SOP Title: Determination of Minimum Inhibitory Concentration of Disinfectant

SOP Title: Determination of Minimum Inhibitory Concentration of Disinfectant

1. Objective:

To lay down the procedure for Determination of Minimum Inhibitory Concentration of Disinfectant.

2. Scope

This procedure is applicable for Determination of Minimum Inhibitory Concentration of Disinfectant in all sections.

3. Responsibility:
   • Quality Control: To prepare and review the SOP. To follow the procedures laid down for Determination of Minimum Inhibitory Concentration of Disinfectant as per this SOP.
   • Quality Assurance Department: To review and approve the SOP and Annexure.

 

4. Accountability:

Head Quality Control Department,  Head Quality Assurance Department.

 

5. Procedure:

 

5.1	Safety Precautions:
5.1.1	Ensure that all glassware’s to be used are properly cleaned and dried.
5.1.2	All the following operations shall be carried out under laminar Air Flow in quality control Lab using aseptic techniques.
5.1.3	Ensure that sterilized pipettes, tubes, media are kept under Laminar air Flow.
5.2	Procedure to Determine the MIC of Disinfectant:
5.2.1	For dilution of cultures, refer SOP: Procedure for Serial Dilution Preparation of Microbial Cultures.
5.2.2	Streak bacterial cultures, Escherichia coli (E. coli) ATCC 8739, Salmonella abony (S. abony) NCTC 6017, Pseudomonas aeruginosa (P. aeruginosa) ATCC 9027 and Staphylococcus aureus (S. aureus) ATCC 6538 SCDA slants from working culture slants. Refer SOP ‘’Maintenance of cultures.
5.2.3	Fungal cultures, Candida albicans (C. albicans) ATCC 10231 and Aspergillus brasiliensis (A. Brasiliensis) ATCC 16404 shall be streaked on SDA slants from working culture slants.
5.2.4	Incubate the bacterial culture slants  at 30°C – 35°C for 24 hours and fungal culture slants at 20 °C – 25 °C for 48 hours in case of C. albicans and 72-120 hours for A. Brasiliensis.
5.2.5	After completion of incubation period observe the slants. If the growth is not satisfactory then again streak the slants. scrape the growth from the slants using a sterile nichrome loop in 10 ml sterile Buffered Sodium Chloride Peptone Solution which is considered as culture stock solution. Thoroughly mix for about 1 minute.
5.2.6	Prepare the dilutions of known disinfectants such as Dettol and Savlon  from 0.5 % to 5 %  i.e. 0.5, 1.0,1.5,2.0,2.5,3.0,3.5,4.0,4.5,5.0. Report in Annexure No. 02.
5.2.7	Eg. For Preparing 0.5%  Dettol solution in 100 ml of water 0.5 ml Dettol + 99.5 ml Sterile Purified Water = 100 ml of 0.5% Dettol solution. Refer Annexure No. 04 for dilution preparations.
5.2.8	For IPA from 50 % to 100% i.e. 50, 60, 70, 80, 90, 100. Label the test tubes as per above dilutions. Using sterile purified water make up to volume to get required concentration. Report in Annexure .
5.2.9	Eg. For Preparing 70%  IPA solution in 100 ml of water 70 ml IPA + 30 ml Sterile Purified Water = 100 ml of 70% IPA solution. Refer Annexure No. 04 for dilution preparations.
5.2.10	Prepared dilution concentrations should be mixed well (Dettol, Savlon and IPA). Dispense these 100 ml disinfectant solutions in 10 tubes of 10 ml each and add to this, 0.1 ml of individual culture stock solution (E. coli, S. abony, P. aeuruginosa, S. aureus, C. albicans and A. brasiliensis.  Shake and keep the tubes standing for 5 minutes. Consider this as Dilution 1.
5.2.11	Mark sets of tubes containing 9 ml of sterile Nutrient broth or SCDM and SDB, according to the dilutions (0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5% and 5.0%) for Dettol and Savlon.
5.2.12	Prepare 50%, 60%, 70%, 80%, 90% and 100% dilution solutions for IPA.
5.2.13	Add 1 ml of each prepared dilution(Dilution 1) of disinfectant containing bacterial culture to the 9 ml of sterile nutrient broth or SCDM and fungal cultures in SDB. Consider this as Dilution 2.
5.2.14	For positive control inoculate 0.1 ml of bacterial culture stock solution of each organism directly in each separate 9.9 ml of nutrient broth or SCDM and fungal cultures in SDB.
5.2.15	Keep one test tube of nutrient broth or SCDM and SDB without inoculation as negative control.
5.2.16	Shake all the tubes and then incubate at 30 to 350 C  for bacteria for 24 hours and at 20 to 25C0 C for fungi 48 hours (C. albicans) and upto 120 hours (A. brasilliensis).
5.2.17	Minimum Inhibitory Concentration of a disinfectant shall be determined depending on the turbidity or clearity of the media in test tubes. If the media is found turbid then it should be considered as bacterial or fungal growth. If found clear then it shall be considered as inhibition of growth.
5.2.18	Observe the inhibition of the growth in tube and make entries of results in Report of Minimum Inhibitory Concentration of Disinfectant Annexure depending on type of disinfectant used.
5.2.19	For Confirmation, prepare SCDA and SDA plates as per SOP. Pipette out 0.1 ml from tubes that showed no visible growth and spread on the SCDA and SDA plates and incubated the plates for 24 hours at 30 – 35° C for bacteria(SCDA) and 48 hours to 120 hours at 20-25° C for fungi (SDA). There should not be any growth on the plates, which confirms Inhibition of specified organisms. Report in Annexure. If growth is observed, discard the plates and perform new study.
5.2.20	Select the concentration of disinfectant for validation of disinfectants which shows growth inhibition at minimum disinfectant concentration for the above microorganisms.
5.2.21	Frequency: Initially (One time) for routine disinfectants or immediately before the introduction of a new disinfectant.

6. Definitions / Abbreviation:

• Definitions:
• Abbreviation:

Abbreviation	Expansion
°C	Degree Centigrade
0.1 N	0.1 Normal
0.1 N	0.1 Normal
0.1 M	0.1 Molar
ml	millilitre
psi	Per square inch
+	Plus
IPA	Isopropyl Alcohol.
SCDA	Soyabean Casein Digest Agar
SDA	Saborauds Dextrose Agar
SDB	Saborauds Dextrose Broth
SCDM	Soyabean Casein Digest Medium

 

*Note – Ready to use SOP available in “DOWNLOAD” Section.