in microbiological assays (Berth, Melvin, 2009). “The goals of testing are to detect possible drugs resistance in common pathogens and assure susceptibility to drugs for particular infections” (James, Mary 2009). According to The University of Melbourne Laboratory Techniques Manual (2002), identification of antimicrobial sensitivity is relevant in patient’s diagnosis and management. According to Mary and James (2009), “the most commonly used testing methods are broth micro-dilution or rapid automated instrument methods.” They further say that manual methods such as disc plate diffusion and gradient diffusion methods that provide for flexibility and possibly cost saving are used. These methods use instruments that are available and easily accessible. Emerging contrivances of resistance push for constant grit of antibiotic concentration. When choosing “empirical antimicrobial agents and detecting resistance in individual bacterial isolates it is important to perform antimicrobial susceptibility testing” (James &Mary, 2009).

Materials and Methods

Disk Plate Diffusion Method

This method is well standardized, simple and practical where a bacterial inoculum is applied to stacks on the surface of a large agar plate. Commercially prepared fixed concentration paper of antibiotic disks are placed on the inoculated agar surface which is then incubated for up to 24 hours at a temperature of 35^oC before the determination results. “These test results are qualitative as the category of susceptibility is derived from tests other than minimal inhibitor concentration” (James and Mary, 2009). This method is simple, does not require specialized equipment, and provides categorical results easily interpreted. The equipment and materials required such as petri dishes and paper discs are widely available and generally accessible for conducting assays.

The Microbiological Antibiotic assay of penicillin

Materials

            Staphylococcus aureus, 3mL nutrient broth (NB), 24h, 35^oC, O₂ (S)

            25mL molten nutrient agar (NA) in bottles at 56^oC in a water bath

            Petri dishes and paper disks (sterile)  

            Penicillin solution 10units/mL (P10)

            Unknown penicillin solution (X)

            Unknown penicillin solution diluted 1:2 (XD)

Procedure of the assay

0.5mL of S. aureus NB culture was added to 25mL of molten NA in a bottle at 50^oC, mixed gently then transferred to a petri dish and set on a level surface to achieve thickness uniformity. The plate dried at 35^oC for 15mins in incubator and a second plate prepared in a similar manner.

Disc was then prepared each at a concentration of penicillin 1.25, 2.5, 5 and 10units/mL. Using a briefly alcohol-flamed forceps, paper disc was held into contact with the solution surface until the disc became wet by capillary action. Same procedure was used to prepare a disc for each of the two unknown penicillin solutions and one in distilled water (DW). (The test solution was roughly assayed and diluted to a level within the standard concentration range.)

The discs were then placed firmly on the surface of the seeded NA plate in positions previously labeled on the bottom of the plate. They were then placed in a room for incubation to allow for the development of zones of inhibitions after which the zones were measured in mm on the reverse of the plates reading the agar and individual reading of the discs recorded.

Results

The size of zone diameters are much influenced by the quantity of the solution applied on the pad. As recorded in the table below, the more the units applied to the pad the larger the zone diameter observed in a proportional influence. The results obtained were plotted as shown below.

Fig. A
Discussion of findings

From the plotted curve, it is possible to establish the estimate of the concentration of penicillin in the unknowns in Units/mL. The results for the undiluted and the 1:2 dilutions were found to be compatible since the diluted concentration (2.511) was half the undiluted concentration (5.623). Pre-incubation time affects the agar if left out longer than it should be, the bacteria may start to die. To control this monitoring of the agar, novel handling and incubation procedures were followed with attention to every step of handling from pouring of the plates to grow out of the test agents. The plates were also dried prior to application of discs to remove the excess moisture that may dilute the antibiotic of the disc, which in the end affects zone size and compromise tests.

Factors such as the unequal exposure of the disc plates to temperatures above or below the room temperature (Davis and Stout, 1971) that may have caused variations in zone diameters during the procedure were investigated. Placing the plates in stacks before they reach room temperature was established to slow and longer cooling time (Davis and Stout, 1971). Varying time interval of pouring agar and placing a pad with antibiotics on to a plate was another major factor. These factors are accounted for and “normalized by interposing reference plates and reference solutions at an approximated interval without involving reference zones” (Davis and Stout, 1971). Associated factors such as agar volume, which should be applied constantly to the pads, agar thickness derived from the amount applied, and dilution errors through drainage effect and time variability were all investigated prior to the main procedure to alleviate any possible errors.

Incubation and storage at room temperatures avert larger fluctuation in temperature, which cannot be controlled but minimized in equivalent plates placed on the stacks for the assay procedure.

Generally, the factors contributing to the variability and possible errors were controlled through proper handling procedures and the choice of conditions coupled with establishment of order and proper operation time. Combination of these factors increased the capacity to control other factors that cannot be controlled by any particular effect. Application of constant volume to pads, accuracy in diluting the test solutions using equipment that are able to provide accurate volumes, agar thickness and minimizing fluctuation in temperature exposure of plates in different positions of the stacks and avoidance of large shifts in temperature were most important controllable factors throughout the procedure. During the assay, there was no attempt to optimize the test conditions, as this was favorable for any repetitive test that could be conducted during the assay. The major controllable factors during the assay were the extent of applying agent concentration to pads, the dilution of test solution, temperature-time exposure and assay timing considerations.

References

Barth L., Weinstein M., Jorgensen H. and Ferrao J. Antimicrobial Sensitivity Testing: A review of General Principles and Contemporary Practice. Clinical Infectious Diseases (2009) 49 (11) 1749-1755

Brock - Biology of Micro organismschp 26

Davis W. and Stout R. Disc Plate Method of Microbiological Antibiotic Assay - Factors Influencing Variability and Error. Applied Microbiology, Oct. 1971, p. 659-665 Vol. 22, No. 4.23

CLSI website http://clsi.org/edu/ accessed on (5^th September 2015)

University of Melbourne Laboratory Techniques Manual

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