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
University of Florida, IFAS, Environmental Horticulture Department, 1533 Fifield Hall,
Gainesville, FL 32611
1
University of Florida, IFAS, Soil and Water Sciences, 1376 Mowry Road, Gainesville, FL 32611
2
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
water would assist management of water quality and treatment in
the horticulture industry. Population density of aerobic bacteria
(expressed as colony forming units per milliliter of the original
sample, CFU/mL) in irrigation water can be monitored to determine the risk of clogged irrigation lines from biofilm (Bucks et al.,
1979). Biofilms are multicellular communities of microorganisms,
and may include bacteria, fungi and algae as well extracellular
polymers produced and excreted by the organisms within the
biofilm. Any wet surface or an interface between air and a highly
eutrophic (nutrient-rich liquid) can host biofilms (Cullimore,
1993). Biofilms are common inside water storage tanks and irrigation lines (Ravina et al., 1997), where they can reduce water
flow, corrode metals, reduce efficacy of sanitation agents, and
serve as sources of pathogens. Biofilms are an important part of
microbial lifecycle and they can serve as both a sink and source
of microbes, depending on the environmental conditions (Cullimore, 1993). Monitoring and controlling biofilms, therefore, is
important in assessing the quality of irrigation water. At present,
growers can send irrigation water samples to a laboratory, where
standard methods are available for bacterial analysis under aseptic conditions (Darbie et al., 2006). However, an onsite test for
aerobic bacteria CFU/mL could provide rapid assessment with a
recommended threshold <10,000 CFU/mL for acceptable risk of
biofilm clogging of drip and mist emitters (Rogers et al., 2003).
We thank the USDA-ARS Floriculture and Nursery Research Initiative, and
industry partners of the Young Plant Research Center (floriculturealliance.org)
and Water Education Alliance for Horticulture (watereducationalliance.org) for
supporting this research.
*Corresponding author; phone: (352) 273-4570; email: [email protected]
340
Total CFU/mL of aerobic bacteria tested at points along the
irrigation system and immediately before and after treatment (for
example, chlorine injection) can serve as a non-specific indicator of disinfection efficacy (APPHA, 1995; Maier et al., 2009).
A 2 to 3 log reduction of bacterial CFU/mL from pre-treatment
to post-treatment indicates acceptable treatment reliability for
disinfection (USEPA, 1999). Quantification of aerobic bacteria
may be more easily accomplished as an onsite test for growers
than quantification of plant pathogens in irrigation water, because
pathogen numbers are often low compared with beneficial or
benign micro-organisms, and quantification of aerobic bacteria
does not require a specialized assay.
Petrifilm™ (3M, Saint Paul, MN) is a plating technology
with a ready-made, dehydrated culture medium with nutrients
and tetrazolium salt that fluoresces living bacteria colonies on a
flat card with a laminate cover slip. Petrifilm is widely used in
food and beverage processing to determine CFU/mL of aerobic
bacteria, yeasts, and molds, and sometimes pathogens such as
pathogenic Escherichia coli or Salmonella spp. depending on
the type of Petrifilm. The Petrifilm technology has been used to
evaluate sanitation processes such as chlorination in post-harvest
handling (Materon, 2003), to identify contaminant sources and
testing water treatment systems in control of human pathogens
(Riordan et al., 2001).
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 agencies. The research objective was to validate the use of Petrifilm to
accurately quantify total aerobic bacteria in horticulture irrigation
water compared with a standard culture substrate. Subirrigation
return water from a Florida nursery was plated in a dilution series
with Petrifilm and R2A agar substrates.
Proc. Fla. State Hort. Soc. 125: 2012.
Materials and Methods
Samples were collected from a subirrigation recovery tank in
a large-scale commercial greenhouse in Apopka, FL. On 4 Nov.
2011 a total of 18 irrigation samples were randomly collected
at 15-min intervals. Samples were collected over a 6-h monitoring period from one subirrigation return tank, during multiple
flooding events. Water quality measurements [pH, electrical
conductivity, dissolved oxygen, and temperature (°C)] were
stable during collection (data not shown). Samples (300 mL)
were collected into sterile 500 mL Whirl-pak bags® (Nasco,
Ocala, FL) and maintained in temperature-controlled vessels at
22 to 25 °C, similar to the initial sample temperature. Samples
were immediately transferred to the laboratory at UF and placed
into culture within 6-h of collection.
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
#09-720-500 (Table 1). Petrifilm (Table 1) preparation followed
manufacturer’s instructions (3M, Saint Paul, MN.). Total aerobic
bacteria for nursery water samples were quantified using 990.12,
Petrifilm Aerobic Count Plate method (AOAC, 1999) or R2A
as described by method 988.18, Aerobic Plate Count Method
(AOAC, 1999). The sample was distributed over the substrate
using the Spread Plate Method 9215 C (APPHA, 1995). Samples
were mixed with a vortex shaker and serial diluted from 100 to
10–8, with sterile saline/peptone diluent, and two replicates per
dilution were used. Cultures were transferred using aseptic procedures (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 period for Petrifilm and R2A agar, respectively. The paired samples
of Petrifilm were compared using ANOVA in PROC GLM using
log-transformed data, with mean comparisons using Tukey’s HSD,
and linear regression with PROC REG in SAS.
Results and Discussion
There were significant effects of substrate type, (P < 0.001),
day (P < 0.001), and their interaction (P < 0.01) on bacteria CFU/
mL (with least-square means shown in Table 2). Bacteria CFU/
mL increased over time. The R2A substrate demonstrated overall
higher bacterial colony counts than Petrifilm. A comparison based
on standard recommendations for incubation periods (3 d for
Petrifilm, and 7 d for R2A (AOAC, 1999) showed that averaged
counts on Petrifilm were lower for most sample times (Table
2). For all methods and measurements, the bacterial CFU/mL
greatly exceeded the recommended threshold of 10,000 CFU/
mL (Rogers et al., 2003). Both methods indicated a high risk of
biofilm formation.
Conversion between bacteria CFU/mL estimates from the two
substrates would depend on the measurement day, based on the
significant interaction between substrate and measurement day.
To convert bacteria CFU/mL from Petrifilm on a given number
of days of incubation to the bacteria CFU/mL estimated with
R2A, the Petrifilm bacteria count CFU/mL (× 104) could be
multiplied by 65.9 / 50.1 = 1.3 for day 3, or 146.2 / 83.1 = 1.79
Proc. Fla. State Hort. Soc. 125: 2012.
Table 1. Ingredients for the two culture substrates.
Ingredients
R2A agar
Petrifilm
Agar
15% (agarose)
20% (guar)
Glucose (anhydrous)
0.05%
Soluble starch
0.05%
Peptone, yeast extract
0.05%
0.02%
Peptone, casein
hydrolysate
0.5%
0.50% to 1.5%
Peptone (other)
0.5% from beef
0.5% from soybean
Magnesium sulfate
0.05%
0.05%
Potassium chloride
0.05%
Potassium (sulfate,
phosphate)
0.3%
0.1%
Sodium alginate
0.5% to 4%
Sodium pyruvate 0.3%
<0.2%
Table 2. ANOVA effects. Effect of substrate (Petrifilm or R2A) and
measurement day (3 or 7) on bacteria CFU/mL. Substrate, day, and
their interaction were significant at P < 0.001. Letters show Tukey’s
HSD comparison at the P = 0.05 level for the substrate × day leastsquare means. Data were log-transformed for ANOVA analysis, and
back-transformed for presentation in this table.
Day 3
Day 7
Least square
(CFU/mL × 104) (CFU/mL × 104)
mean
R2A
65.9 B
146.2 D
98.1
Petrifilm 50.1 A
83.1 C
64.5
Least square mean
57.4
110.2
Std. error
2.9
2.8
on day 7 using least-square means in Table 2. Based on standard
recommendations for incubation periods [3 d for Petrifilm, and 7
d for R2A (AOAC, 1999)], the conversion factor between R2A
and Petrifilm would be 146.2 / 50.1 = 2.92.
Conclusion
This study confirmed that Petrifilm can be used to quantify
aerobic bacteria colonies in irrigation water to estimate its microbiological quality. However, given the effect of substrate and
measurement day on bacteria CFU/mL, a comparison of counts
from Petrifilm to recommended guidelines on R2A required calibration. Based on manufacturer’s suggested incubation time, the
counts on Petrifilm after 3 d can be used to estimate the counts
on R2A after 7 d. Further validation with a known bacterium or
multiple irrigation sources would be helpful to evaluate whether
differences in counts between the substrates is consistent for all
microorganisms of concern. Differences in CFU/mL between
substrates may have resulted from different growth response of
aerobic bacteria species to the nutrient formulations and physical
environment. One limitation of the Petrifilm is its lack of selectivity on specific plant pathogens. This technique as a non-specific
measurement of aerobic bacteria is best suited for quantifying
microbial load, estimating a potential for biofilm formation and
relative efficacy of water treatment systems along points in an
irrigation line, rather than to identify presence or absence of a
specific plant pathogen.
341
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