Office of Water EPA 821

- R

- 14

- 01 1

www.epa.gov September 2014



# Method 1600: Enterococci in Water by Membrane Filtration Using membrane

- Enterococcus Indoxyl

- β

- D

- Glucoside Agar (mEI)

# U.S. Environmental Protection Agency Office of Water (4303T) 1200 Pennsylvania Avenue, NW Washington, DC 20460

EPA

- 821

- R

- 14

- 011Method 1600

i September 2014 

## Acknowledgments

This method was developed under the direction of James W. Messer and Alfred P. Dufour of the U.S. Environmental Protection Agency ’ s (EPA) Human Exposure Research Division, National Exposure Research Laboratory, Cincinnati, Ohio.

The following laboratories are gratefully acknowledged for their participation in the validation of this method in

wastewater effluents:

Volunteer Research Laboratories

 E PA Office of Research and Development, National Risk Management Research Lab: Mark C. Meckes

 U.S. Army Corps of En gineers, Washington Aqueduct: Elizabeth A. Turner, Michael L. Chicoine, and Lisa

Neal

Volunteer Verification Laboratories

 City of Los Angeles Bureau of Sanitation: Farhana Mohamed, Ann Dalkey, Ioannice Lee, Genevieve Espineda, and Zora Bahariance

 Orange Co unty Sanitation District, Environmental Sciences Laboratory: Charles McGee, Michael von Winckelmann, Kim Patton, Linda Kirchner, James Campbell, Arturo Diaz, and Lisa McMath

Volunteer Participant Laboratories

 City of Los Angeles Bureau of Sanitation: Farha na Mohamed, Ann Dalkey, Ioannice Lee, Genevieve Espineda, and Zora Bahariance

 County Sanitation Districts of Los Angeles County (JWPCP): Kathy Walker, Michele Padilla, and Albert

Soof

 County Sanitation Districts of Los Angeles County (SJC): Shawn Thompso n and Julie Millenbach

 Environmental Associates (EA): Susan Boutros and John Chandler

 Hampton Roads Sanitation District (HRSD): Anna Rule, Paula Hogg, and Bob Maunz

 Hoosier Microbiological Laboratories (HML): Don Hendrickson, Katy Bilger, and Lindsey Shelton

 Massachusetts Water Resources Authority (MWRA): Steve Rhode and Mariya Gofhsteyn

 North Shore Sanitation District (NSSD): Robert Flood

 Texas A&M University: Suresh Pillai and Reema Singh

 University of Iowa Hygienic Laboratory: Nancy Hall and Cathy L ord

 Wisconsin State Laboratory of Hygiene (WSLH): Jon Standridge, Sharon Kluender, Linda Peterson, and Jeremy Olstadt

 Utah Department of Health: Sanwat Chaudhuri and Devon ColeMethod 1600

September 2014 ii 

## Disclaimer

The Engineering and Analysis Division, of the Office of Science and Technology, has reviewed and approved this report for publication. The Office of Science and Technology directed, managed, and reviewed the work of DynCorp in preparing this report. Neither the United States Government nor any of its employees, contr actors, or their employees make any warranty, expressed or implied, or assumes any legal liability or responsibility for any third party ’ s use of or the results of such use of any information, apparatus, product, or process discussed in this report, or rep resents that its use by such party would not infringe on privately owned rights. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.

Questions concerning this method or its application should be addres sed to:

Robin K. Oshiro Engineering and Analysis Division (4303T) U.S. EPA Office of Water, Office of Science and Technology 1200 Pennsylvania Avenue, NW Washington, DC 20460 oshiro.robin@epa.gov

202

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- 1053 (facsimile)Method 1600

iii September 2014 

## Table of Contents

Acknowledgments

................................ ................................ ................................ ................................ ........... i

Disclaimer

................................ ................................ ................................ ................................ ...................... ii

1.0 Scope and Application

................................ ................................ ................................ ....................... 1

2.0 Summary of Method

................................ ................................ ................................ .......................... 1

3.0 Definitions

................................ ................................ ................................ ................................ ........ 1

4.0 Interferen ces

................................ ................................ ................................ ................................ ...... 2

5.0 Safety

................................ ................................ ................................ ................................ ................ 2

6.0 Equipment and Supplies

................................ ................................ ................................ .................... 2

7. 0 Reagents and Standards

................................ ................................ ................................ ..................... 3

8.0 Sample Collection, Handling, and Storage

................................ ................................ .......................... 7

9.0 Quality Control

................................ ................................ ................................ ................................ .. 7

10.0 Calib ration and Standardization

................................ ................................ ................................ ....... 12

11.0 Procedure

................................ ................................ ................................ ................................ ........ 12

12.0 Verification Procedure

................................ ................................ ................................ ..................... 13

13.0 Data Analysis and Calculations

................................ ................................ ................................ ........ 14

14.0 Sample Spiking Procedure

................................ ................................ ................................ ............... 14

15.0 Method Performance

................................ ................................ ................................ ....................... 18

16.0 Pollution Prevention

................................ ................................ ................................ ........................ 21

17.0 Waste Management

................................ ................................ ................................ ......................... 21

18.0 References

................................ ................................ ................................ ................................ ........ 22Method 1600

September 2014 iv 

## List of Appendices

Appendices A and B are taken from Microbiological Methods for Monitoring the Environment, Water and Wastes (Reference 18.7).

Appendix A: Part II (General Operations), Section A (Sample Collection, Preservation, and Storage).

Appendix B: Part II (General Operations), Sections C.3.5 (Counting Colonies) and C.3.6 (Calculation of

Results).Method 1600

1 September 2014 

## Method 1600: Enterococci in Water by Membrane Filtration Using

membrane

- Enterococcus Indoxyl

- β

- D

- Glucoside Agar (mEI)

September 2014

1.0 Scope and Application

1.1 Method 1600 describes a membrane filter (MF) procedure for the detection and enumeration of the

enterococci bacteria in water. This is a single

- step method that is a modification of EPA Method 1106.1 (mE

- EIA). Unlike the mE

- EIA method, it does not require the transfer of the membrane filter to

another medium. The modified medium has a reduced amount of triphenyltetrazolium chloride (TTC) and includes indoxyl β

- D

- glucoside, a chromogenic cellobiose analog used in place of esculin. In this procedure, β

- glucosidase

- positive enterococci produce an insoluble indigo blue complex which diffuses

into the surrou nding media, forming a blue halo around the colony.

1.2 Enterococci are commonly found in the feces of humans and other warm

- blooded animals. Although some strains are ubiquitous and not related to fecal pollution, the presence of enterococci in water is an indication of fecal pollution and the possible presence of enteric pathogens.

1.3 Epidemiological studies have led to the development of criteria which can be used to promulgate recreational water standards based on established relationships between h ealth effects and water quality. The significance of finding enterococci in recreational fresh or marine water samples is the direct relationship between the density of enterococci and the risk of gastrointestinal illness associated with swimming in the w ater (References 18.1 and 18.2).

1.4 For method application please refer to Title 40 Code of Federal Regulations Part 136 (40 CFR Part 136).



## 2.0 Summary of Method

2.1 Method 1600 provides a direct count of bacteria in water based on the development of colonies on the surface of the membrane filter (Reference 18.4). A water sample is filtered through the membrane which retains the bacteria. Following filtration, the membrane containing the bacterial cells is placed on a selective medium, mEI agar, and incubated for 24 hours at 41 o C ± 0.5 o C . All colonies greater than or equal to ( > ) 0.5 mm in diameter (regardless of color) with a blue halo are recorded as enterococci colonies. A fluorescent lamp with a magnifying lens is used for counting to give max imum visibility of

colonies.



## 3.0 Definitions

3.1 In Method 1600, enterococci are those bacteria which produce colonies greater than or equal to 0.5 mm in diameter with a blue halo after incubation on mEI agar. The blue halo should not be included in the colony diameter measurement. Enterococci include Enterococcus faecalis (E. faecalis)

, E. faecium

, E.

avium

, E. gallinarium , and their variants. The genus Enterococcus includes the enterococci formerly assigned to the Group D fecal streptococci.Method 1600

September 2014 2 

## 4.0 Interferences

4.1 Water samples containing colloid al or suspended particulate materials can clog the membrane filter and prevent filtration, or cause spreading of bacterial colonies which could interfere with enumeration and identification of target colonies.



## 5.0 Safety

5.1 The analyst/technician must know and observe the normal safety procedures required in a microbiology laboratory while preparing, using, and disposing of cultures, reagents, and materials, and while operating sterilization equipment.

5.2 The selective medium (mEI) and azide

- dextrose broth used in this method contain sodium azide as well as other potentially toxic components. Caution must be exercised during the preparation, use, and disposal of these media to prevent inhalation or contact with the medium or reagents.

5.3 This method does not 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. A reference file of material safety data sheets (MSDSs) should be available to all personnel involved in Method 1600 analyses.

5.4 Mouth

- pipetting is prohibited.



## 6.0 Equipment and Supplies

6.1 Glass lens with magnification of 2

- 5X or stereoscopic microscope

6.2 Lamp, with a cool, white fluorescent tube

6.3 Hand tally or electronic counting device

6.4 Pipet container, stainless steel, aluminum or borosilicate glass, for glass pipets

6.5 Pipets, sterile, T.D. bacteriological or Mohr, glass or plastic, of appropriate volume

6.6 Sterile graduated cylinder s, 100

- 1000 mL, covered with aluminum foil or kraft paper

6.7 Sterile membrane filtration units (filter base and funnel), glass, plastic or stainless steel, wrapped with aluminum foil or kraft paper

6.8 Ultraviolet unit for sanitization of the filter funnel between filtrations (optional)

6.9 Line vacuum, electric vacuum pump, or aspirator for use as a vacuum source (In an emergency or in the field, a hand pump or a syringe equipped with a check valve to prevent the return flow of air, can be used)

6. 10 Flask, filter, vacuum, usually 1 L, with appropriate tubingMethod 1600

3 September 2014 6.11 A filter manifold to hold a number of filter bases (optional)

6.12 Flask for safety trap placed between the filter flask and the vacuum source

6.13 Forceps, straight or curved, with smooth tips to handle filters without damage

6.14 Ethanol, methanol or isopropanol in a small, wide

- mouth container, for flame

- sterilizing forceps

6.15 Burner, Bunsen or Fisher type, or electric incinerator unit for sterilizing loops and needles

6.16 Th ermometer, checked against a National Institute of Standards and Technology (NIST) certified thermometer, or one that meets the requirements of NIST Monograph SP 250

- 23

6.17 Petri dishes, sterile, plastic, 9 x 50 mm, with tight

- fitting lids; or 15 x 60 mm with loose fitting lids; or 15 x 100 mm with loose fitting lids

6.18 Bottles, milk dilution, borosilicate glass, screw

- cap with neoprene liners, 125 mL volume

6.19 Flasks, borosilicate glass, screw

- cap, 250

- 2000 mL volume

6.20 Membrane filters, sterile, white, grid marked, 47 mm diameter, with 0.45  m pore size

6.21 Platinum wire inoculation loops, at least 3 mm diameter in suitable holders; or sterile plastic loops

6.22 Incubator maintained at 41 o C ± 0.5 o C

6.23 Water bath maintained at 50 o C for tempering agar

6.24 Test tubes, 20 x 150 mm, borosilicate glass or plastic

6.25 Caps, aluminum or autoclavable plastic, for 20 mm diameter test tubes

6.26 Test tubes, screw

- cap, borosilicate glass, 16 x 125 mm or other appropria te size

6.27 Autoclave or steam sterilizer capable of achieving 121 o C [15 lb pressure per square inch (PSI)] for 15

minutes



## 7.0 Reagents and Standards

7.1 Purity of Reagents: Reagent grade chemicals shall be used in all tests. Unless otherwise indicated, reagents shall conform to the specifications of the Committee on Analytical Reagents of the American Chemical Society (Reference 18.5). The agar used in preparation of culture media must be of microbiological grade.

7.2 Whenever possible, use commercial culture media as a means of quality control.

7.3 Purity of reagent water: Reagent

- grade water conforming to specifications in: Standard Methods for the Examination of Water and Wastewater (latest edition approved by EPA in 40 CFR Part 136 or 14 1, as applicable), Section 9020 (Reference 18.6).Method 1600

September 2014 4 7.4 Phosphate buffered saline (PBS)

7.4.1 Composition:

Sodium dihydrogen phosphate (NaH 2 PO 4 ) 0.58 g Disodium hydrogen phosphate (Na 2 HPO 4 ) 2.5 g Sodium chloride (NaCl) 8.5 g Reagent

- grade water 1.0 L

7.4.2 Dissolve the reagents in 1 L of reagent

- grade water and dispense in appropriate amounts for dilutions in screw cap bottles or culture tubes, and/or into containers for use as rinse water. Autoclave after preparation at 121 o C (15 PSI) for 15 min. Final pH should be 7.4 ± 0.2.

7.5 mEI Agar

7.5.1 Composition:

Peptone 10.0 g Sodium chloride (NaCl) 15.0 g

Yeast extract 30.0 g

Esculin 1.0 g Actidione (Cycloheximide) 0.05 g

Sodium azide 0.15 g Indoxyl β

- D

- glucoside 0.75 g Agar 15.0 g Reagent

- grade water 1.0 L

7.5.2 Add reagents to 1 L of reagent

- grade water, mix thoroughly, and heat to dissolve completely.

Autoclave at 121 o C (15 PSI) for 15 minutes and cool in a 50 o C water bath.

7.5.3 After sterilization add 0.24 g nalidixic acid (sodium salt) and 0.02 g triphenyltetrazolium chloride (TTC) to the mEI medium and mix thoroughly.

Note : The amount of TTC used in this medium (mEI) is less than the amount used for mE agar in Method 1106.1.

7.5.4 Dispense mEI agar into 9 × 50 mm or 15 × 60 mm petri dishes to a 4

- 5 mm depth (approximately 4

- 6 mL), and allow to solidify. Final pH of medium should be 7.1 ± 0.2. Store in a refrigerator.

7.6 Tryptic soy agar (TSA)

7.6.1 Composition:

Pancreatic digest of casein 15.0 g Enzymatic digest of soybean meal 5.0 g Sodium chloride (NaCl) 5.0 g Agar 15.0 g Reagent

- grade water 1.0 LMethod 1600

5 September 2014 7.6.2 Add reagents to 1 L of reagent

- grade water, mix thoroughly, and heat to dissolve completely.

Autoclave at 121 o C (15 PSI) for 15 minutes and cool in a 50 oC water bath

. Pour the medium

into each 15 × 60 mm culture dish to a 4

- 5 mm depth (approximately 4

- 6 mL), and allow to solidify. Final pH should be 7.3 ± 0.2.

7.7 Brain heart infusion broth (BHIB)

7.7.1 Composition:

Calf brains, infusion from 200.0 g 7.7 g

Beef hear t, infusion from 250.0 g 9.8 g Proteose peptone 10.0 g Sodium chloride (NaCl) 5.0 g Disodium hydrogen phosphate (Na 2 HPO 4 ) 2.5 g

Dextrose 2.0 g Reagent

- grade water 1.0 L

7.7.2 Add reagents to 1 L of reagent

- grade water, mix thoroughly, and heat to diss olve completely. Dispense in 10

- mL volumes in screw cap tubes, and autoclave at 121 o C (15 PSI) for 15 minutes. Final pH should be 7.4 ± 0.2.

7.8 Brain heart infusion broth (BHIB) with 6.5% NaC1

7.8.1 Composition:

BHIB with 6.5% NaC1 is the same as BHIB above (Section 7.7), but with additional NaC1.

7.8.2 Add NaCl to formula provided in Section 7.7 above, such that the final concentration is 6.5% (65 g NaCl/L). Typically, for commercial BHIB media, an additional 60.0 g NaCl per liter of

medium will need to be added to the medium. Prepare as in Section 7.7.2.

7.9 Brain heart infusion agar (BHIA)

7.9.1 Composition:

BHIA contains the same components as BHIB (Section 7.7), with the addition of 15.0 g agar per

liter of BHIB.

7.9.2 Add agar to formu la for BHIB provided in Section 7.7 above. Prepare as in Section 7.7.2. After sterilization, slant until solid. Final pH should be 7.4 ± 0.2.Method 1600

September 2014 6 7.10 Bile esculin agar (BEA)

7.10.1 Composition:

Beef Extract 3.0 g Pancreatic Digest of Gelatin 5.0 g Oxgall 20.0 g

Esculin 1.0 g

Ferric Citrate 0.5 g Bacto Agar 14.0 g Reagent

- grade water 1.0 L

7.10.2 Add reagents to 1 L reagent

- grade water, heat with frequent mixing, and boil 1 minute to dissolve completely. Dispense 10

- mL volumes in tubes for slant s or larger volumes into flasks for subsequent plating. Autoclave at 121 o C (15 PSI) for 15 minutes. Overheating may cause darkening of the medium. Cool in a 50 o C water bath , and dispense into sterile petri dishes. Final pH should be 6.8 ± 0.2. Store in a refrigerator.

7.11 Azide dextrose broth (ADB)

7.11.1 Composition:

Beef extract 4.5 g Pancreatic digest of casein 7.5 g Proteose peptone No. 3 7.5 g

Dextrose 7.5 g Sodium chloride (NaCl) 7.5 g

Sodium azide 0.2 g Reagent

- grade water 1.0 L

7.11.2 Add reagents to 1 L of reagent

- grade water and dispense in screw cap bottles. Autoclave at

121 o C (15 PSI) for 15 minutes. Final pH should be 7.2 ± 0.2.

7.12 Control cultures

7.12.1 Positive control and/or spiking organism (either of the following are acceptable)

 Stock cultures of Enterococcus faecalis ( E. faecalis ) ATCC 19433

 E. faecalis ATCC 19433 BioBalls (bioMerieux, Durham NC)

7.12.2 Negative control organism ( for each organism, either of the following are acceptable)

 Stock cultures of Escherichia coli ( E. coli ) ATCC 11775

 E. coli ATCC 11775 BioBalls ( bioMerieux, Durham NC )

 Stock cultures of Enterobacter aerogenes ( E. aerogenes ) ATCC 13048

 E. aerogenes NCTC 10006 BioBalls ( bioMerieux, Durham NC )Method 1600

7 September 2014 

## 8.0 Sample Collection, Handling, and Storage

8.1 Sampling procedures are briefly described below. Detailed sampling methods can be found in Reference 18.7 (see Appendix A). Adherence to sample preservation procedures and holding time limits is critical to the production of valid data. Samples not collected according to these rules should not be analyzed.

8.1.1 Sampling techniques

Samples are collected by hand or with a sampling device if the sampling site has difficult access such as a dock, bridge, or bank adjacent to a surface water. Composite samples should not be collected, since such samples do not display the range of values found in individual samples. The sampling depth for surface water samples should be 6

- 12 inches below the water surface. Sample containers should be positioned such that the mouth of the container is pointed away from the sampler or sample point. After removal of the container from the water, a small portion of the sample should be discarded to allow for proper mixing before analyses.

8.1.2 Storage t

emperature and handling conditions

Ice or refrigerate water samples at a temperature of <10 o C during transit to the laboratory. Do not freeze the samples. Use insulated containers to assure proper maintenance of storage temperature. Take care that sample bottles are not totally immersed in water during transit or

storage.

8.1.3 Holding time li mitations

Sample analysis should begin immediately, preferably within 2 hours of collection. The maximum transport time to the laboratory is 6 hours, and samples should be processed within 2 hours of receipt at the laboratory.



## 9.0 Quality Control

9.1 Each laboratory that uses Method 1600 is required to operate a formal quality assurance (QA) program that addresses and documents instrument and equipment maintenance and performance, reagent quality and performance, analyst training and certification, and records storage and retrieval. Additional recommendations for QA and quality control (QC) procedures for microbiological laboratories are provided in Reference 18.7.

9.2 The minimum analytical QC requirements for the analysis of samples using Method 160 0 include an initial demonstration of laboratory capability through performance of the initial precision and recovery (IPR) analyses (Section 9.3), ongoing demonstration of laboratory capability through performance of the ongoing precision and recovery (OP R) analysis (Section 9.4) and matrix spike (MS) analysis (Section 9.5, disinfected wastewater only), and the routine analysis of positive and negative controls (Section 9.6), filter sterility checks (Section 9.8), method blanks (Section 9.9), and media

ste rility checks (Section 9.11). For the IPR, OPR and MS analyses, it is necessary to spike samples with either laboratory

- prepared spiking suspensions or BioBalls as described in Section 14.

Note : Performance criteria for Method 1600 are based on the resul ts of the interlaboratory validation of Method 1600 in PBS and disinfected wastewater matrices. The IPR (Section 9.3) and OPRMethod 1600

September 2014 8

(Section 9.4) recovery criteria ( Table 1 ) are valid method performance criteria that should be met, regardless of the matrix bein g evaluated, the matrix spike recovery criteria (Section 9.5,

Table 2 ) pertain only to disinfected wastewaters.

9.3 Initial precision and recovery (IPR)

— The IPR analyses are used to demonstrate acceptable method performance (recovery and precision) and should be performed by each laboratory before the method is used for monitoring field samples. EPA recommends but does not require that an IPR be performed by each analyst. IPR samples should be accompanied by an acceptable method blank (Section 9.9) and appropriate media sterility checks (Section 9.11). The IPR analyses are performed as follows:

9.3.1 Prepare four, 100

- mL samples of PBS and spike each sample with E. faecalis ATCC 19433 according to the spiking procedure in Section 14. Spiking with lab oratory

- prepared suspensions is described in Section 14.2 and spiking with BioBalls is described in Section 14.3. Filter and process each IPR sample according to the procedures in Section 11 and calculate the number of enterococci per 100 mL according to Section 13.

9.3.2 Calculate the percent recovery (R) for each IPR sample using the appropriate equation in Section 14.2.2 or 14.3.4 for samples spiked with laboratory

- prepared spiking suspensions or BioBalls, respectively.

9.3.3 Using the percent recoveries of the four analyses, calculate the mean percent recovery and the relative standard deviation (RSD) of the recoveries. The RSD is the standard deviation divided by the mean, multiplied by 100.

9.3.4 Compare the mean recovery and RSD with the c orresponding IPR criteria in Table 1, below. If the mean and RSD for recovery of enterococci meet acceptance criteria, system performance is acceptable and analysis of field samples may begin. If the mean recovery or the RSD fall outside of the required range for recovery, system performance is unacceptable. In this event, identify the problem by evaluating each step of the analytical process, media, reagents, and controls, correct the problem and repeat the IPR analyses.

Table 1. Initial and Ongoing Precision and Recovery (IPR and OPR) Acceptance Criteria

Performance test Lab

- prepared spike acceptance criteria BioBallTM acceptance criteria Initial precision and recovery (IPR)

Mean percent recovery

Precision (as maximum relative standard deviation) 31%

- 127%

28% 85%

- 106%

14% Ongoing precision and recovery (OPR) as percent recovery 27%

- 131% 78%

- 113%

9.4 Ongoing precision and recovery (OPR)

— To demonstrate on going control of the analytical system, the laboratory should routinely process and analyze spiked PBS samples. The laboratory should analyze one OPR sample after every 20 field and matrix spike samples or one per week that samples are analyzed, whichever occurs more frequently. OPR samples must be accompanied by an acceptab le method blank (Section 9.9) and appropriate media sterility checks (Section 9.11). The OPR analysis is performed as follows:

9.4.1 Spike a 100

- mL PBS sample with E. faecalis ATCC 19433 according to the spiking procedure in Section 14. Spiking with lab oratory

- prepared suspensions is described in Section 14.2 and spiking with BioBalls is described in Section 14.3. Filter and process each OPR sampleMethod 1600

9 September 2014 according to the procedures in Section 11 and calculate the number of enterococci per 100 mL according to Section 13.

9.4.2 Calculate the percent recovery (R) for the OPR sample using the appropriate equation in Section 14.2.2 or 14.3.4 for samples spiked with laboratory

- prepared spiking suspensions or BioBalls, respectively.

9.4.3 Compare the OPR result (percent recovery) with the corresponding OPR recovery criteria in Table 1, above. If the OPR result meets the acceptanc e criteria for recovery, method performance is acceptable and analysis of field samples may continue. If the OPR result falls

outside of the acceptance criteria, system performance is unacceptable. In this event, identify the problem by evaluating each step of the analytical process, media, reagents, and controls, correct the problem and repeat the OPR analysis.

9.4.4 As part of the la

boratory QA program, results for OPR and IPR samples should be charted and updated records maintained in order to monitor ongoing method performance. The laboratory should also develop a statement of accuracy for Method 1600 by calculating the average per cent recovery (R) and the standard deviation of the percent recovery (s r ). Express the accuracy as a
recovery interval from R

- 2s r to R + 2s r
.

9.5 Matrix spikes (MS)

— MS analysis are performed to determine the effect of a particular matrix on enterococci recoveries. The laboratory should analyze one MS sample when disinfected wastewater samples are first received from a source from which the laboratory has not previously analyzed samples. Subsequently, 5% of field samples (1 per 20) from a given disinfe cted wastewater source should include a MS sample. MS samples must be accompanied by the analysis of an unspiked field sample sequentially collected from the same sampling site, an acceptable method blank (Section 9.9), and appropriate media sterility che cks (Section 9.11). When possible, MS analyses should also be accompanied by an OPR sample (Section 9.4), using the same spiking procedure (laboratory

- prepared spiking suspension or BioBalls). The MS analysis is performed as follows:

9.5.1 Prepare two, 100

- mL field samples that were sequentially collected from the same site. One sample will remain unspiked and will be analyzed to determine the background or ambient concentration of enterococci for calculating MS recoveries (Section 9.5.3). The other sa mple will serve as the MS sample and will be spiked with E. faecalis ATCC 19433 according to the spiking procedure in Section 14.

9.5.2 Select sample volumes based on previous analytical results or anticipated levels of in the field sample in order to achieve the recommended target range of enterococci (20

- 60 CFU, including spike) per filter. If the laboratory is not familiar with the matrix being analyzed, it is recommended that a minimum of three dilutions be analyz ed to ensure that a countable plate is obtained for the MS and associated unspiked sample. If possible, 100

- mL of sample should be analyzed.

9.5.3 Spike the MS sample volume(s) with a laboratory

- prepared suspension as described in Section

14.2 or with BioBalls as described in Section 14.3. Immediately filter and process the unspiked and spiked field samples according to the procedures in Section 11.

Note : W hen analyzing smaller sample volumes ( e.g. , < 20 mL), 20

- 30 mL of PBS should be

added to the fun nel or an aliquot of sample should be dispensed into a 20

- 30 mLMethod 1600

September 2014 10

dilution blank prior to filtration. This will allow even distribution of the sample on the

membrane.

9.5.4 For the MS sample, calculate the number of enterococci (CFU/100 mL) according to Section 13 and adjust the colony counts based on any background enterococci observed in the unspiked matrix sample.

9.5.5 Calculate the percent recovery (R) for the MS sample (adjusted based on ambient enterococci in the unspiked sample) using the appropriate equation in Section 14.2.2 or 14.3.4 for samples spiked with laboratory

- prepared spiking suspensions or BioBalls, respectively.

9.5.6 Compare the MS result (percent recovery) with the appropriate method performance criteria in Table 2, below. If the MS recovery meets the acceptance criteria, system performance is acceptable and analysis of field samples from this disinfected wastewater source may continue. If the MS recovery is unacceptable and the OPR sample result associated with this batch of samples is acceptable, a matrix interference may be causing the poor results. If the MS recovery is unacceptable, all associated field data should be flagged.

9.5.7 Acceptance criteria for MS recovery (Table 2) are based on data from spiked disinfected wastewater matrices and are not appropriate for use with other matrices ( e.g. , ambient waters).

Table 2. Matrix Spike Precision and Recovery Acceptance Criteria

Performance test Lab

- prepared acceptance criteria BioBallTM acceptance criteria

Percent recovery for MS 29%

- 122% 63%

- 110%

9.5.8 Laboratories should record and maintain a control chart comparing MS recoveries for all

matrices to batch

- specific and cumulative OPR sample results analyzed using Method 1600. These comparisons should help laboratories recognize matrix effects on method recovery and may also help to recognize inconsistent or sporadic matrix effects from a p articular source.

9.6 Culture Controls

9.6.1 Negative controls

— The laboratory should analyze negative controls to ensure that the mEI agar is performing properly. Negative controls should be analyzed whenever a new batch of media or reagents is used. On an ongoing basis, the laboratory should perform a negative control every day that samples are analyzed.

9.6.1.1 Negative controls are conducted by filtering a dilute suspension of viable E. coli

( e.g. , ATCC 11775) or E. aerogenes ( e.g. , ATCC 13048) and analyzing as described in Section 11. Viability of the negative controls should be demonstrated using a

non

- selective media ( e.g. , nutrient agar or tryptic soy agar).

9.6.1.2 If the negative control fails to exhibit the appropriate response, check and/or replace

the associated media or reagents, and/or the negative control, and reanalyze the appropriate negative control.

9.6.2 Positive controls

— The laboratory should analyze positive controls to ensure that the mEI agar is performing properly. Posi tive controls should be analyzed whenever a new batch of media or reagents is used. On an ongoing basis, the laboratory should perform a positive controlMethod 1600

11 September 2014 every day that samples are analyzed. An OPR sample (Section 9.4) may take the place of a positive co ntrol.

9.6.2.1 Positive controls are conducted by filtering a dilute suspension of viable E. faecalis

( e.g. , ATCC 19433) and analyzing as described in Section 11.

9.6.2.2 If the positive control fails to exhibit the appropriate response, check and/or re place

the associated media or reagents, and/or the positive control, and reanalyze the appropriate positive control.

9.6.3 Controls for verification media

— All verification media should be tested with appropriate positive and negative controls whenever a new batch of media and/or reagents are used. On an ongoing basis, the laboratory should perform positive and negative controls on the verification media with each batch of samples submitted to verifica tion. Examples of appropriate controls for verification media are provided in Table 3

.

Table 3. Verification Controls

Medium Positive Control Negative Control

Bile esculin agar (BEA) E. faecalis E. coli

Brain heart infusion broth (BHIB) with 6.5% NaCl E. faecalis E. coli

Brain heart infusion broth (BHIB) incubated at 45 oC E. faecalis E. aerogenes

9.7 Colony verification

— The laboratory should verify 10 typical colonies (positive) and 10 atypical colonies (negative) per month or 1 typical colony and 1 atypical colony from 10% of all positive samples, whichever is greater. Verification procedures are provided in Section 12.0.

9.8 Filter sterility check

— Place at least one membrane filter per lot of filters on a TSA plate, and incubate

for 24 ± 2 hours at 35 o C ± 0.5 o C . Absence of growth indicates sterility of the filter. On an ongoing basis, the laboratory should perform a filter sterility ch eck every day that samples are analyzed.

9.9 Method blank

— Filter a 50

- mL volume of sterile PBS and place the filter on a mEI agar plate and process according to Section 11.0. Absence of growth indicates freedom of contamination from the target organism. On an ongoing basis, the laboratory should perform a method blank every day that samples are analyzed.

9.10 Filtration blank

— Filter a 50

- mL volume of sterile PBS before beginning sample filtrations. Place the filter on a TSA plate, and incubate for 24 ± 2 hours at 35 o C ± 0.5 o C . Absence of growth

indicat es sterility of the PBS buffer and filtration assembly.

9.11 Media sterility check

— The laboratory should test media sterility by incubating one unit (tube or plate) from each batch of medium (TSA, mEI agar, and verification media) as appropriate and observing for growth. Absence of growth indicates media sterility. On an ongoing basis, the laboratory should perform a media sterility check every day that samples are analyzed.

9.12 Analyst colony counting variability

— Laboratories with two or more ana lysts should compare each analyst ’ s colony counts from one positive field sample per month. Colony counts should be within 10% between analysts. Laboratories with a single analyst should have that analyst perform duplicate colony counts of a single membrane filter each month. Duplicate colony counts should be within 5% for aMethod 1600

September 2014 12 single analyst. If no positive field samples are available, a n OPR sample may be substituted for these

determinations.



## 10.0 Calibration and Standardization

10.1 Check temperatures in incubators twice daily with a minimum of 4 hours between each reading to ensure operation within stated limits.

10.2 Check thermometers at least annually against a NIST certified thermometer or one that meets the requirements of NIST Monograph SP 250

- 23. Check mercury columns for breaks.

10.3 Refrigerators used to store media and reagents should be monitored daily to ensure proper temperature

control.



## 11.0 Procedure

11.1 Prepare the mEI agar as directed in Section 7.5.

11.2 Mark the petri dishes and report forms with sample identification and sample volumes.

11.3 Place a sterile membrane filter on the filter base, grid

- side up and attach the funnel to the base so that the

membrane filter is now held between the funnel and the bas e.

11.4 Shake the sample bottle vigorously about 25 times to distribute the bacteria uniformly, and measure the desired volume of sample or dilution into the funnel.

11.5 Select sample volumes based on previous knowledge of the enterococci level, to prod uce 20

- 60 enterococci colonies on membranes. It is recommended that a minimum of three dilutions be analyzed to ensure that a countable plate (20

- 60 enterococci colonies) is obtained.

11.6 Smaller sample size or sample dilutions can be used to minimize the interference of turbidity or for high bacterial densities. Multiple volumes of the same sample or sample dilutions may be filtered.

Note: When analyzing smaller sample volumes ( e.g. , <20 mL), 20

- 30 mL of PBS or phosphate

- buffered dilution water shoul d be added to the funnel or an aliquot of sample should be dispensed into a dilution blank prior to filtration. This will allow even distribution of the sample on the membrane.

11.7 Filter the sample, and rinse the sides of the funnel at least twice with 20

- 30 mL of sterile buffered rinse

water. Turn off the vacuum and remove the funnel from the filter base.

11.8 Use sterile forceps to aseptically remove the membrane filter from the filter base, and roll it onto the mEI Agar to avoid the formation of bu bbles between the membrane and the agar surface. Reseat the membrane if bubbles occur. Run the forceps around the edge of the filter outside the area of filtration, close to the edge of the dish, to be sure that the filter is properly seated on the agar. Close the dish, invert, and incubate at 41 o C ± 0.5 o C for 24 ± 2 hours.Method 1600

13 September 2014 Note: If the medium is prepared in 15 × 60 mm loose lid petri dishes, they should be incubated in a tight fitting container ( e.g. , plastic vegetable crisper) containing a moistened paper towel to prevent dehydration of the membrane filter and medium.

11.9 After incubation, count and record colonies on those membrane filters containing, if practical, 20

- 60

colonies ≥ 0.5 mm in diameter with a blue halo regardless of colony color as a n enterococci (see Figure

1 ).

Note: When measuring colony size do not include the halo. Use magnification for counting and a small fluorescent lamp to give maximum visibility of colonies.



## 12.0 Verification Procedure

12.1 Colonies ≥ 0.5 mm in diameter of any color having a blue halo after incubation on mEI agar are

considered to be “ typical ” enterococci colonies. Verification of colonies may be required in evidence gathering and it is also recommended as a means of quality control. The verification procedure follows.

Note: W hen evaluating wastewater using Method 1600, it is recommended t hat the false negative rate for each matrix be evaluated through biochemical co nfirmation and results adjusted accordingly, especially if large numbers of atypical colonies are observed in a particular matrix.

12.2 Using a sterile inoculating loop or need le, transfer growth from the centers of at least 10 well

- isolated typical and at least 10 well

- isolated atypical colonies into a BHIB tube and onto a BHIA slant. Incubate broth for 24 ± 2 hours and agar slants for 48 ± 3 hours at 35 o C ± 0.5 o C

.

12.3 After a 24 hour incubation, transfer a loopful of growth from each BHIB tube to BEA, BHIB, and BHIB

with 6.5% NaCl.

12.3.1 Incubate BEA and BHIB with 6.5% NaCl at 35 o C ± 0.5 o C for 48 ± 3 hours.

12.3.2 Incubate BHIB at 45 o C ± 0.5 o C for 48 ± 3 hours.

12.4 Observe all verification media for growth.

12.5 After 48 hour incubation, perform a Gram stain using growth from each BHIA slant. Figure 1 . Enterococci colonies on mEI produce blue halos.Method 1600

September 2014 14 12.6 Gram

- positive cocci that grow and hydrolyze esculin on BEA ( i.e. , produce a black or brown precipitate ), and grow in BHIB with 6.5% NaCl at 35 o C ± 0.5 o C and BHIB at 45 o C ± 0.5 o C are verified

as enterococci.

12.7 Alternately, commercially available multi

- test identification systems ( e.g. , Vitek®) may be used to verify colonies. Such multi

- test identification systems should include esculin hydrolysis and growth in

6.5% NaCl.



## 13.0 Data Analysis and Calculations

Use the following general rules to calculate the enterococci count per 100 mL of sample:

13.1 If possible, select a membrane filter with 20

- 60 colonies ≥ 0.5 mm in diameter (regardless of colony color) with a blue halo. Calculate the number of enterococci per 100 mL according to the following general formula:

Enterococci / 100 mL = Number of enteroco cci colonies

× 100 Volume of sample filtered (mL)

13.2 See general counting rules in Reference 18.7 (see Appendix B).

13.3 Report results as enterococci per 100 mL of sample.



## 14.0 Sample Spiking Procedure

14.1 Method 1600 QC requirements (Section 9.0) include the preparation and analysis of spiked reference (PBS) and matrix samples in order to monitor initial and ongoing method performance. For the IPR (Section 9.3), OPR (Section 9.4), and MS (Section 9.5) analyses it is necessary to spik e samples with either laboratory

- prepared spiking suspensions (Section 14.2) or BioBalls (Section 14.3) as described

below.

14.2 Laboratory

- Prepared Spiking Suspensions

14.2.1 Preparation

14.2.1.1 Stock Culture. Prepare a stock culture by inoculating a TSA slant (or other

non

- selective media) with E. faecalis ATCC 19433 and incubating at 35 o C ± 3 o C for 20 ± 4 hours. This stock culture may be stored in the dark at room temperature for up to 30 days.

14.2.1.2 Undiluted Spiking Suspension . Prepare a 1 % solution of azide dextrose broth

(ADB) by combining 99 mL of sterile phosphate buffered saline and 1 mL of sterile single strength azide dextrose broth in a sterile screw cap bottle or re

- sealable

dilution water container. From the stock culture of E. f aecalis ATCC 19433 in Section 14.2.1.1, transfer a small loopful of growth to the 1 % azide dextrose broth solution and vigorously shake a minimum of 25 times. Disperse the inoculum by vigorously shaking the broth culture and incubate at 35 o C ± 3 o C for 20 ± 4 hours. This culture is referred to as the undiluted spiking suspension and should containMethod 1600

15 September 2014 approximately 1.0 × 10 6

- 1.0 × 10 7 E. faecalis colony forming units (CFU) per mL

of culture.

14.2.1.3 Mix the undiluted spiking suspension (Section 14. 2.1.2) thoroughly by shaking the

bottle a minimum of 25 times and prepare a series of dilutions (4 total) in the following manner:

14.2.1.3.1 Dilution “ A ”

— Aseptically transfer 1.0 mL of the undiluted spiking suspension to 99 mL of sterile PBS and mix thor oughly by shaking the bottle a minimum of 25 times. This is spiking suspension dilution “ A ”

and 1 mL contains 10

- 2 mL of the original undiluted spiking suspension.

14.2.1.3.2 Dilution “ B ”

— Aseptically transfer 1.0 mL of dilution “ A ” to 99 mL of

sterile PB S and mix thoroughly by shaking the bottle a minimum of 25 times. This is spiking suspension dilution “ B ” and 1 mL contains 10

- 4 mL of the original undiluted spiking suspension.

14.2.1.3.3 Dilution “ C ”

— Aseptically transfer 11.0 mL of dilution “ B ” to 99 mL of sterile PBS and mix thoroughly by shaking the bottle a minimum of 25 times. This is spiking suspension dilution “ C ” and 1 mL contains 10

- 5 mL of the original undiluted spiking suspension.

14.2.1.3.4 Dilution “ D ”

— Aseptically transfer 11.0 mL of dilution “ C ” to 99 mL of sterile PBS and mix thoroughly by shaking the bottle a minimum of 25 times. This is spiking suspension dilution “ D ” and 1 mL contains

10

- 6 mL of the original undiluted spiking suspension.

14.2.2 Sample spiking

14.2.2.1 Add 3.0 mL of the spiking suspension dilution “ D ” (Section 14.2.1.3.4) to 100 mL or

PBS or appropriate volume of sample and mix thoroughly by shaking the bottle a minimum of 25 times. The volume of undiluted spiking suspension added to each 100 mL sample is 3.0 x 10

- 6 mL, which is referred to as V spiked per 100 mL sample in Section 14.2.4.1 below. Filter the spiked sample and analyze the filter according to the procedures in Section 11.

14.2.3 Enumeration of spiking suspension

14.2.3.1 Prepare TSA spre ad plates, in triplicate, for spiking suspension dilutions “ B ”

, “ C ”

,

and “ D ”

.

Note: Agar plates must be dry prior to use. To ensure that the agar surface is dry, plates should be made several days in advance and stored inverted at room

temperature or dried using a laminar

- flow hood.

14.2.3.2 Mix dilution “ B ” by shaking the bottle a minimum of 25 times. Pipet 0.1 mL of

dilution “ B ” onto the surface of each TSA plate in triplicate.

14.2.3.3 Mix dilution “ C ” by shaking the bottle a minimum of 25 times. Pipet 0.1 mL of

dilution “ C ” onto the surface of each TSA plate in triplicate.Method 1600

September 2014 16

14.2.3.4 Mix dilution “ D ” by shaking the bottle a minimum of 25 times. Pipet 0.1 mL of

dilution “ D ” onto the surface of each TSA plate in triplicate.

14.2.3.5 Use a sterile bent glass rod or spreader to distribute the inoculum over the surface of

plates by rotating the dish by hand or on a turntable.

Note : Ensure that the inoculum is evenly distribut ed over the entire surface of the plate.

14.2.3.6 Allow the inoculum to absorb into the medium of each plate completely. Invert

plates and incubate at 35 o C ± 0.5 o C for 20 ± 4 hours.

14.2.3.7 Count and record number of colonies per plate. The number of enterococci

(CFU / mL) in the undiluted spiking suspension will be calculated using all TSA plates yielding counts within the countable range of 30 to 300 CFU per plate.

14.2.4 Recovery calculations for spiked samples

14.2.4.1 Calculate the concentration of enterococci (CFU / mL) in the undiluted spiking suspension (Section 14.2.1.2) according to the following equation. Example calculations are provided in Table 4 , below.

Enterococci undiluted spike = ( CFU 1 + CFU 2 + ...+ CFU

n ) / ( V 1 + V 2 + ... + V

n )

Enterococci undiluted spike = Enterococci (CFU / mL) in undiluted spiking suspension

Where,

CFU = Number of colony forming units from TSA plates yielding counts within the

countable range of 30 to 300 CFU per plate

V = V olume of undiluted sample on each TSA plate yielding counts within the countable

range of 30 to 300 CFU per plate

n = Number of plates with counts within the countable range of 30 to 300 CFU per plate

Note: The example calculated numbers provided in the tables below have been rounded at

the end of each step for simplification purposes. Generally, rounding should only

occur after the final calculation.Method 1600

17 September 2014 Table 4. Example Calculations of Laboratory

- prepared Enterococci Spiking Concentration

Examples CFU/plate (triplicate analyses) from TSA plates Enterococci CFU/mL in undiluted spiking suspension ( Enterococci undiluted spike ) a 10

- 5 mL plates 10

- 6 mL plates 10

- 7 mL plates

Example 1 94, 106, 89 9, 11, 28 1, 0, 4 (94+106+89) / (10

- 5 +10

- 5 +10

- 5 ) =

289 / (3.0 x 10

- 5 ) = 9,633,333 =

9.6 x 10 6 CFU /

mL

Example 2 32, 55, 72 8, 5, 3 0, 0, 0 (32+55+72) / (10

- 5 +10

- 5 +10

- 5 ) =

159 / (3.0 x 10

- 5 ) = 5,300,000 =

5.3 x 10 6 CFU /

mL

a Enterococci undiluted spike is calculated using all plates yielding counts within the countable range of 30 to 300 CFU per plate

14.2.4. 2 Calculate true concentration of spiked enterococci (CFU/100 mL) according to the

following equation. Example calculations are provided in Table 5, below.

T spiked Enterococci = ( Enterococci undiluted spike ) x ( V spiked per 100 mL sample )

Where,

T spiked Enterococci = Number of spiked Enterococci (CFU/100 mL)

Enterococci undiluted spike = Enterococci (CFU/mL) in undiluted spiking suspension

V spiked per 100 mL sample = mL of undiluted spiking suspension per 100 mL sample

Table 5. Example Calculations for Determination “ True ” Spiked Enterococci Concentration

Enterococci undiluted spike V spiked per 100 mL sample Tspiked Enterococci

9.6 x 10 6 CFU / mL 3.0 X 10

- 6 mL per 100 mL of sample (9.6 x 10 6 CFU / mL) x (3.0 x 10

- 6 mL / 100 mL) =
28.8 CFU / 100 mL

5.3 x 10 6 CFU / mL 3.0 X 10

- 6 mL per 100 mL of sample (2.8 x 10 6 CFU / mL) x (3.0 x 10

- 6 mL / 100 mL) =
8.4 CFU / 100 mL

14.2.4.2 Calculate percent recovery (R) of spiked enterococci (CFU / 100 mL) according to the following equation. Example calculations are provided in Table 6 , below.

R = 100 x ( N s - N u )

T

Where,

R = Percent recovery

N
s = Enterococci (CFU/100 mL) in the spiked sample (Section 13)

N

u = Enterococci (CFU/100 mL) in the unspiked sample (Section 13)

T = True spiked enterococci (CFU/100 mL) in spiked sample (Section 14.2.4.1)Method 1600

September 2014 18 Table 6. Example Percent Recovery Calculations for Lab

- prepared Spiked Samples

N s (CFU/100 mL) N u (CFU/100 mL) T spiked Enterococci (CFU/100 mL) Percent recovery (R)

42 <1 28.8 100 x (42

- 1) / 28.8

= 142%

34 10 28.8 100 x (34

- 10) / 28.8

= 83%

10 <1 8.4 100 x (10

- 1) / 8.4

= 107%

14.3 BioBallTM Spiking Procedure

14.3.1 Aseptically add 1 BioBallTM to 100 mL of PBS or appropriate volume of sample and mix by vigorously shaking the sample bottle a minimum of 25 times. Analyze the spiked sample according to the procedures in Section 11.

14.3.2 Recovery calculations for samples spiked with BioBalls

— Calculate percent recovery (R) of spiked enterococci (CFU/100 mL) according to the following equation. Example calculations are provided in Table 7, below.

R = 100 x ( N s - N u )

T

Where,

R = Percent recovery

N
s = Enterococci (CFU / 100 mL) in the spiked sample (Section 13)

N

u = Enterococci (CFU / 100 mL) in the unspiked sample (Section 13)

T = True spiked enterococci (CFU / 100 mL) in spiked sample based on the lot mean value provided by manufacturer

Table 7. Example BioBallTM Percent Recovery Calculations

Ns (CFU / 100 mL) Nu (CFU / 100 mL) T (CFU / 100 mL) Percent recovery (R)

24 <1 11.2 100 × (24

- 1) / 32 = 72%

36 10 32 100 × (36

- 10) / 32 = 81%



## 15.0 Method Performance

15.1 Performance Characteristics (Reference 18.4)

15.1.1 Precision

- The degree of agreement of repeated measurements of the same parameter expressed quantitatively as the standard deviation or as the 95% confidence limits of the mean computed from the results of a series of controlled determinations. The precision among laboratories for

marine water and surface water was 2.2% and 1 8.9%, respectively.

15.1.2 Bias

- The persistent positive or negative deviation of the results from the assumed or accepted true value. The persistent positive or negative deviation of the results from the assumed or accepted true value was not significant.Method 1600

19 September 2014 15.1.3 Specificity

- The ability of a method to select and/or distinguish the target bacteria from other bacteria in the same water sample. The specificity characteristic of a method is usually reported as the percent of false positive and fa lse negative results. The specificity for this medium as reported for various environmental water samples was 6.0% false positive and 6.5% false negative.

15.1.4 Multilaboratory variability

- A collaborative study was conducted among fourteen collaborato rs at twelve laboratories to examine the interlaboratory reproducibility of the method. Reproducibility among laboratories (RSDR) for freshwater, marine water, chlorinated secondary effluent, and non

- chlorinated primary effluent ranged from 2.2% for marin e water to 18.9% for freshwater with a low enterococcal density.

15.2 Interlaboratory Validation of Method 1600 in Disinfected Wastewater (Reference 18.3)

15.2.1 Twelve volunteer participant laboratories, two enterococci verification laboratories, and tw o research laboratories participated in the U.S. Environmental Protection Agency ’ s (EPA ’ s) interlaboratory validation study of EPA Method 1600. The purposes of the study were to characterize method performance across multiple laboratories and disinfected wastewater matrices and to develop quantitative quality control (QC) acceptance criteria. A detailed description of the study and results are provided in the validation study report (Reference 18.3). Results submitted by laboratories were validated using a standardized data review process to confirm that results were generated in accordance with study

- specific instructions and the September 2002 version of EPA Method 1600.

15.2.2 Recovery

- Method 1600 was characterized by mean laboratory

- specific recove ries of enterococci from disinfected wastewater samples spiked with BioBallsTM ranging from 77.1% to 114.9%, with an overall mean recovery of 90.8%. Mean laboratory

- specific recoveries of enterococci from PBS samples spiked with BioBalls TM ranged from 88.0% to 105.1%, with an overall mean recovery of 95.4%.

15.2.3 Precision

- Method 1600 was characterized by laboratory

- specific relative standard deviations (RSDs) from disinfected wastewater samples spiked with BioBallsTM ranging from 0% to 69.5%, with an overall pooled, within

- laboratory RSD of 22.6%. For PBS sample s spiked with BioBalls, laboratory

- specific RSDs ranged from 3.1% to 13.7%, with an overall pooled,

within

- laboratory RSD of 8.1%.

15.2.4 False positive confirmation rates

- Method 1600 laboratory

- specific false positive confirmation rates for unspiked d

isinfected/secondary results combined, ranging from 0.0% to 10.0%. For secondary wastewater (excluding disinfected results), only 2 of 123 typical colonies submitted

to verification were non

- enterococci, resulting in a false positive confirmation rate of 1.6%. For disinfected wastewater (excluding secondary results), none of the 66 typical colonies

submitted to verification were non

- enterococci, resulting in a false positive confirmation rate of 0.0%. Since all 2184 typical colonies observed during the s tudy could not be submitted to confirmation, the percent of total colonies that would have resulted in a false positive result was estimated (see Table 6, Reference 18.3). It is estimated that 0.0% and 1.2% of the total

colonies would have resulted in a f alse positive for disinfected wastewater and secondary wastewater, respectively.

15.2.5 False negative rates

- Method 1600 laboratory

- specific false negative rates laboratory

- specific false negative confirmation rates for unspiked disinfected/secondary re sults combined, ranged from 28.6% to 100.0%. For secondary wastewater (excluding disinfected results), 62 of 79Method 1600

September 2014 20

atypical colonies submitted to verification were identified as enterococci, resulting in a false negative confirmation rate of 78.5% for second ary wastewater. For disinfected wastewater (excluding secondary results), eight of eight atypical colonies submitted to verification were identified as enterococci, resulting in a false negative confirmation rate of 100.0% for

disinfected wastewater. Sin ce all 839 atypical colonies observed during the study could not be submitted to confirmation, the percent of total colonies that would have resulted in a false negative result was estimated. It is estimated that 21.2% and 22.8% of the total colonies woul d have resulted in a false negative for disinfected wastewater and secondary wastewater, respectively. The false positive and negative assessments are provided in Table 8

.

Table 8. False Positive and False Negative Assessment for Unspiked Disinfected and Unspiked

Secondary Wastewater Effluents

Matrix Total colonies False positive (FP) assessment False negative (FN) assessment

Typical Atypical Typical

colonies

submitted No. FP

colonies FP
confirmation rate (%) a Estimated

% of total

colonies

that would

have been

a FP b Atypical

colonies

submitted No. FN

colonies FN
confirmation rate (%) c Estimated %

of total

colonies that

would have

been a FN d

Disinfected 391 105 66 0 0.0 0.0 8 8 100.0 21.2

Secondary 1793 734 123 2 1.6 1.2 79 62 78.5 22.8

Disinfected + Secondary 2184 839 189 2 1.1 0.8 87 70 80.5 22.3

a False positive confirmation rate = number of false positive colonies / number of typical colonies submitted

b Percent of total colonies estimated to be false positives = [(total typical colonies FP confirmation rate) / (total number of typical and atypical colonies observed); e.g. , [(1793 ×(2/123))/(1793+734)] × 100 = 1.2%

c False negative confirmation rate = number of false negative colonies / number of aty pical colonies submitted

d Percent of total colonies estimated to be false negatives = [(total atypical colonies* FN confirmation rate) / (total number of typical and atypical colonies observed)] × 100; e.g. , [(734 ×(62/79))/(1793+734)] × 100 = 22.8%

15.2.6 During evaluation of the study results, it was noted that many of the false negatives (atypical colonies submitted to verification which were identified as enterococci) were pink to red in color but simply lacked a blue halo. The predecessor to EPA Method 1600 for enterococci is EPA Method 1106.1 which uses mE and EIA media. For EPA Method 1106.1, pink to red colonies on mE, which produce a brown precipitate after transfer to EIA are considered positive for enterococci. Tetrazolium chloride (TTC), the reagent responsible for producing pink to red enterococci colonies on mE, is also included as a reagent in mEI. A follow

- on study was conducted, for which pink to red colonies without halos from unspiked secondary wastewaters

were submitted to verifi cation. For pink to red colonies without halos that were ≥ 0.5 mm colony size, 54 of 90 colonies submitted were identified as enterococci, resulting in a 60.0%

verification rate.

Results of the verification analyses from the initial study were assessed with pink to red colonies without halos being counted as enterococci. When pink to red colonies without halos areMethod 1600

21 September 2014 counted as enterococci, the estimated percent of total colonies that would have resulted in false positives increases slightly from 0.8% to 2.7%, for combined disinfected and secondary results. More importantly, the estimated percent of total colonies that would have resulted in false negatives decreased from 22.3% to 7.0% for combined disinfected and secondary results and

from 21.2% to 2.9% for disinfected wastewater. The re

- assessment of false positive and false negative initial study results with pink to red colonies without halos counted as enterococci are provided in Table 9

.

Table 9. Re

- Assessment of False Positive and False Negative Initial Study Results with Pink to Red

Colonies without Halos Counted as Enterococci

Matrix (sample no.) Total colonies False positive (FP) assessment False negative (FN) assessment

Typical Atypical Typical

colonies

submitted No. FP

colonies FP

confirmation rate (%) a Estimated

% of total

colonies

that would

have been

a FP b Atypical

colonies

submitted No. FN

colonies FN

confirmation rate (%) c Estimated %

of total

colonies that

would have

been a FN d

Disinfected (Samples 1

- 4) 477 19 69 0 0.0 0.0 4 3 75.0 2.9

Secondary (Samples 5, 6) 2291 236 166 7 4.2 3.8 32 27 84.4 7.9

Disinfected & Secondary (Samples 1

- 6) 2768 255 235 7 3.0 2.7 36 30 83.3 7.0

a False positive confirmation rate = number of false positive colonies / number of typical colonies submitted

b Percent of total colonies estimated to be false positives = [(total typical colonies × FP confirmation rate) / (total number of typical and atypical colonies observed)] × 100; e.g. , [(2291×(7/166))/(2291+236)] × 100 = 3.8%

c False negative confirmation rate = number of false negative colonies / number of atypical colonies submitted

d Percent of total colonies estimated to be false negatives = [(total atypical colonies × FN confirmation rate) / (total number of typical and atypical colonies observed)] × 100; e.g. , [(236 ×(27/32))/(2291+236)] × 100 = 7.9%



## 16.0 Pollution Prevention

16.1 The solutions and reagents used in this method pose little threat to the environment when recycled and managed properly.

16.2 Solutions an d reagents should be prepared in volumes consistent with laboratory use to minimize the volume of expired materials to be disposed.



## 17.0 Waste Management

17.1 It is the laboratory ’ s responsibility to comply with all federal, state, and local regulations governing waste management, particularly the biohazard and hazardous waste identification rules and land disposal restrictions, and to protect the air, water, and land by minimizing and controlling all releases from fumeMethod 1600

September 2014 22 hoods and bench operations. Compl iance with all sewage discharge permits and regulations is also required.

17.2 Samples, reference materials, and equipment known or suspected to have viable enterococci attached or contained must be sterilized prior to disposal.

17.3 Samples preserved wi th HCl to pH <2 are hazardous and must be neutralized before being disposed, or

must be handled as hazardous waste.

17.4 For further information on waste management, consult “ The Waste Management Manual for Laboratory

Personnel ” and “ Less Is Better: Laboratory Chemical Management for Waste Reduction, ” both available from the American Chemical Society ’ s Department of Government Relations and Science Policy, 1155 16th Street NW, Washington, DC 20036.



## 18.0 References

18.1 Cabelli, V. J., A. P. Dufour , M. A. Levin, L. J. McCabe, and P. W. Haberman, 1979. Relationship of Microbial Indicators to Health Effects at Marine Bathing Beaches . Amer. Jour. Public Health.

69:690

- 696.

18.2 Dufour, A.P. 1984. Health Effects Criteria for Fresh Recreational Waters, EPA

- 600/1

- 84

- 004. Office of Research and Development, USEPA.

18.3 USEPA. 2004. Results of the Interlaboratory Validation of EPA Method 1600 (mEI) for Enterococci in Wastewater Effluent. December 2004. EPA 821

- R

- 04

- 008.

18.4 Messer, J.W. and A.P. Dufou r. 1998. A Rapid, Specific Membrane Filtration Procedure for Enumeration of Enterococci in Recreational Water. Appl. Environ. Microbiol

. 64:678

- 680.

18.5 ACS. 2000. Reagent Chemicals, American Chemical Society Specifications . American Chemical Society, N ew York. For suggestions of the testing of reagents not listed by the American Chemical Society, see AnalaR Standards for Laboratory Chemicals , BDH, Poole, Dorset, UK and the United States Pharmacopeia.

18.6 APHA. 1998. Standard Methods for the Examinatio n of Water and Wastewater . 20th Edition. American Public Health Association, Washington D.C.

18.7 Bordner, R., J.A. Winter, and P.V. Scarpino (eds.). Microbiological Methods for Monitoring the

Environment: Water and Wastes , EPA

- 600/8

- 78

- 017. Cincinnati, OH: U.S. Environmental Protection Agency, 1978.

## Appendix A

Part II (General Operations)

Section A (Sample Collect ion, Preservation, and Storage)

### 1 

## Sample Collection 1



### 1.0 Sample Containers

1.1 Sample Bottles: bottles must be resistant to sterilizing conditions and the solvent

action of water. Wide

- mouth borosilicate glass bottles with screw

- cap or ground

glass stopper or heat

- resistant plastic bottles may be used if they can be sterilized without producing toxic materials (see examples A and C in Figure 1). Screw

caps must not produce bacteriostatic or nutritive compounds upon sterilization.

Figure 1 . Suggested sample containers

.



### 1.2 Selection and Cleaning of Bottles: Sample bottles should be at least 125 mL volume for adequate sampling and for good mixing. Bottles of 250 mL, 500 mL, and 1000 mL volume are often used for multiple analyses. Discard bottles which have chips, cracks, and etched surfaces. Bottle closures must be water

- tight. Before use, thoroughly cleanse bottles and closures with detergent and hot water, followed by a hot water rinse to remove all trace of detergent. Then rinse them three times with laboratory

- pure water.

1.3 Dechlorinating Agent: The agent must be placed in the bottle when water and wastewater samples containing residual chlorine are anticipated. Add sodium

thiosulfate to the bottle before sterilization at a c oncentration of 0.1 mL of a 10%

solution for each 125 mL sample volume. This concentration will neutralize approximately 15 mg/L of residue chlorine.

1.4 Chelating Agent: A chelating agent should be added to sample bottles used to collect samples suspecte d of containing >0.01 mg/L concentrations of heavy metals such as copper, nickel or zinc, etc. Add 0.3 mL of a 15% solution of ethylenediaminetetraacetic acid (EDTA) tetrasodium salt, for each 125 mL sample volume prior to sterilization.

1 The text is taken from Part II, Section A, of the EPA publication "Microbiological Methods for Monitoring the

Environment" EPA

- 600/8

- 78

- 017, December 1978.

### 2

1.5 Wrapping Bott les: Protect the tops and necks of glass stoppered bottles from contamination by covering them before sterilization with aluminum foil or kraft

paper.

1.6 Sterilization of Bottles: Autoclave glass or heat

- resistant plastic bottles at 121°C

for 15 minutes. Alternatively, dry glassware may be sterilized in a hot oven at 170°C for not less than two hours. Ethylene oxide gas sterilization is acceptable for plastic containers that are not heat

- resistant. Sample bottles sterilized by gas should be stored overnig ht before being used to allow the last traces of gas to dissipate.

1.7 Plastic Bags: The commercially available bags (Whirl

- pak) (see example B in Figure 1) are a practical substitute for plastic or glass samples bottles in sampling soil, sediment, or bio solids. The bags are sealed in manufacture and opened only at time of sampling. The manufacturer states that such bags are sterilized.

2.0 Sampling Techniques

Samples are collected by hand or with a sampling device if the sampling site has difficult

acce ss such as a bridge or bank adjacent to a surface water.

2.1 Chlorinated Samples: When samples such as treated waters, chlorinated wastewaters or recreational waters are collected, the sample bottle must contain a dechlorinating agent (see section 1.3 abo ve).

2.2 Composite Sampling: In no case should a composite sample be collected for bacteriologic examination. Data from individual samples show a range of values. A composite sample will not display this range. Individual results will give

information ab out industrial process variations in flow and composition. Also, one or more portions that make up a composite sample may contain toxic or nutritive

materials and cause erroneous results.

2.3 Surface Sampling by Hand: A grab sample is obtained using a sample bottle prepared as described in (1) above. Identify the sampling site on the bottle label and on a field log sheet. Remove the bottle covering and closure and protect from contamination. Grasp the bottle at the base with one hand and plunge the bott le mouth down into the water to avoid introducing surface scum (Figure 2). Position the mouth of the bottle into the current away from the hand of the collector and, if applicable, away from the side of the sampling platform. The sampling depth

should be 1 5

- 30 cm (6

- 12 inches) below the water surface. If the water body is static, an artificial current can be created, by moving the bottle horizontally in the direction it is pointed and away from the sampler. Tip the bottle slightly upwards

to allow air to ex it and the bottle to fill. After removal of the bottle from the stream, pour out a small portion of the sample to allow an air space of 2.5

- 5 cm (1

- 2 inches) above each sample for proper mixing of the sample before analyses.

### 3 Tightly stopper the bottle and place on ice (do not freeze) for transport to the laboratory.

Figure 2 . Grab sampling technique for surface waters.



### 3.0 Selection of Sampling Sites and Frequency

These will be described for streams, rivers, estuarine, marine, and recreational waters as

well as domestic and industrial wastewaters.

3 .1 Stream Sampling: The objectives of the initial survey dictate the location, frequency and number of samples to be collected.

3.1.1 Selection of Sampling Sites: A typical stream sampling program includes sampling locations upstream of the area of concern, upstream and downstream of waste discharges, upstream and downstream from tributary entrances to the river and upstream of the mouth of the tributary. For more complex situations , where several waste discharges are involved, sampling includes sites upstream and downstream from the combined discharge area and samples taken directly from each industrial or municipal waste discharge. Using available bacteriological, chemical and disc harge rate data, the contribution of each pollution source can be determined.

3.1.2 Small Streams: Small streams should be sampled at background stations upstream of the pollution sources and at stations downstream from pollution sources. Additional sampl ing sites should be located downstream to delineate the zones of pollution. Avoid sampling areas where stagnation may occur (e.g., backwater of a tributary) and areas located near the inside bank of a curve in the stream which may not be representative of the main

channel.

3.1.3 Large Streams and Rivers: Large streams are usually not well mixed laterally for long distances downstream from the pollution sources. Sampling sites below point source pollution should be established to

### 4 provide desired downstream travel time and dispersal as determined by

flow rate measurements. Particular care must be taken to establish the proper sampling points. Occasionally, depth samples are necessary to determine vertical mixing patterns.

3.2 Estuarine and Marine Sampling: Sampling estuarine and marine waters requires the consideration of other factors in addition to those usually recognized in fresh water sampling. They include tidal cycles, current patterns, bottom

currents and counter

- currents, stratification, seasonal f luctuations, dispersion of discharges and multi

- depth samplings.

The frequency of sampling varies with the objectives. When a sampling program is started, it may be necessary to sample every hour around the clock to establish pollution loads and dispersio n patterns. The sewage discharges may occur continuously or intermittently.

When the sampling strategy for a survey is planned, data may be available from previous hydrological studies done by the Coast Guard, Corps of Engineers,

National Oceanic and Atmo spheric Administration (NOAA), U.S. Geological Survey, or university and private research investigations. In a survey, float studies and dye studies are often carried out to determine surface and undercurrents. Initially depth samples are taken on the bott om and at five feet increments between surface and bottom. A random grid pattern for selecting sampling sites is established statistically.

3.2.1 Estuarine Sampling: When a survey is made on an estuary, samples are often taken from a boat, usually making an end to end traverse of the estuary. Another method involves taking samples throughout a tidal cycle, every hour or two hours from a bridge or from an anchored boat at a number of fixed points.

In a large bay or estuary where many square miles of area a re involved, a grid or series of stations may be necessary. Two sets of samples are usually taken from an area on a given day, one at ebb or flood slack water, and the other three hours earlier, or later, at the half tidal interval. Sampling is scheduled s o that the mid

- sampling time of each run coincides

with the calculated occurrence of the tidal condition.

In location sampling sites, one must consider points at which tributary waters enter the main stream or estuary, location of shellfish beds and

bathi ng beaches. The sampling stations can be adjusted as data accumulate. For example, if a series of stations half mile apart consistently show similar values, some of these stations may be dropped and other

stations added in areas where data shows more varia bility.

### 5 Considerable stratification can occur between the salt water from the sea and the fresh water supplied by a river. It is essential when starting a survey of an unknown estuary to find out whether there is any marked

stratification. This can be don e by chloride determinations at different locations and depths. It is possible for stratification to occur in one part of an estuary and not in another.

On a flood tide, the more dense salt water pushing up into the less dense

fresh river water will cause an overlapping with the fresh water flowing on top. A phenomenon called a salt water wedge can form. As a result, stratification occurs. If the discharge of pollution is in the salt water layer,

the contamination will be concentrated near the bottom at th e flood tide. The flow or velocity of the fresh water will influence the degree of stratification which occurs. If one is sampling only at the surface, it is possible that the data will not show the polluted underflowing water which

was contaminated at the point below the fresh water river. Therefore, where stratification is suspected, samples at different depths will be

needed to measure vertical distribution.

3.2.2 Marine Sampling: In ocean studies, the environmental conditions are most diverse along the coast where shore, atmosphere and the surf are strong influences. The shallow coastal waters are particularly susceptible to daily fluctuations in temperature and seasonal changes.

Sampling during the entire tidal cycle or during a half cycle may be

requ ired. Many ocean studies such as sampling over the continental shelf involve huge areas and no two areas of water are the same.

Selection of sampling sites and depths are most critical in marine waters. In winter, cooling of coastal waters can result in w ater layers which approach 0°C. In summer, the shallow waters warm much faster than the deeper waters. Despite the higher temperature, oxygen concentrations are higher in shallow than in deeper waters due to greater water movement, surf action and photosyn thetic activity from macrophytes and the plankton.

Moving from the shallow waters to the intermediate depths, one observes a moderation of these shallow water characteristics. In the deeper waters,

there is a marked stabilization of conditions. Water temperatures are lower and more stable. There is limited turbulence, little penetration of light, sparse vegetation and the ocean floor is covered with a layer of silts and

sediments.

3.3 Recreational Waters (Bathing Beaches): Sampling sites at bathing be aches or other recreational areas should include upstream or peripheral areas and locations adjacent to natural drains that would discharge stormwater, or run

- off areas

### 6 draining septic wastes from restaurants, boat marinas, or garbage collection areas.

Sam ples of bathing beach water should be collected at locations and times of heaviest use. Daily sampling, preferably in the afternoon, is the optimum frequency during the season. Weekends and holidays which are periods of highest

use must be included in the sampling program. Samples of estuarine bathing waters should be obtained at high tide, ebb tide and low tide in order to determine the cyclic water quality and deterioration that must be monitored during the swimming season.

3.4 Domestic and Industrial Wa ste Discharges: It is often necessary to sample secondary and tertiary wastes from municipal waste treatment plants and various industrial waste treatment operations. In situations where the plant treatment efficiency varies considerably, grab samples are collected around the clock at selected intervals for a three to five day period. If it is known that the process displays little variation, fewer samples are needed. In no case should a composite sample be collected for bacteriological examination. The Nat ional Pollution Discharge Elimination System (NPDES) has established wastewater treatment plant effluent limits for all dischargers. These are often based on maximum and mean values. A sufficient number of samples must be collected to satisfy the permit an d/or to provide statistically sound data and give a fair representation of the bacteriological quality of the discharge.

## Appendix B

Part II (General Operations)

Sections C.3.5 (Counting Colonies) and C.3.6 (Calculation of Results)

### 1 

## Counting Col onies 1



### 1.0 Counting Colonies

Colonies should be counted using a fluorescent lamp with a magnifying lens. The

fluorescent lamp should be nearly perpendicular to the membrane filter. Count colonies individually, even if they are in contact wit h each other. The technician must learn to recognize the difference between two or more colonies which have grown into contact with each other and single, irregularly shaped colonies which sometimes develop on

membrane filters. The latter colonies are usua lly associated with a fiber or particulate material and the colonies conform to the shape and size of the fiber or particulates. Colonies which have grown together almost invariably show a very fine line of contact.

2.0 Calculation of Results

2.1 Select the membrane filter with the number of colonies in the acceptable range and calculate count per 100 mL according to the general formula:

Count per 100 mL = (No. of colonies counted/Volume of sample filtered, in mL) x 100



### 2.2 Counts Within the Acceptable Limits

The acceptable range of colonies that are countable on a membrane is a function of the method. Different methods may have varying acceptable count ranges. All examples in this appendix assume that the acceptable range of counts is between

20

- 60 colonies per membrane.

For example, assume that filtration of volumes of 50, 15, 5, 1.5, and 0.5 mL produced colony counts of 200, 110, 40, 10, and 5, respectively.

An analyst would not actually count the colonies on all filters. By inspection the analys t would select the membrane filter with the acceptable range of target colonies, as defined by the method, and then limit the actual counting to such

membranes.

After selecting the best membrane filter for counting, the analyst counts colonies and applies the general formula as in section 2.1 above to calculate the count/100

mL.

1 The text is largely taken from Part II, Section C, of the EPA publication "Microbiological Methods for Monitoring

the Environment" EPA

- 600/8

- 78

- 017, December 1978. Some examples were kindly provided by Kristen Brenner,

US EPA.

### 2

2.3 More Than One Acceptable Count

2.3.1 If there are acceptable counts on replicate plates, carry counts independently to final reporting units, then calculate the arithmetic me an of these counts to obtain the final reporting value.

Example, if the counts are 24 and 36 for replicate plates of 100 mL each,

then the arithmetic mean is calculated as follows:

( 24 CFU / 100 mL + 36 CFU / 100 mL )

2 

### = 30 CFU / 100 mL



### 2.3.2 I f there is more than one dilution having an acceptable range of counts, independently carry counts to final reporting units, and then average for final reported value.

For example, if volumes of 100, 10, 1 and 0.1 mL produced colony counts

of Too Numerous To Count (TNTC ), 55, 30, and 1, respectively, then two volumes, 10 mL and 1 mL, produced colonies in the acceptable counting

range.

Independently carry each MF count to a count per 100 mL:

55

10 x 100 = 550 CFU/100 mL



### and

30

1 x 100 = 3000 CFU/100 mL



### Calculate the arithmetic mean as in section 2.3.1 above:

( 550 CFU / 100 mL + 3000 CFU / 100 mL )

2 = 1775 CFU / 100 mL



### Report this as 1775 CFU/100 mL.

2.4 If all MF counts are below the lower acceptable count limit, select the most nearly acceptable count.

2.4.1 For example, sample volumes of 100, 10 and 1 mL produced colony counts of 17, 1 and 0, respectively.

### 3 Here, no colony count falls within recommended limits. Calculate on the basis of the most nearly acceptable plate count, 17, and report as 17

CFU/100 mL.

Note that in this case, because no calculations were done (i.e. this is the count for 100 mL), the count is reported as 17 CFU/100 mL rather than an

“estimated count of 17 CFU/100 mL”

2.4.2 A s a second example, assume a count in which sample volumes of 10 and 1 mL produced colony counts of 18 and 0, respectively.

Here, no colony count falls within recommended limits. Calculate on the basis of the most nearly acceptable plate count, 18, and calculate as in

section 2.3.2 above.

18

10 x 100 = 180 CFU/100 mL



### Report this as an estimated count of 180 CFU/100 mL

.

2.5 If counts from all membranes are zero, calculate using count from largest

filtration volume.

For example, sample volumes of 25, 10, and 2 mL produced colony counts of 0, 0, and 0, respectively, and no actual calculation is possible, even as an estimated report. Calculate the number of colonies per 100 mL that would have been reported if there had been one colony on the filter representing the largest

filtration volume. In this e xample, the largest volume filtered was 25 mL and thus

the calculation would be:

1

25 x 100 = 4 CFU/100 mL



### Report this as < (less than) 4 CFU/100 mL.

2.6 If all membrane counts are above the upper acceptable limit, calculate count using

the smallest volume filtered.

For example, assume that the volumes 1, 0.3, and 0.01 mL produced colony counts of TNTC, 150, and 110 colonies, respectively. Since all colony counts are above the acceptable limit, use the colony count from the smallest sample v olume

filtered and estimate the count as:

### 4 110

0

. 01 x 100 = 1,100,000 CFU/100 mL



### Report this as estimated count 1.1 × 10 6 

### CFU/100 mL

2.7 If typical colonies are too numerous to count (TNTC), use upper limit count with

smallest filtration volume.

For example, assume that the volumes 1, 0.3, and 0.01 mL all produced too many typical colonies, and that the laboratory bench record indicated TNTC.

Use the upper acceptable count for the method (60 colonies in this example) as

the basis of calculation w ith the smallest filtration volume and estimate the count

as:

60

0

. 01 x 100 = 600,000 CFU/100 mL



### Report this as > (greater than) 6 ×10 5 

### CFU/100 mL

2.8 If colonies are both above and below the upper and lower acceptable limits (i.e.,

no counts are wi thin the acceptable limits), select the most nearly acceptable

count.

2.8.1 For example, sample volumes of 100, 10 and 1 mL produced colony counts of 64, 6 and 0, respectively.

Here, no colony count falls within recommended limits. Calculate on the

basis of the most nearly acceptable plate count, 64, and report as 64

CFU/100 mL

Note that in this case, because no calculations were done (i.e. this is the count for 100 mL), the count is reported as 64 CFU/100 mL rather than an

“estimated count of 64 CFU/100 mL”

2.8.2 As a second example, assume a count in which sample volumes of 100, 10 and 1 mL produced colony counts of 98, 18, and 0, respectively.

Here, no colony count falls within recommended limits. Calculate on the basis of the most nearly acceptable plate count, 18, and calculate as in

section 2.3.2 above.

18

10 x 100 = 180 CFU/100 mL



### Report this as estimated count 180 CFU/100 mL.

### 5

2.9 If there is no result because of a confluent growth, > 200 atypical colonies (TNTC), lab accident, etc., repor t as No Data and specify the reason.