Appendices 3 - Sanitation District

Transcription

Appendix 3
Microbiology
Appendices
Chapter 3
Microbiology
2014-2015 Biennial Report
Appendix 3.1
Manifold Microbiological Sampling Results
Appendix 3.2
JWPCP Microbiology Laboratory Shoreline and Manifold Station Sampling
Procedure
Appendix 3.3
LACSD Nearshore Bacteriological Sampling Field and Safety Procedures
Appendix 3.4
Total Coliform, Fecal Coliform, and Enterococcus Enumeration by Membrane
Filtration – (Method 349 – Total Coliform, Method 356 – Fecal Coliform, and
Method 357 – Enterococcus) and Fecal Coliform Enumeration by Multiple Tube
Fermentation – (Method 351)
Appendix 3.5
Rain Effects on Fecal Indicator Bacteria Concentrations in Nearshore Water
Samples
Appendix 3.6
Total Coliform, Fecal Coliform, Enterococcus and Shellfish Harvesting Receiving
water Microbiological Results
Appendix 3.7
Spatial and Temporal Trends in Fecal Coliform and Enterococcus Concentrations
Appendix 3.1
Appendix 3.1
All 2014 and 2015 manifold results were used to calculate the 30-day geometric means results
Month
2014 JAN
FEB
MAR
APR
MAY
JUN
JUL
AUG
SEP
OCT
NOV
DEC
2015 JAN
FEB
MAR
APR
MAY
JUN
JUL
AUG
SEP
OCT
NOV
DEC
Total Coliform
120"
90" OUTFALL
OUTFALL
< 289
< 151
< 130
431
< 357
< 209
< 362
< 141
< 288
< 145
< 342
< 204
< 397
< 136
< 194
< 45
< 181
< 73
< 201
< 105
< 150
< 49
< 236
< 125
< 255
807
< 235
1005
< 293
559
< 483
< 197
< 498
< 184
< 432
< 169
< 78
331
< 337
< 153
< 579
< 127
< 463
< 80
< 167
478
< 427
< 136
Fecal Coliform
120"
90" OUTFALL
OUTFALL
< 19
<9
< 17
<9
< 25
< 33
<9
48
< 21
18
< 16
< 11
< 10
53
< 43
< 12
< 17
< 19
< 10
<9
< 16
<9
<9
13
< 14
< 10
< 58
< 13
70
15
19
8
< 13
33
70
26
95
16
46
28
217
11
< 12
< 20
< 29
< 26
< 14
< 10
Manifold samples are grab samples collected once daily.
There are no bacteriological limits in effect at the manifold.
App 3.1 - 1
Enterococcus
120"
90" OUTFALL
OUTFALL
<9
<9
< 10
<9
<9
< 10
<9
<9
<9
<9
<9
<9
<9
<9
<9
<9
<9
<9
<9
<9
<9
<9
<9
<9
<9
<9
<9
< 11
< 10
< 10
<9
< 10
<9
<9
<9
<9
<9
<9
<9
< 10
< 12
<9
<9
<9
<9
< 11
< 10
<9
Appendix 3.2
JWPCP Microbiology Laboratory Shoreline and Manifold Station Sampling
Procedure
Sanitation Districts of Los Angeles County
Laboratories Section
METHOD APPROVAL FORM
Test Code
N/A
Method Name
Shoreline and Manifold Sampling Procedure
Version
13.1.0
Method Date
September 10, 2013
Reasons for
Method Revision
Standard Annual Revisions
Revised by:
Mark Patterson
Laboratory Technician
JWPCP WQL
cl/
Signature
Date
/W/"5.
Debra Leachman
Microbiologist II
JWPCP WQL
Date
Approved by:
Kathy Walker
Supervisor I
JWPCP WQL
9AW/3
Signature
Date
Final Approval:
Jean Lee
Superintendent of
JWPCP Laboratory
Signature
App 3.2 - 1
Date
JWPCP MICROBIOLOGY LABORATORY
SHORELINE AND MANIFOLD STATION SAMPLING PROCEDURE
Introduction
The Los Angeles County Sanitation Districts monitors designated shoreline stations for fecal
indicator bacteria (FIB) levels as part of the receiving water monitoring program described in the
current Joint Water Pollution Control Plant NPDES Permit (Order No. R4-2011-0151, NPDES
No. CA0053813), which became effective on September 1, 2011. This 2011 NPDES permit
continued the previous shoreline bacteriological monitoring program of weekly monitoring
frequency. The shoreline monitoring stations are located along the Palos Verdes Peninsula and
south to San Pedro, coastal areas most likely impacted by the JWPCP outfall system.
The Permit requires the weekly shoreline monitoring program to include sampling from nine
sites: historical District’s shoreline stations S1, S2, S3, S5, S6 and S7, and 1991 NPDES
amended sites SB and SM. The SMBBB-TMDL monitoring program also requires an additional
observation only station SMB-O-9. For additional information on Santa Monica Bay Beaches
Bacterial TMDL, see Santa Monica Bay Beaches Bacterial TMDLs Coordinated Shoreline
Monitoring Plan (4/07/04).
The weekly shoreline bacteriological monitoring program analyzes for total coliform, fecal
coliform and enterococcus indicator bacteria using methods approved in Title 40 CFR for marine
receiving waters. The FIB concentrations are evaluated for compliance with water contact
standards (REC-1) for both single sample and geometric mean criteria. Each bacterial sample
collected must be associated with a recorded set of field observation data and must be analyzed
within the method holding time limit. Repeat sampling is required when a sample exceeds any
single sample criteria.
The District’s shoreline sampling program is coordinated with the environmental monitoring
program, which falls on Mondays, at L.A. County Department of Public Health. Therefore, the
shore samples are routinely collected on Mondays, with few exceptions such as holidays falling
on Mondays, temporary lack of staff, or safety/weather conditions that compromises personnel or
the lab. If samples are not collected on a Monday, every reasonable attempt to reschedule during
the same calendar week (Sunday through Saturday) shall be made.
In addition to the ocean monitoring requirements, daily samples are collected from White Point
manifold pumping station. Effluent samples from the two main discharge lines, 90-inch and 120inch, are analyzed for FIB and chlorine residual.
1.
Scope and application:
1.1
This procedure is applicable for the collection of microbiological samples from all
shoreline samples covered under the current NPDES permit.
JWPCP Water Quality Laboratory
SMSP- 1
Version 13.1.0
App 3.2 - 2
September 10, 2013
1.2
This procedure is also applicable to the collection of microbiological and chlorine
residual samples from the White Point manifold.
1.3
The following stations shall be maintained according to the current amended
NPDES discharge permit. Each sampling and observation-only station has been
assigned a TMDL station identification code. Descriptions of each station include
general directions to the sample site and specific coordinates.
NPDES Site ID: S1
Location: Long Point
Thomas Guide
TMDL Site ID: SMB-7-3 Page: 822 Grid: H5
Coordinates: N33.7386° W118.3940°
Comments: This open beach site is located at 7200 Palos
Verdes Drive South, Rancho Palos Verdes, located along
the beach at Terranea Resort. To access the site turn from
Palos Verdes Drive South into the Terranea driveway and
follow the left perimeter of the parking lot the southeast
corner. By foot, follow the pathway down to the
shoreline. The sample is collected at the end of the path.
NPDES Site ID: S2
Location: Abalone Cove
Thomas Guide
TMDL Site ID: SMB-7-4 Page: 823 Grid: A5
Coordinates: N33.7416° W118.3792°
Comments: This open beach site is located at 6000 Palos
Verdes Drive South, Rancho Palos Verdes. To access the
site, turn from Palos Verdes Drive South into the locked
gate driveway, Abalone Cove Shoreline Rd.
Alternatively, turn into the Abalone Cove parking lot
approximately 100 yards northwest of the site. Follow the
unpaved road down past the nursery school to the
lifeguard tower. Next to the lifeguard tower is a stairway
that leads directly onto the shoreline where the sample is
collected.
SMSP- 2
Version 13.1.0
App 3.2 - 3
September 10, 2013
NPDES Site ID: S3
Location: Portuguese Bend
Thomas Guide
TMDL Site ID: SMB-7-5 Page: 823 Grid: C6
Coordinates: N33.7362° W118.3602°
Comments: This beach site is located along the private
beach within Portuguese Bend Club at 4100 Palos Verdes
Drive South, Ranch Palos Verdes. To access this site,
turn from Palos Verdes Drive South into the Portuguese
Bend Club driveway. Bear right once past the paddle
tennis courts into the parking lot. The shoreline where the
sample is collected is directly in front of the parking lot.
NPDES Site ID: S5
TMDL Site ID: SMB-7-6
Thomas Guide
Page: 853 Grid: G1
Location: Royal Palms County
Beach/ White Point
Coordinates: N33.7177° W118.3220°
Comments: This beach site is located at Royal Palms
County Beach/White Point, 1801 Paseo del Mar, San
Pedro. To access the site, turn from South Paseo del Mar
into the facility and follow the driveway past the kiosk
down to the parking lot. Walk to the right of the lifeguard
stand. The sample is collected just to the right of the jetty.
NPDES Site ID: S6
TMDL Site ID: SMB-7-8
Location: Wilder Addition at Point
Fermin Park
Coordinates: N33.7098° W118.2990°
Comments: This open beach site is located at the Wilder
Addition Park, 825 Paseo del Mar, San Pedro. To access
the site, park on South Paseo del Mar adjacent to South
Meyler Street. Follow the driveway past the public
restroom to the bottom of the lot; go down the steps to
another footpath that leads to a stairway. The sample is
collected at the bottom of the stairway.
Thomas Guide
Page: 854 Grid: A2
SMSP- 3
Version 13.1.0
App 3.2 - 4
September 10, 2013
NPDES Site ID: S7
Location: Cabrillo Beach
Thomas Guide
TMDL Site ID: SMB-7-9 Page: 854 Grid: C2
Coordinates: N33.7092° W118.2831°
Comments: This open beach site is located at outer
Cabrillo Beach, 3720 Stephen M. White Drive. Enter
through Oliver Vickery Circle Way. Follow Breakwater
drive, past the old museum to the lifeguard building. The
sample is collected on the ocean side, directly in front of
the lifeguard building. Weather and tidal condition are
determined at this site.
NPDES Site ID: SB
Location: Bluff Cove
Thomas Guide
TMDL Site ID: SMB-7-2 Page: 792 Grid: F5
Coordinates: N33.7938° W118.4070°
Comments: This open beach site is located at Bluff
Cove: 600 Paseo del Mar, Palos Verdes Estates. To
access the site, park on the 700 block of Paseo del Mar
and follow the footpath down to the base of the trail. The
sample is collected where the path meets the shoreline.
NPDES Site ID: SM
Location: Malaga Cove
Thomas Guide
TMDL Site ID: SMB-7-1 Page: 792 Grid: G3
Coordinates: N33.8034° W118.3959°
Comments: This open beach site is located at 300 Paseo
Del Mar, Palos Verdes Estates. To access the site, turn
from Paseo del Mar into the Malaga Cove International
School parking lot. Follow the asphalt footpath down to
the base of the trail. The sample is collected at the base
of the Malaga Cove sign.
SMSP- 4
Version 13.1.0
App 3.2 - 5
September 10, 2013
NPDES Site ID: SMBO-9
TMDL Site ID: SMBO-9
Thomas Guide
Page: 792 Grid: G3
Location: Storm Drain by PV Swim
Club
Coordinates: N33.8022° W118.3980°
Comments: This site is located at 300 Paseo del Mar,
Palos Verdes Estates. To access the site, turn from Paseo
del Mar into the Malaga Cove parking lot. Access via the
PV Swim/Beach Club. Alternatively, follow the asphalt
footpath down to the base of the trail. The storm drain is
located approximately 50 yards southwest of the Palos
Verdes Swim/Beach Club. This storm drain location is
currently monitored for observational data only.
2.
3.
Summary of Method
2.1
Ocean samples are collected in sterile polypropylene bottles.
2.2
Manifold bacteria samples are collected in Whirl-Pack® sampling bags with
sodium thiosulfate.
2.3
Bacteriological samples are stored in ice during transport to inhibit changes in
microbial population.
2.4
Chlorine residual samples collected at the White Point manifold are analyzed on
site with a HACH DPD kit (SM 4500CL-G).
2.5
Observations are noted for each shoreline site at the time of sampling.
Sample Handling and Preservation
3.1
All bacteriological samples are preserved by being kept in ice during transport.
3.2
Chlorinated bacteriological samples must be treated with sodium thiosulfate to
preserve populations against the effects of chlorine.
3.3
All samples must be handled aseptically to prevent contamination.
3.4
All bacteriological samples must be collected in sterile containers to prevent
contamination.
3.5
Chlorine residual must be analyzed immediately and on-site to prevent loss of
chlorine prior to analysis.
SMSP- 5
Version 13.1.0
App 3.2 - 6
September 10, 2013
4.
5.
6.
Interferences
4.1
Stormy weather can make sampling hazardous or impractical. The samples can be
omitted, provided that such omissions do not occur more than 10 days in any calendar
year. Reasonable effort should be made to sample later in the same day, if conditions
improve.
4.2
Splitting sampling work between two technicians might not be possible on some
days. A single technician, if necessary, may perform the entire run.
Apparatus
5.1
Sterile, wide-mouth polypropylene bottles, 500 mL and 1000 mL in volume
5.2
Sampling poles
5.3
Whirl-Pak® sterilized sampling bags with sodium thiosulfate, 10 mg
5.4
Ice chest, filled with ice
5.5
HACH total chlorine DPD kit
5.6
Two amber 100mL glass bottles
Reagents
6.1
7.
HACH DPD Total Chlorine Reagent pillow or dispenser
Procedure
Ocean Sample Collection and Handling:
7.1
Collect samples that are representative of the water being tested and use aseptic
techniques to avoid sample contamination.
7.2
Collect samples in sterilized polypropylene bottles that have passed the 48-hour
TSB sterility period.
7.3
Place bottle securely in sampling pole and unscrew lid just prior to sampling.
7.4
Lower sample bottle into incoming surf, and with a sweeping motion collect the
ocean water avoiding sand. Leave ample air space in the bottle (at least 2.5 cm) to
facilitate mixing by shaking. Recap bottle.
SMSP- 6
Version 13.1.0
App 3.2 - 7
September 10, 2013
7.5
Collect a scheduled duplicate sample immediately after the initial sample in a
sterile 1000 mL polypropylene bottle to ensure sufficient volume for all tests.
7.6
Label all sample bottles with the sample location, date, time, and the sampler’s
initials.
7.7
All bacteriological samples shall be iced (<10°C) during transport to the laboratory
and processed within 6-hours of collection time.
7.8
If vehicle access to a sampling location is blocked, park in a safe location and access
the site by foot. If there is no foot access and the delay is anticipated to be longer than
30 minutes, complete the remainder of the beach run and return to the missed sample
site.
7.9
A station may be omitted for reasons of personal safety or unreasonable time delay;
however, the technician shall make a reasonable attempt to collect each sample – up
until 15:00 that day. The supervisor shall be informed of the delay.
7.10
In the event of stormy weather that makes sampling hazardous or impractical, the
samples can be omitted, provided that such omissions do not occur more than 10 days
in any calendar year.
7.11
When a sample is omitted, a detailed explanation shall be recorded in the Remarks
portion of the Weekly Shore Station Observations Sheet. The supervisor shall be
informed of the omission immediately.
7.12
All shore samples shall be refrigerated after analyses until the tests results are known
(within 24-hours). In the event the results are lost or questionable, the refrigerated
samples shall be reset. However, data from these samples are not reportable (in
violation of the 6-hour holding period) but are valuable for characterizing the water
quality. Appropriate footnote shall be entered with non-reportable results. The
supervisor shall be informed of the incident.
Daily White Point Manifold Sample Collection and Handling:
7.13
Sampling procedure for daily manifold samples for FIB and total chlorine
residual.
7.13.1 Access the locked manifold security gate and cover with District’s key
#A297.
7.13.2 Open the manifold cover, and place the two rubber tubes that are attached to
the 90-inch and 120-inch pumps into the drain hole. Completely open the two
valves.
7.13.3 Push the buttons to switch on the pumps. The left-hand button is for the
120-inch line, and the right-hand button is for the 90-inch line. The lines
must be cleared of standing water prior to collecting the samples
SMSP- 7
Version 13.1.0
App 3.2 - 8
September 10, 2013
(approximately 5 minutes flush time before collection). See the White
Point Manifold Flow Table (Table 1) for the required pre-sample flush
time for each line.
7.13.4 After the lines are flushed, aseptically open a Whirl-Pak® sampling bag
containing sodium thiosulfate pill, and fill the bag to the 4 oz. fill line with
the 120-inch line sample. Close the bag securely and label it with the sample
name, date and time, and sampler’s initials. Gently agitate filled bag to
dissolve and mix the sodium thiosulfate pill into the sample. Keep the sample
iced.
7.13.5 Immediately after the bacteriological sample is collected, collect the chlorine
residual sample from the 120-inch line by filling the pre-labeled 100 mL
amber glass bottle. Record the collection time and analyze the sample for
chlorine residual immediately (DPD Method SM4500-Cl G).
7.13.6 Repeat steps 4.1.4 and 4.1.5 for the 90-inch line.
7.13.7 Duplicate chlorine residual analysis is done daily. Also, any sample with
chlorine residual of 0.4mg/L or greater must be verified by a second analysis.
7.13.8 Chlorine residual of 1.0 mg/L or greater must be reported to the Assistant
Superintendent Treatment Plant Operations who is in charge of the
chlorination processes. On weekends and holidays, the Alarm Center must be
notified.
7.13.9 Close the valves and confirm the pumps are off before locking the manifold
cover. Lock the access gate upon departure.
7.13.10 The analyst shall record sample collection times, when the samples were
received by the lab and name of sampling analyst on the Daily
Bacteriological & Chlorine Residual Sample Collection form.
Weekly Shore Station Run:
7.14
The shore run is split by two technicians due to numerous sites for various
sampling and field data purposes. The long run covers SM, SMB-O-9, SB, S1 and
S2. The short run covers S7, S6, S5 (including manifold and rain gauge) and S3.
7.15
The technicians take the electronic tablet to input the shore run observations.
Upon returning to the lab, the data is synchronized with the LIMs.
7.16
The Microbiology truck is used for the long runs, and a Process Control truck is
borrowed for the short runs. In an event where a truck is not available, a plant
truck shall be obtained from the mechanic shop.
7.17
Splitting runs might not be possible on some days, e.g. insufficient staff. The
technician must be prepared to cover the entire run when necessary.
SMSP- 8
Version 13.1.0
App 3.2 - 9
September 10, 2013
7.18
Cell phones shall be carried out on the shore runs by both technicians. A spare
cell phone is available from Process Control Lab.
Weekly Shore Station Observation:
7.19
Weekly Shore Station Observations for specified parameters shall be recorded at the
same time bacteriological samples are collected. Observations are selected from the
drop-down menu options with standardized descriptions. All sections shall be
completely filled out prior to submittal. Remarks shall be worded succinctly and
uniformly. If there are problems with the electronic tablet, the analyst must fill out a
temporary Weekly Shore Station Observation Sheet manually. The following
guidelines shall be maintained:
7.20
Air Temperature, Wind, Tide and Weather – These observations pertain to conditions
as recorded at S7, Cabrillo Beach Life Guard Station, at time of sampling.
7.20.1 Air Temperature – Taken with a hand-held thermometer (Fahrenheit) at the
sampling site.
7.20.2 Wind Speed and Direction – Taken with a hand held wind meter (MPH),
away from obstructions and aimed towards the direction of the wind. If no
wind is detected enter none detected in the wind category.
7.20.3 Tide – Calculated by Tides and Currents Program. Report the tide height in
feet and the tidal action as ebbing or flooding, based on the time S7 sample is
taken.
7.20.4 Weather – Select description of the weather at time of sampling.
7.20.5 Rain during sampling – Select yes or no.
7.21
Time – The time each sample is collected.
7.22
Water Color – The general color of the water in the sampling area.
7.23
Turbidity – The clarity of the water when sampled.
7.24
Odor – The presence or absence of odor at the sampling site. When odor is
detected, the description of the odor and its possible source shall be recorded in
the Remarks section.
7.25
Sewage Grease – Sewage grease shall be quantified as cubic centimeters per 100
feet of beach (cc/100ft), bagged and transported to the laboratory.
7.26
Fecal Matter – Possible source of origin shall be recorded in the Remarks section.
7.27
Sewage Garbage –Description shall be recorded in the Remarks section.
SMSP- 9
Version 13.1.0
App 3.2 - 10
September 10, 2013
7.28
Tar/Oil – Tar and bilge oil shall be quantified as cubic centimeters per 100 feet
(cc/100ft).
7.29
Garbage – Quantify non-sewage garbage present on and/or offshore near
sampling site.
7.30
Kelp Deposits – Quantify kelp deposits on and/or offshore, near sampling site.
7.31
Bathers in Water – Approximate number of persons in direct contact with ocean
water.
7.32
Bathers Onshore – Approximate number of persons at or near the shoreline.
7.33
Rain gauge – The rain gauge is located at the Paseo del Mar pumping station just
north of White Point, at Paseo del Mar. The rain gauge shall be checked when
rainfall is noted in the past 24-hours. The following guidelines shall be
maintained:
7.33.1 Access the locked security gate to Paseo del Mar pumping station with
District’s key #A297.
7.33.2 Lift off the funnel and insert the graduated dipstick into the center cylinder.
7.33.3 Read the dipstick to the hundredth of an inch. Record the rainfall and the time
it was measured, e.g. 0.12 inches @ 09:15.
7.33.4 When the rainwater collected in the center cylinder overflows into the
housing cylinder, carefully remove the center cylinder and discard the
rainwater. Carefully pour the excess rainwater in the housing cylinder into the
emptied center cylinder and immerse the dipstick to measure. Add 2 inches to
this measurement for the full rain measurement. The center cylinder holds 2
inches of water when full.
7.33.5 Discard the rainwater and reassemble the rain gauge, storing the dipstick
upside-down in the main housing cylinder.
7.33.6 Lock access gate upon departure.
7.34
Remarks – Additional comments, clarifications, and quantifications (e.g. sewage
grease) are entered in this section. Presence of diatomaceous scum, approximate
number of birds or mammals in the sampling area, unusual runoff situations,
explanation of odors, approximate distance of boats within 200 yards of shore are
examples of noteworthy remarks.
7.35
Observations By – Enter the first name initial and last name of the technician.
Once the analyst returns from the field, the electronic tablet data is synchronized
with the LIMs system.
SMSP- 10
Version 13.1.0
App 3.2 - 11
September 10, 2013
8.
Calculations: not applicable.
9.
Quality Assurance guidelines
9.1
9.2
Each shore run will include one sample for duplicate analysis
9.1.1
Follow the list in the back of the bench book to determine the proper sample
for duplicate analysis each week.
9.1.2
Sample number for duplicate analysis is planned in order to assure that
samples are all checked by duplicate analysis with equal frequency.
9.1.3
To collect for duplicate analysis, use a 1L sterile polypropylene bottle to
collect the designated sample. Extra volume is necessary for the extra testing.
Quarterly manifold pump maintenance to check flow rate:
9.2.1
Turn the valve to the 90-inch line completely open and fill the 20-liter carboy
to the 20-liter mark.
9.2.2
Record the time it takes to fill the carboy to 20 L.
9.2.3
Empty the carboy and repeat the procedure with the 120-inch line.
9.2.4
Compare the recorded times to the White Point Manifold Flow Table
(Table 1) to determine if the flow rates are in the acceptable range.
9.2.5
If the flow rate is not within the acceptable limit, a work request must be
immediately submitted to the JWPCP Maintenance section. Microbiology
Group supervisor shall be informed of the problem.
9.2.6
Record the flow rate check in the designated section of the Microbiology
Laboratory Bacteriological Notebook.
10.
Method Performance: not applicable.
11.
References
11.1
County Sanitation Districts of Los Angeles County, Joint Water Pollution Control
Plant NPDES Permit (NPDES Permit No. CA0053813), 09/01/11
11.2
Santa Monica Bay Beaches Bacterial TMDLs Coordinated Shoreline Monitoring
Plan, Technical Steering Committee, City of Los Angeles and County of Los
Angeles, 4/07/04.
SMSP- 11
Version 13.1.0
App 3.2 - 12
September 10, 2013
11.3
Thomas Bros. Maps, The Thomas Guide Los Angeles County 2000 Edition, 1999.
SMSP- 12
Version 13.1.0
App 3.2 - 13
September 10, 2013
TABLE 1
WHITE POINT MANIFOLD FLOW TABLE
The flow rate timetable is based on the following parameters:
• sample piping is ¾ inch thinwall tube
• I.D. is 0.680 inches
• test carboy is 20 liters (5.25 gallons)
• 90-inch line: length of 76.81 ft. and volume of 1.44895 gal.
• 120-inch line: length of 44.86 ft. and volume of 0.84631 gal.
Pre-sample Flush
TIME
min:sec
gpm
00:45
00:50
00:55
01:00
01:05
01:10
01:15
01:20
01:25
01:30
01:35
01:40
01:45
01:50
01:55
02:00
02:05
02:10
02:15
02:20
02:25
7.05
6.34
5.76
5.28
4.88
4.53
4.23
3.96
3.73
3.52
3.34
3.17
3.02
2.88
2.76
2.64
2.54
2.44
2.35
2.26
2.19
ft/s
6.22
5.60
5.09
4.67
4.31
4.00
3.73
3.50
3.30
3.11
2.95
2.80
2.67
2.55
2.44
2.33
2.24
2.15
2.07
2.00
1.93
90-inch
120-inch
02:03
02:17
02:31
02:45
02:58
03:12
03:26
03:39
03:53
04:07
04:21
04:34
04:48
05:02
05:15
05:29
05:43
05:56
06:10
06:24
06:38
01:12
01:20
01:28
01:36
01:44
01:52
02:00
02:08
02:16
02:24
02:32
02:40
02:48
02:56
03:04
03:12
03:20
03:28
03:36
03:44
03:52
WRITE
WORK
ORDER!
FAIL!
White Point Manifold Sample Maintenance
Manifold flow rate is required to be at least 2.00 ft/s, based on a previous NPDES permit
specification. A work order must be requested prior to such reduced flow speed. When the flow test
does not fill the 20-liter carboy within 1 minute and 54 seconds, a work request must be submitted to
the JWPCP Maintenance section. The EID number is 348MDF00 for manifold structures and
outfalls. The Microbiology Supervisor shall be informed of the problem before submittal.
SMSP- 13
Version 13.1.0
App 3.2 - 14
September 10, 2013
Appendix 3.3
LACSD Nearshore Bacteriological Sampling Field and Safety Procedures
CSDLAC
NEARSHORE BACTERIOLOGICAL
SAMPLING FIELD AND SAFETY
PROCEDURES
Using R/V Phaon
DMS #1265842-v4-PhaonNearPRO.doc modified 08 Oct. 2015
INTRODUCTION
Nearshore bacteriological sampling is conducted at nine stations along the Palos Verdes Peninsula. Six
“Inshore” stations (IL2-IL7) are located along the 9.1 m contour and are typically sampled five times per
month and at least once per week. The sampling run is extended to include three “Offshore” sites (6C, 8C,
9C) once per month. Separate surface and bottom water samples are collected at each Inshore station and
surface samples only are taken at the Offshore sites. All samples are collected with a Niskin bottle.
Samples are kept on ice and returned to the Microbiology Laboratory at JWPCPWQL for analysis. A
minimum of two people is required to conduct the sampling.
MOBILIZATION
Prior to each day's sampling and before leaving the dock check to see that the following equipment and
supplies are on board or taken down to the survey vessel:
1. Fifteen 500 ml and two 1 L autoclaved Nalgene bottles
2. Ice chest and ice (ice is stored in the Ocean Sentinel freezer)
3. Pre-printed adhesive labels
4. Alcohol wash carboy with approximately 10 L of 50:50 ethanol/seawater solution
5. Wash bucket
6. Niskin bottle (1.2 L) with messenger (plus spares of each)
7. Bucket thermometer
8. Ocean Nearshore Station Observations sheets
9. Phaon Cell phone (located on Ocean Sentinel)
Make sure there are an adequate number of sampling bottles available for the bacteriological run. Twelve
500 ml bottles are required for the regular sampling run (six stations) and 15 bottles are required for the
extended run (nine stations). At least two 1 L bottles may be needed for the Split samples. Split samples
are taken in lieu of replicates and are split in the laboratory for testing of two analytes. Autoclaved
sample bottles are obtained from the JWPCPWQL Microbiology Laboratory. Check with the
Microbiology staff to insure that the bottles have been properly sterilized and are ready for sampling.
Processed bottles will show black indicator lines on the autoclave tape and must be checked by
Microbiology staff for sterilization two days after the date written on top of the bottle.
Pre-printed adhesive labels should be made up at least the day before the scheduled run. Labeling
instructions are below.
Upon arrival at the Phaon, an operator will set up the vessel per the posted "Boat Set-Up and Safety
Procedures”. Once the boat is set up, make sure all necessary gear is on board and operational. There are back
ups for all gear if necessary. The alcohol wash should be checked for sufficiency; enough to cover half the
Niskin bottle. If a sufficient level is not met, add more ethanol to the existing alcohol wash solution. Note:
Alcohol is flammable and care should also be taken to avoid eye contact when transferring the alcohol
1
App 3.3 - 1
between the carboy and the wash bucket and while immersing the Niskin bottle. A fresh batch of alcohol
wash solution should be made, minimally, on an annual basis in July.
Personal floatation vests are available to anyone desiring their use. The boat operator may require
floatation vests be worn if he/she deems conditions warrant their use. Noise cancellation headphones are
available for engine noise reduction.
SAMPLING PROCEDURES
During a regular sampling run, surface and bottom samples are to be taken at each of the six, 9.1 m,
Inshore stations (IL2-IL7). During the extended sampling run, surface samples only are taken at the
Offshore stations 6C, 8C, and 9C, which are along the 60 m contour. The sampling crew must depart on
the run as soon as possible after the start of working hours. Samples must be received at the Microbiology
Lab no later than 1200 hours and within six hours of the time the first sample is taken. Four hours should
be considered the goal.
Sampling typically begins at the station furthest from port. During an extended run the offshore sites (9C,
8C, 6C) are sampled in-route to station IL2. Weather and sea conditions may occasionally require the
reverse order. Stations are to be located by means of DGPS. Visual line-ups and fathometer readings are
to be used to confirm position. All Inshore stations, as established in the permit, are located along the 9.1
m depth contour. However, if kelp is present at a site, sampling may take place further offshore at the outside
edge of the kelp canopy. Foul weather, rough seas, or the presence of hazardous reefs may also
necessitate sampling slightly offshore of the 9.1 m contour.
In conditions of strong current or high winds the boat is to be anchored during sampling. The cap rail is
very narrow, so care should be taken when walking fore and aft from anchor deployment and retrieval. To
the extent practical (and with the exception of conditions discussed in the preceding paragraph), the anchor
should be placed so as to position the vessel in 9.1 m (30 feet) depth at mean lower low water or at the outer
edge of the kelp bed.
Surface water temperature (°C) is to be determined by means of a bucket thermometer at each station.
Surface samples are to be taken 0.5 m below the surface and bottom samples are to be taken within two
meters of the seabed. Samples are taken with a Niskin bottle attached to a line weighted with a salmon
ball.
The Niskin bottle is to be completely immersed in an alcohol wash prior to sampling and between stations.
Care is to be taken to assure that the valves are exposed to the alcohol disinfectant wash. Disinfection
is not necessary between surface and bottom samples taken at each station. The surface sample is to be
taken before the bottom sample. If samples are not taken in this order the Niskin bottle must be rewashed
in disinfectant between the samples.
Upon arriving at station, prepare the Niskin bottle to take a sample by opening both the bottom and top caps
and attaching the lines to the triggering mechanism. Attach the Niskin bottle to the line approximately one
meter above the salmon ball. Lower salmon ball into the water just below the water surface; placing the
salmon ball just below the surface will prevent excessive swinging motion caused by back and forth listing
of the vessel. The messenger can then be attached to the line above the Niskin bottle. While holding the
messenger, which is attached to the line, lower the bottle to the respective depth. The bottle is triggered to
close by dropping the messenger, which will slide down the line and make contact with the triggering
mechanism. Care should be taken that the messenger is properly attached by sliding it up and down on the
line prior to release. Fill a sterile 500 ml or 1 L Nalgene sample bottle to the shoulder of the container. Upon
2
App 3.3 - 2
completing sampling at a station, carefully bring the salmon ball back on board the vessel. Remove the
Niskin bottle and place the opened sampler in the disinfectant alcohol wash, exposing top and bottom to
the alcohol.
Each sample is to be labeled by means of an adhesive label placed on the side of the bottle. The label is to
list the station name and sampling date, time, depth (Surface or Bottom), split (Yes or No), and personnel.
Samples are to be refrigerated or kept on ice (ideally at 4°C) until delivery to the Microbiology
Laboratory.
An Ocean In/Offshore Station Observations Sheet is to be completed for each sampling run. All boxes of
the Station Observations Sheet must be completed.
The Observations Sheet also serves as a Chain of Custody form when samples are turned over to the
JWPCP Microbiology Lab. See a completed example below:
3
App 3.3 - 3
DEMOBILIZATION
All over-the-side gear must be washed with fresh water upon return to the dock and stowed carefully for the
next sampling.
Samples are to be transported to the lab as soon as possible upon arrival back to the dock. Samples must be
received at the Microbiology Lab no later than 1200 hours and within six hours of the time the first
sample is taken. Fours hours should be considered the goal.
A copy of the Ocean Nearshore Observations Sheet is to be given to the Microbiology Lab with the
appropriate signatures. The original data sheet is filed in the Marine Biology Lab.
On a weekly basis the Ocean Nearshore Observations Sheet will be checked for accuracy and signed
by a QA/QC officer. At the end of the month the Field Sheets are transcribed electronically to files
LEMMYY.xls (MM= two digit month code, YY= two digit year code). The file is then sent to an
Engineering Technician [at the time of this writing, Lysa Gaboudian ([email protected]) and Brittany
Lui ([email protected])]. These are to be incorporated into a JWPCP Monthly Report. The data is also
reported annually in the Annual Palos Verdes Ocean Monitoring Report data dump and biennially in the
Biennial Palos Verdes Monitoring Report for trends and spatial analysis.
4
App 3.3 - 4
LABEL PROCEDURES
The label file for sample bottles is preformatted and located at the OMRG DM5 site Ocean Monitoring\
labels\bactee and is pre-organized for each month. These monthly files can be modified for each
upcoming month’s of sampling by inserting the new date, personnel, and sample split designation as
needed. Save changes with new file name that includes the current year.
These labels are to be printed only on the PolyPaper Laser Paper label sheets. Avery brand label sheets
are not to be used, as they cannot be easily removed from bottles after sampling. The PolyPaper labels are
found in the MBL drawer underneath the printer. Additional supply of this paper can be purchased thru
the San Jose Creek’s stock room from VWR (item # 6314-0020).
The Microbiology Laboratory no longer requires replicate samples. In lieu of this the larger bottles
previously used for the replicates samples are now used as a Split sample. The following replicate regime
will still be used for the Split samples:
5
App 3.3 - 5
June (Split samples)
Inshore sampling #1………….IL2B and IL5S
Inshore sampling #2………….IL7S
Inshore sampling #3………….9C---example of sequence only
Inshore sampling #4………….IL6B
July (Split samples)
Inshore sampling #4………… 6C---example of sequence only
Inshore sampling #5………….IL3S and IL7B
August (Split samples)
Inshore sampling #2………….8C----example of sequence only
September (Split samples)
Inshore sampling #1…………. IL2S
Inshore sampling #2…………..9C---example of sequence only
Inshore sampling #3…………. IL4B and IL6S
Inshore sampling #4…………. IL5B
Inshore sampling #5…………. IL3S and IL7B
October (Split samples)
November (Split samples)
Inshore sampling #1……….….IL2S
Inshore sampling #5…………..IL3S and IL7
December (Split samples)
Inshore sampling #5………….9C---example of sequence only
6
App 3.3 - 6
January (Split samples)
Inshore sampling #1……….….6C---example of sequence only
Inshore sampling #5…………..IL3S and IL7B
February (Split samples)
Inshore sampling #5……….....8C---example of sequence only
March (Split samples)
Inshore sampling #1……….….9C---example of sequence only
April (Split samples)
Inshore sampling #1………….IL2B and 5S
Inshore sampling #5……...…..6C---example of sequence only
May (Split samples)
Inshore sampling #3…………. 8C---example of sequence only
7
App 3.3 - 7
Appendix 3.4
Total Coliform, Fecal Coliform, and Enterococcus Enumeration by Membrane
Filtration – (Method 349 – Total Coliform, Method 356 – Fecal Coliform, and
Method 357 – Enterococcus) and Fecal Coliform Enumeration by Multiple Tube
Fermentation – (Method 351)
Test Code
349
Method Name
Total Coliform Enumeration by Membrane Filtration
Version
13.1.0
Method Date
September 10, 2013
Reasons for
Method Revision
Revised by:
Tuan Lai
JWPCP WQL
Date
Debra Leachman
Microbiologist II
JWPCP WQL
'
*VW\ b
Date
Approved by:
Kathy Walker
Supervisor I
JWPCP WQL
ignatun
() £&J
9U o f 11
Date
Final Approval:
Jean Lee
Superintendent of
JWPCP Laboratory
Signature
App 3.4 - 1
Date
349. TOTAL COLIFORM ENUMERATION BY MEMBRANE FILTRATION
INTRODUCTION
The total coliform group is defined as facultative anaerobic, gram-negative, non-spore-forming,
rod-shaped bacteria that typically develop red colonies with a partial or entire metallic sheen
surface within 24 h at 35ºC on an Endo-type medium containing lactose. This sheen production
results from a lactose fermentation by-product (acetaldehyde) and the Schiff’s reagent, present in
the mEndo media (SM 9222B). Wastewater, fresh and marine receiving water may be analyzed
for total coliforms using the membrane filtration method. The membrane filtration method
allows for greater reproducibility than the multiple tube fermentation method and confirmed
results can be obtained in 24 hours.
1.
2.
Scope and Application
1.1
This procedure is applicable for use with treated wastewater, fresh and marine
receiving waters.
1.2
Solids and other background interference present in the sample must be
considered when determining the applicability of the test.
1.3
The two-step enrichment procedure must be used to aid recovery of stressed
coliforms from secondary chlorinated wastewater effluent.
1.4
Results ranging from 1 to 9 are reported to 1 significant figure, and results 10 or
greater are rounded to 2 significant figures.
Summary of Method
2.1
Sample aliquot is filtered through a membrane filter.
2.2
The filter is rolled onto an absorbent pad saturated with lauryl tryptose broth and
incubated for 1.5 to 2 h at 35ºC. This constitutes the enrichment step.
2.3
The filter is transferred to mEndo agar and incubated for the remaining 20 to 22 h
at 35 ± 5ºC. Total incubation time is 22 to 24 h.
2.4
The filter is examined under fluorescent light and magnification. All red colonies
exhibiting green or golden metallic sheen are counted as total coliform.
2.5
Results are reported as total coliform colony forming units (cfu) per 100 mL of
sample.
349-1
Version 13.1.0
App 3.4 - 2
September 10, 2013
3.
4.
3.1
Sampling schedules must be planned to ensure that testing is done immediately
after the samples are delivered to the lab, ideally within 2 h. Samples collected in
the field that are not analyzed immediately must be iced or refrigerated at < 8°C
during transit to the lab. The holding time for samples may not exceed 6 h.
3.2
After each sample is collected the analyst records various observations, including
time of collection, electronically onto a PDA. When the analyst returns to the
laboratory a hotsync is performed on the PDA, and an observation sheet(s) is
printed, signed and filed. The signed observation sheet is the chain of custody
form for the samples.
3.3
Once the samples arrive back to the laboratory, an analyst must measure and
record the temperature of a randomly selected sample, using a Raytek® laser
temperature probe or other similar external device.
3.4
Samples are collected in polypropylene bottles that have been thoroughly washed
and sterilized. Prior to use, sample bottles must pass a 48 h TSB sterility check.
3.5
Bottle size should allow for adequate sample volume with sufficient headspace for
thorough mixing. Appropriate labeling must include sample location, date/time
and sampler’s initials.
3.6
Shore samples must be held refrigerated until final results are known.
3.7
Chlorinated effluent samples must be chlorine neutralized for bacteriological
analysis. Sterile Whirl- pak® bags that contain 10 mg sodium sulfate pill are used
to collect chlorinated samples. Total chlorine residual is analyzed from a separate
grab without sodium sulfate pill to measure the effluent chlorine level and to
ensure the sodium sulfate pill will neutralize the chlorine concentration present in
the sample.
Interferences
4.1
The presence of solids and turbidity may cause clogging of the membrane filter.
If the solids interference cannot be diluted out because coliform density is low,
then the multiple tube fermentation test must be substituted.
4.2
Samples with high background concentrations may overgrow coliform organisms.
Reducing sample aliquots or setting serial dilutions may reduce this interference.
349-2
Version 13.1.0
App 3.4 - 3
September 10, 2013
4.3
5.
Bacterial clumping may result in false low coliform determinations. This effect
may be reduced by vigorous agitation of the sample prior to analysis.
Apparatus
5.1
Incubator, capable of holding a temperature range of 35 ± 0.5ºC
5.2
Waterbath, capable of holding a temperature range of 44.5± 0.2ºC
5.3
Stereoscopic dissecting microscope (10× or 15×) and cool white fluorescent lamp
5.4
Hand tally (periodically calibrated)
5.5
Vacuum source
5.6
Vacuum filter flasks, safety coated (2 liter and 1 liter in sequence)
5.7
Sterilized filtration units, funnel and filter holder
5.8
UV sterilizer with short-wave (254 nm) germicidal lamp
5.9
Bunsen burner
5.10
Ethanol in wide mouth cup for flaming forceps
5.11
Forceps with flat ends
5.12
Sterilized graduated cylinders (50 mL)
5.13
Sterile pipets, 1 and 10 mL, cotton plugged, graduated or serological
5.14
Sterile petri dishes, loose lids, 60 × 15 mm
5.15
Manifold for multiple filtration units
5.16
Sterile membrane filters with grid, white, 47 mm diameter, 0.45µm pore diameter,
e.g., Millipore type HA
5.17
Sterile absorbent pads for enrichment step, i.e., Pall® 47 mm sterile absorbent
pads.
5.18
Sterilized Guth dispensing bottle(s)
5.19
Platinum loop / needle or sterile, disposable, plastic loop / needle
349-3
Version 13.1.0
App 3.4 - 4
September 10, 2013
6.
5.21
12 × 75 mm sterile disposable test tubes with snap caps
5.22
16 × 125 mm disposable test tubes with screw caps
5.23
Test tube racks for holding 13 mm / 16 mm tubes
5.24
Aluminum foil
5.25
Plastic disposable transfer pipets
5.26
Pipet bulb
5.27
Sterilized amber reagent bottle for ONPG reagent
5.28
Reagent bottles for toluene and 0.85% NaCl physiological saline
Media/Reagents
6.1
Thoroughly review MSDS sheets for health and safety concerns prior to media or
reagent handling.
6.2
Laboratory prepared media should be recorded in the Equipment/Media
Preparation Laboratory Notebook, including all appropriate information such as
manufacturer, lot, expiration date, quantity of dehydrated media used, quantity
prepared, additives, pH, autoclave cycle, controls and analyst initials.
6.3
Laboratory Pure Water, Deionized Water. Water that has passed through a
reverse osmosis system and deionizing resins so that it meets the Standard
Methods performance specifications for “medium-quality” reagent water
(Standard Methods Online Editions, 1080C, Table 1080:II). Additionally, it must
pass the Standard Methods performance specifications for water used in
microbiological testing (Standard Methods Online Editions, 9020B.4.d, Table
9020: II).
6.4
Phosphate Buffered Solution. Sterile buffered solution is used to rinse down
filtration unit between subsequent aliquots of the same sample. Phosphate
buffered solution is also used for dilution blank preparation.
6.4.1
Stock Phosphate Buffer Solution. Add 34.0 g potassium dihydrogen
phosphate (KH 2 PO 4 ) to 500 mL laboratory pure water and mix. Adjust
pH to 7.2 ± 0.5 with NaOH and bring volume to 1 L. Label the bottle with
reagent name, manufacturer’s name, lot number, preparation date,
expiration date, and analyst initials. Autoclave at 121ºC for 15 min. After
349-4
Version 13.1.0
App 3.4 - 5
September 10, 2013
completely cooling bottle to room temperature, aseptically inoculate 20
mL of stock phosphate buffer solution into a 100 mL TSB media bottle.
Incubate the TSB bottle at 35 ± 0.5ºC for 24 and 48 h. Record sterility
information in the Equipment/Media Preparation Book. Discard solution
if the TSB bottle becomes turbid. Store refrigerated and handle
aseptically. Discard if turbidity is present or after 3 months of use.
6.4.2
Magnesium Chloride Solution. Add 81.1 g magnesium chloride
MgCl 2 ·6H 2 O to 1 L laboratory pure water and mix to dissolve. Label the
bottle with reagent name, manufacturer’s name, lot number, preparation
date, expiration date and analyst initials. Autoclave at 121ºC for 15 min.
After completely cooling bottle to room temperature, aseptically inoculate
20 mL of magnesium chloride solution into a 100 mL TSB media bottle.
6.4.3
Working Phosphate Buffered Solution. Add 50 mL magnesium chloride
solution to 10 L of laboratory pure water in a carboy and mix thoroughly.
Dispense the laboratory pure water containing magnesium chloride into
1 L or 2 L reagent bottles and cap loosely. Suspend a biological indicator
in a separate 2 L bottle filled with laboratory pure water. This bottle is
designated exclusively for quality control use and should be rotated to
different areas in the autoclave with each batch. The label information
should include the reagent name, preparation date, expiration date, analyst
initials and bottle number, which identifies the bottle position in the
autoclave. Cover each bottle cap with foil, label and autoclave tape.
Autoclave at 121ºC for 60 min and record bottle positions in the
Equipment/Media Preparation Book. Cool and tighten caps. Label and
incubate biological indicator at 58-62ºC for 24 and 48 h (follow
manufacturer’s directions). After completely cooling bottles to room
temperature, aseptically add 1.25 mL stock phosphate buffer solution per 1
L sterile laboratory pure water containing magnesium chloride. Mix by
inverting bottles several times. Record all preparation information in
Equipment/Media Preparation Book. Final pH 7.2 ± 0.1.
6.4.4
Each batch of phosphate buffer is tested for sterility. Randomly select one
bottle from the batch and aseptically inoculate 20 mL of phosphate
buffered water into a 100 mL TSB media bottle. Also check the sterility
of the stock phosphate solution by aseptically inoculating 10 mL of
solution into a 50 mL TSB media bottle. Incubate the inoculated TSB
bottles at 35 ± 0.5ºC for 24 and 48 h. Record sterility information in the
Equipment/Media Preparation Book. If the TSB bottles becomes turbid,
check biological indicator result, autoclave records and TSB batch QA.
349-5
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App 3.4 - 6
September 10, 2013
Discard the entire batch. Discard stock phosphate solution if it fails QA.
Store working phosphate buffer away from direct sunlight for up to three
months. Discard if turbidity develops before three months.
6.4.5
Phosphate Buffer 9.0 mL Dilution Blanks: Add 1.25 mL stock phosphate
buffer solution and 5.0 mL magnesium chloride solution per 1 L laboratory
pure water and mix thoroughly. Adjust pH with 0.1N or 1.0N NaOH to
achieve a final pH of 7.2 ± 0.1. Record all preparation information in the
Equipment/Media Preparation Book. Dispense into calibrated 9.0 mL test
tubes and label racks with reagent name, preparation date, expiration date
and analyst initials. Attach autoclave tape to each rack and place a
biological indicator to one of the racks, autoclave at 121ºC for 15 min.
Record all autoclave information in Equipment/Media Preparation Book.
Once phosphate buffered blanks have cooled to room temperature take the
final pH using several phosphate buffer blanks. Visually check each
tube’s calibration mark to ensure each tube has 9.0 ± 0.2 mL, and tighten
caps.
6.4.6
Each batch of phosphate buffer blanks is tested for sterility. Randomly
select 2 tubes from the batch and aseptically pour into a 100 mL TSB
media bottle. Incubate the TSB bottle at 35 ± 0.5ºC for 24 and 48 h.
Record sterility information in the Equipment/Media Preparation Book. If
the TSB bottle becomes turbid, check autoclave records and biological
indicator result. Discard the entire batch if quality assurance tests indicate
problem to be autoclave related. If everything is normal, retest another 2
tubes from the batch in TSB. If the TSB is negative, keep the entire batch.
If the TSB bottle turns positive, discard the entire batch. Store phosphate
buffer blanks away from direct sunlight and discard if turbidity develops
before the expiration date of 3 months for screw cap and 2 weeks for slip
cap tubes.
6.5
Lauryl Tryptose Broth: lactose broth media used in the enrichment step to aid in
recovery of stressed coliforms. Add 35.6 g lauryl tryptose (Difco) to 1 L of
laboratory pure water and mix to dissolve. Transfer to smaller reagent bottles
(i.e., 100 mL and 250 mL) and label bottles with reagent name, preparation date,
expiration date and analyst initials. Autoclave at 121ºC for 15 min. Cool and
tighten caps. Store at room temperature away from direct sunlight for up to three
months. Final pH 6.8 ± 0.2. Positive and sterility controls are incubated for 24 h
and 48 h at 35 ± 0.5ºC. Record all preparation information in the
Equipment/Media Preparation Book.
6.6
mEndo agar LES. Confirmatory plate media used to select for total coliforms.
Add 51.0 g mEndo (Difco) to 1 L of laboratory pure water containing 20 mL 95%
non-denatured ethanol and mix to dissolve. Continue mixing and bring to a boil.
Cool to approximately 55ºC and dispense 5-mL into 60 × 15 mm sterile petri
349-6
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App 3.4 - 7
September 10, 2013
dishes with a sterile dispenser, such as a Unispense automatic dispenser with
sterilized tubing. Dispensed medium should be a depth of 4 to 5 mm. Dispensed
plates are covered to protect them from light and cooled until solidified. Prepared
plates should be repackaged and labeled with reagent name, preparation date,
expiration date and analyst initials. Store in refrigerator for up to two weeks.
Final pH 7.2 ± 0.2. Prepare positive and negative controls as well as a minimum
of 3 sterility controls or 1% of the plates set for each batch of media and incubate
for 24 h at 35 ± 0.5ºC. Record all preparation information in the
Equipment/Media Preparation Book. Note: mEndo is considered a respiratory
irritant. Ethanol is highly flammable and should be handled with care. Nondenatured ethanol stock is to be stored in an inventory-controlled locked cabinet.
Consult MSDS for details.
6.7
Tryptic Soy Agar (TSA) slants: all-purpose growth media. Add 40 g of
dehydrated media to 1L laboratory pure water and mix to dissolve. Continue
mixing and bring to a boil. Cool to approximately 55oC, dispense 5 mL into
16 × 125 mm screw cap test tubes, and loosely cap. Label racks with reagent
name, preparation date, expiration date and analyst initials. Autoclave at 121oC
for 15 minutes. Pour out a tube for final pH before media solidifies. Tilt racks so
that the agar forms a slant length of approximately 6.3 cm and cool to solidify.
Slant racks may also be used. Final pH 7.3 ± 0.2. Tighten down caps and store
away from direct light at room temperature for up to 3 months. Prepare a positive
control along with a minimum of 2 sterility controls or 1% of the tubes set for
each batch of media and incubate for 24 h at 35 ± 0.5oC. Record all preparation
information in the Equipment/Media Preparation Book.
6.8
1.0 M monosodium phosphate solution. Buffering agent for ONPG solution.
Dissolve 13.8 g NaH 2 PO 4 ·H 2 O in approximately 80 mL of laboratory pure water,
and adjust to pH 7.0 using appropriate concentrations of NaOH and bring volume
to 100 mL. Label the bottle with reagent name, preparation date, expiration date
and analyst initials. Store at refrigerated for 3 months. NOTE: Refrigerated
reagent may crystallize and should be warmed up to 37oC before use (do not
overheat). Record all preparation information in the Equipment/Media
Preparation Book.
6.9
ONPG (o-nitrophenyl-β-D-galactopyranoside) solution. Reagent used to detect
the enzyme β-D-galactosidase. Dissolve 0.24 g o-nitrophenyl-β-Dgalactopyranoside (ONPG) in 45 mL laboratory pure water that has been warmed
to 37oC. Add 15 mL warmed 1.0M monosodium phosphate solution and mix to
dissolve (solution must be colorless). Filter sterilize with Millex 0.22 μm filter
Syringe Driven Unit. Label the bottle with reagent name, preparation date,
expiration date and analyst initials. Store at 2-8°C in sterilized amber reagent
bottle for up to 2 weeks. Warm sufficient reagent in a sterile ONPG tube to 37oC
prior to use (do not overheat). Prepare positive and negative controls, along with
sterility for each batch of ONPG solution and incubate for 24 h at 35 ± 0.5oC.
349-7
Version 13.1.0
App 3.4 - 8
September 10, 2013
7.
6.10
BBLTM DrySlide Oxidase test. Commercially prepared test system used to
determine the oxidase reaction of bacteria. Each disposable 2"×2" slide has four
plastic-film reaction areas which contain the substrate N,N,N,- tetramethyl-pphenylenediamine dihydrochloride and ascorbic acid, a stabilizing agent.
Examine each slide before use. The reaction area should be colorless. Store at
15-30oC and protect slides from light. Opened slide packets have an expiration
date of one week. Positive and negative controls are used with each set of
verifications.
6.11
Physiological saline, 0.85%. Dissolve 8.5 g NaCl in approximately 500 mL
laboratory pure water. Bring volume up to 1 L in a 1-L volumetric flask and
transfer to 1-L reagent bottle. Label the bottle with reagent name, preparation
date, expiration date and analyst initials. Autoclave at 121oC for 15 minutes.
Store refrigerated for up to 3 months. Warm to 37oC prior to use. Perform
sterility control once physiological saline solution has cooled. Aseptically
inoculate 20 mL of physiological saline solution into a 100 mL TSB media bottle.
information in the Equipment/Media Preparation Book.
6.12
Toluene, analytical grade. Aids in cell lysis, enhancing the liberation of
β−galactosidase enzyme from coliform bacteria. Stock toluene is stored in a
flammables storage cabinet. A small amount (no more than 40 mL), is labeled
with a flammables warning and stored in the laboratories dehydrated media and
reagents cabinet.
6.13
Biological indicator, Bacillus stearothermophilus spore suspension ampules. An
unused ampule serves as a positive control, labeled and placed in the water bath
on a monthly basis or sooner if a new lot is used. Store unused ampules at 2-8oC.
Procedure
7.1
Sample aliquots and dilutions are predetermined and based on sample type. The
optimal sample volume will yield 20-80 colonies per plate. The laboratory is
currently using 0.5, 5.0, 20, and 50 mL aliquots to ensure that a range of 1-16,000
total coliform/100 mL may be calculated from marine receiving waters.
7.2
Aseptically insert sterile absorbent pads onto lid side of prepared mEndo plates
and saturate with lauryl tryptose broth (approx. 1.5 mL). Label the bottom of each
mEndo plate with the sample location and volume. Include a blank with each
sample. Stack the labeled dishes starting with the largest aliquot and end with a
sample blank on top. Cover plates until processing begins.
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7.3
Aseptically set up to three sterile filtering apparatus into a manifold unit that is
attached to a vacuum and waste collection system.
7.4
Aseptically transfer sterile phosphate buffered water to a sterile Guth bottle and
label with reagent name. Record the phosphate buffer identification information
in the laboratory data book.
7.5
Using flamed forceps, place a membrane filter, grid side up, on the mesh plate of
the filter base (forceps tip should only touch outer rim of filter). Reseat and lock
on filtering funnel. Rinse the interior of the funnel thoroughly with phosphate
buffered water and draw a steady but continuous vacuum through the filter. Do
not pull excess air through the filter membrane.
7.6
Using flamed forceps, carefully remove the blank membrane filter and place it
grid-side up onto the lauryl tryptose broth-saturated absorbent pad. Manipulate
the filter to ensure air is not trapped under the filter. This method is used for
transferring blank and sample membrane filters.
7.7
Shake each sample bottle vigorously (approx. 25 times) prior to processing.
7.8
Each membrane filter should be pre-wet with buffer before adding sample. This
allows the analyst to examine the filter to ensure it is seated correctly and not
damaged.
7.9
Sample volumes of less than 10 mL should not be dispensed directly onto a filter.
Dispense sample into filter funnel containing a small amount of buffer (~10 mL)
and swirl for an even distribution.
7.10
Dispense the smallest volume or highest dilution of sample into each filtering
funnel, and draw a steady vacuum through the filter, ensuring that there is no
excess moisture left on the filter. Thoroughly rinse down the filtering unit with at
least 30 mL of phosphate buffer and draw through. Remove the filter aseptically
and transfer grid side up onto the lauryl tryptose broth-saturated pad until all of
the dilutions are processed. Continue to process sample aliquots ending with the
largest or least dilute sample volume.
7.11
After each sample has been processed, rinse the filtering units thoroughly with hot
water followed by deionized water, and dry with lint-free wipes. The clean
filtering units are sanitized for immediate reuse only. Place them in a UV
sterilizing unit for a minimum of 4 min.
7.12
After all samples are processed, separately filter positive (Escherichia coli) and
negative (Pseudomonas aeruginosa) controls. Incubate these plates with the
processed samples. Use the appropriate dose that will yield individual colonies
with characteristic colonial morphology on mEndo agar.
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8.
7.13
Place the inverted plates containing filtered samples in a covered tray containing a
wet paper towel to maintain a humid environment. Label the container with test
name, date and time. Incubate at 35 ± 0.5ºC for 22 to 24 hr.
7.14
After initial incubation period, lift filter from the pad using sterile forceps and
reposition grid side up onto the mEndo agar surface. Inspect for complete contact
of filter surface to agar. Invert plates, return to container and continue incubation
for a total of 22-24 h at 35 ± 0.5ºC.
7.15
Duplicate analyses are included with each batch processed and the duplicate
schedule for the routine inshore, shoreline and manifold monitoring stations are
scheduled on a monthly basis at a frequency of at least 10% of sample type.
Calculations
8.1
Counting Colonies
8.1.1
After 24 h incubation, starting with the blank plate, examine for
contamination or any notable changes on the filter or media. Examine and
count plates set for each sample, starting with the highest dilution or
lowest volume filtered, i.e., blank, 10-1, 1 mL, etc.
8.1.2
Use a stereoscopic microscope (10×-15×) with a white fluorescent light
source to yield maximum visibility for identifying and counting coliform
colonies. Adjust light to maximize sheen on colony surface.
8.1.3
A total coliform colony is characterized as a dark red colony with a partial
or entire metallic green or golden sheen to its surface. This sheen is a
result of an acid aldehyde complex (by-product of lactose fermentation)
combining with the Schiff's reagent in the mEndo media. Count colonies
using magnification and a fluorescent light source, record all colonies
demonstrating this characteristic.
8.1.4
A separate nuclei or a fine line of contact can discern individual colonies
that have grown into each other. Irregularly shaped colonies may result
from turbidity or artifacts in the sample.
8.1.5
The use of a hand tally (counter) may aid in keeping track of higher
counts, but it is important to calibrate the tally regularly.
8.1.6
Record all counts and any notable information in the laboratory bench
book. Comments should include unusual conditions on the filter such as
the presence of solids, artifacts, or high background counts. The condition
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of the growth on the filter should also be noted. This may include poor
sheen development, confluent growth, confluent areas or the presence of
too many colonies to obtain an accurate reading. A record of positive and
negative controls must be associated with each set of samples.
8.1.7
8.2
All incubated media must be autoclaved prior to disposal. Liquid waste
can be dumped down the drain, and disposable materials can be discarded
with the laboratory trash.
Due to the possible inhibitory effect of colony crowding, and in order to maximize
the confidence level of a total coliform concentration, statistically determined
minimum and maximum ranges are used to calculate final results. Sample
aliquots producing counts within the ideal countable range of 20-80 total coliform
colonies must be used. If more than one aliquot yields a countable coliform range,
then these results are calculated individually but reported as an arithmetic mean.
The number of total coliform colonies from a sample dilution or volume can be
used in the following equation to determine colony forming units per 100 mL of
original sample volume.
Total Coliform Colonies Counted × 100 = Total Coliform cfu/100 mL
Volume of Sample Filtered (mL)
8.3 If there are no filters containing the countable range and the counts are less than 20,
add all counts and divide by total volume. Report as CFU per 100 mL. Filters with
inhibitory effects are reported as an estimated count and are excluded from the
calculation.
8.4
8.3.1
If no colonies are present on any plate, divide one by the sum of all
representative aliquots or dilutions (mL), multiply by 100 and report result
as less than that value per 100 mL.
8.3.2
If the number of colonies is greater than the countable range maximum of
80 colonies from the smallest volume filtered, calculate the result using 80
and report the result as greater than that value. If the colony count is
greater than 80, and the colonies are distinct, uncrowded and of
characteristic morphology, the exact count may be used to calculate the
result. Such a result is noted as an estimated count.
8.3.3
Plates with a total bacteria count exceeding 200 colonies (total coliform
and non-total coliform colonies), are recorded as TNTC (too numerous to
count) and not used to calculate a final concentration.
For QA purposes, a sampling location may require duplicate sampling and
analysis. This requires a split analysis of a single sample. A separate result is
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calculated from each split analysis and the geometric mean is calculated. The
single geometric mean result is used for reporting.
9.
8.5
Because verifications are part of the JWPCP Microbiology Laboratory quality
assurance program and not applied to an entire sample count, percent verified
results are not used to adjust reported results.
8.6
Concentrations of indicator organisms, such as total coliform, are expressed as
colony forming unit (CFU) per 100 mL sample. The calculation is based on the
theory that one characteristic total coliform colony represents one total coliform
bacterium in the original sample.
Quality Assurance
9.1
Verifications
9.1.1
Method validation is routinely applied to 5% of positive samples by
verifying a target number of 10 colonies per aliquot used in the sample
calculation. Although this total coliform membrane filtration test is based
on a lactose fermentation reaction signaled by the formation of colonial
sheen production (SM 9222B), the combined cytochrome oxidase/onitrophenyl-β-D-galactopyranoside verification method was selected
because it verifies a coliform bacteria based on the presence of the
coliform specific enzymes cytochrome oxidase and β-D galactosidase.
9.1.2
Perform biochemical verification on 5% of each type of sample set per
month for typical coliform colonies. For atypical coliform colonies, set
verifications on at least one sample per month. In order to evaluate false
positive and false negative trends adequately, atypical verifications may be
substituted for typical verifications as needed. Atypical colonies have no
metallic sheen and can be dark red, mucoid, or nucleated.
9.1.3
For each sample to be verified, select colonies from mEndo plate(s) that
have been used to report the final result. Using sterilized needles or loops,
pick approximately 10 isolated typical coliform colonies from one
quadrant of the plate(s) and streak onto individual TSA slants. Using the
single quadrant approach improves the chance for randomizing colony
selection. Systematically add additional quadrants as necessary.
9.1.4
Include both positive and negative controls with each set of verifications.
Streak Escherichia coli (positive control) and Pseudomonas aeruginosa
(negative control) onto TSA slants and include along with test colonies.
Label rack with I.D. numbers, sample date, set date, location, control
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information and analyst initials. Record corresponding information in
bacteriological data book. Incubate at 35 ± 0.5ºC for 18-24 hours.
9.1.5
9.1.6
Cytochrome Oxidase Test
9.1.5.1
The cytochrome oxidase (CO) test is used to determine the
presence of the cytochrome oxidase enzyme. With the exception
of Plesiomonas shigelloides, the entire Enterobacteriaceae family
is cytochrome oxidase negative. Therefore, if a colony yields a
positive CO reaction, the colony is not verified as a total coliform
and the ONPG verification step is unnecessary.
9.1.5.2
Remove sufficient DrySlides from the packet and examine prior
to use. The reaction areas should be colorless to light green.
Using a sterile loop, remove a portion of the growth from each
TSA slant and rub the inoculum directly onto the reaction area of
the DrySlide Oxidase slide (each reaction area can accommodate
up to 4 tests).
9.1.5.3
Examine the reaction area for the appearance or absence of a
dark purple color within 20 seconds. Oxidase-positive organisms
produce a dark purple color within 20 seconds while oxidasenegative organisms produce no color change or a change to light
gray within the 20-second test period. Note: Color development
after 20 seconds should be disregarded. Any unclear results
should be repeated using a new reaction site on the slide.
9.1.5.4
Positive and negative controls must be run with each verification
set. Escherichia coli is negative for cytochrome oxidase and
Pseudomonas aeruginosa is positive for cytochrome oxidase.
Control results deviating from the expected reactions negate the
verification test and an immediate review of the verification
process should be done.
9.1.5.6
Record cytochrome oxidase results for each colony in the
bacteriological data book. Any comments or unusual results
should be noted in the comment section.
ONPG Test
9.1.6.1
The ONPG test is used to demonstrate the presence or absence of
the enzyme β-galactosidase using the organic substrate onitrophenyl-β-D-galactopyranoside (ONPG). This lactoseinduced enzyme in lactose fermenting bacteria (coliform group)
is detected when the colorless ONPG reagent is hydrolyzed to
349-13
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liberate a yellow chromogenic compound, o-nitrophenol (ONP).
A positive ONPG reaction (yellow) which corresponds with a
negative cytochrome oxidase reaction verifies the original colony
as total coliform bacteria.
9.1.7
9.1.6.2
Aseptically pour a sufficient quantity of 0.85% saline solution in
a sterile plastic disposable test tube. Place tube and the bottle of
ONPG reagent into an incubator and warm to approximately
37oC. Arrange 12 × 75 mm sterile plastic disposable test tubes
with caps in a rack and label with numbers, sample date, set date,
location and analyst initial. Remove caps and add 0.25 mL of
warmed saline solution to each test tube. Using a sterile loop,
remove a loopful of growth from each TSA slant and emulsify
the growth into each corresponding numbered test tube with
saline. Add one drop of toluene to each test tube. Cover the rack
with aluminum foil and gently agitate rack. Incubate at 35 ±
0.5ºC for five minutes. Note: toluene is toxic and flammable and
should be handled with care. Stock toluene is to be stored in the
flammables storage cabinet. Consult the Material Safety Data
Sheet (MSDS) for additional safety precautions.
9.1.6.3
After approximately 5 minutes, remove the test rack containing
saline, sample and toluene from the 35oC incubator. Remove the
foil covering and add 0.25 mL of warmed ONPG solution to each
tube. Recap each tube, gently agitate the rack, and reincubate at
35 ± 0.5ºC for a maximum of 24 hours; a positive reaction may
develop as quickly as 30 minutes after the ONPG reagent is
added.
9.1.6.4
After 24-hour incubation period read each tube for the ONPG
reaction. A positive reaction is indicated by a yellow color
development while a negative reaction remains colorless.
Escherichia coli is positive and Pseudomonas aeruginosa is
negative for ONPG. Control results deviating from the expected
reactions negate the verification test and an immediate review of
the verification procedure should be done. Note: An extra heavy
Pseudomonas inoculum may result in a positive ONPG result
due to some β-galactosidase production.
9.1.6.5
Record ONPG results for each colony in the bacteriological data
book. Any comments or unusual results should be noted in the
comment section.
Autoclave all contaminated tubes, DrySlides, loops or other contaminated
devices or media prior to disposal.
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September 10, 2013
9.1.8
A sample colony that is negative for cytochrome oxidase and positive for
the ONPG test verifies that colony to be a total coliform. Calculate the %
verification for each sample as follows:
(No. of Colonies With “-“ CO and “+” ONPG rxn) × (100) = % verified total
number of colonies tested
coliform
9.1.9
Record all results in the bacteriological data book and on the Total
Coliform Verification Checklist. Note: Any TSA slants which have
negative growth should be reported as such and not included into the
percent verification formula.
9.1.10 A pattern of low percentage verifications may indicate a problem for some
agent of the membrane filtration procedure. Review initial sample data
and notify the supervisor if this occurs.
9.1.11 Verifications are part of the JWPCP Microbiology Laboratory quality
assurance program. Percent verified results are not used to adjust
reporting results as stated in Standard Methods.
9.2
Record purchased sterile equipment, reagent and media product information in the
Laboratory Supply Inventory Log. Information should include manufacturer,
lot number, expiration date, quantity, date received and date in-use, and additional
QA testing required or performed in-house.
9.3
When using a commercial dehydrated product for media preparation, always
follow manufacturer's directions for preparation, unless otherwise directed by the
SOP. No media is put into use prior to passage of all quality assurance testing.
9.4
New reagent lots recorded in the Supply Inventory notebook shall include
Manufacturer, Lot Number, Manufacturer’s Expiration Date, Received Date,
Opened Date and Analyst Initials. This is done primarily for Quality Assurance
tracking purposes.
9.5
Review JWPCP Microbiology Quality Assurance Guidelines for Standardization
of Accumet AB15 pH Meter.
9.6
Review JWPCP Microbiology Quality Assurance Guidelines for media
preparation. Positive (Escherichia coli) and negative (Pseudomonas aeruginosa)
control cultures are set with each batch of media prepared.
.
9.7
Record reagent and batch media preparation information in the laboratory media
bench book.
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September 10, 2013
9.8
Post-filtering is a quality assurance procedure that is performed once a month to
ensure that no carryover exists between subsequent filtering. An extra mEndo
plate is included with the sample plates and labeled. After the last aliquot of a
sample is filtered through, another membrane is placed onto the holder and the
filtering unit is rinsed down with phosphate buffer. This filter is then rolled onto
the pad and incubated with the other samples. Colonies present after incubation
indicate that the analyst must improve the rinsing technique between aliquots.
9.9
Single and multiple analysts should count total coliform colonies from the same
membrane filter plate once per month. The multiple analysts’ counts should agree
within 10% and the single analyst counts should agree within 5%. Record all
results in the JWPCP Bacteriological Bench Book. If the repeatability of the
counts exceeds the acceptable percentage, notify the supervisor and/or the Quality
Assurance Project Manager immediately.
9.10
Duplicate analyses are to be included with each batch processed. To ensure all
stations are included, the routine inshore, shoreline and manifold monitoring
stations are scheduled on a monthly basis at a frequency of at least 10% of sample
type.
9.10.1 The filter is rolled onto to lauryl tryptose broth-saturated absorbent pads.
Incubate these plates with the other samples.
9.10.3 The two results obtained (duplicate one and duplicate two) are entered into
the LIMS.
9.10.4 Report unusual results or out of control response sheets, within 24 h, to the
laboratory supervisor.
10.
9.11
Proper and consistent labeling of racks and tubes are essential for accurate data
reporting.
9.12
Multiple analysts should count total coliform colonies on the same membrane
filter once a month and each individual’s counts should agree within 10%.
9.13
Participation in the Laboratory Quality Assurance Section monthly program for
total and fecal membrane filtration is required.
Method Performance
10.1
The laboratory shall participate in the annual Laboratory Proficiency Testing for
Total Coliform isolation by the Membrane Filtration Method.
349-16
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September 10, 2013
11.
References
11.1
Standard Methods for the Examination of Water and Wastewater. Online Edition,
Methods 9222 A, 9222 B.
11.2
Microbiological Methods for Monitoring the Environment Water and Wastes, R.
Bordner and J. Winters, Eds. 1978. EPA 600/8-78-017, pp. 108-113.
11.3
JWPCP Microbiology Quality Assurance Appendices, 2013. Los Angeles County
Sanitation Districts-JWPCP Microbiology Laboratory.
11.4
Changes in Calculation and Reporting Procedures, Los Angeles County Sanitation
Districts-JWPCP Microbiology Internal Memo, January 8, 2004.
11.5
Water Microbiology Laboratory Field Procedures, Millipore Corporation
Publication, 1992. Catalog # AD323.
11.6
Difco Manual, 10th Edition, Difco Laboratories, 1984. pp. 294-296.
11.7
Biochemical Tests for Identification of Medical Bacteria, 2000 Ed. J. MacFadden,
The Williams and Wilkins Company, Baltimore, Maryland, 1976. pgs. 71-78,
160-169.
11.8
BBL DrySlide Oxidase package insert. Becton Dickinson and Company, 1999.
349-17
Version 13.1.0
App 3.4 - 18
September 10, 2013
Test Code
356
Method Name
Fecal Coliform Enumeration by Membrane Filtration
Version
13.1.0
Method Date
September 10, 2013
Reasons for
Method Revision
Revised by:
Tuan Lai
JWPCP WQL
7/te/&
Signature
Date
Debra Leachman
Microbiologist II
JWPCP WQL
Ol
12* I (3
Date
Approved by:
Kathy Walker
Supervisor I
JWPCP WQL
Signature
Final Approval:
Jean Lee
Superintendent of
JWPCP Laboratory
\ Am ?
OA)A£,
U
/
Signature
App 3.4 - 19
1
9/ao /(3
Date
-eJL
Date
356. FECAL COLIFORM ENUMERATION BY MEMBRANE FILTRATION
INTRODUCTION
Fecal coliform bacteria are a subgroup of the total coliform group. Fecal coliforms are defined
as aerobic or facultative anaerobic, gram negative, lactose fermenting, non-spore-forming, rodshaped bacteria capable of growth at 44.5ºC. When grown on mFC medium, fecal coliforms
ferment lactose and produce an acid (SM 9222D). This acid then reacts with the aniline dye
present in the medium to produce characteristic blue colonies. Wastewater and fresh and marine
receiving waters may be analyzed for fecal coliforms using the membrane filtration method. The
present membrane filtration method allows for greater reproducibility than the multiple tube
fermentation method, and confirmed results can be obtained in 24 hours.
1.
2.
3.
1.1
This procedure is applicable for use with treated wastewater, marine and fresh
receiving waters.
1.2
Solids and other background interferences present in the sample must be
1.3
Summary of Method
2.1
2.2
The filter is rolled directly onto the surface of an mFC agar plate.
2.3
The plate is sealed in a waterproof pouch. The pouch is then submerged in a
44.5 ± 0.2ºC water bath for 24 ± 2 h.
2.4
The filter is examined under fluorescent light and magnification. All blue-colored
colonies are counted as fecal coliform.
2.5
Results are reported as fecal coliform colony forming units (cfu) per 100 mL of
sample.
356-1
App 3.4 - 20
Version 13.1.0 September 10, 2013
4.
3.1
the field that are not analyzed immediately must be iced or refrigerated at < 8°C
during transit to the lab. The holding time for samples may not exceed 6 h.
3.2
time of collection, electronically onto a PDA. When the analyst returns back to
the laboratory a hotsync is performed on the PDA, and an observation sheet(s) is
3.3
Once the samples arrive back to the laboratory, an analyst must measure and
record the temperature of a randomly selected sample, using a Raytek® laser
temperature probe or other similar external device.
3.4
3.5
Bottle size should allow for adequate sample volume with sufficient headspace
for thorough mixing. Appropriate labeling must include sample location,
date/time and sampler’s initials.
3.6
Shore samples must be held refrigerated until final results are known.
3.7
If plant effluent has been chlorinated, then the addition of sodium thiosulfate to
the collection bottle is mandatory. A 0.1 mL dose of a 10% solution of sodium
thiosulfate will neutralize approximately 10 ppm residual chlorine. The use of
commercially prepared sterile Whirl-pak® bags containing thiosulfate pills is
especially convenient. To ensure that no chlorine remains, a chlorine residual
analysis should be run on the neutralized sample prior to fecal coliform analysis.
If the original chlorine level exceeds the neutralizing capacity of the sodium
thiosulfate, then the sample must be re-collected with enough sodium thiosulfate
to completely neutralize the chlorine. If a high chlorine residual is suspected, a
chlorine residual determination should be made prior to the neutralization step.
Interferences
4.1
If the solids interference cannot be diluted out because the fecal coliform density
is low, then the multiple tube fermentation method must be substituted.
4.2
Samples with high background concentrations may overgrow fecal coliform
organisms. Reducing sample aliquots or setting serial dilutions may reduce this
interference. The addition of rosolic acid to the mFC media is used to inhibit
background growth.
356-2
App 3.4 - 21
4.3
5.
Bacterial clumping may result in false low fecal coliform determinations. This
effect may be reduced by vigorous agitation of the sample prior to analysis.
Apparatus
5.1
Circulating water bath, or other incubator capable of maintaining a constant
temperature of 44.5 ± 0.2ºC
5.2
Stereoscopic dissecting microscope (10× or 15×) and daylight type fluorescent
lamp
5.3
Hand tally (periodically calibrated)
5.4
Vacuum source
5.5
Vacuum filter flasks – safety coated (2 liter and 1 liter in sequence)
5.6
Sterilized filtration units - funnel and filter holder
5.7
UV sterilizer with short-wave germicidal lamp (254 nm)
5.8
Bunsen burner
5.9
Ethanol in wide mouth cup for flaming forceps
5.10
5.11
5.12
5.13
Sterile petri dishes, loose lids, 60 × 15 mm
5.14
Manifold for multiple filtration units.
5.15
Sterile membrane filters with grid, white, 47 mm diameter, 0.45 µm pore
diameter, i.e., Millipore® type HA
5.16
Sterilized Guth dispensing bottle(s).
5.17
Heat sealable pouches and sealer
5.18
Incubator capable of holding temperature of 35 ± 0.5ºC
356-3
App 3.4 - 22
6.
5.19
Platinum loop / needle or sterile, disposable, plastic loop / needle
5.20
16 × 125 mm disposable test tubes with screw caps
5.21
Test tube racks for holding 16 mm tubes
Media/Reagents
6.1
reagent handling.
6.2
6.3
Methods performance specifications for medium quality reagent water (Standard
Methods Online Editions,1080C, Table 1080:II). Additionally, it must pass the
Standard Methods performance specifications for water used in microbiological
testing (Standard Methods Online Editions, 9020B.4.d, Table 9020:II).
6.4
Phosphate Buffered Solution. Sterile buffered solution used to rinse down
filtration unit between subsequent aliquots of the same sample. Phosphate
buffered solution is also used for dilution blank preparation.
6.4.1
phosphate (KH2PO4) to 500 mL laboratory pure water and mix. Adjust
expiration date and analyst initials. Autoclave at 121ºC for 15 min. After
completely cooling bottle to room temperature, aseptically inoculate
20 mL of stock phosphate buffer solution into a 100 mL TSB media bottle.
6.4.2
(MgCl2·6H2O) to 1 L laboratory pure water and mix to dissolve. Label the
356-4
App 3.4 - 23
6.4.2.1
After completely cooling bottle to room temperature, aseptically
inoculate 20 mL of magnesium chloride solution into a 100 mL
TSB media bottle. Incubate the TSB bottle at 35 ± 0.5ºC for 24
and 48 h. Record sterility information in the Equipment/Media
Preparation Book. Discard solution if the TSB bottle becomes
turbid. Store refrigerated and handle aseptically. Discard if
turbidity is present or after 3 months of use.
6.4.3
Autoclave at 121ºC for 60 min and record bottle positions in
Equipment/Media Preparation Book. Cool and tighten caps. Label and
incubate biological indicator at 58-62ºC for 24 and 48 h (following
6.4.4
buffered water into a 100 mL TSB media bottle. Also check the sterility
solution into a 50 mL TSB media bottle. Incubate the TSB bottles at 35 ±
0.5ºC for 24 and 48 h. Record sterility information in the
Equipment/Media Preparation Book. If the TSB bottle becomes turbid,
6.4.5
Phosphate Buffer 9.0 mL Dilution Blanks. Add 1.25 mL stock phosphate
buffer solution and 5.0 mL magnesium chloride solution per 1 L
laboratory pure water and mix thoroughly. Adjust pH with 0.1 N or 1.0 N
NaOH to achieve a final pH of 7.2 ± 0.1. Record all preparation
information in the Equipment/Media Preparation Book. Dispense into
calibrated 9.0 mL test tubes and label racks with reagent name,
preparation date, expiration date and analyst initials. Attach autoclave
356-5
App 3.4 - 24
tape to each rack and place a biological indicator to one of the racks,
autoclave at 121ºC for 15 min. Record all autoclave information in
Equipment/Media Preparation Book. Once phosphate buffered blanks
have cooled to room temperature take the final pH using several phosphate
buffer blanks. Visually check each tube’s calibration mark to ensure each
tube has 9.0 ± 0.2 mL, and tighten caps.
6.4.6
6.5
6.6
select 2 tubes from the batch and aseptically pour into a 100 mL TSB
media bottle. Incubate the TSB bottle at 35 ± 0.5ºC for 24 and 48 h.
Record sterility information in the Equipment/Media Preparation Book. If
the TSB bottle becomes turbid, check autoclave records and biological
problem to be autoclave related. If turbidity is not present, retest another
2 tubes from the batch in TSB. If the TSB is negative, keep the entire
batch. If the TSB bottle turns positive, discard the entire batch. Store
phosphate buffer blanks away from direct sunlight and discard if turbidity
develops before the expiration date of 3 months for screw cap and 2 weeks
for slip cap tubes.
mFC Agar. Used to detect and enumerate fecal coliform from water samples
using membrane filtration method. Add 52.0 g M-FC agar (Difco) to 1 L of
laboratory pure water and mix to dissolve. Continue mixing and bring to a boil.
Once media begins to boil, add 10 mL of a 1% solution of rosolic acid in 0.2 N
NaOH. Continue heating for 1 min. Do not autoclave. If necessary, adjust to
pH 7.4 with 1 N HCl. Cool to approximately 55ºC and dispense 5-mL into
60 × 15 mm sterile petri dishes with a sterile dispenser, such as a Unispense
automatic dispenser. Dispensed medium should be a depth of 4 to 5 mm.
Dispensed plates are covered to protect them from light and cooled until
solidified. Prepared plates are repackaged and labeled with reagent name,
preparation date, expiration date and analyst initials. Store in refrigerator for up
to two weeks. Final pH 7.4 ± 0.2. Prepare positive and negative controls as well
as a minimum of 3 sterility controls or 1% of the plates set for each batch of
media and incubate for 24 h at 44.5 ± 0.2ºC. Record all preparation information
in the Equipment/Media Preparation Book. Note: mFC is considered a
respiratory irritant and a possible carcinogen. Consult MSDS for details.
6.5.1
Rosolic Acid, 1% Solution. Add 0.1 g rosolic acid to 10 mL of stock 0.2
N NaOH. Mix well. Prepare fresh reagent for each batch of mFC agar.
6.5.2
Stock 0.2 N NaOH. Add 1 g NaOH to 125 mL laboratory pure water.
Mix to dissolve. Store in labeled reagent bottle for up to 3 months.
Lauryl Tryptose Broth. Media used in initial step of fecal coliform verification.
Add 35.6 g (Difco) to 1 L of laboratory pure water and mix to dissolve. Dispense
10 mL into each 16 × 125 mm screw cap tube containing an inverted fermentation
356-6
App 3.4 - 25
vial and label racks with reagent name, preparation date, expiration date and
analyst initials. Autoclave at 121°C for 15 min. Cool and tighten caps. Store at
room temperature away from direct sunlight for up to three months. Final pH
6.8 ± 0.2. Positive, negative and sterility controls are incubated for 24 and 48 h at
35 ± 0.5ºC. Record all preparation information in the Equipment/Media
Preparation Book.
7.
6.7
EC Broth (Escherichia coli broth). Media used in final step of fecal coliform
verification. Add 37.0 g (Difco) to 1 L of laboratory pure water and mix to
dissolve. Dispense 10 mL into each 16 × 125 mm screw cap tube containing an
inverted fermentation vial and label racks with reagent name, preparation date,
expiration date and analyst’s initials. Autoclave at 121ºC for 15 min. Cool and
tighten caps. Store at room temperature away from direct sunlight for up to three
months. Final pH 6.9 ± 0.2. Positive, negative and sterility controls are incubated
for 24 h at 44.5 ± 0.2ºC. Record all preparation information in the
Equipment/Media Preparation Book.
6.8
Biological Indicator. Bacillus stearothermophilus spore suspension ampules. An
unused ampule serves as a positive control, labeled and placed in the water bath
on a monthly basis or sooner if a new lot is used. Store unused ampules at 2-8oC.
Procedure
7.1
currently using 0.5, 5.0, 20, and 50 mL aliquots to ensure that a range of 1-12,000
fecal coliform/100 mL may be calculated from marine receiving waters.
7.2
Label the bottom of each mFC plate with sample location and volume. Include a
blank with each sample. Stack the labeled dishes starting with the largest aliquot
and end with a sample blank on top. Cover plates until processing begins.
7.3
7.4
Aseptically transfer sterile phosphate buffered water to a sterile Guth bottle and
label with reagent name. Record the phosphate buffer identification information
in the laboratory data book.
7.5
the filter base (forceps tip should only touch outer rim of filter). Reseat and lock
on filtering funnel. Rinse the interior of the funnel thoroughly with phosphate
buffered water and draw a steady but continuous vacuum through the filter. Do
356-7
App 3.4 - 26
8.
7.6
grid-side up onto the mFC agar surface. Manipulate the filter to ensure air is not
trapped under the filter. This method is used for transferring blank and sample
membrane filters.
7.7
Shake each sample bottle vigorously prior to processing.
7.8
allows the analyst to examine the filter to ensure it is seated correctly and not
damaged.
7.9
Sample volumes of less than 10 mL should not be dispensed directly onto a filter.
Dispense sample into filter funnel containing a small amount of buffer (~10 mL)
7.10
excess moisture left on the filter. Thoroughly rinse down the filtering unit with at
least 30 mL of phosphate buffer and draw through. Remove the filter aseptically
and transfer grid side up onto the mFC agar. Continue to process sample aliquots
ending with the largest or least dilute sample volume.
7.11
filtering units are sanitized for immediate reuse only. Place them in a UV
7.12
After all samples are processed, separately filter positive (Escherichia coli) and
negative (Pseudomonas aeruginosa) controls. Incubate these plates with the
processed samples. Use the appropriate dose that will yield individual colonies
with characteristic colonial morphology on mFC agar.
7.13
Place the inverted plates containing filtered samples in waterproof bags. Label
the bags with date and time. Seal and submerge bags in a circulating water bath
at 44.5 ± 0.2ºC for 24 ± 2 h.
7.14
The sample plates must be incubated within 30 min of processing.
7.15
Duplicate analyses are included with each batch processed, and the routine
inshore, shoreline and manifold monitoring stations are scheduled on a monthly
basis at a frequency of at least 10% of sample type.
Counting Colonies
356-8
App 3.4 - 27
8.1
After 24 h incubation, starting with the blank plate, examine for contamination or
any notable changes on the filter or media. Examine and count plates set for each
sample, starting with the highest dilution or lowest volume filtered, i.e., blank,
10-1, 1 mL, etc.
8.2
Use a stereoscopic microscope (10× or 15×) with daylight fluorescent light source
to yield maximum visibility for identifying and counting fecal coliform colonies.
8.3
Remove bagged plates from the water bath. Review and record positive and
negative control reactions.
8.4
Fecal coliform bacteria are various shades of blue. As the fecal coliforms ferment
lactose and grow, they produce an acid, which reacts with the aniline dye in the
media to produce a blue color. Count colonies using magnification and a
fluorescent light source, record all colonies demonstrating this morphology.
Colonies exhibiting a gray coloration should not be counted as fecal coliforms.
Non fecal coliforms are gray to cream-colored, and atypical colonies are gray to
green-colored.
8.5
Individual colonies that have grown into each other can be discerned by a separate
nuclei or a fine line of contact. Irregularly shaped colonies may result from
turbidity or artifacts in the sample.
8.6
The use of a hand tally (counter) may aid in keeping track of higher counts, but it
is important to calibrate the tally regularly.
8.7
Record all counts and any notable information in the laboratory bench book.
Comments include unusual conditions on the filter such as the presence of solids,
artifacts, or high background counts. The condition of the growth on the filter
should also be noted. This may include confluent growth, confluent areas or the
presence of too many colonies to obtain an accurate reading. A record of positive
and negative controls must be associated with each set of samples.
8.8
All incubated media must be autoclaved prior to disposal. Liquid waste can be
dumped down the drain, and disposable materials can be discarded with the
laboratory trash.
8.9
Due to the possible inhibitory effect of colony crowding, and in order to
maximize the confidence level of a fecal coliform concentration, statistically
determined minimum and maximum ranges are used to calculate final results.
Sample aliquots producing counts within the ideal countable range of 20-60 fecal
coliform colonies must be used. If more than one aliquot yields a countable fecal
coliform range, then these results are calculated individually but reported as an
arithmetic mean. The number of fecal coliform colonies from a sample dilution or
volume can be used in the following equation to determine colony forming units
per 100 mL of original sample volume.
356-9
App 3.4 - 28
Fecal Coliform Colonies Counted × 100 = Fecal Coliform cfu/100 mL
8.10
If a countable range of 20-60 fecal coliform colonies is not obtained from any of
the sample aliquots tested, then total all fecal coliform counts on all filters and
report as number CFU per 100 mL.
8.10.1 If the colony count(s) on one or more aliquots is less than 20, add all
numbers and divide by total volume. Report as CFU per 100 mL. Filters
with inhibitory effects are reported as an estimated count and are excluded
from the calculation.
8.10.2 If no colonies are present on any plate, divide one by the sum of all
representative aliquots or dilutions (mL), multiply by 100 and report result
as less than that value per 100 mL.
8.10.3 If the number of colonies is greater than the countable range maximum of
8.10.4 Plates with a total bacteria count exceeding 200 colonies (fecal coliform
and non-coliform colonies) are recorded as TNTC (too numerous to count)
and not used to calculate a final concentration.
8.11
9.
Concentrations of indicator organisms, such as fecal coliform, are expressed as
colony forming unit (cfu) per 100 mL sample. The calculation is based on the
theory that one characteristic fecal coliform colony represents one fecal coliform
bacterium in the original sample.
Quality Assurance
9.1
Perform biochemical verification on 5% of each type of sample set per month for
typical fecal colonies. For atypical fecal coliform colonies, set verifications on at
least one sample per month. In order to evaluate false positive and false negative
trends adequately, atypical verifications may be substituted for typical
verifications as needed.
9.1.1
For each sample to be verified select colonies from the mFC plate(s) that
have been used to report the final result. Using sterilized needles or loops,
pick approximately 10 isolated typical fecal coliform colonies from one
quadrant of the plate(s) and inoculate into individual LTB tubes. Using
the single quadrant approach when selecting typical colonies for
356-10
App 3.4 - 29
verification improves the chance for randomizing colony selection.
Systematically add additional quadrants as necessary.
9.1.2
Include both positive and negative controls with each set of verifications.
Inoculate Escherichia coli (positive control) and Pseudomonas aeruginosa
(negative control) into LTB tubes and include along with test colonies.
Label rack with I.D. numbers, sample date, set date, location, control
information and analyst initials. Incubate at 35ºC for 24 h and examine
for gas production (a positive reaction). Reread at 48 h if the 24 h reading
is negative.
9.1.3
Record LTB results for each colony in the bacteriological data book. Any
comments or unusual results should be noted in the comment section.
9.1.4
Transfer growth from each positive LTB tube and the negative control to a
corresponding EC broth tube. Label rack with I.D. numbers, sample date,
set date, location, control information and analyst initials. Incubate in a
circulating waterbath at 44.5 ± 0.2ºC for 24 h. Gas production in EC tubes
verifies the presence of fecal coliform bacteria.
9.1.5
Record EC results for each colony in the in the bacteriological data book.
Any comments or unusual results should be noted in the comment section.
9.1.6
Autoclave all contaminated tubes, loops or other contaminated devices or
media prior to disposal.
9.1.7
A sample colony that is positive for LTB growth and positive for gas
production in EC tubes verifies that colony to be a fecal coliform.
Calculate the % verification for each sample.
9.1.8
Record all results in the bacteriological data book and on the Fecal
Coliform Verification Checklist.
9.1.9
A pattern of low percentage verifications may indicate a problem with the
membrane filtration procedure. Review initial sample data and notify the
supervisor if this occurs. Review trends or changes in verification rates.
9.1.10 Verifications are part of the JWPCP Microbiology Laboratory quality
assurance program. Percent verified results are not used to adjust
reporting results as stated in Standard Methods.
9.2
Duplicate analyses are included with each batch processed. To ensure all stations
are included, the routine inshore, shoreline and manifold monitoring stations are
scheduled on a monthly basis at a frequency of at least 10% of sample type. The
two results obtained (duplicate one and duplicate two) are entered into LIMS.
356-11
App 3.4 - 30
Report unusual results or out of control response sheets within 24 h to the
9.3
ensure that no carryover exists between subsequent filtering. An extra M-FC
plate is prepared with the sample plates and labeled. After the last aliquot of a
sample is filtered through, another membrane is placed on the holder and the
filtering unit is rinsed down with phosphate buffer. The filter is then rolled onto
the agar and incubated with the other samples. Colonies present after incubation
indicate that the analyst must improve the rinsing technique between aliquots.
9.4
Single and multiple analysts should count fecal coliform colonies from the same
9.5
Record purchased sterile equipment, reagent and media product information in the
Laboratory Supply Inventory Log. Information should include manufacturer,
lot number, expiration date, quantity, date received and date in-use, and additional
QA testing required or performed in-house.
9.6
When using a commercial dehydrated product for media preparation, always
follow manufacturer's directions for preparation, unless otherwise directed by the
SOP. No media is put into use prior to passage of all quality assurance testing.
9.7
New reagent lots recorded in the Supply Inventory notebook shall include
Manufacturer, Lot Number, Manufacturer’s Expiration Date, Received Date,
Opened Date and Analyst Initials. This is done primarily for Quality Assurance
tracking purposes.
9.8
Review JWPCP Microbiology Quality Assurance Guidelines for Standardization
of Accumet AB15 pH Meter.
9.9
preparation. Positive (Escherichia coli) and negative (Pseudomonas aeruginosa)
control cultures are set with each batch of media prepared.
9.10
Record reagent and batch media preparation information in the laboratory media
bench book.
9.11
Proper and consistent labeling of racks and tubes are essential for accurate data
reporting.
356-12
App 3.4 - 31
9.12
10.
Method Performance
10.1
11.
Participation in the Laboratory Quality Assurance Section monthly program for
quality assurance for total and fecal membrane filtration is required.
Fecal Coliform isolation by the Membrane Filtration Method.
References
11.1.
Standard Methods for the Examination of Water and Wastewater, Online
Editions, 9022 A, 9222 D.
11.2.
Microbiological Methods for Monitoring the Environment Water and Wastes, R.
Bordner and J. Winters, Eds. 1978. EPA 600/8-78-017.
11.3.
JWPCP Microbiology Quality Assurance Appendices, 2013. Los Angeles County
11.4.
Changes in Calculation and Reporting Procedures, Los Angeles County Sanitation
Districts, JWPCP Microbiology Internal Memo, January 8, 2004.
11.5.
Water Microbiology Laboratory Field Procedures, Millipore Corporation
Publication, Catalog # AD323, 1992.
356-13
App 3.4 - 32
Test Code
357
Method Name
Enterococcus Enumeration by Membrane Filtration
Version
13.1.0
Method Date
July 9, 2013
Reasons for
Method Revision
Standard Annual Revisions/Removal of the reference to the addition of
indoxyl B-D glucoside to the mEI agar
Revised by.
Susan Mizoguchi
Senior Laboratory Technician
JWPCP WQL
01,/Aijti
Date
Debra Leachman
Microbiologist II
JWPCP WQL
Date
Approved by:
Kathy Walker
Supervisor I
JWPCP WQL
Final Approval:
Jean Lee
Superintendent of
JWPCP Laboratory
it-
Signature 77
/ ) A
)&?_
"\
Date
^
App 3.4 - 33
0\-£x,
357. ENTEROCOCCUS ENUMERATION BY MEMBRANE FILTRATION
INTRODUCTION
The enterococcus indicator group is historically defined as a subgroup of the fecal streptococci
(members of the genus Streptococcus), which inhabit the GI tract of warm-blooded animals. The
enterococci are characterized as gram-positive ovoid bacteria that hydrolyze indoxyl ß-D
glucoside and grow at an elevated temperature of 41º C. Since this SOP reference is EPA-821R-06-009, Method 1600: Membrane Filter Test Method for Enterococci in Water, members of
the enterococcus group are referred to using the genus Enterococcus, i.e., Enterococcus faecalis,
Enterococcus faecium, Enterococcus gallinarum, and Enterococcus avium. Bergey's Manual
(1989) and ATCC have also reclassified the enterococcus group to the genus Enterococcus from
Streptococcus. Enterococci presence in recreational waters, both fresh and marine, have been
correlated to swimming-associated gastroenteritis and use of membrane filtration methods have
been approved for their recovery. The EPA method 1600 employs the selective medium, mEI
agar, for enumerating enterococci in 24 hours. Wastewater and marine and fresh receiving
waters may be analyzed for the enterococcus group using this method.
1.
2.
1.1.
This procedure is applicable for use with wastewater and fresh and marine
receiving waters.
1.2.
Solids and other background interference present in the sample must be
1.3.
Summary of Procedure
2.1.
2.2.
The filter is rolled onto an mEI agar plate and incubated at 41 ± 0.5ºC for 24 h.
2.3
The filter is examined under cool, white fluorescent light and magnification. All
colonies exhibiting any blue halo regardless of colony color are counted as
enterococci. Colonies must be greater than or equal to 0.5 mm in diameter,
independent of the blue halo.
2.4
Results are recorded as Enterococcus colony forming units (CFU) per 100 mL of
sample.
357-1
App 3.4 - 34
Version 13.1.0 July 09, 2013
3.
4.
3.1
the field that are not analyzed immediately must be iced or refrigerated at
temperature < 8°C during transit to the lab. The holding time for samples may
not exceed 6 h.
3.2
time of collection, electronically onto a PDA. When the analyst returns back to
the laboratory a hotsync is performed on the PDA, and an observation sheet(s) is
3.3
Once the samples arrive back at the laboratory, an analyst must measure and
record the temperature of a randomly selected sample, using a Raytek®
laser temperature probe or other similar external device.
3.4
3.5
Bottle size should allow for adequate sample volume with sufficient headspace
for thorough mixing. Appropriate labeling must include sample location,
date/time, and sampler’s initials.
3.6
Shore samples must be refrigerated and held until the final results are known.
3.7
If plant effluent has been chlorinated, then the addition of sodium thiosulfate to
the collection bottle is mandatory. A 0.1 mL dose of a 10% solution of sodium
thiosulfate will neutralize approximately 10-ppm residual chlorine. The use of
commercially prepared sterile Whirl-pak© bags with thiosulfate pills is especially
convenient. To ensure that no chlorine remains, a chlorine residual analysis
should be run on the neutralized sample prior to coliform analysis. If the original
chlorine level exceeds the neutralizing capacity of the sodium thiosulfate, then the
sample must be re-collected with enough sodium thiosulfate to completely
neutralize the chlorine. If high chlorine residual is suspected, a chlorine residual
determination should be made prior to the neutralization step.
Interferences
4.1
Reducing aliquot volumes or setting serial dilutions may reduce this interference.
357-1
App 3.4 - 35
Version 13.1.0 July 09, 2013
5.
6.
4.2
Samples with high background concentrations may overgrow enterococcus
organisms. Reducing aliquot volumes or setting serial dilutions may reduce this
interference.
4.3
Bacterial clumping may result in false low Enterococcus determinations. This
effect may be reduced by vigorous agitation of the sample prior to analysis.
Apparatus
5.1
Incubators capable of holding temperatures of 41 ± 0.5ºC, 35 ± 0.5ºC, and
45 ± 0.5ºC
5.2
Stereoscopic dissecting microscope (10× or 15×) and cool, white fluorescent lamp
5.3
Hand tally
5.4
Vacuum source
5.5
Vacuum filter flasks - safety coated (2 liter and 1 liter in sequence)
5.6
Sterilized filtration units - funnel and filter holder
5.7
UV sterilizer with short-wave (254 nm) germicidal lamp
5.8
Bunsen burner
5.9
Ethanol in wide mouth cup used for flaming forceps
5.10
5.11
5.12
5.13
Sterile disposable petri dishes, loose lids, 60 × 15 mm
5.14
Manifold used for multiple filtering units
5.15
Sterile membrane filters with grid, white, 47 mm diameter, 0.45 µm pore diameter
e.g., Millipore® type HA
5.16
Sterilized Guth dispensing bottle
Media/Reagents
357-1
App 3.4 - 36
Version 13.1.0 July 09, 2013
6.1
Methods performance specifications for “medium-quality” reagent water
(Standard Methods Online Editions 1080C, Table 1080:II). Additionally, it must
pass the Standard Methods performance specifications for water used in
microbiological testing (Standard Methods Online Editions 9020B.4.d, Table
9020:II).
6.2
Phosphate Buffered Solution. Sterile buffered water used to rinse down filtration
unit between subsequent aliquots of the same sample. Phosphate buffered
solution is also used for dilution blank preparation.
6.2.1
pH to 7.2 with 1 N NaOH and bring volume to 1 L. Label bottle with
information in the Equipment/Media Preparation Book. Store refrigerated
and handle aseptically. Discard if turbidity is present or after 3 months of
use.
6.2.2
(MgCl2·6H20) to 1 L laboratory pure water and mix to dissolve. Label
After completely cooling bottle to room temperature, aseptically inoculate
20 mL of magnesium chloride solution into a 100 mL TSB media bottle.
information in the Equipment/Media Preparation Book. Store refrigerated
and handle aseptically. Discard if turbidity is present or after 3 months of
use.
6.2.3
357-1
App 3.4 - 37
Version 13.1.0 July 09, 2013
equipment/media preparation book. Cool and tighten caps. Label and
incubate biological indicator at 58-62ºC for 24 and 48 h (following
6.2.4
buffered water into a 100 mL TSB media bottle. Also, check the sterility
solution into a 50 mL TSB media bottle. Incubate the TSB bottles at 35 ±
0.5ºC for 24 and 48 h. Record sterility information in the
Equipment/Media Preparation Book. If the TSB bottles become turbid,
6.2.5
Phosphate Buffered 9.0 mL Dilution Blanks. Add 1.25 mL stock
phosphate buffer solution and 5.0 mL magnesium chloride solution per
1 L laboratory pure water and mix thoroughly. Adjust pH with 0.1 N or
1.0 N NaOH to achieve a final pH of 7.2 ± 0.1. Record all preparation
information in Equipment/Media Preparation Book. Dispense into
calibrated 9.0 mL test tubes and label racks with reagent name,
preparation date, expiration date and analyst initials. Attach autoclave
tape to each rack and place a biological indicator to one of the racks,
autoclave at 121ºC for 15 min. Record all autoclave information in
equipment/media preparation book. Once phosphate buffered blanks have
cooled to room temperature take the final pH using several phosphate
buffer blanks. Check the tubes to ensure each tube is 9.0 ± 0.2 mL.
6.2.6
select 2 tubes from the batch and aseptically pour into a 50 mL TSB media
bottle. Incubate the TSB bottle at 35 ± 0.5ºC for 24 and 48 h. Record
sterility information in the Equipment/Media Preparation Book. If the
TSB bottle becomes turbid, check autoclave records and biological
problem to be autoclave related. If everything is normal, retest another 2
tubes from the batch in TSB. If the TSB is negative, keep the entire batch.
If the TSB bottle turns positive, discard the entire batch. Store phosphate
buffer blanks away from direct sunlight and discard if turbidity develops
before the expiration date of 3 months for screw cap and 2 weeks for slip
cap tubes.
357-1
App 3.4 - 38
Version 13.1.0 July 09, 2013
6.3
mEI Agar: Plate media used to isolate and differentiate enterococci from water by
membrane filtration. Add 72 g of dehydrated mEI agar (Difco) to 1 L of
laboratory pure water and mix to dissolve. Continue mixing and bring to a boil.
Autoclave at 121ºC for 15 min. Remove and cool to 55ºC. Add 0.24 g of
nalidixic acid sodium salt (C12H11N2NaO3) and 0.02 g dehydrated 2,3,5-triphenyltetrazolium chloride (Sigma) to the mEI medium and mix to dissolve completely
(consult the MSDS for the proper handling of nalidixic acid). Dispense 4-5 mL
into 60 × 15 mm sterile petri dishes and cool. Final pH 7.1 ± 0.2. Set positive
and negative controls and a minimum of 3 sterility plates for each batch of media
prepared and incubate at 41ºC ± 0.5ºC for 24 h. Re-bag prepared plates, label and
store refrigerated for up to 2 weeks. NOTE: Effective August 1, 2013, the
addition of indoxyl ß-D glucoside to the mEI agar will no longer be required.
Refer to the “Media Comparison for Enterococcus Analysis by EPA Method
1600” (DM # 2712601) for a detailed explanation.
6.4
Brain Heart Infusion Agar. A complete medium for cultivating a variety of
fastidious microorganisms and used in the verification procedure for enterococci.
Add 52 g of dehydrated BHI agar (Difco) to 1 L laboratory pure water and mix to
dissolve. Continue mixing and bring to a boil. Autoclave at 121ºC for 15 min.
Remove and cool to 50ºC. Dispense 15 mL into 100 × 15 mm sterile petri dishes
and cool. Final pH 7.4 ± 0.2. Set positive and negative controls and a sterility
plate for each batch of media prepared and incubate at 35ºC ± 0.5ºC for 48 h. Rebag prepared plates, label and store refrigerated for up to 2 weeks.
6.5
Brain Heart Infusion Broth. A highly nutritive broth medium used for cultivating
a variety of fastidious microorganisms including Streptococcus and used in the
verification procedure for enterococci. Add 37 g of dehydrated BHI (Difco) to
1 L laboratory pure water and mix to dissolve. Dispense 3 mL into 13 × 100 mm
screw cap tubes and cap loosely. Autoclave at 121ºC for 15 min. Remove, cool
and tighten caps. Final pH 7.4 ± 0.2. Set positive and negative controls and a
sterility check for each batch prepared and incubate at 35ºC ± 0.5ºC for 24 h and
45ºC ± 0.5ºC for 48 h. Store away from sunlight at 25ºC for up to 3 months.
6.6
Brain Heart Infusion Broth with 6.5 % NaCl. A highly selective broth medium
used in the verification procedure for enterococci. Add 37 g of dehydrated BHI
(Difco) and 60 g of sodium chloride (NaCl) to 1 L of laboratory pure water to
achieve a final concentration of 6.5% NaCl. In this example, Difco BHI is preformulated with 5 g NaCl per Liter. Mix to dissolve. Dispense 3 mL into
16 × 100 mm screw cap tubes and cap loosely. Autoclave at 121ºC for 15 min.
Remove, cool and tighten caps. Final pH 7.4 ± 0.2. Set positive and negative
controls and a sterility check for each batch prepared and incubate at 35º C ±
0.5ºC for 48 h. Store away from sunlight at 25ºC for up to 3 months.
6.7
Bile Esculin Agar. A differential medium used in the verification procedure for
enterococci for the isolation and presumptive identification of Group D
357-1
App 3.4 - 39
Version 13.1.0 July 09, 2013
streptococci. Add 43.5 g of dehydrated BEA (Difco) to 1 L laboratory pure water
and mix to dissolve. Continue mixing and bring to a boil. Cool to 50ºC and
dispense 5 mL into 16 × 125 mm screw cap tubes. Autoclave at 121ºC for 15
min. After autoclaving, tilt racks so that the agar forms a slant length of
approximately 6.3 cm., cool to solidify and tighten caps. Final pH 6.8 ± 0.2. Set
positive and negative controls and a sterility check for each batch prepared and
incubate at 35ºC ± 0.5ºC for 48 h. Store refrigerated for up to 3 months.
7.
Procedure
7.1
currently using 0.5, 5.0, 20, and 50 mL aliquots to insure that a range of 1-12,000
Enterococcus/100 mL may be calculated.
7.2
Label the bottom of each mEI plate with the sample location and volume. Stack
the labeled dishes starting with the largest aliquot and end with a sample blank on
top.
7.3
7.4
Aseptically transfer the sterile buffered water to a sterile Guth bottle. Record the
phosphate buffer identification information in the laboratory bench book.
7.5
the filter base (forceps tip should only touch outer rim of filter). Reseat and lockon filtering funnel. Rinse the interior of the funnel thoroughly with phosphate
buffered water, and draw a steady but continuous vacuum through the filter. Do
7.6
grid-side up onto the mEI agar surface. Manipulate the filter to ensure air is not
trapped under the filter. This method is used for transferring blank and sample
membrane filters.
7.7
Shake each sample bottle vigorously prior to processing.
7.8
allows the analyst to examine the filter to insure it is seated correctly and not
damaged.
7.9
Sample volumes of less than 10 mL are not dispensed directly onto a filter.
Dispense sample into filter funnel, containing a small amount of buffer (~10 mL)
357-1
App 3.4 - 40
Version 13.1.0 July 09, 2013
7.10
excess moisture left on the filter. Thoroughly rinse down the filtering funnels
with at least 30 mL of phosphate buffer and draw through. Remove the filter
aseptically and transfer grid side up onto the mEI agar surface. Continue to
process sample aliquots ending with the largest or least dilute sample volume.
7.11
filtering units are sterilized for immediate reuse only. Place them in a UV
7.12
After all the samples are processed separately filter positive (Enterococcus
faecalis) and negative (Pseudomonas aeruginosa) controls. Incubate these
controls with the processed samples. Use the appropriate dose that will yield
individual colonies with characteristic colonial morphology on mEI agar.
7.13
Place the inverted plates containing filtered samples in a covered tray containing a
wet sponge or paper towel to maintain a humid environment. Label the container
with test name, date and time. Incubate at 41 ± 0.5ºC for 24 ± 2h.
8. Calculations
8.1
Counting Colonies
8.1.1
After 24 h incubation, starting with the blank plate, examine for
contamination or any notable changes on the filter or media. Examine and
count plates set for each sample, starting with the highest dilution or
lowest volume filtered, i.e., blank, 10-1, 1 mL, etc.
8.1.2
Use of a stereoscopic microscope (10×-15×) with a cool, white fluorescent
light source will yield maximum visibility in identifying and counting
enterococci colonies.
8.1.3
An Enterococcus colony is characterized as any colony surrounded by a
blue halo, regardless of colony color. The blue halo around the colony is
due to the ability of Enterococcus to cleat the chromogenic substrate
indoxyl ß-D glucoside present in the mEI medium. Colonies must be
greater than or equal to 0.5 mm in diameter, independent of the blue halo.
Count colonies using magnification and a fluorescent light source, record
all colonies demonstrating this morphology.
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App 3.4 - 41
Version 13.1.0 July 09, 2013
8.2
8.1.4
A separate nuclei or a fine line of contact can discern individual colonies
that have grown into each other. Irregularly shaped colonies may result
from turbidity or artifacts in the sample.
8.1.5
The use of a hand tally (counter) may aid in keeping track of higher
counts; however, the tally must be calibrated regularly.
8.1.6
Record all counts and any notable information in the laboratory bench
book. Comments should include unusual conditions on the filter such as
the presence of solids, artifacts, or high background counts. The condition
of the growth on the filter should also be noted. This may include
confluent growth, confluent areas, or the presence of too many colonies to
obtain an accurate reading (> 60 colony forming units). A record of
positive and negative controls must be associated with each set of samples.
Calculations
8.2.1
Due to the possible inhibitory effect of colony crowding (and in order to
maximize the confidence level of an enterococcus concentrations),
statistically determined minimum and maximum ranges are used to
calculate the final results. Sample aliquots producing counts within the
ideal countable range of 20-60 enterococci colonies must be used. The
number of enterococci colonies from a sample dilution or volume can be
used in the following equation to determine colony-forming units (CFU)
per 100 mL of original sample volume.
Enterococci Colonies Counted × 100 = Enterococcus cfu/100 mL
8.2.2
If the colony count(s) on one or more aliquots is less than 20, add all
numbers and divide by total volume, excluding plates with possible
inhibitory effects. Such a result is noted as an estimated count.
8.2.3
If no colonies are present on any plate, divide one by the sum of all
representative aliquots or dilutions (mL); multiply by 100 and report result
as less than that value.
8.2.4
If the number of colonies is greater than the countable range maximum of
357-1
App 3.4 - 42
Version 13.1.0 July 09, 2013
8.3
9
8.2.5
Plates with a total bacteria count exceeding 200 colonies (enterococci and
non enterococci colonies) are recorded as TNTC (too numerous to count)
and not used to calculate a final concentration.
8.2.6
Concentrations of indicator organisms, such as enterococci, are expressed
as colony-forming unit (CFU) per 100 mL of sample. The calculation is
based on the theory that one characteristic enterococcus colony represents
one enterococcus bacterium in the original sample. Refer to EPA Method
1600 (EPA-821-R-06-009), Appendix B on General Operations, Counting
Colonies and Calculation of Results.
For QA purposes, a sampling location may require duplicate sampling and
analysis. This requires a split analysis of a single sample. A separate result is
calculated from each split analysis and the geometric mean is calculated. The
single geometric mean result is used for reporting.
Quality Assurance
9.1
Verifications
9.1.1
Perform biochemical verifications on 5% of each type of sample set per
month.
9.1.2
Select well-isolated “typical” colonies (preferably 10 colonies) from the
membrane filter on the mEI agar. The inclusion of colonies possessing
marginally typical traits in the routine verification process will improve
analyst confidence and ability.
9.1.3
Using a sterile inoculating loop, transfer cells that represent a well-isolated
colony to a BHI agar plate. Incubate the plate for 24 ± 2 and 48 ± 3 h at
35 ± 0.5ºC.
9.1.4
Prepare a Gram stain using growth from the BHI agar plate and read under
1000× magnification with immersion oil. Fecal streptococci (enterococci)
are Gram-positive, ovoid cocci (0.5 to 1.0 µm) occurring in pairs and short
chains.
9.1.5
Select a well-isolated colony from the agar plate and transfer to a glass
slide. Aseptically place a drop of hydrogen peroxide on the colony and
look for oxidation (“bubbling”). Fecal streptococci (enterococci) are
negative for the catalase reaction.
9.1.6
Transfer growth from plates exhibiting fecal streptococci (enterococci)
characteristics (catalase negative, Gram-positive, ovoid cocci) into a BHI
broth tube. Incubate the tube for 24 ± 2 h at 35 ± 0.5ºC.
357-1
App 3.4 - 43
Version 13.1.0 July 09, 2013
9.1.7
Transfer a loopful of growth from the BHI broth to each of the following
media: bile esculin agar (incubate at 35 ± 0.5ºC for 48 ± 3 h); brain-heart
infusion broth (incubate at 45 ± 0.5ºC for 48 ± 3 h); and brain-heart
infusion broth with 6.5% NaCl (incubate at 35 ± 0.5ºC for 48 ± 3 h).
9.1.8
Gram-positive cocci that grow in BHI broth at 45ºC with a positive result
on BEA at 35ºC verifies that the colony is a fecal streptococci. A positive
result for a culture grown on BEA is evidenced by the production of a
black-brown precipitate. Growth in BHI broth at 45ºC and in BHI broth
with 6.5% NaCl at 35ºC confirms that the colony belongs to the
enterococcus group.
9.1.9
A positive (Enterococcus faecalis) and negative (Psuedomonas
aeruginosa) control culture is set with each sample verification.
9.1.10 Results and the percent verification rate are recorded in the bacteriological
laboratory data notebook.
9.1.11 Review trends or changes in verification rates.
9.1.12 Because verifications are part of the JWPCP Microbiology Laboratory
quality assurance program and not applied to an entire sample count,
percent verified results are not used to adjust reported results.
9.2
9.3
Media preparation
9.2.1
preparation. Positive (Enterococcus faecalis) and negative (Pseudomonas
aeruginosa or Escherichia coli) control cultures are set with each batch of
media prepared.
9.2.2
When using a commercial dehydrated product for media preparation,
follow manufacturer’s directions for preparation, unless directed by the
SOP. No media is put into use prior to the passage of all quality assurance
testing.
9.2.3
Review JWPCP Microbiology Quality Assurance Guidelines for
Standardization of Accumet AB15 pH Meter.
9.2.4
Record reagent and batch media preparation information in the laboratory
media bench book.
insure that no carryover of bacteria exists between individual sample analyses.
An extra mEI plate is included with the sample plates and labeled. After the last
357-1
App 3.4 - 44
Version 13.1.0 July 09, 2013
aliquot of a sample is filtered through, another membrane filter is place onto the
holder and the filtering unit is rinsed down with phosphate buffer. This filter is
then rolled onto the extra mEI agar plate and incubated with the other sample
plates. Colonies present after incubation indicate that the analyst must improve
the rinsing technique between aliquots. Record the results in the JWPCP
Bacteriological Bench Book.
10.
9.4
Single and multiple analysts should count enterococcus colonies from the same
9.5
Duplicate analyses are to be included with each batch processed. To ensure all
stations are included, the routine inshore, shoreline and manifold monitoring
stations are scheduled on a monthly basis at a frequency of at least 10% of sample
type. Refer to QA Appendices for Duplicate sample analyses. The two results
obtained (duplicate one and duplicate two) are entered into the LIMS.
9.6
Report unusual results, or out-of-control response sheets, within 24 h, to the
Method Performance
10.1
11.
the isolation of Enterococcus by the Membrane Filtration Method.
References
11.1
Method 1600: Enterococci inWater by Membrane Filtration Using membrane
Enterococus Indoxyl-B-D-Glucoside Agar (mEI) Membrane Filter Test Method
for Enterococci in Water, A. Dufour and W. Messer, Eds. July 2006. EPA-821R-06-009.
11.2
Microbiological Methods for Monitoring the Environment - Water and Wastes, R.
Bordner and J. Winters, Eds. 1978. EPA 600/8-78-017.
11.3
Standard Methods for the Examination of Water and Wastewater, Online Edition,
Methods 1000, 9000.
11.4
Comparison of Current Enterococcus 48h Method 9230C with 24h Method 1600.
Internal Report, JWPCP Microbiology Laboratory, 2002. Los Angeles County
Sanitation Districts.
357-1
App 3.4 - 45
Version 13.1.0 July 09, 2013
11.5
JWPCP Microbiology Quality Assurance Appendices. JWPCP Microbiology
Laboratory, 2013. Los Angeles County Sanitation Districts
11.6
Changes in Calculation and Reporting Procedures. JWPCP Microbiology Internal
Memo, 2004. Los Angeles County Sanitation Districts.
357-1
App 3.4 - 46
Version 13.1.0 July 09, 2013
Test Code
351
Method Name
Fecal Coliform Enumeration by Multiple Tube Fermentation
Version
13.1.0
Method Date
September 10, 2013
Reasons for
Method Revision
Revised by.
Mark Patterson
JWPCP WQL
Signature
Debra Leachman
Microbiologist II
JWPCP WQL
O
? CriJ/ /
Date/
CA
/W
3>
IV2o ( <£>
Date
Approved by:
Kathy Walker
Supervisor I
JWPCP WQL
Signature
Date
Signature
Date
Final Approval:
Jean Lee
Superintendent of
JWPCP Laboratory
App 3.4 - 47
351. FECAL COLIFORM ENUMERATION BY MULTIPLE TUBE FERMENTATION
INTRODUCTION
The fecal coliform is a subgroup of the total coliform indicator group of bacteria. Separation
into this subgroup is based upon the fecal coliform’s ability to ferment lactose at an elevated
temperature of 44.5ºC, as well as at 35ºC, the optimum growth temperature for total coliform.
Members of the fecal coliform are found in the feces of various warm-blooded animals, and have
been used as a more definitive indicator for recent fecal pollution. The fecal coliform subgroup
defines primarily Escherichia coli, and occasionally thermotolerant Klebsiella sp. Receiving
waters and wastewaters may be monitored using the multiple tube fermentation method. This
method employs the 5-tube fermentation setup. A mathematically derived index based on the
number and combination of positive and negative tubes per dilution is reported as the Most
Probable Number (MPN)/100 mL or MPN/g dry wt.
1.
2.
3.
1.1.
This procedure is applicable for use with wastewater, seawater, and receiving
waters.
1.2.
Results ranging from 1 to 9 are reported with 1 significant figure and results 10 or
greater are reported to 2 significant figures.
1.3
A fecal coliform presumptive, confirmed, or completed Most Probable Number
index may be calculated from a 5-tube MPN table (Standard Methods Online). This
MPN table is used to determine, within 95% confidence limits, an MPN index for a
combination of positive results from 5 plants of a 3 decimal dilution series.
Summary of Procedure
2.1.
A preliminary 5-tube MPN setup of Lauryl Tryptose Broth (LTB) is inoculated
and incubated at 35ºC for 24 and 48 h to determine total coliform density.
2.2.
The result of each LTB tube is recorded. Each positive LTB is transferred to
E. coli (EC) broth for incubation at 44.5ºC for 24 h.
2.3.
Results are recorded. Fecal coliform densities are determined from the MPN
tables provided by Standard Methods Online on the basis of positive EC tubes.
App 3.4 - 48
4.
3.1.
Start bacteriological examination of water samples promptly after collection.
Collected samples that are not analyzed immediately must be iced or refrigerated
at < 8 ºC. The holding time for samples may not exceed 6 h.
3.2.
Collect samples for bacteriological examination in polypropylene bottles that
have been thoroughly cleaned, rinsed and sterilized. Prior to use, sample bottles
must pass a 48 h tryptic soy broth sterility check. Allow bottle size for an
adequate sample with sufficient headspace for thorough mixing. Label with
sample description, date, time, and sampler’s initials.
3.3
If there is a possibility that the sample has residual chlorine, the sample container
must already contain a reducing agent. A 0.1 mL aliquot of a 10% solution of
sodium thiosulfate, Na2S2O3, will neutralize approximately 10 ppm of residual
chlorine in the sample. The use of commercially pre-sterilized thiosulfate sample
bags is especially convenient (i.e., Nasco Whirl-pak®). To ensure that chlorine
neutralization is achieved, a chlorine residual analysis should be performed on the
sample prior to continuing total coliform procedure.
Interferences
4.1
5.
Background organisms or toxic constituents in 10 mL volumes of sample may
inhibit fecal coliform growth. Smaller aliquots or serial dilutions may reduce the
toxicity to the point of no effect.
Apparatus
5.1.
Culture tubes, 16 × 150 mm and 18 × 150 mm, (Kimble® or Pyrex®) or similar
disposable borosilicate tubes and slip caps
5.2.
Disposable culture tubes, 16 × 125 mm, and screw caps
5.3.
Test tube racks to hold 16 mm and 18 mm OD tubes
5.4.
Shell vials, ¼" diameter, or Durham tubes
5.5.
Sterile disposable cotton plugged serological pipettes, 10 mL, wide tip
5.6.
Pipette bulb or pipette dispenser
5.7.
Sterile plastic disposable loop or nichrome wire loop
5.8.
Sterile cotton tip applicators
5.9.
Bunsen burner
App 3.4 - 49
6.
5.10.
Incubator, capable of holding temperature of 35 ± 0.5ºC
5.11.
Circulating water bath capable of maintaining 44.5 ± 0.2ºC
Media/Reagents
6.1
reagent handling.
6.2
6.3
Methods performance specifications for “medium-quality” reagent water (SM
21st Edition Online 1080C, Table 1080:II). Additionally, it must pass the
Standard Methods performance specifications for water used in microbiological
testing (SM 21st Edition Online (2006) 9020B.4.d, Table 9020:II).
6.4
Phosphate Buffered Solution. Sterile buffered solution used for dilution blank
preparation.
6.4.1
reagent name, manufacturer’s name, lot number, preparation date, and
6.4.1.1
Magnesium Chloride Solution. Add 81.1 g magnesium
chloride MgCl2·6H2O to 1 L laboratory pure water and mix to
dissolve. Label the bottle with reagent name, manufacturer’s
name, lot number, preparation date, expiration date and analyst
initials. Autoclave at 121ºC for 15 min. After completely
cooling bottle to room temperature, aseptically inoculate 20
mL of magnesium chloride solution into a 100 mL TSB media
bottle. Incubate the TSB bottle at 35 ± 0.5ºC for 24 and 48 h.
App 3.4 - 50
Record sterility information in the Equipment/Media
Preparation Book. Discard solution if the TSB bottle becomes
turbid. Store refrigerated and handle aseptically. Discard if
turbidity is present or after 3 months of use.
6.4.1.2
Working Phosphate Buffered Solution. Add 50 mL
magnesium chloride solution to 10 L of laboratory pure water
in a carboy and mix thoroughly. Dispense the laboratory pure
water containing magnesium chloride into 1 L or 2 L reagent
bottles and cap loosely. Suspend a biological indicator in a
separate 2 L bottle filled with laboratory pure water. This
bottle is designated exclusively for quality control use and
should be rotated to different areas in the autoclave with each
batch. The label information should include the reagent name,
preparation date, expiration date, analyst initials and bottle
number, which identify the bottle position in the autoclave.
Cover each bottle cap with foil, label and autoclave tape.
the Equipment/Media Preparation Book. Cool and tighten
caps. Label and incubate biological indicator at 58-62ºC for 24
and 48 h (follow manufacturer’s directions). After completely
cooling bottles to room temperature, aseptically add 1.25 mL
stock phosphate buffer solution per 1 L sterile laboratory pure
water containing magnesium chloride. Mix by inverting
bottles several times. Record all preparation information in
6.4.1.3
Each batch of phosphate buffer is tested for sterility.
Randomly select one bottle from the batch and aseptically
inoculate 20 mL of phosphate buffered water into a 100 mL
TSB media bottle. Also check the sterility of the stock
phosphate solution by aseptically inoculating 10 mL of solution
into a 50 mL TSB media bottle. Incubate the inoculated TSB
bottles at 35 ± 0.5ºC for 24 and 48 h. Record sterility
information in the Equipment/Media Preparation Book. If the
TSB bottle becomes turbid, check biological indicator result,
autoclave records and TSB batch QA. Discard the entire batch.
Discard stock phosphate solution if it fails QA. Store working
phosphate buffer away from direct sunlight for up to three
6.4.1.4
Phosphate Buffer 9.0 mL Dilution Blanks: Add 1.25 mL stock
phosphate buffer solution and 5.0 mL magnesium chloride
solution per 1 L laboratory pure water and mix thoroughly.
Adjust pH with 0.1N or 1.0N NaOH to achieve a final pH of
7.2 ± 0.1. Record all preparation information in the
App 3.4 - 51
Equipment/Media Preparation Book. Dispense into calibrated
9.0 mL test tubes and label racks with reagent name,
preparation date, expiration date and analyst initials. Attach
autoclave tape to each rack and place a biological indicator to
one of the racks, autoclave at 121ºC for 15 min. Record all
autoclave information in Equipment/Media Preparation Book.
Once phosphate buffered blanks have cooled to room
temperature take the final pH using several phosphate buffer
blanks. Visually check each tube’s calibration mark to ensure
each tube has 9.0 ± 0.2 mL, and tighten caps.
6.4.1.5
Each batch of phosphate buffer blanks is tested for sterility.
Randomly select 2 tubes from the batch and aseptically pour
into a 100 mL TSB media bottle. Incubate the TSB bottle at 35
± 0.5ºC for 24 and 48 h. Record sterility information in the
Equipment/Media Preparation Book. If the TSB bottle
becomes turbid, check autoclave records and biological
indicator result. Discard the entire batch if quality assurance
tests indicate problem to be autoclave related. If everything is
normal, retest another 2 tubes from the batch in TSB. If the
TSB is negative, keep the entire batch. If the TSB bottle turns
positive, discard the entire batch. Store phosphate buffer
blanks away from direct sunlight and discard if turbidity
develops before the expiration date of 3 months for screw cap
and 2 weeks for slip cap tubes.
6.5
Lauryl Tryptose Broth. Presumptive test media for the total coliform group. Add
35.6 g Lauryl Tryptose (Difco) to 1 L laboratory pure water and mix to dissolve.
Dispense 10 mL volumes into 16 x 150 culture tubes containing an inverted gas
collection vial; cap tubes. Autoclave at 121ºC for 15 min. Final pH is 6.8 ± 0.2.
Prepare positive and negative controls as well as a minimum of 3 sterility controls
or 1% of the tubes set for each batch of media and incubate for 24 h at 35 ± 0.5ºC.
Record all preparation information in the Equipment/Media Preparation Book.
The holding time is 2 weeks for slip cap tubes and 3 months for screw cap tubes
at room temperature.
6.6
Double Strength Lauryl Tryptose Broth. Double strength presumptive test media
used for the 10 mL sample inoculum. Add 71.2 g of Lauryl Tryptose (Difco) to 1
L laboratory pure water and mix to dissolve. Dispense 10 mL volumes into 18 x
150 culture tubes containing an inverted gas collection vial; cap tubes. Autoclave
at 121ºC for 15 min. Final pH is 6.8 ± 0.2. Prepare positive and negative controls
as well as a minimum of 3 sterility controls or 1% of the tubes set for each batch
of media and incubate for 24 h at 35 ± 0.5ºC. Record all preparation information
in the Equipment/Media Preparation Book. The holding time is 2 weeks for slip
cap tubes and 3 months for screw cap tubes at room temperature.
App 3.4 - 52
6.7
7.
EC Broth, confirmatory test transfer medium for fecal coliforms. Add 37.0 g of
EC media (Difco) to 1 L laboratory pure water and mix to dissolve. Dispense 10
mL volumes into 16 x 150 culture tubes containing an inverted gas collection vial;
cap tubes. Autoclave at 121ºC for 15 min. Final pH is 6.9 ± 0.2. Prepare positive
and negative controls, as well as a minimum of 3 sterility controls (or 1% ) for
each batch of media and incubate for 24 h at 35 ± 0.5ºC. Record all preparation
information in the Equipment/Media Preparation Book. The holding time is 2
weeks for slip cap tubes and 3 months for screw cap tubes at room temperature.
Procedure
7.1
Homogenation of receiving and wastewater samples: Collect sample in a sterile
container, leaving enough headspace for adequate mixing. Shake sample
vigorously (at least 25 times). Proceed to section 7.3 for inoculation of sample
dilutions.
7.2
Homogenation of biosolids, sludge, sediment, and slurry samples: Collect sample
in a sterile container. Homogenization procedures are based on whether the
sample is a liquid or a solid (liquid samples are generally defined as samples
containing 7% or less total solids/dry weight):
7.2.1
Liquid sample. Using a sterile graduated cylinder, measure 300 mL of
sample. Transfer to a sterile blender and blend on high speed
(approximately 50 to 60 volts) for one to two minutes. Measure the pH of
the homogenized sample using a pH indicator strip (pH range 5-10). If
necessary, adjust the pH to 7.0-7.5 by adding 1.0 N hydrochloric acid
(HCl) or 1.0 N sodium hydroxide (NaOH). Record any adjustments in the
biosolids bench book. NOTE: When adjusting the pH, do not exceed the
homogenized sample volume by greater than 5% (15 mL).
7.2.2
Solid sample. Weigh 30.0 ± 0.1 g of well-mixed sample in a sterile dish.
Whenever possible, the sample tested should contain all the material that
will be included in the biosolids. For example, if wood chips are part of
the biosolids compost, some mixing or grinding may be needed to achieve
homogeneity before testing. Large pieces of wood that are not easily
ground may be discarded prior to homogenizing. Transfer the sample to a
sterile blender. Make a 1:10 dilution by adding 270 ml sterile phosphate
buffer to the blender. Cover and blend on high speed (approximately 50 to
60 volts) for one to two minutes. This is considered the homogenized
sample. If necessary, adjust the pH to 7.0-7.5 by adding 1.0 N
hydrochloric acid (HCl) or 1.0 N sodium hydroxide (NaOH). Record any
adjustments made in the biosolids bench book. NOTE: When adjusting
the pH, do not exceed the homogenized sample volume by greater than
5% (15 mL).
App 3.4 - 53
8
7.3
Set up a 5-tube MPN rack (NOTE: For samples containing greater than 7 % total
solids, please refer to EPA Method 1680: Fecal Coliforms in Biosolids by
Multiple Tube Fermentation Procedure for dilution information). This is achieved
by placing 5 LTB tubes per dilution row in a rack that is labeled with the
appropriate sample identification and date. Using a sterile wide mouth pipette,
dispense 10 mL volumes into the first row of concentrated (2X) LTB. Dispense 1
mL volumes of the sample and 1 mL of a 1:10, 1:100, and 1:1000 sample
dilutions into single strength LTB. The series of dilutions may be carried out
further if necessary. Incubate inoculated tubes at 35 ± 5ºC for 24 ± 2h.
7.4
After the initial 24 ± 2 h incubation, shake the rack and examine each tube for gas
production. Any gas bubble with turbidity present is considered positive evidence
for the presence of coliform. Record presumptive results. Place EC medium
tubes into a plastic rack that is labeled with appropriate sample information.
Transfer an inoculum from the positive LTB tubes to EC medium by using a
sterile loop or cotton swab. Reincubate all negative LTB tubes for an additional
24 h at 35 ± 5ºC for 24 ± 2h and incubate the inoculated EC tubes at 44.5 ± 0.2ºC
for 24 ± 2 h.
7.5
After 24 h incubation, gently shake the racks of LTB and EC tubes. Examine
both sets of tubes for the presence of growth and gas. Record the results. Gas
formation in the EC tubes constitutes a positive confirmatory test for the presence
of fecal coliform. Transfer positive 48 h LTB tubes to a properly labeled rack of
EC tubes and incubate for 24 h. LTB tubes that are negative after 48 ± 3h are
discarded.
7.6
Record results and obtain an MPN value based on the confirmatory EC results.
Calculations
8.1
Record fecal coliform concentration as MPN/100 mL for liquid samples or MPN/g
dry wt. for sample with > 7% solids (NOTE: Please refer to Standard Method 2540 G
for determining percent total solids. The percent total solids will be used to
determine MPN/g dry wt.). MPN values for a variety of positive and negative tube
combinations are given in Standard Methods Online 9221.C., table 9221: IV. When
more than three dilutions are used, select the highest dilution that gives positive
results in all five tubes tested and the two next succeeding dilutions.
8.2
For combinations not appearing in the MPN index, use the MPN calculator developed
by Albert Klee.
8.3
The distribution of positive and negative culture tubes should follow a logical pattern.
Repeated occurrence of illogical patterns should be reviewed and reported
immediately.
App 3.4 - 54
8.4
9
All samples and results from the analyses should be entered into the LIMS.
Quality Assurance
9.1
Test one pure culture of known positive reaction with each sample set.
9.2
Duplicate analyses should be performed on 10% of all samples, or at least once for
every batch of samples, whichever is more frequent. Calculate the logarithmic
average of both samples and report the average in the LIMS.
9.3
Review JWPCP Microbiology Quality Assurance Guidelines for media preparation.
9.4
Follow aseptic procedures for sample collection. Refer to a standard reference such
as Standard Methods or the EPA manual, Microbiological Methods for Water and
Wastewater for detailed sampling instructions.
If fermentation media are stored in a refrigerator, bring the media to room
temperature before use. Media with gas collected in the fermentation vials must be
discarded to eliminate false positive results.
9.5
9.6
All data results and quality assurance information must be recorded in a bound bench
book.
10 Method Performance
10.1
Fecal Coliform isolation by the Multiple Tube Fermentation method.
11 References
11.1
Standard Methods for the Examination of Water and Wastewater, Online Edition,
Methods 9221 A, 9221 C, 9221 E.
11.2
Microbiological Methods for Monitoring the Environment, Water and Wastes, R.
Bordner and J. Winters, Eds. 1978. EPA-600/8-78-017
11.3
Difco Manual, Difco Laboratories, 1984. .
11.4
JWPCP Microbiology Quality Assurance Guidelines, 2013. Los Angeles County
11.5
Method 1680: Fecal Coliforms in Sewage Sludge (Biosolids) by Multiple-Tube
Fermentation using Lauryl Tryptose Broth (LTB) and EC Medium. U.S.
Environmental Protection Agency, July 2006.
App 3.4 - 55
11.6
A computer program for the determination of the most probable number and its
confidence limits. A. J. Klee 1993. Journal of Microbiological Methods. 18:91-98.
App 3.4 - 56
Appendix 3.5
Samples
Appendix 3.5
Samples
Figure 3.5.1 shows the effects of rainfall on the average levels of fecal indicator bacteria (FIB) at
shoreline and inshore sites, respectively. All 1972-2015 FIB data were used in preparation of this figure.
Figure 3.5.1 Shoreline and Inshore FIB Response to Rain
Average concentrations of total coliform, fecal coliform and enterococcus FIB at shoreline and inshore. Prepared
using all 1972-2015 data separated into periods of rain (defined as days with >0.1 inch of rain), 1-day post-rain, 2day post-rain, 3-day post-rain, trace rain, no rain, and all days.
App 3.5 - 1
The “rain day” average levels were calculated using FIB sampling results from all shoreline sites on days
when there was 0.1 inch or more of rain recorded at either the Sanitation Districts’ rain gauge at White Point, or,
prior to 1991, from the L.A. Civic Center rain gauge. The long time frame provides a large sample size (shoreline
rain day sample size for total coliform (TC) = 4,821, fecal coliform (FC) = 701, and enterococcus (ENT) = 1,167,
nearshore rain day sample size TC = 1,923, FC = 432, and ENT = 841). Similarly, all FIB sampling results for all
days 1, 2 or 3 days following these rain day events were used to calculate the 1-day, 2-day and 3-day post rain,
respectively, average levels. The “trace rain” average levels used all FIB sampling results for all days with any
amount of measured rainfall below 0.1 inches, as long as these days did not fall in the 3-day period following a
rain event as defined above (shoreline trace rain sample size for TC = 1,588, FC = 322, and ENT = 369, nearshore
trace rain sample size TC = 704, FC = 210, and ENT = 276). The “no rain” average levels excluded days with
any amount or rain, as well as three days following any day with 0.1 inches or more of rain. The “all days”
average levels were calculated using every FIB sample result (shoreline sample size for TC = 78,894, FC = 9,996,
and ENT = 16,526, nearshore sample size TC = 42,607, FC = 7,960, and ENT = 16,170). The 0.1 inches rainfall
amount was selected because the County Health Department advises avoiding water contact for 72-hours (3-days)
following a rain event of 0.1 inches or more.
Figure 3.5.1 shows that at shoreline and inshore sites total coliform and enterococcus average levels are
about an order of magnitude higher on rain days than on ‘no rain’ days. At the shoreline total coliform averaged
700 CFU/100 ml on rain days and 84 CFU/100 ml on “no rain” days, and enterococcus averaged 191 CFU/100 ml
on rain days and 13 CFU/100 ml on “no rain” days. At the inshore total coliform averaged 281 CFU/100 ml on
rain days and 45 CFU/100 ml on “no rain” days, and enterococcus averaged 24 CFU/100 ml on rain days and 2
CFU/100 ml on “no rain” days. Both figures show the recovery that occurs as FIB levels return to ‘no rain’ levels,
usually within three days following rain events.
An assessment of the 1972-2015 FIB sample results with AB411 water contact standards determined that
62% (501 of 811 occurrences) of single sample maximum (SSM) exceedances at shore sites are associated with
rain, and 65% (59 of 91 occurrences) of SSM exceedances at inshore sites are associated with rain. In both cases
most of the exceedances occur on the actual rain day. These percentages are even more notable because 82% of
all FIB sampling is done during dry weather. During dry weather the chances of SSM exceedances of total
coliform, fecal coliform, and enterococcus SSM standards are 0.12%, 0.44%, and 1.6%, respectively at shore
stations, and 0.08%, 0.001%, and 0.004%, respectively at inshore stations. By comparison, on days with more
than 0.1 inch of rain, chances of SSM exceedances increased to 1.4%, 2.5%, and 25%, respectively at shore
stations, and to 0.73%, 0%, and 3.0%, respectively at inshore stations (only a very limited amount of fecal
coliform sampling was done at inshore sites, explaining why this value is zero).
The JWPCP began continuous year-round disinfection of effluent in the 1980s. Once disinfection was
continuous, FIB levels at the inshore sites dropped further, and subsequently, rain explains nearly all observations
of elevated FIB at inshore sites. The incidence of SSM exceedances at shoreline sites did not change significantly
in response to continuous disinfection, suggesting that dry weather runoff, birds, marine mammals, and other
animals observed at the shoreline, as well as trash, bathers, decaying material from multiple sources, etc.,
continued to be the predominant sources of FIB detected at the shoreline. Figure 3.5.2 compares the average level
of each FIB between rain days (days with >0.1 inch of rain), and dry weather (days with <0.1 inches of rain in the
previous 3 days). A Baseline period (B; 1972) is compared with subsequent periods of primary (P; 1973-1983),
partial secondary (PS; 1984-2002), full secondary (FS; 2003-2013) treatment, and the current (Current; 20142015) period. There was no routine sampling for fecal coliform or enterococcus prior to 1984. Overall, levels of
total coliform, fecal coliform and enterococcus were 8.3, 4.9, and 14.6 times higher, respectively, on rain days
(days when rainfall was > 0.1 inches) than in dry weather.
App 3.5 - 2
Figure 3.5.2 Temporal Trends in Rain Effects
Average concentrations of shoreline total coliform measured on rain days (>0.1 inch) and in dry weather. Data are
presented for a Baseline period of primary treatment (B, 1972), and for periods of increasing treatment; Advanced
Primary (AP, 1973-1983), Partial Secondary (PS, 1984-2002), and Full Secondary (FS, 2003-2013), and for the
current reporting period (Current, 2014-2015).
App 3.5 - 3
Appendix 3.6
Total Coliform, Fecal Coliform, Enterococcus and Shellfish Harvesting Receiving
water Microbiological Results
Appendix 3.6
Total Coliform Receiving Water Microbiological Results
All 2014 and 2015 shoreline and inshore results for Total Coliform were used to determine monthly
maximum single sample and 30-day geometric mean maximum values [Rain data included]
Single Sample Monthly Maximum Values
S1
S2
S3
Month
62
12
18
2014 JAN
11
7
200
FEB
12
31
200
MAR
13
4
200
APR
11
8
18
MAY
280
4
18
JUN
130
18
18
JUL
130
12
24
AUG
420
110
200
SEP
560
18
200
OCT
110
24
97
NOV
100
330
200
DEC
2015 JAN
60 < 7 < 15
25 < 6 < 60
FEB
5 < 7 < 60
MAR
6 < 7 < 60
APR
MAY < 6 < 3 < 15
JUN < 41 < 3 < 8
50 < 6 < 16
JUL
41 < 5 < 16
AUG
84 < 13 < 62
SEP
63 < 12 < 130
OCT
54 < 12 < 61
NOV
31 < 52 < 71
DEC
Shore Stations
S5
S6
110
180
140
110
110
400
55
36
32
98
100
8
700
110
120
1000
3200
27
2800
150
230
71
120
2000
66 < 116
62 < 66
62
86
< 25
32
< 13
43
< 19 < 43
78 < 25
88 < 68
80 < 44
114 < 26
94 < 36
44
99
<
<
<
<
<
<
<
S7
33
42
310
180
27
150
150
36
200
73
73
270
23
15
61
32
23
20
39
22
33
19
25
65
<
<
<
<
<
<
<
<
<
<
<
SB
16
1
12
400
44
120
44
200
27
2800
17
91
4
3
3
11
7
32
18
40
25
50
21
20
<
<
<
<
<
<
<
SM
250
75
160
270
47
55
91
110
110
200
270
540
147
100
52
59
35
28
58
75
76
52
111
100
IL2
< 1
5
11
3
2
3
3
11
8
3
2
Inshore Stations (Surface)
IL3 IL4 IL5 IL6 IL7
7
1
1
1
1
5
1
3
1
3
4
11
1
7
20
3 < 1
3 < 1
8
4
1
7
1
1
9
4
2
4
3
8
4
4
1
1
7
3
3
3
3
12
1 < 1 < 1 < 18
5
7
1
1
8
5
1 < 1 < 1
3
2400
<
<
<
<
<
<
<
<
<
<
<
<
2.2
1.8
2.1
2
1.2
1.2
2.3
3
3.3
2.4
2.3
14
3400
<
<
<
<
<
<
<
<
<
<
<
<
3.2
2.5
2.2
2
1.7
1.8
2.9
3.4
3.4
2
2
20
3600
<
<
<
<
<
<
<
<
<
<
<
<
1
1
2
1.8
1
1.3
2
1.3
1.3
2
2.4
7.7
6600
<
<
<
<
<
<
<
<
<
<
<
<
1.2
1.2
1.3
1.3
1.8
1.6
1.3
1.6
1.3
1
1
15
6200
12000
<
<
<
<
<
<
<
<
<
<
<
<
1.3
1
1.9
1.7
1
1.6
1.6
1.6
1.3
1
1
26
IL2
3
1
7
8
12
1
7
4
9
5
25
<
<
<
<
<
<
<
<
<
<
<
<
1
1.2
2.8
2.2
1.5
1.2
1.3
2.1
3.6
1.8
2
23
Inshore Stations (Bottom)
IL3 IL4 IL5 IL6 IL7
8
5
3
1
4
12
1
1
2
3
5
18
8
8
27
3
20
1
1
7
8
3
1
3
20
1
4
8
3
3
3
4
3
1
3
10
4
24 18
5
9
3
9
4
8
7
8
5
3
12
8
3
5
1
4
700
<
<
<
<
<
<
<
<
<
<
<
<
1.8
1.3
2.3
3.5
1.9
1.9
2.3
1.8
2.9
3.3
2.6
15
3000
<
<
<
<
<
<
<
<
<
<
<
<
2.7
5.4
4.6
2.3
2
1.7
1.6
1.8
3.6
3.5
4.9
51
2000
<
<
<
<
<
<
<
<
<
<
<
<
2
2
2.6
4.8
1.6
1.9
1.4
1.4
1.9
1.7
1.7
17
1800
<
<
<
<
<
<
<
<
<
<
<
<
1.6
1.6
2.8
2.3
1
1.7
1.7
3.1
3.8
1.9
2.2
19
2800
<
<
<
<
<
<
<
<
<
<
<
<
1.9
1.1
2.6
2
1.3
1.6
1.2
3.6
3.8
1.6
1.3
28
30-day Geometric Mean Monthly Maximum Values
2014 JAN < 19 < 3 < 6
12 < 4
9 < 1 < 4 < 1 < 1 < 1 < 1 < 1 < 1 < 1 < 1 < 1 < 1 < 1
11 < 10
10 < 1
5 < 1 < 2 < 1 < 1 < 1 < 1 < 1 < 2 < 1 < 1 < 1
FEB < 8 < 2 < 8
4 < 1 < 2 < 1 < 1 < 1 < 1 < 1 < 2 < 1 < 1 < 1
MAR < 1 < 1 < 7 < 10 < 14 < 10 < 1
APR < 1 < 1 < 8 < 3 < 3 < 6 < 1 < 6 < 1 < 1 < 1 < 1 < 1 < 1 < 1 < 1 < 1 < 1 < 1
MAY < 1 < 1 < 3 < 2 < 2 < 4 < 2 < 7 < 1 < 1 < 1 < 1 < 1 < 1 < 1 < 1 < 1 < 1 < 1
JUN < 4 < 1 < 3 < 4 < 2 < 6 < 4 < 4 < 1 < 1 < 1 < 1 < 1 < 1 < 1 < 1 < 1 < 1 < 1
10 < 3 < 20 < 2 < 4 < 1 < 1 < 1 < 1 < 1 < 1 < 1 < 1 < 1 < 1 < 1
JUL < 14 < 2 < 3
7 < 9 < 4 < 9 < 2 < 2 < 1 < 2 < 1 < 1 < 1 < 1 < 1 < 1 < 2 < 2 < 1
AUG < 20 < 2
17 < 12 < 2 < 15 < 2 < 7 < 1 < 2 < 1 < 1 < 1 < 1 < 2 < 1 < 1 < 2 < 1
SEP < 32 < 3
20 < 3 < 6 < 1 < 5 < 1 < 1 < 1 < 1 < 1 < 1 < 2 < 1 < 1 < 1 < 1
OCT < 25 < 2 < 20
9 < 1 < 1 < 1 < 1 < 1 < 1 < 2 < 1 < 1 < 1 < 1
NOV < 4 < 2 < 10 < 15 < 4 < 8 < 1
25 < 5 < 11 < 7 < 2
14 < 2 < 2 < 2 < 3 < 4 < 3 < 3 < 4 < 3 < 2 < 3
DEC < 9 < 3
2015 JAN
21
54 < 71
45
72 < 53
22
107 < 8 < 11 < 5 < 12 < 15 < 15 < 11 < 23 < 10 < 10 < 14
21
31 < 25 < 34
36 < 63 < 21
76 < 1 < 2 < 1 < 1 < 1 < 1 < 2 < 3 < 2 < 3 < 6
FEB
98 < 20 < 15 < 64
67
12 < 3
112 < 1 < 2 < 1 < 1 < 1 < 2 < 2 < 3 < 1 < 3 < 6
MAR
15 < 4 < 8
23
27
16 < 11
69 < 1 < 3 < 1 < 1 < 1 < 2 < 1 < 3 < 3 < 2 < 2
APR
24 < 5 < 8
13
37
15 < 7
50 < 1 < 2 < 2 < 1 < 2 < 2 < 2 < 3 < 4 < 3 < 3
MAY
36 < 6 < 7
46
32
8 < 5
59 < 1 < 2 < 1 < 1 < 2 < 2 < 2 < 3 < 2 < 3 < 3
JUN
16 < 8 < 8
42
9
18 < 7
65 < 2 < 2 < 3 < 2 < 1 < 2 < 1 < 3 < 3 < 3 < 4
JUL
13 < 7
23
63
55
47 < 30
70 < 2 < 2 < 3 < 2 < 1 < 2 < 2 < 6 < 3 < 3 < 3
AUG
18 < 68
28
62 < 63
47 < 9
52 < 4 < 7 < 2 < 1 < 1 < 2 < 2 < 6 < 1 < 1 < 2
SEP
24
59
45
118 < 34
27 < 15
93 < 7 < 17 < 4 < 4 < 6 < 7 < 5 < 5 < 4 < 4 < 4
OCT
22 < 54
265
62 < 37 < 20
93 < 3 < 9 < 4 < 3 < 3 < 3 < 3 < 7 < 3 < 5 < 3
NOV < 29
9 < 41
188 < 35 < 17 < 23
54 < 2 < 2 < 1 < 1 < 1 < 1 < 4 < 6 < 2 < 3 < 4
DEC < 2
TOTAL COLIFORM BATHING WATER LIMITS (Only in effect at Inshore Stations)
A) Single sample shall not exceed 10,000/100 mL as a result of wastes discharged.
B) 30-day Geometric Mean shall not exceed 1,000/100 mL as a result of wastes discharged.
C) No single sample greater than 1000/100 mL when fecal coliform/total coliform ratio exceeds 0.1 as a result of wastes discharged.
Shore station sampling is conducted for monitoring purposes only, not compliance determination.
App 3.6 - 1
6400
<
<
<
<
<
<
<
<
<
<
<
<
1.6
1.5
4.3
3.1
3.9
3.9
1.6
2.6
4.9
2.3
2.3
24
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
1
1
1
1
1
1
1
1
2
1
1
3
16
2
2
3
2
2
6
4
2
4
2
4
Appendix 3.6 (Continued)
Fecal Coliform Receiving Water Microbiological Results
All 2014 and 2015 shoreline and inshore results for Fecal Coliform were used to determine monthly
S1
S2
Month
29
8
2014 JAN
1
1
FEB
1
5
MAR
3
1
APR
MAY < 1 < 1
100
1
JUN
130
9
JUL
50
3
AUG
120
4
SEP
220
4
OCT
62
4
NOV
11
7
DEC
2015 JAN
24
56
30
6
FEB
900
18
MAR
4
2
APR
30 < 2
MAY
13
13
JUN
22
50
JUL
27
2
AUG
4
50
SEP
220
4
OCT
1
11
NOV
1
4
DEC
S3
15
19
16
20
1
13
7
16
52
12
16
94
9
17
4
8
4
2
70
30
50
58
64
20
Shore Stations
S5
S6
40
74
16
20
11
17
9
3
17
12
13
1
110
46
17
5
220
8
330
7
19
15
24
75
64
40
6
8
300
62
11
2
13
9
80
17
140
4
50
300
11
4
360
26
86
15
15
5
S7
SB
17
1
9
1
25 < 1
98
3
7
11
26
17
95
4
16
5
21
4
28
3
19 < 1
24
8
110
67
17
4
30
13
50 < 2
4
2
23
2
70
4
240
4
8
4
44
2
9
20
4
20
SM
11
8
7
26
8
9
4
5
24
15
25
25
12
8
240
8
11
8
30
80
22
15
21
11
IL2
< 1
1
1
3
< 1
1
2
1
3
< 1
< 1
25
< 1
< 2
2
2
2
4
4
< 2
2
1
< 1
< 1
IL3 IL4 IL5 IL6 IL7
3 < 1
1 < 1 < 1
4
1
3 < 1 < 1
1
1 < 1 < 1
4
< 1 < 1 < 1
1 < 1
4 < 1 < 1 < 1 < 1
< 1
1 < 1
1 < 1
< 1
1 < 1 < 1 < 1
4 < 1 < 1 < 1
1
1 < 1
1 < 1 < 1
4
3
1 < 1
1
2
1 < 1 < 1 < 1
17 29 64 150 82
2
1
1 < 1 < 1
< 2
2 < 2 < 2 < 2
9 < 2 < 2 < 2 < 2
2
2 < 2 < 2 < 2
< 2 < 2 < 2 < 2
7
< 2 < 2 < 2 < 2 < 2
2
2
8
7
8
4
2 < 2
2 < 2
2
2 < 2 < 2
2
1
1 < 1
1
1
3 < 1 < 1 < 1
1
3
1 < 1 < 1 < 1
30-day Geometric Mean Monthly Maximum Values
2014 JAN < 19 < 3 < 6
12 < 4
9 < 1 < 4 < 1 < 1 < 1 < 1 < 1
11 < 10
10 < 1
5 < 1 < 2 < 1 < 1 < 1
FEB < 8 < 2 < 8
4 < 1 < 2 < 1 < 1 < 1
MAR < 1 < 1 < 7 < 10 < 14 < 10 < 1
APR < 1 < 1 < 8 < 3 < 3 < 6 < 1 < 6 < 1 < 1 < 1 < 1 < 1
MAY < 1 < 1 < 3 < 2 < 2 < 4 < 2 < 7 < 1 < 1 < 1 < 1 < 1
JUN < 4 < 1 < 3 < 4 < 2 < 6 < 4 < 4 < 1 < 1 < 1 < 1 < 1
10 < 3 < 20 < 2 < 4 < 1 < 1 < 1 < 1 < 1
JUL < 14 < 2 < 3
7 < 9 < 4 < 9 < 2 < 2 < 1 < 2 < 1 < 1 < 1
AUG < 20 < 2
17 < 12 < 2 < 15 < 2 < 7 < 1 < 2 < 1 < 1 < 1
SEP < 32 < 3
20 < 3 < 6 < 1 < 5 < 1 < 1 < 1 < 1 < 1
OCT < 25 < 2 < 20
9 < 1 < 1 < 1 < 1 < 1
NOV < 4 < 2 < 10 < 15 < 4 < 8 < 1
25 < 5 < 11 < 7 < 2
14 < 2 < 2 < 2 < 3 < 4
DEC < 9 < 3
2015 JAN < 7 < 6
25 < 7 < 6 < 8 < 10 < 8 < 2 < 2 < 2 < 3 < 3
9 < 4 < 4 < 6 < 10 < 2 < 2 < 2 < 2 < 2 < 2
FEB < 10 < 9
MAR < 43 < 3 < 9 < 17 < 7 < 7 < 4 < 15 < 2 < 3 < 2 < 2 < 2
APR < 8 < 2 < 5 < 24 < 5 < 10 < 4 < 4 < 2 < 3 < 2 < 2 < 2
6 < 3 < 8 < 2 < 4 < 2 < 2 < 2 < 2 < 2
MAY < 8 < 2 < 5
13 < 4 < 5 < 2 < 5 < 2 < 2 < 2 < 2 < 2
JUN < 14 < 3 < 2
36 < 3 < 14 < 2 < 9 < 2 < 2 < 2 < 3 < 3
JUL < 9 < 11 < 7
12
44 < 7 < 42 < 3
34 < 2 < 2 < 2 < 3 < 3
AUG < 7 < 7
35 < 2 < 3 < 2 < 2 < 2
SEP < 7 < 5 < 12 < 13 < 9 < 10 < 3
9 < 1 < 1 < 1 < 1 < 1
OCT < 8 < 4 < 7 < 22 < 4 < 8 < 2
NOV < 8 < 3 < 23 < 45 < 7 < 11 < 2 < 10 < 1 < 1 < 1 < 1 < 1
DEC < 1 < 2 < 15 < 19 < 4 < 4 < 4 < 6 < 1 < 1 < 1 < 1 < 1
FECAL COLIFORM BATHING WATER LIMITS (Only in effect at Inshore Stations)
A) Single sample shall not exceed 400/100 mL as a result of wastes discharged.
B) 30-day Geometric Mean shall not exceed 200/100 mL as a result of wastes discharged.
App 3.6 - 2
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
1
1
1
1
1
1
1
1
1
1
1
3
2
2
2
2
3
3
3
3
2
1
1
1
IL2 IL3 IL4 IL5 IL6 IL7
< 1
4
2
1
1 < 1
1
5
1 < 1
1
1
< 1
1
1
1
3
1
< 1
1
3
1
1
1
1 < 1 < 1 < 1
1
1
< 1 < 1 < 1
1
1 < 1
1
1
2
1
1 < 1
1
3
4
13
3
3
3
1 < 1 < 1 < 1
4
3
1 < 1 < 1
1
3
17
1
2 < 1
1
2
8
16 24 23 42 58
1
1 < 1 < 1
1 < 1
4 < 2
2 < 2 < 2
2
< 2
23
3 < 2 < 2 < 2
< 2
2
4
2 < 2
4
2
2
2
14
2
3
2
2 < 2 < 2 < 2
2
4
4
2
4
2
8
3
4
4
2
2
2
< 2
8
4
2
2 < 2
1
9
1
4
1 < 1
1
3
3
1
3
4
1
5
1 < 1
1
1
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
1
1
1
1
1
1
1
1
2
2
2
3
2
2
2
2
2
2
2
3
2
1
1
1
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
1
2
2
1
1
1
1
1
1
1
1
4
3
2
4
4
2
2
2
2
4
2
2
2
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
1
1
1
1
1
1
1
2
1
1
1
3
2
2
2
2
2
2
2
2
3
2
1
1
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
1
1
1
1
1
1
1
2
2
1
1
2
2
2
2
2
3
3
2
2
2
1
1
1
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
1
1
1
1
1
1
1
1
1
1
1
3
2
2
2
2
2
2
2
2
2
1
1
1
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
1
1
1
1
1
1
1
1
2
1
1
3
2
2
2
3
2
2
3
3
2
1
1
1
Enterococcus Receiving Water Microbiological Results
All 2014 and 2015 shoreline and inshore results for Enterococcus were used to determine monthly
S1
S2
Month
2014 JAN
4
4
3 < 1
FEB
1
11
MAR
1 < 1
APR
1
1
MAY
16
2
JUN
17 < 1
JUL
9
3
AUG
19
4
SEP
18
1
OCT
NOV < 1 < 1
3
15
DEC
2015 JAN
11
40
4
2
FEB
640
35
MAR
9 < 1
APR
40
4
MAY
14 < 1
JUN
2 < 2
JUL
3
1
AUG
1
11
SEP
16
7
OCT
4
NOV < 1
1 < 1
DEC
S3
2
4
18
18
4
4
4
5
12
18
4
18
18
9
3
1
3
4
3
9
12
38
18
5
Shore Stations
S5
S6
40
64
32
64
5
27
1
7
5
7
21 < 1
10
1
15
4
120
3
440
3
3
1
29
120
440
110
9
17
57
60
1
13
3
4
5
13
25
4
10
110
4
5
280
56
34
30
7
5
S7
SB
SM
IL2 IL3 IL4 IL5 IL6 IL7 IL2
3 < 1
19 < 1 < 1 < 1
1
1
1 < 1
12 < 1
23 < 1 < 1 < 1
1 < 1
1 < 1
16
1
8
1 < 1
1 < 1 < 1
1 < 1
96
46
20 < 1 < 1 < 1 < 1 < 1 < 1 < 1
3
4
7 < 1 < 1 < 1 < 1
1 < 1 < 1
9
3
4 < 1 < 1 < 1 < 1
1 < 1 < 1
33
1 < 1 < 1 < 1 < 1
1 < 1 < 1 < 1
7
15
3 < 1
1 < 1 < 1 < 1 < 1 < 1
11
1
15
8 < 1 < 1 < 1 < 1 < 1 < 1
37
1
8 < 1 < 1 < 1 < 1 < 1 < 1 < 1
4 < 1
20 < 1 < 1 < 1 < 1 < 1 < 1 < 1
33
10
8
84
11 < 1
4
12 52
5
40
12
26 < 1 < 1 < 1
1
1
1 < 1
6
1
12
1 < 1
1 < 1 < 1 < 1 < 1
23
9
60 < 1
1 < 1 < 1 < 1 < 1 < 1
5
1
7 < 1
1 < 1 < 1 < 1 < 1 < 1
5
1
8 < 1
1 < 1 < 1 < 1 < 1 < 1
5
1
4 < 1 < 1 < 1
1 < 1 < 1 < 1
7
1
13 < 1 < 1
3 < 1 < 1
1 < 1
8
4
18 < 1 < 1 < 1
1 < 1 < 1 < 1
3
1
3 < 1 < 1 < 1 < 1 < 1 < 1 < 1
110
12
24 < 1
1 < 1 < 1 < 1 < 1
1
4
96
87 < 1
1 < 1 < 1 < 1
1 < 1
7
8
11 < 1 < 1 < 1 < 1 < 1 < 1 < 1
Monthly Maximum 30-day Geometric Mean
2014 JAN < 6 < 1 < 1 < 19 < 5 < 3 < 1 < 4 < 1 < 1 < 1 < 1 < 1
FEB < 2 < 1 < 2 < 13 < 7 < 2 < 1 < 10 < 1 < 1 < 1 < 1 < 1
MAR < 1 < 2 < 4 < 10 < 11 < 7 < 1 < 4 < 1 < 1 < 1 < 1 < 1
APR < 1 < 2 < 4 < 1 < 3 < 4 < 3 < 5 < 1 < 1 < 1 < 1 < 1
MAY < 1 < 1 < 4 < 1 < 3 < 3 < 2 < 4 < 1 < 1 < 1 < 1 < 1
JUN < 2 < 1 < 3 < 5 < 2 < 2 < 2 < 3 < 1 < 1 < 1 < 1 < 1
JUL < 2 < 1 < 3 < 4 < 1 < 6 < 1 < 1 < 1 < 1 < 1 < 1 < 1
AUG < 3 < 1 < 2 < 4 < 1 < 4 < 2 < 2 < 1 < 1 < 1 < 1 < 1
SEP < 6 < 2 < 4 < 11 < 2 < 3 < 2 < 4 < 2 < 1 < 1 < 1 < 1
OCT < 6 < 1 < 6 < 11 < 2 < 4 < 1 < 3 < 2 < 1 < 1 < 1 < 1
5 < 1 < 1 < 1 < 1 < 1
NOV < 2 < 1 < 5 < 11 < 2 < 4 < 1
9 < 2 < 1 < 1 < 2 < 2
DEC < 1 < 3 < 6 < 4 < 4 < 3 < 2
2015 JAN < 2 < 7 < 6 < 11 < 8 < 6 < 4 < 9 < 2 < 1 < 1 < 2 < 2
FEB < 3 < 6 < 5 < 8 < 9 < 5 < 3 < 3 < 1 < 1 < 1 < 1 < 1
MAR < 13 < 2 < 3 < 8 < 4 < 3 < 2 < 14 < 1 < 1 < 1 < 1 < 1
APR < 5 < 1 < 2 < 6 < 10 < 3 < 2 < 3 < 1 < 1 < 1 < 1 < 1
MAY < 7 < 1 < 1 < 1 < 3 < 3 < 1 < 3 < 1 < 1 < 1 < 1 < 1
JUN < 13 < 1 < 2 < 2 < 2 < 2 < 1 < 3 < 1 < 1 < 1 < 1 < 1
JUL < 2 < 1 < 2 < 4 < 3 < 2 < 1 < 5 < 1 < 1 < 1 < 1 < 1
3 < 4 < 1 < 5 < 1 < 1 < 1 < 1 < 1
AUG < 1 < 1 < 2 < 6
SEP < 1 < 2 < 3 < 3 < 3 < 2 < 1 < 7 < 1 < 1 < 1 < 1 < 1
OCT < 2 < 4 < 5 < 9 < 4 < 7 < 2 < 2 < 1 < 1 < 1 < 1 < 1
NOV < 2 < 4 < 10 < 21 < 7 < 7 < 5 < 20 < 1 < 1 < 1 < 1 < 1
6 < 5 < 5 < 11 < 1 < 1 < 1 < 1 < 1
DEC < 1 < 2 < 4 < 11
ENTEROCOCCUS BATHING WATER LIMITS (Only in effect at Inshore Stations)
A) Single sample shall not exceed 104/100 mL as a result of wastes discharged.
B) 30-day Geometric Mean shall not exceed 35/100 mL as a result of wastes discharged.
App 3.6 - 3
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
1
1
1
1
1
1
1
1
1
1
1
2
2
1
1
1
1
1
1
1
1
1
1
1
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
IL3 IL4 IL5 IL6 IL7
1 < 1 < 1 < 1
1
< 1 < 1 < 1 < 1
1
1
1
1
1
4
< 1 < 1 < 1 < 1 < 1
< 1 < 1 < 1 < 1 < 1
< 1 < 1 < 1 < 1 < 1
< 1 < 1 < 1 < 1 < 1
1 < 1
4
1
3
1 < 1 < 1 < 1 < 1
< 1 < 1 < 1 < 1
1
< 1 < 1 < 1 < 1 < 1
20
1
3
4
16
4
1 < 1 < 1
1
< 1 < 1
1 < 1
1
3
1 < 1 < 1 < 1
< 1 < 1 < 1 < 1 < 1
< 1 < 1 < 1
1 < 1
< 1 < 1 < 1 < 1 < 1
1 < 1 < 1 < 1 < 1
< 1 < 1 < 1 < 1 < 1
1 < 1 < 1 < 1 < 1
< 1 < 1 < 1 < 1 < 1
1
1 < 1 < 1 < 1
< 1 < 1 < 1 < 1 < 1
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
1
1
1
1
1
1
1
1
1
1
1
2
2
1
1
1
1
1
1
1
1
1
1
1
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
1
1
1
1
1
1
1
1
1
1
1
2
2
1
1
1
1
1
1
1
1
1
1
1
Shellfish Harvesting Standards
All 2014 and 2015 shoreline and inshore results were used to determine 6-month median and percent
greater than 230/100 ml values [Rain data included]
COMPLIANCE WITH SHELLFISH HARVESTING LIMIT (Six - Month Median)
Month
2014 JAN
FEB
MAR
APR
MAY
JUN
JUL
AUG
SEP
OCT
NOV
DEC
2015 JAN
FEB
MAR
APR
MAY
JUN
JUL
AUG
SEP
OCT
NOV
DEC
S1
12
12
12
10
9
9
9
12
13
20
22
20
20
18
18
10
13
12
12
12
10
14
8
7
S2
4
4
4
4
4
4
4
4
4
4
4
11
12
13
14
7
5
4
4
4
4
8
10
12
S3
18
18
18
18
18
18
18
18
18
18
18
18
21
23
18
17
12
5
7
8
8
12
17
18
Shore Stations
S5
S6
55
30
31
39
31
42
29
34
30
42
28
24
29
19
29
18
25
18
30
18
30
15
33
20
30
20
20
19
20
21
17
21
17
21
17
16
17
15
16
21
21
18
24
20
39
20
43
20
S7
9
10
12
16
18
14
16
17
12
16
17
21
21
14
12
11
8
7
8
11
14
16
18
17
SB
4
4
4
1
1
1
3
8
11
10
8
8
8
7
4
5
4
4
4
4
7
7
7
6
SM
48
45
47
47
47
37
37
43
47
43
49
55
46
39
38
41
38
38
48
51
48
50
45
41
IL2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
IL3 IL4 IL5 IL6 IL7
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
3
1
1
1
1
3
1
1
1
1
3
1
1
1
1
2
1
1
1
1
IL2
1
1
1
1
1
1
1
1
1
1
1
2
1
2
1
1
1
1
1
1
1
1
1
1
IL3 IL4 IL5 IL6 IL7
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
1
1
1
1
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
3
1
1
1
3
5
1
1
1
3
5
1
1
1
1
4
1
1
1
1
1
1
1
1
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
3
1
1
1
1
4
1
1
1
1
5
1
1
1
1
COMPLIANCE WITH SHELLFISH HARVESTING LIMIT (Six - Month Percent over 230/100 mL)
11%
0%
4%
10%
12%
0%
0%
15%
0% 0% 0% 0% 0% 0% 0% 0% 0% 0%
2014 JAN
11%
0%
4%
7%
12%
0%
0%
15%
0% 0% 0% 0% 0% 0% 0% 0% 0% 0%
FEB
11%
0%
4%
4%
15%
4%
0%
15%
0% 0% 0% 0% 0% 0% 0% 0% 0% 0%
MAR
11%
0%
4%
4%
15%
4%
4%
19%
0% 0% 0% 0% 0% 0% 0% 0% 0% 0%
APR
7%
0%
0%
0%
15%
4%
4%
15%
0% 0% 0% 0% 0% 0% 0% 0% 0% 0%
MAY
4%
0%
0%
0%
4%
4%
4%
8%
0% 0% 0% 0% 0% 0% 0% 0% 0% 0%
JUN
4%
0%
0%
4%
4%
4%
4%
4%
0% 0% 0% 0% 0% 0% 0% 0% 0% 0%
JUL
4%
0%
0%
4%
8%
4%
4%
4%
0% 0% 0% 0% 0% 0% 0% 0% 0% 0%
AUG
7%
0%
0%
7%
4%
0%
4%
4%
0% 0% 0% 0% 0% 0% 0% 0% 0% 0%
SEP
12%
0%
0%
11%
4%
0%
4%
0%
0% 0% 0% 0% 0% 0% 0% 0% 0% 0%
OCT
12%
0%
0%
11%
4%
0%
4%
4%
0% 0% 0% 0% 0% 0% 0% 0% 0% 0%
NOV
8%
4%
0%
11%
7%
4%
4%
8%
3% 3% 3% 3% 3% 3% 3% 3% 3% 3%
DEC
2015 JAN
8%
8%
0%
10%
11%
12%
4%
12%
3% 3% 3% 3% 3% 3% 3% 3% 3% 3%
8%
8%
0%
10%
7%
12%
4%
12%
3% 3% 3% 3% 3% 3% 3% 3% 3% 3%
FEB
8%
8%
0%
11%
11%
12%
4%
15%
3% 3% 3% 3% 3% 3% 3% 3% 3% 3%
MAR
4%
8%
0%
7%
11%
12%
0%
15%
3% 3% 3% 3% 3% 3% 3% 3% 3% 3%
APR
4%
8%
0%
7%
14%
12%
0%
12%
3% 3% 3% 3% 3% 3% 3% 3% 3% 3%
MAY
4%
4%
0%
7%
11%
8%
0%
8%
0% 0% 0% 0% 0% 0% 0% 0% 0% 0%
JUN
4%
0%
0%
4%
8%
0%
0%
4%
0% 0% 0% 0% 0% 0% 0% 0% 0% 0%
JUL
4%
0%
0%
4%
11%
0%
0%
4%
0% 0% 0% 0% 0% 0% 0% 0% 0% 0%
AUG
0%
0%
0%
0%
7%
0%
0%
0%
0% 0% 0% 0% 0% 0% 0% 0% 0% 0%
SEP
4%
0%
4%
7%
11%
0%
0%
4%
0% 0% 0% 0% 0% 0% 0% 0% 0% 0%
OCT
4%
0%
4%
11%
7%
0%
4%
4%
0% 0% 0% 0% 0% 0% 0% 0% 0% 0%
NOV
4%
0%
4%
11%
7%
0%
4%
4%
0% 0% 0% 0% 0% 0% 0% 0% 0% 0%
DEC
SHELLFISH HARVESTING LIMITS (Only in effect at Inshore Stations)
A) 6-month median shall not exceed 70/100 mL as a result of wastes discharged.
B) No more than 10% >230/100 mL in any 6 month period as a result of wastes discharged.
App 3.6 - 4
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
3%
3%
7%
6%
7%
7%
3%
3%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
3%
3%
3%
3%
3%
3%
0%
0%
0%
0%
0%
0%
0%
Offshore Microbiological Sampling Sites
All 2014 and 2015 offshore results from monthly microbiological sampling are shown
[Rain data included]
Month
Offshore Stations
Total Coliform
6C
1
1
1
5
1
1
1
1
1
1
1
8C
1
1
1
1
1
1
1
2
1
1
1
Fecal Coliform
9C
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
5
1
1
6C
1
1
1
1
1
1
1
1
1
1
1
120
1
1
2
2
2
2
2
2
2
1
1
1
8C
1
1
1
1
1
1
1
1
1
1
1
150
1
1
2
2
2
2
2
2
2
1
1
1
9C
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
1
1
1
Enterococcus
6C
1
1
1
1
1
1
1
1
1
1
1
70
1
1
1
1
1
1
1
1
1
1
1
1
8C
9C
2014 JAN
<
<
<
<
<
<
<
< 1
< 1
<
<
<
<
< 1
< 1
<
FEB
<
<
<
<
<
<
< 1
<
MAR
20
<
<
<
<
<
< 1
< 1
APR
<
<
<
<
<
<
< 1
< 1
<
MAY
<
<
<
<
<
<
< 1
< 1
<
JUN
<
<
<
<
<
<
< 1
< 1
<
JUL
<
<
<
<
<
< 1
<
AUG
1
<
<
<
<
<
<
< 1
< 1
<
SEP
<
<
<
<
<
<
< 1
< 1
<
OCT
<
<
<
<
<
<
< 1
< 1
<
NOV
5600
8600
<
<
DEC
66
1
2015 JAN
< 1
< 1
<
<
<
<
<
< 1
< 1
<
<
<
<
< 1
< 1
< 1
FEB
1
<
<
<
<
<
<
<
< 1
<
MAR
1
1
1
< 1
<
<
<
<
<
< 1
< 1
< 1
APR
< 1
<
<
<
<
<
< 1
< 1
< 1
MAY
< 1
<
<
<
<
< 1
< 1
< 1
JUN
<
<
< 1
< 1
JUL
2
7
< 1
<
<
<
<
<
< 1
< 1
< 1
AUG
< 1
<
<
<
<
<
< 1
< 1
< 1
SEP
200 < 200 < 200 <
<
<
<
< 1
< 1
OCT
< 1
< 1
<
<
<
<
< 1
< 1
< 1
NOV
< 1
<
<
<
<
< 1
< 1
< 1
DEC
1
OFFSHORE RECEIVING WATER LIMITS:
A) Total coliform single sample shall not exceed 10,000/100 mL as a result of wastes discharged.
B) Fecal coliform single sample shall not exceed 400/100 mL as a result of wastes discharged.
C) Enterococcus single sample shall not exceed 104/100 mL as a result of wastes discharged.
D) No single sample greater than 1000/100 mL when fecal coliform/total coliform ratio exceeds 0.1 as a
result of wastes discharged.
NOTE: Since only one sample per month is required at Offshore Stations, compliance with geometric mean
standards is not assessed.
App 3.6 - 5
Appendix 3.7
Spatial and Temporal Trends in Fecal Coliform and Enterococcus Concentrations
Appendix 3.7
Spatial and Temporal Trends in Fecal Coliform Concentrations
No Fecal coliform data available prior to 1984
Mean fecal coliform concentrations from shoreline (S1, S2, S3, S5, S6, S7, SB, and SM), inshore (IL2, IL3, IL4,
IL5, IL6, IL7), and offshore (9C, 8C, 7C) sampling locations (Figure 3.1) from 1984 through 2015. Spatial trends
are depicted by depth and the relative location of shoreline geographic landmarks (CB = Cabrillo Beach, WP =
White Point, PB = Portuguese Bend, AC = Abalone Cove, BC = Bluff Cove, and MC = Malaga Cove). The outfall
discharge zone is in 60 meters of water off White Point. Temporal trends are assessed by comparison of three
discrete sampling periods (1984-2002, 2003-2013, and 2014-2015). No fecal coliform data are available for the
period prior to 1984, when the JWPCP only provided primary treatment. [Rain data excluded].
App 3.7 - 1
Appendix 3.7
Spatial and Temporal Trends in Enterococcus Concentrations
No Enterococcus data available prior to 1984
Mean enterococcus concentrations from shoreline (S1, S2, S3, S5, S6, S7, SB, and SM), inshore (IL2, IL3, IL4,
IL5, IL6, IL7), and offshore (9C, 8C, 7C) sampling locations (Figure 3.1) from 1984 through 2015. Spatial trends
are depicted by depth and the relative location of shoreline geographic landmarks (CB = Cabrillo Beach, WP =
White Point, PB = Portuguese Bend, AC = Abalone Cove, BC = Bluff Cove, and MC = Malaga Cove). The outfall
discharge zone is in 60 meters of water off White Point. Temporal trends are assessed by comparison of three
discrete sampling periods (1984-2002, 2003-2013, and 2014-2015). No enterococcus data are available for the
period prior to 1984, when the JWPCP only provided primary treatment. [Rain data excluded].
App 3.7 - 2

Appendices 3 - Sanitation District

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