# **Quality Assurance/Quality Control Manual**

**Ohio Water Microbiology Laboratory**

***By* Donna S. Francy, Rebecca N. Bushon, Erin E. Bertke, Amie M. G.
Brady, Christopher M. Kephart, Christina A. Likirdopulos, and Donald M.
Stoeckel\
**

**Updated November 2006**

######## [ABSTRACT](#abstract-1)

[**INTRODUCTION**](#introduction)

#### **Purpose and scope**

#### **Organizational structure**

[**GENERAL LABORATORY QUALITY ASSURANCE/QUALITY-CONTROL
PRACTICES**](#general-laboratory-quality-assurancequality--control-practices)

**Analytical methods**

**Training**

**Safety**

**Laboratory materials and equipment**

> **General sterility and cleanliness**
>
> **Autoclaves**
>
> **Laboratory water**
>
> **Analytical balances**
>
> **Hoods**
>
> **Specific conductance, pH, and turbidity meters**
>
> **Micropipettors**
>
> **Vacuum pump**
>
> **Thermometers, incubators, water baths, refrigerators, and freezers**
>
> **Microscope and centrifuge**

##### **Sample management and documentation**

[**METHODS OF ANALYSIS, MEDIA AND REAGENT PREPARATION, AND ANALYTICAL
QUALITY-CONTROL
PROCEDURES**](#methods-of-analysis-media-and-reagent-preparation-and-analytical-quality-control-procedures)

**Fecal-indicator bacteria**

**Enteric viruses**

**Coliphage**

***Cryptosporidium* and *Giardia***

#### **Actinomycetes**

[**REFERENCES**](#references)

**APPENDIXES**

###### [**OWML Service request form**](http://oh.water.usgs.gov/micro/qcmanual/appenda.pdf)

> **AA**. [**Equipment QC Log
> Sheet**](http://oh.water.usgs.gov/micro/qcmanual/appendaa.pdf)

A.  [**Sample log
    sheet**](http://oh.water.usgs.gov/micro/qcmanual/appendb.pdf)

B.  [**MI agar
    preparation**](http://oh.water.usgs.gov/micro/qcmanual/appendc.pdf)

C.  [**Analysis of *E. coli* and total coliforms using Colilert
    Quantitray**](http://oh.water.usgs.gov/micro/qcmanual/appendd.pdf)

D.  [**Presence/absence analysis of *E. coli* and total coliforms using
    Colilert, large volume
    samples**](http://oh.water.usgs.gov/micro/qcmanual/appende.pdf)

E.  [**mTEC agar and urea-phenol solution
    preparation**](http://oh.water.usgs.gov/micro/qcmanual/appendf.pdf)

###### [**Modified mTEC agar preparation**](http://oh.water.usgs.gov/micro/qcmanual/appendg.pdf)

###### [**mEI agar preparation**](http://oh.water.usgs.gov/micro/qcmanual/appendh.pdf)

F.  [**Analysis of *Clostridium perfringens* in environmental water
    samples**](http://oh.water.usgs.gov/micro/qcmanual/appendi.pdf)

G.  [**Media quality-control
    procedures**](http://oh.water.usgs.gov/micro/qcmanual/appendj.pdf)

H.  [**Media and buffer quality-control log
    sheet**](http://oh.water.usgs.gov/micro/qcmanual/appendk.pdf)

I.  [**Expendable supplies request
    form**](http://oh.water.usgs.gov/micro/qcmanual/appendl.pdf)

J.  [**Buffer
    preparation**](http://oh.water.usgs.gov/micro/qcmanual/appendm.pdf)

K.  **[RT-PCR elution
    protocol](http://oh.water.usgs.gov/micro/qcmanual/appendn.pdf)\
    N1. [Inhibitor removal
    protocol](http://oh.water.usgs.gov/micro/qcmanual/appendn1.pdf)**

> **N2. [RT-PCR
> analysis](http://oh.water.usgs.gov/micro/qcmanual/appendn2.pdf)**

L.  **[Coliphage detection by USEPA Method 1602: Single-Agar Layer
    (SAL](http://oh.water.usgs.gov/micro/qcmanual/appendo.pdf))\
    O1. [NWIS coding for coliphage
    results](http://oh.water.usgs.gov/micro/qcmanual/appendo1.pdf)**

M.  [**QC for Method 1602: Single-Agar Layer
    (SAL)**](http://oh.water.usgs.gov/micro/qcmanual/appendp.pdf)

N.  [**Coliphage detection by USEPA Method 1601: Two-Step
    Enrichment**](http://oh.water.usgs.gov/micro/qcmanual/appendq.pdf)

O.  [**QC for Method 1601: Two-Step
    Enrichment**](http://oh.water.usgs.gov/micro/qcmanual/appendr.pdf)

###### **[Filtration and elution of samples by USEPA Method 1623 -- ultrafiltration method](http://oh.water.usgs.gov/micro/qcmanual/appendt.pdf)** 

###### [**Detection of Actinomycetes in water**](http://oh.water.usgs.gov/micro/qcmanual/appendu.pdf)

**V. [Master Cycler QC instructions and
form](http://oh.water.usgs.gov/micro/qcmanual/appendv.pdf)**

**TABLES**

**1****. Current laboratory personnel and qualifications**

**2. Acceptance criteria for laboratory water quality-assurance checks**

**3. Acceptance criteria for laboratory thermometers**

**4. Acceptance criteria for laboratory refrigerators, freezers,
incubators, and water baths**

**5. Methods for fecal-indicator bacteria analysis used by the OWML**

**6. Information on media, buffered-dilution water, and reagents
prepared and stored in the OWML**

# **Quality Assurance/Quality Control Manual**

# 

# **Ohio Water Microbiology Laboratory**

# 

# **ABSTRACT**

**The U.S. Geological Survey (USGS), Ohio Water Microbiology Laboratory
(the OWML) provides water-quality data on microorganisms of public
health significance for a variety of projects within the USGS.
Currently, the OWML analyzes samples for and provides training on
bacterial indicators, coliphage, enteric viruses, and two protozoan
pathogens---*Cryptosporidium* and *Giardia*.**

**Quality-assurance and quality-control (QA/QC) practices for the
operation of OWML are described in this manual. The Laboratory Manager,
Laboratory Coordinator, Chemical Hygiene Officer, and laboratory and
field staff are responsible for implementing QA/QC procedures. This
includes correctly following methods of analysis, media and reagent
preparation and storage, and analytical quality-control procedures. A
sample management and documentation system involves the use of service
request forms and login ID's for each sample. A laboratory information
management system (LIMS) has been implemented to store sample login
information and results. Laboratory equipment maintenance and
calibration records are also stored in the LIMS.**

# **INTRODUCTION**

**The USGS Ohio Water Center Ohio Water Microbiology Laboratory (the
OWML) mainly provides analytical data for projects within the Ohio
Center. With the growth of microbiological monitoring within the WRD,
however, the OWML has begun analyzing samples for other Water Center
programs by request. Samples are collected to determine the presence of
microbiological organisms of public health significance in ground waters
and surface waters for a variety of study objectives. For example, some
local studies are done to judge compliance with standards for protection
of public health in swimmable or drinkable waters. Other studies
investigate the occurrence, distribution, and trends of pathogenic
organisms and indicators in surface and ground waters and relate these
to environmental and water-quality factors.**

**The OWML fulfills analytical requirements of the WRD by analyzing
environmental samples for bacterial indicators, coliphage, enteric
viruses, and two protozoan pathogens---*Cryptosporidium* and *Giardia*.
OWML personnel provide assistance for project planning and training on
three major groups of microorganisms of public health significance in
the United States: bacteria, viruses, and protozoa. As funds become
available for expansion, the OWML plans to add other analytical methods
and types of microorganisms to its analytical list. The OWML is not
involved in method development at the present time, but instead tests
new methods developed by others for applicability to ambient monitoring
programs.**

**The OWML is committed to providing quality microbiological analytical
services to the USGS. The quality assurance/quality control (QA/QC)
program is designed to ensure the production of scientifically sound,
legally defensible data of known and documented quality. The
effectiveness of this program relies on clearly defined objectives,
well-documented procedures, and management support.**

## Purpose and Scope

**The purpose of this manual is to identify and document practices and
standard operating procedures for those activities of the OWML that
affect quality of data. The manual provides OWML personnel and customers
with general descriptions of quality practices and goals to aid in the
interpretation of data. This manual is intended to be an unpublished,
dynamic document that will be frequently updated as laboratory
activities expand or change.**

## Organizational Structure

**The Laboratory Manager (1) oversees the daily operations of the OWML,
(2) directs technical personnel in the proper performance of laboratory
procedures and the reporting of results, (3) ensures that appropriate
methods are used, (4) plans activities leading to testing and
modification of analytical procedures, and (5) designs and implements a
comprehensive QA/QC program. The Laboratory Manager is responsible for
initiating the QA/QC program, providing information and training to the
staff, and periodically reviewing QA/QC activities.**

**The Laboratory Coordinator oversees the daily operations of the OWML,
ensures that the equipment is properly maintained and calibrated, orders
supplies and equipment, and oversees and performs analytical work. The
Laboratory Coordinator implements the QA/QC program in the daily tasks
of conducting analyses, performing quality control checks, and
calculating and reporting results.**

**The Chemical Hygiene Officer oversees safety operations in the
laboratory with assistance from the Laboratory Manager and Laboratory
Coordinator.**

**The laboratory and field staffs are responsible for correctly
implementing collection and analysis procedures and for identifying and
working with supervisors to correct and avoid potential problems.**

**Table 1. Current laboratory personnel and qualifications.**

+--------------+--------------+-----------+----------------------------+
| **NAME**     | **LABORATORY | **USGS    | **Education/experience**   |
|              | TITLE**      | TITLE**   |                            |
+--------------+--------------+-----------+----------------------------+
| **Donna      | **Mic        | **Hy      | **B.A. Biology, M.S.       |
| Francy**     | robiology/** | drologist | Environmental Science,**   |
|              |              | GS-13**   |                            |
|              | **Water      |           | **Certified Clinical       |
|              | Quality      |           | Microbiologist**           |
|              | Specialist** |           |                            |
|              |              |           | **15 years experience in   |
|              |              |           | water quality and          |
|              |              |           | environmental              |
|              |              |           | microbiology**             |
+--------------+--------------+-----------+----------------------------+
| **Rebecca    | **Laboratory | **Hydr    | **B.S. Biology**           |
| Bushon**     | Manager**    | ologist** |                            |
|              |              |           | **10 years experience in   |
|              |              | **GS-12** | microbiology**             |
+--------------+--------------+-----------+----------------------------+
| **Don        | **Project    | **Hy      | **B.S. Microbiology**      |
| Stoeckel**   | Chief/       | drologist |                            |
|              | Special      | GS-12**   | **M.S. Environmental       |
|              | Projects**   |           | Science**                  |
|              |              |           |                            |
|              |              |           | **Ph.D. Soil Science**     |
|              |              |           |                            |
|              |              |           | **10 years experience in   |
|              |              |           | microbiology**             |
+--------------+--------------+-----------+----------------------------+
| **Chris      | **Molecular  | **StuTra  | **B.S. Microbiology**      |
| Kephart**    | Analyst**    | in(Hyd)** |                            |
|              |              |           | **Working on M.S.          |
|              |              | **GS-9**  | Environmental Science**    |
|              |              |           |                            |
|              |              |           | **7 years experience in    |
|              |              |           | microbiology**             |
+--------------+--------------+-----------+----------------------------+
| **Amie       | **Laboratory | **Hy      | **B. S. Environmental      |
| Brady**      | C            | drologist | Science**                  |
|              | oordinator** | GS-9**    |                            |
|              |              |           | **B.S. Plant Biology**     |
|              |              |           |                            |
|              |              |           | **M.S. Environmental       |
|              |              |           | Science**                  |
|              |              |           |                            |
|              |              |           | **7 years experience in    |
|              |              |           | microbiology**             |
+--------------+--------------+-----------+----------------------------+
| **Christina  | *            | **Hy      | **B.A. Chemistry**         |
| Li           | *Specialized | drologist |                            |
| kirdopulos** | Lab Analyst/ | GS-9**    | **M.S.P.H. Environmental   |
|              | Chemical     |           | Sciences & Engineering**   |
|              | Hygiene      |           |                            |
|              | Officer**    |           | **7 years experience in    |
|              |              |           | microbiology**             |
+--------------+--------------+-----------+----------------------------+
| **Erin       | **General    | **Bi      | **B.S. Environmental       |
| Bertke**     | Lab          | ologist** | Science**                  |
|              | Analyst**    |           |                            |
|              |              | **GS-7**  | **3 years experience in    |
|              |              |           | microbiology**             |
+--------------+--------------+-----------+----------------------------+

# **GENERAL LABORATORY QUALITY ASSURANCE/QUALITY- CONTROL PRACTICES**

**An overview of analytical methods, training policies, safety,
laboratory maintenance, sample management, and data documentation is
given in this section.**

## Analytical methods

**The methods used by the OWML can be categorized into four groups:
compliance, official, provisional, and experimental. The United States
Environmental Protection Agency (USEPA) and others in the research
community are continuously developing new methods for detecting and
quantifying microbiological pathogens and indica­tors in water;
therefore, several types of methods for target organisms may be
currently in use at the OWML.**

**Compliance methods are those published by USEPA in the Federal
Register and are used to determine compliance with standards for
protection of public health in swimmable or drinkable waters. Analytical
methods for fecal-indicator bacteria are often in this group because
they are straightforward, quantitative, and routinely used.**

**Official methods are those noncompliance methods published by
water-analysis authorities such as American Public Health Association,
the U.S. Environmental Protection Agency, or the USGS. Official methods
should be well established, have known levels of bias and variability,
and be relatively easy to apply in field opera­tions or have holding
times long enough to allow shipping to a central laboratory for
analysis.**

**Provisional methods are published methods that are still being
validated by the method developer, usually the USEPA. For these methods,
the method developer establishes precision and accuracy and ensures the
methods are adequately tested. Because methods for detection of protozoa
are complex, qualitative to semiquantitative, expensive, and very time
consuming, these methods are often provisional.**

**Experimental methods are unpublished methods that are currently being
testing to establish QA/QC practices and determine applicability to
ambient monitoring programs.**

## Training

**The Laboratory Manager and Laboratory Coordinator are responsible for
ensuring that laboratory employees receive proper training in analytical
methods and laboratory procedures and for documenting any training
received. In particular, laboratory employees will be trained in sterile
technique before handling samples for microbiological analysis. A new
employee will receive orientation and skills training. New or
established employees may receive training on new methods given by the
method developer. The Laboratory Coordinator will maintain training
records for microbiological methods on file by employee; this includes
on-the-job training as certification of proficiency in microbiology.**

**The Laboratory Coordinator, Chemical Hygiene Officer, and Water Center
Safety Officer provide safety orientation to new employees and safety
education to all employees. The employee orientation covers general
safety issues, emergency procedures, standard-safety operations, the
chemical-hygiene plan, hazardous-waste management, waste disposal, and
location of safety equipment.**

## Safety

**Detailed laboratory safety practices and responsibilities are
described in the Chemical Hygiene Plan. Safety activities include
safeguards to avoid electric shock; prevent fire; prevent accidental
chemical spills; and minimize microbiological dangers, facility
deficiencies, and equipment failures.**

**Laboratory personnel that are isolating microorganisms from natural
sources must be made aware that pathogens may be present in
environmental samples. Technicians are to wear disposable gloves and lab
coats when handling samples that are likely to contain pathogens. Safety
glasses are worn if there is a chance of projectiles, aerosols, or other
foreign matter entering the eye. This includes when using
positive-pressure air to blow out any remaining liquid during the
ultrafiltration process for *Cryptosporidium* and *Giardia.* Laboratory
personnel will receive immunizations for pathogens on a project-specific
basis. Each project sending samples to the OWML is required to have a
project safety plan\--copies are available for OWML employees.
Immunizations are offered to all OWML workers for Hepatitis A virus,
Hepatitis B virus, and tetanus.**

**Safety equipment is tested at regular intervals. Safety showers and
eyewash stations are tested annually and recorded in the LIMS. Fire
extinguishers are inspected annually. The Chemical Hygiene Officer
maintains a list of chemicals and arranges for a contract for disposal
of hazardous waste.**

## Laboratory materials and equipment

**The Laboratory Manager sets policies for preventive maintenance and
calibration of laboratory materials and equipment. Two QA/QC logbooks
are kept in the laboratory bookshelf with records of quality-assurance
checks of materials and equipment up through September 30, 2006. The
logbooks are for (1) equipment -autoclaves, balances, pipettors, hoods,
and thermometers; and (2) laboratory water. Examples of equipment log
sheets are in [Appendix
AA](http://oh.water.usgs.gov/micro/qcmanual/appendaa.pdf). Results of
quality-assurance checks of materials and equipment starting in FY 04
are stored in the LIMS. Quality-control checks that are required LIMS
entries are listed in italics below.**

-   ***The Laboratory Manager or Laboratory Coordinator must review
    QA/QC quarterly reports from LIMS to ensure procedures are followed
    and problems are properly addressed.***

**For some pieces of equipment, the use of daily logbooks to record
operating times and other types of frequent entries are required. A
daily logbook is kept with the autoclaves and the water-quality meters
(pH, specific conductance, and turbidity).**

### **General sterility and cleanliness**

**The sterility and cleanliness of the laboratory is necessary to ensure
the integrity of samples and analytical procedures.**

-   **Traffic through the laboratory is restricted to those doing work
    in the laboratory, especially when analytical work is being done.**

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-   **The countertops are wiped down with surface disinfectants, such as
    Conflikt (Decon Labs, Inc., King of Prussia, PA) or 70 percent
    ethanol, before and after use.**

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-   **Antimicrobial soap is available at various laboratory sinks to
    facilitate hand washing before and after laboratory work.**

**Clean and sterile glassware that is free of detergent residue is
crucial to ensure valid results in microbiology.**

-   **Dirty dishes are placed on a moveable laboratory cart after use
    and are not to be stored on countertops. Dishes are washed in a
    dishwasher or by hand with hot water and laboratory-grade
    phosphate-free detergent. Dishes are rinsed with tap water and then
    deionized water.**

### **Autoclaves** 

**Sterilization is the process that eliminates living organisms from
substances or objects. The OWML is equipped with three autoclaves for
sterilization of glassware, reagents, media, and disposables---two
medium-sized autoclaves (Market Forge) that are operated in the side
laboratory and one large autoclave (Consolidated) that is operated in
the warehouse.**

-   **Dishes that need to be sterilized are wrapped in aluminum foil or
    kraft paper and placed in the autoclave for moist heat
    sterilization. Clean and sterile dishes are stored in closed
    cupboards until use.\
    **

-   **The autoclaves are operated at 15 lb/in^2^ steam pressure,
    producing an inside temperature of 121 to 124^o^C (American Public
    Health Association, 1998, Section 9020B). Do not overload the
    autoclave. Autoclave time depends on the type and amount of
    equipment as follows:**

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-   **Glassware and up to 250 mL of liquid---15 minutes**

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-   **500 to 2,000 mL liquid---30 minutes**

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-   **Greater than 2,000 mL to 6,000 mL liquid---15 minutes per 1,000
    mL**

-   **Greater than 6,000 mL liquid---90 minutes**

-   **Carbohydrate-containing media---15 minutes (no more than 250 mL
    volumes)**

[]{.mark}

-   **Contaminated materials and discarded cultures---30 minutes, allow
    autoclave chamber pressure to decrease, then run for a 60 minute
    cycle**

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-   **Operating temperature and pressure are checked once a week.
    Heat-sterilizing tape is used with each run to identify supplies
    that have been properly sterilized and checks the performance of the
    autoclave. *The performance is also checked monthly by using spore
    indicators and recorded in the LIMS.***

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-   **If the autoclave does not reach the specified temperature or fails
    the spore indicator test, service the autoclave and re-sterilize all
    glassware and reagents that were insufficiently sterilized.**

**For the two medium-sized autoclaves, general maintenance is as
follows:**

-   **The autoclaves are operated using deionized water.**

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-   **At the end of the day, autoclaves are drained. Twice a month,
    autoclaves are cleaned with mild soap, rinsed with water, and
    drained. The condensate holding tank is drained daily or as needed.
    *The cleaning date is recorded in the LIMS.***

-   **Twice a year, have a contractor inspect and calibrate the
    autoclaves and perform preventive maintenance. *Preventive
    maintenance dates are recorded in the LIMS.***

-   **Twice a year, clean the chambers with 10% muriatic acid and flush
    well with water. *Cleaning dates are recorded in the LIMS.***

**For the large autoclave, general maintenance is as follows:**

-   **Once a month, clean chamber with water and liquinox. *Cleaning
    dates are recorded in the LIMS.***

-   **Twice a year, have a contractor perform preventive maintenance and
    inspection, clean and service the generator, clean the door gasket
    and head ring, apply graphite to the door gasket, oil the door hinge
    pins, and lubricate the door hub. P*reventive maintenance dates are
    recorded in the LIMS.***

-   **Twice a year, clean the chamber with 10% muriatic acid and flush
    well with water. *Cleaning dates are recorded in the LIMS.***

### **Laboratory water**

**The OWML has three types of laboratory water:**

**(1) Type III deionized water ("deionized water") produced from City of
Columbus tap water for general laboratory use. The deionized water unit
and tap are stored in the warehouse. The system is described in Francy
and others (1998). The vendor changes the cation and anion columns,
moves forward the standby mixed-bed column, installs a new standby tank,
and changes the carbon filter when the red service light illuminates.
*Maintenance checks are recorded in the LIMS*.**

**(2) Reagent-grade water produced using a Millipore MilliQ system
("MilliQ water"). Deionized water is used as source water for the MilliQ
system. Reagent water is used for cultivation media and additives (mTEC,
MI, mEI, antibiotic stocks, and others) as well as for preparation of
reagents for sensitive procedures (elutions, PCR, hybridization, and
others). The MilliQ cartridges are changed by OWML laboratory personnel
when the service light blinks and the display message reads "EXCH.
CARTRIDGES." *Indicate the date of cartridge change in the LIMS.***

**(3) Deionized water is stored in a laboratory carboy ("stored water")
and used for rinsing of dishware and other supplies.**

**A variety of quality-control checks are routinely done on the three
types of water and may differ depending on the type of water. Acceptance
criteria are listed in table 2. For deionized water, two levels of
acceptance criteria are listed---(1) a warning level wherein the system
is inspected and constituents are retested and (2) a shut-down level.
For MilliQ water, only a shut-down level is listed in table 2. For
stored water, if criteria are not met, the container is cleaned out,
refilled, and retested.**

-   ***Quarterly checks of specific conductance and turbidity are done
    on all three types of water and recorded in the LIMS.* Instructions
    for performing this check are in the back of the equipment QA/QC
    logbook.**

-   ***Quarterly checks of bacterial growth are done on the MilliQ water
    and recorded in the LIMS.* Instructions for performing this check
    are in the back of the equipment QA/QC logbook.**

-   ***A blank of deionized water is submitted to the National Water
    Quality Laboratory (NWQL) annually and analyzed for low level
    nutrients (Schedule 1217), and total-organic carbon (Labcode 114),
    and the results are recorded in the LIMS.* We no longer analyze a
    blank for trace elements and low-level major ions because the need
    for these low-level analyses is project specific.**

-   **The stored deionized water carboy is to be emptied completely and
    cleaned with Liquinox and water every other week. *Record cleanings
    in the LIMS.***

**Table 2. Acceptance criteria for laboratory water quality-assurance
checks**

\[Adopted from USEPA (1978), APHA (1998), and ASTM (1999); NA is not
applicable; constituents highlighted in gray are no longer required
tests\]

  ------------------------- --------------- ----------- ------------ --------------
  ** **                     **DEIONIZED**               **MILLIQ**   **STORED**

  **ACTION**                **warning**     **shut      **shut       **clean and
                                            down**      down**       refill**

  **Specific conductance    **3**           **5**       **2**        **3**
  (μs/cm)**                                                          

  **Turbidity**             **1**           **5**       **1**        **1**

  **Heterotrophic plate     **NA**          **NA**      **\<1**      **NA**
  count (colonies/mL)**                                              

  **Total organic carbon    **0.2**         **10**      **NA**       **NA**
  (mg/L)**                                                           

  **Sodium (mg/L)**         **0.1**         **1**       **NA**       **NA**

  **Nutrients individual    **0.1**         **1**       **NA**       **NA**
  (mg/L)**                                                           

  **Heavy metals,           **1**           **10**      **NA**       **NA**
  individual (Cd, Cr, Cu,                                            
  Ni, Pb, Zn) (μg/L)**                                               

  **Other trace elements    **3**           **50**      **NA**       **NA**
  (μg/L)**                                                           
  ------------------------- --------------- ----------- ------------ --------------

###  

### **Analytical balances** 

**Analytical balances are used for accurate weighing of reagents and
media*. They are checked and calibrated annually by the manufacturer's
service technician, and the results are recorded in the LIMS*. Balances
must rest on a firm, level surface. Balance trays are wiped off after
each use with water or a surface disinfectant, such as Conflikt or 70
percent ethanol.**

### **Hoods**

**The Ohio Water Center has four types of hoods--- (1) two biosafety
cabinets, (2) a laminar-flow hood, (3) a hazardous-waste fume hood, and
(4) a PCR workstation. To ensure proper use of hoods 1, 2, and 3, "quick
check" criteria for properly running each hood are posted on the
hoods.**

-   ***The operation of all hoods are checked and certified by a
    qualified inspector annually and recorded in the LIMS*.**

**The biosafety and laminar flow hoods have magnehelic pressure gauges
(MAG) that are used to monitor operation of the hoods. When using these
hoods, check to make sure the pressure gauge is reading at a level
approximately equal to the annually recorded MAG level on the
calibration sticker. A significant increase in pressure indicates that
the filters are dirty whereas a significant decrease in pressure
indicates an electrical problem.**

**The biosafety cabinets, laminar-flow hood, and PCR workstation (Hoods
1, 2, and 4) must be free from contamination by live organisms.**

-   **The working surfaces of the laminar-flow hood, the biosafety
    cabinets, and the PCR workstation (Hoods 1, 2, and 4) are wiped down
    with a surface disinfectant, such as Conflikt or 70 percent ethanol
    before and after general use. When working with pathogenic
    organisms, the hoods are wiped down with Dispatch Hospital Cleaner
    Disinfectant towels with bleach (Caltech Industries Inc.,
    Midland, MI) followed with 70 percent ethanol. For the biosafety
    cabinets, be sure to lift up the work surface and clean under this
    area periodically.**

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-   **The biosafety cabinets and PCR workstation (Hoods 1 and 4) have
    ultraviolet bulbs for germicidal purposes. The ultraviolet lights in
    the biosafety cabinets and PCR workstation are cleaned quarterly by
    wiping the bulbs with a soft cloth. C*leaning dates are recorded in
    the LIMS.* A bulb that is dull in the center needs to be replaced.
    *Record the bulb change in the LIMS. ***

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```
-   **Biannually, nonselective agar plates are exposed to airflow in the
    laminar-flow hood, the biosafety cabinets, and PCR workstation for 1
    hour (Hoods 1, 2, and 4). The plates are incubated at 35^o^C for 24
    hours and examined for contamination. *The results are record in the
    LIMS.***

**The hazardous-waste fume hood (Hood 3) must be checked to ensure that
it is operating properly.**

-   ***Check the operation of the hazardous-waste fume hood (Hood 3)
    quarterly by use of fume cartridges and record results in the
    LIMS*.**

### **Specific conductance, pH, and turbidity meters**

**With each use of the specific conductance, pH, or turbidity meter,
calibrate the instrument according to the manufacturer's instructions
(kept with the meter). Use a calibrated solution that is within the
range of the water sample to be measured. Label specific conductance and
pH buffer solutions with the date opened and discard working solution
weekly. Each piece of equipment has a daily logbook; record all
calibrations in the appropriate logbook.**

**Micropipettors**

**Micropipettors are used for the accurate delivery of small volumes*.
***

-   **Pipettors are sent to the manufacturer annually for cleaning,
    preventative maintenance, calibration, and adjustment, if necessary.
    *Preventive maintenance dates are recorded in the LIMS.*
    Preventative maintenance includes a new seal and piston cleaning
    annually, and a new shaft and reconditioned piston every 3 years.**

**Vacuum pump**

**The vacuum pump is mainly used for membrane filtration*.* The oil is
changed in the pump every 2 years. *Record the oil change in the
LIMS.***

### **Thermometers, incubators, water baths, refrigerators, and freezers**

**Thermometers are kept in three areas and are inventoried according to
storage and use: (1) extra thermometers for general laboratory use,
including the National Institute of Standards and Technology (NIST)
thermometer (2) daily-use water-bath, incubator, and refrigerator
thermometers, (3) digital thermometers, and (4) back-lab thermometers.**

-   **The NIST thermometer is calibrated and certified annually by an
    outside service technician. *Certification dates are recorded in the
    LIMS. ***

-   ***Extra and daily-use thermometers are checked quarterly against
    the NIST thermometer. Results are recorded in the LIMS and
    acceptance criteria are listed in table 4.* Criteria are based on
    use.**

-   **Digital thermometers are checked quarterly against the NIST
    thermometer and are calibrated annually by the manufacturer.
    *Results and calibration dates are recorded in the LIMS.***

+--------------+---------------+------------+----------------+------+
| **Table 3.   |               |            |                |      |
| Acceptance   |               |            |                |      |
| criteria for |               |            |                |      |
| laboratory   |               |            |                |      |
| th           |               |            |                |      |
| ermometers** |               |            |                |      |
+--------------+---------------+------------+----------------+------+
| *            | **Thermometer | **         | **Test         | **Cr |
| *Thermometer | i             | Location** | Temperature**  | iter |
| use**        | dentification |            |                | ia** |
|              | (will vary)** |            |                |      |
+--------------+---------------+------------+----------------+------+
| **NIST**     | **NIST**      | **Drawer** | **Certified**  | *    |
|              |               |            |                | *Cer |
|              |               |            |                | tifi |
|              |               |            |                | ed** |
+--------------+---------------+------------+----------------+------+
| **Daily      | **L**         | **W/B 1**  | **48°C**       | **±  |
| use**        |               |            |                | 1    |
|              |               |            |                | °C** |
| **water      |               |            |                |      |
| baths**      |               |            |                |      |
+--------------+---------------+------------+----------------+------+
|              | **S**         | **W/B 2**  | **37°C and     | **±  |
|              |               |            | 70°C**         | 1    |
|              |               |            |                | °C** |
+--------------+---------------+------------+----------------+------+
|              | **O**         | **W/B 3**  | **36°C**       | **±  |
|              |               |            |                | 1    |
|              |               |            |                | °C** |
+--------------+---------------+------------+----------------+------+
|              | **T**         | **W/B 5**  | **70°C**       | **±  |
|              |               |            |                | 1    |
|              |               |            |                | °C** |
+--------------+---------------+------------+----------------+------+
|              | **K**         | **W/B 6**  | **36°C and     | **±  |
|              |               |            | 48°C**         | 1    |
|              |               |            |                | °C** |
+--------------+---------------+------------+----------------+------+
| **Daily      | **U**         | **Inc 5**  | **35°C**       | **±  |
| use**        |               |            |                | 1    |
|              |               |            |                | °C** |
| **           |               |            |                |      |
| incubators** |               |            |                |      |
+--------------+---------------+------------+----------------+------+
|              | **R**         | **Inc 6**  | **35°C**       | **±  |
|              |               |            |                | 1    |
|              |               |            |                | °C** |
+--------------+---------------+------------+----------------+------+
|              | **Q**         | **Inc 7**  | **35°C**       | **±  |
|              |               |            |                | 1    |
|              |               |            |                | °C** |
+--------------+---------------+------------+----------------+------+
| **Daily      | **M**         | **Refrig   | **0°C**        | **±  |
| use**        |               | 1**        |                | 1    |
|              |               |            |                | °C** |
| **ref        |               |            |                |      |
| rigerators** |               |            |                |      |
+--------------+---------------+------------+----------------+------+
|              | **N**         | **Refrig   | **0°C**        | **±  |
|              |               | 2**        |                | 1    |
|              |               |            |                | °C** |
+--------------+---------------+------------+----------------+------+
|              | **V**         | **Refrig   | **0°C**        | **±  |
|              |               | 3**        |                | 1    |
|              |               |            |                | °C** |
+--------------+---------------+------------+----------------+------+
|              | **B**         | **Refrig   | **0°C**        | **±  |
|              |               | 4**        |                | 1    |
|              |               |            |                | °C** |
+--------------+---------------+------------+----------------+------+
|              | **W**         | **Refrig   | **0°C**        | **±  |
|              |               | 5**        |                | 1    |
|              |               |            |                | °C** |
+--------------+---------------+------------+----------------+------+
| **Digital**  | **Dig F, Dig  | **Drawer** | **0°C, 36°C,   | **±  |
|              | G, Dig H**    |            | and 48°C**     | 0.3  |
|              |               |            |                | °C** |
+--------------+---------------+------------+----------------+------+
| ####         | **F**         | **Freezer  | **30°C and     | **±  |
| ### Back lab |               | 5, Back    | 0°C**          | 1    |
|              |               | lab**      |                | °C** |
+--------------+---------------+------------+----------------+------+
|              | **X**         | **W/B 4,   | **51°C**       | **±  |
|              |               | back lab** |                | 0.1  |
|              |               |            |                | °C** |
+--------------+---------------+------------+----------------+------+
|              | **C**         | **back     | **0°C**        | **±  |
|              |               | lab**      |                | 1    |
|              |               |            |                | °C** |
+--------------+---------------+------------+----------------+------+

**There are 7 incubators, 6 water baths, 5 refrigerators and 5 freezers
in use in the laboratory. Temperature settings and criteria for
acceptance are dependent on use (table 3). Approximately 11
aluminum-block blue field incubators and 2 double-chamber gray
incubators are used for laboratory and field operations.**

-   ***The temperatures of the laboratory incubators, water baths, and
    refrigerators are checked quarterly with NIST-calibrated laboratory
    thermometers and recorded in the LIMS. During use, the incubators,
    water baths, and refrigerators are checked daily and recorded on a
    designated daily log sheet.***

-   ***The temperatures of the freezers are checked quarterly with
    NIST-calibrated laboratory thermometers and recorded in the LIMS.*
    The -70ºC freezers are equipped with automatic alarms that are set
    to respond when temperatures rise above -60ºC; the alarm system
    sounds during regular working hours and is attached to a telephone
    notification system after hours.**

-   ***The operating temperatures of microbiological aluminum-block
    incubators are checked annually (or in preparation for a major
    study) and recorded on the outside of each incubator and in the
    LIMS*. During periods of heavy use, the temperatures are checked and
    recorded weekly.**

-   **The two --70^o^C freezers (freezers 3 and 4) are used to store
    samples and microbiological cultures. *A filter is cleaned and fans
    behind the filter are checked by laboratory personnel for operation
    quarterly and dates are recorded in the LIMS*. *The condenser is
    dusted or vacuumed every 6 months and recorded in the LIMS. A
    temperature chart is changed after a single pass around the chart
    (weekly). ***

-   **Water baths are filled with distilled water and are cleaned with
    mild soap quarterly, or more often as needed. *Record quarterly
    cleanings in the LIMS.***

+--------------+-----------------------+-------------------------------+
| **Table 4.   |                       |                               |
| Acceptance   |                       |                               |
| criteria for |                       |                               |
| laboratory   |                       |                               |
| re           |                       |                               |
| frigerators, |                       |                               |
| freezers,    |                       |                               |
| incubators,  |                       |                               |
| and          |                       |                               |
| waterbaths** |                       |                               |
+--------------+-----------------------+-------------------------------+
| **Equipment  | **Use**               | **Acceptance criteria**       |
| iden         |                       |                               |
| tification** |                       |                               |
+--------------+-----------------------+-------------------------------+
| **INC 2**    | ***Actinomycetes***   | **28°C ± 1.0°C**              |
+--------------+-----------------------+-------------------------------+
| **INC 3**    | **Hybridization       | **51°C ± 1.0°C, 80°C ±        |
|              | oven**                | 1.0°C**                       |
+--------------+-----------------------+-------------------------------+
| **INC 4**    | **Fungi, general      | **36°C ± 1°C**                |
|              | use**                 |                               |
+--------------+-----------------------+-------------------------------+
| **INC 5**    | **Method 1601/1602,   | **35°C ± 1.0°C**              |
|              | transfer cultures,    |                               |
|              | general use**         |                               |
+--------------+-----------------------+-------------------------------+
| **INC 6**    | **Method 1601/1602,   | **35°C ± 1.0°C, 42°C ±        |
|              | *Clostridium*,        | 0.5°C**                       |
|              | transfer cultures,    |                               |
|              | general use**         |                               |
+--------------+-----------------------+-------------------------------+
| **INC 7**    | **Method 1601/1602,   | **35°C ± 1.0°C**              |
|              | transfer cultures,    |                               |
|              | general use**         |                               |
+--------------+-----------------------+-------------------------------+
| **W/B 1**    | **Melt and temper     | **48°C ± 3.0°C**              |
|              | agar**                |                               |
+--------------+-----------------------+-------------------------------+
| **W/B 2**    | **Method 1602, heat   | **37°C ± 1.0°C, 70°C ±        |
|              | treatments**          | 3.0°C**                       |
+--------------+-----------------------+-------------------------------+
| **W/B 3**    | **Grow hosts/1602**   | **36°C ± 1.0°C, 48°C ±        |
|              |                       | 1.0°C**                       |
+--------------+-----------------------+-------------------------------+
| **W/B 4**    | **Shaking bath for    | **51°C ± 0.2°C**              |
|              | hybridization**       |                               |
+--------------+-----------------------+-------------------------------+
| **W/B 5**    | **Heat treatments**   | **70°C ± 3.0°C**              |
+--------------+-----------------------+-------------------------------+
| **W/B 6**    | **Grow hosts/1602**   | **36°C ± 1.0°C, 48°C ±        |
|              |                       | 1.0°C**                       |
+--------------+-----------------------+-------------------------------+
| **REFRIG 1** | **Sample storage**    | **1 to 4°C**                  |
+--------------+-----------------------+-------------------------------+
| **REFRIG 2** | **Sample storage**    | **1 to 4°C**                  |
+--------------+-----------------------+-------------------------------+
| **REFRIG 3** | **Reagent storage**   | **1 to 4°C**                  |
+--------------+-----------------------+-------------------------------+
| **REFRIG 4** | **Sample storage**    | **1 to 4°C**                  |
+--------------+-----------------------+-------------------------------+
| **REFRIG 5** | **Reagent/ media      | **1 to 4°C**                  |
|              | storage**             |                               |
+--------------+-----------------------+-------------------------------+
| **FREEZ 1**  | **Reagent storage**   | **-20°C to -30°C**            |
+--------------+-----------------------+-------------------------------+
| **FREEZ 2**  | **Ice packs,          | **-20°C to -30°C**            |
|              | biological sample     |                               |
|              | storage**             |                               |
+--------------+-----------------------+-------------------------------+
| **FREEZ 3**  | **Bacteria stocks,    | **Shelf 1 -70°C ± 10°C**      |
|              | virus stocks,         |                               |
|              | samples**             | **Shelf 2 -70°C ± 10°C**      |
|              |                       |                               |
|              |                       | **Shelf 3 -60°C ± 10°C**      |
|              |                       |                               |
|              |                       | **Shelf 4 -60°C ± 10°C**      |
|              |                       |                               |
|              |                       | **Shelf 5 -60°C ± 10°C**      |
+--------------+-----------------------+-------------------------------+
| **FREEZ 4**  | **Sample storage**    | **-70°C ± 10°C**              |
+--------------+-----------------------+-------------------------------+
| **FREEZ 5**  | **Probes,             | **-20°C to -30°C**            |
|              | hybridization         |                               |
|              | reagents**            |                               |
+--------------+-----------------------+-------------------------------+
| **FIELD      | **Membrane            | **35°C ± 1.0°C, 44.5°C ±      |
| INCUBATORS** | filtration**          | 0.5°C, 41.5°C ± 0.5°C**       |
+--------------+-----------------------+-------------------------------+

### **Microscope**

**There are two Zeiss microscopes used to perform microscopy. The Zeiss
Axio Imager microscope has the capability to perform fluorescence
microscopy and differential interference contrast (DIC). Additionally,
it is equipped with a digital camera. This microscope is used for
specialized laboratory work and for enumerating *Cryptosporidium*
oocysts and *Giardia* cysts by USEPA Method 1623. The microscope has
three objectives: 20X, 40X and 100X (oil), as well as stage and ocular
micrometers. Furthermore, the microscope is equipped with
excitation/band-pass filters for the immunofluorescence assay (FA) and
4', 6-diamidino-2-pheylindole (DAPI) analysis. The microscope also has
optics for DIC analysis under the 100X objective. It is kept in a room
capable of being almost completely darkened.**

**The older Zeiss microscope is used for general laboratory work, Gram
Stains, and Actinomycetes analysis. It is equipped for fluorescence
microscopy, so it can also be used to enumerate *Cryptosporidium*
oocysts and *Giardia* cysts, if necessary. It has five objectives: 10X,
25X, 40X, 63X and 100X (oil), as well as stage and ocular micrometers.**

-   **The mercury bulb power supply for the Zeiss Axio Imager has an
    automated timer to monitor the number of hours the bulb has been on.
    After 150-200 hours, the mercury bulb should be changed. *Note: the
    maximum life expectancy of the bulb is 300 hours.* Call the
    manufacturer's local representative or a professional company for
    assistance. The bulb must be disposed of in accordance with legal
    regulations, not in domestic waste. Contact the Carl Zeiss
    microscopy service for assistance. *Record the mercury bulb number
    and date of installation in the LIMS.***

-   **The mercury bulb power supply for the older Zeiss microscope has a
    log book to record the number of hours the bulb has been in use. As
    explained above, the bulb should be changed after 150-200 hours and
    the appropriate information recorded in the LIMS.**

***The cleaning procedures are the same for both microscopes.***

-   **The microscope is cleaned by a professional company and the ocular
    micrometer is calibrated yearly by the company or the manufacturer's
    local representative. *Record this maintenance in the LIMS*.**

-   **After each use, clean the objectives and stage with lens paper and
    lens cleaner. A Q-tip and lens cleaner can be used to help remove
    oil from the end of the objective, as well as keep the oculars
    clean. Keep the dust protection cover on the microscopes while not
    in use and let the lamp housing cool before putting on the cover.**

-   **As needed, blow dust off of the microscope (especially the
    condenser, field aperature, and the oculars) with compressed air.**

### **Centrifuges**

**There are three types of centrifuges that are used to perform
separation of particles by centrifugal force. The refrigerated floor
centrifuge is used to concentrate *Cryptosporidium* oocysts and
*Giardia* cysts by USEPA Method 1623, for DNA extraction, and to process
concentrated samples. The ultracentrifuge is used to isolate virus
particles eluted from water samples and for concentrating bacterial
spores. Two microcentrifuges are used for processing bacterial
extractions, purifications, concentrations, and phase separations.**

#### **Refrigerated floor centrifuge**

-   **Each run of the centrifuge is recorded in the centrifuge log
    book.**

-   **The temperature is monitored quarterly with the digital
    thermometer (acceptance criteria is 4[+]{.underline} 3ºC).**

-   **The buckets are disinfected with dilute bleach and dechlorinated
    quarterly.**

-   **Rotors and adapters are checked for deterioration, as needed.**

-   **Lubrication is done annually, or as needed.**

-   ***All maintenance is recorded in the LIMS*.**

#### **Ultracentrifuge**

-   **Each run of the centrifuge is recorded in the centrifuge log
    book.**

-   **The rotor and buckets are disinfected with dilute bleach and
    dechlorinated quarterly.**

-   **Lubrication of the O-ring with vacuum grease, and lubrication of
    the buckets and cap mating surfaces with Spinkote lubricant are done
    quarterly.**

-   **The O-rings on the buckets are replaced twice a year.**

-   **The vacuum pump oil is changed every 2 years.**

-   ***All maintenance is recorded in the LIMS.***

#### **Microcentrifuge (2)**

-   **The chamber and the rotor are cleaned with soap and water
    quarterly.**

-   **The air intake and exhaust vents are cleared from obstructions
    quarterly.**

-   **Lubrication of the drive shaft and threads and O-ring with vacuum
    grease is done quarterly.**

-   ***All maintenance is recorded in the LIMS.***

### **Thermal Cyclers**

**There are two thermal cyclers used to perform the polymerase chain
reaction (PCR). The PCR is done to amplify microbial DNA targets through
a series of temperatures changes. Numerous projects use the PCR for a
variety of applications including microbial source tracking, detection
and/or quantification of enteric viruses, and detection and/or
quantification of bacterial indicators. The thermal cycler used for
end-point PCR performs the PCR to completion so that the amplified DNA
products can be subsequently detected using gel electrophoresis. The
thermal cycler used for real-time quantitative PCR (QPCR) detects and
quantifies the targeted DNA sequence as it is being amplified.**

**End-point PCR thermal cycler**

-   **The temperature is monitored twice a year ([Appendix
    V](http://oh.water.usgs.gov/micro/qcmanual/appendv.pdf)).**

-   **Results of the temperature QC are kept in the Master Cycler QC
    logbook.**

-   ***Temperature monitoring dates are recorded in the LIMS.***

> **QPCR thermal cycler**

-   **Background calibration is performed monthly.**

-   **A pure-spectra assay is done twice a year.**

-   **A RNase P verification run is done annually.**

-   **A regions of interest (ROI) plate is run as needed (following lamp
    replacement or if the system is jostled).**

-   ***All quality assurance checks are recorded in the LIMS.***

## Sample Management and Documentation

**Samples for the bacterial indicators, *E. coli*, enterococci, fecal
coliform, and total coliforms, are most commonly processed and analyzed
in the field; however, they may be done in the OWML. Holding times are 6
hours for compliance purposes and 24 hours for noncompliance purposes
(American Public Health Association, 1998, Section 9060 B.) Adhering to
a 6-hour holding time for all bacteriological samples, however, is
highly recommended.**

**Samples for *Clostridium perfringens*, coliphage, and enteric viruses
are processed and analyzed in the OWML; samples are kept on ice and
processed within 48 hours of sample collection. Samples for the analysis
of coliphage that arrive chilled within 48 to 96 hours from collection
are acceptable, but the results are qualified. Samples for
*Cryptosporidium* and *Giardia* are processed in the OWML within 48
hours of sample collection. Although the samples for *Cryptosporidium*
and *Giardia* are not kept on ice during transport, they are stored in
the refrigerator upon receipt in the OWML.**

**All requests for laboratory analysis must be submitted using a service
request form ([Appendix
A](http://oh.water.usgs.gov/micro/qcmanual/appenda.pdf)). The following
categories must be filled out when requesting sample analysis: station
name, site number, date/time of sample collection, medium code, Water
Center user code, and project number. The field personnel must check off
requested analyses and make prior arrangements with laboratory
personnel. Upon receipt of the sample in the laboratory, personnel will
fill in Received By and Date Received on the front of the Service
Request form.**

**Laboratory personnel will then enter sample information on to a log
sheet ([Appendix
B](http://oh.water.usgs.gov/micro/qcmanual/appendb.pdf)) and log the
sample into LIMS, which will assign a login ID. There is one sample
logbook that contains a log sheet, service request forms, and results
bench sheets. Laboratory personnel will write the login ID on the
Service Request form (front and back) and on the sample bottle (or
filter cartridge).**

**The Service Request form is routed to the analyst, who will enter
sample login ID, processing times, and analytical information on a
separate Results Worksheet ([Appendix
A](http://oh.water.usgs.gov/micro/qcmanual/appenda.pdf)). Samples are
analyzed within 24 hours of receipt and are stored in the laboratory
refrigerators until processing.**

**Upon completion of the analysis, the analyst writes final results on
the back of the Service Request form. A second analyst routinely checks
the calculations of the analyst performing the work. The results are
entered into the LIMS and then transmitted to the person requesting the
analysis by email in a format that can be uploaded into NWIS. The
Service Request form and Results Worksheet are then filed together in
the sample logbook.**

# **METHODS OF ANALYSIS, MEDIA AND REAGENT PREPARATION, AND ANALYTICAL QUALITY-CONTROL PROCEDURES**

**Methods of analysis, media and reagent preparation and storage, and
analytical quality-control procedures are discussed in this section.
Because microbiological analyses measure constantly changing living
organisms, the methods are inherently variable. Some quality-control
tools used by chemists, therefore, may not be available to the
microbiologist (American Public Health Association, 1998, Section 9020
A).**

**References of published microbiological methods are kept in a notebook
in the laboratory. Media-preparation instructions and method summaries
written by the OWML are kept in the reference notebook and furnished as
Appendixes to this document.**

## Fecal-indicator bacteria

**The methods used for analysis of fecal-indicator bacteria are those of
the USGS, USEPA, and APHA and others (table 5). All fecal-indicator
bacteria methods used by the OWML are compliance or official methods.**

**Table 5. Methods for fecal-indicator bacteria analysis used by the
Ohio Water Center Microbiology Laboratory (OWML) \[DW is drinking water,
RW is recreational water\]**

+------------+--------------+------------------+-----------------------+
| **         | **METHOD**   | **TYPE OF        | **REFERENCE**         |
| BACTERIA** |              | METHOD**         |                       |
+------------+--------------+------------------+-----------------------+
| **Total    | **mENDO      | **C              | **Britton and Greeson |
| c          | method**     | ompliance---DW** | (1987)**              |
| oliforms** |              |                  |                       |
|            |              | **               | **APHA (1998) Section |
|            |              | Official---other | 9222B**               |
|            |              | waters**         |                       |
+------------+--------------+------------------+-----------------------+
|            | **MI         | **Official---all | **USEPA (2000a and    |
|            | method**     | waters**         | 2002a)**              |
|            |              |                  |                       |
|            |              |                  | **([Appendix          |
|            |              |                  | C](http://oh.wat      |
|            |              |                  | er.usgs.gov/micro/qcm |
|            |              |                  | anual/appendc.pdf))** |
+------------+--------------+------------------+-----------------------+
|            | **Colilert   | **C              | **Idexx Corp.,        |
|            | method**     | ompliance---DW** | Westbrook, ME**       |
|            |              |                  |                       |
|            |              | **               | **APHA (1998) Section |
|            |              | Official---other | 9223**                |
|            |              | waters**         |                       |
|            |              |                  | **(Appendix           |
|            |              |                  | [D](http://oh.w       |
|            |              |                  | ater.usgs.gov/micro/q |
|            |              |                  | cmanual/appendd.pdf), |
|            |              |                  | [E](http://oh.wat     |
|            |              |                  | er.usgs.gov/micro/qcm |
|            |              |                  | anual/appende.pdf))** |
+------------+--------------+------------------+-----------------------+
| **Fecal    | **mFC        | **C              | **Britton and Greeson |
| c          | method**     | ompliance---DW** | (1987)**              |
| oliforms** |              |                  |                       |
|            |              | **               | **APHA (1998) Section |
|            |              | Official---other | 9222D**               |
|            |              | waters**         |                       |
+------------+--------------+------------------+-----------------------+

+------------+--------------+------------------+-----------------------+
| ***E       | **mTEC       | **               | **USEPA (1985)**      |
| scherichia | method**     | Compliance---RW, |                       |
| coli***    |              | DW**             | **([Appendix          |
|            |              |                  | F](http://oh.wat      |
|            |              |                  | er.usgs.gov/micro/qcm |
|            |              |                  | anual/appendf.pdf))** |
+------------+--------------+------------------+-----------------------+
|            | **MI         | **Official---all | **USEPA (2000a and    |
|            | method**     | waters**         | 2002a)**              |
+------------+--------------+------------------+-----------------------+
|            | **Colilert   | **C              | **Idexx Corp.,        |
|            | method**     | ompliance---DW** | Westbrook, ME**       |
|            |              |                  |                       |
|            |              | **               | **APHA (1998) Section |
|            |              | Official---other | 9223**                |
|            |              | waters**         |                       |
+------------+--------------+------------------+-----------------------+
|            | **Modified   | **Official**     | **USEPA (2000b and    |
|            | mTEC**       |                  | 2002b)**              |
|            |              |                  |                       |
|            |              |                  | **([Appendix          |
|            |              |                  | G](http://oh.wat      |
|            |              |                  | er.usgs.gov/micro/qcm |
|            |              |                  | anual/appendg.pdf))** |
+------------+--------------+------------------+-----------------------+

+------------+--------------+------------------+-----------------------+
| **Ent      | **mEI        | **C              | **USEPA (1997)**      |
| erococci** | method**     | ompliance---RW** |                       |
|            |              |                  | **([Appendix          |
|            |              | **               | H](http://oh.wat      |
|            |              | Official---other | er.usgs.gov/micro/qcm |
|            |              | waters**         | anual/appendh.pdf))** |
+------------+--------------+------------------+-----------------------+
| ***C       | **Modified   | **Official**     | **USEPA (1996),       |
| lostridium | mCP method** |                  | modified by OWML      |
| perf       |              |                  | ([Appendix            |
| ringens*** |              |                  | I](http://oh.wat      |
|            |              |                  | er.usgs.gov/micro/qcm |
|            |              |                  | anual/appendi.pdf))** |
+------------+--------------+------------------+-----------------------+

**Reagents and media for fecal-indicator analysis are prepared according
to the methods and are labeled to indicate media, date prepared, and
analyst. Each lot of media is quality-control tested by using a pure
culture of the target bacterium or a sewage sample as a positive
control; for modified mTEC, Colilert, and MI agars, negative controls
are also required ([Appendix
J](http://oh.water.usgs.gov/micro/qcmanual/appendj.pdf)). Fresh sewage
samples are obtained from the Olentangy Wastewater Treatment Plant as
needed. Stock cultures of the positive and negative controls are kept on
slants in the refrigerator and transferred once a month. *Transfer dates
are recorded in the LIMS.* When preparing positive and negative controls
to be sent to other Water Centers, stock cultures must be transferred
within a week before use.**

**Results are recorded in the "Media and Buffer" logbook on
quality-control sheets ([Appendix
K](http://oh.water.usgs.gov/micro/qcmanual/appendk.pdf)); documentation
of preparation procedures is also kept in this logbook. Media storage
requirements and holding times are strictly followed (table 6). Requests
for media, buffered-dilution water, and reagent preparation by project
personnel are made using the "Expendable supplies request forms"
([Appendix L](http://oh.water.usgs.gov/micro/qcmanual/appendl.pdf)).**

**The type of buffered-dilution water used by the OWML is phosphate
buffer with magnesium chloride dilution water (U.S. EPA, 2000b).
Instructions for preparation are listed in [Appendix
M](http://oh.water.usgs.gov/micro/qcmanual/appendm.pdf).**

**Table 6. Information on media, buffered-dilution water, and reagents
prepared and stored in the Ohio Water Cente Microbiology Laboratory
(OWML).**

+--------------+---------------+---------------+-----------------------+
| **TYPE OF    | **SOURCE**    | **STORAGE**   | **HOLDING TIME**      |
| ME           |               |               |                       |
| DIA/BUFFER** |               |               |                       |
+--------------+---------------+---------------+-----------------------+
| **mENDO      | **Difco,      | *             | **Expiration date for |
| agar**       | Detroit, MI** | *Desiccator** | agar kits**           |
|              |               |               |                       |
|              |               |               | **3 days as plates**  |
+--------------+---------------+---------------+-----------------------+
| **MI agar**  | **OWML**      | **R           | **6 months in         |
|              |               | efrigerator** | dilution bottles**    |
|              |               |               |                       |
|              |               |               | **2 weeks as plates** |
+--------------+---------------+---------------+-----------------------+
| **MI agar**  | **Becton      | **Cabinet**   | **As specified by     |
|              | Dickinson,    |               | manufacturer**        |
|              | Cockeysville, |               |                       |
|              | MD**          |               | **2 weeks as plates** |
+--------------+---------------+---------------+-----------------------+
| **Colilert** | **Idexx       | **Cabinet**   | **As specified by     |
|              | Corp.,        |               | manufacturer**        |
|              | Westbrook,    |               |                       |
|              | ME**          |               |                       |
+--------------+---------------+---------------+-----------------------+
| **mFC agar** | **Difco,      | **Cabinet**   | **As specified by     |
|              | Detroit, MI** |               | manufacturer**        |
|              |               |               |                       |
|              |               |               | **3 days as plates**  |
+--------------+---------------+---------------+-----------------------+
| **mTEC       | **OWML**      | **R           | **6 months in         |
| agar**       |               | efrigerator** | dilution bottles**    |
|              |               |               |                       |
|              |               |               | **2 weeks as plates** |
+--------------+---------------+---------------+-----------------------+
| **mTEC       | **Becton      | **Cabinet**   | **As specified by     |
| agar**       | Dickinson,    |               | manufacturer**        |
|              | Cockeysville, |               |                       |
|              | MD**          |               | **2 weeks as plates** |
+--------------+---------------+---------------+-----------------------+
| **Modified   | **OWML**      | **R           | **6 months in         |
| mTEC**       |               | efrigerator** | dilution bottles**    |
|              |               |               |                       |
|              |               |               | **2 weeks as plates** |
+--------------+---------------+---------------+-----------------------+
| **Modified   | **Becton      | **Cabinet**   | **As specified by     |
| mTEC**       | Dickinson,    |               | manufacturer**        |
|              | Cockeysville, |               |                       |
|              | MD**          |               |                       |
+--------------+---------------+---------------+-----------------------+
| **mEI**      | **OWML**      | **R           | **6 months in         |
|              |               | efrigerator** | dilution bottles**    |
|              |               |               |                       |
|              |               |               | **3 days to 2 weeks   |
|              |               |               | as plates\***         |
+--------------+---------------+---------------+-----------------------+
| **mCP agar** | **OWML**      | **R           | **6 months in         |
|              |               | efrigerator** | dilution bottles**    |
|              |               |               |                       |
|              |               |               | **1 month as plates** |
+--------------+---------------+---------------+-----------------------+
| **Phosphate  | **OWML**      | **Cabinet     | **1 year (unopened)** |
| buffer with  |               | (unopened)**  |                       |
| magnesium    | **Hardy       |               | **2 weeks (after      |
| chloride     | Diagnostics,  | *             | opening)**            |
| ([Appendix   | CA**          | *Refrigerator |                       |
| M](http://   |               | (after        |                       |
| oh.water.usg |               | opening)**    |                       |
| s.gov/micro/ |               |               |                       |
| qcmanual/app |               |               |                       |
| endm.pdf))** |               |               |                       |
+--------------+---------------+---------------+-----------------------+
| *            | **OWML**      | **R           | **6 months or until   |
| *Urea-phenol |               | efrigerator** | it is no longer a     |
| solution     |               |               | straw-yellow color**  |
| ([Appendix   |               |               |                       |
| F](http://   |               |               |                       |
| oh.water.usg |               |               |                       |
| s.gov/micro/ |               |               |                       |
| qcmanual/app |               |               |                       |
| endf.pdf))** |               |               |                       |
+--------------+---------------+---------------+-----------------------+

**\* If reagents that are added after autoclaving are filter sterilized,
the longer holding time is applied.**

**Analytical quality-control samples for fecal-indicator bacteria by
membrane filtration (mENDO, MI, mFC, mTEC, modified mTEC, mEI, and mCP
agar methods) include the following:**

-   **Filter blank---a 50-100 mL aliquot of sterile buffered water is
    plated before the sample to confirm the sterility of equipment and
    supplies.**

-   **Procedure blank---a 50-100 mL aliquot of sterile buffered water is
    plated after every fifth sample to measure the effectiveness of the
    analyst's rinsing technique or presence of incidental contamination
    of the buffered water.**

-   **A sewage sample is plated daily when *C. perfringens* analysis is
    done to evaluate the test procedure and to ensure anaerobic culture
    conditions.**

-   **For MI, positive and negative controls are plated every 10 samples
    to ensure proficiency with the method and evaluate the integrity of
    the medium. Positive and negative controls include the following:**

```{=html}
<!-- -->
```
-   **Positive controls of *E. coli* and *Serratia marcescens***

-   **Negative controls of *Pseudmonas* ATCC 10145 (unable to grow on MI
    and ensures the selectively of the agar) and *Providencia
    alcalifaciens* (grows on MI but will not fluoresce and ensures
    target colonies are correctly identified).**

> **For some projects, a sewage sample is plated with each batch of MI
> plates at the time of sample analysis to evaluate the effectiveness of
> cefsulodin (an antibiotic added at the time of plate preparation).**

**Analytical quality-control samples for Colilert include the
following:**

-   **Positive (*E. coli*) and negative-control (*Pseudomonas*) cultures
    are included with every 20^th^ sample to evaluate the test procedure
    and aid in interpretation of results.**

## Enteric viruses

**RT-PCR and cell-culture methods are recommended for the detection of
enteric viruses in water. To prepare samples for RT-PCR and cell
culture, attached viruses are eluted from a 1MDS filter with beef
extract (pH 9.5), concentrated using celite (pH 4.0), and eluted with
sodium phosphate (pH 9.5). These steps are done in the OWML, and a
protocol is included as [Appendix
N](http://oh.water.usgs.gov/micro/qcmanual/appendn.pdf).**

**The RT-PCR method was written and laboratory tested by USEPA and is
experimental (G. Shay Fout, U.S. Environmental Protection Agency,
written commun., 1997; Fout and others, 2003). Viruses are isolated from
the eluate by ultracentrifugation through a sucrose gradient, and trace
contaminants are removed by extraction with a sol­vent mixture ([Appendix
N1](http://oh.water.usgs.gov/micro/qcmanual/appendn1.pdf)). An aliquot
of the concentrate is used for RT-PCR, wherein any target viral RNA is
converted to DNA (by reverse transcriptase) and amplified by the
polymerase chain reaction. The RT-PCR products confirmed by
hybridization. The enteric viruses detected by use of this method
include enterovirus, hepatitis A virus, rotavirus, reovirus, and
calicivirus (including Norwalk-like virus).**

**The OWML consists of a 1,000 ft^2^ main laboratory and a 300 ft^2^
limited-use laboratory. Virus elutions, inhibitor removal, and reaction
preparations for RT-PCR are done in the main lab, along with media
preparation, membrane filtration, incubation, and culture maintenance.
The limited-use lab is the only area in the building in which PCR
products are handled. Gel electrophoresis and hybridization are
performed in this room. To avoid contamination of incoming samples,
staff that have entered the limited-use lab are not allowed to reenter
the main lab unless they have showered and changed their clothes.
Equipment and supplies are also not to be transferred into the main lab,
unless adequate sterilization and decontamination procedures have been
followed.**

**A list describing QC samples for all stages of sample preparation and
analysis by the RT-PCR method are listed in [Appendix
N2](http://oh.water.usgs.gov/micro/qcmanual/appendn2.pdf). A check list
to be used when interpreting all results of RT-PCR analyses is also
contained in [Appendix
N2](http://oh.water.usgs.gov/micro/qcmanual/appendn2.pdf).**

**Cell-culture analysis is not done at the OWML; these samples need to
be sent to a contract laboratory for analysis. The recommended
cell-culture method is an experimental method and was modified from U.S.
Environmental Protection Agency (1996) by USEPA (G. Shay Fout, U.S.
Environmental Protection Agency, written commun., 1999). The sample
eluate is added to a monolayer of a continuous cell line derived from
African Green monkey kidney cells. Each cell culture is examined
microscopically for the appearance of cytopathic effects (CPE) for a
total of 14 days; if CPE is not observed in 14 days, a second passage is
done. Results are reported as most probable number of infectious units
per volume of water. The following QC samples are included in
cell-culture analysis:**

-   **A negative control, containing cells and sodium phosphate buffer,
    is incubated in a roller bottle with each batch of samples
    inoculated for the first passage.**

```{=html}
<!-- -->
```
-   **A positive control, containing cells, sodium phosphate buffer, and
    polio virus vaccine, is incubated in a 75 cm^2^ flask with each
    batch of samples inoculated for the first passage and the second
    passage.**

**Media and reagent preparations are done in the OWML or at the contract
laboratory and are prepared and stored per method instructions. The
contract laboratory is required to strictly follow the QA/QC guidelines
listed in the method documentation (G. Shay Fout, U.S. Environmental
Protection Agency, written commun., 1997 and 1999).**

## Coliphage

**The method currently in use for quantitative coliphage analysis by the
OWML is the USEPA Method 1602, single-agar layer (SAL) procedure (USEPA,
2001b) ([Appendix
O](http://oh.water.usgs.gov/micro/qcmanual/appendo.pdf)). This method is
generally most suitable for quantification of coliphage in surface-water
samples. Antibiotic-resistant *E. coli* CN-13 (resistant to nalidixic
acid) and *E. coli* F-amp (resistant to streptomycin and ampicillin) are
used as bacterial hosts for somatic and F-specific coliphage,
respectively. Procedures for quality-control samples are described in
[Appendix O](http://oh.water.usgs.gov/micro/qcmanual/appendo.pdf).
Results are recorded on a QC log form ([Appendix
P](http://oh.water.usgs.gov/micro/qcmanual/appendp.pdf)).**

**The method currently in use for qualitative determination of coliphage
in larger sample volumes at the OWML is the USEPA Method 1601, two-step
enrichment method (USEPA, 2001a). Sample volumes of 1 L are recommended
for detec­tion of coliphage using this method. Because the SAL method is
impractical for sample volumes above 100 mL, the two-step enrichment
method is often used for ground-water sample analysis. A summary of
Method 1601 can be found in [Appendix
Q](http://oh.water.usgs.gov/micro/qcmanual/appendq.pdf). Results from
quality-control samples are recorded on a QC log form ([Appendix
R](http://oh.water.usgs.gov/micro/qcmanual/appendr.pdf)).**

***Results from coliphage QC samples are recorded in the LIMS.*
Coliphage QC log forms are kept in the Sample Logbook with the Service
Request Forms. A second book, coliphage QC log book, contains
information on media sterility, additional quality-control samples, and
coliphage stock enumeration results. Information on host culture strains
is maintained in LIMS.**

# 

## Cryptosporidium and Giardia

**USEPA method 1623 (USEPA, 2001c) is the USEPA-recommended method for
detection of *Cryptosporidium* oocysts and *Giardia* cysts in water.
This provisional method involves filtration, immunomagnetic separation,
staining with fluorescent antibody, and microscopic evaluation. At the
present time, the OWML has the capability to complete the sample
processing stepsfiltration and concentration ([Appendix
T](http://oh.water.usgs.gov/micro/qcmanual/appendt.pdf)). The OWML is
developing the capability for immunomagnetic separation and
microscopy.**

## Actinomycetes

**The Actinomycetes are a large group of filamentous gram-positive
bacteria that resemble fungi because they produce mycelium and dry
spores, called conidia. (Madigan and others, 2000). They are considered
nuisance organisms for those in the water industry, as they are one of
two types of organisms that impart an earthy-musty odor to waters. The
odors are caused by two compounds formed during normal actinomycete
development, geosmin and 2-methylisoborneol (American Public Health
Association, 1998).**

**The method used for isolation of Actinomycetes from water in the OWML
is based on a published method (American Public Health Association,
1998) and a method provided by a commercial supplier of Actinomycetes
medium (Difco, Detroit Michigan, The Difco Manual, 2001). A double agar
layer method is used ([Appendix
U](http://oh.water.usgs.gov/micro/qcmanual/appendu.pdf)). Stock cultures
of *Streptomycetes albus* are used as positive controls. The stock
culture is transferred every two months and transfers are recorded in
the LIMS.**

# **REFERENCES**

**American Public Health Association, American Water Works Association,
and Water Pollution Control Federation, 1998, Standard methods for the
examination of water and wastewater (20th ed.): Washington, D.C.,
American Public Health Association \[variously paginated\].**

**American Society for Testing and Materials, 1999, Annual Book of ASTM
Standards, Section 11, Water and Environmental Technology, Designation:
D 1193-99, p. 107-109.**

**Britton , L.J., and Greeson, P.E., eds., 1987, Methods for collection
and analysis of aquatic biological and microbiological samples: U.S.
Geological Survey Techniques of Water-Resources Investigations, book 5,
chap. A4, 363 p.**

**Fout, G.S., Martinson, B.C., Moyer, M.W.N., and Dahling, D.R., 2003, A
multiplex reverse transcription-PCR method for detection of human
enteric viruses in groundwater: Applied and Environmental Microbiology,
v. 69, no. 6, p. 3158-3164.**

**Francy, D.S., Jones, A.L., Myers, D.N., Rowe, G.L., Eberle, M., and
Sarver, K.M., 1998, Quality-assurance/quality-control manual for
collection and analysis of water-qual­ity data in the Ohio District, U.S.
Geological Survey: U.S. Geolog­ical Survey Water-Resources Investigations
Report 98-4057, 71 p.**

**Francy, D.S., Helsel, D.L., and Nally, R.A., 2000, Occurrence and
distribution of micro­biological indicators in ground water and stream
water: Water Environment Research, v. 72, no. 2, 152 p.**

**Ijzerman, M.M., and Hagedorn, C., 1992, Improved method for coliphage
detection based on β-galactosidase induction: Journal of Virological
Methods, v. 40, p. 31-36.**

**Madigan, M. T., Martinko, J.M., and Parker, J., Brock, Biology of
Microoganisms---Ninth Edition: Prentice Hall, Upper Sadlle River, NJ, p.
519.**

**U.S. Environmental Protection Agency, 1978, Microbiological methods
for monitoring the environment---water and wastes: Cincinnati, Ohio,
U.S. Environmental Protection Agency, EPA-600/8-78-017, 338 p.**

**\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_1985, Test methods for *Escherichia
coli* and enterococci in water by the membrane filtered procedure:
Cincinnati, Ohio, Environmental Monitoring and Support Laboratory, EPA
600/4-85/076, 24 p.**

**\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_1996, EPA Information Collection
Rule microbial laboratory manual: Washington, D.C., U.S. Environmental
Protection Agency, EPA/600/R-95/178.**

**\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_1997, Method 1600---Membrane filter
test method for enterococci in water: U.S. Environmental Protection
Agency, Office of Water, Washington, D.C., EPA-821-R-97-004, 9 p.**

**\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_2000a, Membrane filter method for
the simultaneous detection of total coliforms and *Escherichia coli* in
drinking water: U.S. Environmental Protection Agency, Office of Research
and Development, EPA 600-R-00-013.**

**\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_2000b, Improved enumeration methods
for the recreational water quality indicators: enterococci and
*Escherichia coli*: U.S. Environmental Protection Agency, Office of
Science and Technology, EPA/821/R-97/004.**

**\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_2001a, Method 1601: Male-specific
(F+) and somatic coliphage in water by two-step enrichment procedure:
Washington, D.C., U.S. Environmental Protection Agency,
EPA-821-R-01-030.**

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_2001b, Method 1602: Male-specific (F+)
and somatic coliphage in water by single agar layer (SAL) procedure:
Washington, D.C., U.S. Environmental Protection Agency,
EPA-821-R-01-029.

**\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ 2001c, Method
1623*Cryptosporidium* and *Giardia* in water by filtration,
immunomagnetic separation, and fluorescent antibody: Washington, D.C.,
U.S. Environmental Protection Agency, EPA 821-R-01-025.**

**\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ 2002a, Method 1604---Total
coliforms and *Escherichia coli* in water by membrane filtration using a
simultaneous detection technique (MI medium): Washington D.C., EPA
821-R-02-024, 14 p.**

**\_\_\_\_\_\_\_\_\_\_\_\_\_\_ 2002b, Method 1603---*Escherichia coli*
in water by membrane filtration using modified membrane-thermotolerant
*Escherichia coli* agar: Washington, D.C., EPA 821-R-02-23, 9 p.**

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