Author: James Henter
Peer Reviewer: Min-Seung Kang
Professional Reviewer: Ulrike Lorenz
Abstract
Background: Keeping school environments as clean as possible is important for student health. In this two-year study, the quantity and variety of bacteria were measured in a local Pre-K-8 school in Year 1 and in a local high school in Year 2. In both years, the effectiveness of cleaning products to kill bacteria was measured.
Methods: In Year 1, 10 surfaces across a Pre-K-8 school were swabbed twice using sterile swabs, both before students arrived and after a regular school day. Bacteria were grown in pre-plated nutrient agar petri dishes placed in a small incubator under a heat lamp. In Year 2, bacterial counts were explored by swabbing five surfaces across a high school. Using methods similar to those in Year 1, bacteria were grown on two kinds of pre-plated petri dishes: nutrient agar and blood agar. In both experiments after seven days, bacterial cultures were plated onto slides, stained for analysis, and viewed under a microscope for identification. In both years, three surfaces that had grown a large quantity and variety of bacteria were then swabbed again to test the effectiveness of nine different cleaning products. After five days, the zone of inhibition in millimeters around each cleaning product was recorded.
Results: In both Years 1 and 2, variations in colony counts were observed across all surfaces. Microscope analysis showed a wide variety of both gram-positive and gram-negative cocci, bacilli, spirilla, and fungi. In Year 2, colony counts were generally higher on blood agar than on nutrient agar. In Year 1, three of nine cleaning products tested were fully effective, meaning that they killed both gram negative and gram-positive bacteria as well as fungi across all surfaces tested. Four of the cleaning products were partially effective, meaning that they inhibited the growth of some bacterial colonies but not all of them, and two cleaning products were ineffective. In Year 2, four of the nine cleaning products tested were fully effective, four were partially effective, and one was ineffective. With few exceptions, the most effective cleaners in both years all contained quaternary ammonium compounds. Clorox bleach was also fully effective in Year 2, but not Year 1.
Conclusion: Clorox bleach and quaternary ammonium compounds were the most effective against the large quantity of both gram-positive and gram-negative bacteria observed in these two school environments. Since almost all of the cleaning products tested in this study kill viruses as well as bacteria, these findings have important implications for keeping school environments clean in general as well as during the global COVID-19 pandemic.
Introduction
Illnesses spread quickly across school populations. As a result, cleanliness is always an issue of concern for school administrators (Ridenhour et al., 2011). This study was conducted over two years, with the ultimate goal of assessing the quantity and type of bacteria in school settings and determining the effectiveness of cleaning products to kill those bacteria. Over the course of two years, this study tested 13 different cleaning products, 11 of which are supposedly effective against both viruses and bacteria (information available on the manufacturers’ websites). Thus, even though the findings in the present study only describe bacteria, they can be generalized for viruses as well. Importantly, in both Years 1 and 2, the schools provided samples of their own cleaning products to be tested alongside several household cleaners.
In Year 1, the study had two hypotheses. The first was that surfaces would have a larger quantity and variety of bacteria growing on them in the late afternoon than in the morning, reflecting that most surfaces were cleaned in the evening after students had left the building. Second, it was hypothesized that the three cleaning products provided by the school would be more effective than the six household cleaners tested, because it was assumed that the school would be using more powerful, industrial-grade products. Building on what was learned in Year 1, the Year 2 study also had two hypotheses. The first was that cleaning products containing quaternary ammonium compounds would be more effective than other regular household cleaning products specifically because they were the only cleaning products that effectively killed all bacteria in Year 1. The second hypothesis was that, across all locations swabbed, a larger quantity and variety of bacteria would grow on blood agar than on nutrient agar because blood agar is more likely to grow organisms that require the nutrients and growth factors found in blood (Drancourt et al, 2003); these blood agar-preferring organisms, in turn, are likely to be present in a typical high school environment.
Materials and Methods
School Environments
The Year 1 study was conducted in a local private Pre-K through eighth grade school with approximately 200 students. Ten different locations were swabbed with a sterile cotton swab: the water spigot in the gym water fountain; the water fountain push button in the gym; a basketball from the gym; a flush button in the girls’ bathroom; an interior boys’ bathroom door; a mouse in the computer lab; a chair in the computer lab; xylophone handles in the music room; a stairwell railing; and the microwave open button in the lunchroom. Two of the 10 surfaces (the two bathrooms) were described by the school as “high-touch” surfaces, broadly defined as surfaces that are handled many times throughout the day by various users. These were cleaned daily by school custodians. Three of the surfaces were cleaned weekly but with no set schedule (stairwell railing, water fountain spigot in gym, water fountain push button in gym). The remaining places were not regularly cleaned by custodial staff (basketball in the gym, mouse in the computer lab, chair in the computer lab, xylophone handles in the music room, and the microwave open button in the lunchroom).
In Year 2, the study was conducted in a local public high school with approximately 1,200 students. Five different locations were swabbed: a bathroom flush in a boys’ bathroom; an interior boys’ bathroom door; a stairwell railing in a communal area; a water fountain in a communal area; and a lunch table in a communal area. All five surfaces were cleaned daily by custodial staff.
Collecting Samples
In Year 1, the 10 locations were swabbed twice, first in the morning before students arrived and again in the late afternoon after students had left for the day. In Year 2, all five locations in the local high school were swabbed three different times; each swabbing date was several weeks apart. Two swabs were taken for each location, one for the blood agar and one for the nutrient agar. Swabs were taken in the late afternoon after students had left for the day.
Growing Bacteria
In Year 1, bacteria were grown in pre-plated nutrient agar petri dishes whereas in Year 2, bacteria were grown in both pre-plated nutrient and blood agar petri dishes. In both years the petri dishes were placed in a small, homemade incubator under a heat lamp, which maintained an average temperature of 87° in Year 1 and 91° in Year 2. Colony counts and notes about the physical appearance of the bacteria were recorded daily.
After seven days, bacterial cultures were plated onto slides using inoculating loops, which were sterilized with a candle. Cultures were then stained for analysis using a commercially available Gram Stain kit (Hardy Diagnostics, Santa Maria, CA). Plastic safety goggles, surgical disposable face masks, surgical gloves, and disposable lab coats were worn during this process. The resulting slides were viewed under a Celestron LCD Digital #44340 microscope (Celestron, Torrance, CA) for identification (De la Maza, 1997).
Effectiveness of Cleaning Products
In both years, the effectiveness of various cleaning products was tested for three locations that had shown the most bacterial growth in the petri dishes. In Year 1, these were the microwave open button, the basketball in the gym, and the water spigot in the gym. In Year 2, these were the bathroom flush in the boys’ bathroom, the interior boys’ bathroom door, and the water fountain in a communal area. Whatman Filter Paper circles (diameter=½ inch) dipped into the cleaning products were placed using small tongs into petri dishes containing fresh cultures from those locations. After five days, the Kirby Bauer method was used to measure the zone of inhibition around each cleaning product in millimeters.
In Year 1, nine cleaning products were tested: household Clorox bleach, diluted 1 part bleach:32 parts water; Parson’s ammonia, diluted 1:1 with water; Mr. Clean Antibacterial Multipurpose Cleaner; tea tree oil, diluted 1 part tea tree oil:12 parts water; rubbing alcohol (70% isopropyl alcohol); Lysol Antibacterial Kitchen Cleaner; Betco Rest Stop (provided by the school); Fresh Breeze-TB (provided by the school); and Purell Professional Surface Disinfectant (provided by the school). Table 1 lists the cleaning products, including those provided by the school, and their active ingredients.
In Year 2, nine different cleaning products were tested: household Clorox bleach, diluted 1 part bleach:4 parts water; white vinegar, diluted 2:1 with water; Mr. Clean Antibacterial Multipurpose Cleaner; tea tree oil, diluted 1 part tea tree oil:12 parts water; rubbing alcohol (70% isopropyl alcohol); Lysol Kitchen Pro Antibacterial Cleaner; 409 Multi-Surface Cleaner; Spartan Clean on the Go Super HDQL10 (provided by the school); and Spartan Clean on the Go by Peroxy (provided by the school) (see Table 2 for a list of these products and their active ingredients).
Results
Year 1
In Year 1, variations in bacterial colony counts were observed across surfaces not regularly cleaned by the school but with no clear pattern (range: 3 – 20 individual colonies; see Figure 1). Both areas cleaned daily (the bathrooms) had higher colony counts in the morning, before students arrived, than in the evening (Figure 2). Of the three areas cleaned weekly, only the stairwell railing was measurably cleaner in the morning than in the evening (Figure 2).
Microscope analyses showed a wide variety of both gram-positive (eight of eight places swabbed) and gram-negative (five of eight places swabbed) cocci (including diplococci, staphylococci, and streptococci), bacilli, spirilla, and fungi (see Table 3). Fungi were found on the water fountain spigot in the gym; the basketball; the flush button in the girls’ bathroom; and the stairwell railing. Interestingly, the surfaces that had gram-negative bacteria were either cleaned weekly (but not daily) by custodial staff or not cleaned at all (see Table 3).
Of the nine cleaning products tested, the three products that contained quaternary ammonium compounds were effective in all three petri dishes: Lysol Antibacterial Kitchen Cleaner (average zone of inhibition=4.7mm), Betco Rest Stop (average zone of inhibition=5.3mm), and Fresh Breeze-TB (average zone of inhibition=3.3mm) (see Table 1). Four products tested showed only partial effectiveness, meaning that they inhibited the growth of some bacterial colonies but not all of them: tea tree oil (average zone of inhibition=4.0mm), rubbing alcohol (average zone of inhibition=1.7mm), Mr. Clean (average zone of inhibition=3.0mm), and ammonia (average zone of inhibition=1.3mm). Two showed no effectiveness (Clorox bleach and Purell Professional Surface Disinfectant; the latter was a cleaning product provided by the school).
Year 2
In Year 2, variations in colony counts were observed across all surfaces (range: 2 – 392 individual colonies; see Figure 3A-3C and Table 4). Colony counts were lowest on the stairwell railing (for both nutrient and blood agar petri dishes across all three swabs) and highest for the interior bathroom door. In general, colony counts were higher on blood agar petri dishes (average colony count for three separate swabs=124.2) than on nutrient agar petri dishes (average=67). Microscope analyses showed a wide variety of both gram-positive (five of five places swabbed) and gram-negative (five of five places swabbed) cocci (including diplococci, staphylococci, and streptococci) and fungi (five of five places swabbed) (see Table 5). In addition, all five locations had gram-positive bacilli, but only one location had gram-negative bacilli. However, across the 15 separate petri dishes, gram-positive bacteria were far more common than gram-negative.
Of the nine cleaning products tested, four were effective in all six petri dishes: bleach (average zone of inhibition=7.3 mm), Lysol Kitchen Pro Antibacterial Cleaner (average zone of inhibition=7 mm), 409 Multi-Surface Cleaner (average zone of inhibition=7 mm), and Spartan Clean On the Go by Peroxy (average zone of inhibition=2.7 mm). Four products tested showed only partial effectiveness (tea tree oil, rubbing alcohol, Mr. Clean, and white vinegar), and one showed no effectiveness (Spartan Clean on the Go Super HDQL10, a product provided by the school). However, with the exception of the ineffective Spartan Clean on the Go Super HDQL10, all eight other cleaning products killed the bacteria growing on the interior bathroom door. With the exception of bleach, the most effective cleaners all contained quaternary ammonium compounds (see Table 2); however, the ineffective compound, Spartan Clean on the Go Super HDQL10, also contained quaternary ammonium compounds.
Discussion
This study, which measured the type and quantity of bacteria in two different school environments, found a large quantity and variety of both gram-positive and gram-negative bacteria and fungi across all of the school surfaces tested. In Year 2, for most surfaces tested, colony counts were far higher for the blood agar petri dishes than for the nutrient agar petri dishes, probably reflecting the ability of many common human pathogens to grow more easily on blood agar than nutrient agar (Drancourt et al, 2003). This may reflect the fact that a typical high school environment is most likely to have microorganisms that more easily infect humans. Fungi were also present on all the areas tested. The findings suggest that to reduce the number of bacteria on school surfaces, more frequent cleaning or more effective cleaning products are needed.
In terms of the overall effectiveness of cleaning products, it is important to note that only seven of the 13 different products tested over the course of this two-year study were fully effective. In Year 1, four of the cleaning products tested were partially effective (see Table 1), probably reflecting their usefulness against certain growths. For instance, tea-tree oil, an antifungal agent, was very effective on surfaces that contained mold, but ineffective for bacteria. Surprisingly, two products were completely ineffective: diluted Clorox bleach and Purell Professional Surface Disinfectant. It is possible that the Clorox bleach was too diluted, and that undiluted bleach would have been more effective. It is also possible that the Clorox bleach had expired. Notably, the Purell Professional Surface Disinfectant was provided by the school, suggesting that the school would be advised to discontinue use of this ineffective product. The three products that contained quaternary ammonium compounds were effective in all three petri dishes, suggesting that these agents were the most effective against bacteria and fungi in this school environment.
In Year 2, it was found that of the cleaning products tested, four were partially effective (see Table 2), again probably reflecting their usefulness against certain pathogens but not others. Surprisingly, one product containing quaternary ammonium compounds was completely ineffective: Spartan Clean on the Go Super HDQL10, which was provided by the school. Given that the manufacturer’s instructions for this product require extreme dilution (½ ounce of Super HDQL10 per gallon of water), and that its main ingredient (quaternary ammonium compounds) was effective in all the other products tested, it is likely that the product was too diluted. These findings suggest that the school should alter the dilution level of this product. Finally, Clorox bleach and the three other products that contained quaternary ammonium compounds were effective in all six petri dishes, suggesting that bleach and quaternary ammonium compounds were both most effective against bacteria and fungi in this school environment.
Though these preliminary results are interesting and useful, the study has several limitations. First, bacteria were grown in a homemade incubator under a heat lamp. Professional equipment would likely have given more accurate results. Second, microorganisms were identified by a high school student using a Celestron LCD digital microscope whose maximum magnification was 400x. Identification would likely have been more accurate if conducted by a more experienced microbiologist with a more powerful microscope. Furthermore, microscope analyses were only conducted once, and the effectiveness of cleaning products was only tested once. Future studies should test surfaces and the effectiveness of cleaning products multiple times to ensure the accuracy of the results. Future studies should also consider testing different dilutions of the cleaning products to see which might be most effective. Despite these limitations, the study demonstrates that significant bacterial growth occurs in school environments despite regular cleaning, and that more frequent cleaning or more effective cleaning products are needed. This study provides a simple template that schools could follow to carefully assess the effectiveness of the cleaning products they use as well as their cleaning regimen.
Though school sanitation has always been an important area of concern (Ridenhour et al., 2011), the global COVID-19 pandemic has made this issue particularly important. As students and faculty return to school, school administrations are focused on making schools as clean as possible. Since schools have limited budgets, they will want to make sure they are only buying effective products. In this study, spanning two years and two different school environments, quaternary ammonium compounds were the only similar ingredient in all of the effective cleaning products with the exception of Clorox bleach, whose active ingredient is sodium hypochlorite. While one product containing quaternary ammonium compounds was ineffective, it is likely that the product was overly diluted (Spartan Clean on the Go Super HDQL10). However, quaternary ammonium compounds are not considered environmentally-friendly, and carry a risk of toxicity that may prevent them from being used on “high-touch” areas, particularly in a school (Grillitsch et al., 2006). This may limit their use in school environments. Nevertheless, it is possible that various schools will put environmental concerns aside in order to try to make schools as safe and germ-free as possible during the COVID-19 pandemic. In the interim, diluted bleach may provide the safest and most cost-effective option.
TABLES AND FIGURES
Table 1. Size of zone of inhibition (in millimeters) for cleaning products, Year 1
| Product | Active Ingredient | Basketball | Water Spigot | Microwave |
Bleach | Sodium hypochlorite | — | — | — |
Ammonia | Ammonium hydroxide | — | 2 | 2 |
Mr. Clean | Sodium hydroxide | 5 | 4 | — |
Tea Tree Oil | terpinen-4-ol | 7 | 5 | — |
Rubbing Alcohol | 70% isopropyl alcohol | — | 1 | 4 |
Lysol Antibacterial Kitchen Cleaner | Quaternary ammonium compounds | 2 | 6 | 6 |
Betco* | Quaternary ammonium compounds | 5 | 5 | 6 |
Purell Professional Surface Disinfectant* | 29.4% Ethyl alcohol | — | — | — |
Fresh Breeze* | Quaternary ammonium compounds | 3 | 4 | 3 |
Products listed in red text were effective in all three petri dishes.
*Provided by the school
Table 2. Zone of Inhibition (in Millimeters) for Cleaning Products, Year 2
| Product | Active Ingredient | Water Fountain (Nutrient Agar) | Bathroom Flush(Blood Agar) | Interior Bathroom Door (Blood Agar) |
Bleach (diluted 1:4) | Sodium hypochlorite | 2 | 15 | 5 |
White Vinegar(diluted 2:1) | Acetic acid | — | — | 1.5 |
Mr. Clean | Sodium hydroxide | 6 | — | 3 |
Tea Tree Oil(diluted 1:12) | terpinen-4-ol | — | — | 5 |
Rubbing Alcohol | 70% isopropyl alcohol | — | 3 | 3 |
Lysol Kitchen Pro Antibacterial Cleaner | Quaternary ammonium compounds | 14 | 3 | 4 |
409 Multi-Surface Cleaner | Quaternary ammonium compounds | 1 | 18 | 2 |
*Spartan Clean on the Go Super HDQL10 | Four different quaternary ammonium compounds | — | — | — |
*Spartan Clean On the Go by Peroxy | Hydrogen peroxide; undeceth-3; C9-11 pareth-6; quaternary ammonium compounds | 3 | 1 | 4 |
Products listed in red text were effective in all three petri dishes.
*Provided by the school. Diluted according to the manufacturer’s instructions.
Table 3. Microscope Identification of Bacterial Colonies, Year 1
| Gram Positive Cocci | Gram Positive Bacilli | Gram Positive Spirilla | Gram Negative Cocci | Gram NegativeBacilli | Gram Negative Spirilla | Fungi | |||
| Bathroom Flush Button (evening) | X | X | — | — | — | — | X | ||
| Interior Bathroom Door (evening) | X | — | — | — | — | — | — | ||
| Water Fountain Spigot in Gym (evening) | X | — | — | X | — | — | X | ||
| Water Fountain Button (morning) | X | — | — | — | — | — | — | ||
| Stairwell Railing (morning) | X | — | — | X | — | X | X | ||
| Microwave Open Button(morning) | X | X | — | — | — | — | — | ||
| Basketball in Gym(morning) | X | — | X | — | — | — | — | ||
| Chair in Computer Lab (morning) | X | — | — | X | X | — | — | ||
| Computer Lab Mouse (evening) | X | — | — | X | — | — | — | ||
| Basketball in Gym(evening) | X | X | — | X | — | — | X |
Orange text: cleaned daily by custodial staff; Green text: cleaned weekly by custodial staff; Blue text: not cleaned regularly by custodial staff
Table 4. Colony Counts from Three Separate Swabs, Year 2
| Location | Average Colony Count | Range |
| Bathroom Flush(nutrient agar)* | 3 | 2 – 4 |
| Bathroom Flush(blood agar) | 153 | 89 – 281 |
| Interior Bathroom door(nutrient agar) | 48 | 24 – 95 |
| Interior Bathroom door(blood agar) | 303 | 171 – 392 |
| Stairwell Railing(nutrient agar) | 9 | 3 – 13 |
| Stairwell Railing(blood agar) | 22 | 15 – 34 |
| Water Fountain(nutrient agar)* | 195 | 179 – 211 |
| Water Fountain(blood agar) | 126 | 2 – 211 |
| Lunchroom Table(nutrient agar) | 80 | 56 – 112 |
| Lunchroom Table(blood agar) | 17 | 2 – 32 |
*average of two swabs rather than three, due to faulty petri dishes
Table 5. Microscope Identification of Bacterial Colonies, Year 2
Figure 1. Colony Counts for Surfaces Not Regularly Cleaned by Custodial Staff, Year 1
Figure 2. Colony Counts for Surfaces Cleaned Daily or Weekly, Year 1
Figure 3. Colony Counts for School Surfaces, Year 2
References
De la Maza LM, Pezzlo MT, Baron EJ, Eds. (1997). Color Atlas of Diagnostic Microbiology. Mosby Press, MO.
Drancourt M, Carrieri P, Gévaudan M-J, Raoult D (2003). Blood Agar and Mycobacterium Tuberculosis: the End of a Dogma. J Clin Microbiol 41: 1710-1711.
Grillitsch B, Gans O, Kreuzinger N, Scharf S, Uhl M, Fuerhacker M (2006). Environmental Risk Assessment for Quaternery Ammonium Compounds: a Case Study from Austria. Water Sci Technol 54: 111-18.
Ridenhour BJ, Braun A, Teyrasse T, Goldsman D (2011). Controlling the Spread of Disease in Schools. PLoS One 6: e29640.
