Refereed Manuscript

Proc. Fla. State Hort. Soc. 125:340–342. 2012.

The Use of Petrifilms to Quantify Aerobic Bacteria in Irrigation Water

Dustin P. Meador1, Paul R. Fisher*1, Max Teplitski2 1University of Florida, IFAS, Environmental Horticulture Department, 1533 Fifield Hall, Gainesville, FL 32611 2University of Florida, IFAS, Soil and Water Sciences, 1376 Mowry Road, Gainesville, FL 32611

Additional index words. algae, disinfestation, heterotrophic, plate count, sanitation, sanitizer, water quality, water treatment A protocol was developed using Petrifilms™ to quantify the population densities of aerobic bacteria in irrigation water as an onsite monitoring technique. To validate this method, quantification of colony-forming units (CFU/mL) of aerobic bacteria in recirculated irrigation water was compared between Petrifilm and standard culture substrate, with 18 subsamples of 300 mL and 9 log dilutions. A Petrifilm is a dehydrated medium containing guar, xanthan gums, nutrients, and triphenyl tetrazolium chloride on a card with a plastic cover. The comparison method used the APPHA Standard Methods (#9215C.6c) for Heterotrophic Plate Count, using Spread Plate Method with R2A substrate. The substrate (Petrifilm or R2A), day (3 or 7), and their interaction significantly affected bacteria CFU/mL. Estimated CFU/mL (×104) using the Petrifilms increased from 50.1 on measurement day 3 to 83.1 at day 7, whereas use of the R2A substrate resulted in higher values of 65.9 at day 3 to 146.2 at day 7 (standard error = 4.0). Comparison of the estimated CFU/mL from Petrifilm to R2A required a conversion factor of 130%, 172%, or 292% when comparing counts from day 3 for both substrates, day 7 for both substrates, or day 7 R2A/day 3 Petrifilm, respectively. The results validate use of Petrifilm as a substrate for quantifying bacterial density in irrigation water, although a calibration curve is needed to interpret the results.

An onsite method to monitor microbial density in irrigation Total CFU/mL of aerobic bacteria tested at points along the water would assist management of water quality and treatment in irrigation system and immediately before and after treatment (for the horticulture industry. Population density of aerobic bacteria example, chlorine injection) can serve as a non-specific indica- (expressed as colony forming units per milliliter of the original tor of disinfection efficacy (APPHA, 1995; Maier et al., 2009). sample, CFU/mL) in irrigation water can be monitored to deter- A 2 to 3 log reduction of bacterial CFU/mL from pre-treatment mine the risk of clogged irrigation lines from biofilm (Bucks et al., to post-treatment indicates acceptable treatment reliability for 1979). Biofilms are multicellular communities of microorganisms, disinfection (USEPA, 1999). Quantification of aerobic bacteria and may include bacteria, fungi and algae as well extracellular may be more easily accomplished as an onsite test for growers polymers produced and excreted by the organisms within the than quantification of plant pathogens in irrigation water, because biofilm. Any wet surface or an interface between air and a highly pathogen numbers are often low compared with beneficial or eutrophic (nutrient-rich liquid) can host biofilms (Cullimore, benign micro-organisms, and quantification of aerobic bacteria 1993). Biofilms are common inside water storage tanks and ir- does not require a specialized assay. rigation lines (Ravina et al., 1997), where they can reduce water Petrifilm™ (3M, Saint Paul, MN) is a plating technology flow, corrode metals, reduce efficacy of sanitation agents, and with a ready-made, dehydrated culture medium with nutrients serve as sources of pathogens. Biofilms are an important part of and tetrazolium salt that fluoresces living bacteria colonies on a microbial lifecycle and they can serve as both a sink and source flat card with a laminate cover slip. Petrifilm is widely used in of microbes, depending on the environmental conditions (Cul- food and beverage processing to determine CFU/mL of aerobic limore, 1993). Monitoring and controlling biofilms, therefore, is bacteria, yeasts, and molds, and sometimes pathogens such as important in assessing the quality of irrigation water. At present, pathogenic Escherichia coli or Salmonella spp. depending on growers can send irrigation water samples to a laboratory, where the type of Petrifilm. The Petrifilm technology has been used to standard methods are available for bacterial analysis under asep- evaluate sanitation processes such as chlorination in post-harvest tic conditions (Darbie et al., 2006). However, an onsite test for handling (Materon, 2003), to identify contaminant sources and aerobic bacteria CFU/mL could provide rapid assessment with a testing water treatment systems in control of human pathogens recommended threshold <10,000 CFU/mL for acceptable risk of (Riordan et al., 2001). biofilm clogging of drip and mist emitters (Rogers et al., 2003). Many standardized industrial processes are monitored with Petrifilm and are recognized as a proven technology for food safety by the USDA, USEPA and international regulatory agen- cies. The research objective was to validate the use of Petrifilm to We thank the USDA-ARS Floriculture and Nursery Research Initiative, and accurately quantify total aerobic bacteria in horticulture irrigation industry partners of the Young Plant Research Center (floriculturealliance.org) and Water Education Alliance for Horticulture (watereducationalliance.org) for water compared with a standard culture substrate. Subirrigation supporting this research. return water from a Florida nursery was plated in a dilution series *Corresponding author; phone: (352) 273-4570; email: [email protected] with Petrifilm and R2A agar substrates.

340 Proc. Fla. State Hort. Soc. 125: 2012. Materials and Methods Table 1. Ingredients for the two culture substrates. Ingredients R2A agar Petrifilm Samples were collected from a subirrigation recovery tank in Agar 15% (agarose) 20% (guar) a large-scale commercial greenhouse in Apopka, FL. On 4 Nov. Glucose (anhydrous) 0.05% 2011 a total of 18 irrigation samples were randomly collected Soluble starch 0.05% at 15-min intervals. Samples were collected over a 6-h monitor- Peptone, yeast extract 0.05% 0.02% ing period from one subirrigation return tank, during multiple Peptone, casein flooding events. Water quality measurements [pH, electrical hydrolysate 0.5% 0.50% to 1.5% conductivity, dissolved oxygen, and temperature (°C)] were Peptone (other) 0.5% from beef 0.5% from soybean stable during collection (data not shown). Samples (300 mL) Magnesium sulfate 0.05% 0.05% were collected into sterile 500 mL Whirl-pak bags® (Nasco, Potassium chloride 0.05% Ocala, FL) and maintained in temperature-controlled vessels at Potassium (sulfate, 22 to 25 °C, similar to the initial sample temperature. Samples phosphate) 0.3% 0.1% were immediately transferred to the laboratory at UF and placed Sodium alginate 0.5% to 4% into culture within 6-h of collection. Sodium pyruvate 0.3% <0.2% The Reasoner’s 2A medium (R2A) (Remel Labs, Lenexa, KS) was prepared by dissolving 15.2 g of the dehydrated mix into 1 L of purified boiled water and adjusted to pH 7.2 at 25 °C, autoclaved and cooled to 45 to 50 °C in a water bath prior to pouring into sterile 50-mm-diameter petri dishes (Fisherbrand Table 2. ANOVA effects. Effect of substrate (Petrifilm or R2A) and measurement day (3 or 7) on bacteria CFU/mL. Substrate, day, and #09-720-500 (Table 1). Petrifilm (Table 1) preparation followed their interaction were significant atP < 0.001. Letters show Tukey’s manufacturer’s instructions (3M, Saint Paul, MN.). Total aerobic HSD comparison at the P = 0.05 level for the substrate × day least- bacteria for nursery water samples were quantified using 990.12, square means. Data were log-transformed for ANOVA analysis, and Petrifilm Aerobic Count Plate method (AOAC, 1999) or R2A back-transformed for presentation in this table. as described by method 988.18, Aerobic Plate Count Method Day 3 Day 7 Least square (AOAC, 1999). The sample was distributed over the substrate (CFU/mL × 104) (CFU/mL × 104) mean using the Spread Plate Method 9215 C (APPHA, 1995). Samples R2A 65.9 B 146.2 D 98.1 were mixed with a vortex shaker and serial diluted from 100 to Petrifilm 50.1 A 83.1 C 64.5 10–8, with sterile saline/peptone diluent, and two replicates per Least square mean 57.4 110.2 dilution were used. Cultures were transferred using aseptic pro- Std. error 2.9 2.8 cedures (APPHA, 1995). Plates were incubated in darkness ranging from 25 to 27 °C in a growth chamber at 70% RH. Colonies were counted on both media at day 3 and 7, to satisfy the recommended incubation pe- on day 7 using least-square means in Table 2. Based on standard riod for Petrifilm and R2A agar, respectively. The paired samples recommendations for incubation periods [3 d for Petrifilm, and 7 of Petrifilm were compared using ANOVA in PROC GLM using d for R2A (AOAC, 1999)], the conversion factor between R2A log-transformed data, with mean comparisons using Tukey’s HSD, and Petrifilm would be 146.2 / 50.1 = 2.92. and linear regression with PROC REG in SAS. Conclusion Results and Discussion This study confirmed that Petrifilm can be used to quantify There were significant effects of substrate type, P( < 0.001), aerobic bacteria colonies in irrigation water to estimate its mi- day (P < 0.001), and their interaction (P < 0.01) on bacteria CFU/ crobiological quality. However, given the effect of substrate and mL (with least-square means shown in Table 2). Bacteria CFU/ measurement day on bacteria CFU/mL, a comparison of counts mL increased over time. The R2A substrate demonstrated overall from Petrifilm to recommended guidelines on R2A required cali- higher bacterial colony counts than Petrifilm. A comparison based bration. Based on manufacturer’s suggested incubation time, the on standard recommendations for incubation periods (3 d for counts on Petrifilm after 3 d can be used to estimate the counts Petrifilm, and 7 d for R2A (AOAC, 1999) showed that averaged on R2A after 7 d. Further validation with a known bacterium or counts on Petrifilm were lower for most sample times (Table multiple irrigation sources would be helpful to evaluate whether 2). For all methods and measurements, the bacterial CFU/mL differences in counts between the substrates is consistent for all greatly exceeded the recommended threshold of 10,000 CFU/ microorganisms of concern. Differences in CFU/mL between mL (Rogers et al., 2003). Both methods indicated a high risk of substrates may have resulted from different growth response of biofilm formation. aerobic bacteria species to the nutrient formulations and physical Conversion between bacteria CFU/mL estimates from the two environment. One limitation of the Petrifilm is its lack of selectiv- substrates would depend on the measurement day, based on the ity on specific plant pathogens. This technique as a non-specific significant interaction between substrate and measurement day. measurement of aerobic bacteria is best suited for quantifying To convert bacteria CFU/mL from Petrifilm on a given number microbial load, estimating a potential for biofilm formation and of days of incubation to the bacteria CFU/mL estimated with relative efficacy of water treatment systems along points in an R2A, the Petrifilm bacteria count CFU/mL (× 104) could be irrigation line, rather than to identify presence or absence of a multiplied by 65.9 / 50.1 = 1.3 for day 3, or 146.2 / 83.1 = 1.79 specific plant pathogen.

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