Quality Assurance Project Plan

**Growth rate of biofilm organisms in a distribution system simulator**

**Prepared by:**

XXX

Address

Cincinnati, T&E facility

Telephone

**Table of Contents**

> I. Project Management 3
>
> A. Distribution List 3
>
> B. Project Organization 3
>
> C. Problem Definition/Background 4
>
> D. Project Description and Schedule 4
>
> E. Quality Objectives and Criteria for Measurement Data 5
>
> F. Special Training Requirements 5
>
> G. Documentation and Records 5
>
> II\. Measurement/Data Acquisition 5
>
> A. Experimental Design 5
>
> B. Sampling Methods Requirements 6
>
> C. Sample Handling and Custody Requirements 10
>
> D. Analytical Methods Requirements 11
>
> E. Quality Control/Quality Assurance Requirements 11
>
> F. Instrument/Equipment Testing, Inspection, and Maintenance
> Requirements\
> 12
>
> G. Instrument Calibration and Frequency 12
>
> H. Inspection/Acceptance Requirements for Supplies and Consumables 12
>
> I. Data Acquisition Requirements (Non-Direct Measurements) 12
>
> J. Data Management 12
>
> III\. Assessment/Oversight 13
>
> A. Assessments and Response Actions 13
>
> B. Reports to Management 13
>
> IV\. Data Validation and Usability 13
>
> A. Data Review, Validation, and Verification Requirements 13
>
> B. Validation and Verification Methods 13
>
> C. Reconciliation with Data Quality Objectives 14
>
> References 15
>
> Appendix 1 16

**List of Tables**

Table 1. Distribution system simulator operating conditions 6

Table 2. Sample collection schedule for microbiological samples 8

Table 3. Sample collection schedule for chemical analyses 9

Table 4. Methods for analysis of water and biofilm samples 11

**I. Project Management**

**A. Distribution List**

XX , Principal Investigator, Microbial Contaminants Control Branch
(MCCB), Water Supply Water Resources Division (WSWRD)

XX, Acting Chief, MCCB, WSWRD

XX, Quality Assurance Manager, WSWRD

XX, Acting Chief and Project Manager, Water Quality Management Branch
(WQMB), WSWRD

XX, Biological Science Technician, MCCB, WSWRD

XX, Engineering Technician, WQMB, WSWRD

XX, Biological Science Technician, MCCB, WSWRD

Indicates individuals who are responsible for implementation the quality
assurance plan.

**B. Project Organization**

XX, the principal investigator, is responsible for plan development,
planning and overall coordination and implementation of the project,
data management and report preparation.

XX, the acting chief, of MCCB, WSWRD, is responsible for supervision of
project staff affiliated with the MCCB.

XX, the quality assurance manager, is responsible for ensuring that this
quality assurance plan meets Division acceptance criteria.

XX, as Project Manager and Work Assignment Manager for the Distribution
System Simulator (DSS) is responsible for ensuring that the DSS is
operated in accordance with the criteria identified in this QA project
plan. As Acting Chief of the WQMB, XX, is responsible for supervision of
project staff affiliated with the WQMB.

XX, the engineering technician, is responsible for ensuring that the DSS
is operated in accordance with quality assurance guidelines, labeling of
sample vessels, collection of samples, analysis of water samples and
record keeping.

XX and XX, the biological science technicians, are responsible for
preparation of sample vessels and equipment, analysis of samples for
microorganisms and record keeping.

**C. Problem Definition/Background**

Research is needed to identify the factors that affect the growth of
biofilms and the survival of opportunistic pathogens in drinking water
distribution systems. There is also strong evidence of the interactions
between water quality following treatment and regrowth of bacteria
within distribution systems. Bench scale reactor work indicates that the
rate of attachment and growth of heterotrophic biofilms is influenced by
the amount of bioavailable carbon and materials of construction. This
study seeks to evaluate the rate of attachment and biofilm growth in a
distribution system simulator under defined water quality conditions.

**D. Project Description and Schedule**

NRMRL has constructed a unique pilot-scale distribution system simulator
at its Test and Evaluation facility (T&E). The simulator consists of 6
independent 90-foot loops of 6-inch diameter ductile iron pipe. Each
loop can be operated to represent a point in the distribution system
with a different travel time and the loops can be configured in any
parallel or series arrangement (Figure 1). The quality of water entering
a loop may be modified by process modifications or by adding specific
amounts of known compounds. An initial set of experiments will be run to
examine the dependence of biofilm growth rate on the amount of organic
carbon in the feed water. Periodic sampling of the biofilm coupons will
provide information on the amount and rate of biofilm growth as a
function of organic carbon and residual disinfectant.

**E. Quality Objectives and Criteria for Measurement Data**

This is a basic research project NRMRL Category IV. Data collected as a
result of this work will be used to estimate attachment and growth rates
of heterotrophic bacteria within a simulated water distribution system.
Water quality parameters such as the amount of organic carbon as well as
other nutrients and oxidizing agents can influence microbial growth.
Also, the materials of construction may also affect microbial attachment
and growth. Therefore, water quality parameters, specifically organic
carbon concentrations (two levels 1.0 1.0 mg/L and 5.0 1.0 mg/L) will be
quantified to determine if variation of this parameter affects
heterotrophic biofilm growth. Similarly, materials used in the
construction of distribution systems will also be evaluated for growth
of heterotrophic biofilms on a limited basis (ductile iron and PVC
substrates). Results from this work are intended to provide information
regarding growth trends rather than for the production of data for model
development.

**F. Special Training Requirements**

Specialized training or certification is not required for any of the
procedures involved in this project.

**G. Documentation and Records**

A log book will be used for recording operating conditions, sample dates
and times. Laboratory bench sheets will also be used for recording
sample handling and assay results. Electronic data files will be used
for archiving operating data and analytical results. A final project
report in the form of a presentation and/or written report will be
generated.

**II. Measurement/Data Acquisition**

**A. Experimental Design**

A single test loop of the distribution system simulator (DSS) located at
the T&E will be used for this project. Specific operating conditions
will be set and maintained for at least one full week prior to sample
collection to ensure that system operation is consistent. Water supplied
to the loop system will be obtained from the City of Cincinnati water
distribution system which will be augmented with sodium hypochlorite and
sodium acetate in accordance with the operating conditions listed below.
A series of slides will be used as a fixed substrate for biofilm
formation and growth. These slides will be submerged in the recycle
water tank of the loop system, and retrieved at specified time
intervals. Biofilms from these samples will be assayed for total
heterotrophic bacteria using R2A agar spread plates.

Operating conditions:

The following operating conditions will be evaluated:

Hydraulic retention time will be maintained at 24 4 h.

Water velocity will be maintained at 0.3 0.1 m/s (turbulent flow
regime).

pH will be as received, i.e., not altered (approximately 8.0 0.3).

**Table 1. Distribution system simulator operating conditions**

  ------------------------- ----------- ----------- ----------- -----------
  Parameter                 Series 1    Series 2    Series 3    Series 4

  Free Cl~2~ residual       0.2 0.2     2.0 0.2     2.0 0.2     0.2 0.2
  (mg/L)                                                        

  Temperature (C)           25 3        25 3        25 3        25 3

  Organic carbon (mg/L)     1.0 1.0     1.0 1.0     5.0 1.0     5.0 1.0
  ------------------------- ----------- ----------- ----------- -----------

Free residual chlorine concentrations will be adjusted by addition of a
10 - 7% sodium hypochlorite solution to achieve the stated conditions.

Loop water temperatures will be adjusted by indirect cooling through a
shell and tube heat exchanger to achieve the stated conditions.

Organic carbon concentrations of the feed water will be adjusted by
addition of sodium acetate or another defined soluble carbon source to
achieve the stated conditions.

Operating conditions of the loop will be recorded daily including flow,
recirculation rate, pH, ORP, dissolved oxygen, turbidity, free chlorine
residual and water temperature.

**B. Sampling Methods Requirements**

*Microbiological samples*:

Samples for microbiological analyses will be collected from the loop
discharge (bulk water samples). All liquid and slide samples will be
collected in duplicate. Biofilms will be collected from slides supported
in a polycarbonate holder. All slides in the holder will be 7.5 0.2 X
2.5 0.1 cm with a total exposed surface area of 37.5 2 cm^2^ constructed
of ductile iron or a non-pourous media (such as glass or
polyvinylchloride, PVC). Slides inserted into the holder will be steam
sterilized for 15 minutes at 121 C. PVC slides will be sterilized by
exposure to short wavelength ultraviolet light and then inserted into
the steam sterilized polycarbonate holder. After operating conditions
have been stabilized and maintained for a minimum of seven days, the
sterile holder with slides will be inserted into the recirculation tank
of the DSS loop and the time noted. Slide and water samples will be
collected for analysis according to the schedule shown in Table 2.
Slides will be aseptically removed from the system and placed into new
sterile 50 mL tubes containing 45 mL of phosphate buffered dilution
water with 0.1 mL of a 10% sodium thiosulphate solution. These will be
assayed within four hours of sample collection for cultureable
heterotrophs in accordance with section 9215 A and C of "Standard
Methods for the Examination of Water and Wastewater 20^th^ Edition," or
held under refrigeration at 42 C for no more than 24 h prior to
initiation of assays. The surfaces of each slide will be scraped using a
flame sterilized stainless steel spatula, and sonicated for 30 s at low
power. Liquid samples will be collected in machine cleaned and steam
sterilized polypropylene bottles.

**Table 2. Sample collection schedule for microbiological samples** ^†^

  ------------------- ------ --------- --------------- -------- --------------
  Sample              Num.   Code      Container       Volume   Time (h)

  Loop Water          2      L2D-0     Polypropylene   100 mL   0

  Biofilm: PVC slide  2      L2U-2     Cent. tube      50mL     2

  Biofilm: iron slide 2      L2S-2     Cent. tube      50 mL    2

  Biofilm: PVC slide  2      L2U-8     Cent. tube      50 mL    8

  Biofilm: iron slide 2      L2S-8     Cent. tube      50 mL    8

  Biofilm: PVC slide  2      L2U-24    Cent. tube      50 mL    24

  Biofilm: iron slide 2      L2S-24    Cent. tube      50 mL    24

  Biofilm: PVC slide  2      L2U-48    Cent. tube      50 mL    48

  Biofilm: iron slide 2      L2S-48    Cent. tube      50 mL    48

  Biofilm: PVC slide  2      L2U-72    Cent. tube      50 mL    72

  Biofilm: iron slide 2      L2S-72    Cent. tube      50 mL    72

  Loop Water          2      L2D-168   Polypropylene   100 mL   168

  Biofilm: PVC slide  2      L2U-168   Cent. tube      50 mL    168

  Biofilm: iron slide 2      L2S-168   Cent. tube      50 mL    168

  Loop Water          2      L2D-336   Polypropylene   100 mL   336

  Biofilm: PVC slide  2      L2U-336   Cent. tube      50 mL    336

  Biofilm: iron slide 2      L2S-336   Cent. tube      50 mL    336

  Loop Water          2      L2D-504   Polypropylene   100 mL   504

  Biofilm: PVC slide  2      L2U-504   Cent. tube      50 mL    504

  Biofilm: iron slide 2      L2S-504   Cent. tube      50 mL    504
  ------------------- ------ --------- --------------- -------- --------------

^†^Heterotrophic plate counts, critical measurements, duplicate assays,
duplicate samples.

Upon collection of the last set of samples for a series, the next set of
operating conditions for a given series will be implemented.

*Chemical analysis*:

In addition to the above, water samples will also be collected for
analyses according to the following schedule:

**Table 3. Sample collection schedule for chemical analyses**

  ----------------------- ----------- ----------------- ------------------
  Parameter               Number      Volume (mL)       Time (h)

  Metals\*                1           250               0

  Non-Metals\*            1           250               0

  Organic carbon ^†^      2           45                0

  Metals\*                1           250               168

  Non-Metals\*            1           250               168

  Organic carbon ^†^      2           45                168

  Metals\*                1           250               336

  Non-Metals\*            1           250               336

  Organic carbon ^†^      2           45                336

  Metals\*                1           250               504

  Non-Metals\*            1           250               504

  Organic carbon ^†^      2           45                504
  ----------------------- ----------- ----------------- ------------------

\*Assays considered to be non-critical, collected in pre-cleaned high
density polyethylene bottles.

^†^Critical assays, duplicate samples, collected in carbon free brown
borosilicate glass vials.

As noted above, water samples destined for analysis of metals and
non-metal constituents will be collected in new pre-cleaned high density
polyethylene disposable containers and assayed within 14 days of
collection. Samples collected for total organic carbon analysis are to
be collected in new carbon free brown borosilicate glass vials with
phenolic caps, and will be assayed within 7 days of collection. Sample
bottles destined for microbiological assays will be polypropylene. These
are machine cleaned with detergent, rinsed with hot water, deionized
water, and steam sterilized. A small volume (0.5 mL) of a sterile 10%
sodium thiosulphate solution will be added to these bottles to reduce
any residual oxidant in the sample. Slides used for collection of
biofilms are described above. These will be collected in new pre-cleaned
sterile 50 mL polypropylene tubes containing 45 mL of sterile phosphate
buffer water with 0.1 mL of a 10% sodium thiosulphate solution. Water
samples will be collected from valved sample taps located on the
discharge line of the loop recycle tank. Slide samples will be collected
from the polycarbonate sample holder described above and in accordance
with the sample schedule (Table 2). All samples will be assayed as soon
as possible following collection. When assays cannot be completed within
four hours of collection, they will be held at 42 C, and assayed within
96 hours of collection. Excess samples will be discharged to the public
wastewater collection system. Spent sample vessels will be discarded
(metals, non-metals, TOC) or re-used (slides, polypropylene bottles) as
appropriate.

**C. Sample Handling and Custody Requirements**

All sample vessels will be labeled according to the following code:

Loop number: Loop 2 = L2

Sample location: Discharge = D, Glass slide = G, PVC slide = P, Iron
slide = I

Elapsed time: 0 through 504 h

Date: collection date

For example, L2D-336-020804 designates a sample from loop #2 collected
from the discharge tap at an elapsed time of 336 hours on February 8,
2004.

Analysis of collected samples will be conducted at the T&E facility and
at the AWBERC facility laboratories. Specifically, residual chlorine
analysis will be conducted on site at the time of sample collection. TOC
analyses will be conducted at the T&E facility, normally within hours of
sample collection. To ensure that labeled samples are transferred to the
analyst in a timely fashion following collection, the samples destined
for TOC analysis are placed in the refrigerator in the instrumentation
laboratory of the T&E facility. This ensures that the samples are held
under refrigeration prior to sample analysis. All other samples are
placed in a cooler and transported to the AWBERC facility. Samples for
metals and non-metals analysis are placed in the refrigerator in room
B22, along with a sample custody sheet. Samples for microbiological
analysis are placed in room 379.

**D. Analytical Methods Requirements**

**Table 4. Methods for analysis of water and biofilm samples**

  ----------------------------------------------------------------------------
  Critical           QC Procedure  Frequency    Acceptance    Corrective
  Measurement                                   Criteria      Actions
  ------------------ ------------- ------------ ------------- ----------------
  HPC, Method 9215 C Growth        Each new lot growth with   Discard media
  ^1^ using R2A agar control,      of media     no atypical   batch, remake,
                     known HPC                  colony        rerun experiment
                     colonies                   morphology    
                     grown on R2A                             
                     agar                                     

                     Precision     Each Sample  Any one of 3  measurements not
                     Assessment    assessed in  counts must   meeting this
                                   triplicate   have a RPD    requirement are
                                                \<70% from    outliers.
                                                other two     
                                                samples       

                     Sterility     Each         No growth     Check for
                     check         sampling                   sources of
                                                              contamination,
                                                              reanalyze
                                                              sterility check,
                                                              assess sample
                                                              results

  pH, Method         Accurcy       See Facility See Facility  Recalibrate pH
  4500-H^+^ B ^1^                  Manual       Manual        electrode using
                                   Section      Section 7.5.1 buffers
                                   7.5.1                      

  Residual Chlorine, Accuracy      Daily,       No color      Recalibrate
  Method 4500-Cl G                 Deionized    development   colorimeter.
  ^1^                              water blank                

  Organic carbon,    Accuracy      See Section  See Section   Recalibrate
  Method 415.1 ^2^                 II E.        II E.         organic carbon
                                                              instrument.
  ----------------------------------------------------------------------------

^1^ American Public Health Association, American Water Works
Association, Water Environment Federation. 1998. Standard Methods for
the Examination of Water and Wastewater 20^th^ ed. Washington, D.C.

^2^ U.S. Environmental Protection Agency. 1983. Methods for Chemical
Analysis of Water and Wastes. EPA-600/4-79-020 Revised March 1983.
Washington, D.C.

**E. Quality Control/Quality Assurance Requirements**

The quality assurance checks for operation of the DSS include
calibration and standardization of flow meters, thermocouples, pressure
indicators, and on-line monitors which include pH, turbidity,
oxidation-reduction potential, residual oxidant, and dissolved oxygen.
The nature and frequency of these checks are identified in the
Distribution System Simulator Facility Manual. The QC checks for
heterotrophic plate count (HPC) assays, pH, and residual chlorine
measurements are presented in Table 4. For organic carbon analysis,
prior to initiating sample analysis a four point calibration is run
using standard solutions containing 0.0, 0.5, 3.0, and 5.0 mg/L of a lab
control samples(LCS) . A surrogate tap water is analyzed with a
tap+spike. After every five samples a1.0 mg/L LCS or a 3.0 mg/L standard
check is run. Following an analytical series, carbon free water is
injected into the instrument.

**F. Instrument/Equipment Testing, Inspection, and Maintenance
Requirements**

DSS equipment testing, inspection and maintenance will be in accordance
with the specifications identified in the Distribution System Simulator
Facility Manual. Testing inspection and maintenance of equipment
required for completion of analytical measurements will be conducted as
needed to ensure proper operation, however given the limited duration of
this study it is not anticipated that inspections will be required
outside of the normal quality assurance checks identified above.

**G. Instrument Calibration and Frequency**

The protocols and frequencies of calibration of the instruments, gauges,
and meters associated with the DSS are identified in the Distribution
System Simulator Facility Manual. Calibration and standarization of the
organic carbon analyzer are identified in Section II. E. of this
document.

**H. Inspection/Acceptance Requirements for Supplies and Consumables**

Supplies and consumables will be inspected upon receipt by the
biological science or engineering technicians. Acceptance of these will
be based upon visually determining that received material is consistent
with project requirements, packaging is in tact or there is no obvious
damage to the received materials. Items identified as damaged or
contaminated will be declined.

**I. Data Acquisition Requirements (Non-Direct Measurements)**

Use of non-direct measurements is not anticipated. However, if such
measurements are used, the source of this information will cited.

**J. Data Management**

DSS operating data will be recorded at the time of sample collection in
a bound notebook. Organic carbon and chemical analyses will be recorded
electronically and archived by the analysts, in a database or
spreadsheet format. Laboratory bench sheets will be used to record
conditions and results of microbiological tests (see attachment). These
sheets will be archived in a binder. Operating conditions and analytical
results will be entered into an electronic spreadsheet (Lotus 1-2-3 or
equivalent) and archived. Data transferred to the spreadsheet will be
reviewed for accuracy and consistency with recorded values. Data
reduction and manipulation will be via transfer of electronic data files
to alternate spreadsheet formats.

**III. Assessment/Oversight**

**A. Assessments and Response Actions**

This is a basic research project. No assessments of the research are
planned.

**B. Reports to Management**

This work will be conducted over a relatively brief period of time. A
final report detailing the research completed from this effort is
planned. The principle investigator is responsible for preparation of
the final report which will be in the form of a presentation (oral or
poster) and/or a written article for submission to a scientific journal.
Review of this output will be at the Branch and Division level via
normal ORD clearance procedures.

**IV. Data Validation and Usability**

**A. Data Review, Validation, and Verification Requirements**

System operations data will be collected daily and reviewed on a weekly
basis to determine if operating conditions are within range of the
quality assurance criteria. If these conditions are found to be outside
the ranges as specified in this document, appropriate actions will be
taken to bring systems operations within specifications. Additionally,
data obtained while the system was operating outside of specified ranges
will be flagged, and will be reported as out of range.

All samples assayed for critical parameters will be conducted in
duplicate and compared against known standards. Analysis of duplicate
samples will be used as a measure of precision while accuracy of
individual measurements will be determined based upon comparison of
analytical results to known standards.

**B. Validation and Verification Methods**

The quality assurance objectives of this project are based upon project
requirements and are designed to ensure that the data generated during
the project are of known and acceptable quality to achieve the projects
technical objectives.

Variables being monitored for this project are identified in Section II.
D. of this document. Validated methods to be used for analysis of total
organic carbon, free residual chlorine, and total heterotrophic bacteria
are consistent with standard protocols as described in Standard Methods
for the Examination of Water and Wastewater, 1998. Calibration of the
monitors used for determinations of pH, and residual chlorine will be
completed weekly, in accordance with the Facility Manual (1996). Samples
submitted for HPC analysis will be analyzed in triplicate spiking such
samples is not considered feasible, however, for each lot of media
produced control plates will be incubated and inspected for growth.
Similarly, dilution water sans sample will be used to inoculate R2A
media and will be inspected for growth. These "control" plates will be
used to verify sterility of the media and dilution water. Growth of
microbes on R2A media will be verified by inoculation of the R2A agar
plates with water samples containing heterotrophic bacteria. Failure of
the media to produce colonies following incubation as described in
Section II B. indicates that the growth medium may be flawed. If this
occurs, data collected during this phase of the experiment will be
flagged, the experiment will be terminated, and repeated with freshly
prepared media.

Systems operating data will be collected daily (5 days/week) and
recorded by the engineering technician or a designee. Similarly, the
engineering technician or a designee will collect all samples according
to the schedule described in Section II. C. Recorded information will be
transmitted to the principle investigator or a designee and will be
archived in a spreadsheet format. Manually recorded information will be
retained in a file folder and/or a record book which will be maintained
by the principle investigator or a designee. Similarly, results obtained
from the analysis of all samples will be archived in a spreadsheet
format and manually recorded results will be retained as above.

**C. Reconciliation with Data Quality Objectives**

Results from individual analyses will be calculated in accordance with
the appropriate standard method. For samples obtained from coupons,
results will be calculated on the basis of surface area.

*Precision*:

For HPC determinations, countable R2A plates (30-300 colonies/plate)
will be recorded and the sample standard deviation will be computed
according to the following equation:

standard deviation

Where:

*n* = the number of analyses

*x* = each value from an HPC analysis for a given sample.

If any one of the three values has a relative percent difference \>70%
from either of the other two values, that value will be treated as an
outlier. Relative percent difference will be calculated according to the
following equation:

relative percent difference =

Where:

*s* = sample value

*d* = duplicate value

*Accuracy*:

By definition, visible colonies formed on R2A media following a 7-8 day
incubation period at 20-28^o^C will be considered to have developed from
individual cells. To ensure that the R2A media will support growth, a
dilute culture of laboratory grown heterotrophic bacteria will be plated
out on each lot of media and checked for growth. If no growth occurs, or
if colony morphology is atypical, the media will be discarded. To ensure
that sterility is maintained, sterility controls will be used. For
biofilm samples this means that a sterile coupon will be treated as a
biofilm sample, and analyzed for HPC. Cell growth on these samples
indicates that sterility was not maintained, and data collected from
that sample session must be adjusted to account for such growth.

*Completeness*:

Objectives for data capture are 80% for the critical parameters.
Completeness is defined as the ratio of the number of valid measurements
to the total number of measurements planned.

**References**

American Public Health Association, American Water Works Association,
Water Environment Federation. 1998. Standard Methods for the Examination
of Water and Wastewater 20^th^ ed. Washington, D.C.

Facility Manual for NRMRL\'s Experimental Pipe Loop System, NRMRL
internal document. Sam Hayes, NRMRL, Cincinnati, OH 1996.

U. S. Environmental Protection Agency. USEPA. 1983. Methods for the
Analysis of Water and Wastes. EPA-60014-79-020 Office of Technology
Transfer, Washington, D.C.

**Appendix 1**

Distribution System Simulator (Ductile Iron Pipe)

[Microbiological Analysis Laboratory Bench Sheet]{.smallcaps}

Sample Collection Date:\_\_\_\_\_\_\_\_\_\_\_\_\_
Time:\_\_\_\_\_\_\_\_\_\_\_Analyst:\_\_\_\_\_\_\_\_\_\_\_

Plates Inoculated
on:\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_Time:\_\_\_\_\_\_\_\_\_\_\_Analyst:\_\_\_\_\_\_\_\_\_\_\_

Plates Enumerated
on:\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_Time:\_\_\_\_\_\_\_\_\_\_\_Analyst:\_\_\_\_\_\_\_\_\_\_\_

Incubation Temperature, in:\_\_\_\_\_\_\_\_\_\_\_\_C;
out:\_\_\_\_\_\_\_\_\_\_\_\_C

  ---------- ------------- ---------- --------- --------- --------- --------
                                      R2A                           
                                      Colony                        
                                      Forming                       
                                      Units                         

  Lab. No.   Sample I.D.   Dilution   Plate A   Plate B   Plate C   Avg.

                                                                    

                                                                    

                                                                    

                                                                    

                                                                    

                                                                    

                                                                    

                                                                    

                                                                    

                                                                    

                                                                    

                                                                    

                                                                    

                                                                    
  ---------- ------------- ---------- --------- --------- --------- --------

Include media control and dilution water control samples for each
analytical series.