Developing a Performance Standard for Adequate Sanitization of Wire‑Bar Lids

Abstract

The wire-bar lids on rodent cages are an integral part of the microenvironment and as such can impact rodent health and wellbeing. The Guide for the Care and Use of Laboratory Animals recommends changing wire-bar lids every other week but does not include a predetermined performance standard. To develop a sanitization performance standard, we evaluated the bacterial and other cellular burden of wire-bar lids over 4 wk. The results show no significant difference in ATP or bacterial burden over 3 wk of continuous use in conventional cages with standard rodent pelleted or high-fat diet or in IVC with an irradiated diet.

Both the 7th and 8th editions of the Guide for the Care and Use of Laboratory Animals state that “In general, enclosures and accessories, such as tops, should be sanitized at least once every two weeks,” with an acknowledgment that different cage types or housing conditions may alter the frequency of cleaning.^3,4 However, neither edition describes a performance standard nor are such standards available in the literature for laboratory animal housing. A prior study found the degree of contamination in both mouse IVC and conventional filter-topped cages did not differ between 14 and 90 d when using corncob bedding and when nonsterilized feed was added to the feed hopper weekly.⁸ To compare our housing conditions with those in the previous study, we conducted a pilot study to evaluate any difference between changing wire-bar lids (WBL) every 2 wk compared with 4 wk for mice housed in conventional cages.⁷ The results suggested that a difference was unlikely, but the study lacked power. We therefore expanded our study to achieve sufficient statistical power to evaluate sanitization in IVC and measure the occurrence of microbes.

Sanitization consisting of cleaning and disinfection, is an important component of the animal program in that it provides an environment free of organic debris and with an acceptable concentration of microbes. Disinfection reduces the concentration of microbes through either a microbiocidal mechanism, such as detergent or heat, and may provide an environment that is suboptimal for microbial growth. For appropriate disinfection or sterilization, the surfaces must be cleaned to remove organic debris from the animals and the environment. This organic debris can consist of residual compounds or cleaning supplies, microbes, epithelial cells, urine, feces, blood, or saliva. A clean and disinfected environment is key to supporting reproducibility of studies by eliminating cage level variables.

Evaluation of the efficacy of the sanitization program can be measured by “microbiologic culture or the use of organic material detection systems (e.g., ATP bioluminescence), and/or by confirming the removal of artificial soil applied to equipment surfaces before washing.”⁴ ATP is an energy molecule found in all living cells and can be used as an indicator of bacterial and other cellular burdens to measure the hygienic effect in a specified area. Organic debris such as food residue, urine, and feces may contains small amounts of ATP.¹³ The amount of ATP in a sample can be determined by using a luminometer to measure the emission of light from the reaction of ATP with the luciferase enzyme and expressed as relative light units (RLU). Bacteria and other microbial contamination on surfaces can be measured by the use of agar contact plates. Currently ATP concentration standards are available for the food industry and hospital infection control. These studies suggest that a well-cleaned surface results in fewer than 250 RLU and possibly as low as 100 RLU and that a poorly cleaned surface can generate more than 1000 RLU.^1,5,6,9 Two studies have suggested 1000 RLU as a reasonable cutoff value in a laboratory animal facility.^2,8 The inherent differences between human hospitals and laboratory animal facilities suggests that developing a performance standard for sanitizing laboratory animal caging is critical to ensuring a robust animal care and use program.

Materials and Methods

Animals.

C57BL/6 background mice (28 male, 103 female) were either obtained from a commercial vendor (32 female C57Bl/6JOlaHsd; Harlan, Horst, Netherlands) or bred inhouse. Mice were housed either singly or in groups of as many as 5 mice per cage on the basis of preliminary data that showed that bacterial burden and RLU counts did not differ significantly due to sex or number of mice per cage.⁷ All mice were free from mouse viral pathogens, ecto- and endoparasites, and Pneumocystis and Helicobacter spp. Mouse age varied between 11 and 40 wk, and all mice were allowed to acclimate for at least 1 wk prior to beginning experimental studies or a nonstandard diet. Cage density was in accordance with the 8th edition of the Guide and Swedish regulations.^4,10 All animals had free access to food and water and were on protocols approved by the regional animal welfare committee in accordance with Swedish regulatory requirements.¹⁰

Animal husbandry.

Mice were housed in conventional cages or IVC (Macrolon Eurostandard type 3.0, Tecniplast, Buguggiate, Italy) in AAALAC-accredited facilities. Cages contained hardwood bedding (J Rettenmaier and Sönhe, Rosenburg, Germany) with nesting material (Ancare, Bellmore, WA), shredded paper (Papyrus, Göteborg, Sweden), gnawing sticks (Tapvei, Paekna, Estonia), and a shelter (LBS, Horley, United Kingdom). Conventional cages were kept on a rack with WBL without a filter top and not autoclaved prior to use; IVC were closed by using a lid and autoclaved prior to use. Mice in conventional cages received either nonsterile standard diet (R70, Lantmännen Lantbruk, Kimstad, Sweden) or a nonsterile high-fat diet (D12492, Research Diets, New Brunswick, NJ). Mice in IVC received an irradiated diet (R36, Lantmännen Lantbruk, Kimstad, Sweden). Standard diet in the conventional and IVC cages was added to the feed hopper at the time of housing, with additional food added as needed weekly. High-fat diet was changed completely each week, with all old diet discarded. Water was provided by using a water bottle, which was changed weekly. Conventional cage bottoms containing the bedding and enrichment were changed weekly (female mice) or every third week (singly housed male mice). IVC were changed every other week. All cage changes were performed in laminar air-flow cabinets.

Animal holding room temperatures were maintained between 20 and 23 °C, with relative humidity between 40% and 60%, 20 air changes hourly, and a 12:12-h light:dark cycle. All facility personnel wore dedicated shoes, socks, and scrubs and donned respiratory protection when working outside of the laminar air-flow cabinet.

Sanitization assessment.

Twenty cages from each of the 3 housing conditions (IVC, conventional cage with R70 diet, and conventional cage with high-fat diet) were sampled weekly for 4 wk for ATP concentration and presence of aerobic bacteria and molds. The initial (day 0) test occurred when the cages received clean WBL. Measurements were made from a variety of cages (n = 60) containing male or female and group- or single-housed mice.

The SystemSURE Plus Luminometer (Hygiena, Watford, United Kingdom), sterile premoistened Ultrasnap swabs, and test tubes (Medical Packaging Corporation, Camarillo, CA) was used to assess ATP concentration (RLU count). A swab was used to sample the test area, placed in a test tube, and inserted into the luminometer. All ATP tests were done on the horizontal areas inside the feed hopper, in contact with food. Each WBL was swabbed in 2 locations (Figure 1) for each test, with a total of 6 sites for the 3 assessments.

Figure 1.

Typical wire bar lid, with the sampling areas for (A) yeast and fungi (Y and F), (B) aerobic bacterial culture (TPC), and (C) ATP measurement indicated.

Microbial samples were taken using Hygicult Total Plate Count (TPC) and Yeast and Fungi (Y and F) slides (Orion Diagnostica Oy, Espoo, Finland). The TPC slides were pressed against the left side wall within the feed and on the wire bars of the feed hopper, inside the cage. The Y and F slides were pressed against the right side wall within the feed and at the bottom of the feed hopper, inside the cage (Figure 1). The sample-containing TPC slides were incubated at 36 °C for 1 d, and the Y and F slides were incubated at 28 °C for 4 d.

Activity and ergonomic assessments.

Time studies of 4 staff recorded the time required for handling WBL to determine an average time associated with each task. The assessment of the workplace ergonomics surrounding the tasks was conducted by using the European Union-endorsed KIM1 guidance from the Swedish Work Environment Authority.^11,12

Statistical analysis.

The numbers of CFU and RLU (mean ± SEM) corresponding to each time point were evaluated by using one-way ANOVA (Analyse-it Software, Leeds, United Kingdom). A P value less than 0.05 was considered statistically significant.

Results

ATP analysis.

Although variable in all 3 groups, the increase in RLU count did not differ significantly over time in either the mouse contact zone or the noncontact side of the WBL. The noncontact zone had lower RLU values than the corresponding contact zone at all sample time points (data not shown). The RLU average was less than 100 RLU at all time points for both IVC and static cages with high-fat diet, whereas the average RLU for the conventional cages with standard feed remained less than 250 RLU at all time points (Figure 2).

Figure 2.

Relative light unit (RLU) counts (mean ± SEM) over time for static cages with nonsterile feed (black bars), IVC with irradiated feed (gray bars), and conventional cages with irradiated high-fat diet (white bars).

Microbial growth analysis.

The majority of cages showed no fungal growth; 5 high-fat diet cages yielded sparse growth during week 4. This parameter was not evaluated statistically due to the inability to quantify fungal growth. The bacterial contamination analysis of the nonmouse contact area remained low throughout the study and did not differ significantly over time (Figure 3 B). In fact, microbial analysis of the nonmouse contact area did not differ among the 3 groups at any time point, suggesting that the key variable leading to increased bacterial counts was contact with mice. Interestingly, no bacteria were cultured from the noncontact area on week 1 but bacteria were cultured prior to the addition of food on week 0. The microbial counts of the mouse contact area differed significantly (P < 0.05) between weeks 0 and 1 but did not differ from week 1 to week 4 (Figure 3 A).

Figure 3.

CFU counts (mean ± SEM) over time for conventional cages with nonsterile feed (black bars), IVC with irradiated feed (gray bars), and conventional cages with irradiated high-fat diet (white bars) from the (A) mouse contact area and (B) noncontact area.

Labor assessment.

The timed activity study (Figure 4) demonstrated that for every 100 WBL, only 4 min (3% of total handling) was devoted to transporting racks to and from the cage wash, whereas 20 min (14%) was required for loading and unloading the WBL into the washing racks and placing them into the cagewash machine. The remaining 83% of the time was associated with changing the WBL—approximately 1 min per cage to replace the prior WBL with a clean one filled with food and to add a new water bottle; 124 min, approximately 25% of a technician's daily work time, was required to change an average room. The time associated with decreasing the change frequency from every 2 wk to every 4 wk for a facility with 2000 mouse cages saves 48 h each month or approximately 30% of a technician's monthly duty time.

Figure 4.

Time (min) associated with changing 100 wire-bar lids (WBL).

The ergonomic risk assessment demonstrated that the tasks associated with WBL represent a physical overload. A typical WBL containing food and water weighs 2.2 kg, whereas a complete conventional cage weighs an additional 1.1 kg. While changing the 100 WBL in a typical animal room in our facility, staff move more than 930 kg of cages and accessories. An IVC weighs 4.2 kg, increasing the total weight moved during the changing of 100 IVC WBL to 1100 kg. The repeated twisting and lifting of these weights rank the full task of cage change as a physical overload.¹¹

Discussion

The daily husbandry tasks associated with maintaining cage microenvironments require a significant investment of personnel time and energy resources for washing the cages and accessories. The changing of WBL in a typical room of 100 cages might comprise 25% of an average technician's working time. Dramatic savings in personnel time and operating costs can be realized if the time between cleanings can be lengthened without adversely affecting the animals’ health. A prior study suggested that decreasing the cage-change frequency while maintaining low RLU values did not reduce animal wellbeing.² Standards for cleanliness in laboratory animal housing are currently unavailable despite their availability for hospital sanitization. These hospital standards were developed initially in regard to bacterial burdens to prevent nosocomial infections but have been refined to assess organic and bacterial burdens measured as ATP concentration and expressed as RLU.^1,5

The average RLU counts in the current study fall below the 250-RLU cutoff recommended as a sanitization standard, and the averages for IVC were below the increased stringency of 100 RLU. Determining RLU counts is a rapid measure of replicating microbes as well as urine, feces, residual disinfectant, and cellular debris.^9,13 To characterize the component of the RLU value from bacteria, we conducted bacterial cultures concurrent with RLU analysis. Following the same trend as the RLU, the culturable bacterial burden increased over time but remained low and did not differ between wk 1 through 4. Interestingly, the increased handling due to changing the high-fat diet weekly did not significantly increase either RLU or CFU counts, suggesting that appropriate aseptic technique minimizes contamination of the WBL. In addition, the major contributor to increased counts are the animals within the cage rather than handling by technicians or room-level contamination, given that the values did not differ significantly between nonrodent contact areas in the closed IVC and open static cages. Mold growth occurred only at the final testing point and in a limited number of cages containing the high-fat diet. This result was most likely due to accumulation of diet residue.

Our study demonstrates that neither bacterial growth nor ATP concentration differed significantly between 1 and 4 wk of use by mice. This finding suggests that changing the WBL every 4 wk (instead of 2) will minimally affect mouse wellbeing. This trend was seen with several mouse housing schemes: nonsterile feed in conventional cages, irradiated feed in IVC, and irradiated high-fat diet in conventional cages.