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 EP A 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”) interpretatio n occurs with samples containing < 1 PFU/ 100mL. 1.3 The Fast Phage tests are designed to meet the sensi tivity 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 co liphage positive samples in less than 8 hours. The fluorescent detection is a presumptive positiv e result that has no equivalent status in coliphage detection using Method 1601. 

## 2.0 Summary of Fast Phage Method and 1601 Modificat ions

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 coli phage 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 F amp for male-specific coliphages, are supplied as readyto-use freeze dried tablets at a level of 2 x 10 7 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 t emperature in a water bath. 2.4 The E. coli culture tablets are manufactured and supplied in a standar dized 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 ad dition to autoclaved and tempered spot agar provides a convenient reagent and a simplified preparat ion 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 rea d as much as 25 hours later. 2.7 STEP-2 media, also supplied pre-dispensed in dissolvable po uch, 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 pla te). The rapid fluorescence indication is based on the principle of inducing intracellular ß-galact osidase production during E. coli growth in both STEP-1 and STEP-2. The enzyme is released extrace llularly 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, ind icating the presence of coliphage. 2.8 The combined Method 1601 modifications allow the detecti on 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 a llows for a more rapid response to a contamination event but does not have a status of Meth od 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 testi ng 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 pouc h. 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 μ L 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 STE P-1 culture. Transfer STEP-1 enrichment, 10mL for somatic coliphage and 1mL fo r male specific coliphage, to STEP-2. Heat in 38±1°C water bath 0.5 hour for somatic co liphage 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 sa mple is predicted to be positive for coliphage, absence of fluorescence at 3 hours indica tes a negative prediction for coliphage. Fluorescence in positive samples may appear a s early as 1 hour (6th hour from initial STEP-1 start). Fluorescent positive STEP-2 medi a is transferred by 10 uL loop to a prepared agar spot plate and dried into the plate and in cubated 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 bacterio phages specific to E. coli and coliform bacteria and indicative of fecal contaminat ion. These coliphage infect via attachment to the E. coli F pilli. 3.3 Plaque- A region of no cell growth in an otherwise un iform bacterial lawn that is formed by a phage lysing adjoining host bacteria. 

## 4.0 Interferences

4.1 Antibiotic resistant (nalidixic acid or ampicillin/str eptomycin) coliform bacteria in excess of 1-10 million CFU/mL capable of producing extracellular ß-galact osidase enzyme may produce false positive results. While the Fast Phage STEP media use s 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 i nterferences are not viewed as a detriment to sanitary water testing due to the unlikely high level s 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 conf irmation step. Bacteria may grow over the lysis zone and obscure visualization, resulting in a fa lse negative interpretation. Generally, when bacteria have overgrown lysis zones, they appear as ra ised colonies or a confluent growth of raised colonies. As a result, they are distinguishable from t he surrounding lawn of host bacteria. If this problem is noted, follow the procedure described in Sect ion 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 pee lable 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 i f 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 disposa l.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 wh en working with potentially harmful samples. 5.6 Fast Phage does not purport to address all of the safe ty 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 sa fety procedures required in a laboratory that handles biohazardous material while preparing, using, an d 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 prio r to disposal or re-use. Field and laboratory staff collecting and analyzing environmen tal samples are under some risk of exposure to pathogenic microorganisms. Staff should apply safety pro cedures 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 t he 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 inf ormation on laboratory safety can be found in Section 15.0 Waste Management. 5.8 Samples may contain high concentrations of biohazardo us 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 fa ce after exposure to media known or suspected to contain pathogenic microorganisms. Laborat ory personnel must change gloves after handling raw sewage or any other items which may ca rry 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 desi ccated 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 chlori nated waters. 6.7 Positive control, Ф X174 for somatic coliphage and MS2 for male-specific coli phage, supplied with Fast Phage or quality control cultures, (ATCC 13706-B1, ATCC 15597-B1). 6.8 Antibiotic, nalidixic acid 0.1g for somatic coliphage an d Ampicillin/Streptomycin 0.015g each, freeze dried rehydrated with 10mL water to make prescr ibed mg/mL stock.Nov.28, 2012 Page 5

6.9 Autoclave, Refrigerator, Thermometer and other Labora tory 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, froz en for use 6.20 Bubble wrap 

## 7.0 Reagents and Standards

7.1 Fast Phage STEP-1 and STEP-2 media in dissolvable pouche s (25 pouches per foil zip bag), 28 E. coli tablets in blister, and antibiotic freeze dried are s upplied 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 FPLU S]-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 Na 2 S 2 O 3 ) 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 re agents 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 Meth ods 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 disinfecti on 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 Meth ods for the Examination of Water and Wastewater Section 9060 B or appropriate regulatory requirem ent. 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 af ter collection. 

## 9.0 Quality Control

9.1 QA Program- Each laboratory that uses Fast Phage is required to ope rate a formal quality assurance (QA) program. The minimum QA requirements con sist 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 wate r and field samples to evaluate and document data quality, and analysis of positive controls and m ethod blanks as tests of continued acceptable performance. Spiked sample results are compared to accept ance criteria based on EPA Method 1601. Specific quality control (QC) requirements are provid ed below. General recommendations on QA and QC for facilities, personnel, laboratory equipme nt, instrumentation, and supplies used in microbiological analyses are provided in the USEPA Mi crobiology 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 Φ X174 (somatic) coliphage supplied with test kits (section 6. 7). All other spiked QC samples must be spiked with enumerated sewage f iltrate or equivalent. QA/QC procedures should be consistent with practices delineate d in the 5 th addition of the Manual for the Certification of Laboratories Analyzing Drinking Wat er . 9.2.1 Initial demonstration of capability (IDC). The laboratory shall demonstrate the ability to generate acceptable performance with this method by perf orming an IDC test before analyzing any field samples. The procedure for performi ng the IDC is described in Section 9.3. IDC tests must be accompanied by a method blank (S ection 9.2.2) for each coliphage type. 9.2.2 Method blanks. The laboratory shall analyze method blanks (reagent w ater 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 me thod blank should be spotted onto the lawn of host bacteria immediately following the posit ive control spot.Nov.28, 2012 Page 7 9.2.3 Positive controls. The laboratory shall analyze positive controls to ens ure that stock coliphage suspensions, host bacterial cultures, and growt h media are performing properly (Section 9.5). The laboratory shall inoculate one positi ve 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 fo r 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 bas is after every 20 th 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 o f 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 con taining 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 avail able. The laboratory should also participate in available interlaboratory performance stud ies conducted by local, state, and federal agencies or commercial organizations. The l aboratory should review results, correct unsatisfactory performance, and record correc tive actions. 9.2.8 The specifications contained in this method can be me t 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 analy sis of field samples or to evaluate acceptable performance of a method modification. IDC test sample s 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 sampl es should be used. 9.3.2 IDC samples must be spiked in “bulk” for each coliphage ty pe at concentrations as specified in Table 1. For example, for 100mL somatic IDC sa mples, 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 s piking suspension enumeration procedure and Sections 9.11 and 9.12 for spike volume calcula tions.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 proced ure (Section11). See Table 1 for the minimum number of samples that must be positive for ea ch set of 10 IDC samples. If system performance is unacceptable, identify and correc t 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 sampl es. 9.4.2 If coliphage, or any potentially interfering organism s are found in the blank, analysis of additional samples must be halted until the source of co ntamination 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 re collected (if holding times have been violated) and reanalyzed. Samples from a batch th at 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 (Se ction 6.7), Ф X174 (ATCC 13706-B1) for somatic coliphage and MS2 (ATCC 15597-B1) for male -specific coliphage, previously reconstituted with 10mL reagent wat er 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 i n Section 9.2.4. The laboratory shall spike and analyze field samples from each ground water sourc e 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 enume rated sewage filtrate or equivalent. 9.6.1 The ongoing MS sample must be collected and analyzed a t 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 v olume 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 sam ple results associated with this batch of samples are acceptable, a matrix interference m ay 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 positiv e. 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 ty pe. Spike the bulk samples at concentrations according to Table 2. For example, for 100m L somatic ODC samples, spike 300mL of reagent water with 4.5 PFU of somatic coli phage to achieve a target spike concentration of 1.5 PFU per sample, based on stock coli phage enumeration. See Section 9.10 for enumeration of coliphage stock and Sections 9.11 a nd 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 sampl e volume being evaluated. If not, method performance is unacceptable. Identify and c orrect 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 e valuation 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 modifie d 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 (mal e-specific and somatic) are required. 9.8.2 Samples must be spiked in “bulk” for each coliphage type. S pike the bulk samples at concentrations according to Table 3. For example, for 100m L somatic expanded-MS samples, spike 1.0 L of ground water with a target spike dose of 15 PFU of somaticNov.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 met hod 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 t he two-step enrichment procedure (Section 11). Table 3 specifies the minimum number of sample s that must be positive for each set of 10 Expanded MS samples. 9.9 Raw Sewerage and preparation for IDC, ODC, and Expande d MS Test 9.9.1 The double agar layer (DAL) procedure is used to enumerat e stock suspensions of somatic and male specific coliphage for use in spiking quality c ontrol samples. Use sewage filtrate prepared as follows: 9.9.1.1 Collect approximately 100mL of raw sewage in a 250mL collect ion bottle. 9.9.1.2 Transport to the laboratory on ice. Analysis of raw s ewage 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 min utes. This will make the filtration process easier. 9.9.1.4 Remove a sterile, 20mL syringe from its package, asepti cally remove plunger from barrel, and attach a 0.45 μm low-protein-binding fil ter disk to the syringe barrel. 9.9.1.5 Pipet 10 to 15mL of supernatant from settled sewage into t he syringe barrel. 9.9.1.6 Hold the assembly over a sterile 15mL polypropylene tube wi th screw-cap or snap-cap, insert the plunger into the syringe barrel, an d push the sewage through the filter into the sterile tube. If filter clogs, ch ange it as necessary and continue to filter sewage until at least 10mL of filtered sewag e is obtained in the 15mL polypropylene tube (filtration may require use of numerous f ilters). 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 tha n 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 neces sary 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 ant ibiotics (Section 7.5) is used as the diluents and as the method blank. 9.9.3 Aseptically add 9.0mL of TSB without antibiotics (Sectio n 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 setti ng (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 setti ng (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 se tting (if available) or until well mixed. 9.9.7 Add 1.0mL of TSB without antibiotics (Section 7.3.1.1) to t he 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 laye r (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 doubl e-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 simult aneous 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 appropriat e 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 chlori de 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 the n autoclave 15Nov.28, 2012 Page 12 minutes at 121 o C and 15 psi. Place in a 45 to 48 o C water bath to temper the prepared medium until used. 9.10.1.2 To one flask add 3 mL of nalidixic acid solution (sect ion 6.8) and swirl to mix. To second flask add 3 mL of ampicillin/streptomycin so lution (section 6.8) and swirl to mix. 9.10.1.3 Dispense 17 mL of the nalidixic acid TSA to 100 mm petri di sh (Section 6.12) on a level surface. Allow to cool and air dry for 10 m inutes. 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 36 o C 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 ai r 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 i ncubating at least 1 plate at 36 o C 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 sec tion 11.1.2. 9.10.2.2 Add an F amp E.coli tablet to TSB with ampicillin/streptomycin as describe d 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 (s ection 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 (secti on 6.8) and swirl to mix. To second flask add 1mL of ampicillin/streptomycin sol ution (section 6.8) and swirl to mix. 9.10.3.3 18 tubes are necessary to enumerate four dilution volumes i n duplicate for each phage type. The 18 tubes also includes an additional metho d blank tube for each phage type. Add 5mL of each prepared 0.7% TSA to 9 tubes. Th ere should be 9 tubes with nalidixic acid labeled for somatic coliphage determination and there should be 9 tubes labeled for male specific coliphage deter mination. Tubes should remain in water bath. 9.10.4 As a precaution against contamination, disinfect a wo rkspace near the water bath with a 1:100 dilution of household bleach and allow to dry. If wo rkspace can be corroded byNov.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 t he following information is identifiable:

• Dilution of stock filtrate or method blank

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

• Coliphage type (somatic for the E. coli CN-13 bacterial host or male-specific for the E. coli F amp 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 spik ing suspension until ready to plate. 9.10.6 Preparation of plates for enumeration of somatic coliph age 9.10.6.1 With the top agar tube still in the water bath, aseptica lly inoculate a top agar tube containing nalidixic acid with 100 μ L of cultured E. coli CN-13 (section 9.10.2.3). 9.10.6.2 Immediately add 500 μ L (0.5mL) of undiluted coliphage stock or sewage filtrat e. 9.10.6.3 Mix the inoculum by rolling the tube briefly in palm of han d. 9.10.6.4 Pour contents into one of the two bottom agar plates m arked “undiluted, E. coli CN-13, somatic." 9.10.6.5 Duplicate analysis—Repeat Sections 9.10.6.1 through 9.10.6.4 for t he duplicate. 9.10.6.6 Repeat Sections 9.10.6.1 through 9.10.6.5 for each dilution volum e. 9.10.7 Preparation of somatic coliphage method blank 9.10.7.1 With the nalidixic acid top agar tube still in the wate r bath, aseptically inoculate a top agar tube containing nalidixic acid with 100 μ L of log-phase E. coli CN-13 (section 9.10.2.3). 9.10.7.2 Immediately add 500 μ L (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 han d. 9.10.7.4 Pour contents into a naladixic acid bottom agar plate m arked “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/stre ptomycin and log-phase E. coli F amp (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/streptom ycin and log-phase E. coli F amp (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 sam ples. 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 diam eter) in lawn of host bacteria after 16 to 24 hours of incubation are plaques. Count the number of pl aques 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 co liphage spiking suspension will be calculated using DAL plates that yield plaque coun ts 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 th e total number of plaques on a plate will be above the ideal range. If the count exceed s the upper range or if the plaques are not discrete, results should be recorded as “too n umerous 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 TN TC or all zeros. (See equation in Section 9.11.5) 9.11.3 Sum the undiluted sample volumes used to inoculate all repl icate 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 sa mple 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, excludi ng dilutions with all TNTC or all zeros (0)

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

• n = number of useable countsNov.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 approximat ely 682 PFU per mL, the 1 : 10 dilution contains approximately 68.2 PFU per mL, the 1 :100 dil ution 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 co ntrol 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 specifi ed 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 v olume: 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 necessar y 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 sam ples 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 aliquo ts dispensed. Each 100 mL sample should contain approximately 1.5 PFU. 9.13 Use the enumerated somatic and male-specific stocks t o 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 grad uations to check temperature of incubator. The mercury should not be separated. Alterna tively, because of safety issues regarding mercury thermometers, a thermometer that meets the r equirements of NIST Monograph SP-250-23 can be used. 10.2 Check temperature of incubator twice daily at least 4 ho urs apart or use constant recording device. Record measurements in a log book. 10.3 Check temperatures in water baths, refrigerators, -20 o C freezers daily to ensure operation within stated limits of Fast Phage protocol and record meas urements in a log book. 10.4 Follow Methods for the Examination of Water and Wa stewater Section 9030 A for other laboratory equipment specifications such as pipets, graduat ed cylinders, dilution bottles, refrigerator, autoclaves. 10.5 Check balances annually using ASTM-certified Class 2 re ference 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 t he 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, o r 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, d issolve 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 rehyd rated 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 A gar, TSB 9g (section 6.10) and Bacto Agar 2.25g (section 6.11)) and bri ng to boil. Autoclave for 15 minutes. Place TSA in water bath 45-48°C water bath to te mper 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 sol idify 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 o r 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 peeli ng blister foil and adding tablet to water. Do not touch tablet. Replace cap and swirl to d issolve tablet. Store sample refrigerated until ready to test. If shipping to a te st 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 preca utions 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. Al low 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 F amp 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, tra nsfer a 10μL loop of STEP-1 enrichment from each sample on to a spot plate (sectio n 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 i nhibition 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 col iphage/100mL). A light box may aid in the evaluation and interpretation of lytic zon es. 11.6.3 Questionable spots may be picked/scooped into 0.5mL TSB (se ction 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 s ample. 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 d issolved 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 un der 366nm UV light. If coliphage are absent the sample will not fluoresce after 3 hours a nd will appear turbid. If coliphage are present, the media will fluoresce in 1-3 hours and t he samples are presumptivepositive. Samples may also appear clear (non-turbid), an d or turn blue. Confirmation of presumptive positive by spot plate is required, steps 11.7.4 an d 11.7.5.
11.7.4 Transfer a 10μL loop of STEP-2 samples to a spot plate. A llow plate to air dry to absorb liquid. This may take 30 minutes. Incubate spot plate inve rted 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 pos itive (> 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, reco rd 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 hour s STEP-2 (8 hours from sample start in STEP-1), sample is interpreted “predicted absence of col iphage [type tested]”. 12.2 If the sample is fluorescent at any time up to 3 hours S TEP-2, sample is interpreted “predicted presence of coliphage [type tested]”. 12.3 Positive fluorescent samples may also be clear (non-t urbid) or turn blue; but fluorescence is the determinative factor in determining positive.

Identical negative samples viewed under UV light an dambient room light. UV results show negative nonfluorescent results compared to positive fluorescen ce results. The same samples viewed in ambient light show the negative sample as turbid yellow, while th e positive sample may appear greenish with more clari ty.

PositivePresence of coliphage Negative – Absence of ColiphageNov.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 a s not found (<1PFU coliphage/100 mL [type tested]). See exam ple photographs below.

The photograph of a male-specific Step-1 plaque confirmat ion 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 ampl ified culture. PC is a coliphage morphology where the amplified host grow as a circular spot on th e agar but have a surrounding zone of inhibition into the E. coli seeded in the agar. Sample 8 has a micro-plaque morpho logy 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 morphologie s 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 descr ibed in Sections 9.10-9.12. 

## 13.0 Method Performance

13.1 A shared sample evaluation was conducted with 4 laborato ries proficient in coliphage analysis and meeting IDC and ODC for method 1601 and Fast Phage. 13.2 Regionally collected waste water samples, Arizona (A Z), Wisconsin (WI) and Massachusetts (MA), were sent to the co-ordinating laboratory in M A. Samples were filtered and sent to 4 collaborating laboratories in AZ, New Jersey (NJ), Ve rmont (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 was te 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 individua l analyses. In 24 of 24 experimental cycles, 12 cycles each somatic and male-specific, expan ded-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 we re 89% male-specific positive, 107 of 120 samples, by Fast Phage modification com pared to 96% Method 1601 positive, 115 positive of 120 samples. The maximum difference between an y Fast Phage and any Method 1601 comparative cycle was -40% which occurred 1 of 12 insta nces of male-specific coliphage. The maximum difference was -30% in one of 12 cycles of so matic 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) a nalysis of the comparability results, section 13.5, do not show significant differences. The somatic X 2 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 th e 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 un filtered spots as method comparison and filtered spots as the referee method. 13.8 Specificity of the somatic coliphage Fast Phage metho d 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 negati ve rate of 4.6% and a false positive rate of 17.4%. The observed method had an agreement with the r eference 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 ha d an observed method agreement of 99% with a false negative rate of 0.9% and a false positive rat e 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 me thod 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 expecte d 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 SciencesNov.28, 2012 Page 22

16.2 Standard Methods for the Examination of Water and Wa stewater, 21 st Edition 16.3 EPA Method 1601: Male-specific (F+) and Somatic Coliphag e in Water by Two-step Enrichment Procedure, April 2001 16.4 Manual for the Certification of Laboratories Analyzin g Drinking Water, EPA 815-B-97-001. 16.5 Microbiological Methods for Monitoring the Environme nt, 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 soma tic 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 indic ated 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 signific ant 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 sa mples, n=10 replicates at 4 differentNov.28, 2012 Page 23

laboratories, diluted into geographically diverse groun d 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 ind icate 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 r esults compared to the confirmation steps of Method 1601 are shown in Table 17.3 and compared in 4 fold ta bles below. The results indicate a 93% overall agreement with EPA Method 1601 confirmation pl ate using filtered method. There was a 4.6 % false negative error and a 17.4% false positi ve 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 ka ppa statistic k=0.78 indicate substantial agreement between Fast Phage method and the referenc e 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 re sults recultured as positive; FN indicates falsenegative, i.e., negative results recultured as positive; F P 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 110Nov.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 P hage positive results compared to the confirmation steps of Method 1601 are shown in Table 17.4 an d compared in 4 fold tables below. The results indicate a 98% agreement with the EPA Meth od 1601 confirmation plate using filtered method with a 2.7 % false negative error and a 0% fals e positive error. This compares to the EPA Method 1601 un-filtered method that had a 98% agreement wi th 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 an d the reference Method 1601Nov.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 re sults recultured as positive; FN indicates falsenegative, i.e., negative results recultured as positive; F P 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 120Nov.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 s omatic 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 mal e 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 t here 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