Testing Disinfectants in the Food Factory

Phenol Coefficient Method Anavella Gaitan Herrera

1. Introduction

Contamination of foods by the environment has direct public health and keeping quality significance. The food factory environment (with raw materials and processing) governs the numbers and types of microorganisms in finished products (1). Use of the appropriate sampling procedures permits us to discover the magnitude and type of contamination. Microbiological sampling allows objective evaluation of the disinfectants and the sanitation practices and procedures used in the food factory (2; see Note 1).

Disinfectants are antimicrobial pesticides that are primarily used on inanimate surfaces (such as floors, walls, and countertops) to kill infectious bacteria, fungi, and viruses. Antimicrobial pesticides are substances used to kill or suppress the growth of harmful microorganisms on inanimate objects and surfaces (3). Products intended for the control of microorganisms in or on people or animals are considered drugs, not pesticides, and are therefore regulated by the Food and Drug Administration (FDA). Antimicrobial pesticides (4,5) are divided into two broad use categories:

1. Non-public health products include those used to control the growth of algae, odor-causing bacteria, and microorganisms causing spoilage, deterioration, and fouling of materials. Examples include antimicrobials used in cooling towers, paints, and paper products (6).

2. Public health products are intended to control microorganisms infectious to people. Examples include sterilants, which are used to destroy or eliminate all forms of micro-bial life including fungi, viruses, and all forms of bacteria and their spores; disinfectants, which are used to destroy or irreversibly inactivate infectious fungi and bacteria, but not necessarily their spores; and sanitizers, which are used to reduce, but not necessarily eliminate microorganisms. Examples range from sterilants used to treat surgical instruments to disinfectants applied to hospital floors, walls, and bed linens and sanitizers used on carpets or in laundry additives (6).

From: Methods in Molecular Biology, vol. 268: Public Health Microbiology: Methods and Protocols Edited by: J. F. T. Spencer and A. L. Ragout de Spencer © Humana Press Inc., Totowa, NJ

Products making pesticidal claims are registered by the Environmental Protection Agency (EPA). In the early 1990s, the Office of Pesticide Programs (OPP) implemented the Antimicrobial Product Testing Program to test the antimicrobial effectiveness and chemical composition of the EPA-registered products (7,8).

In 1881, Robert Koch developed the first method for evaluating the activity of disinfectants (9). In 1897, Kroing and Paul noted that the activity of disinfectants could be compared under controlled conditions (temperature and concentration) (9). In 1903, Rideal and Walker developed the phenol coefficient method (10). The novelty of this method was the standardization of components (test methods, recovery medium, microorganisms, and disinfectant stock solution). This method continues to be used today with some modifications (3). Ideally, food, finished product container, and product contact surfaces of the equipment should be practically free from microorganism (see Note 2).

The purposes of microbiological monitoring can be summarized as follows (6):

1. Determination of the frequency required for execution of sanitation cycles.

2. Determination of environmental contamination by foodborne pathogens.

3. Evaluation of designs of food processing facilities and equipment in terms of sanitation.

4. Discovery of environmental sources of spoilage organisms in response to shelf life problems.

5. Determination of the efficiency of sanitation procedures and cycles.

6. Determination of the frequency required for special maintenance procedures (changing of air filters to reduce airborne mold contamination.

The Phenol Coefficient Method given here is applicable to testing disinfectants miscible with water or for standard resistance of test bacteria. The 95% confidence limits are ± 12% (3).

2. Materials 2.1. Culture Media

1. Nutrient broth (see Note 3): Boil 5 g beef extract, 5 g NaCl and 10 g peptone in 1 L H2O for 20 min and adjust to pH 6.8. Filter through paper. Place 10-mL portions in 20 x 150-mm test tubes. Autoclave for 20 min at 121°C.

2. Synthetic broth:

a. Solution A: dissolve 0.05 g L-cystine, 0.37 g DL-methionine, 0.4 g L-arginine-HCl, 0.3 g DL-histidine-HCl, 0.85g L-lysine-HCL, 0.21 g L-tyrosine, 0.5 g DL-threonine, 1.0 g dl-valine, 0.8 g L-leucine, 0.44 g DL-isoleucine, 0.06 g glycine, 0.61 g DL-serine, 0.43 g dl-alanine, 1.3 g L-glutamic acid-HCl, 0.45 g L-aspartic acid, 0.26 g DL-phenylalanine, 0.05 g DL-tryptophan, and 0.05 g L-proline in 500 mL H2O containing 18 mL 1 N NaOH.

b. Solution B: dissolve 3.0 g NaCl, 0.2 g KCl, 0.1 g MgSO4-7 H2O, 1.5 g KH2PO4, 4.0 g Na2HPO4, 0.01 g thiamine-HCl, and 0.01 g niacinamide in 500 mL H2O. Mix solution A and solution B, dispense in 10-mL portions in test tubes (16 x 150 mm), and autoclave (20 min at 121°C). Before using for daily transfers of test cultures, aseptically add 0.1 mL sterile 10% glucose solution per test tube. Grow cultures with tube slanted 8° from horizontal.

3. Nutrient agar: Dissolve Bacto Agar to 1.5% in nutrient broth (or synthetic broth) and adjust to pH 7.2 or 7.4, add 10-mL portions per tube (13 x 100 mm), autoclave (20 min at 121°C), and slant.

4. Letheen broth (see Note 4): Dissolve 0.7 g lecithin and 5.0 g polysorbate 80 or Tween-80 in 400 mL of hot H2O and boil until clear. Add 600 mL solution of 5.0 g beef extact, 10.0 g peptone, and 5.0 g NaCl in H2O and boil for 10 min. Adjust pH to 7.0 ± 0.2 (with 1 N NaOH or 1 N HCl). Filter through coarse paper, transfer 10-mL portions to test tubes (16 x 150 mm), and autoclave (20 min at 121°C).

5. Thioglycolate medium USP XX (see Note 4): Mix 0.5 g L-cystine, 0.75 g agar-agar, 2.5 g NaCl, 5.5 g glucose-H2O, 5.0 g H2O-soluble yeast extract, and 15.0 g pancreatic digest of casein with 1 L H2O. Heat in H2O bath to disolve, add 0.5 g sodium thioglycolate (or 0.3 g thioglycolate acid), and adjust to pH 7.1 ± 0.2 (with 1 N NaOH). Filter if necessary when hot (without boiling) through moistened filter paper. Add 1.0 mL of 0.1% sodium resarzurin solution (freshly prepared). Transfer 10 mL medium to 16 x 150-mm tubes and autoclave (20 min at 121 °C). Cool to 25°C and store at 20-30°C (protected from light).

2.2. Reagents and Apparatus

1. Test organism (see Note 2):

a. Salmonella typhi, ATCC 6539: Maintain stock culture on nutrient agar slants and transfer monthly. Incubate new stock transfer for 2 d at 2-5°C. Inoculate tubes of nutrient broth, from stock culture and make at least four consecutive daily transfers (<30) in nutrient broth, incubating at 37°C (before using culture for testing). Culture the organism for 22-26 h in nutrient broth at 37°C in test tubes. Shake and let settle for 15 min before using.

b. Staphylococcus aureus, ATCC 6538: Use 22-26-h cultures of S. aureus, having at least the resistance indicated in Table 1 at 20°C. Maintain stock culture on nutrient agar slants. Incubate both 18-24 h at 37°C. (Store slants at 2-5°C). Inoculate Trypticase soy agar (TSA) plates, and Mannitol Slants Agar (MSA). Incubate overnight at 37°C. Colonies of S. aureus are round, shiny, and yellow. Gram staining shows Gram positive cocci. Store the cultures for 30 d (2-5°C).

c. Pseudomonas aeruginosa ATCC 15442: Use 22-26-h cultures of P. aeruginosa, having at least the resistance indicated in Table 2, at 20°C. Inoculate Cystina trypticase agar (CTA). Incubate tube 18-24 h at 37°C and store at 2-5°C. Inoculate TSA and Pseudosel Petri plates; incubate overnight at 37°C. Colonies of P. aeruginosa are flat, greenish-yellow, and opaque. Gram staining shows Gram-negative rods. Store cultures for 30 d (2-5°C).

2. Phenol stock solution (see Note 5): Solution should be 5% w/v.

a. Weigh 50 g phenol (USP), which congeals at >40°C, in beaker.

b. Dissolve in H2O, rinse solution in 1-L volumetric flask, and dilute to volume.

c. Standardize with 0.1 N KBr-KbrO3 solution (see Subheading 2.2.3.).

d. Transfer 25 mL stock solution to 500 mL volumetric flask and dilute to volume with H2O.

e. Transfer 15-mL aliquot of diluted solution to 500 mL I2 flask and add 30 mL standard KBr-KBr03 solution.

f. Add 5 mL HCl and immediately insert stopper. Shake frequently (for 30 min) and let stand for 15 min.

Table 1

Phenol Coefficient Method of Testing Disinfectants Against Staphylococcus aureus"

Table 1

Phenol Coefficient Method of Testing Disinfectants Against Staphylococcus aureus"

Phenol dilution

5 min

10 min

15 min

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