Nov.28, 2012 Page 1 

Fast Phage Test 

Presence/Absence for Coliphage in Ground Water 

with Same Day Positive Prediction 

ATP Case No. D09-0007, Version 009 

November 28, 2012 

1.0 Scope and Application 

1.1 The Fast Phage tests are method modifications of EPA Method 1601. The modifications described 

herein are specifically for detection of somatic and male-specific coliphages in ground water. 

Coliphages are fecal contamination indicators listed in the EPA Ground Water Rule. 

1.2 Presence (or “positive”) interpretation occurs with 100mL samples containing ≥1 plaque forming 

units (PFU/100mL). Absence (or “negative”) interpretation occurs with samples containing < 1 

PFU/ 100mL. 

1.3 The Fast Phage tests are designed to meet the sensitivity defined in the Ground Water Rule for 

100mL samples. 

1.4 The Fast Phage test modifications detect coliphages in 16-30 hours comparably to Method 1601 

(40-60 h). A rapid fluorescent transfer step can predict coliphage positive samples in less than 8 

hours. The fluorescent detection is a presumptive positive result that has no equivalent status in 

coliphage detection using Method 1601. 

2.0 Summary of Fast Phage Method and 1601 Modifications 

2.1 Fast Phage kits are made for either somatic or male-specific coliphages. Kit contents are specific 

for the coliphage type being detected and are not to be interchanged. Kits are similar in test format 

and in Method 1601 procedural modification. 

2.2 The STEP-1 media is LB based nutrient rich relative to TSB used in Method 1601 and this allows 

for rapid E. coli growth. Exponential growth rapidly amplifies any coliphage present in a water 

sample. Coliphage propagation at 5 hours is equivalent to Method 1601 at 24 hours. STEP-1 

media is supplied pre-dispensed in convenient single use dissolvable pouches. 

2.3 E. coli, CN-13 for somatic coliphage and Famp for male-specific coliphages, are supplied as readyto-use freeze dried tablets at a level of 2 x 107

 colony forming units ± 0.5 log. On addition to the 

media dissolved in the water sample, the E. coli tablet disperses and the bacteria are in logarithmic 

growth by the time the test sample has been brought to temperature in a water bath. 

2.4 The E. coli culture tablets are manufactured and supplied in a standardized form that makes them 

convenient for rapid and consistent assay performance both in enrichment and spot plate 

performance. 
Nov.28, 2012 Page 2 

2.5 Freeze dried antibiotic for solution preparation and addition to autoclaved and tempered spot agar 

provides a convenient reagent and a simplified preparation procedure. 

2.6 The agar spot plates are made with E. coli host cultured from the supplied tablet. Coliphage lysis

on the plates can be observed within 11 hours but can be read as much as 25 hours later. 

2.7 STEP-2 media, also supplied pre-dispensed in dissolvable pouch, is a rapid fluorescent indication 

which predicts coliphage presence in the enriched STEP-1 media. STEP-2 allows for a same day (6 

– 8 hours from STEP-1 start) prediction of positive plaque formation (that are confirmed the 

following morning with plaques on corresponding spot plate). The rapid fluorescence indication is 

based on the principle of inducing intracellular ß-galactosidase production during E. coli growth in 

both STEP-1 and STEP-2. The enzyme is released extracellularly after coliphage infection, 

replication, and subsequent E. coli cell lysis. The extracellular enzyme reacts with a fluorescent 

substrate (MUG-GAL) in STEP-2 to cause fluorescence, indicating the presence of coliphage. 

2.8 The combined Method 1601 modifications allow the detection of coliphage in 16-30 hours by 

conventional plaque formation. The rapid fluorescence prediction of plaque formation is a same 

day indicator of presumptive fecal contamination and allows for a more rapid response to a 

contamination event but does not have a status of Method 1601 result. 

2.9 The Fast Phage method overview is as follows: 

2.9.1 Prepare spot plates from an agar preparation by using E. coli tablet cultured into TSB 

from supplied tablets and antibiotic in advance of testing samples. 

2.9.2 Collect water samples and neutralize disinfectant as needed. 

2.9.3 Measure 100mL into vented vessel and add STEP-1 media pouch. 

2.9.4 Let media pouch dissolve for about 10 minutes. Add E. coli tablet to vessel and gently 

swirl. 

2.9.5 Incubate vessel for 0.5 hours at 38±1°C in a water bath. 

2.9.6 Transfer vessel to air incubator at 39±1°C for 4.5 hours. 

2.9.7 STEP-1 enrichment samples are transferred by 10 μL loop to a prepared agar spot plate. 

The sample is allowed to absorb and dry into the plate and the plate is incubated inverted 

at 39±1°C and read the next day, 11 to 25 hours later. 

2.9.8 For same day prediction of the presence of coliphage, dissolve STEP-2 media pouch in 

100mL sterile water 15 minutes prior to completing 5 hour STEP-1 culture. Transfer 

STEP-1 enrichment, 10mL for somatic coliphage and 1mL for male specific coliphage, to 

STEP-2. Heat in 38±1°C water bath 0.5 hour for somatic coliphage and 1.0 hour for 

male-specific coliphage, and transfer to air incubator at 39±1°C for a total of 3 hours from 

transfer. Fluorescence 366nm in samples indicates the sample is predicted to be positive 

for coliphage, absence of fluorescence at 3 hours indicates a negative prediction for 

coliphage. Fluorescence in positive samples may appear as early as 1 hour (6th hour from 

initial STEP-1 start). Fluorescent positive STEP-2 media is transferred by 10 uL loop to a 

prepared agar spot plate and dried into the plate and incubated inverted at 39°C and read 

the next day, 11 to 25 hours later. 
Nov.28, 2012 Page 3 

2.9.9 Samples that produce visible plaque(s) from either STEP-1 or STEP-2 contain ≥ 1 

coliphage PFU/100mL. Samples that produce even and uniform E. coli growth spots 

contain <1 PFU/100 mL (coliphage not detected). 

3.0 Method Definitions 

3.1 Somatic Coliphage- A group of DNA bacteriophages specific to E. coli and coliform bacteria and 

indicative of fecal contamination. These coliphage infect via attachment to the E. coli cell wall. 

3.2 Male-Specific Coliphage- A group of DNA and RNA bacteriophages specific to E. coli and 

coliform bacteria and indicative of fecal contamination. These coliphage infect via attachment to 

the E. coli F pilli. 

3.3 Plaque- A region of no cell growth in an otherwise uniform bacterial lawn that is formed by a 

phage lysing adjoining host bacteria. 

4.0 Interferences 

4.1 Antibiotic resistant (nalidixic acid or ampicillin/streptomycin) coliform bacteria in excess of 1-10 

million CFU/mL capable of producing extracellular ß-galactosidase enzyme may produce false 

positive results. While the Fast Phage STEP media uses antibiotic to provide a favorable 

competitive growth environment for the host E. coli, it may not completely suppress the growth of 

large quantities of enzyme producing coliform. These interferences are not viewed as a detriment to 

sanitary water testing due to the unlikely high levels of contamination and due to the potential 

pathogenicity of the interfering bacteria. 

4.2 During the enrichment phase of the two-step enrichment procedure, other bacteria in the water 

sample can grow and interfere with the spot-test confirmation step. Bacteria may grow over the 

lysis zone and obscure visualization, resulting in a false negative interpretation. Generally, when 

bacteria have overgrown lysis zones, they appear as raised colonies or a confluent growth of raised 

colonies. As a result, they are distinguishable from the surrounding lawn of host bacteria. If this 

problem is noted, follow the procedure described in Section 11.6.3. 

5.0 Safety 

5.1 Refer to Fast Phage MSDS, the E. coli tablets are contained in a foil bag and protective peelable 

blister packaging. 

5.2 Follow good laboratory practices and appropriate safety precautions when handling bacterial 

cultures. Samples may contain high concentrations of biohazardous microorganisms and should be 

handled with appropriate personal protective equipment such as gloves, lab coat, safety glasses, 

and face shield. Perform work in a biosafety hood if available. Do not mouth pipet. 

5.3 Completed Fast Phage tests and culture contents should be autoclaved prior to disposal. 

5.4 Follow local state and country regulations for disposal. 
Nov.28, 2012 Page 4 

5.5 The biohazards and the risk of infection by pathogens associated with handling raw sewage are 

high in this method. Use good laboratory practices when working with potentially harmful samples. 

5.6 Fast Phage does not purport to address all of the safety issues associated with its use. It is the 

responsibility of the laboratory to establish appropriate safety and health practices prior to use of 

this method. The analyst must know and observe the safety procedures required in a laboratory that 

handles biohazardous material while preparing, using, and disposing of cultures, reagents, and 

materials. The analyst must use proper safety procedures while operating sterilization equipment. 

Equipment and supplies that have come into contact with biohazardous material or are suspected of 

containing biohazardous material must be sterilized prior to disposal or re-use. Field and 

laboratory staff collecting and analyzing environmental samples are under some risk of exposure to 

pathogenic microorganisms. Staff should apply safety procedures used for handling pathogens to 

all samples. 

5.7 The laboratory is responsible for maintaining a current awareness file of Occupational Safety and 

Health Administration (OSHA) regulations regarding the safe handling of the chemicals specified 

in this method. A reference file of material safety data sheets should be made available to all 

personnel involved in these analyses. Additional information on laboratory safety can be found in 

Section 15.0 Waste Management. 

5.8 Samples may contain high concentrations of biohazardous agents and must be handled with gloves. 

Any positive reference materials also must be handled with gloves in an appropriate laboratory 

hood. The analyst must never place gloves near the face after exposure to media known or 

suspected to contain pathogenic microorganisms. Laboratory personnel must change gloves after 

handling raw sewage or any other items which may carry pathogenic microorganisms. 

5.9 Laboratory personnel must wear UV protective eyeglasses or a face shield when operating the 6 

watt 366 nm UV Lamp. 

6.0 Equipment and Supplies 

6.1 Air incubator capable of maintaining 39± 1°C. 

6.2 Waterbath capable of maintaining 38±1°C and 45°C to 48°C. 

6.3 Fast Phage STEP-1 and STEP-2 dissolvable pouches in desiccated foil zip bags. 

6.4 Fast Phage E. coli tablet in peelable blister. 

6.5 Fast Phage vessels with vented caps and with 100mL fill line. 

6.6 Optional thiosulfate tablet blisters for use with chlorinated waters. 

6.7 Positive control, ФX174 for somatic coliphage and MS2 for male-specific coliphage, supplied with 

Fast Phage or quality control cultures, (ATCC 13706-B1, ATCC 15597-B1). 

6.8 Antibiotic, nalidixic acid 0.1g for somatic coliphage and Ampicillin/Streptomycin 0.015g each, 

freeze dried rehydrated with 10mL water to make prescribed mg/mL stock. 
Nov.28, 2012 Page 5 

6.9 Autoclave, Refrigerator, Thermometer and other Laboratory Apparatus described in Standard 

Methods for the Examination of Water and Wastewater Section 9030. 

6.10 Tryptic Soy Broth- EMD or equivalent 

6.11 Bacto Agar – BD Difco Laboratory or equivalent 

6.12 Plastic Petri Dishes- 100 mm 

6.13 Hand held 366 nm UV Lamp - VWR 36582-158 or equivalent 

6.14 Light box – VWR 21475-460 or equivalent 

6.15 Test Tubes 16x100mm or 18x100mm with culture caps. 

6.16 Equipment for collection and transport of samples 

6.17 Bottles for collection of water—Sterile, wide-mouth, polypropylene, bottles or carboys with screw 

caps 

6.18 Ice chest—Igloo, Coleman, styrofoam box or equivalent 

6.19 Ice 

6.19.1 Wet ice—purchased locally, or 

6.19.2 Ice packs—Blue Ice, UTek cat. no. 429, or equivalent, frozen for use 

6.20 Bubble wrap 

7.0 Reagents and Standards 

7.1 Fast Phage STEP-1 and STEP-2 media in dissolvable pouches (25 pouches per foil zip bag), 28 E. 

coli tablets in blister, and antibiotic freeze dried are supplied as a kit manufactured by Charm 

Sciences and supplied as Catalogue # Fast-Phage-[SOM] or [FPLUS]-25K. The kit is supplied 

with test vessels with caps. The refrigerated shelf life of the desiccated reagents are at least 6 

months from production date. 

7.2 The catalogue number for Fast Phage transparent vessel with vented cap and 100mL fill line. 

Catalogue # 615-013. Or alternatively a non-fluorescent round jar with 150mL or greater with a 

breathable cap. 

7.3 Fast Phage STEP-1 and STEP-2 pouches/E. coli tablets and vessels may be ordered as kits in 25, 

100, 500 or 5000 quantities: Catalogue # Fast Phage-[SOM or FPLUS]-25; Fast Phage-[SOM or 

FPLUS]-100; Fast Phage-[SOM or FPLUS]-500;or Fast Phage-[SOM or FPLUS]-5000. 

7.4 The catalogue number for thiosulfate tablets (blisters of 20 tablets containing 10mg each Na2S2O3) 

is EC-THIO-2—BP. The thiosulfate tablet shelf life is 18 months from packaging when stored at 

room temperature. 
Nov.28, 2012 Page 6 

7.5 Tryptic Soy Broth (TSB) and Bacto agar are routine reagents found in microbiology labs and are 

supplied separately from Fast Phage kit. 

8.0 Sample Collection, Preservation and Storage 

8.1 Water sample is collected according to Standard Methods for the Examination of Water and 

Wastewater Section 9060 A or as as appropriate. If the Fast Phage vessel is used, it must be filled 

to at least the 100 mL level. 

8.2 Sodium thiosulfate appropriate to neutralizing disinfection halides, or a tablet, containing 10 mg 

thiosulfate, may be aseptically added to 100mL sample to neutralize chlorine or other halogen 

treated waters. 

8.3 Preservation and Storage should follow Standard Methods for the Examination of Water and 

Wastewater Section 9060 B or appropriate regulatory requirement. If transporting sample to 

laboratory, it is recommended the sample should be held at less than 10°C. Samples should be 

analyzed as soon as possible but not more than 30 hours after collection.

9.0 Quality Control 

9.1 QA Program- Each laboratory that uses Fast Phage is required to operate a formal quality 

assurance (QA) program. The minimum QA requirements consist of an initial demonstration of 

laboratory capability through performance of the initial demonstration of capability (IDC) test 

(Section 9.3), ongoing analysis of spiked reagent water and field samples to evaluate and document 

data quality, and analysis of positive controls and method blanks as tests of continued acceptable 

performance. Spiked sample results are compared to acceptance criteria based on EPA Method 

1601. Specific quality control (QC) requirements are provided below. General recommendations on 

QA and QC for facilities, personnel, laboratory equipment, instrumentation, and supplies used in 

microbiological analyses are provided in the USEPA Microbiology Methods Manual, Part IV, C 

(Reference 16.5). 

9.2 General QC requirements—Positive control samples may be spiked with pure cultures of MS2 

(male-specific) or ΦX174 (somatic) coliphage supplied with test kits (section 6. 7). All other 

spiked QC samples must be spiked with enumerated sewage filtrate or equivalent. QA/QC 

procedures should be consistent with practices delineated in the 5th addition of the Manual for the 

Certification of Laboratories Analyzing Drinking Water. 

9.2.1 Initial demonstration of capability (IDC). The laboratory shall demonstrate the ability to 

generate acceptable performance with this method by performing an IDC test before 

analyzing any field samples. The procedure for performing the IDC is described in Section 

9.3. IDC tests must be accompanied by a method blank (Section 9.2.2) for each 

coliphage type. 

9.2.2 Method blanks. The laboratory shall analyze method blanks (reagent water sample 

containing no coliphage) to demonstrate freedom from contamination. The procedures and 

criteria for analysis of a method blank are described in Section 9.4. At a minimum, the 

laboratory shall analyze one method blank per spot plate (Section 11.6). In an effort to 

determine if cross-contamination is an issue, the method blank should be spotted onto the 

lawn of host bacteria immediately following the positive control spot. 
Nov.28, 2012 Page 7 

9.2.3 Positive controls. The laboratory shall analyze positive controls to ensure that stock 

coliphage suspensions, host bacterial cultures, and growth media are performing properly 

(Section 9.5). The laboratory shall inoculate one positive control spot per spot plate 

(Section 11.6). If multiple spot plates are inoculated with samples on the same day, a 

single enriched positive control sample may be used to inoculate multiple spot plates on 

that day. 

9.2.4 Matrix spikes (MS). The laboratory shall analyze one set of MS samples for each 

coliphage type when samples are first received from a ground water source for which the 

laboratory has never before analyzed samples (Section 9.6). In addition, the laboratory 

shall analyze one set of MS samples on an ongoing basis after every 20th field sample for 

each ground water source. For example, when a laboratory receives the first sample from a 

source, the laboratory must obtain additional aliquots of the field samples to be used for 

the MS test. When the laboratory receives the 20 th field sample from this site, additional 

aliquots of this sample must be collected and spiked. MS samples should be collected at 

the same time as routine field samples. 

9.2.5 Ongoing demonstration of capability (ODC). The laboratory shall demonstrate that the 

analytical system is in control on an ongoing basis through analysis of ODC samples 

(Section 9.7). The laboratory shall analyze one set of ODC samples after every 20 field 

and MS samples or one per week, whichever occurs more frequently. 

9.2.6 Record maintenance. The laboratory shall maintain records to define the quality of data 

that are generated. The laboratory shall maintain a record of the date and results of all QC 

samples described in Section 9.2. A record of sterility check, IDC, ODC, and MS sample 

results must be maintained. In addition, a log book containing reagent and material lot 

numbers should be maintained along with samples analyzed using each of the lots. 

9.2.7 Performance studies. The laboratory should periodically analyze external QC samples, 

such as performance evaluation (PE) samples, when available. The laboratory should also 

participate in available interlaboratory performance studies conducted by local, state, and 

federal agencies or commercial organizations. The laboratory should review results, 

correct unsatisfactory performance, and record corrective actions. 

9.2.8 The specifications contained in this method can be met if the analytical system is 

maintained under control. 

9.3 Initial demonstration of capability (IDC)—The IDC test is performed to demonstrate acceptable 

performance with the method as written prior to analysis of field samples or to evaluate acceptable 

performance of a method modification. IDC test samples must be spiked with enumerated sewage 

filtrate or equivalent. IDC tests must be accompanied by analysis of a method blank (Section 9.4). 

9.3.1 A total of 10 spiked reagent water samples per coliphage type (male-specific and somatic) 

are required for the IDC test. 100mL reagent water samples should be used. 

9.3.2 IDC samples must be spiked in “bulk” for each coliphage type at concentrations as 

specified in Table 1. For example, for 100mL somatic IDC samples, spike 1.0-L of 

reagent water with 15 PFU to achieve the target spike concentration of 1.5 PFU per 

sample. Please refer to Section 9.10 for the coliphage spiking suspension enumeration 

procedure and Sections 9.11 and 9.12 for spike volume calculations. 
Nov.28, 2012 Page 8 

Table 1. Initial demonstration of laboratory capability (IDC) 

Coliphage type Target spike 

concentration 

(PFU per sample) 

“Bulk” volume 

to be spiked 

Total 

Number of 

Plaques to 

add to Bulk 

Volume 

Minimum 

number of 

positive samples 

out of 10 

Male Specific 1.3 PFU/100mL 1.0 L 13 5 

Somatic 1.5 PFU/100mL 1.0 L 15 5 

9.3.3 Mix the spiked reagent water for 20 minutes and, dispense 10 aliquots into individual 

containers. 

9.3.4 Analyze the spiked IDC samples according to the procedure (Section11). See Table 1 for 

the minimum number of samples that must be positive for each set of 10 IDC samples. If 

system performance is unacceptable, identify and correct the problem and repeat the IDC 

test. 

9.4 Method blank—performed at the frequency specified in Section 9.2.2. 

9.4.1 Prepare and analyze a reagent water sample containing no coliphage using the same 

procedure as used for analysis of the field or QC samples. 

9.4.2 If coliphage, or any potentially interfering organisms are found in the blank, analysis of 

additional samples must be halted until the source of contamination is eliminated, and a 

repeat of the method blank analysis shows no evidence of contamination. Any sample in a 

batch associated with a contaminated blank should be recollected (if holding times have 

been violated) and reanalyzed. Samples from a batch that proves to have no coliphage in 

its blank may be reported. 

9.5 Positive controls—performed at the frequency specified in Section 9.2.3. 

9.5.1 To one vessel, add 1mL of supplied Positive Control (Section 6.7), ФX174 (ATCC 

13706-B1) for somatic coliphage and MS2 (ATCC 15597-B1) for male-specific 

coliphage, previously reconstituted with 10mL reagent water for at least 10 minutes and 

stored refrigerated for up to 3 weeks. Analyze positive control sample by the procedure as 

described in Section 11. 

9.6 Matrix spike (MS) analyses for ongoing assessment of method performance—The laboratory 

shall analyze MS samples according to the frequency in Section 9.2.4. The laboratory shall spike 

and analyze field samples from each ground water source to assess method performance in each 

matrix. For each coliphage type and sample volume, at a minimum, one out of three MS samples 

must be positive. MS test samples must be spiked with enumerated sewage filtrate or equivalent. 

9.6.1 The ongoing MS sample must be collected and analyzed at the same time as the unspiked 

field sample from the same source. 

9.6.2 MS samples must be spiked in “bulk” for each coliphage type and sample volume. Spike 

the bulk samples at concentrations according to Table 2. See Section 9.10 for enumeration 

of coliphage stock and Sections 9.11 and 9.12 for spiking volume calculations. 
Nov.28, 2012 Page 9 

9.6.3 Analyze MS samples according to the procedure (Section 11). 

9.6.4 One or more MS samples out of three must be positive for method performance to be 

considered acceptable for that ground water source. If the matrix spike results are 

unacceptable and the ODC sample and positive control sample results associated with this 

batch of samples are acceptable, a matrix interference may be causing the poor results. 

9.7 Ongoing demonstration of capability (ODC)—Performed at the frequency specified in Section 

9.2.5. To demonstrate that the laboratory is in control for each coliphage type, at a minimum, one 

out of three reagent water spiked samples must be positive. ODC test samples must be spiked with 

enumerated sewage filtrate or equivalent. 

9.7.1 ODC samples must be spiked in “bulk” for each coliphage type. Spike the bulk samples at 

concentrations according to Table 2. For example, for 100mL somatic ODC samples, 

spike 300mL of reagent water with 4.5 PFU of somatic coliphage to achieve a target spike 

concentration of 1.5 PFU per sample, based on stock coliphage enumeration. See Section 

9.10 for enumeration of coliphage stock and Sections 9.11 and 9.12 for spiking volume 

calculations. 

9.7.2 Mix the spiked reagent water by swirling the container and dispense three aliquots into 

individual containers. 

9.7.3 Analyze the spiked ODC samples according to Section 11. One or more out of three 

samples must be positive for each coliphage type and sample volume being evaluated. If 

not, method performance is unacceptable. Identify and correct the problem and perform 

another ODC test before continuing with the analysis of field samples. 

Table 2. MS and ODC sample spiking requirements for ongoing evaluation of method 

performance 

Coliphage type Target spike 

concentration 

(PFU per sample) 

“Bulk” volume 

to be spiked 

Total 

Number of 

Plaques to 

add to Bulk 

Volume 

Minimum 

number of 

positive samples 

out of 3 

Male-Specific 1.3 PFU/100mL 300 mL 3.9 1 

Somatic 1.5 PFU/100ml 300 mL 4.5 1 

9.8 Expanded MS test— If IDC and Expanded MS test performance is equal to or better than the 

criteria set forth in Tables 1 and 3, then the modified version of the method is acceptable. 

Expanded MS analyses for evaluation of modified method performance are as follows. Please 

note: the expanded MS test must be accompanied by an unspiked matrix sample. If the unspiked 

matrix sample tests positive, the Expanded MS test must be re-run. 

9.8.1 A total of 10 spiked field samples per coliphage type (male-specific and somatic) are 

required. 

9.8.2 Samples must be spiked in “bulk” for each coliphage type. Spike the bulk samples at 

concentrations according to Table 3. For example, for 100mL somatic expanded-MS 

samples, spike 1.0 L of ground water with a target spike dose of 15 PFU of somatic 
Nov.28, 2012 Page 10 

coliphage. See Section 9.10 for enumeration of coliphage stock and Sections 9.11 and 9.12 

for spiking volume calculations. 

Table 3. Expanded MS test requirements for evaluation of method modification 

performance 

Coliphage type Target spike 

concentration 

(PFU per sample) 

“Bulk” volume 

to be spiked 

Total 

Number of 

Plaques to 

add to Bulk 

Volume 

Minimum 

number of 

positive samples 

out of 10 

Male Specific 1.3 PFU/100mL 1.0 L 13 4 

Somatic 1.5 PFU/100mL 1.0 L 15 5 

9.8.3 Mix the spiked ground water by swirling the container. For each coliphage type dispense 

ten 100mL aliquots into individual containers. 

9.8.4 Analyze the spiked ground water samples according to the two-step enrichment procedure 

(Section 11). Table 3 specifies the minimum number of samples that must be positive for 

each set of 10 Expanded MS samples. 

9.9 Raw Sewerage and preparation for IDC, ODC, and Expanded MS Test 

9.9.1 The double agar layer (DAL) procedure is used to enumerate stock suspensions of somatic 

and male specific coliphage for use in spiking quality control samples. Use sewage filtrate 

prepared as follows: 

9.9.1.1 Collect approximately 100mL of raw sewage in a 250mL collection bottle. 

9.9.1.2 Transport to the laboratory on ice. Analysis of raw sewage filtrate should begin 

within 24 hours of collection. 

9.9.1.3 Allow the raw sewage to settle at 4°C ± 1°C for 15-20 minutes. This will make 

the filtration process easier. 

9.9.1.4 Remove a sterile, 20mL syringe from its package, aseptically remove plunger 

from barrel, and attach a 0.45 μm low-protein-binding filter disk to the syringe 

barrel. 

9.9.1.5 Pipet 10 to 15mL of supernatant from settled sewage into the syringe barrel. 

9.9.1.6 Hold the assembly over a sterile 15mL polypropylene tube with screw-cap or 

snap-cap, insert the plunger into the syringe barrel, and push the sewage through 

the filter into the sterile tube. If filter clogs, change it as necessary and continue 

to filter sewage until at least 10mL of filtered sewage is obtained in the 15mL 

polypropylene tube (filtration may require use of numerous filters). 

9.9.1.7 Cap the tube, label with source, date, and initials, and store the filtrate at 4±1°C 

until ready to assay. If filtrate is stored more than 24 hours, it must be re-titered 

before use. 
Nov.28, 2012 Page 11 

9.9.2 A minimum of four different volumes/dilutions are necessary for the (DAL) enumeration 

of the sewage filtrate: 

• Undiluted 

• 0.1 Dilution 

• 0.01 Dilution 

• 0.001 Dilution 

Additional dilutions may be necessary. TSB without antibiotics (Section 7.5) is used as the 

diluents and as the method blank. 

9.9.3 Aseptically add 9.0mL of TSB without antibiotics (Section 7.5) into each of four (or more) 

sterile dilution tubes (Section 6.15). Label them as “0.1,” “0.01,” “0.001,” “method 

blank,” etc. 

9.9.4 Add 1.0mL of the coliphage stock or sewage filtrate to the tube of TSB labeled “0.1.” Cap 

the tube and vortex for 5 seconds on a medium-high setting (if available) or until well 

mixed. 

9.9.5 Add 1.0mL of the well-mixed 0.1 dilution to a tube with 9 mL of TSB labeled “0.01". Cap 

the tube and vortex for 5 seconds on a medium-high setting (if available) or until well 

mixed. 

9.9.6 Add 1.0mL of the well-mixed 0.01 dilution to a tube with 9 mL of TSB labeled “0.001.” 

Cap the tube and vortex for 5 seconds on a medium-high setting (if available) or until well 

mixed. 

9.9.7 Add 1.0mL of TSB without antibiotics (Section 7.3.1.1) to the tube labeled “method 

blank.” Cap the tube and vortex for 5 seconds on a medium-high setting (if available) or 

until well mixed. 

9.10 Coliphage spiking suspension enumeration by double agar layer (DAL) procedure—In this 

procedure, a tube of molten 0.7% TSA “top agar” with added host bacteria is inoculated with 

coliphage stock and will be poured into a 1.5% TSA “bottom agar” plate. Four Method 1601: 

Two-Step Enrichment Procedure April 2001 dilutions of sewage filtrate will be analyzed in 

duplicate for each coliphage type. As a result, nine double-agar layer plates will be required for 

each coliphage type: two plates per dilution (undiluted, 0.1, 0.01, and 0.001) and one method blank 

plate. The procedures 9.10.1 – 9.10.12 are written for the simultaneous performance of somatic 

and male-specific coliphage. A lab may choose to test for only one type of coliphage and therefore 

may modify the procedure to enumerate only the appropriate type of coliphage. Please note: 

Laboratories are not permitted to modify or omit any aspects of the coliphage stock enumeration 

procedure (Section 9.10). As a result, magnesium chloride or calcium chloride must not be 

added the sample or media. 

9.10.1 Bottom layer agar preparation 

9.10.1.1 Prepare two flasks of 300 mL 1.5% TSA by adding 4.5 g agar (section 6.11) 

and 9.0 g TSB (section 6.12) to 300ml. Heat to boil and then autoclave 15 
Nov.28, 2012 Page 12 

minutes at 121oC and 15 psi. Place in a 45 to 48oC water bath to temper the 

prepared medium until used. 

9.10.1.2 To one flask add 3 mL of nalidixic acid solution (section 6.8) and swirl to mix. 

To second flask add 3 mL of ampicillin/streptomycin solution (section 6.8) and 

swirl to mix. 

9.10.1.3 Dispense 17 mL of the nalidixic acid TSA to 100 mm petri dish (Section 6.12) 

on a level surface. Allow to cool and air dry for 10 minutes. Cover and label as 

somatic bottom layer. Store bagged and refrigerated up to 2 weeks. Before use, 

check for media sterility by incubating at least 1 plate at 36oC for 48-72 hours 

and observing for growth. 

9.10.1.4 Dispense 17 mL of the ampicillin/streptomycin TSA to 100 mm petri dish 

(Section 6.12) on a level surface. Allow to cool and air dry for 10 minutes. 

Cover and label as male-specific bottom layer. Store bagged and refrigerated up 

to 2 weeks. Before use, check for media sterility by incubating at least 1 plate at 

36oC for 48-72 hours and observing for growth. 

9.10.2 Culture preparation. 

9.10.2.1 Add an CN-13 E.coli tablet to TSB with nalidixic acid as described in section 

11.1.2. 

9.10.2.2 Add an Famp E.coli tablet to TSB with ampicillin/streptomycin as described in 

section 11.1.2. 

9.10.2.3 Incubate tubes with E.coli added in 38°C water bath for 4-5 hours until growth 

is visibly turbid. 

9.10.3 0.7% top agar preparation. 

9.10.3.1 Prepare two flasks of 100mL 0.7% TSA by adding 0.7 g agar (section 6.11) 

and 3.0g TSB (section 6.10) to 100ml. Heat to boil and then autoclave 15 

minutes at 121°C and 15 psi. Place in a 45 to 48°C water bath to temper the 

prepared medium until used. 

9.10.3.2 To one flask add 1mL of nalidixic acid solution (section 6.8) and swirl to mix. 

To second flask add 1mL of ampicillin/streptomycin solution (section 6.8) and 

swirl to mix. 

9.10.3.3 18 tubes are necessary to enumerate four dilution volumes in duplicate for each 

phage type. The 18 tubes also includes an additional method blank tube for each 

phage type. Add 5mL of each prepared 0.7% TSA to 9 tubes. There should be 9 

tubes with nalidixic acid labeled for somatic coliphage determination and there 

should be 9 tubes labeled for male specific coliphage determination. Tubes 

should remain in water bath. 

9.10.4 As a precaution against contamination, disinfect a workspace near the water bath with a 

1:100 dilution of household bleach and allow to dry. If workspace can be corroded by 
Nov.28, 2012 Page 13 

bleach, use an ethanol solution of 70% or greater or use an EPA registered disinfectant or 

sanitizer appropriate for viruses. 

9.10.5 Assemble 9 each of 1.5% TSA bottom agar plates (Section 9.10.1.3 and 9.10.1.4) and 

allow them to reach room temperature. Label so that the following information is 

identifiable: 

• Dilution of stock filtrate or method blank 

• Bacterial host (E. coli CN-13 or E. coli Famp) 

• Coliphage type (somatic for the E. coli CN-13 bacterial host or male-specific for 

the E. coli Famp bacterial host) 

• Date 

• Time 

Please note: The following steps are critical. To ensure viability of bacterial host and 

coliphage, do not add bacterial host and coliphage spiking suspension until ready to plate. 

9.10.6 Preparation of plates for enumeration of somatic coliphage 

9.10.6.1 With the top agar tube still in the water bath, aseptically inoculate a top agar 

tube containing nalidixic acid with 100μL of cultured E. coli CN-13 (section 

9.10.2.3). 

9.10.6.2 Immediately add 500μL (0.5mL) of undiluted coliphage stock or sewage filtrate. 

9.10.6.3 Mix the inoculum by rolling the tube briefly in palm of hand. 

9.10.6.4 Pour contents into one of the two bottom agar plates marked “undiluted, E. coli 

CN-13, somatic." 

9.10.6.5 Duplicate analysis—Repeat Sections 9.10.6.1 through 9.10.6.4 for the duplicate. 

9.10.6.6 Repeat Sections 9.10.6.1 through 9.10.6.5 for each dilution volume. 

9.10.7 Preparation of somatic coliphage method blank 

9.10.7.1 With the nalidixic acid top agar tube still in the water bath, aseptically inoculate 

a top agar tube containing nalidixic acid with 100μL of log-phase E. coli CN-13 

(section 9.10.2.3).

9.10.7.2 Immediately add 500μL (0.5mL) of TSB from the “method blank” dilution tube. 

9.10.7.3 Mix the inoculum by rolling the tube briefly in palm of hand. 

9.10.7.4 Pour contents into a naladixic acid bottom agar plate marked “method blank, E. 

coli CN-13, somatic. 
Nov.28, 2012 Page 14 

9.10.8 "Preparation of plates for enumeration of male-specific (F+) coliphage—Repeat Section 

9.10.6 using bottom and top agar containing ampicillin/streptomycin and log-phase E. coli 

Famp (section 9.10.2.3) 

9.10.9 Preparation of the male-specific (F+) coliphage method blank—Repeat Section 9.10.7 

using top and bottom agar containing ampicillin/streptomycin and log-phase E. coli Famp

(section 9.10.2.3) 

9.10.10 Store undiluted coliphage stock or sewage filtrate at 4±1°C for use in preparing new 

dilutions for positive controls, IDC, ODC, and MS samples. 

9.10.11 After the top agar hardens, cover, invert the plates and incubate for 16 to 24 hours at 

36±1°C. 

9.10.12 Circular zones of clearing (typically 1 to 10 mm in diameter) in lawn of host bacteria after 

16 to 24 hours of incubation are plaques. Count the number of plaques on each plate. 

Please note: The use of a light box (Section 6.14) to evaluate results is recommended. 

9.11 Calculate the PFU / mL for each coliphage type. 

9.11.1 The number of plaque forming units (PFU) per mL in the coliphage spiking suspension 

will be calculated using DAL plates that yield plaque counts within the desired range of 

zero to 300 PFU per plate for male-specific (F+) coliphage and zero to 100 PFU per plate 

for somatic coliphage. There may be occasions when the total number of plaques on a 

plate will be above the ideal range. If the count exceeds the upper range or if the plaques 

are not discrete, results should be recorded as “too numerous to count” (TNTC). 

9.11.2 For each coliphage type, sum the number of PFU from all dilutions with plaques (on either 

of the duplicate plates), excluding dilutions with all TNTC or all zeros. (See equation in 

Section 9.11.5) 

9.11.3 Sum the undiluted sample volumes used to inoculate all replicate plates at all dilutions 

having useable counts (as defined above). (See equation in Section 9.11.5) 

9.11.4 Divide the sum of the PFU by the sum of the undiluted sample volume to obtain PFU/ mL 

in the spiking suspension. (See equation in Section 9.11.5)

9.11.5 The equation for Sections 9.11.1 through 9.11.4 is as follows: 

Undiluted spiking suspension PFU / mL = (PFU1 + PFU2... PFUn)/(V1 + V2.... Vn) 

Where: 

• PFU = number of plaque forming units from plates of all countable sample 

dilutions (dilutions with 1 or more PFU per plate, excluding dilutions with all 

TNTC or all zeros (0) 

• V = volume of undiluted sample in all plates with countable plaques 

• n = number of useable counts 
Nov.28, 2012 Page 15 

Example DAL data 

Dilution PFU / plate (for each 

duplicate plate) 

Volume of undiluted spiking 

suspension (mL) 

Undiluted TNTC, TNTC 0.5mL 

1:10 35, 37 0.05mL 

1:100 3, 0 0.005mL 

1:1000 0, 0 0.0005mL 

Example: (35 + 37 + 0 + 3)/(0.05 + 0.05 + 0.005 + 0.005) = 75/0.11 = 682 PFU / mL. In this 

example, the undiluted spiking suspension contains approximately 682 PFU per mL, the 1 : 10 

dilution contains approximately 68.2 PFU per mL, the 1 :100 dilution contains approximately 6.82 

PFU per mL, and the 1 : 1000 dilution contains approximately 0.682 PFU per mL. 

9.12 Calculation for preparing IDC, ODC, MS, and positive control spikes 

9.12.1 Use a dilution of the QC sample spiking suspension that will result in a bulk spike volume 

between 0.1 and 3.0 mL for the spike concentration specified in IDC Section 9.3, ODC 

Section 9.7, or MS Sections 9.6 or 9.8. 

9.12.2 Use the following equation to determine the spiking volume: 

S= (T)(B)/(C) 

where, 

S = Spike volume (mL) 

T = Target number of coliphage per sample (PFU) 

B = Number of samples that will be spiked (only necessary when multiple QC samples are 

spiked in bulk) 

C = Concentration (PFU/mL) in the dilution to be used for spiking 

9.12.3 For example, for 100mL, somatic IDC samples (Section 9.3.2): T) A spike dose of 1.5 

PFU is needed per 100mL sample B) A total of ten, 100mL samples will be spiked at the 

same time C) The 1 : 100 dilution contains 6.82 PFU / mL. The equation would be solved 

as follows: 

S= (1.5 PFU)(10)/(6.82 PFU / mL)=2.19mL 

As a result, approximately 2.2mL of the 1:100 dilution would be spiked into the 1L bulk 

sample. The 1L bulk sample would be mixed and ten, 100mL aliquots dispensed. Each 100 

mL sample should contain approximately 1.5 PFU. 

9.13 Use the enumerated somatic and male-specific stocks to spike the two-step enrichment IDC, ODC, 

MS, and positive control (PC) samples as described in this Section 9. 
Nov.28, 2012 Page 16 

10.0 Calibration and Standardization 

10.1 Use NIST traceable thermometer calibrated to 0.5°C graduations to check temperature of 

incubator. The mercury should not be separated. Alternatively, because of safety issues regarding 

mercury thermometers, a thermometer that meets the requirements of NIST Monograph SP-250-23 

can be used. 

10.2 Check temperature of incubator twice daily at least 4 hours apart or use constant recording device. 

Record measurements in a log book. 

10.3 Check temperatures in water baths, refrigerators, -20oC freezers daily to ensure operation within 

stated limits of Fast Phage protocol and record measurements in a log book. 

10.4 Follow Methods for the Examination of Water and Wastewater Section 9030 A for other 

laboratory equipment specifications such as pipets, graduated cylinders, dilution bottles, 

refrigerator, autoclaves.

10.5 Check balances annually using ASTM-certified Class 2 reference weights. 

10.6 Calibrate spectrophotometer prior to each use. Use TSB without antibiotics as the blank. 

10.7 Adhere to all quality control measures set forth in the EPA Manual for the Certification of 

Laboratories Analyzing Drinking Water and Standard Methods 9020. 

11.0 Procedure 

11.1 Prepare Spot Plates - In advance of testing samples, or during the incubation time of the samples, 

prepare spot plates for plaque detection. 

11.1.1 Make TSB (section 6.10) (Tryptic Soy Broth 3g/100mL) tubes, dissolve and add 10 mL 

into 16 x 150 culture tubes with screw caps. Autoclave TSB tubes for 15 minutes. TSB 

tubes may be refrigerated 4 -8°C for 3 months. 

11.1.2 To 10mL sterilized Tryptic Soy Broth add 0.1 mL of rehydrated antibiotic solution 

(section 6.8) and add one E. coli tablet (section 6.4). Gently swirl to disperse tablet.

11.1.3 Place in water bath 38±1°C for 4-5 hours until visibly turbid.

11.1.4 While E. coli is culturing, in 500mL flask prepare 300mL TSA (Tryptic Soy Agar, TSB 

9g (section 6.10) and Bacto Agar 2.25g (section 6.11)) and bring to boil. Autoclave for 15 

minutes. Place TSA in water bath 45-48°C water bath to temper and prevent 

solidification. 

11.1.5 Add 3mL of respective rehydrated antibiotic solution to 300mL 45-48°C tempered agar. 

Swirl to mix. 

11.1.6 Add 6mL of E. coli culture, 11.1.3, to 300 mL TSA. Swirl to mix sample and pipet 20mL 

to 100mm petri dishes (section 6.12). Allow plates to solidify and air dry for 10 minutes. 

Store spot plates inverted and refrigerated in sealed containers. Plates may be used for up 

to 5 days. 
Nov.28, 2012 Page 17 

11.2 Collect sample: If collecting from tap, run water for several minutes prior to collecting water 

sample. Add water sample to the 100mL vessel fill line or alternatively measure with a sterile 

100mL graduated cylinder and add water to sample. 

11.2.1 If applicable, add thiosulfate tablet (section 6.6) by peeling blister foil and adding tablet to 

water. Do not touch tablet. Replace cap and swirl to dissolve tablet. Store sample 

refrigerated until ready to test. If shipping to a test laboratory, ship refrigerated. 

11.2.2 NOTE: Fast Phage Kits are coliphage specific. Media pouches, antibiotic and E. coli are 

not interchangeable between kits. Take appropriate precautions to segregate kit 

components and to add correct kit components for the type of coliphage tested. 

11.3 Add the STEP-1 Fast Phage (section 6.3) media pouch for somatic or for male-specific coliphage 

to the measured water sample in the supplied vessel. Allow it to dissolve over 10 minutes with 

periodic gentle swirling. 

11.4 Add one E. coli tablet (section 6.4), either CN-13 for somatic coliphage or Famp for male- specific 

coliphage, preparation per test sample. Swirl to break up tablet and allow particulates to settle. 

11.5 Incubate the vessel contents for 0.5 hours in a 38+1°C water bath and then transfer for 4.5 hours 

to a 39+1°C static incubator. Total incubation time 5.0 hours. 

11.6 To complete the Fast Phage Method 1601 modification, transfer a 10μL loop of STEP-1 

enrichment from each sample on to a spot plate (section 11.1.6). Allow plate to air dry to absorb 

liquid. This may take 30 minutes. 

11.6.1 Incubate spot plate inverted overnight, 11-25 hours, in 39+1°C incubator. 

11.6.2 Observe the spots for each sample. If the spots show inhibition of bacterial lawn, or clear 

zone with no growth, or small or micro-plaques in the sample spot, or if STEP-2 

fluorescent (11.7.3) with confirmatory spot plate lysis (11.7.4), record sample as positive 

(> 1 PFU coliphage/100mL) for coliphage. If spot has uniform E. coli growth on the agar 

lawn and smooth edges, record as not found (<1PFU coliphage/100mL). A light box may 

aid in the evaluation and interpretation of lytic zones. 

11.6.3 Questionable spots may be picked/scooped into 0.5mL TSB (section 6.10) with a blunt end 

of a Pasteur pipet, allowed to equilibrate for 5 minutes, and 10μL spotted on to another 

plate. Samples may also be filtered through a 0.45μm low-protein-binding filter and 

spotted to remove interference from bacteria in the sample. Samples spots should be 

allowed air dry and then incubate overnight. Observe as previously to determine positive 

or not found. 

11.7 For same day fluorescent presumptive-positive prediction, fifteen minutes prior to completion of 

the 5.0 hours STEP-1incubation, add STEP-2 media pouch to 100mL RO/DI or 100mL reagent 

water in a transparent Fast Phage vessel. 

11.7.1 On completion of the 5.0 hours incubation, transfer STEP-1 culture, 10mL for somatic 

coliphage or 1.0mL for male-specific coliphage, to the dissolved STEP-2 media. 
Nov.28, 2012 Page 18 

11.7.2 Incubate the STEP-2 vessel contents in a 38+1°C water bath, 0.5 hours for somatic 

coliphage and 1.0 hours for male specific coliphage, and then transfer to a 39±1°C static 

incubator for a total STEP-2 incubation time 3.0 hours from transfer. 

11.7.3 At hourly intervals, the STEP-2 vessels are observed under 366nm UV light. If coliphage 

are absent the sample will not fluoresce after 3 hours and will appear turbid. If coliphage 

are present, the media will fluoresce in 1-3 hours and the samples are presumptivepositive. Samples may also appear clear (non-turbid), and or turn blue. Confirmation of 

presumptive positive by spot plate is required, steps 11.7.4 and 11.7.5. 

11.7.4 Transfer a 10μL loop of STEP-2 samples to a spot plate. Allow plate to air dry to absorb 

liquid. This may take 30 minutes. Incubate spot plate inverted overnight, 11-25 hr, in a 

39±1°C incubator. 

11.7.5 Observe the spots from fluorescent positive samples. If either the STEP-1 spot result, or 

the STEP-2 spots show inhibition of bacterial lawn, or clear zone with no growth, or small 

or micro-plaques in the sample spot, record sample as positive (> 1 PFU 

coliphage/100mL) . If both the STEP-1 and the STEP-2 spot result are uniform E. coli

growth on the agar lawn and with smooth edges, record as not found (<1 PFU 

coliphage/100mL). 

12.0 Data Analysis and Calculations (Interpretation) 

For Rapid Prediction of plaque formation observe STEP-2 vessel in the dark under a long UV 366 

nm light (section 6.13) as follows: 

12.1 If the sample is not fluorescent, and is turbid at 3 hours STEP-2 (8 hours from sample start in 

STEP-1), sample is interpreted “predicted absence of coliphage [type tested]”. 

12.2 If the sample is fluorescent at any time up to 3 hours STEP-2, sample is interpreted “predicted 

presence of coliphage [type tested]”. 

12.3 Positive fluorescent samples may also be clear (non-turbid) or turn blue; but fluorescence is the 

determinative factor in determining positive. 

Identical negative samples viewed under UV light and 

ambient room light. UV results show negative nonfluorescent results compared to positive fluorescence 

results. The same samples viewed in ambient light 

show the negative sample as turbid yellow, while the 

positive sample may appear greenish with more clarity.

PositivePresence of 

coliphage 

Negative – 

Absence of 

Coliphage 
Nov.28, 2012 Page 19 

12.4 Spot plates should be observed the next day 11-25 hours after STEP-1 (section 11.6) and 

fluorescent positive STEP-2 loop transfer (section 11.7.4). Observe the spots for each sample. If 

the either spot show inhibition of bacterial lawn, or clear zone of no growth, or small or microplaques in the sample spot, record sample as positive (> 1 PFUcoliphage/100mL) for coliphage 

[type tested]. If spots have uniform E. coli growth on the agar lawn and smooth edges, record as 

not found (<1PFU coliphage/100 mL [type tested]). See example photographs below. 

The photograph of a male-specific Step-1 plaque confirmation plate shows ten different samples 

spotted from E. coli Step-1 enrichment samples onto a lawn of E. coli seeded agar and Positive 

and Negative Controls. Zones of inhibition observed in samples 5, 8 and PC (positive control) 

indicate positive coliphage samples. Note that sample 5 is a coliphage morphology showing almost 

a clear zone of inhibition with no host from the amplified culture. PC is a coliphage morphology 

where the amplified host grow as a circular spot on the agar but have a surrounding zone of 

inhibition into the E. coli seeded in the agar. Sample 8 has a micro-plaque morphology with a 

faint-spotty circle of amplified host and a zone of inhibition into the E. coli seeded host. 
Nov.28, 2012 Page 20 

The table below are photographs of some plaque morphologies that may be seen on spot plates : 

 Clear 

inhibitory zone 

Zone of 

inhibition 

surrounding 

amplified host 

growing on agar 

surface 

Microplaques in 

amplified host 

growing on agar 

surface with inhibition 

of edge of amplified 

host and E. coli

seeded in agar 

Microplaques on edge 

of amplified host and 

seeded E. coli growing 

in ager 

Inhibitory zone with 

inner faint circular 

growth of amplified 

host 

Microplaques creating 

a speckled amplified 

host growth with a very 

rough surface 

12.5 Enumeration of coliphage QC spiking suspensions is described in Sections 9.10-9.12. 

13.0 Method Performance 

13.1 A shared sample evaluation was conducted with 4 laboratories proficient in coliphage analysis and 

meeting IDC and ODC for method 1601 and Fast Phage. 

13.2 Regionally collected waste water samples, Arizona (AZ), Wisconsin (WI) and Massachusetts 

(MA), were sent to the co-ordinating laboratory in MA. Samples were filtered and sent to 4 

collaborating laboratories in AZ, New Jersey (NJ), Vermont (VT) and WI. Double layer agar 

analyses were performed day of receipt and used by each lab to determine spike levels to meet 

expanded-MS criteria. 

13.3 The receiving laboratory local ground water was spiked to target expanded-MS levels and ten 

replicates each were evaluated using Method 1601 and Fast Phage. In total 4 different regionally 

diverse ground waters were spiked with 3 different waste waters to generate n=120 somatic and 

n=120 male-specific analyses of Fast Phage and Method 1601. 
Nov.28, 2012 Page 21 

13.4 Section 17 provides details of the comparative individual analyses. In 24 of 24 experimental 

cycles, 12 cycles each somatic and male-specific, expanded-MS criteria presented in Table 3 were 

met by both methods. 

13.5 There were 77 % somatic positive, 92 positive of 120 samples, by Fast Phage method compared to 

85% positive by Method 1601, 102 positive of 120 samples. There were 89% male-specific 

positive, 107 of 120 samples, by Fast Phage modification compared to 96% Method 1601 positive, 

115 positive of 120 samples. The maximum difference between any Fast Phage and any Method 

1601 comparative cycle was -40% which occurred 1 of 12 instances of male-specific coliphage. 

The maximum difference was -30% in one of 12 cycles of somatic coliphage. In these lower Fast 

Phage detections the expanded-MS criteria of Method 1601 were met. 

13.6 Cochran–Mantel–Haenszel (CMH) Chi square (corrected) analysis of the comparability results, 

section 13.5, do not show significant differences. The somatic X2 gave a population overlap of 

P=0.13 which became P=0.29 when the one 30% different cycle was excluded. The male specific 

X

2

 gave a population overlap P=0.06 which became P= 0.36 when the one 40% different cycle was 

excluded. 

13.7 Specificity of Fast Phage results was determined by re-culturing the Fast Phage spot culture per 

the confirmation method section 12.3 of Method 1601 using unfiltered spots as method comparison 

and filtered spots as the referee method. 

13.8 Specificity of the somatic coliphage Fast Phage method had an observed method agreement of 94% 

with a false negative rate of 4.6% and false positive rate of 13.6%. Fast Phage had an observed 

reference method agreement of 93% with a false negative rate of 4.6% and a false positive rate of 

17.4%. The observed method had an agreement with the reference method of 97% with a false 

negative rate of 2.3% and a false positive rate of 4.6%.

13.9 Specificity of the male specific Fast Phage method had an observed method agreement of 99% with 

a false negative rate of 0.9% and a false positive rate of 0%. Fast Phage had an observed 

reference agreement of 98% with a false negative rate of 2.7% and a false positive rate of 0%. The 

observed method had an agreement with the referee method of 98% with a false negative rate of 

1.8% and a false positive rate of 0%. 

14.0 Pollution Prevention 

14.1 Additional minimization of pollutants is not an expected result from use of the Fast Phage method 

in comparison to other phage detection methods. 

15.0 Waste Management 

15.1 Dispose in accordance with good laboratory practices. Vessels and contents should be autoclaved 

prior to disposal. Follow local, state and regional disposal regulations. 

16.0 References 

16.1 Fast Phage MSDS- Charm Sciences 
Nov.28, 2012 Page 22 

16.2 Standard Methods for the Examination of Water and Wastewater, 21st Edition 

16.3 EPA Method 1601: Male-specific (F+) and Somatic Coliphage in Water by Two-step Enrichment 

Procedure, April 2001 

16.4 Manual for the Certification of Laboratories Analyzing Drinking Water, EPA 815-B-97-001. 

16.5 Microbiological Methods for Monitoring the Environment, EPA-600 / 8-78-017, December 1978. 

http://nepis.epa.gov . 

17.0 Method Comparison 

17.1 Method comparison data, Table 17.1, of 120 Fast Phage somatic and Method 1601 tests using 3 

geographically diverse filtered waste water samples, n=10 replicates at 4 different laboratories, 

diluted into geographically diverse ground water indicated 85% positive samples by Method 1601, 

and 77% positive samples by Fast Phage. Fisher exact test of each cycle and CMH Chi square 

analysis of entire group P=0.13 do not indicate significant difference between the methods. 

Table 17.1: Comparability Study- Somatic Coliphage 

Waste 

Location 

Ground water 

Location PFU/100mL 

EPA 

1601 

Positive 

n 

Fast 

Phage 

Positive 

n 

Fluorescence 

Positive 

n 

Total 

Samples 

n 

MA WI 1.4 9 7 7 10 

MA NJ 1.4 9 10 10 10 

MA VT 0.8 6 7 5 10 

MA AZ 1.1 9 6 6 10 

AZ WI 1.3 10 8 8 10 

AZ NJ 1.2 10 8 8 10 

AZ VT 0.8 6 5 4 10 

AZ AZ 1.2 8 8 8 10 

WI WI 1.1 10 9 9 10 

WI NJ 1.1 8 10 8 10 

WI VT 1.1 8 7 7 10 

WI AZ 1.3 9 7 7 10 

Sum 

%Positive 

102 92 87 120 

85% 77% 73% 

17.2 Method comparison data, Table 17.2, of 120 Fast Phage and Method 1601 male-specific tests 

using 3 geographically diverse filtered waste water samples, n=10 replicates at 4 different 
Nov.28, 2012 Page 23 

laboratories, diluted into geographically diverse ground water indicated 96% positive samples by 

Method 1601, and 89% positive samples by Fast Phage. Fisher exact test of each test cycle and 

CMH chi square analysis of entire group P=0.06 do not indicate significant difference between the 

methods. 

Table 17.2: Comparability Study- Male specific Coliphage 

Waste 

Location 

Ground water 

Location PFU/100mL 

EPA 

1601 

Positive 

n 

Fast 

Phage 

Positive 

n 

Fluorescence 

Positive 

n 

Total 

Samples 

n 

MA WI 1.0 10 10 9 10 

MA NJ 1.5 10 10 10 10 

MA VT 1.2 10 8 7 10 

MA AZ 1.3 10 9 9 10 

AZ WI 1.2 10 10 8 10 

AZ NJ 1.0 10 10 10 10 

AZ VT 1.0 10 10 10 10 

AZ AZ 1.3 9 9 9 10 

WI WI 0.9 10 9 9 10 

WI NJ 1.5 10 10 10 10 

WI VT 0.3 10 6 8 10 

WI AZ 1.2 6 6 6 10 

Sum 

%Positive 

115 107 105 120 

96% 89% 88% 

17.3 The somatic selectivity of the Fast Phage positive results compared to the confirmation steps of 

Method 1601 are shown in Table 17.3 and compared in 4 fold tables below. The results indicate a 

93% overall agreement with EPA Method 1601 confirmation plate using filtered method. There 

was a 4.6 % false negative error and a 17.4% false positive error. This compares to the EPA unfiltered method that had a 97% overall agreement with the the filtered method with a 2.3% false 

negative error and a 4.6% false positive error. The kappa statistic k=0.78 indicate substantial 

agreement between Fast Phage method and the reference Method 1601. 
Nov.28, 2012 Page 24 

Table 17.3 Selectivity Study- Somatic Coliphage 

Waste 

Location

Ground 

Water 

Location

Fast 

Phage 

Negative 

n 

Fast 

Phage 

Positive 

n 

Unfiltered spot 

re-picked to EPA 

1601-n 

Filtered spot repicked to EPA 

1601-n 

Unfiltered EPA 

1601 vs Filtered 

EPA 1601-n 

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

MA WI 3 7 3 7 0 0 3 7 0 0 3 7 0 0 

MA NJ 0 10 0 10 0 0 0 10 0 0 0 10 0 0 

MA VT 3 7 2 7 1 0 3 6 0 1 2 6 0 2 

MA AZ 4 6 4 6 0 0 3 6 1 0 3 6 1 0 

AZ WI 2 8 2 6 0 2 2 6 0 2 4 6 0 0 

AZ NJ 2 8 0 8 2 0 0 8 2 0 0 10 0 0 

AZ VT 5 5 la la la la la la la la la la la la 

AZ AZ 2 8 2 8 0 0 2 8 0 0 2 8 0 0 

WI WI 1 9 1 9 0 0 1 9 0 0 1 9 0 0 

WI NJ 0 10 0 10 0 0 0 10 0 0 0 10 0 0 

WI VT 3 7 3 6 0 1 3 6 0 1 4 6 0 0 

WI AZ 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 

n=number TP indicates true-positive, i.e., positive results recultured as positive; FN indicates falsenegative, i.e., negative results recultured as positive; FP indicates false-positive, i.e., positive results 

recultured as negative. la = laboratory accident -QC samples disqualified the selectivity portion of this 

cycle 

 Fast Phage Results 

 + - Total 

Somatic EPA- 

unfiltered

+ 84 4 88

- 3 19 22

Total 87 23 110
Nov.28, 2012 Page 25 

 Fast Phage Results 

 + - Total 

Somatic EPA- 

filtered

+ 83 4 87

- 4 19 23

Total 87 23 110

 Somatic EPA- unfiltered 

+ - Total 

Somatic EPA  

filtered

+ 86 2 88

- 1 21 22

Total 87 23 110

17.4 The male-specific coliphage selectivity of the Fast Phage positive results compared to the 

confirmation steps of Method 1601 are shown in Table 17.4 and compared in 4 fold tables below. 

The results indicate a 98% agreement with the EPA Method 1601 confirmation plate using filtered 

method with a 2.7 % false negative error and a 0% false positive error. This compares to the EPA 

Method 1601 un-filtered method that had a 98% agreement with the independent method with a 

1.8% false negative error and a 0% false positive error. The kappa statistics k=0.90 indicate 

almost perfect agreement between Fast Phage method and the reference Method 1601 
Nov.28, 2012 Page 26 

Table 17.4 Selectivity Study- Male Specific Coliphage 

Waste 

Location

Ground 

Water 

Location 

Fast 

Phage 

Negative 

n 

Fast 

Phage 

Positive 

n 

Unfiltered spot repicked to EPA 1601 

n 

Filtered spot repicked to EPA 1601 

n 

Unfiltered EPA 1601 

vs Filtered EPA 1601 

n 

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

MA WI 0 10 0 10 0 0 0 10 0 0 0 10 0 0 

MA NJ 0 10 0 10 0 0 0 10 0 0 0 10 0 0 

MA VT 2 8 1 8 1 0 0 8 2 0 0 9 1 0 

MA AZ 1 9 1 9 0 0 1 9 0 0 1 9 0 0 

AZ WI 0 10 0 10 0 0 0 10 0 0 0 10 0 0 

AZ NJ 0 10 0 10 0 0 0 10 0 0 0 10 0 0 

AZ VT 0 10 0 10 0 0 0 10 0 0 0 10 0 0 

AZ AZ 1 9 1 9 0 0 1 9 0 0 1 9 0 0 

WI WI 1 9 1 9 0 0 1 9 0 0 1 9 0 0 

WI NJ 0 10 0 10 0 0 0 10 0 0 0 10 0 0 

WI VT 4 6 4 6 0 0 3 6 1 0 3 6 1 0 

WI AZ 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 

n=number TP indicates true-positive, i.e., positive results recultured as positive; FN indicates falsenegative, i.e., negative results recultured as positive; FP indicates false-positive, i.e., positive results 

recultured as negative. 

 Fast Phage Results 

 + - Total 

Male-specific EPA- 

unfiltered

+ 107 1 108

- 0 12 12

Total 107 13 120

 Fast Phage Results 

 + - Total 

Male-specific EPA- 

filtered 

+ 107 3 110

- 0 10 10

Total 107 13 120
Nov.28, 2012 Page 27 

 Male specific EPA- unfiltered 

 + - Total 

Male-specific EPA  

filtered

+ 108 2 110

- 0 10 10

Total 108 12 120

17.5 17.5 Data for the positive prediction rate of the rapid somatic fluorescence prediction are presented 

in 4 fold table below. The overall prediction rate of the somatic fluorescence relative to the Fast 

Phage result was 93% with a false negative error of 7.5% and false positive error of 7.4%. This 

shows value as a rapid same day predictor of a positive result, while requiring negative results to 

be confirmed by the plaque assay. 

Fast Phage Somatic Fluorescence 

Prediction 

 + - Total 

Fast Phage 

Somatic Result

+ 86 7 93 

- 2 25 27 

Total 88 32 120 

17.6 Data for the positive prediction rate of the rapid male specific fluorescence prediction are presented 

in 4 fold table below. The overall prediction rate of the male specific fluorescence relative to the 

Fast Phage result was 95% with a false negative error of 1.9% and false positive error of 26.7%. 

The false positive rate is likely overstated because there are only a few actual negative samples in 

the analysis. The results show value as a rapid same day predictor of a positive result, while 

requiring negative results to be confirmed by the plaque assay. 

Fast Phage male specific 

fluorescent prediction 

 + - Total 

Fast Phage 

result

+ 103 2 105 

- 4 11 15 

Total 107 13 120