ATP Study Report of Modified Method 1601 (Fast Phage) for Somatic and 

Male-Specific Coliphage 

ATP Case Number- 09-0007 

August 22, 2012 

TECHNICAL SUPPORT CENTER 

OFFICE OF GROUND WATER AND DRINKING WATER 

U. S. ENVIRONMENTAL PROTECTION AGENCY 

CINCINNATI, OHIO 45268 

 
Background 

Introduction: 

The information and data supplied here were generated to demonstrate the comparability of Fast 

Phage (modified Method 1601) to reference Method 1601, a US Environmental Protection 

Agency (USEPA) approved method for compliance monitoring of somatic and male-specific 

coliphage detection in groundwater and other waters. Coliphages are fecal indicator options 

specified in the 2006 Ground Water Rule (GWR) Regulations. 

Original Method 1601 is USEPA approved for compliance monitoring of public groundwater 

systems as required by the GWR. This document describes the Alternative Test Procedure (ATP) 

study design and data that were used in the decision process for evaluation of Fast Phage. 

Fast Phage is manufactured by Charm Sciences, Inc. (CSI). Data for this study report were 

generated in accordance with the CSI study plan of October 15, 2010. The study plan was 

approved by the USEPA Office of Ground Water and Drinking Water's Technical Support 

Center as compliant to the performance-based method system (PBMS) outlined for method 

modifications in Method 1601. In addition, the study was compliant with guidance issued in 

USEPA Microbiological Alternate Test Procedure Protocol for Drinking Water, Ambient Water 

and Wastewater - version September 2010. 

Justification: 

Fast Phage method shortens and simplifies hands-on aspects of Method 1601 that will allow 

water testing facilities to easily train and perform a coliphage detection method in about one-half 

the time of Method 1601. 

Detection of coliphages as a viral fecal contamination indicator is one of three recommended 

options of the GWR. Under current regulations individual states must choose and adopt a fecal 

indicator option to be in compliance with the rule. However, there are a number of public health 

officials that recommend screening of water samples for both bacterial and viral indicators 

because in combination they provide for a broader range of public health protection. Existing 

methods for coliphage detection are time-consuming, labor intensive, and therefore more costly 

than bacterial indicator tests, so it is unlikely that states would choose a coliphage method for 

ground water screening. However, coliphages are an important viral fecal contamination 

indicator. Over 50% of the water borne illnesses in the US over the last 20 years have been viral 

in etiology. Fast Phage modifications of Method 1601 make coliphage detection faster, simpler, 

and convenient, and provide a low cost water testing alternative for laboratories that will 

increase the probability that individual states may broaden their public health assessment of the 

water supply above and beyond bacterial indicators. 

Method Summary 

Fast Phage is a commercial kit containing a number of modifications of Method 1601 to detect 

coliphages in groundwater. The method components are manufactured by CSI and complete 

protocol instructions can be obtained from the company located at 659 Andover St., Lawrence, 

MA 01843, Phone 978-687-9200 Fax 978-687-9216, or website www.charm.com. The 

modified method involves a 5 hour abbreviated coliphage amplification in a 100mL water 
sample in a dosed, pre-packaged enrichment media that is inoculated with a ready-to-use freeze 

dried host E. coli tablet. There are different test kits for somatic and male-specific coliphage 

detection that use host E. coli specific to the coliphage type and consistent with the hosts used in 

Method 1601 (strain CN-13 for somatic coliphage and Famp for male-specific coliphage). The 

enrichment media used in these kits contain the antibiotic(s) to which that particular host E. coli

is resistant so as to promote growth of the host while inhibiting growth of other environmental 

bacteria in the sample. The enrichment broth is LB broth based to promote a faster logarithmic 

growth phase of the host culture than the tryptic soy broth (TSB) used in Method 1601. 

The coliphage amplified 5 hour enrichment (STEP-1) is spotted onto a soft agar plate seeded 

with host E. coli. Spots from samples that produce wholly lytic zones, small plaques or 

microplaques within 11-24 hours, are coliphage positive, while spots from samples containing no 

lytic areas are coliphage negative, containing <1 plaque forming units(PFU)/100mL. 

The first step 5 hour enrichment culture is also used in the same day prediction of the spot plate 

result. An aliquot of amplified enrichment is transferred to a second media (STEP-2) that has 

been previously rehydrated with 100mL reagent or sterile laboratory water. The second media 

contains a fluorogenic detection mechanism that is activated by phage induced lysis of the host 

E. coli. Samples that fluoresce under 366nm UV light within 3.0 hours of enrichment broth 

transfer are presumptive positive for coliphage and should be spot plated for confirmation. 

The Fast Phage media contains inducers that promote intracellular production of the enzyme ßgalactosidase. E. coli cells that are ruptured during lytic coliphage infection and replication, 

release the enzyme extracellularly. The extracellular enzymes cleave a fluorogenic enzyme 

substrate, MUG-gal (4-methylumbelliferyl-d-galactopyranoside) included in the second media to 

produce the fluorescent signal through the liberation of MUG. 

Following the assay scheme of Method 1601, the first step enrichment (STEP-1) of the Fast 

Phage assay is spotted onto an agar plate seeded with host E. coli. The Fast Phage modification 

kit provides a freeze dried E. coli host tablet (cultured in TSB for 4-5 hours) to seed the 45-48oC 

tempered molten agar, as well as a convenient freeze dried antibiotic to simplify spot agar 

preparation. Spotted enrichment media are allowed to dry and the plates are incubated overnight. 

Samples that show zones of inhibition, spots or pinpoint lytic spots after 11-24 hours of plate 

incubation contain coliphage. If the water sample produced an early warning fluorescence during 

the second rapid indicator step (STEP-2) that second step enrichment is also spot plated and 

incubated overnight. Samples that show zones of inhibition, spots or pinpoint lysis spots after 

11-24 hours of spot plate culture contain coliphage. Samples that have a uniform and confluent 

spot of host E. coli growth in STEP-1 enrichment and non-fluorescent in STEP-2 are negative for 

coliphage and contain < 1 PFU in the 100mL sample. 

The combined modifications of the Fast Phage test allow negative coliphage determination in as 

little as 16 hrs which is 24-48 hours shorter than Method 1601. Presumptive positive coliphage 

detection is as early as 5-8.0 hours with fluorescence development during the rapid indicator 

step. Fluorescent positive samples and all STEP-1 enrichment samples are confirmed positive or 

negative by the spot plate method. 
Study Objectives and Design 

Main Objectives: 

The main objective of the ATP study was to demonstrate the equivalence of the Fast Phage, 

modified Method 1601, to USEPA Method 1601, an approved reference method for detection of 

coliphages as stipulated in the 2006 GWR. CSI conducted the ATP study to facilitate their 

request for nationwide approval from the USEPA for Fast Phage to support coliphage screening 

under the GWR. 

Data Quality Objectives: 

CSI's objective for the study was to provide USEPA with data compliant to the approved study 

plan. Specifically, four third-party laboratories performed replicate side-by-side analyses of 

groundwater samples spiked with waste water filtrate, following Method 1601 and the spiking 

procedures specified for method modification stipulated in Method 1601, to provide a basis of 

comparison for the Fast Phage method. 

Study Design: 

The design for the Fast Phage ATP study was based on PBMS criteria detailed in Method 1601 

for national approval of method modifications consistent with protocol detailed in USEPA 

Microbiological Alternate Test Procedure Protocol for Drinking Water, Ambient Water and 

Wastewater, version 2010. Three geographically diverse non-chlorinated, primary stage, 0.45μm 

filtered waste waters were used as sources of wild somatic and male-specific coliphages for the 

study. The coliphages were enumerated in each waste water filtrate sample by double agar layer 

(DAL) technique and then diluted to 1.3-1.5 plaque forming unit (PFU) per 100mL groundwater 

sample as specified in expanded matrix spikes of Method 1601. By Method 1601 specification, 

the inoculation of coliphages into the test groundwater must result in at least five of ten replicate 

samples being positive for coliphage. This spike level has been shown in Method 1601 

collaborative study to be sufficiently low to result in at least some negative results in the sample 

replicates. 

For this ATP study two different coliphage methods, Fast Phage and USEPA Method 1601 were 

compared for somatic and male-specific detection in groundwater. Ten replicate 100mL samples 

for Fast Phage and ten replicate 100mL samples for Method 1601 were aliquoted from 2.2L 

locally sourced groundwaters inoculated with low level coliphages from a common waste water 

filtrate. To each of the ten Fast Phage samples a STEP-1 media pouch was dissolved, followed 

by addition of a host E. coli tablet, and samples were incubated 0.5 hour in a water bath at 38±1o 

C followed by 4.5 hours in a dry incubator at 39±1o C. Afterwards, a 10μL loopful of each 

enriched Fast Phage sample was applied and dried onto host specific E. coli seeded soft spot agar 

plates for overnight incubation at 39±1o C. Additionally, the enriched STEP-1 cultures were also 

transferred, 1mL for male-specific coliphage and 10mL for somatic coliphage, to respective 

100mL Fast Phage STEP-2 fluorescent media and incubated in a water bath at 38±1o C, 30 

minutes for somatic coliphage and 1 hour for male-specific coliphage, and then 2-2.5 hours in 

dry incubator 39±1o C. Samples that fluoresced at anytime during the 3 hours predicted 

coliphage presence and had a 10μL loopful applied and dried onto host E. coli seeded agar 

plates for overnight incubation at 39±1o C. The ten replicate Method 1601 samples had the 

various pre-prepared media components added and expodential-growth cultured E. coli from 
slant. These were incubated 24 hours and then had a 10μL loopful applied and dried onto host 

E. coli seeded soft agar plates for overnight incubation at 36±1o C as specified in Method 1601. 

Sensitivity was determined by comparing the number of positive lytic responses from Fast Phage 

soft agar spot plates with the number of positive lytic responses from Method 1601 soft agar spot 

plates. Precision and specificity was determined by testing the 120 somatic and 120 malesspecific Fast Phage results with the described Method 1601 confirmation procedure. 

Predictability of the fluorescent step was determined by comparing the STEP-2 fluorescent result 

to the Fast Phage lytic response. Statistical tests, sensitivity and precision were calculated as 

specified in Section 8 of the 2010 Microbiologic ATP Protocol. 

Study Implementation 

Study Management: 

CSI coordinated approval of the study plan with USEPA and sourced the four participant 

laboratories. CSI acted as the coordinating laboratory for the collection and distribution of waste 

water filtrate for spiking, and the collection of data from the laboratories as well as the 

submission of the study report. The four laboratories conducting the method evaluations were 

Tetratech Environmental Consultants-Clancy Laboratory, St. Albans, VT; American Water, 

Delran, NJ; University of Arizona, Tucson, AZ; and Wisconsin Department of Hygiene, 

Madison, WI. 

Type of Study: 

The Fast Phage method was evaluated according to the PBMS criteria as specified for national 

approval in Method 1601. This involved sharing waste water filtrate samples for spiking with a 

minimum of three different participant laboratories and spiking groundwater to coliphage levels 

as specified in Method 1601. In addition, the Fast Phage method was compared to Method 1601 

to provide side-by-side comparison data as specified in the USEPA Microbiological Alternative 

Test Protocol. 

Study Schedule 

Study data for the information presented in this report was collected over the months of May 

through October of 2011. On a coordinated schedule, collected waste water filtrate was 

distributed by CSI to all four laboratories. The laboratories determined either the somatic or 

male-specific coliphage titer in the waste water filtrate and spiked and performed each coliphage 

detection method using a locally obtained groundwater within the same week as receiving the 

filtrate. 

Matrix and spiking source 

The test matrix was local groundwater collected by each testing laboratory. The coliphage 

spiking sources were primary, non-chlorinated sewerage obtained from waste water treatment 

plants. Waste water samples were acquired from diverse geographical locations around the 

United States, specifically Arizona, Wisconsin, and Massachusetts, to provide variation of wildtype coliphages for the study. 
Preliminary testing: 

Preliminary test cycles which followed the approved study protocol were initiated by each 

participant laboratory in May 2011 for somatic coliphage and again in August 2011 for male 

specific coliphage. For these trials, a primary non-chlorinated waste water sample from each 

laboratory’s own geographical location was collected, filtered, and used as the coliphage spiking 

source into their local groundwater. This was to qualify each laboratory under the specifications 

and quality control criteria of Method 1601 Section 14.1 and also to support qualifying 

extended-matrix-spike performance with the Fast Phage modification. The preliminary test 

cycles were completed by each laboratory on their own schedule and without sharing waste 

water filtrate. All laboratories properly met the expanded spiking performance criteria of 

Method 1601 as well as the ongoing demonstration of capability (ODC) with proper quality 

control tests. 

Numbers and Types of Analyses: 

Six waste water samples were collected and filtered for use as shared coliphage spiking sources 

during the study. Three waste water sample filtrates were used for somatic coliphage spiking 

and three filtrates were used for male-specific coliphage spiking. Each filtrate was spiked by the 

laboratories into their locally collected groundwater which represented the four geographical 

locations of Vermont, New Jersey, Wisconsin, and Arizona. Each testing cycle included ODC 

performance following Method 1601 to verify proper coliphage detection in the groundwater 

samples. 

Sample Collection Distribution and Storage:

Sewage samples were collected at waste water treatment facilities. CSI collected samples in 

Massachusetts, while personnel at U. of Arizona and Wisconsin Department of Hygiene 

collected samples in their respective states. Sewage samples were chilled on ice immediately 

after collection, then filtered and quantified for coliphage titer using the DAL technique. 

Coliphage titer determination occurred on the day of collection just prior to same day shipping to 

CSI by FedEx priority delivery in a sealed cooler packed with frozen gel packs and received 

within 24 hours of collection. Upon receipt at CSI, samples were immediately transferred to a 

refrigerator and regulated at 1- 4° C. Within 48 hours of receipt of out-of-state waste water, 

samples were quantified by DAL to verify no more than 50% degradation of coliphage titer as 

specified by Method 1601 waste water spiking criteria. Samples were filtered (25mm 0.45μm 

low protein binding filter),split and shipped on wet gel ice by Federal Express for overnight 

delivery to the collaborating laboratories. The laboratories received the filtrate sample, 

quantified coliphage titer and refrigerated for next day spiking experiments. The filtrate was 

stored at refrigeration temperatures throughout the experiment except for short periods when 

being used, such as during the initial coliphage quantification by DAL. Quantification of 

coliphages was also repeated by participant laboratories on the day of spiking the groundwater 

and at CSI for end of the test week titer determination (96 hour after shipping the filtered sample 

from CSI to the laboratories). The various DAL results were used to demonstrate that the filtrates 

used in the study met the Method 1601 specifications for waste water samples used for spiking. 

Spiking Enumeration: 

To determine the initial titer of coliphages in the original sewage filtrate spiking suspension, 

double agar layer (DAL) analyses were performed in accordance with the requirements of 
Method 1601 and PBMS for national approval of method modification. DAL and enumeration 

calculations were performed exactly as specified in Methods 1601 and 1602. 

Sample Waters and Spiking Procedure: 

For comparability testing, a local untreated groundwater was collected from local wells into a 

clean container by each laboratory. Collected groundwaters were verified negative for coliphage 

presence by using the groundwater matrix as a negative control in Method 1601, as a negative 

control in Method 1601 ODC, and as negative control in the Fast Phage method. The locally 

collected groundwater was spiked to the target DAL using the shared waste water filtrate and 

was calculated using the recipient laboratory’s initial DAL determination. The reported study 

spike level is based on the amount of waste water added to the groundwater and the laboratory’s 

second DAL quantification performed on the day of spiking. Waste water was added to 2.2 L of 

groundwater and mixed by stir bar for 20 minutes prior to splitting 100mL samples alternately 

between Method 1601 and Fast Phage. 

Data Reporting and Validation 

Quality Assurance/Quality Control: 

QA/QC procedures were followed in this study per the study plan, USEPA ATP guidance 

document, Method 1601, and the QA/QC guidelines for certified drinking water laboratories. 

Detailed protocols and recording sheets were supplied to participant laboratories following 

review and approval by USEPA. In addition, the laboratories received weekly log sheets to 

document equipment calibrations, temperatures, and procedural times; log sheets were returned 

to the coordinating laboratory for review and collating. 

Control cultures: 

For each lot of medium, analytical procedures were checked by testing with known positive 

cultures as noted below: 

Coliphage somatic type ФX174 (ATCC- 13706-B1) 

Coliphage male-specific type MS2 (ATCC- 15597-B1) 

Control Blanks: 

Control Blanks were used for each test run in both the test and reference methods. Blanks 

consisted of a reverse/osmosis or distilled water and autoclaved for sterility. 

Comparability Study 

DAL determinations: 

The results of the DAL determinations of the shared waste water samples are shown in Table 1. 

Throughout the comparison study, the WI and the VT laboratories as a trend obtained lower 

PFU/mL counts than did the NJ and AZ laboratories and the coordinating MA laboratory. DAL 

results also indicated that the male-specific coliphage was less stable over the week of testing 

than was the somatic coliphage and that the male-specific coliphage titer trended lower during 

overnight shipping on gel ice. The end-of-week male-specific DAL titer determinations 

completed by the MA laboratory were almost 50% degraded after 96 h storage post-filtering 

whereas the somatic DAL titers were more stable and showed only 15–30% degradation. 
Table 1- Double Agar Layer (DAL) Coliphage Enumeration Performed on Shared Water 

Samples to Demonstrate Compliance with Waste Water Spiking Specifications of USEPA 

Method 1601 

Cycle #

Waste Water 

Source 

Coliphage 

Type Lab 

Collection 

DAL 

PFU/mL 

Receipt 

DAL 

PFU/mL 

Day 

Before 

Assay 

DAL 

PFU/mL 

Day of 

Assay 

DAL 

PFU/mL 

End of 

Week 

Assay 

DAL 

PFU/mL 

S1 Lawrence, MA somatic MA 1382 nd 1354 1171 973 

 S1-1 VT na na 1636 1727 na 

 S1-2 NJ na na 1216 1164 na 

 S1-3 AZ na na 1009 820 na 

 S1-4 WI na na 901 856 na 

S2 Tucson, AZ somatic MA na 7273 6455 6636 5182 

 S2-1 VT na na 11727 8454 na 

 S2-2 NJ na na 6487 6667 na 

 S2-3 AZ 5909 na 9000 8545 na 

 S2-4 WI na na 3909 4090 na 

S3 Madison, Wisc somatic MA na 4090 3400 3636 3636 

 S3-1 VT na na 4636 4181 na 

S3-2 NJ na na 4505 4054 na 

S3-3 AZ na na 4636 4727 na 

S3-4 WI 2909 na 2727 2454 na 

F+1 Lowell, MA F+ MA 2685 nd 2720 2387 1018 

F+1-1 VT na na 946 1180 na 

F+1-2 NJ na na 1892 2306 na 

F+1-3 AZ na na 2189 2351 na 

F+1-4 WI na na 738 604 na 

F+2 Madison, Wisc F+ MA na 4009 3649 3387 1645 

F+2-1 VT na na 865 847 na 

F+2-2 NJ na na 3919 3108 na 

F+2-3 AZ na na 5649 5855 na 

F+2-4 WI 2820 na 1468 1658 na 

F+3 Tucson, AZ F+ MA na 279 251 243 120 

F+3-1 VT na na 140 38 na 

F+3-2 NJ na na 204 223 na 

F+3-3 AZ 205 na 142 119 na 

F+3-4 WI na na 101 79 na 

DAL—double agar layer, ID—identification, NA indicates measurement is not applicable to the noncollecting 

laboratories.NP indicates not performed because the collecting laboratory was also the coordinating laboratory and 

therefore no shipment of sample took place. DAL calculations are a single numeric calculation based on duplicate 

plates of 4-log serial dilutions of a single test sample. Collection DAL was performed by the laboratory collecting 

the sample and on the day of collecting sample. Receipt DAL was performed by the coordinating laboratory on 

receipt of sample to verify < 50% loss of PFU/mL over shipment. Day-before-assay DAL was performed 

on split waste water samples on day of receipt of sample, and when compared with collecting laboratory collection

DAL, demonstrated < 50% loss of PFU/mL. Day-of-assay DAL was performed on split waste water sample on the 

day of spiking into groundwater and took place within 72 h of receipt of sample. End-of-week assay DAL was 

performed on waste water sample by the coordinating laboratory at the end of study week. 
 The waste water filtrate therefore met the USEPA Method 1601 specifications for raw sewage 

filtrate because the filtrate titers were determined within 24 h of collection, did not degrade by 

more than 50% after collection, and were re-titered and used within 72 h of receipt by the 

laboratories. The laboratories obtained their own groundwater source and spiked it with the 

filtered sewage at the prescribed target PFU spike levels (1.5 pfu/100 mL for somatic coliphages 

and 1.3 pfu/100 mL for male-specific coliphages) as specified by Method 1601 for PBMS 

validation. They used their day-before-assay DAL results (Table 1) in the spiking calculations. 

The DAL determination was based on duplicate plates of 4-log serial dilutions of the waste 

water. The laboratories spiked the 2.2 L of ground water based on the calculated day-beforeassay coliphage concentration, mixed, and then aliquoted 100mL test samples alternatively 

between the Fast Phage and Method 1601 and completed each sample as per method protocol. 

The laboratories also repeated the DAL assay of the waste water sample that same day; the 

reported spiked coliphage PFU/100mL based on this day-of-assay DAL result is shown in Table 

1. The global day-to-day DAL repeatability within labs was 15% of the average whereas the 

reproducibility of DAL results of the same samples shared among the labs was 40% of the 

average. 

Coliphage method comparisons of somatic coliphage: 

Results of the somatic coliphage method comparison are shown in Table 2. The range of somatic 

coliphage spike levels (0.8–1.4 PFU/100mL) met the target spiking level of < 1.5 PFU/100mL 

specified by Method 1601. The Fast Phage, complete through plaque confirmation, identified 

77% (92 of 120 samples) as positive, compared with 85% (102 of 120 samples) identified as 

positive by Method 1601. The 8 h fluorescence prediction, a component of the Fast Phage, 

identified 73% (87 of 120 samples) as positive. At least 5 positives in 10 replicates at the 

targeted PFU/100 mL were detected in all somatic cycles, which met the PBMS acceptance 

criteria described in Method 1601, Section 14.2, for national approval of method modifications. 

The comparative Method 1601 performance likewise met the extended matrix spike QC criteria 

as described in Section 9 of the method. In all cases, internal positive and negative controls and 

concurrent Method 1601 ODC in all the laboratories met quality specifications for reporting 

results. In any one somatic cycle, the maximum difference observed in the number of plaquepositive samples by Method 1601 and the 16- to 24 h modified method was 30%. The 

comparability table included the actual spike level, PFU/100mL as calculated from the amount of 

filtered sewage added, and the day-of-assay DAL result (Table 1) performed on the day of 

spiking. 

 
Table 2: Somatic Coliphage Comparability Study of (n=10 replicates per Cycle) 

Cycle # 

Spiked 

PFU/ 

100mL 

48 hour 

EPA 1601 

Positive-n 

16-24 hour 

Fast Phage 

Positive-n 

8 Hour 

Fluorescent

Positive-n 

S1-1 1.4 9 7 7 

S1-2 1.4 9 10 10 

S1-3 0.8 6 7 5 

S1-4 1.1 9 6 6 

S2-1 1.3 10 8 8 

S2-2 1.2 10 8 8 

S2-3 0.8 6 5 4 

S2-4 1.2 8 8 8 

S3-1 1.1 10 9 9 

S3-2 1.1 8 10 8 

S3-3 1.1 8 7 7 

S3-4 1.3 9 7 7 

Sum 102 92 87 

%Positive of n=120 85% 77% 73% 

DAL—double agar layer, ID—identification, n—number 

Spiked level was made by each laboratory by spiking shared waste water (Table 1) into local groundwater. The 

PFU/100mL was calculated based on volume of waste water added to 2.2 L and using each laboratory day-of-assay 

DAL PFU/mL of waste water to calculate plaques added to ground water. 

Coliphage method comparisons of male-specific coliphage: 

Table 3 shows results of the male-specific coliphage method comparison. The range of malespecific coliphage spike levels was 0.9 to 1.5 PFU/100mL, targeting the specified USEPA 

Method 1601 spiking level of < 1.3 PFU/100mL and simulating the range of studied sample 

spikes from the Method 1601 collaborative study. The Fast Phage, complete through plaque 

confirmation, identified 89% (107 of 120 samples) as positive, compared with 96% (115 of 120 

samples) identified as positive by Method 1601. The 8 h fluorescence prediction identified 88% 

(105 of 120 samples) as positive. At least 5 positives in 10 replicates at the targeted PFU/100mL 

were detected in all male-specific cycles, which met the PBMS acceptance criteria described in 

Method 1601, Section 14.2, for national approval of method modifications. The comparative 

Method 1601 performance likewise met the extended matrix spike QC criteria as described in 

Section 9 of the method. In all cases, internal positive and negative controls and concurrent 

Method 1601 ODC in all the laboratories met quality specifications for reporting results. In any 

one male-specific cycle, the maximum difference observed in the number of plaque positives by 

method 1601 and the 24-h Fast Phage, was 40%. The comparability table included the actual 

spike level, PFU/100mL as calculated from the amount of filtered sewage added, and the day-ofassay DAL result (Table 1) performed on the day of spiking.
Table 3: Male-Specific Coliphage Comparability Study of n=10 Replicates per Cycle 

Cycle # 

Spiked 

PFU/ 

100mL 

48 hour 

EPA 1601

Positive-n 

16-24 hour 

Fast Phage

Positive-n 

8 Hour 

Fluorescent 

Positive-n 

F+1-1 1.0 10 10 9 

F+1-2 1.5 10 10 10 

F+1-3 1.2 10 8 7 

F+1-4 1.3 10 9 9 

F+2-1 1.2 10 10 8 

F+2-2 1.0 10 10 10 

F+2-3 1.0 10 10 10 

F+2-4 1.3 9 9 9 

F+3-1 0.9 10 9 9 

F+3-2 1.5 10 10 10 

F+3-3 0.3 10 6 8 

F+3-4 1.2 6 6 6 

Sum 115 107 105 

%Positive of n=120 96% 89% 88% 

DAL—double agar layer, ID—identification, n—number 

Spiked level was made by each laboratory by spiking shared wastewater (Table 1) into local groundwater. The 

PFU/100mL was calculated based on volume of waste water added to 2.2 L and using each laboratory day-of-assay 

DAL PFU/mL of waste water to calculate plaques added to groundwater. 

Comparability Study Data Analysis and Discussion 

Fisher Exact Tests: 

Because of the number of sample replicates (n =10), Fisher exact tests were performed on all 

comparative method somatic and male-specific test cycles reported in Tables 2 and 3 to 

determine if there were significant differences between the Fast Phage and the reference method. 

Fisher exact tests did not indicate a significant difference between any one comparative set of 

data. 

Cochrane-Mantel-Haenszel Chi-square Tests: 

When the data sets were considered as whole data sets with a single degree of freedom, 

Cochran-Mantel-Haenszel chi-square analysis delivered probabilities of p=0.13 (somatic) and 

p=0.06 (male-specific) that the 12 data sets were homologous. These probabilities do not indicate 

significant differences between the Fast Phage and the reference method (p < 0.05). Although not 

significant, the p=0.13 and p=0.06 may indicate a lower trend in positive determinations. When 

probabilities were recalculated excluding the single most different study cycle from the chisquare determinations, the resulting probabilities—p=0.29 (somatic) and p=0.38 (malespecific)—indicated that one study cycle’s strong influence on the overall p determination and 

further supported the conclusion that the methods were not significantly different. Excluding one 

of 10 method comparison cycles in determining a probability of method difference is consistent 

with acceptable Microbial Alternative Test Protocol comparability requirements that at least 80% 

of comparison cycles be equivalent. 
Precision Study 

All Fast Phage spot plate circular spot areas (with or without a lytic zone) were transferred to 

TSB broth and re-spotted to a fresh Method 1601 agar spot plate to calculate the precision of the 

Fast Phage in terms of agreement with Method 1601. This experiment determined the associated 

false-negative and false-positive results of the modified method. Results were scored provided 

the negative control and positive control samples of the transferred plaques tested 

appropriately. Appropriate QC results occurred in all test cycles except for a single somatic 

coliphage cycle in which the negative controls tested positive; those results were recorded as a 

laboratory accident. Tables 4 and 5 show the precision-study results for somatic coliphage and 

male-specific coliphage, respectively. In these analyses, the re-spotted sample without filtration 

was considered the compared method, and the sample with filtration was considered the 

reference method. In the two-by-two contingency table scoring, a true-positive produced a lytic 

zone in both the compared and reference methods, a true-negative produced an intact lawn 

of bacteria in both the compared and reference methods, a false negative did not produce a lytic 

zone in the Fast Phage but produced a lytic zone on the USEPA method, and a false-positive 

produced a lytic zone in the Fast Phage but not on the USEPA method. 

Somatic Coliphage Precision Study Results and Discussion. 

Somatic coliphage precision experiment analysis depicted in Table 4 showed a 94% observed 

agreement with the compared method and a 93% observed agreement with the reference method. 

False-negative rates for both the compared and reference methods were 4.6%, whereas falsepositive rates were 13.6% for the compared method and 17.4% for the reference method. These 

false rates can be compared with those of the unfiltered USEPA method versus the filtered 

USEPA method, which had false-negatives of 2.3% and false positives of 4.6%. The somatic 

coliphage false-positive rates in the Fast Phage analysis were consistent with earlier published 

evaluation data but might be overstated because of the small number of true-negative samples 

in the study. For example, if the negative control and matrix negative control data were included 

in the analysis, the false-positive rate of the Fast Phage to Method 1601 decreased from 17.4 

to 8.9%, which more closely approximated the compared performance of the unfiltered and 

filtered USEPA methods. The smaller number of negative samples was a consequence of the 1.5-

pfu/100 mL somatic coliphage spike level specified for the PBMS comparison. 

The limited number of false results in the tables did not allow Yates or Pearson chi-squared 

analysis to determine if the observed differences were significant, but a f=0.78 indicated the 

modified method precision was not significantly different from the reference method with 

f=0.92. The odds ratio of the modified method was about half that of the comparative method, 

indicating about twice the probability of a false result; again, however, this analysis was 

influenced by the smaller number of negative samples and was not considered significantly 

different from the reference method by Breslow–Day analysis. The false negative rate of < 5% 

and the overall 93–94% agreement of the Fast Phage with method 1601 were measures of 

equivalence between the methods. 
Table 4: Somatic Precision Study Showing Number of Fast Phage Somatic Coliphage Positive and 

Negative Plaque Results Picked into TSB Broth and Re-cultured to EPA Method 1601 Unfiltered 

(Compared Method) and Filtered (Reference Method). 

Cycle # 

Fast Phage 

Negative 

n 

 Fast 

 Phage 

Positive 

n 

Unfiltered Spot Repicked to EPA 

1601 

(Compared 

Method)-n 

Filtered Spot Repicked to EPA 1601 

(Reference Method)-

n 

Unfiltered EPA 1601 

compared to Filtered EPA 

1601 

n 

TN TP FN FP TN TP FN FP TN TP FN FP

S1-1 3 7 3 7 0 0 3 7 0 0 3 7 0 0

S1-2 0 10 0 10 0 0 0 10 0 0 0 10 0 0

S1-3 3 7 2 7 1 0 3 6 0 1 2 6 0 2

S1-4 4 6 4 6 0 0 3 6 1 0 3 6 1 0

S2-1 2 8 2 6 0 2 2 6 0 2 4 6 0 0

S2-2 2 8 0 8 2 0 0 8 2 0 0 10 0 0

S2-3 5 5 la la la la la la la la la la la la

S2-4 2 8 2 8 0 0 2 8 0 0 2 8 0 0

S3-1 1 9 1 9 0 0 1 9 0 0 1 9 0 0

S3-2 0 10 0 10 0 0 0 10 0 0 0 10 0 0

S3-3 3 7 3 6 0 1 3 6 0 1 4 6 0 0

S3-4 3 7 2 7 1 0 2 7 1 0 2 8 0 0

Total 28 92 19 84 4 3 19 83 4 4 21 86 1 2

Sensitivity (SE) 95% 95% 97%

Specificity (SP) 86% 83% 95%

Efficiency* (EFF) 94% 93% 97%

Predictive value of positive test (PVP) 97% 95% 99%

Predictive value of negative test (PVN) 83% 83% 91%

False positive rate (FP) 13.6% 17.4% 4.6%

False negative rate (FN) 4.6% 4.6% 2.3%

Cohen’s Kappa (κ) 0.8045†

0.7801‡0.9162†

Observed agreement (PO) 0.94 0.93 0.97

Positive agreement (PA) 0.96 0.95 0.98

Negative agreement (NA) 0.84 0.83 0.93

ID—identification, n—number, PVN—predictive value of negative test, PVP—predictive value of positive test, 

QC—quality control, TSB—tryptic soy broth, USEPA—US Environmental Protection Agency 

*Represents the correct classification rate 

†This kappa indicates almost perfect statistical agreement.

‡This kappa indicates substantial statistical agreement. 

LA indicates laboratory accident, i.e., QC samples disqualified the selectivity portion of this cycle. 

TN indicates true-negative, i.e., negative results recultured as negative; TP indicates true-positive, i.e., positive 

results recultured as positive; FN indicates false-negative, i.e., negative results recultured as positive; FP indicates 

false-positive, i.e., positive results recultured as negative. Inter-rater calculations of sensitivity, specificity, overall 

agreement, and Cohen’s kappa are based on summed TN, TP, FP, and FN in two by-two contingency table 

comparability analysis. 
Table 5: Male-Specific Precision Study Showing Number of Fast Phage Male-Specific Coliphage 

Positive and Negative Plaque Results Picked into TSB Broth and Re-cultured to EPA Method 1601 

Unfiltered (Compared Method) and Filtered (Reference Method). 

Cycle # 

Fast 

Phage 

Negative 

n 

 Fast 

 Phage 

Positive

n 

Unfiltered Spot Repicked to EPA 1601 

(Compared Method)-

n 

Filtered Spot Repicked to EPA 1601 

(Reference Method)-

n 

Unfiltered EPA 1601 

compared to Filtered EPA 

1601-n 

TN TP FN FP TN TP FN FP TN TP FN FP

F+1-1 0 10 0 10 0 0 0 10 0 0 0 10 0 0

F+1-2 0 10 0 10 0 0 0 10 0 0 0 10 0 0

F+1-3 2 8 1 8 1 0 0 8 2 0 0 9 1 0

F+1-4 1 9 1 9 0 0 1 9 0 0 1 9 0 0

F+2-1 0 10 0 10 0 0 0 10 0 0 0 10 0 0

F+2-2 0 10 0 10 0 0 0 10 0 0 0 10 0 0

F+2-3 0 10 0 10 0 0 0 10 0 0 0 10 0 0

F+2-4 1 9 1 9 0 0 1 9 0 0 1 9 0 0

F+3-1 1 9 1 9 0 0 1 9 0 0 1 9 0 0

F+3-2 0 10 0 10 0 0 0 10 0 0 0 10 0 0

F+3-3 4 6 4 6 0 0 3 6 1 0 3 6 1 0 

F+3-4 4 6 4 6 0 0 4 6 0 0 4 6 0 0

Total 13 107 12 107 1 0 10 107 3 0 10 108 2 0

Sensitivity (SE) 99% 97% 98%

Specificity (SP) 100% 100% 100%

Efficiency* (EFF) 99% 98% 98%

Predictive value of positive test 

(PVP)

100% 100% 100%

Predictive value of negative test 

(PVN)

92% 77% 83%

False positive rate (FP) 0% 0% 0%

False negative rate (FN) 0.9% 2.7% 1.8%

Cohen’s Kappa (κ) 0.9554†0.8560†0.9000†

Observed agreement (PO) 0.99 0.98 0.98

Positive agreement (PA) 0.995 0.986 0.991

Negative agreement (NA) 0.96 0.87 0.91

ID—identification, n—number, PVN—predictive value of negative test, PVP—predictive value of positive test, 

TSB—tryptic soy broth, USEPA—US Environmental Protection Agency 

*Represents the correct classification rate 

†This kappa indicates almost perfect statistical agreement.

TN indicates true-negative, i.e., negative results recultured as negative; TP indicates true-positive, i.e., positive 

results recultured as positive; FN indicates false-negative, i.e., negative results recultured as positive; FP indicates 

false-positive, i.e., positive results recultured as negative. Inter-rater calculations of sensitivity,specificity, overall 

agreement, and Cohen’s kappa are based on summed TN, TP, FP, and FN in two-by-two contingency table 

comparability analysis. 

Male-specific Coliphage Precision Results and Discussion. 

Male-specific coliphage precision analysis depicted in Table 5 showed a 99% observed 

agreement with the compared method and a 98% observed agreement with the reference method. 

False-negative rates of the modified method were 0.9% in the compared method analysis and 
2.7% in the reference method analysis. False-positive rates were 0% for both the compared and 

reference method analyses. These false rates can be compared with those of the unfiltered versus 

filtered two-by-two contingency table analysis, which had false-negatives of 1.8% and falsepositives of 0%. The false positives in the current analyses could not be accurately determined 

because of the small number of negative samples in the study data. The smaller number of 

negative samples was caused by the spike level of 1.3 pfu/100 mL specified in USEPA Method 

1601 for PBMS comparison. The false-negative rate of < 3% and the overall 98–99% agreement 

of the Fast Phage with method 1601 were measures of equivalence between the methods. 

Fluorescence Prediction Study 

Fluorescence Prediction of Positive. 

Table 6 shows the predictability of the same-day fluorescence warnings for both coliphage types. 

Fluorescence detected 73% of the samples positive for somatic coliphage whereas the Fast Phage 

plaque process found 77% of the samples positive. Fluorescence detected 86% of the samples 

positive for male-specific coliphage whereas the Fast Phage plaque process found 89% of the 

samples positive. In the table scoring, a true-positive fluorescence produced a positive plaque on 

the spot plate, a true-negative fluorescence produced a negative plaque on the spot plate, a falsenegative did not fluoresce but produced a positive plaque on the spot plate, and a false-positive 

fluoresced but produced a negative plaque on the spot plate. 

Somatic coliphage fluorescent prediction discussion. 

Comparative table analysis showed an observed somatic agreement of the fluorescent result and 

the Fast Phage of 93%, with a false-negative rate of 7.5% and a false-positive rate of 7.4%. 

The false-negative rate of the fluorescence prediction was higher than that of the Fast Phage 

complete with plaque confirmation relative to method 1601. This indicated an elevated risk of 

relying solely on a fluorescent negative result without the plate confirmation. The positive 

fluorescent results were highly correlative to a positive plate confirmation, with a predictive 

value of a positive result of 98%. 

Male-specific coliphage prediction discussion. 

The male-specific fluorescent prediction showed 95% overall agreement compared with the Fast 

Phage plaque result, with a false-negative rate of 1.9% and a false-positive rate of 

27%. The high calculated false-positive fluorescence level could have been influenced by the 

small number of negative results in this data set. However, the false-positive rate was higher than 

the false-positive rate of the completed Fast Phage with plaque confirmation relative to the 

USEPA methods and as such indicated the importance of performing plate confirmation. The 

male-specific fluorescent positive results were highly correlative to a positive plate confirmation, 

with a predictive value of a positive result 96%. 
Table 6: Fluorescence Result of Somatic and Male-specific Modified Methods at 8 hours Compared to 

Modified Method Plaque (Fast Phage Negative and Positive) result after pick and overnight confirmation 

of Plaque. 

Somatic 

Cycle # 

Fast 

Phage 

Negative

n 

 Fast 

 Phage 

Positive

n 

Fluorescence 

Compared to 

Somatic Fast 

Phage-n 

MaleSpecific 

Cycle # 

Fast 

Phage 

Negative

n 

 Fast 

 Phage 

Positive 

n 

Fluorescence 

Compared to 

Male-specific Fast 

Phage- n 

TN TP FN FP TN TP FN FP

S1-1 3 7 3 7 0 0 F+1-1 0 10 0 9 1 0

S1-2 0 10 0 10 0 0 F+1-2 0 10 0 10 0 0

S1-3 3 7 3 5 2 0 F+1-3 2 8 2 7 1 0

S1-4 4 6 4 6 0 0 F+1-4 1 9 1 9 0 0

S2-1 2 8 2 8 0 0 F+2-1 0 10 0 8 2 0

S2-2 2 8 2 8 0 0 F+2-2 0 10 0 10 0 0

S2-3 5 5 4 5 1 0 F+2-3 0 10 0 10 0 0

S2-4 2 8 2 8 0 0 F+2-4 1 9 1 9 0 0

S3-1 1 9 1 9 0 0 F+3-1 1 9 1 9 0 0

S3-2 0 10 0 8 2 0 F+3-2 0 10 0 10 0 0

S3-3 3 7 2 6 1 1 F+3-3 4 6 2 6 0 2

S3-4 3 7 2 6 1 1 F+3-4 4 6 4 6 0 0

Total 28 92 25 86 7 2 13 107 11 103 4 2

Sensitivity (SE) 92% Sensitivity (SE) 98%

Specificity (SP) 93% Specificity (SP) 73%

Efficiency*(EFF) 93% Efficiency* (EFF) 95%

Predictive value of positive test 

(PVP)

98% Predictive value of positive test 

(PVP)

96%

Predictive value of negative test 

(PVN)

78% Predictive value of negative test 

(PVN)

85%

False positive rate (FP) 7.4% False positive rate (FP) 26.7%

False negative rate (FN) 7.5% False negative rate (FN) 1.9%

Cohen’s Kappa (κ) 0.7982† Cohen’s Kappa (κ) 0.7576†

Observed agreement (PO) 0.93 Observed agreement (PO) 0.95

ID—identification, n—number, PVN—predictive value of negative test, PVP—predictive value of positive test 

*Represents the correct classification rate 

†This kappa indicates substantial statistical agreement. 

TN indicates true-negative, i.e., non-fluorescent cultured as negative; TP indicates true-positive, i.e., fluorescent 

positive results cultured as positive; FN indicates false-negative, i.e., negative fluorescent results cultured as 

positive; FP indicates false-positive, i.e., positive fluorescent results cultured as negative. Inter-rater calculations of 

sensitivity, specificity, overall agreement, and Cohen’s kappa are based on summed TN, TP, FP, and FN in two-bytwo contingency table comparability analysis. 

Conclusion 

The Fast Phage method met the PBMS acceptance criteria described in Method 1601, Section 

14.2, for national approval of method modifications. The comparative USEPA method met the 

extended matrix spike QC criteria specified in Section 9 of Method 1601 and met all QC and 

ODC criteria for accepting data. The PBMS criteria are based on the target spike level and the 

lower limit of qualitative positive results required to demonstrate that variability in modified 

results does not exceed the original collaborative study results of method 1601. In that study the 
average positive rate ranged from 60 to 90% with average spike levels of 1.0 to 1.4 PFU/100 mL, 

which were consistent with the spike levels and positive rates obtained by the Fast Phage and 

Method 1601 in this study. 

Performing the actual reference method to generate comparative data is not part of PBMS criteria 

as described in USEPA Method 1601 but was part of this alternative test protocol required by 

the agency as part of the current study design. The ATP protocol considers the two comparative 

methods as unrelated. The current study design is a hybrid of two study designs—PBMS and 

ATP—and therefore 120 comparative somatic and male-specific method analyses using split 

samples were performed. The modified methods (Fast Phage somatic and male-specific) did not 

show statistically significant differences from the reference method 1601, using any of the study 

design criteria, PBMS, or ATP comparability and precision. When ATP design comparability 

criteria were used, none of the modified method results for the 24 cycles differed significantly 

from the method 1601 results using Fisher‘s exact tests. Taken as a single degree of freedom 

with CMH chi-square analysis, the differences between comparison cycles of the two methods 

were not significant, Precision study evaluation also did not indicate significant differences 

between the original method and the Fast Phage modifications. 

The results of these studies indicate that the Fast Phage method is equivalent to Method 1601 in 

performance, sensitivity and precision and may be used at a national level for detecting 

coliphages in 100mL water samples and may be used in compliance with the Ground Water 

Rule.