sop for Disinfectant Efficacy Test

1.0 OBJECTIVE

1.1 To lay down Procedure for Disinfectant Efficacy Test.

2.0 SCOPE

2.1 This procedure applicable for Disinfectant Efficacy Test of Microbiology Laboratory

3.0 RESPONSIBILITY

3.1 Microbiologist QC

4.0 ACCOUNTABILITY

4.1 QC Manager

5.0 PROCEDURE

5.1 Requirement
5.1.1 Disinfectant
5.1.2 Measuring Cylinder
5.1.3 Sterile Petri Plates
5.1.4 Sterile Saline solution
5.1.5 Sterile & Molten cooled Soybean casein digest agar
5.1.6 Sterile & Molten cooled Sabouraud dextrose agar

5.2 Methodology
5.2.1 From a recently grown stock culture of each of the test organisms subculture on

the surface of respective media slants as given below.
Table 1

Organism	Medium	Incubation temperature and duration
Salmonella sp.	SCDA	30-35ºC for 24 to 48 hours
Bacillus subtilis	SCDA	30-35ºC for 24 to 48 hours
Aspergillus niger	SDA	20-25°C for 48 to 72 hours
Candida albicans	SDA	20-25°C for 48 to 72 hours
Pseudomonas aeruginosa	SCDA	30–35^OC for 24 to 48 hours
Staphylococcus aureus	SCDA	30–35⁰C for 24 to 48 hours

5.2.2 Transfer 1 ml of sterile normal saline solution to the freshly grown culture slant, mix properly and transfer

the content of the slant to a tube containing 10 ml sterile normal saline & mix it properly. Similarly prepare the

culture suspension of other cultures. The concentration of culture suspension to be prepared, should be not less

than 106 cfu/ml. To suspend spores of Aspergillus niger 0.05 per cent polysorbate 80 may be added to the saline.
5.2.3 Determine the number of cfu per ml in each suspension by a ten-fold serial dilution.
5.2.4 Transfer 1.0 ml of culture suspension to a tube containing 9.0 ml of sterile saline solution to obtain 10-1 dilution and mix properly.
5.2.5 Transfer 1.0 ml of the 10-1 dilution to a tube containing 9.0 ml of sterile saline solution to obtain test dilution 10-2 and mix properly.
5.2.6 Similarly serially dilute the culture suspension to obtain dilution of 10-3, 10-4, 10-5, 10-6, 10-7 and 10-8.
5.2.7 Plate 1.0 ml from test dilution 10-5, 10-6, 10-7 and 10-8 in duplicate into 90 mm sterile Petri plates in duplicate.
5.2.8 Aseptically pour in each plate about 20 ml sterilized Soyabean Casein Digest agar medium for bacterial culture

and sterilized Sabouraud Dextrose agar for fungal culture, previously liquefied and cooled to around 450C.
5.2.9 Carry out the same dilution method for all the cultures and label accordingly with dilution number and test organism.
5.2.10 Allow to solidify the plates under LAF, invert and incubate individually as specified in above table.
5.2.11 Count the number of colonies and find out the concentration of culture suspension and tabulates the results in specified format.
5.2.12 Apply the above known culture cells (NLT 106 cfu) individually to a surface of 25 cm2.
5.2.13 Allow to air dry the applied cultures at room temperature.
5.2.14 Prepare the disinfectant solution to be tested as per SOP for Preparation of Disinfectant Solution

SOP No. FPPL/MB/018-00 and apply on the surface of artificially contaminated area.
5.2.15 Take a swab immediately after 5 min, 10 min and after 15 min., and then proceed the swab testing.
5.2.16 Media, to be used in swab testing, should be having following neutralizing agents to neutralize the residual disinfectant effect :

Table 2

Organism	Medium	Incubation temperature and duration
Salmonella sp.	SCDA	30-35ºC for 24 to 48 hours
Bacillus subtilis	SCDA	30-35ºC for 24 to 48 hours
Aspergillus niger	SDA	20-25°C for 48 to 72 hours
Candida albicans	SDA	20-25°C for 48 to 72 hours
Pseudomonas aeruginosa	SCDA	30–35^OC for 24 to 48 hours
Staphylococcus aureus	SCDA	30–35⁰C for 24 to 48 hours

5.2.7 Incubate the plates as specified in Table 1 and tabulate the result in specified format.
5.2.8 The tested sample is effective at specified concentration if not less than 3 log reduction is achieved in 10 minutes for each culture.

5.3 PRECAUTION
5.3.1 Artificially contaminated area should be cleaned immediately after completion of work.
5.3.2 Use hand gloves and nose mask during testing.
5.4 FREQUENCY: After 6 Month
5.5 ACCEPTANCE CRITERIA
Not less than three log reduction in 10 minutes.

6.0 ABBREVIATIONS