Microbiology Methods for Drinking Water Laboratories. Erica Fox

Transcription

1 Microbiology Methods for Drinking Water Laboratories Erica Fox

2 Origins of Drinking Water Bacteriological Testing 1854 Cholera epidemic in London Dr. John Snow determined source of Cholera to be public water supply Traced nearly all deaths to water from the Broad Street pump, which supplied water from a public well dug 3 feet from an old cesspit Cholera epidemic stopped after removal of pump handle Showed connection between fecal contamination of drinking water and disease

3 Origins of Drinking Water Bacteriological Testing 1885 Bacterium coli commune described by Theodor Escherich Found in higher numbers than enteric pathogens in fecal material Suggested that detection of B. coli would indicate presence of fecal matter Later renamed Escherichia coli, this organism remains the main indicator of fecal pollution 3

4 Regulations Safe Drinking Water Act (SDWA), 1974 passed by Congress to protect public health by regulating public drinking water supplies National Primary Drinking Water Regulations standards set by Environmental Protection Agency (EPA), as required by SDWA Found in Code of Federal Regulations (CFR) Part 141 Establish regulated contaminants and Maximum Contaminant Levels (MCLs) if applicable, as well as approved methods for analysis Microbiological contaminants include Total coliforms (includes Fecal coliforms and E. coli)

5 Regulations National Primary Drinking Water Regulations Revised Total Coliform Rule (RTCR) Establishes MCL for E. coli and uses E.coli and total coliforms to initiate find and fix approach to address fecal contamination that could enter distribution system. Requires public water systems to perform assessments to identify sanitary defects and take action to correct them Surface Water Treatment Rule (SWTR) HPC are regulated under the SWTR with a limit of 500 cfu/ml Virginia a Primacy state enforces national regulations Virginia Waterworks Regulations: 12 VAC Virginia certifies drinking water laboratories through Division of Consolidated Laboratory Services (DCLS) Labs are required to comply with EPA Manual for the Certification of Laboratories Analyzing Drinking Water 5

6 Regulations To comply with regulations, certified laboratories must use an approved method. Each of the methods presented today is listed below with the applicable regulation and method reference: Surface Water Treatment Rule, CFR SimPlate - IDEXX SimPlate HPC Test Method for Heterotrophs in Water Revised Total Coliform Rule, CFR ONPG-MUG - SM 9223 B Ground Water Rule, CFR ONPG-MUG - SM 9223 B Long Term 2 Enhanced Surface Water Treatment Rule, CFR Quanti-Tray - SM 9223 B

7 Critical Elements for Microbiology (from EPA Lab Cert Manual, Chapter V) Personnel Laboratory Equipment and Supplies General Laboratory Practices Analytical Methodology Sample Collection, Handling and Preservation Quality Assurance Records and Data Reporting Action Response to Laboratory Results 7

8 Personnel Supervisor Bachelor s degree in microbiology, biology or equivalent Minimum 2 weeks training at federal/state agency or academic institution in drinking water microbiological analysis or 80 hours on-the-job training in water microbiology at certified laboratory Analyst At least 3 months bench experience in water, milk, or food microbiology Training acceptable to the state in drinking water microbiology analysis and at least 30 days of on-the-job training in drinking water microbiology under an experienced analyst Demonstrate acceptable results on unknown samples before analyzing compliance samples

9 Laboratory Equipment and Supplies ph meter Accuracy with ± 0.1 units Use buffers once Standardize before each use with ph 4.0 and 7.0 or 7.0 and 10.0 buffers Record slope monthly Maintained according to manufacturer s instructions Balance Readability of 0.1 g Sensitivity of 0.1 g for a load of 150 g and 1 mg for load of 10 g or less Readability and Sensitivity checks performed monthly Maintenance and calibration performed annually Thermometers Graduated in 0.5 C increments Exception: 1 C for Refrigerator Calibration checked annually, discard thermometer if differs more than 1 degree

10 Laboratory Equipment and Incubator Must maintain a constant temperature of 35 ± 0.5 C and provide a source of humidity Thermometers on top and bottom shelves of the use area Record temperature twice each day in use, separated by at least 4 hours Supplies 10

11 Laboratory Equipment and Supplies Autoclave Maintain sterilization temperature during cycle Complete cycle within 45 minutes when minute sterilization used Record date, contents, initials,sterilization time & temperature, total time in autoclave each use Maintenance performed annually Maximum registering thermometer used during each cycle Check automatic timer each quarter with stop watch Spore strips or spore ampules should be used monthly as bioindicators to confirm sterilization. 11

12 Laboratory Equipment and Supplies Conductivity Meter Suitable for checking reagent-grade water Readable in micromhos/cm or microsiemens/cm Calibrate monthly Refrigerator Maintain temperature of 1 5 C Record temperature once each day in use Inoculating equipment Sterile or presterilized disposable loops and sticks should be used 12

13 Laboratory Equipment and Supplies Pipets Presterilized plastic or glass 10 ml or less must be accurate to within 2.5% Reseal packages between use periods Glassware and Plasticware Borosilicate glass or clear, non-toxic plastic Tube closures should be screw caps with non-toxic liners, stainless steel, plastic or aluminum Sample Containers Wide mouth plastic or glass bottles or sterile plastic bags with sodium thiosulfate 13

14 General Laboratory Practices Sterilization procedures Autoclave at 121 C for recommended time Media should be removed immediately after end of cycle Sample Containers Randomly test 1 container from each batch or lot for sterility using 25 ml nonselective broth (such as Tryptic Soy Broth), incubate for 48 hours & check for growth Reagent grade water Quality should meet criteria for Conductivity (<2.0 micromhos/cm), Metals (Pb, Cd, Cr, Cu, Ni, Zn) less than 0.05 mg/l per contaminant and no greater than 0.1 mg/l collectively, Total Chlorine Residual (<0.1 mg/l), and HPC (<500 CFU/ml) Glassware washing Use distilled or deionized water in final rinse Inhibitory residue test should be performed prior to initial use of detergent or whenever different washing procedure or detergent is used; at the very least, every five years ph residue check 14

15 Analytical Methodology Media Discard media by manufacturer s expiration date Store prepared medium in dark at recommended temperature For lab-prepared media, record date prepared, type, lot #, volume, sterilization time & temperature, final ph, expiration date, and initials For commercially-prepared media, record date received, type, lot # and ph verification if specified by method, and expiration date Each batch of lab-prepared and each lot of commercially-prepared media should be checked before use for sterility and with positive & negative control cultures Run positive & negative controls each quarter for commercially-prepared media with shelf-lives longer than 90 days Examples of control cultures: Total coliforms Positive: Escherichia coli Negative: Pseudomonas aeruginosa Fecal coliforms Positive: Escherichia coli Negative: Enterobacter aerogenes E. coli Positive: Escherichia coli Negative: Enterobacter aerogenes 15

16 Analytical Methodology Analysis For compliance samples, use only methods specified in Revised Total Coliform Rule, Surface Water Treatment Rule and Groundwater Rule Laboratory must be certified for all methods used on compliance samples Minimum of 1 total coliform method and 1 E. coli method Recommend certification in second total coliform method if one method cannot be used due to interference in some drinking water samples Shake samples at least 25 times before analyzing Tolerance of dilution buffer should be ± 2 ml for volumes of 90 or 99 ml RTCR sample volume analyzed must be 100 ml 16

17 Sample Collection, Handling and Preservation Sample collectors should be trained in aseptic sampling procedures Sampling Locations must be representative of distribution system Sample taps must be free of attachments (aerator, strainer, hose) Use only cold water taps Flush tap until steady temperature Collect at least 100 ml, but allow 1 inch air space Sample Icing RTCR: recommended to hold drinking water samples at < 10 C during transport SWTR: must hold at < 10 C during transport, sample temperature should be recorded upon receipt at lab 17

18 Sample Collection, Handling and Preservation Sample Holding/Travel Time (all times are from collection to placing sample in incubator) Total coliforms in drinking water: 30 hours Total coliforms & E.coli in source water: 8 hours E. coli under Ground Water Rule: 30 hours Sample Information Form Record information after collection: system name; sample identification and site location; sample type; date & time of collection; analysis requested; disinfectant residual; sampler name 18

19 Quality Assurance Laboratory should prepare & follow a Quality Assurance (QA) plan Analyze a set of Proficiency Testing (PT) samples once every 12 months for each method for which laboratory is certified Participate in laboratory audit every three years 19

20 Records and Data Reporting Laboratory should keep thorough and accurate records QA Plan should describe procedures used for record retention Laboratory should maintain easily accessible records for 5 years (includes raw data, calculations and QC data) Electronic data should be backed up and should state how long you will retain records 20

21 Records and Data Reporting Data should be recorded in ink; changes should be lined through and dated & initialed Record date & time of sample receipt and name of person receiving sample, along with any comments on sample condition Record laboratory sample identification, date & time analysis begins, initials of analyst, method used, media lot number, items noted as QC and results Maintain preventive maintenance and repair records for all instruments and equipment for five years 21

22 Action Response to Laboratory Results RTCR samples: laboratory must promptly notify appropriate authority of positive Total coliform (in excess of 5%) or an E. coli result E. coli positive results: water system must notify state by end of day when notified of result 22

23 Basic Aseptic Technique Disinfect bench tops prior to analysis Long hair should be pulled back Wash hands prior to analysis Take every precaution not to contaminate sterile bottles or media during analysis by talking, coughing, sneezing, etc Do not touch any surfaces that are sterile (interiors of bottles or bottle tops, tips of pipets, etc.) Keep sterile pipets, bottles, etc. closed until ready to use Use care when removing pre-sterilized pipets from bulk bags, to avoid contaminating inside of bag

24 Break 24

25 Total Coliforms/E. coli by Colilert and Quanti-Tray

26 Coliforms The term total coliforms refers to a broad group of bacteria that belong to the taxonomic family Enterobacteriaceae. This group includes the genera Enterobacter, Klebsiella, Citrobacter, and Escherichia. Total coliforms are present in the environment and are used as an indicator to determine the efficacy of treatment plant operation and the integrity of the distribution system. In the ONPG-MUG method, total coliforms are defined as any bacteria possessing the enzyme β-d-galactosidase. 26

27 Coliform Group Total Coliforms Enterobacter Klebsiella Citrobacter Escherichia Klebsiella Citrobacter Escherichia Escherichia coli 27

28 Escherichia coli Escherichia coli (commonly abbreviated as E. coli) is a member of the fecal coliform group. E. coli is found in the digestive tract of warm blooded animals and is considered an indicator of fecal contamination. In the ONPG-MUG method, E. coli is defined as bacteria giving a positive total coliform response and possessing the enzyme β- glucuronidase. 28

29 Total Coliforms/E. coli by Colilert and Quanti-Tray Method Summary These methods allow for the simultaneous detection of total coliform bacteria and E. coli through the use of nutrient indicators ortho-nitophenyl-β-d-galactopyranoside (ONPG) and 4-methyl-umbelliferyl-β-Dglucuronide (MUG). Coliform bacteria and E. coli produce enzymes that can metabolize ONPG and MUG respectively.

30 Total Coliforms/E. coli by Colilert and Quanti-Tray Method Summary When ONPG is metabolized, a yellow color change occurs, indicating the presence of total coliforms. 30

31 Total Coliforms/E. coli by Colilert and Quanti-Tray Method Summary When MUG is metabolized, a fluorescent product is produced, indicating the presence of E. coli. 31

32 Total Coliforms/E. coli by Colilert and Quanti-Tray Method Summary Colilert is a Presence/Absence test. Quanti-Tray is an enumeration test. 32

33 Total Coliforms/E. coli by Colilert and Quanti-Tray Helpful Hints Make sure media is not discolored or caking before opening snap pack. If a sample is yellow or fluorescing, but is less intense than the comparator, it can be reincubated, but do not exceed a total of 28 hours incubation. 33

34 Total Coliforms/E. coli by Colilert and Quanti-Tray Interferences Some non-coliform bacteria produce small amounts of the enzyme β-d-galactosidase. These bacteria are suppressed, and will not produce a positive response unless 10 4 CFU/mL are present. A combination of hydrogen sulfide and high levels of either manganese or iron may cause a greenish-black to black color change to occur after incubation. Some water samples may have background color. If there is background color, compare the inoculated sample to a control containing only sample. 34

35 Total Coliforms/E. coli by Colilert and Quanti-Tray Interferences Some samples may fluoresce without having the yellow color. According to the manufacturer, an ONPG(-)/ MUG(+) test (colorless with fluorescence) is considered a nonspecific reaction as it does not meet the test definition of a total coliform or E. coli. Therefore this test would be interpreted as negative for both total coliforms and E. coli. Per the method, only samples that test positive for total coliforms should be checked under UV light for E. coli. 35

36 Total Coliforms/E. coli by Colilert and Quanti-Tray Colilert media QC requirements Media Fluorescence Check Each lot of media must be checked for fluorescence prior to use. Media dissolved in sterile reagent grade water should not fluoresce. Positive/Negative Control Check Each lot of media must be checked for selectivity and proper performance prior to use, and every 3 months while in use. Media dissolved in sterile reagent grade water and inoculated with the following cultures should exhibit the appropriate reactions after a 24 hour incubation at 35 ± 0.5 C. Culture Reaction E. coli Positive yellow color, fluorescence K. pneumoniae or E. aerogenes Positive yellow color, no fluorescence P. aeruginosa No color, no fluorescence 36

37 Total Coliforms/E. coli by Colilert and Quanti-Tray Vessel QC requirements Vessel sterility check Each lot of vessels must be checked for sterility prior to use. Add 25mL of single strength Tryptic Soy Broth to a randomly selected vessel and incubate for 48 hours at 35 ± 0.5 C. If no growth is present, the vessel lot may be put into service. Vessel volume check Each lot of vessels must be checked for volume accuracy prior to use. Fill a Class A 100mL graduated cylinder with reagent grade water and transfer to a randomly selected vessel. The level of water should be at the 100mL mark on the vessel for the lot to be placed in service. Vessel fluorescence check A randomly selected vessel should be checked for fluorescence using the UV light box prior to use. Vessel should not exhibit any fluorescence. 37

38 Total Coliforms/E. coli by Colilert and Quanti-Tray Quanti-Tray QC requirements Quanti-Tray sterility check Each lot must be checked for sterility prior to use. Add 25mL of single strength tryptic soy broth to a randomly selected Quanti-Tray, seal, and incubate for 48 hours at 35 ± 0.5 C. If no growth is present, the Quanti-Tray lot may be put into service. Quanti-Tray Sealer QC check The sealer is checked each month for proper sealing. Add 100mL of a solution of water and a dark dye to a Quanti- Tray, and run through the sealer. Check for leaks between the wells and on edges of the tray. 38

39 Total Coliforms/E. coli by Colilert

40 Total Coliforms/E. coli by Colilert Materials: Sterile sample vessels Colilert media Sterile Reagent Grade Water Sterile pipettes Vessel rack Incubator, 35 ± 0.5 ºC UV light 40

41 Total Coliforms/E. coli by Colilert Shake sample thoroughly (at least 25 times) Use sterile pipet to draw volume of sample down to 100 ml. Tap Colilert media snap pack on counter to settle contents. Snap pack open, and empty media into sample vessel. 41

42 Total Coliforms/E. coli by Colilert Replace cap and shake sample vigorously. Media does not have to be completely dissolved in the sample prior to incubation. Place samples in a rack, and incubate at 35 ± 0.5 C for hours. 42

43 Total Coliforms/E. coli by Colilert After incubating for hours, the bottles are read using a Colilert Presence/Absence comparator for reference. 43

44 Total Coliforms/E. coli by Colilert Samples that show a yellow color equal to or greater than that of the Comparator are total coliform positive. Samples with no color are total coliform negative. 44

45 Total Coliforms/E. coli by Colilert Total coliform positive (yellow) samples and the Comparator are checked with UV light. Samples that show fluorescence equal to or greater than that of the Comparator are E. coli positive. Samples with no fluorescence are E. coli negative. 45

46 Total Coliforms/E. coli by Colilert Questions?

47 Total Coliform/E. coli by Quanti-Tray

48 Total Coliform/E. coli by Quanti- Tray This method is used for the detection of total coliform bacteria and E. coli in source water and ground water. This method is approved for the analysis of samples for Long Term 2 Enhanced Surface Water Treatment Rule (LT2) and Ground Water Rule compliance. 48

49 Total Coliform/E. coli by Quanti-Tray Quanti-Tray sizes trays are available in two different The 51-well Quanti-Tray contains only large wells, and is appropriate for samples containing less than CFU (colony forming units) per ml. The 97-well Quanti-Tray /2000 contains 49 large and 48 small wells. This tray is appropriate for samples containing less than CFU per ml. Samples may be diluted as needed 49

50 Total Coliform/E. coli by Quanti-Tray Materials: Package of sterile Quanti- Tray trays Sterile sample bottles Colilert media Sterile dilution water (90 and 99 ml) Sterile pipettes Quanti-Tray sealer and inserts Incubator, 35 ± 0.5 ºC UV light MPN table 50

51 Total Coliform/E. coli by Quanti-Tray Procedure Preheat Quanti-Tray sealer. Shake sample thoroughly (at least 25 times) Use sterile pipet to draw volume of sample down to 100 ml. Sample may be diluted using sterile deionized water if necessary. Tap Colilert media snap pack on counter to settle contents. Snap pack open, and empty media into sample bottle. 51

52 Total Coliform/E. coli by Quanti-Tray Replace cap and mix sample until media dissolves. Media needs to be completely dissolved in the sample prior to pouring into the Quanti-Tray. Open Quanti-Tray by squeezing in at the sides while pulling the foil tab away. Pour sample into tray. Procedure 52

53 Total Coliform/E. coli by Quanti-Tray Procedure Fit Quanti-Tray in rubber insert and slide into the preheated sealer. 53

54 Total Coliform/E. coli by Quanti-Tray The Quanti-Tray will go through the sealer and the sealed Quanti-Tray will come out the back. Incubate sealed trays at 35 ± 0.5 C for hours. Procedure 54

55 Total Coliform/E. coli by Quanti-Tray Wells with a yellow color equal to or greater than that of the Quanti-Tray Comparator are total coliform positive. Count the number of positive wells, and refer to the provided Quanti- Tray MPN table to find the total coliform MPN. Reading Samples 55

56 Total Coliform/E. coli by Quanti-Tray Reading Samples Trays with total coliform positive (yellow) wells and the Comparator are checked for fluorescence with a UV light. Wells that show fluorescence equal to or greater than that of the Comparator are E. coli positive. Count the number of positive wells, and refer to the provided MPN table to find the E. coli MPN. The number of positive wells is converted to a Most Probable Number result in the Quanti-Tray or Quanti-Tray /2000 MPN Table. The MPN is multiplied by the appropriate dilution factor, if needed. 56

57 Total Coliform/E. coli by Quanti-Tray MPN table for Quanti-Tray /

58 Total Coliform/E. coli by Quanti-Tray MPN table for Quanti-Tray 58

59 Total Coliform/E. coli by Quanti- Tray Questions?

60 HPC by SimPlate

61 HPC by SimPlate Summary This method is used for the quantification of heterotrophic bacteria in drinking and raw water and can be used for raw, in process and finished water samples. This method is approved for the analysis of samples for Surface Water Treatment Rule compliance.

62 Heterotrophs Population of microorganisms that commonly occur in drinking water. Include bacteria, yeasts and molds. Dependent upon organic carbon source for growth. Growth in distribution system often found near dead ends or where loss of disinfectant residual occurs. 62

63 Why HPC? Used to verify adequate disinfection in distribution system in lieu of monitoring for disinfectant residual, or when residual is low. Monitoring of distribution system for biofilm growth. Monitoring for nitrification in distribution system when chloramines are used. Evaluating effectiveness of treatment process. 63

64 Consideration of SimPlate for HPC as replacement for HPC Pour Plate method Approved by EPA in late 2002 for compliance monitoring of drinking water samples under the Surface Water Treatment Rule, as an alternative to the HPC Pour Plate Method. SimPlate for HPC has the potential to reduce staff time, while getting similar results. 64

65 How does it work? SimPlate media contains multiple enzyme-substrates, each targeted to a different bacterial enzyme. When bacteria metabolize the enzyme substrates provided by the media, they produce a fluorescence that is visible under UV light. 65

66 SimPlate for HPC Analysis Materials: Sleeve of sterile plates Bottle of dehydrated SimPlate media Sterile reagent grade water Sterile pipettes Sterile phosphate buffered dilution water Incubator, 35 ± 0.5 ºC UV light 66

67 SimPlate for HPC Analysis Shake sample thoroughly (at least 25 times) Use sterile pipet to transfer 1 ml total volume of sample into middle of SimPlate, then touch tip of pipet off on dry portion of plate. Sample can be diluted using sterile phosphate buffered dilution water as needed. 67

68 SimPlate for HPC Analysis 9 ml of SimPlate media is aseptically transferred from the media bottle to the center of the base plate. Sample and media mix and begin to spread out over the surface of the plate. 68

69 SimPlate for HPC Analysis The plate is gently swirled, dispersing the sample and media to the wells. Gently tapping the plate on the counter helps bring any remaining air bubbles to the surface. 69

70 SimPlate for HPC Analysis Air bubbles will frequently remain on the surface of the wells after tapping the plate. After waiting a minute or two for the bubbles to pop, the excess media is poured off the base plate. 70

71 SimPlate for HPC Analysis After incubating for hours, the plates are read under a UV light. A blank plate is shown next to a raw plate, and both are upside down to facilitate viewing of fluorescing wells. 71

72 SimPlate for HPC Analysis The number of positive wells is converted to a Most Probable Number result in the SimPlate for HPC MPN Table. The MPN is multiplied by the appropriate dilution factor, if needed. 72

73 SimPlate for HPC Analysis Lessons learned Up to 10 samples and one blank can be run out of one bottle of prepared SimPlate media. Prepared media that has been refrigerated must be prewarmed before next use. Sterile phosphate buffered dilution water may be used to dilute raw water samples prior to analysis. Although air bubbles in the wells do not interfere with the test, we have found that it can make interpretation of fluorescence more difficult. To help prevent this interference, we have found that the majority of bubbles popped if the plates are allowed to sit a minute or two after tapping, prior to pouring off the sample. 73

74 SimPlate for HPC Analysis Lessons learned Swirling the plates should be done GENTLY to avoid spilling excess media. Gently tapping the plate on the counter can help pop bubbles up out of the wells. Use care with the plates while they are inverted. Bumping, dropping, or setting the plates down roughly can result in the sample being knocked out of the wells. Any amount of fluorescence greater than the blank is considered positive when counting wells. Keeping the lid on or reading plates upside down can help prevent background fluorescence from interfering with reading. 74

75 SimPlate for HPC Analysis Quality Control Analysis QC: A blank is analyzed with each set of samples. If more than one bottle of media is needed for a set, a blank should be run for each bottle, and the samples that correspond to that bottle/blank should be noted. Media QC: All lots of media must be checked for proper performance prior to being placed in service. If the lot is in service for more than a quarter, the media check must be repeated. Analyze a plate using raw water or inoculated sterile water as the sample. Incubate for 48 ± 3 hours at 35 ± 0.5 C. The sample must produce growth for the media to be put in service. Check for sterility by incubating an uninoculated plate (blank) for 48 ± 3 hours at 35 ± 0.5 C. No growth should be observed. Sterile Reagent Grade Water QC: All batches of sterile water must be confirmed sterile prior to being placed in service. Add 50mL of sterile water to 50mL of Double Strength Tryptic Soy Broth and incubate for 48 hours. Check for turbidity indicating bacterial growth. 75

76 HPC by SimPlate Questions?

77 Thank you for your attention! Questions? For more information, please contact: Erica Fox,