PREPARATION OF ANTIBIOTIC SUSCEPTIBILITY TEST (AST) DISKS

Spread the love

Antibiotic susceptibility disks arespecially made paper disks which are impregnated with known concentrations of antimicrobial agents or antibiotics. They are used in the laboratory to test and determine the efficacy of a known drug against a known pathogenic microorganism prior to antibiotic therapy. Paper disks used for susceptibility studies are special type of paper that has the ability to uniformly absorb sufficient volumes of the antibiotic solution being prepared. The paper disks are usually cut into various sizes that range from 1-10 mm in diameter.

Whatman filter papers are the most preferred type of paper used for preparing susceptibility disks due to their high absorbent ability. Some of the problems associated with the unavailability or procurement cost of conventional antibiotic disks for susceptibility studies in the hospital, local health center in the community or research laboratories in academic institutions could actually be addressed in a more sustainable approach if these disks can be locally produced by those that often use them.  Apart from ensuring that antibiograms are appropriate and available prior to antimicrobial chemotherapy in the face of an infection, local production of antibiotic disks will also guarantee the uninterrupted local supply of these all important biomedical tool (antibiotic disks) for susceptibility studies in the laboratory or for other vital medical research purposes.

Local production of antibiotic disks is cost effective especially in the developing economies that still rely heavily on the direct importation of these disks from developed countries for susceptibility studies. If quality antibiotic disks with potency that meets international criteria can be locally produced and encouraged, then the medical cost associated with their importation including the time it takes for it to arrive the wanting countries can be channeled to meet other important medical needs.  In addition, local production of quality antibiotic/susceptibility disks will help to ensure their constant supply for medical laboratories and hospitals that make use of them in these countries.

Susceptibility disks for antibiogram can be produced locally using the following formular:

V = Final volume of the drug

C = Concentration of the drug

W = Weight of the antibiotic powder

P = Potency of the drug

The above formular helps the researcher to calculate the concentration of antibiotics that will be inoculated or added to each of the paper disks for the preparation of antibiotic susceptibility disks. Some of the materials required for the preparation of susceptibility disks include: Whatman filter paper, hot air oven, Petri dish, quality control organisms (e.g. Escherichia coli ATCC 25922), standard antibiotic powders (e.g. ampicillin), culture media (e.g. nutrient agar), hole puncher or perforator, micropipette and commercially available susceptibility disks (from Oxoid, UK) that will serve as positive control disk. Paper disk that has not been impregnated with antibiotic solution can serve as the negative control disk.    

PROCEDURE FOR THE PREPARATION OF ANTIBIOTIC PAPER DISK

  1. Perforate or cut out the paper disk into a particular size one by one from the adsorbent paper (in this case Whatman filter paper) using the hole puncher.
  • Label the paper disk with the name of the drug, then place all cut paper disks inside a glass Petri dish and cover.
  • Sterilize the Petri dish containing the paper disk in the hot air oven at 50oC for 30-60 mins.
  • Use the formular above to prepare the antibiotic solution.
  • To impregnate the paper disk with the antibiotic solution, there are two methods:
  • Pipetting method: In the pipette delivery method, a fixed volume and/or concentration of the antibiotic solution is mechanically delivered and loaded onto each of the paper disks one after the other using a micropipette.
  • Immersion method: In the immersion method, all the cut paper disks are soaked in the antibiotic solution with known concentration, and the disks were allowed to dry.
  • Place the prepared susceptibility disks in clean Petri dish and allow to dry in the incubator at 37oC for about 3 hrs.
  • Load the susceptibility disks onto sterile vials/phials and store in a cool dry place away from light. Note: Desiccants should be placed at the bottom of the vials before loading the susceptibility disks, and a cotton wool should be used to separate both from coming into contact.
  • Perform a standard susceptibility test with the locally prepared susceptibility disks on the quality control organisms (e.g. Escherichia coli ATCC 25922) using the Kirby-Bauer disk diffusion method as per the CLSI criteria. Similar test should also be performed using the commercially available susceptibility disks (from Oxoid, UK) as positive control. Disk without antibiotic should serve as negative control.
  • Incubate plates at 37oC for 18-24 hrs, and measure the inhibition zone diameters (IZDs).
  1. Evaluate the performance of the locally prepared disks using standard IZDs made available by the CLSI criteria.

FURTHER READING

Ashutosh Kar (2008). Pharmaceutical Microbiology, 1st edition. New Age International Publishers: New Delhi, India. 

Block S.S (2001). Disinfection, sterilization and preservation. 5th edition. Lippincott Williams & Wilkins, Philadelphia and London.

Courvalin P, Leclercq R and Rice L.B (2010). Antibiogram. ESKA Publishing, ASM Press, Canada.

Denyer S.P., Hodges N.A and Gorman S.P (2004). Hugo & Russell’s Pharmaceutical Microbiology. 7th ed. Blackwell Publishing Company, USA. Pp.152-172.

Finch R.G, Greenwood D, Norrby R and Whitley R (2002). Antibiotic and chemotherapy, 8th edition. Churchill Livingstone, London and Edinburg.

Russell A.D and Chopra I (1996). Understanding antibacterial action and resistance. 2nd edition. Ellis Horwood Publishers, New York, USA.

Be the first to comment

Leave a Reply

Your email address will not be published.


*