Assessment and Mitigation of Bacterial and Fungal Contamination in Refrigerator Waterlines

Three experiments were conducted on the recovery of microorganisms associated with refrigerator water lines. In the first experiment, between 2 and 3 logs CFU/ml were recovered from 100 different refrigerators’ residential water and ice lines. In a second experiment, plastic tubing segments cut from commercial refrigerator waterline systems were inoculated with the bacterium Pseudomonas aeruginosa and a fungal strain of Aspergillus flavus. Either 0.02% peracetic acid (PAA) exposure for 2 min or 0.03% PAA exposure for 1 min resulted in no recoverable bacteria cells, however, 0.25% PAA contact for 2 min was needed to eliminate detection of fungal cells. The third experiment tested sanitation of a water system inoculated with a mixed culture of P. aeruginosa and A flavus with a water filter cartridge filled with 3.5% PAA. The 3.5% concentration was determined using a computer simulation for mixing during a cleaning cycle that would yield a minimum concentration of at least 0.25% throughout the water system. After loading a water filter cartridge containing 3.5% PAA, allowing water to flow into the system for 15 s, and then allowing the system to sit for 5 min, no viable P. aeruginosa or A flavus cells were recovered up to 10 days after treatment.

Introduction

Drinking Regulations

In 1974, U.S. Congress passed the Safe Water Drinking Act (SWDA) to protect public health by assigning the authority to set drinking water standards for public water systems to the U.S. Environmental Protection Agency [1]. The SWDA was amended in 1986 and 1996 to enhance the original law and provide additional protection for microbiological contaminates and chemical disinfectant byproducts [1]. The later amendment also added a process of continuously evaluating unregulated contaminates to determine if they need future regulation through SWDA [2]. Specifics on the legal limits and treatment techniques for microorganisms, disinfectants, disinfectant byproducts, inorganic and organic chemicals and radionuclides are described in the National Primary Drinking Water Regulations [1], [2]. Although these laws are comprehensive and designed to evolve over time, contamination of drinking water can still occur. Treated potable drinking water that passes through an improperly maintained distribution system or contaminated water line may become contaminated. In many cases, the source of contamination in distribution systems is biofilms.

Wingender and Flemming [3] indicated that biofilms form on all surfaces in water treatment, distribution, and storage systems, and this includes consumer water lines and plumbing situations. These same researchers indicated that biofilms not only serve as a continuous source of microbiological contamination in water distribution systems but also may be a source of chemical hazards from biofilm-associated metabolites of health significance, such as hydrogen sulfide, nitrite and endotoxins. Biofilms have also been reported to reduce water quality by affecting color, turbidity and odor, and these quality defects are more readily detected by consumers than invisible and harmful contaminates [3].

Water that contains microbiological and chemical contaminants plays an important role in public health because it may be consumed directly as food or indirectly as a cooling medium, such as ice.The World Health Organization (WHO) declared that ice consumed or ice that comes in contact with food should be held to the same quality and safety level as the drinking water [4]. The Centers for Disease Control and Prevention (CDC) has reported gastroenteritis outbreaks due to the consumption of contaminated ice in different regions of the world [2]. Burnett et al. [5] found that ice contamination may occur from the main water supply, facility plumbing, including backflow from drains, and irregular cleaning of ice machines and delivery systems. Freezing does not eliminate microorganisms but can reduce their proliferation in food and water [6]. Hampikyan et al. [7] reported that coliforms were detected in 12.4% water, 67.6% ice chest, and 51.4% ice samples collected from food service establishments located in different regions of Istanbul, Turkey. On the other hand, no E. coli was detected in water, ice chest, and ice samples in all categories: restaurant, bar, or fish market, while coliform-type bacteria ranked highest in all categories. Cleaning, disinfecting and maintenance of delivery systems is necessary to improve the microbiological quality of water and ice [7], [8].

Waterline Biofilms

In addition to drinking water and ice, dental water lines have been cited as a common niche for biofilm development [9]. While dental water line contamination might be attributed to oral microbes, numerous studies have found that the primary contaminants of dental water lines are bacteria normally found in potable water [10]–[12]. The formation of bacterial biofilms in dental water lines when exposed to only municipal water was confirmed after six weeks of exposure to municipal water [13]. Several conditions allow the formation of biofilms within waterlines exposed to potable water. The majority of microbes living in biofilm communities present in dental waterlines are gram-negative bacteria of the same varieties that survive in small numbers in municipal water systems [14]. Thus, refrigerator waterlines would be susceptible to the same gram-negative bacteria. When biofilms form in waterlines, bacteria grow and shed, transferring to water as it passes through the line. Factors contributing to biofilm formation in waterlines include laminar flow conditions, which result in slower flow rates near the waterline wall and greater surface area exposure to equal volumes of water when passing through smaller-diameter tubes or pipes. Mills [13] illustrated the surface area effect by calculating that a 25.4 cm (10-inch) diameter main water line has four square inches exposed to 100 ml of water while 1.59 mm (1/16^th inch) diameter waterline tubing will have 400 square inches of surface area exposed. The greater surface area will also expose more bacteria to these surfaces allowing the opportunity for attachment to initiate biofilm formation. In fact, mixed species biofilms form in dental water lines and can harbor pathogens such as mycobacteria, Pseudomonas aeruginosa, Legionella pneumophila and others [15]. Al-Hiyasat et al. [16] recovered greater than one log of Pseudomonas aeruginosa from 26 (86.7%) of 30 teaching facility dental units sampled at the beginning of the working day [16]. In a separate study of 30 public facilities in northern Italy, heterotrophic plate counts, Pseudomonsa aeruginosa, Legionella pneumophila and amoebae were detected in 100%, 16.67%, 86.67%, and 60% of the samples, respectively [9].

Waterline Sanitation

Various sanitation systems for dental lines have been developed and investigated for efficiency of biofilm removal. Two commercial continuous sanitation systems included EDTA, benzalkonium chloride, and chloramine-T as active ingredients and reduced the bacterial load of dental lines to below detection [17]. A 3% Peroxy Ag⁺ solution (H₂O₂ 3% based disinfectant with 0.001% Ag⁺⁾ diluted to a final concentration of 600 ppm reduced Legionella pneumophila serogroup 2 logs after 60 min, 4 logs after 75 min and 5 logs after 15 h of exposure [18]. Flushing with 200 ml of a commercial hypochlorous-acid-based sanitizing agent (Cleancert™) was found to reduce biofilms on dental unit water lines in a practicing dental college setting [19] and on new dental lines on which biofilms were allowed to develop for 10 days [20]. Peracetic acid is an organic peroxide commonly used as an antimicrobial in water purification, and as disinfectant in the healthcare. Peracetic acid is also commonly used on fresh produce, meat and poultry and in the food and beverage industries. The current study was undertaken to enumerate microorganisms recovered from commercial refrigerator water lines and to determine the efficacy of various antimicrobial treatments (exposure time and concentration) for reducing microbiological populations on water-line surfaces. The National Sanitation Foundation (NSF) examined 14 common kitchen objects, including the water dispenser and ice dispenser in refrigerators, to determine the presence of four types of microorganisms: E. coli, Salmonella, yeast and mold, and Listeria. While E. coli, Salmonella, and Listeria were not detected in either of these two items, both the water and ice dispensers ranked among the top six items with concerning levels of yeast and mold. Here is the order in which these items ranked from highest to lowest in germ count in terms of yeast and mold levels [21]: 1.Refrigerator water dispenser 2.Rubber spatula 3.Blender 4.Refrigerator vegetable compartment 5.Refrigerator ice dispenser 6.Refrigerator meat compartment

Materials and Methods

Experiment 1: Microbiological Population of Water in Home and Workplace Tap Refrigerator Water and Ice

One hundred water samples were collected over the span of 8 weeks from 100 locations in South Carolina, U.S. Water samples were acquired from the various kitchen tap lines, refrigerator water dispense lines, and ice makers in private homes and workplaces. Sample collection from both the tap and dispenser included allowing the lines to run for 5 s before filling a sterile 120 ml container. Each sample taken from an icebox in a sterile 120 ml container. Labeled samples from each were then sealed and kept under refrigeration until transport to the lab for analysis. On the day of testing, samples were removed from the refrigerator and allowed to warm to room temperature for approximately 1 h. Serial dilutions of each sample were prepared using 0.1% sterile phosphate buffered saline (PBS) and plated onto 3M Petrifilm Aerobic Plate Count agar (APC). Plates were incubated at 35 °C for 48 h and counted with a bacterial colony counter (model 3325; Leica Quebec Darkfield).

Yeast and mold counts were enumerated by plating serial dilutions of samples onto Dichloran Rose Bengal Chloramphenicol Agar (DRBC) and plates were incubated at 25 °C for approximately five days. After incubation, colony forming units (cfu) were counted and converted to log₁₀ cfu/ml.

Accumet Model 10 (Fisher Scientific) digital pH meters were used to determine sample pH. Two pH meter electrodes, connected to two separate meters, were standardized, and then inserted directly in the samples simultaneously, and the results were recorded.

Experiment 2: Reduction of Bacterial and Fungal Contamination on Waterline Tubing

To determine the levels and contact time of sanitizer needed to reduce bacteria and mold in refrigerator waterlines small pieces of refrigerator waterline tubing were used first. These pieces of tubing were inoculated with bacteria (Pseudomonas) and mold (Aspergillus) then treated with different concentrations and contact times of peraectic acid after which the remaining populations of bacteria and mold were determined.

Inoculum Preparation

Pseudomonas aeruginosa (ATCC 109246) from the American Type Culture Collection (ATCC), was reconstituted and grown on tryptic soy agar (TSA). A loopful of Pseudomonas was removed from TSA and inoculated into Tryptic Soy Broth (TSB) overnight in a shaker water bath at 30 °C. After the overnight incubation, the Pseudomonas aeruginosa culture was centrifuged and washed three times at 7000 RPM for 10 min with sterile 0.1% peptone water (Fisher Scientific). The washed inoculum was diluted with a 0.1% peptone solution to a final concentration which was estimated by reading absorbance of 0.503 at 600 nm for a 0.500 McFarland standard (1.5 × 10⁸ CFU/ml) in a sterile 50 ml Falcon tube.

Aspergillus flavus (ATCC 9643) from Microbiologics Inc. was plated onto DRBC for 10 days at 25 °C. Mature mycelia were recovered with PBS using a sterile loop and then centrifuged and washed three times with sterile 0.1% PBS at 7000 RMP for 10 min, then further diluted with PBS to a final concentration absorbance (–0.502 spectrophotometer at 600 nm to prepare a standard McFarland inoculum).

Inoculation of Internal Water Line Tubing

Pieces of polyethylene tubing from a commercial standard refrigerator internal water line were cut approximately 20 cm in length and submerged into inoculum for vigorous shaking for 30 min. For the tubing inoculated with Pseudomonas aeruginosa, each inoculated piece was placed into its own individual sterile beaker with 100 ml 0.1% peptone with the use of sterile tweezers. Beakers were covered with parafilm and then placed on a rocker for 24-h incubation at room temperature.

For tubing inoculated with Aspergillus flavus, each piece was placed in a petri dish with 10 ml sterile 10-fold diluted of stock 10X PBS to give a 1 × PBS solution. The dishes were then covered with a lid and allowed to incubate at room temperature for approximately 10 days.

Sanitizer Tests

For Pseudomonas aeruginosa, 10 ml of peracetic acid (PAA) at concentrations of 0.01%, 0.02%, and 0.03% were prepared in 15 ml centrifuge tubes using sterile deionized water.

One at a time, pieces of plastic tubing were removed from the inoculation plate with sterile tweezers and placed in the various concentrations of peracetic acid treatment for 1, 1.5, 2, or 3 min.

After treatment, each piece was then placed in 5 ml sterile 0.1% peptone water and shaken vigorously for 1 min, and then 1 ml aliquot of the rinse solution was serially diluted and plated in duplicate on TSA. The TSA plates were incubated at 30 °C for approximately two days before colonies were counted with a bacterial colony counter (model 3325; Leica Quebec Darkfield), and populations were converted to cfu/ml.

For Aspergillus flavus, 10 ml of PAA concentrations of 0.25%, 0.5%, and 1.0% were prepared in 15 ml centrifuge tubes using sterilized water. Plastic tubing exposed to Aspergillus flavus was subjected to the PAA treatments in the same manner as described for Pseudomonas aeruginosa. After treatment, each piece was placed in 5 ml sterile PBS and vigorously shaken 1 min before 1 ml aliquot of the rinse solutions were serially diluted and plated in duplicate on DRBC and incubated at room temperature for 5 days after which the number of the colonies were counted using a bacterial colony counter (model 3325; Leica Quebec Darkfield) and results reported as cfu/ml and converted to log₁₀ cfu/ml. The different peracetic acid concentrations were prepared fresh before each use and kept in a dark environment until used.

Experiment 3: Inoculation and Sanitation Test of Refrigerator Water Systems

The Pseudomonas aeruginosa and Aspergillus flavus inoculums were prepared in the same manner described in Experiment 2, excluding the wash and dilution of Pseudomonas aeruginosa in which 1 × PBS was used. The refrigerator water system had a 710 ml holding tank, a 236 ml filter, an 11.2 ml volume valve and a total of 284 cm (length)/49.1 ml (volume) of tubing. Tubing internal diameter varied between 4.07 and 5.72 mm. Tubing was made from a blend of low and high-density polyethylene, and the valve and the tank were constructed from polypropylene.

During each of the three replications, duplicate mixtures of 160 ml of Aspergillus flavus inoculum were added to 160 ml Pseudomonas aeruginosa inoculum with 500 ml PBS to create an 820 ml bacterial/fungal cocktail mix for inoculating two refrigerator water tank systems. One water system was treated with a PAA solution, and the other with sterile water.

Water tank systems for each unit were inoculated with the bacterial/fungal mix by disconnecting and filling the tank through inlet/outlet lines attached to the tank. Inoculated water tank systems were held at 21 °C for 24 h, after which one system was treated with peracetic acid, and the other was treated with tap water. Then, a cassette containing 236 ml of either sterile water or a 3.5% PAA solution with red food dye was placed into the filter module, after which the system was turned on for about 15 s until the red food dye added to the PAA began to exit the water outlet. The water flow was then stopped, and the system was allowed to sit for 5 min. A new filter was placed in the cassette, and the system was turned on, and 6 litres were run through the system as previously described per the manufacturer’s recommendation with a new filter installation. A total of 10 water system samples were collected on days 0, 5 and 10 after inoculation, with 500 ml of water running out of the system between each sample.

The target concentration of 0.25% PAA for contact with all wet surfaces was determined as optimal in Experiment 2. The volume of the refrigerator filter was 236 ml, and the total volume of the system was 826 ml. A computer simulation accounted for water mixing and dilution once the freshwater line was turned on and flushed the filter, finding that a concentration 14 times the target of 0.25% PAA was required to obtain the 0.25% concentration throughout the system. Thus, 236 ml of a 3.5% PAA solution was created from the stock 15% to fill an empty refrigerator filter cartridge. The computer simulation was done using commercially available Computational Fluid Dynamics (CFD) software (Simcenter STAR-CCM+). The computer simulation was used to determine the initial concentration (X) of PAA needed in the refrigerator filter so that once fresh water was flushed through the system for a specific period of time to distribute the PAA, the concentration of PAA throughout the system would be at least the concentration (Y) necessary for effective sanitation. The higher concentration of PAA in the filter was necessary because the introduction of water into the line diluted the PAA as it passed through the cold-water storage tank, water valves, and other components of the refrigerator’s water system. In the worst-case water system arrangement (236 ml filter volume and 826 ml downstream system volume) and knowing the flow rate of water through the system, the simulation determined that to achieve the goal as described, the initial concentration needed to be equal to 14Xs that initial concentration to achieve the targeted concentration throughout the system and use a distribution flush of 15 s.

After inoculation with 820 ml of a mixed culture of Aspergillus flavus and Pseudomonas aeruginosa containing ~8 log of each microorganism, the water systems were allowed to sit for 24 h for bacterial attachment.

DAY 0

Delivery of peracetic acid to water line system and sample collection:

The filter cartridge was filled with PAA, and a water line was attached to the refrigerator unit/tap water connection and then turned on for 15 s to allow the PAA to be diluted and spread through the whole system.

The PAA was allowed to sit in the water line system for five min, during which time a new refrigerator water filter was inserted into the unit prior to sample collection. After the 5-min wait time, the system was flushed with 6 liters of tap water per the manufacturer’s protocol when installing a new water filter. After the flushing step, a total of 10 water samples were collected, with 500 mL of water being passed through the system between each sample collection. Samples were then plated for enumeration of bacteria on TSA medium and fungus on DRBC medium. The system was then detached from the water flow and left undisturbed for incubation at 21 °C for five days.

Day 5

The water line was attached to the refrigerator unit/tap water connection, then one water sample was collected, after which 500 ml of water was run through the system, and then another water sample was collected. This procedure was repeated with 500 ml being run between sample collections until a total of 10 samples were collected. Samples were then plated for enumeration of bacteria on TSA medium and Fungus on DRBC medium as previously described.

Water flow was then detached from the system, allowing it another five days of undisturbed incubation at 21 °C.

Day 10

The sample collection procedure and microorganism enumeration were repeated as described for Day 5.

Statistical Analysis

Experiment 1 was a randomized sampling of 100 home refrigerators with simple statistics of mean, standard deviation, maximum and minimum values calculated using PCSAS [22]. Experiment 2 was replicated 3 times on different days using different cultures of P. aeruginosa and A. flavus. Average microbial populations after exposure to various treatments were compared using linear contrasts of treatment means. Where significant differences were detected (P < 0.05), treatment means were separated using the pdiff statement of SAS. The data were analyzed using the general linear model procedure of SAS [21].

Results and Discussion

Experiment 1: Microbiological Population of Water in Home and Workplace Tap, Refrigerator Water and Ice

When water and ice samples were collected from household refrigerators and water lines, the average aerobic plate count (APC) recovered from water collected from refrigerator water lines was found to be nearly 20 times higher than the APC recovered from tap water supplying the refrigerator, and 3 times higher than the APC found in ice. The APC recovered from ice collected from home refrigerators was 6.5 times higher than the APC recovered from tap water (Table I). These findings agree with those reported by Hampikyan et al. [7] who reported that that water had higher counts (by 0.7 log cfu/ml) of APC than ice served at restaurants. In water distribution systems, Fleming et al. [23] estimated that 95% of the biomass in the system is attached to the surfaces while 5% is in the water. Higher flow rate water systems limit biofilm development and result in thinner layers less likely to release microbial cells into the bulk water [24]–[26]. Municipal water distribution systems have flow velocities in the range from 0.2 to 0.5 m/s [27] while home refrigerator linear flow rates are much higher in the range from 1.0–2.0 m/s depending on the water system pressure. Refrigerator water systems remain stagnant for most of the time allowing for biofilm development. Drinking water stored in polyethylene containers had greater biofilm growth in 48 h compared to containers made of galvanized steel [28] and LeChevallier et al. [29] reported higher drinking water bacterial counts in water drawn from a tap turned on after being closed overnight. This scenario mimics that of a refrigerator water system that remain off for overnight hours then is activated. The pH of the water recovered from the refrigerator water system and the tap water feeding the refrigerator did not differ (Table I). However, the pH of the ice from the refrigerator was slightly lower than tap water. This phenomena may be due to the precipitation of salts as reported by Berg & Rose [30] who reported the pH of phosphate solutions dropped after freezing due to the precipitation of dibasic salts.

Table I. Aerobic Plate Count, Yeast and Mold and pH of Refrigerator Water, Ice and Tap Water from 100 Household Refrigerators

		Tap water	Refrigerator water	Ice
Aerobic plate count (log CFU/ml)1	Mean	1.6c	2.9a	2.5b
	sd2	2.2	3.4	3.0
	Range	0–3.0	0–4.0	0–3.8
Yeast and mold (log CFU/ml)	Mean	0.9b	2.6a	2.3a
	sd	1.6	3.0	2.9
	Range	0–2.5	0–3.5	0–3.5
pH	Mean	7.28a	7.28a	7.15b
	sd	0.39	0.52	0.41
	Range	6.4–8.75	6.40–9.72	5.75–7.87

Experiment 2: Reduction of Bacterial and Fungal Contamination on Waterline Tubing

In the current study, no detectable Pseudomonas aeruginosa viable cells were detected after exposure to 0.01% peracetic acid for 2 min and 0.02% peracetic acid for 1.5 min (Table II). Peracetic acid is formed from a mixture of acetic acid and hydrogen peroxide that react to give a compound similar to acetic acid with an extra oxygen molecule [31]. Peracetic acid is a relatively safe sanitizing agent approved for food contact surfaces and as a sterilizing agent in medical facilities. It is effective in removing biofilms even when used at cold temperatures and has advantages compared to other sanitizers by not reacting with proteins to produce toxic compounds and having a low environmental impact [32]. It’s mode of action against microorganisms is as an oxidizing agent to denature microbial enzymes and membrane proteins. Peracetic acid was the most effective sanitizer in removing Listeria monocytogenes biofilms compared to chlorohexidine, sodium and a mixture of organic acids (lactic acid 30%, citric acid 3%, ascorbic acid 3%, and salts of fatty acids 7% in water [33]. Rossoni and Gaylarde [34] reported that a 0.025% concentration of peracetic acid was 90% effective in reducing adhesion of Escherichia coli and Pseudomonas fluorescens and Staphylococcus aureus by 50% [33]. Furthermore, peracetic acid is favored over some other sanitizers, such as chlorine, in the European Union because it breaks down to carbon dioxide and water over time, making it less toxic and more environmentally-friendly. Wastewater treatment with PAA have resulted in minimal toxic, mutagenic and genotoxic by-produces compared to chlorine-based disinfectants [35]. When various antimicrobials were used to reduce the number of microorganisms on the surface of refrigerator water line tubing, Pseudomonas fluorescens biofilms on polystyrene were found to be completely inhibited after exposure to 1.61% peracetic acid for 15 min [36].

Table II. Population of Pseudomonas aeruginosa Recovered from Inoculated Refrigerated Water Tubing Exposed to Various Peracetic Acid Concentrations for Different Times

Peracetic acid concentration (%)	Time (min)	Aerobic plate count (log CFU/ml) 1
		Mean	sd2	Range
0	0	7.6a	0.25	7.3–8.1
0.01	1.0	2.7b	1.86	0–4.1
0.01	1.5	1.0c	1.7	<1.0–3.0
0.01	2.0	<1.0c	–	<1.0–<1.0
0.01	3.0	<1.0c	–	<1.0–<1.0
0.02	1.0	<1.0c	1.7	<1.0–3.4
0.02	1.5	<1.0c	–	<1.0–<1.0
0.02	2.0	<1.0c	–	<1.0–<1.0
0.02	3.0	<1.0c	–	<1.0–<1.0
0.03	1.0	<1.0c	–	<1.0–<1.0
0.03	1.5	<1.0c	–	<1.0–<1.0
0.03	2.0	<1.0c	–	<1.0–<1.0
0.03	3.0	<1.0c	–	<1.0–<1.0

Experiment 3: Inoculation and Sanitation Test of Refrigerator Water Systems

Since the sanitizing solution volume would be contained in the filter, this system was a “worst-case” scenario compared to other systems in that the volume of the filter was relatively small compared to total volume of the system to be sanitized. The inoculum mixture was enumerated on TSA for Pseudomonas aeruginosa and DRBC for A. flavus with the resulting populations of 2.6 × 10⁷ and 5.5 × 10⁵, respectively.

No detectable Pseudomonas aeruginosa or Aspergillus flavus were recovered from any samples taken from the PAA treated water system after day 0, 5 or 10. However, between 2.9 and 4.6 log CFU/ml of Pseudomonas aeruginosa and 3.0 to 3.2 log cfu/ml of Aspergillus flavus were recovered in water drawn from untreated water systems (Fig. 1). There was a slight decrease in bacterial cells recovered from 4.6 to 2.9 log CFU/ml between days 1 to day 10. The mold population of water from the untreated system maintained a consistent population of around 3 logs over the 10 days of sampling. Interestingly, there was also about a 0.5 log decrease in bacterial population in between water samples 1 and 10 on each day of sampling, while the mold did not display the same trend (Fig. 1).

Fig. 1. Population of Pseudomonas aeruginosa and Aspergillus flavus recovered from an inoculated refrigerator water system after 0, 5, and 10 days without treatment with peracetic acid. Ten water samples were taken each day, with 500 L of water passed through the system between each sample. Standard deviation for Pseudomonas aeruginosa ranged from 0.82 to 1.16, standard deviation for Aspergillus flavus ranged from 0.85 to 0.93. 1,1 = day 1, sample 1; 1,2 = day 1 sample 2, etc.

Conclusions

Numerous studies have focused on the microbiological quality of ice from ice manufacturing plants and retail outlets [37], restaurants, bars, and food service [7], [38], [39], or vending machines [40]. However, these previous studies did not evaluate water and ice from home refrigerator systems and while most of the researchers recommended some level of sanitation as a solution, solutions for reducing counts were not necessarily included in the research design. The current project conducted a survey of water and ice contamination in home refrigerators to determine the level and type of microbiological contamination, and then further examined efficacy of solutions and delivery of antimicrobials treatments in commercial refrigerator water lines. Findings demonstrate that water in home refrigerator lines has higher contamination than tap water leading into the refrigerator and ice made by the refrigerator; however, microbial loads can be reduced using dilute PAA (1.61% for 15 min).