Antibiotic Sensitivity Test.pptx

ANTIBIOTIC SENSITIVITY TEST
Tube dilution and agar plate method.
Filter paper and cup plate method.
Ditch-plate method.
Phenol coefficient method.
Kelsey Sykes method.

1. Antibiotic Sensitivity Test Presented By- Heera V. Karemore Assistant Professor M.Pharm

2. ANTIBIOTIC SENSITIVITY TESTING OBJECTIVES • To utilize specific monitoring techniques to evaluate the susceptibility of a microbe to different antibiotics. • To distinguish the range of activity of an antibiotic. • To recognize and define advantages and limitations of two different susceptibility testing procedures.    

3. ANTIBIOTIC SENSITIVITY TEST Tube dilution and agar plate method. Filter paper and cup plate method. Ditch-plate method. Phenol coefficient method. Kelsey Sykes method.

4. 1. Tube dilution and agar plate method: The chemical agent is incorporated into nutrient broth or agar medium and inoculated with the test microorganisms. These tubes are incubated at 30 to 35°C for 2 to 3 days and then the results in the form of turbidity or colonies are observed. The results are recorded and the activity of the given disinfectant is compared as shown in the figure.

5. Clear Slight Moderate More Turbid Turbid Turbid a ) Tube dilution method No Colonies Approx.100 Approx.500 Approx.1000 Colonies Colonies Colonies b ) Agar plate dilution method

6. 2. Filter paper and cup plate method: In these methods, the agar is melted, cooled at 45°C, inoculated with the test microorganisms and poured into a sterile Petri plate. In the cup-plate method, when the inoculated agar has solidified, holes about 9 mm in diameter are cut in the medium with a sterile cork borer. The antimicrobial agent is directly placed in the holes. In the filter paper and cylinder plate method, the antimicrobial agent is applied to the surface of the solidified, inoculated agar by using a filter paper disc and cylinder respectively. The zone of inhibition is observed after Incubation at 30 to 35°C for 2 to 3 days. The diameter of the zone of inhibition gives an indication of the relative activities of different antimicrobial substances against the test microorganisms. Cup plate method

7. 3 . Ditch-plate method: A solution of the antimicrobial substance is carefully run into the ditch which is prepared in an agar plate. A loopful of each test microorganism is then streaked outwards from the ditch on the apar surface. Microorganisms resistant to the antimicrobial grow right upto the ditch whereas susceptible microorganisms show a zone of inhibition adjacent to the ditch or centre of plate. The width of the inhibition zone gives an indication of the relative activity of the antimicrobia1 substance against the various test microorganisms. Ditch-plate method

8. 4.Phenol coefficient method. The test is for the measurement of the bactericidal activity of a chemical compound in relation to phenol. This test is carried out by measuring the concentration at which a chemical is equal in effectiveness to phenol. Staphylococcus aureus or Salmonella typhi is used for this test which is inoculated at 20-37°C for 2-3 days. Phenol coefficient is calculated by: Phenol coefficient is equal to concentration of chemical upon concentration of phenol. If a chemical is equal in effectiveness to phenol at the same concentration then its phenol coefficient is = 1. If the concentration of the chemical is less than phenol then the phenol coefficient is greater than 1. Example: Phenol dilution = 1/50 and the maximum effective dilution for disinfectant (x) = 1/ 450. Then phenol coefficient = 9. It is determined by two methods: Rideal Walker Method Chick Martin Test

9. 1.Rideal Walker Method It is the ratio of the dilution of the disinfectant that kills a microorganism to the dilution of phenol that kills the organism at the same time under identical conditions. The test is based on the principle of dilutions of the test disinfectant is compared with the standard dilutions of the phenol (usually 1 in 95) further activity against Salmonella typhi. Procedure: Preparation of sample:  The disinfectant fluids sample is mixed thoroughly and is withdrawn from the middle of the sample without forming an air bubble. Apparatus:  A loop, 4 mm in internal diameter is made at end of a 0.376 mm wire of platinum or platinum-iridium alloy, 38 mm long from the loop to the holder. The loop is bent at such an angle to the length of the wire as will facilitate in removal vertically from the surface of the liquid by the loop horizontal.

10. Reagents: Broth:  Broth is prepared with the mixture of the following ingredients: Meat extract (Microbiological grade) 20 g. Peptone (Microbiological grade) 20 g. Sodium Chloride (Reagent Quality) 10 g. Distilled Water: 1000 ml. Dissolved the solids in distilled water then sodium hydroxide is added to neutralize the solution and boiled to bring down phosphates and filter while hot. pH 7.6 is adjusted with normal hydrochloric acid. The broth is then sterilized by autoclaving at 15 Ibs with pressure for 20 minutes. It is then filtered and placed in 5 ml quantities in sterilized broth tubes and is sterilized by autoclaving at 15 Ibs pressure for 10 minutes. Test Organism:  The test organism Salmonella typhi (NCTC 786) is used. This culture is maintained by a weekly sub-culture on a nutrient agar slope and incubated in the sub-culture for 24 hours at 37°C and then stored in the refrigerator at a temperature below 22°C. For the purpose of the test, a little of the growth from the most recent sub-culture in nutrient agar slope is placed in a tube of R.W. broth and incubated for 23 hours at 38°C. A standard loopful is then transferred to a second tube and incubated. This is done for at least three times before a test is carried out.

11. ii. Chick Martin Test This test is carried out in the presence of organic matter like 3% human feces or dried yeast. This test is a modified method of RW test or qualitative suspension test. Procedure Serial dilution of the test solution and phenol is prepared in distilled water. Prepared 5% solution of phenol. To this 5% yeast suspension is added after 30 minutes. Set up a row of nine test tubes. To each test tube added 2.5 ml of diluted disinfectant at 30 seconds intervals. Allow the reaction to proceed at 20°C (room temperature) in a water bath. To this solution, Salmonella typhi is added. After a contact time of 30 minutes, the above mixture is transferred to the freshly prepared 10 ml of broth. Set up two rows of 10 ml nutrient broth, one for each dilution of disinfectant under test and of the Phenol (i.e. two at each disinfectant and phenol dilution). The test tubes are incubated at 37°C for 48 hours. Finally, the presence of microorganisms is calculated.

12. Thank you !