Monday, June 18, 2012

Antibiotic Susceptibility test and Minimum Inhibitory Concentration tests


A. DISC DIFFUSION METHOD FOR THE ANTIMICROBIAL SUSCEPTIBILITY TESTING

principle:
A standardized inoculum of bacteria is swabbed onto the surface of a Mueller Hinton agar (MHA) plate. Filter paper disc impregnated with antimicrobial agents are placed on the agar. After overnight incubation, the diameter of the zone of inhibition is measured around each disc. By referring to the tables in the CLSI disc diffusion standard, a qualitative report of susceptible, intermediate or resistant is obtained.

Quality control:
      A. QC strains
     1.  Escherichia coli ATCC 25922     2. Staphylococcus aureus ATCC 25923
     3. Enterococcus faecalis ATCC 29212           
B. Monitoring accuracy
      1. Test QC strains by following routine procedure, and record results. Record lot number and expiration date of discs and agar.
      2. Compare to expected results (CLSI QC tables). Note any out of control result and document; proceed with corrective action, if necessary.
      3. Perform daily and weekly QC testing.
PROCEDURE:
  1. Brings agar plates and canisters of discs to room temperature before use. Agar plates may be removed from refrigerator and placed in a 35 C ambient air incubator with lids slightly ajar to evaporate excess moisture. Do not leave in incubator for longer than 30 min.
  2. Inoculum preparation
Using a loop or swab, transfer colonies as follows
    1. Direct colony suspension method: - pick several colonies from a fresh (18 – 24 hr) nonselective agar plate to broth or 0.9% NaCl.
    2. log phase method
a.       Pick four or five isolated colonies to 3.0 to 5.0 ml of broth.
b.      Incubate at 35 C for 2 to 8 hr until growth reaches the turbidity at or above that of a 0.5 McFarland standards.
    1. For either the log phase or direct colony suspension method, vortex well and adjust turbidity visually with sterile broth or 0.9% NaCl to match a 0.5% McFarland standard.
  1. Inoculation of agar plates
    1. Within 15 minutes of adjusting turbidity, dip a sterile cotton swab into the inoculum and rotate against the wall of the tube above the liquid to remove excess inoculum.
    2. Swab entire surface of agar plate three times, rotating plates approximately 60˚ between streaking to ensure even distribution. Avoid hitting the slides of the plate to avoid aerosols. Finally, run swab around the edge of the agar to remove any excess moisture.
    3. Allow inoculated plate to stand for 3 to 15 min before applying discs.
  2. Application of discs
    1. Apply disc to agar surface with dispenser or manually with a sterile forceps.
    2. Apply gentle pressure with sterile forceps or needle to ensure complete contact of disc with agar.
    3. Do not place discs closer than 24mm from center to center (no more than 12 discs on 150 mm plates and 5 discs on 100 mm plates.
    4. Do not relocate a disc once it has made contact with agar surface. Instead, place a new disc in another location on the agar.
  3. Incubation
    1. Invert plates and incubate within 15 min of disc application.           
    2. Incubate for 16 to 18 at 35˚C in an ambient air incubator.
  4. Reading plates
    1. Read plates only if lawn of growth is confluent or nearly confluent.
    2. Hold inverted plate a few inches above a black nonreflecting surface.
    3. Illuminate plate with reflected light.
    4. Use a sliding caliper or ruler held on the back of the plate to measure the diameter of inhibition zone to nearest whole millimeter.
    5. When measuring zones for sulfonamides, trimethoprim, or trimethoprim- sulfamethoxazole, disregard light growth (20% less of lawn of growth) and measure edge of the more obvious margin of the zone.
    6. Discrete colonies growing within the inhibition zone may represent a mixed culture or resistant variants; subculture single colonies from the primary culture plate, re-identify, and retest for susceptibility. If the discrete colonies are still apparent, measure the colony – free inner zone.
G. Interpretation and Reporting
Use criteria specified by the CLSI to interpret the zone of inhibition for each         antimicrobial agents and report categorical result as either susceptible(S), intermediate (I), or resistant (R).

Figure 1: Antibiotic Susceptibility test of a bacterial isolate.


precautions
The following common sources of error should be investigated to verify that:
  • Zone diameters were measured and transcribed correctly;
  • The turbidity standard has not expired, is stored properly, meets performance requirements, and was adequately mixed prior to use;
  • All materials used were within their expiration dates and stored at the proper temperature;
  • The incubator is at proper temperature and atmosphere;
  • Other equipment used (e.g., pipettors) are functioning properly;
  • Discs are stored desiccated and at proper temperature;
  • The control strain has not changed and is not contaminated;
  • Inoculum suspensions were prepared and adjusted correctly; and inoculum for the test was prepared from a plate incubated for the correct length of time and in no case more than 24 hours old.

Friday, June 15, 2012

RLABB Inauguration program


RLABB in KCMS

Research Laboratory for Biotechnology and Biochemistry (RLABB) is now in Kantipur College of Medical Science (KCMS), Sitapaila, Kathmandu. Some of photographs of Inauguration of RLABB-KCMS are presented here.

Inauguration of RLABB-KCMS Branch by Honorable Minister of Environment, Science and Technology in presence of other respected delegates from different sectors.


MOU (Memorandum of Understanding) signed between Executive Director of RLABB, Prof. Dr. Vishwanath P. Agrawal and Principal, KCMS, Mr. Kedar Ji Kandel on June 07, 2012 in presence of Honorable Minister of Environment, Science and Technology and other respected delegates from different sectors at KCMS premises.


Invited chief guest, Honorable Minister of Environment, Science and Technology and other respected delegates from different sectors at KCMS premises (From left: Dr. Ganesh Agrawal, Head, Central department of Chemistry, Dean, TU, Head, KCMS, Executive Director, RLABB, Honorable Minister, Principle, KCMS and NESOM president.


Invited guests and respected delegates attending the program.



Lab visit


NOTE: All interested scientists and students are heartily welcomed to work in RLABB-KCMS branch.

Bacteria in Photos

Bacteria in Photos