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MicroChem Laboratory is a custom contract laboratory specialized in efficacy testing of disinfectants and other antimicrobial products. We offer a range of disinfectant testing and consulting services.

 

 

MicroChem Laboratory recently attended the 2006 ASM Biodefense Research Meeting and Poster Session in Washington DC to talk about acidified (diluted) bleach.  Dr. Miner was interviewed by ASM, MicrobeWorld Radio, and Bloomberg at the meeting.

Click the following link to read online article:

Vinegar increases killing power of bleach by Jim Sliwa, ASM

 

Below are the actual procedures and results from the studies conducted at MicroChem Laboratory:

 

A Commonly Available Household Sterilant

 Norman Miner, Ph.D., Valerie Harris, B.S., Jennifer Dunham, B.S., Brandon Musgrove, B.A., and Sara Stumph, B.A.

 MicroChem Laboratory, Euless ( Dallas ), TX 76040

Introduction

Sodium hypochlorite (NaOCl) in the form of laundry bleach is available in most households.  The concentrate is about 5.25% to 6.00% NaOCl, and the pH value is about 12.  Sodium hypochlorite is stable for many months at this high alkaline pH value.  Laundry bleach is commonly diluted about 10 to 25-fold with tap water to about 2000 to 5000 ppm free available chlorine for use as an environmental surface disinfectant, without regard to the pH value of the diluted bleach.  However, the pH value is very important for the antimicrobial effectiveness of bleach.  At alkaline pH values of about 8.5 or higher, more than 90% of the bleach is in the form of the chlorite ion (OCl-), which is relatively ineffective antimicrobially.  At acidic pH values of about 6.8 or lower, more than 80% of the bleach is in the form of hypochlorite (HOCl).  HOCl is about 80 to 200 times more antimicrobial than OCl-.

These studies show the antimicrobial effectiveness of bleach diluted with tap water to a pH of about 11, as compared with bleach similarly diluted with tap water, and then acidified with 5% household vinegar to a pH of about 6.0.  Bacillus subtilis ATCC 19659 was used as a non-pathogenic surrogate for B. anthracis, and Aspergillus niger ATCC 6275 was used as the test fungus. 

Methods

 Unglazed porcelain cylinders, and braided polyester suture loops, collectively called “carriers”, were soaked in a soil-extract nutrient both culture of B. subtilis, and then dried for 72 hrs, and tested according to the methods of the Association of Analytical Chemists (AOAC) Sporicidal Test 966.04.  After drying, the cylinders and suture loops each carried about 10^5 to 10^6 colony forming units (CFU) of B. subtilis.  Cylinders were soaked in a culture of A. niger, dried for 30.0 min at 35 C, exposed to the test disinfectants, transferred to Sabouraud Broth (SAB), incubated and scored for growth or no growth.  The cylinders carried about 10^5 CFU of A. niger .  Laundry bleach was diluted 25-fold with tap water to about 2000 ppm free available chlorine (FAC), pH 11.1, or similarly diluted with tap water and then acidified with 5% vinegar to a pH value of about 6.0.  The B. subtilis- or A. niger-labeled carriers were exposed to the diluted bleach at pH 11, or pH 6 for various exposure times at ambient temperature. 

Results  

Bleach at 2000 ppm FAC disinfected more carriers labeled with B. subtilis at pH 6.5 than at pH 11.1.  The results for B. subtilis are shown in Table 1.   

Test Chemistry

Carrier Type

Exposure Time

Sterile Carriers Per Total Tested

Percent Sterile Carriers

2000 ppm FAC, pH 11.1

Cylinders

2.5 min

0/40

0%

5.0 min

0/40

0%

10.0 min

0/40

0%

20.0 min

1/40

2.5%

Polyester Suture Loops

2.5 min

0/40

0%

5.0 min

0/40

0%

10.0 min

0/40

0%

20.0 min

0/40

0%

2000 ppm FAC, pH 6.5

Cylinders

2.5 min

33/40

82%

5.0 min

34/40

85%

10.0 min

39/40

98%

20.0 min

40/40

100%

Polyester Suture Loops

2.5 min

5/40

12%

5.0 min

10/40

25%

10.0 min

26/40

65%

20.0 min

34/40

85%

Bleach at 2645 ppm disinfected more carriers labeled with A. niger at pH 5.8 than at pH 11.3.  These results are shown in Table 2.   

Table 2.  The percentage of a group of cylinders labeled with A. niger disinfected after exposure to bleach at pH 11.3 or pH 5.8 after a 10.0 or 20.0 min exposure time.   

Test Chemistry

Carrier Type

Exposure Time

Sterile Carriers Per Total Tested

Percent Sterile Carriers

2645 ppm FAC, pH 11.3

Cylinders

10.0 min

17/20

85%

20.0 min

19/20

95%

2645 ppm FAC, pH 5.8

Cylinders

10.0 min

20/20

100%

20.0 min

20/20

100%

Discussion

In the event of an emergency involving Bacillus anthracis spores contaminating such environmental surfaces as counter tops, desk and table tops, and floors, for example, virtually every household has a sporicidal sterilant available in the form of diluted, acidified bleach.  Bacterial spores dried in their culture medium onto a porous surface are considered the most resistant form of all types of microbes, and a disinfectant that can kill such spores would be expected to also be able to kill all types of vegetative bacteria, fungi, mycobacteria (TB), and all types of viruses.  Bacillus subtilis is widely published as a non-pathogenic surrogate as resistant to antimicrobial chemicals as Bacillus anthracis.

A convenient formula to produce a solution of acidified bleach is 1.0 cup (8.0 oz.) of concentrated bleach (approx. 5.25% NaOCl) added to 1.0 gallon (128 oz.) of tap water, and then add 1.0 cup of 5 % distilled white cooking vinegar.  Follow the warning directions on the bleach label.  Do not add cleaning solutions containing ammonia to bleach.  After an exposure of 20.0 or 30.0 min, rinse the surfaces to remove the bleach.  Use acidified bleach within about 8.0 hrs, and then discard to a sanitary drain. 

Acidified bleach at a pH value of about 6.5 or less is a more potent antimicrobial chemical than the same concentration of bleach at an alkaline pH value.  Dry spores of B. subtilis , a non-pathogenic surrogate for B. anthracis , were killed within 20.0 min by bleach at 2000 ppm FAC at pH 6.5, but not at pH 11.1.  For maximum antimicrobial activity, it is important to not only dilute the bleach to about 2000 to 2500 ppm FAC, but also to lower the pH value of the diluted bleach to about 6.5 or less where most of the chemistry is hypochlorous acid (HOCl).   

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