Epa method 1600

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Analytical Methods

mEI agar method for enterococci: U.S. Environmental Protection Agency Method 1600.
Updated October 2007

The mEI agar method is a one-step membrane-filtration method that allows the detection of enterococci in 24 hours with an incubation at 41°C (U.S. Environmental Protection Agency, 2006a). It is recommended for use in place of the old enterococci method, the mE-EIA method, which is a two-step method that takes 48 hours to complete with an incubation at 35°C (U.S. Environmental Protection Agency, 2006b). The mEI method can be done in the field or laboratory.

THEORY
The mEI medium is similar to the mE medium except that it contains a reduced amount of triphenyltetrazaolium chloride (TTC) and contains a substrate, indoxyl b-D-glucoside, that turns blue when cleaved by an enzyme present in enterococci (b-glucosidase). All colonies with any blue halo are recorded as enterococci, regardless of colony color. Magnification with a dissecting microscope is used for counting to give maximum visibility of colonies.

USE
The mEI test for enterococci can be applied to potable, fresh, estuarine, marine, and shellfish-growing waters.

MEDIA
The mEI agar medium is available from Government Scientific Source, Inc. (800/248-8030, Cat 214885 (100 g) or Cat 214881 (500 g)). Indoxyl β-D-glucoside, nalidixic acid, and 1% presterilized TTC must also be purchased if the medium is made from dehydrated ingredients. Indoxyl β-D-glucoside can be purchased from Sigma-Aldrich Corp. (800/325-3010, Cat I3750-2G (2g) or Cat I3750-1G (1g)). Nalidixic acid can be purchased from Hach (800/227-4224, Cat 24071-24) and needs to be dissolved in 10 N NaOH that can be purchased from Fisher Scientific (800/766-7000, Cat SS255-1 (1 L)). The 1% presterilized TTC can be purchased from Hach (Cat 24060-42).

(See preparation instructions for details on media composition and preparation (Appendix H)).

Pre-poured plates can be purchased in lots of 20 plates from Fisher Scientific (Cat B15045) or VWR (Cat 900006-316). Other quantities and plate sizes are available from these manufacturers.  

Use phosphate buffered dilution water and 0.45 µm membrane filters.  Buffer can be purchased from Hardy Diagnostics (800/266-2222, Cat D699 (99mL) or Cat U193 (500mL)). (See buffer preparation (Appendix M)).

REFERENCES
U.S. Environmental Protection Agency, 2006a, Method 1600—Enterococci in water by membrane filtration using membrane-Enterococcus Indoxyl-b-D-Glucoside agar (mEI): Washington, D.C., EPA/821/R-06/009, 42 p.

U.S. Environmental Protection Agency, 2006b, Method 1106.1—Enterococci in water by membrane filtration using membrane-Enterococcus Esculin Iron Agar (mE-EIA): Washington, D.C., EPA 821/R-06-008, 42 p.

These documents can be obtained at http://www.epa.gov/waterscience/methods/method/biological/

NWIS PARAMETER CODES:
90909 Enterococci on mEI agar (preferred method), colonies per 100 mL

31649 Enterococci on mE agar (acceptable), colonies per 100 mL

Sours: https://oh.water.usgs.gov/OWML/micro_methods_mEI_agar.htm

Comparison of Enterococcus species diversity in marine water and wastewater using Enterolert and EPA Method 1600

EPA Method 1600 and Enterolert are used interchangeably to measure Enterococcus for fecal contamination of public beaches, but the methods occasionally produce different results. Here we assess whether these differences are attributable to the selectivity for certain species within the Enterococcus group. Both methods were used to obtain 1279 isolates from 17 environmental samples, including influent and effluent of four wastewater treatment plants, ambient marine water from seven different beaches, and freshwater urban runoff from two stream systems. The isolates were identified to species level. Detection of non-Enterococcus species was slightly higher using Enterolert (8.4%) than for EPA Method 1600 (5.1%). E. faecalis and E. faecium, commonly associated with human fecal waste, were predominant in wastewater; however, Enterolert had greater selectivity for E. faecalis, which was also shown using a laboratory-created sample. The same species selectivity was not observed for most beach water and urban runoff samples. These samples had relatively higher proportions of plant associated species, E. casseliflavus (18.5%) and E. mundtii (5.7%), compared to wastewater, suggesting environmental inputs to beaches and runoff. The potential for species selectivity among water testing methods should be considered when assessing the sanitary quality of beaches so that public health warnings are based on indicators representative of fecal sources.

Sours: https://pubmed.ncbi.nlm.nih.gov/23840233/
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Enterococci in Water by Membrane Filtration Using membrane-Enterococcus Indoxyl-B-D-Glucoside Agar (mEI)July 2006
WATER
Membrane Filtration
Microbiological
USEPA, 2006, Method 1600: Enterococci in water by membrane filtration using membrane-Enterococcus Indoxyl-ß-D-Glucoside Agar (mEI): U.S. Environmental Protection Agency Report 821-R-06-009, 42 p.
This method provides a direct count of bacteria in water based on the development of colonies on the surface of the membrane filter. 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 41oC + 0.5oC. 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 maximum visibility of colonies.
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 B-D-glucoside, a chromogenic cellobiose analog used in place of esculin. In this procedure, B-glucosidase-positive enterococci produce an insoluble indigo blue complex which diffuses into the surrounding media, forming a blue halo around the colony.
This method describes a membrane filter procedure for the detection and enumeration of the enterococci bacteria in water.
Water samples containing colloidal 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.
The minimum analytical QC requirements for the analysis of samples using this method include an initial demonstration of laboratory capability through performance of the initial precision and recovery (IPR) analyses, ongoing demonstration of laboratory capability through performance of the ongoing precision and recovery (OPR) analysis and matrix spike (MS) analysis (disinfected wastewater only), and the routine analysis of positive and negative controls, filter sterility checks, method blanks, and media sterility checks. For the IPR, OPR and MS analyses, it is necessary to spike samples with either laboratory-prepared spiking suspensions or BioBalls.
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 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.
Ice or refrigerate water samples at a temperature of
Analysis preferably within 2 hours of collection; max. transport time to lab is 6 hours, and samples should be processed within 2 hours of receipt at lab.
Less than $50
Sours: https://www.nemi.gov/methods/method_summary/9790/
EPA Method 5035 Technical Presentation
United States Environmental Protection Agency Office of Water Washington DC 20460 EPA821-R-02-022 September 2002 vvEPA Method 1600: Enterococci in Water by Membrane Filtration Using membrane-Enterococcus Indoxyl-p-D-Glucoside Agar (mEI)
------- U.S. Environmental Protection Agency Office of Water (4303T) 1200 Pennsylvania Avenue, NW Washington, DC 20460 EPA-821-R-02-022
------- 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. 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, contractors, 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 represents that its use by such party would not infringe on privately owned rights. This document combines the information previously published in Method 1600: Membrane Filter Test Method for Enter ococci in Water (EPA-821-R-97-004) (Reference 18.8) and Improved Enumeration Methods for the Recreational Water Quality Indicators: Enterococci and Escherichia coll (EPA/821/R-97/004) (Reference 18.7). Mention of trade names or commercial products does not constitute endorsement or recommendation for use. Questions concerning this method or its application should be addressed 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 [email protected] 202-566-1075 202-566-1053 (facsimile)
------- Introduction EPA has been increasingly concerned with the public health risks of infectious diseases caused by microbial organisms in our nation's beaches. In response to this problem, EPA has established the Beaches Environmental Assessment Closure and Health (BEACH) Program. This analytical method is published for use in the BEACH Program. In 1986, EPA issued a revision to its bacteriological ambient water quality criteria recommendations to include new indicator bacteria, E. coll and enterococci, which provide a better correlation with swimming-associated gastrointestinal illness than the previous criteria recommendations for fecal coliform bacteria. These revised criteria are useful to public health officials because they enable quantitative estimates of illness rates associated with swimming in polluted water. This method is a revision of EPA's previous enterococci method, used since 1985 in ambient water quality monitoring. It reduces analysis time to 24 hours and improves analytical quality. The method has been validated in single- and multi-laboratory studies and has undergone peer review.
------- Table of Contents 1.0 Scope and Application 1 2.0 Summary of Method 1 3.0 Definitions 1 4.0 Interferences 1 5.0 Safety 2 6.0 Equipment and Supplies 2 7.0 Reagents and Standards 3 8.0 Sample Collection, Preservation, and Storage 5 9.0 Quality Control 6 10.0 Calibration and Standardization 6 11.0 Procedure 6 12.0 Data Analysis and Calculations 7 13.0 Method Performance 7 14.0 Reporting Results 7 15.0 Verification Procedure 7 16.0 Pollution Prevention 8 17.0 Waste Management 8 18.0 References 8
------- Method 1600: Enterococci in Water by Membrane Filtration Using membrane-Enterococcus lndoxyl-J3-D-Glucoside Agar (mEI) September 2002 1.0 Scope and Application 1.1 This method describes a membrane filter (MF) procedure for the detection and enumeration of the enterococci bacteria in water. 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. 1.2 The enterococci test measures the bacteriological quality of recreational waters. Epidemiological studies have led to the development of criteria which can be used to promulgate recreational water standards based on the established relationship between health effects and water quality. The significance of finding enterococci in recreational water samples is the direct relationship between the density of enterococci in the water and swimming-associated gastroenteritis studies of marine and fresh water bathing beaches (Reference 18.3, Reference 18.4). 1.3 The test for enterococci can be applied to potable, fresh, estuarine, marine, and shellfish growing waters. 1.4 Since a wide range of sample volumes or dilutions can be analyzed by the MF technique, a wide range of enterococci levels in water can be detected and enumerated. 2.0 Summary of Method 2.1 The MF method provides a direct count of bacteria in water based on the development of colonies on the surface of the membrane filter (Reference 18.5). 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 h at 41°C. All colonies (regardless of color) with a blue halo are recorded as enterococci colonies. Magnification and a small fluorescent lamp are used for counting to give maximum visibility of colonies. 3.0 Definitions 3.1 In this method, enterococci are those bacteria which produce colonies with a blue halo after incubation on mEI agar. Enterococci include Streptococcus faecalis, Streptococcus faecium, Streptococcus avium, and their variants. 4.0 Interferences 4.1 Water samples containing colloidal or suspended particulate materials can clog the membrane filter and prevent filtration, or cause spreading of bacterial colonies which could interfere with identification of target colonies. September 2002
------- Method 1600 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 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 Graduated cylinders, 100-1000 mL, covered with aluminum foil or kraft paper and sterile. 6.7 Membrane filtration units (filter base and funnel), glass, plastic or stainless steel, wrapped with aluminum foil or kraft paper and sterilized. 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 tubing. A filter manifold to hold a number of filter bases is optional. 6.11 Flask for safety trap placed between the filter flask and the vacuum source. 6.12 Forceps, straight or curved, with smooth tips to handle filters without damage. 6.13 Ethanol, methanol or isopropanol in a small, wide-mouth container, for flame-sterilizing forceps. 6.14 Burner, Bunsen or Fisher type, or electric incinerator unit for sterilizing loops and needles. 6.15 Thermometer, checked against a National Institute of Standards and Technology (NIST) certified thermometer, or one that meets the requirements of NIST Monograph SP 250-23. 6.16 Petri dishes, sterile, plastic, 9 x 50 mm, with tight-fitting lids. 6.17 Bottles, milk dilution, borosilicate glass, screw-cap with neoprene liners, marked at 99 mL for 1:100 dilutions. Dilution bottles marked at 90 mL or tubes marked at 9 mL may be used for 1:10 dilutions. 6.18 Flasks, borosilicate glass, screw-cap, 250-2000 mL volume. 6.19 Membrane filters, sterile, white, grid marked, 47 mm diameter, with 0.45 ± 0.02 (im pore size. 6.20 Inoculation loops, at least 3 mm diameter, and needles, nichrome or platinum wire, 26 B & S gauge, in suitable holders. Sterile disposable applicator sticks or plastic loops are alternatives to inoculation loops. 6.21 Incubator maintained at 41 ± 0.5°C. 6.22 Waterbath maintained at 50°C for tempering agar. September 2002 2
------- Method 1600 6.23 Test tubes, 20 x 150 mm, borosilicate glass or plastic. 6.24 Caps, aluminum or autoclavable plastic, for 20 mm diameter test tubes. 6.25 Test tubes, screw-cap, borosilicate glass, 16 x 125 mm or other appropriate size. 6.26 Whirl-Pak® bags. 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 18.9). 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 Water: Reagent water conforming to Specification Dl 193, reagent water conforming Type II, Annual Book of ASTM Standards (Reference 18.1). 7.4 Buffered Dilution Water 7.4.1 Composition: Sodium Dihydrogen Phosphate 0.58 g Sodium Monohydrogen Phosphate 2.50 g Sodium Chloride 8.50 g Reagent-Grade Distilled Water 1.0 L 7.4.2 Preparation: Dissolve the ingredients in 1 L of reagent water in a flask 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°C (15 Ib pressure) for 15 min. Final pH should be 7.4 ± 0.2. 7.5 Phosphate Buffered Dilution Water (Reference 18.2) 7.5.1 Composition of Stock Phosphate Buffer Solution: Phosphate dihydrogen phosphate 34.0 g Reagent-Grade distilled water 500.0 mL 7.5.2 Preparation: Adjust the pH of the solution to 7.2 with 1 N NaOH, and bring the volume to 1 L with reagent-grade distilled water. Sterilize by filtration or autoclave at 121°C (15 Ib pressure) for 15 min. 7.5.3 Preparation of Stock Magnesium Chloride Solution: Add 38 g anhydrous MgCl2 or 81.1 g MgCl2-6H2O to 1 L reagent-grade distilled water. Sterilize by filtration or autoclave at 121°C (15 Ib pressure) for 15 min. 7.5.4 Storage of Stock Solutions: After sterilization, store the stock solutions in the refrigerator until used. Handle aseptically. If evidence of mold or other contamination appears, the affected stock solution should be discarded and a fresh solution should be prepared. 7.5.5 Working Phosphate Buffered Dilution Water: Mix 1.25 mL of the stock phosphate buffer and 5 mL of the MgCl2 stock per liter of reagent-grade distilled water. Dispense in 3 September 2002
------- Method 1600 appropriate amounts for dilutions in screw-cap bottles or culture tubes, and/or into containers for use as rinse water. Autoclave at 121°C (15 Ib pressure) for 15 min. Final pH should be 7.0 ± 0.2. 7.6 mEI Agar 7.6.1 Composition of Basal Medium (mE Agar, Difco 0333) Peptone 10.0 g Sodium Chloride 15.0 g Yeast Extract 30.0 g Esculin 1.0 g Actidione (Cycloheximide) 0.05 g Sodium Azide 0.15g Agar 15.0 g Reagent-Grade Distilled Water 1.0 L 7.6.2 Preparation of mEI Medium: Add 71.2 g of dehydrated basal medium plus 0.75 grams of indoxyl P-D glucoside to 1 L of reagent grade water in a flask and heat to boiling until ingredients dissolve. Autoclave at 121°C (15 Ib pressure) for 15 min and cool in a 50°C water bath. 7.6.3 Reagents Added After Sterilization: Mix 0.24 g nalidixic acid in 5 mL reagent-grade sterile distilled water, add a few drops of 0. IN NaOH to dissolve; add to the mEI medium. Add 0.02 g triphenyltetrazolium chloride separately to the mEI medium and mix. 7.6.4 Alternately, the following solutions may be used: 7.6.4.1 Nalidixic acid: Add 0.48 g of nalidixic acid and 0.4 mL 10 N NaOH to 10 mL of reagent-grade distilled water and mix. Filter-sterilize the solution, and add 5.2 mL per liter of medium. 7.6.4.2 Triphenyltetrazolium chloride (TTC): Add 0.1 g of TTC to 10 mL of reagent- grade distilled water, and warm to dissolve. Filter-sterilize the solution, and add 2 mL per liter of medium. 7.6.5 Preparation of mEI Agar Plates: Pour the mEI agar into 9x50 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. 7.7 Brain Heart Infusion Broth (BHIB) (Difco 0037, BD 4311059) 7.7.1 Composition: Calf Brain Infusion 200.0 g Beef Heart Infusion 25 0.0 g Peptone lO.Og Sodium Chloride 5.0 g Disodium Phosphate 2.5 g Dextrose 2.0 g Reagent-Grade Distilled Water 1.0 L 7.7.2 Preparation: Dissolve 37 g of dehydrated brain heart infusion in 1 L of reagent grade water. Dispense in 8-10 mL volumes in screw-cap tubes and autoclave at 121°C (15 Ib pressure) for 15 min. If the medium is not used the same day as prepared and sterilized, September 2002 4
------- Method 1600 heat in boiling water bath for several min to remove absorbed oxygen, and cool quickly without agitation, just prior to inoculation. The final pH should be 7.4 ± 0.2. 7.8 Brain Heart Infusion Broth (BHIB) with 6.5% NaC 1 7.8.1 Composition: BHIB with 6.5% NaCl is the same as BHIB above, but with additional NaCl. 7.8.2 Preparation: Add 60.0 g NaCl per liter of medium. Since most commercially available dehydrated media contain sodium chloride, this amount is subtracted from the 65 g per liter required to make a final concentration of 6.5% NaCl. 7.9 Brain Heart Infusion Agar (BHIA) (Difco 0418, BD4311065) 7.9.1 Composition: BHIA contains the same components as BHIB above with the addition of 15.0 g of agar per L of BHIB. 7.9.2 Preparation: Suspend 52 g dehydrated BHIA in 1 L of reagent-grade distilled water. Heat to boiling until the ingredients are dissolved. Dispense 10 mL of medium in screwcap test tubes, and sterilize for 15 min at 121°C (15 Ib pressure). After sterilization, slant until solid. Final pH should be 7.4 ± 0.2. 7.10 Bile Esculin Agar (BEA) (Difco 0879) 7.10.1 Composition: Bacto Beef Extract 3.0 g Bacto Peptone 5.0g Bacto Oxgall 40.0 g Bacto Esculin l.Og Ferric Citrate 0.5 g Bacto Agar 15.0g Reagent-Grade Distilled Water 1.0 L 7.10.2 Preparation: Add 64.0 g dehydrated BEA to 1 L reagent-grade distilled water, and heat to boiling to dissolve completely. Dispense 10-mL volumes in tubes for slants or larger volumes into flasks for subsequent plating. Autoclave at 121°C (15 Ib pressure) for 15 min. Overheating may cause darkening of the medium. Cool in a 50°C waterbath, and dispense into sterile petri dishes. Final pH should be 6.6 ± 0.2. Store in a refrigerator. 8.0 Sample Collection, Preservation, and Storage 8.1 Sampling procedures are described in detail in the USEPA microbiology methods manual, Section II, A (Reference 18.2). Adherence to sample preservation procedures and holding time limits is critical to the production of valid data. Samples shall not be analyzed if these conditions are not met. 8.1.1 Storage Temperature and Handling Conditions Ice or refrigerate bacteriological samples at a temperature of 1-4°C during transit to the laboratory. Use insulated containers to assure proper maintenance of storage 5 September 2002
------- Method 1600 temperature. Take care that sample bottles are not totally immersed in water during transit or storage. 8.1.2 Holding Time Limitations Examine samples as soon as possible after collection. Do not hold samples longer than 6 h between collection and initiation of analyses. 9.0 Quality Control 9.1 See recommendations on quality control for microbiological analyses in the USEPA microbiology methods manual, Part IV, C (Reference 18.2). 10.0 Calibration and Standardization 10.1 Check temperatures in incubators daily 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. 11.0 Procedure 11.1 Prepare the mEI agar as directed in 7.6. 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 base. 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 pollution level, to produce 20-60 enterococci colonies on membranes. Sample volumes of 1-100 mL are normally tested at half log intervals (e.g., 100, 30, 10, 3 mL). 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, and the results may be combined. 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 bubbles between the membrane and the agar surface. Reseat the membrane if bubbles occur. Run the forceps around the edge of the filter to be sure that the filter is properly seated on the agar. Close the dish, invert, and incubate at 41 ± 0.5°C for 24 h. 11.9 After incubation, count and record colonies on those membrane filters containing, if practical, 20-60 colonies with a blue halo regardless of colony color as an enterococci. Use magnification for counting and a small fluorescent lamp to give maximum visibility of colonies. September 2002
------- Method 1600 Figure 1. Enterococci on mEI Agar. Colonies with a blue halo are considered to be enterococci. 12.0 Data Analysis and Calculations 12.1 Use the following general rules to calculate the enterococci count per 100 ml of sample: 12.1.1 Select the membrane filter with an acceptable number of colonies (regardless of colony color) with a blue halo (20-60). Calculate the number of enterococci per 100 mL according to the following general formula: Number of enterococci colonies Enterococci/100 mL = X 100 Volume of sample filtered (mL) 12.1.2 See the USEPA microbiology methods manual, Part II, Section C, 3.5, for general counting rules (Reference 18.2). 13.0 Method Performance 13.1 Specificity - The specificity of the medium used in this method is 6.0% false positive and 6.5% false negative for various environmental water samples (Reference 18.6). The false positive rate was calculated as the percent of colonies which reacted typically, but did not verify as members of the enterococcus group. The false negative rate was calculated as the percent of all verified enterococcus colonies not reacting typically. 13.2 Bias - The persistent positive or negative deviation of the results from the assumed or accepted true value is not significant (Reference 18.6). 13.3 Precision - The precision among laboratories for marine water and surface water was 2.2% and 18.9% (Reference 18.6). 14.0 Reporting Results 14.1 There should be at least three volumes tested per sample. Report the results as enterococci per 100 mL of sample. 15.0 Verification Procedure 15.1 Colonies of any color having a blue halo after incubation on mEI agar can be verified as enterococci. Verification of colonies may be required in evidence gathering and it is also recommended as a means of quality control for the initial use of the test and for changes in 7 September 2002
------- Method 1600 sample sites, lots of commercial media, or major ingredients in media compounded in the laboratory. The verification procedure follows. 15.2 Using a sterile inoculating needle, transfer cells from the centers of at least 10 well-isolated typical colonies into a BHIB tube and onto a BHIA slant. Incubate broth tubes for 24 h and slants for48hat35±0.5°C. 15.3 After a 24 h incubation, transfer a loopful of material from each BHIB tube to BEA, BHIB and BHIB with 6.5% NaCl. 15.3.1 Incubate the BEA and BHIB with 6.5% NaCl at 35 ± 0.5°C for 48 h. 15.3.2 Incubate the BHIB at 45 ± 0.5°C for 48 h. 15.4 Observe for growth. 15.5 After 48 h incubation, apply a Gram stain to growth from each BHIA slant. 15.6 Gram-positive cocci that grow and hydrolyze esculin on BEA (i.e., produce a black or brown precipitate), and grow in BHIB at 45 ± 0.5°C and BHIB with 6.5% NaCl at 35 ± 0.5°C are verified as enterococci. 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 and 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 fume hoods and bench operations. Compliance 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 with HC1 to pH
Sours: https://nepis.epa.gov/Exe/ZyPURL.cgi?Dockey=P10099DH.TXT

1600 epa method

&EPA United States Environmental Protection Agency Method 1600: Enterococci in Water by Membrane Filtration Using membrane- Enterococcus Indoxyl-p-D-Glucoside Agar (mEI) July 2006
------- U.S. Environmental Protection Agency Office of Water (4303T) 1200 Pennsylvania Avenue, NW Washington, DC 20460 EPA-821-R-06-009
------- 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 • EPA Office of Research and Development, National Risk Management Research Lab: Mark C. Meckes U.S. Army Corps of Engineers, Washington Aqueduct: Elizabeth A. Turner, Michael L.
------- 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, contractors, 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 represents 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 addressed 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 [email protected] 202-566-1075 202-566-1053 (facsimile)
------- Table of Contents 1.0 Scope and Application 1 2.0 Summary of Method 1 3.0 Definitions 2 4.0 Interferences 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 Calibration and Standardization 12 11.0 Procedure 12 12.0 Verification Procedure 13 13.0 Data Analysis and Calculations 14 14.0 Sample Spiking Procedure 15 15.0 Method Performance 19 16.0 Pollution Prevention 23 17.0 Waste Management 23 18.0 References 23 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: Enterococci in Water by Membrane Filtration Using membrane-Enterococcus Indoxyl-p-D-Glucoside Agar (mEI) July 2006 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 p-D-glucoside, a chromogenic cellobiose analog used in place of esculin. In this procedure, p-glucosidase-positive enterococci produce an insoluble indigo blue complex which diffuses into the surrounding 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 health 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 water (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°C ± 0.5°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 maximum visibility of colonies. July 2006
------- Method 1600 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. 4.0 Interferences 4.1 Water samples containing colloidal 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 cylinders, 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) July 2006
------- Method 1600 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 tubing 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 Thermometer, 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, 9x50 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 (im 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°C ± 0.5°C 6.23 Waterbath maintained at 50°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 appropriate size 6.27 Autoclave or steam sterilizer capable of achieving 121°C [15 Ib 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 141, as applicable), Section 9020 (Reference 18.6). July 2006
------- Method 1600 7.4 Phosphate buffered saline (PBS) 7.4.1 Composition: Sodium dihydrogen phosphate (NaH2PO4) 0.58 g Disodium hydrogen phosphate (NajHPO^ 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°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 0.15g Indoxyl p-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°C (15 PSI) for 15 minutes and cool in a 50°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 9x50 mm or 15 x 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. Og Enzymatic digest of soybean meal 5.0 g Sodium chloride (NaCl) 5.0 g Agar 15.Og Reagent-grade water l.OL 7.6.2 Add reagents to 1 L of reagent-grade water, mix thoroughly, and heat to dissolve completely. Autoclave at 121°C (15 PSI) for 15 minutes and cool in a 50°C waterbath. Pour the medium into each 15 x 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. July 2006 4
------- Method 1600 7.7 Brain heart infusion broth (BHIB) 7.7.1 Composition: Calf brains, infusion from 200.0 g 7.7 g Beef heart, infusion from 250.0 g 9.8 g Proteose peptone lO.Og Sodium chloride (NaCl) 5.0 g Disodium hydrogen phosphate (NajHPO^ 2.5 g Dextrose 2.0 g Reagent-grade water l.OL 7.7.2 Add reagents to 1 L of reagent-grade water, mix thoroughly, and heat to dissolve completely. Dispense in 10-mL volumes in screw cap tubes, and autoclave at 121°C (15 PSI) for 15 minutes. Final pH should be 7.4 ± 0.2. 7.8 Brain heart infusion broth (BHIB) with 6.5% NaCl 7.8.1 Composition: BHIB with 6.5% NaCl is the same as BHIB above (Section 7.7), but with additional NaCl. 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 formula 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. July 2006
------- Method 1600 7.10 Bile esculin agar (BEA) 7.10.1 Composition: Beef Extract 3.0g Pancreatic Digest of Gelatin 5.0 g Oxgall 20.0 g Esculin l.Og Ferric Citrate 0.5 g Bacto Agar 14.0 g Reagent-grade water l.OL 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 slants or larger volumes into flasks for subsequent plating. Autoclave at 121°C (15 PSI) for 15 minutes. Overheating may cause darkening of the medium. Cool in a 50°C waterbath, 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 l.OL 7.11.2 Add reagents to 1 L of reagent-grade water and dispense in screw cap bottles. Autoclave at 121°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 # 1943 3 BioBalls (BTF Pty, Sydney, Australia) 7.12.2 Negative control organism (either of the following are acceptable) • Stock cultures ofEscherichia coli (E. coli) ATCC #11775 • E. coli ATCC # 11775 BioBalls (BTF Pty, Sydney, Australia) July 2006
------- Method 1600 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 temperature and handling conditions Ice or refrigerate water samples at a temperature of
Sours: https://nepis.epa.gov/Exe/ZyPURL.cgi?Dockey=P1002D7W.TXT
VOC Sampling Tutorial: Ground \u0026 Surface Water

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