Factors influencing disinfections, antiseptics an their evaluation

Factors influencing disinfections, antiseptics an their evaluation

1. UNIT III 3.2 Factors influencing disinfections, antiseptics an their evaluation Presented by: Mohammad Abuzar( M. Pharm ) Assistant Professor School of Pharmacy AIKTC, New Panvel .

2. CONTENTS 2

3. 3 INTRODUCTION Disinfectant evaluation – Tests used to assess bacteriostatic and bactericidal activity of disinfectants Bacteriostatic activity – ability of an disinfectant to stop the growth of microorganisms Bactericidal activity – ability to kill the organism

4. 4 Tests for disinfectant activity WWW.PHARMANOTES.ORG

5. 5 SUBSTANCE TESTED BACTERIOSTATIC TESTS BACTERICIDAL TESTS Liquid disinfectants         Serial dilution in fluid media End-point or extinction time methods Serial dilution in solid media Counting methods Cup-plate, fish-spine bead and filter paper methods Turbidometric assessment Gradient-plate method ‘In use’ and other tests Ditch-plate technique Additional in vivo tests can be applied Semi-solid antibacterial formulations, e.g. creams, ointments, pastes and gels   Cup-plate methods Modified end-point or extinction time methods The ditch-plate technique Additional in vivo tests can be applied (e.g. skin tests) Solid disinfectants, disinfectant powders Inhibition on seeded agar Modified end-point or   extinction time methods Aerial disinfectants Use of slit-sampler in test chamber

6. 6 Tests applied to liquid disinfectants Bacteriostatic activity Serial dilution in fluid media Minimum Inhibitory Concentration (MIC) – the minimum concentration preventing detectable growth is taken as the measurement of bacteriostatic activity MIC varies with inoculum size, medium used and incubation conditions.

7. 7 Serial dilution in solid media   A suitable volume of double strength nutrient agar Diluted with an equal volume of bacteriostatic solution Poured into sterile petri plate   Surface is dried by incubating at 37 ⁰ C for 1 h Drops of the test organism are placed on the dried surface   Incubated for 2 to 3 days A separate petri dish is used for each concentration of bacteriostatic

8. 8 Cup-plate, fish-spine and filter paper methods Agar medium is melted, cooled suitably and inoculated with the test organism Poured into a sterile petri dish Cup-plate: holes about 8 mm in diameter are cut in the agar with the help of a sterile cork borer Antimicrobial agent is placed in the holes

9. 9 Fish-spine bead, filter paper and cylinder method

10. 10 In all the cases, zones of inhibition may be observed   The diameter of zones of inhibition gives rough indication of   The relative activities of different antimicrobial substances against the test organism   The effect of different concentrations of antimicrobial substance

11. The gradient - plate method Agar is streaked in the same line as the slope of the agar (along the concentration gradient) and reincubated . Approximate MIC is obtained from the equation MIC=CX(x/y)mg/ml C= concentration, in mg/ml, in total volume ( i.e , volume of wedges A and B) X=Length of growth, in cm Y= total length of possible growth, in cm 11

12. 12 The ditch-plate technique

13. 13 Carrier test A carrier such as a silk or catgut thread is contaminated by submersion in a liquid culture of the test organism T he carrier is then dried and brought in contact with the disinfectant for a given exposure time Cultured in a nutrient broth No growth indicates activity of the disinfectant tested whereas growth indicates a failing. By multiplying the test concentrations of the disinfectant and the contact times, a potentially active concentration-time relationships of the disinfectant is obtained. Limitations The number of bacteria dried on a carrier is hard to standardize   The survival of the bacteria on the carrier during drying is not constant.

14. 14 The AOAC Use – Dilution Test ( American Association of Official Analytical Chemists)     A carrier-based test Organisms: Salmonella cholerasuis , S. aureus and P. aeruginosa Carriers (stainless steel cylinders) are meticulously cleaned, sterilized, cooled and inoculated with a test organism by immersing in one of the culture suspensions The cylinders are drained on filter paper, dried at 37°C for 40 minutes, exposed to the use-dilution of the disinfectant for 10 minutes. After transfer from the disinfectant, the treated test surfaces are incubated in the neutralizing growth medium for 48 hours The number of tubes showing growth of the target microorganism is recorded. To “PASS" a 60 carrier test, at least 59 of the 60 surfaces tested must demonstrate complete disinfection (no detectable growth of the target microorganism in the test tubes containing neutralizing growth medium) To "PASS" a 10 carrier test, complete disinfection must take place on all test surfaces.

15. 15 Suspension tests A sample of the bacterial culture is suspended into the disinfectant solution After exposure it is verified by subculture whether this inoculum is killed or not. Suspension tests are preferred to carrier tests as the bacteria are uniformly exposed to the disinfectant. Types A. Qualitative suspension tests: Loopful of bacterial suspension brought into contact with the disinfectant A loopful of this mixture cultured for surviving organisms. Results expressed as ‘growth’ or ‘no growth’.

16. 16 B. Quantitative suspension tests.   The number of surviving organisms (B) is counted and compared to the original inoculum size (A).   Microbicidal effect (ME) = Log (A) - Log (B)   ME = 1 → killing of 90% of the initial number   ME = 2 → 99% killed.   A generally accepted requirement is:   ME ≥ 5 →99.999% of the germs are killed.

17. 17 Capacity test The ability to retain activity in the presence of an increasing load is the capacity of the disinfectant.   In a capacity test, the disinfectant is challenged repeatedly by successive additions of bacterial suspension until its capacity to kill has been exhausted.   Capacity tests simulate the practical situations of housekeeping and instrument disinfection.   Best known capacity test is the Kelsey-Sykes test (Kelsey and Sykes, 1969).

18. 18 Kelsy -Sykes ‘In-use’ tests A triple challenge test, designed to determine concentrations of disinfectant that will be effective in clean and dirty conditions. Organisms: 4 organisms (S. aureus, E.coli, Psedomonas aeruginosa and Proteus vulgaris) Three successive loads of bacteria (additions) Transfer 1ml at 0, 10, and 20 mins Temp: 20 ⁰ C Calibrated pipette for subculture rather than loop Clean and dirty conditions Assessment (kill or not) Sets that contain two or more negative cultures are recorded as a negative result. The disinfectant passes at the dilution tested if negative results are obtained after the first and second challenges. The third challenge is not included in the pass/fail criterion but positive cultures serve as inbuilt controls. If there are no positive cultures after the third challenge, a lower concentration of the disinfectant may be tested.

19. 19 Time from start (min) Procedure Inoculate 3ml of the disinfectant dilution with 1ml of bacterial suspension in broth, yeast or serum and shake gently (this gives a bactericide/bacteria reaction mixture) 8 Remove sample from above reaction mixture with a 50 dropper pipette. Transfer 1 drop to each of 5 tubes of liquid recovery media, or 5 drops to the surface of a nutrient agar plate 10 To bactericide or bacteria reaction mixture, prepared at start (time 0), add a second 1 ml of bacterial suspension Kelsy -Sykes ‘In-use’ tests

20. 20 Tests on aerial disinfectants A closed room of approximately cubic dimensions and 1000cu ft capacity is used   Fans incorporated to ensure uniform mixing of bacteria and bactericide   Staphylococcus albus (non clumping strain) is used as the test organism   Dispersion of organism is done using collision inhaler   Air samples are taken using a slit sampler at suitable intervals   The room should be initially free from extraneous microorganisms   Temperature and humidity of air controlled   Cyclopentanol-1-carboxylic acid is chosen as the reference standard for air disinfection

21. 21 WWW.PHARMANOTES.ORG Tests applied on solid disinfectants Disinfectant powder is mixed with an inert substance such as talc or kieselguhr to form a disinfectant powder   Powder is dusted onto inoculated plates   Inert diluent is used as the control   Alternatively Rideal Walker coefficient can also be used   A weighed sample is shaken with distilled water at 18 ⁰ C for 3 min The suspension is used for the Rideal -walker test

22. 22 Bacteriostatic tests – evaluate the property of stopping the growth of microorganisms   Bactericidal tests – evaluate the killing property   Bactericidal tests   Serial dilution in solid media   Serial dilution in liquid media   Cup plate   Ditch plate   Gradient plate   Aerial disinfectants evaluated by using a slit sampler and Staph albus as test organism   Solid disinfectants – sprayed over the surface of a inoculated plate Summary

23. 23 W.B. Hugo and A.D. Russel: Pharmaceutical Microbiology, Blackwell Scientific publications, Oxford London. Prescott and Dunn., Industrial Microbiology, 4th edition, CBS Publishers & Distributors, Delhi. Pelczar , Chan Kreig , Microbiology, Tata McGraw Hill edn . Malcolm Harris, Balliere Tindall and Cox: Pharmaceutical Microbiology. Rose: Industrial Microbiology. Probisher , Hinsdill et al: Fundamentals of Microbiology, 9th ed. Japan Cooper and Gunn’s: Tutorial Pharmacy, CBS Publisher and Distribution. Peppler : Microbial Technology. I.P., B.P., U.S.P.- latest editions. Ananthnarayan : Text Book of Microbiology, Orient-Longman, Chennai Edward: Fundamentals of Microbiology. 12. N.K.Jain : Pharmaceutical Microbiology, Vallabh Prakashan , Delhi REFERENCES

24. 24