The document describes a minimum inhibitory concentration (MIC) test to determine the effectiveness of BIO-CLEANTM against various microorganisms. It lists the microorganism strains tested, concentrations of BIO-CLEANTM, and the procedure which includes preparing microorganism cultures, making dilutions to a standardized concentration, mixing with BIO-CLEANTM samples of varying concentration, and observing results to determine the lowest concentration that inhibits microbial growth. Controls without BIO-CLEANTM are also included to validate the testing method.

1. MINIMUM INHIBITORY CONCENTRATION TEST : MIC TEST

2. MATERIALS AND METHODS Microorganisms Concentration of BIO-CLEANTM Procedure and assay

3. MICROORGANISMS The American Type Culture Collection strain [ATCC] Department of Medical Sciences Thailand strain [DMST] Escherichia coli ATCC 25922 Salmonella Typhimurium DMST 15674 Salmonella Enteritidis DMST 15676 Enterococcus faecium DMST 17660 Enterococcus faecalis ATCC 29212

4. MICROORGANISMS (CONT.) Enterococcus faecalis ATCC 51299 Enterobacter aerogenes DMST 13048 Bacillus cereus ATCC 11778 Pseudomonas aeruginosa ATCC 27853

5. CONCENTRATION OF BIO-CLEANTM The following BIO-CLEANTM concentrations were tested : 1,000 ppm 900 ppm 800 ppm 700 ppm 600 ppm 500 ppm 400 ppm 300 ppm 200 ppm 100 ppm

6. PROCEDURE AND ASSAY Preparation of microorganisms Cultivated microorganisms in Tryptic soy broth (TSB) and incubated 37 ° C at 24 hours. Microorganisms suspension to McFarland’s pattern of 105 cfu/ml was prepared.

7. FLOW CHART (PREPARATION OF MICROORGANISM) Compared with 0.5 McFarland = 10 5 cfu/ml ten fold dilution with PSS for Mcfarland Culture in TSB medium Slant agar Streak on NA agar and incubated at 37 o C; 24 hrs Picked 3 colonies 10 -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8 Incubated at 37 o C ; 24 hrs

8. PROCEDURE AND ASSAY (CONT.) Preparation of Mcfarland 105 cfu/ml To standardized the inoculum density is used (0.5 McFarland standard)

9. PREPARATION OF 0.5 MCFARLAND

10. PREPARATION OF 0.5 MCFARLAND

11. FLOW CHART (PREPARATION OF 0.5 MCFARLAND) OD 625 = 0.8-0.10 0.5 McFarland BaCl 2 + H 2 SO 4 (Dilution) 1:1 1:2 1:3 1:4 1:5

12. PROCEDURE AND ASSAY (CONT.) Controls Control test tube 1 = 1 ml TSB + 1 ml microorganism Control test tube 2 = 1 ml TSB + 1 ml BIO-CLEANTM Control test tube 3 = 2 ml TSB C1 C2 C3

13. PROCEDURE AND ASSAY (CONT.) Sample 1 ml BIO-CLEANTM (start 1,000-100ppm) 1 ml microorganism (105 cfu/ml) 1,000 ppm

14. PROCEDURE AND ASSAY (CONT.) --- -+- -++ Report C1 C2 C3 1,000 ppm 900 ppm 800 ppm 700 ppm 600 ppm 500 ppm 400 ppm 300 ppm 200 ppm 100 ppm