NISTIR 6752 

NATIONAL BIOMONITORING SPECIMEN BANK 

NIST-CHARLESTON LABORATORY:

Clean Room and Specimen Bank Protocols 

 

 Rebecca S. Pugh 

Barbara J. Porter 

Paul R. Becker 

 and Stephen A. Wise

 

 
NISTIR 6752 

 

NATIONAL BIOMONITORING SPECIMEN BANK 

NIST-CHARLESTON LABORATORY:

Clean Room and Specimen Bank Protocols 

 

 Rebecca S. Pugh1 

Barbara J. Porter2

Paul R. Becker1

and Stephen A. Wise2 

1

National Institute of Standards and Technology 

Chemical Science and Technology Laboratory 

Charleston, SC 29412 

2

National Institute of Standards and Technology 

Chemical Science and Technology Laboratory 

Gaithersburg, MD 20899 

January 2002 

 

U.S. Department of Commerce 

Donald L. Evans 

 National Institute of Standards and Technology 

 Arden L. Bement, Jr., Director

 
CONTENTS 

 

 page 

List of Tables.......................................................................................................ii 

List of Figures..................................................................................................... ii 

Disclaimer................................................................................................ ......... iii 

INTRODUCTION..................................................................................... 1 

 National Biomonitoring Specimen Bank................................................... 1 

NBSB-CHARLESTON LABORATORY.......................................................... 3 

 Location........................................................................................ 3

 Class 100/10,000 Facility.................................................................... 3 

ANTE-ROOM.......................................................................................... 9 

SPECIMEN BANK.................................................................................... 11 

 Liquid Nitrogen Piping System............................................................. 11 

 Archiving Samples: The ASTRO System..................................................11 

 Biological Dry Shippers..................................................................... 15 

CLEAN ROOM........................................................................................ 21 

 Hood Usage/Cleaning of Teflon and Titanium............................................ 21 

 Cryogenic Homogenization.................................................................. 21 

 

SAFETY................................................................................................ 29 

 Hurricane Evacuation......................................................................... 29

 Datatron........................................................................................ 32 

MAINTENANCE...................................................................................... 33 

REFERENCES......................................................................................... 36 

APPENDICES......................................................................................... 38 

A. National Biomonitoring Specimen Bank Data Sheets............................... 38 

B. Instructions for Cleaning Teflon and Titanium Supplies............................ 46 

C. NOS/CCEHBR Laboratory Safety Manuals and Forms............................. 48 

Chemical Hygiene Plan..................................................................49 

Emergency Preparedness Plan........................................................ 62 

Job Hazard Analysis Form.............................................................. 73 

Chemical Inventory for Room 227..................................................... 74 
LIST OF TABLES 

 

 page 

1. Projects within the National Biomonitoring Specimen Bank at NIST...................... 1 

2. Equipment located in the Specimen Bank at NBSB-Charleston............................ 12 

3. Equipment located in the Clean Room at NBSB-Charleston............................... 22 

4. Safety equipment located in the NBSB-Charleston........................................... 30 

LIST OF FIGURES 

1. NBSB-Charleston located within the NOS/CCEHBR Laboratory.......................... 4 

2. NBSB-Charleston Floor Plan...................................................................... 5 

3. The Hollings Marine Laboratory (HML), First Floor......................................... 6 

4. Clean Room Air Flow............................................................................. 8 

5. ASTRO ‘Enter New Specimens and Aliquots’ Screen....................................... 14 

6. ASTRO ‘Standard EPI Information’ Screen................................................... 16 

7. ASTRO Posted Shipment Detail Report........................................................ 17 

8. MVE Biological Dry Shippers; Cryo Shipper XC (left) and 

SC14/2V (right)............................................................................... 19 

9. Holding Time of Biological Dry Shippers...................................................... 20 

10. TS-250 Teflon Disk Mill........................................................................ 24 

11. Teflon Materials Needed for Homogenization............................................... 25 

12. Compressed Air Smashing Machine............................................................. 26 

13. Samples of Tissue in Fragment Form and Powder Form..................................... 28 

14. NBSB-Charleston Safety Features............................................................... 31 

15. NBSB-Charleston Cleanliness Levels........................................................... 34 
DISCLAIMER 

Certain commercial equipment or instruments are identified in this paper to specify adequately 

the experimental procedures. Such identification does not imply recommendations or 

endorsement by the National Institute of Standards and Technology nor does it imply that the 

equipment or instruments are the best available for the purpose. 
1

INTRODUCTION 

NATIONAL BIOMONITORING SPECIMEN BANK 

The National Institute of Standards and Technology (NIST) has been involved in 

the long term banking of biological specimens for over 20 years. The U.S. National 

Biomonitoring Specimen Bank (NBSB) was initiated in 1979 from the pilot 

Environmental Specimen Bank Program under the sponsorship of the Environmental 

Protection Agency (EPA) (Wise and Zeisler, 1985). It is maintained at the NIST 

Research Reactor Building in Gaithersburg, Maryland. The NBSB is an archive that 

provides for the long-term storage of well documented and preserved environmental 

specimens. Details of the NBSB have been described elsewhere (Wise and Koster, 1995; 

Wise et al., 1993). Specimens have been collected through numerous research projects 

that have been developed in collaboration with several different national and international 

agencies. These projects are summarized in Table 1. 

Table 1. Projects within the National Biomonitoring Specimen Bank at NIST 

 

 

Project Sponsoring Organization/Agency Specimen Type 

Environmental Specimen Bank Environmental Protection Agency (EPA) Human liver, mussels 

National Status and Trends 

 Specimen Bank Project 

National Oceanic and Atmospheric 

Administration (NOAA) 

Mussels/oysters, 

sediment, fish muscle 

 and fish liver 

Alaska Marine Mammal 

 Tissue Archival Project 

NOAA, U.S. Geological Survey/Biological 

Resources Division (USGS/BRD), Minerals 

Management Service (MMS) 

Marine mammal tissues 

(kidney, liver, blubber)

National Marine Mammal NMFS/Office of Protected Resources (NOAA) Marine mammal tissues

 Tissue Bank (kidney, liver, blubber)

Trace Nutrients in Human Diet International Atomic Energy Agency (IAEA) Total human diet 

 Project US Department of Agriculture (USDA) 

 Food and Drug Administration (FDA) 

Seabird Tissue Archival and USGS/BRD, USFWS/NWR Seabird eggs 

 Monitoring Project 

 
2

Specimen banking provides researchers the ability to look at environmental trends over 

long periods of time through retrospective analysis of archived samples. Over the last ten 

years, the specimen bank inventory has included primarily marine samples, with the 

majority of the tissues from marine mammals (Becker et al. 1997). Two ongoing 

environmental monitoring programs that have provided a large percentage of these 

samples are the Alaska Marine Mammal Tissue Archival Project (AMMTAP) and the 

Marine Mammal Health and Stranding Response Program (MMHSRP). 

The AMMTAP was initiated in 1987 with sponsorship from the U.S. Department 

of the Interior, Minerals Management Service (MMS). The project is now conducted as a 

collaboration between the U.S. Geological Survey/Biological Resources Division 

(USGS/BRD) and NIST. The goal of this program is to establish a representative 

collection of marine mammal tissues taken during Alaska Native subsistence hunts for 

future contaminant analyses and documentation of long-term trends in environmental 

quality (Zeisler et al., 1992). A detailed description of the project and protocols have 

been published (Becker et al., 1991; 1993). 

The National Marine Mammal Tissue Bank (NMMTB) was initiated in 1987 by 

the National Marine Fisheries Service (NMFS) to collect marine mammal tissues and 

bank them for long-term storage at the NBSB. In 1992, the NMMTB was formally 

established by Federal Legislation (Public Law 102-587) and a portion of tissues 

collected by NMFS’s MMHSRP are archived in the NMMTB. The MMHSRP obtains 

specimens from marine mammal strandings and from animals taken incidentally during 

commercial fishing operations and emphasizes contaminant monitoring and information 

management. Detailed descriptions of this project have been published (Lillestolen et al. 

1993 and Becker et al. 1994). 

A third program that currently contributes to the NBSB is the Seabird Tissue 

Archival and Monitoring Program (STAMP). This project was initiated in 1998 with 

financial support from the U.S. Department of the Interior, USGS/BRD and is conducted 

as a collaboration among USGS/BRD, U.S. Fish and Wildlife Service Alaska Maritime 

National Wildlife Refuge (USFWS/NWR), and NIST. This program was established to 

archive a representative collection of seabird tissues from representative Alaskan colonial 

seabird species for future contaminant analyses and documentation of long-term trends in 

environmental quality. Currently, seabird eggs from common murre (Uria aalge) and 

thick-billed murre (Uria lomvia) colonies are collected and banked but additional species 

and types of specimens will be collected in the future. A description of this project and 

protocols have been published (York et al. 2001). 

 
3

NBSB - CHARLESTON LABORATORY 

In 1995, an agreement was signed between NIST and NMFS to establish the 

National Marine Analytical Quality Assurance Program (NMAQAP). Through 

interlaboratory comparisons and reference material development, the NMAQAP is able 

to assess and improve the quality of analytical measurements in the marine environment 

as well as allow for the improvement in the capabilities to assess trends in marine 

environmental quality by expanding environmental specimen banking activities. As part 

of this agreement, NIST established a satellite facility of the NBSB in Charleston, South 

Carolina. 

The NBSB-Charleston Laboratory is dedicated to banking only marine 

environmental specimens, currently through the AMMTAP, MMHSRP and STAMP 

programs. Because specimen banking must ensure that the sample does not become 

contaminated or change in chemical composition during collection, processing, storage 

and homogenization procedures, it is important that all protocols be followed precisely. 

The NBSB-Gaithersburg facility (hereafter, NBSB-Gaithersburg) and the satellite 

specimen bank in Charleston (hereafter, NBSB-Charleston), will maintain identical 

operating procedures for all aspects of the NBSB, but the following information will 

specifically describe those of NBSB-Charleston. 

LOCATION 

NBSB-Charleston is located at 219 Fort Johnson Road, Charleston, South 

Carolina, on the Fort Johnson Marine Resources Center complex in NOAA’s National 

Ocean Service (NOS), Center for Coastal Environmental Health and Biomolecular 

Research (CCEHBR) building (Fig. 1). The NIST space is approximately 185.81 m2 

(2,000 ft2) which includes office space, associated storage space, analytical laboratories 

and NBSB-Charleston. The NBSB-Charleston consists of three rooms, the Ante Room, 

8.55 m2

 (92 ft2), the Specimen Bank, Room 227B, 39.02 m2 (420 ft2) and the Clean 

Room, Room 227A, 33.45 m2 (360 ft2

)(Fig. 2). 

 The Hollings Marine Laboratory (HML) will be completed in the fall of 2001. 

This facility is located on an approximately 8-acre site within the Fort Johnson Marine 

Resources Center. The HML will promote collaborative and interdisciplinary scientific 

research through a multi-disciplinary team of scientists. This team includes NIST, 

NOAA/National Ocean Service, South Carolina Department of Natural Resources, 

University of Charleston, and Medical University of South Carolina. The NIST space 

will be approximately 743.22 m2

 (8,000 ft2) and will include office space, analytical 

laboratories, a Standard Reference Material production facility and the NBSB. (Fig. 3). 

CLASS 100/10,000 FACILITY 

The NBSB-Charleston has been designed to contain a Class 100 sample 

processing area (Clean Room) and a Class 10,000 cryogenic storage room (Specimen 

5

LN2

Freezer

LN2

Freezer

-80 °C

Freezer Cabinet Balance Bio Hood

Teflon

Fume 

Hood

Small

Disk

Mill

ANTE ROOM

ROOM 227BCLASS 10000SPECIMEN BANK

ROOM 227ACLASS 100CLEAN ROOM

Figure 2. NBSB-Charleston Floor Plan

LN2

Freezer

LN2

Freezer

LN2

Freezer

LN2

Freezer

LN2

Freezer

Cabinet

Acid 

Storage

-80 °C

Freezer

CompressedAirSmasher

ManualSmasher

Large

Disk

Mill

Laminar

Flow

Hood

ASTRO

Database

Vinyl Door

LN2

Freezer

LN2

Freezer

-80 °C

Freezer Cabinet Balance Bio Hood

Teflon

Fume 

Hood

Small

Disk

Mill

ANTE ROOM

ROOM 227BCLASS 10000SPECIMEN BANK

ROOM 227ACLASS 100CLEAN ROOM

Figure 2. NBSB-Charleston Floor Plan

LN2

Freezer

LN2

Freezer

LN2

Freezer

LN2

Freezer

LN2

Freezer

Cabinet

Acid 

Storage

-80 °C

Freezer

CompressedAirSmasher

ManualSmasher

Large

Disk

Mill

Laminar

Flow

Hood

ASTRO

Database

Vinyl Door

7

Bank). The clean laboratories control the concentration of airborne particles to specified 

limits and are essential in minimizing contamination of samples during processing. The 

clean room provides a clean sample preparation work area for materials to be analyzed 

for organic and inorganic constituents. At present, organic analyses are performed 

primarily for chlorinated pesticides, polycyclic aromatic hydrocarbons, and 

polychlorinated biphenyls; the inorganic analyses center on the measurement of trace and 

ultratrace quantities of elements, with particular attention being given to metals. 

The normal Class 100 clean room is designed to provide clean filtered air, exhaust 

particulate matter as fast as possible and maintain Class 100 air quality when there is a 

minimum of activity in the room. Class 100 air is defined as a measurement of no more 

than 3,530 particles per m3 (100 particles per ft3

) which are 0.5 μm in diameter or larger. 

Class 10,000 air is then defined as a measurement of no more than 353,000 particles per 

m3 (10,000 particles per ft3

) which are 0.5 μm in diameter or larger. These standard 

classes of air cleanliness for airborne particulate levels in clean rooms are established by 

the Federal Standard 209E document (U.S. General Services Administration, 1992; 

Institute of Environmental Sciences and Technology, 1997). The NBSB Class 100 room 

is achieved by driving air vertically downward through high efficiency particulate air 

(HEPA) filters and capturing return air in two of the side walls at ground level. The air is 

then recirculated back through the HEPA filters (Fig. 4) and is exchanged several times 

per hour. The airflow in NBSB-Charleston is non-unidirectional. Federal Standard 209E 

defines this as ‘airflow which does not meet the definition of unidirectional airflow’. In 

other words, it allows the vertical downflow of air to change directions to horizontal at 

working height. This provides an efficient shielding against any cross-contamination. 

As mentioned above, the purpose of the NBSB is the long-term preservation of 

specimens that are representative of the environment or organism immediately prior to 

collection. A major concern of specimen banking efforts is that the samples are 

collected, processed, and stored under conditions that minimize contamination of 

specimens or any other changes in their chemical composition (Wise and Koster, 1995). 

Clean technique allows the sample to be processed while minimizing contamination and 

protects the sample’s integrity from possible extraneous addition of the chemical analytes 

of interest. Clean technique is often confused with sterile technique, which is used to 

prevent the transfer of a bacteriological or viral species to an individual or medium which 

does not contain this agent. 

 Since it is impossible to point out every source of contamination that may be 

encountered, some of the most obvious sources will be described to provide a general 

awareness of the problem. For example, a 1 μg flake of stainless steel contains 

approximately 100 ng of nickel (National Bureau of Standards, 1986). The natural 

occurrence of nickel in human liver is at levels of 1 ng/g to 2 ng/g of sample. 

Approximately 1 g of sample is used for analysis; therefore, a μg flake of stainless steel 

in the sample could produce an analytical result which is 100 times higher than the true 

value of nickel in the sample. 

In the clean room the air, implements, bench tops, working personnel, and 

processing protocols are all potential sources of contamination. The air may contain 

vapors of contaminants as well as particles of dust, cigarette ash, and wear particles from 

motors. Cigarette ash contains relatively high amounts of cadmium and organic 
8

Class 100 Clean RoomTeflon-lined Fume Hood

LN2

Freezer

Biohazard

Hood

Cabinet

Balance

DiskMill Smasher

Work Bench

Air Ducts - Ground Level

Figure 4. Clean Room Air Flow

DiskMill

Vertical Laminar Flow 

Hood

- HEPA Filters Class 100 Clean Room

Teflon-lined Fume Hood

LN2

Freezer

Biohazard

Hood

Cabinet

Balance

DiskMill Smasher

Work Bench

Air Ducts - Ground Level

Figure 4. Clean Room Air Flow

DiskMill

Vertical Laminar Flow 

Hood

- HEPA Filters
9

compounds. Cigarette smoke also contains trace quantities of hundreds of organic 

compounds. Implements and working surfaces may be contaminated with chemicals 

used for cleaning and salt and oils from human contact. Also, a common contaminant 

introduced by human hands is gold from jewelry. 

 The dust-free vinyl gloves that are used should be changed frequently as they are 

easily contaminated. For example, picking up a pen to record a mass contaminates the 

gloves. Grasping a Teflon bottle, adjusting eyeglasses, touching one’s face, touching the 

outside of the bag that contains the clean Teflon sheets or bags also contaminates the 

gloves. Therefore, caution should be taken at all times while inside the clean areas. 

 Each of the three rooms that make up the NBSB-Charleston serves a different 

purpose. It is important to know the function and the types of equipment that are used in 

each room. 

ANTE-ROOM 

The Ante-Room, a Class 10,000 room, is used to change into proper ‘clean’ 

garments before entering the Specimen Bank and Clean Room. Disposable laboratory 

frocks, hoods, and shoe covers are provided in this room and must be worn at all times 

while in the NBSB. The material used to make these garments is non-woven Tyvek, a 

highly effective contamination control fabric that is resistant to penetration by airborne 

particles. These garments are processed and packaged in a clean environment by the 

manufacturer and are disposable but can be worn several times before soiling and tears 

occur. 

Extreme caution must be taken while working in NBSB-Charleston to prevent 

further contamination of samples; therefore the following precautions should be taken 

before entering the Ante-Room: 

1. Do not use cosmetic substances such as excessive makeup, alcohol based 

perfumes, or after shave products; 

2. Avoid clothing with excessive lint, such as, sweaters, turtlenecks, torn and 

frayed clothing, or any item that has been worn in a dusty environment; 

3. Do not bring in items that may cause further contamination, such as a lead 

pencil, dirty chemical bottles, paper towels, cardboard boxes, paper products, 

or other particulate products; 

4. Do not smoke, chew gum or tobacco, bring in food or drink, or bring personal 

items into the NBSB; and 

5. Do not use pencils or erasers, only use ballpoint pens in the NBSB. 

The following gowning and de-gowning procedures must be followed in the Ante-Room 

before entering and exiting NBSB-Charleston: 

 
10

Gowning Procedure: 

1. Wash hands thoroughly before entering the Ante-Room;

2. Upon entering, step onto the sticky mat to remove loose particles on street 

shoes;

3. Place disposable shoe covers carefully over shoes;

4. Place the hood over the head, making sure all hair is inside the hood;

5. Choose the closest fit frock size and put it on, making sure to close all snaps 

and secure the hood inside of the frock;

6. If facial hair is present, place a beard cover on, over the hood, again making 

sure all hairs are inside the cover; and

7. Step onto the second sticky mat and enter the Specimen Bank.

De-gowning Procedure: 

1. From the Specimen Bank, step into the Ante-Room, remove gloves, and 

dispose of them in the trash can provided; 

2. Remove the beard cover, if applicable, and dispose of it in the trash can; 

3. Remove the hood and inspect it for heavy soiling or damage. If it is dirty or 

damaged, discard it in the trash can. If it is clean, hang it on the hook next to 

the Specimen Bank door; 

4. Remove the frock and inspect it. If it is dirty or damaged, discard it in the 

trash can; if not, hang it on the hook underneath the top shelf; and 

5. Remove the shoe covers and again, inspect for damage or excessive amount of 

dirt. If dirty or damaged, discard the covers in the trash can; if clean, place 

them underneath the frock on the floor. 

Some of the cleaning supplies are stored in the Ante-Room. These include a clean-room 

mop, water bucket, sticky roller, lint-free cloths, and an aerosol can of isopropyl alcohol, 

Deconahol. The proper use of these cleaning materials is discussed in detail in the 

Maintenance Section. All items that are brought into NBSB-Charleston must be wiped 

down with Deconahol and a lint free cloth. If an item is extremely dirty, the vacuum 

cleaner located in the Specimen Bank must first be used before wiping down with the 

lint-free cloth. 

 
11

SPECIMEN BANK 

The Specimen Bank is a Class 10,000 cryogenic storage facility. Samples that are 

collected for the AMMTAP, MMHSRP, and STAMP projects are archived in liquid 

nitrogen vapor-phase freezers (-150 °C) that are located in this room. Along with the 

liquid nitrogen freezers, there are several other pieces of equipment that are either used or 

stored inside of the Specimen Bank (these are listed in Table 2). 

LIQUID NITROGEN PIPING SYSTEM 

All freezers in the specimen bank are connected to a liquid nitrogen in-line piping 

system. This system provides vacuum insulated piping along with a cryovent device 

located on the exterior of the building to ensure that only liquid is maintained in the lines, 

not nitrogen gas. The system is monitored by a Watlow-manufactured series 965 

microprocessor-based controller. The controller is located on the wall inside the 

specimen bank and is set to vent only enough gas to allow the piping to remain full of 

liquid. There are four liquid nitrogen pipes that enter from the ceiling into the specimen 

bank with two valves on each pipe. These valves provide the liquid nitrogen to all eight 

freezers: six -150 °C vapor phase and two -80 °C ultra-cold electric freezers. The ultracold electric freezers are connected to the piping system for the liquid nitrogen back-up 

system. The liquid nitrogen for the Specimen Bank and Clean Room operations is stored 

behind the CCEHBR building in a 5.7 m3 (1,500 gallon) vertical storage tank. The 

Datatron System 3001: Monitoring, Alarming, and Data Management System is used to 

ensure correct temperatures and liquid nitrogen levels are maintained for each freezer. In 

general, a node is located above each freezer that has three probes/channels to monitor 

the top, middle, and bottom temperatures inside the freezer. See the ‘Safety’ section of 

this manual for a full description of this system. 

ARCHIVING SAMPLES: THE ASTRO SYSTEM 

The operating procedures for storage of specimens at the NBSB-Charleston 

facility are identical to those used at the Gaithersburg facility except for the use of the 

Archival Specimen Tracking Retrieval Operation (ASTRO) database system at 

Charleston. ASTRO is a PC-based graphical program that resembles other Windowsbased applications allowing one to point and mouse click to communicate with the 

software. It was designed for the Centers for Disease Control and Prevention (CDC) to 

ensure that sample collections are properly maintained and managed. The ASTRO 

system automates data entry and data query operations to facilitate admission of 

specimens, cataloging of collections, specimen retrieval, specimen tracking, data sharing, 

allocation and tracking of available storage space, and overall storage facility 

management (Ayal, 1999). A user’s guide (Ayal, 1999) has been produced that 

introduces the database. The following information describes the use of ASTRO 

specifically for NBSB-Charleston and the system used to assign the NIST Storage 

Number which is needed for the ASTRO database. 
12

 
13

Once marine mammal tissues have been collected using the NBSB protocols, the 

180 mL Teflon jars that the tissues are stored in are placed in one of the -150 °C 

biological dry shippers and transported to NBSB-Charleston. Individual NBSB field data 

sheets (see Appendix A) are completed for each animal sampled and are shipped with the 

samples. Once the shipper is received, the samples are checked to ensure that they are 

still in a frozen condition and that they are properly marked and correspond with the 

information on the field data sheets. A NIST Storage Number is then assigned to each 

tissue that is archived in the specimen bank (See Appendix A). 

 MM 16 L 240 C

After all storage numbers have been assigned, a ‘New Shipment’ is created in ASTRO. 

This screen contains the following information: 

Who shipped the samples, 

Date shipped, 

Date received at NBSB-Charleston, 

Contents of the shipment, 

Location where the samples will be stored (Charleston), 

Project type (NMMTB), 

Collection type (AMMTAP, MMHSRP, STAMP, etc.). 

The Teflon jars are then placed in pre-labeled cardboard tubes (racks). Five Teflon jars 

will fit in each cardboard tube and each individual tube contains only one tissue type (i.e.; 

liver, kidney, etc.). An inventory of the current available space for each tube is printed 

from ASTRO so that the samples can be placed in the appropriate tubes. After the new 

shipment is created the ‘Enter New Specimens and Aliquots’ screen appears (Fig. 5). 

This screen allows you to enter the following detailed information about each sample: 

Local ID (Animal ID Number), 

Specimen ID (NIST Storage Number), 

Specimen Source (condition of the animal–Code, 1, 2 or 3), 

Collection Date, 

Aliquot ID (subsample, A and B), 

Aliquot Container Type (Teflon jar, Teflon bag, etc.), 

Aliquot Type (tissue type), 

Program: MM-AMMTAP 

 NM-MMHSRP 

 ST-STAMP 

Year of 

the 

Program

Tissue Type: 

L-Liver 

K-Kidney 

B-Blubber

nth tissue in the bank 

for that program 

Location of tissue: 

C-Charleston 

G-Gaithersburg

15

Condition (condition of the sample), 

Quantity/Unit of Measurement (mass of tissue), 

Distribution Destination (Charleston, Gaithersburg, Temporary space), 

and 

Storage Location (Freezer/Rack/Box/XY Location) 

Once those data are entered, the ‘Standard EPI (epidemiological) Information’ screen 

appears (Fig. 6). This screen allows entering of values for: 

Age (neonate, sub-adult, adult, etc.), 

Race (species of animal), 

Sex (Male, Female, Unknown), and 

Geographic Location (where the animal was sampled) 

The tissue samples that are collected for the NBSB are divided in two portions, A and B. 

Portion A is collected for long-term storage (i.e., decades) and portion B is used for 

multiple analyses for different analytes. Additionally, these portions are stored in 

separate freezers to minimize the possibility of sample loss due to equipment failure. 

Currently, a plan is being implemented to store all ‘A’ portions at NBSB-Gaithersburg 

and all ‘B’ portions at NBSB-Charleston. After the samples have been properly stored, 

the shipment is posted in ASTRO and a ‘Posted Shipment Detail Report’ is printed (Fig. 

7). This report provides the following information: 

Shipping information, 

Project, 

Collection, 

NIST storage number, 

Aliquot number, 

Animal ID number, 

Collection date, 

Storage location information, 

Tissue type, 

Tissue weight, 

Condition of the sample, 

Unique ID number assigned by ASTRO, and 

User ID 

A copy of each shipment report is then placed in a binder for permanent storage. The 

field data sheet and NIST Storage data sheet information is entered into a Paradox 

database. This database is a computerized duplicate of the hardcopy sheets.


18

BIOLOGICAL DRY SHIPPERS 

The biological dry shippers are designed to transport biological samples at cryogenic 

temperatures and provide a reasonable holding time. There are primarily two sizes of shippers 

that are used, the MVE Cryo Shipper XC, which holds 10 L of liquid nitrogen and the MVE 

SC14/2V, which holds 8.7 L of liquid nitrogen (Fig. 8). The static holding time of these shippers 

are estimated at 14 d and 25 d, respectively (Fig. 9). To properly charge a shipper, the following 

instructions must be followed: 

1. Place a lab coat or nonabsorbent apron, a pair of safety goggles and a pair of 

loose insulating cryogenic gloves on, 

2. Remove the lid of the dry shipper and fill 3⁄4 of the way with liquid nitrogen, 

3. Replace the lid and let the shipper stand overnight (24 h), 

4. Remove the lid again and fill the shipper 1⁄2 of the way with liquid nitrogen, 

5. Let stand for 3 h to 4 h and pour off excess liquid nitrogen. 

All excess liquid nitrogen must be removed by pouring it out of the shipper in order to transport 

the shipper as a non-hazardous item. In addition, Teflon jars and bags and glass jars may crack if 

there is direct contact with the liquid, therefore, it is important to make sure all liquid nitrogen is 

removed. 
19

Figure 8. MVE Biological Dry Shippers; Cryo Shipper XC (left) and SC14/2V (right)

21

CLEAN ROOM 

The Clean Room is a Class 100 clean area that is used for cleaning Teflon supplies (i.e., cryomills, jars, smashers, etc.) and for preparation of samples for analyses. The cleanliness level of 

this room is critical because it is in this room that samples are exposed to airborne particles 

through the homogenization process. There are many pieces of equipment needed for the 

preparation before and during homogenization and these items, along with other equipment used 

in the Clean Room, are listed in Table 3. 

HOOD USAGE/CLEANING OF TEFLON AND TITANIUM 

A special Teflon-lined exhaust hood is provided for the cleaning of supplies. Cleaning 

procedures for Teflon and titanium materials are posted near the hood (see Appendix A). 

Always write the date that the solvent/acid bottle was opened on the label of the bottle. HPLCgrade water is used for all water rinses. The chemicals can be used for approximately 6 to 8 

cleaning sessions before disposal. See the Chemical Hygiene Plan (Appendix C) for proper 

solvent and acid disposal procedures. Do not pour any solvent, acid, or strong alkali solutions 

down the sink in the Clean Room; it is not constructed to withstand such solutions. 

CRYOGENIC HOMOGENIZATION 

Cryogenic homogenization is a process that transforms a solid frozen tissue into a 

particulate powder and provides identical (i.e., homogenous) sample aliquots. Identical sample 

aliquots are necessary to allow for valid comparison of data obtained by various researchers and 

analytical techniques. The Teflon disk mill is recommended as an effective device for size 

reduction and homogenization of biological tissue. Further, operation at cryogenic temperatures 

reduces loss of volatile components and changes in composition during the size reduction step. 

 Cryogenic homogenization is performed in the Clean Room following strict clean room 

procedures. All equipment used during the homogenization process is made of titanium or 

Teflon. This reduces the sources of contamination that are unavoidable to two types of 

materials. Additionally, the titanium and Teflon equipment is precleaned, as mentioned above, 

to eliminated any surface contamination. The following list of equipment and instructions 

pertain to the homogenization of a single sample (i.e., liver, kidney, blubber). 

 

Equipment 

 The following pieces of equipment are used for cryogenic homogenization and should be 

checked to see that everything is in proper working condition the day before homogenizing is 

conducted: 

 Liquid Nitrogen (-150 °C) Vapor Phase Freezer 

 Compressed Air Smasher 

 Air Compressor 
22

Table 3. Equipment located in the Clean Room at NBSB-Charleston.

Equipment Type Quantity Purpose

MVE XLC 1211 Liquid Nitrogen (- 150° C) 1 Working freezer for cryogenically homogenizing materials Vapor Phase FreezerTS-250 Disk Mill 1 Grind small particles of tissue into powder, for use with small Teflon disk millTS-Specialized 250 Disk Mill 1 Grind small particles of tissue into powder, for use with medium and large

 Teflon disk mills

Manual Smashing Machine 1 Smash large tissues into particles during the homogenization processSolvent Storage Cabinet 1 Store ethyl alcohol and chloroform, used for cleaning Teflon materialsAcid Storage Cabinet 1 Store hydrochloric and nitric acids, used for cleaning Teflon materialsNodes for the Datatron System 3001: Monitoring, 3 One node each to monitor temperatures in working freezer, temperatures inAlarming, and Data Management System upright freezer located in the hallway and % humidity in the roomTeflon Heat Sealer 1 Seal Teflon bagsTeflon-lined Fume Hood 1 Teflon and titanium materials are cleaned under this hoodNuaire Biological Safety Hood 1Vertical Laminar Airflow Hood 1 Cleaned Teflon and titanium materials are placed in the hood to dryMarble Table and Analytical Balance 1 Measure weights of homogenized aliquotsBalance 1 Measure samples >1 kgMet One Particle Counter 1 Measures airborne particles in a number of size ranges

Desiccator Cabinet 1Polypropylene Storage Cabinet 1 Store clean Teflon materialsDiaphragm Liquid Pump 1 Transfer chemicals/water from storage tank to bottles for cleaning supplies
23

TS-250 Disk Mill Shaker (Fig. 10) 

 Lint Free Cloths 

Items which require cleaning and cooling to liquid nitrogen temperature before homogenizing is 

conducted: 

 Teflon disk mill (Fig. 11) 

 Teflon smasher (Fig. 11) 

Teflon jars (15mL) or bags 

 Teflon scraper and scoop (Fig. 11) 

 Teflon handled titanium bladed knife 

 Stainless steel plates for shaker (Fig. 10) 

Homogenizing (Individual Sample) 

Locate the sample that is to be homogenized and collect all items in the second list. 

Make sure all equipment has been cleaned properly following the NIST cleaning procedures (See 

Appendix B). With a pre-cut lid label, properly label 24 15-mL Teflon jars with the NIST 

Storage ID Number and the Aliquot Number (B001, B002,..., B024). Preweigh all Teflon jars 

with lids and labels in place and record the masses in a laboratory notebook. Put all of the small 

items (Teflon jars, scraper, scoop, and knife) in a Teflon-lined plastic tray and place the tray in 

the LN2 freezer to cool overnight. Place the Teflon smasher, disk mill, and shaker plates at the 

bottom of the same freezer but place them on small boxes so they can be easily lifted out of the 

freezer. 

 On the day of homogenizing, remove the sample that is to be homogenized from storage 

and follow the steps below: 

 

1. Remove the puck (lid) from the Teflon smasher and place it inside the freezer. 

2. Remove sample (tissue) from Teflon jar and place approximately 150 g to 200 g inside 

the Teflon smasher. 

3. Place the puck of the smasher on top of the fragments and place the smasher at the 

bottom of the compressed air smasher. 

4. Remove the safety bar and press the UP button until the 90 lb weight is released onto the 

Teflon smasher. This will fracture the sample into small fragments. (Fig. 12) 

5. Press the DOWN button until the claw has clamped onto the 90 lb weight. 

6. Press the UP button until the safety bar can be replaced. 

7. Quickly put the smasher back inside the freezer. 

8. Remove the lid and inner puck from the disk mill and place them inside the freezer. 

9. Remove the puck from the smasher and with the Teflon spatula, carefully scrape the 

fragments from the bottom of the puck into the disk mill. Scrape remaining pieces off of 

the bottom plate of the smasher into the disk mill. Set the smasher aside in the freezer. 

10. Scrape all pieces inside the disk mill into the outer ring area. Do not force tissue 

fragments into this area, if a fragment is too large, place it back in the smasher 
24

Figure 10. TS-250 Teflon Disk Mill

Clamp/Arm

Steel Plates

Teflon Disk Mill
25

Teflon Disk Mill Teflon Smasher

Teflon Spatula and Scoop

Lid

Puck

Ring

Lid/Puck

Base

Ring

Figure 11. Teflon Materials Needed for Homogenization

Mill

Teflon Disk Mill Teflon Smasher

Teflon Spatula and Scoop

Lid

Puck

Ring

Lid/Puck

Base

Ring

Figure 11. Teflon Materials Needed for Homogenization

Mill
26

Figure 12. Compressed-Air Smashing Machine

90 lb weight

Safety Bar

Control
27

and repeat Steps 2 through 7. Once all pieces are placed loosely in the outer ring, place 

the inner puck back in the center of the disk mill and place the lid securely on top. 

11. Remove the shaker plates from the freezer and place the bottom plate on the TS-250 disk 

mill shaker. Quickly place the disk mill onto the shaker plate, place the top shaker plate 

on top of the disk mill, and fasten the shaker clamp, making sure it is on tight (Fig. 10). 

12. Close the lid and set the timer for 3 minutes. Turn the machine on and wait for it to stop. 

13. Once the machine has stopped, place the shaker plates and the Teflon disk mill back in 

the freezer, remove the mill lid, and scrape any tissue remaining on the lid into the disk 

mill. Verify that the tissue is sufficiently homogenized and has completely turned into 

powder. If the tissue is still in fragments there is probably a piece of tissue that is 

jammed between the mill and the ring (Fig. 13). Remove the puck from the disk mill and 

move the fragments back into the inner ring area with the spatula until the inner ring is 

loose enough to be removed from the disc mill completely. Move all pieces towards the 

center of the disk mill and replace the inner ring, making sure there are no pieces 

underneath the ring. Repeat steps 8, 9,10, and 11. Repeat these steps until the tissue is in 

powder form (Fig. 13). If there are any hard pieces found after repeating the steps several 

times, the equipment or material is too warm to work with and you must stop 

homogenizing and wait several hours for all materials to cool down. 

14. Using the Teflon scoop, fill all jars in numerical sequence, making sure the lids are 

placed on tightly. Weigh the jars and record the masses in the laboratory notebook. 

Place the jars in a storage box and return to the LN2 permanent freezer for long-term 

storage. 

15. Fill out the sample storage form including the homogenization date, the masses of each 

sample, any comments necessary, and identify the permanent storage locations. 

16. Update the Paradox computer file that contains the sample storage data form with the 

same new information and the ASTRO database. 

Note: When removing the lids (puck) to the Teflon disk mill and smasher, place them upsidedown inside the freezer so that the side of the lid that touches the tissue does not come in contact 

with any part of the freezer or other items in the freezer. Also, all work should be completed 

with the equipment inside of the working freezer, except when transferring Teflon equipment to 

and from the smahing unit and the TS-250 disk mill. Because the cold temperatures of the 

equipment and the tissue are critical, it is important to work quickly to keep these items inside 

the freezer as much as possible. 

 
28

Tissue in Powder Form Tissue in Fragment Form

Figure 13. Sample of Tissue in Fragment Form and Powder Form
29

SAFETY 

NBSB-Charleston has been equipped with several safety features throughout each of the 

three rooms to ensure safe laboratory operating procedures (Table 4, Figure 14). In addition, the 

CCEHBR Laboratory Safety Committee has written a Laboratory Chemical Hygiene Plan and 

Emergency Preparedness Plan that are strictly enforced throughout the entire laboratory. (See 

Appendix C). A Job Hazard Analysis Form has also been designed (See Appendix C). This 

form must be filled out for each individual laboratory and it must be posted outside of the 

laboratory. This form includes information on the work activities that are completed in the 

laboratory and what Personal Protective Equipment (PPE) is required to be worn during those 

activities. All personnel who work in that laboratory must sign this form. Also, a list of 

chemicals that are used in the laboratory must be posted outside the entrance of that laboratory 

(See Appendix C). In addition to those two lists, Material Safety Data Sheets (MSDS) for all 

chemicals used in each laboratory must be placed in an easily accessible area near the chemicals. 

In NBSB-Charleston, these sheets have been posted just inside the Clean Room on the wall. 

HURRICANE EVACUATION 

 

The Atlantic hurricane season is from June 1 to November 30. Special actions must be 

taken in order for NBSB-Charleston to be prepared for a hurricane evacuation. If a mandatory 

evacuation is announced, the following steps must be followed before leaving the CCEHBR 

building: 

1. The computer in the Specimen Bank and in the NIST Office must have the latest back up 

of files on zip disk. These include the ASTRO database inventory and all Paradox files 

with completed field data sheets for all samples inventoried. These disks must be taken 

out of the building by authorized personnel. 

2. A hard copy of the NBSB inventory must be printed from the ASTRO database for all 

freezers. This will ensure that if a freezer has a mechanical breakdown and the inside of 

the freezer does not maintain cryogenic temperatures, those samples can be labeled 

properly. This hard copy must be taken out of the building. 

3. All -180 °C liquid nitrogen freezers must be filled with liquid nitrogen just before 

evacuation. 

4. All temperatures and liquid nitrogen levels must be recorded in the black NIST 

Laboratory notebook and taken with personnel out of the building. 

5. All binders with AMMTAP, MMHSRP, and STAMP Field and NBSB Storage Data 

Sheets must be taken with personnel out of the building. Also, binders that contain 

information on miscellaneous items that are stored in the NBSB must also be taken. 

6. The Cryo-Cyl 230LP must be filled with liquid nitrogen and brought into the Specimen 

Bank. 

7. All computers and other electrical equipment must be covered with plastic in case of 

water leaks due to roof damage. 
30
31

LN2

Freezer

LN2

Freezer

-80 °C

Freezer Cabinet Balance Bio Hood

Teflon

Fume 

Hood

15 Small

Disk

Mill

ANTE ROOM

ROOM 227BCLASS 10000SPECIMEN BANK

ROOM 227ACLASS 100CLEAN ROOM

14

2

5

Figure 14. NBSB-Charleston Safety Features

Large

Disk

Mill

-80 °C

Freezer

Manual Smasher

1

Acid 

Storage

9

Laminar

Flow

Hood

11

10

34

3

4 

13

12

9

LN2

Freezer

LN2

Freezer

LN2

Freezer

LN2

Freezer

LN2

Freezer

3

CompressedAirSmasher

Cabinet

6

6

6

6

6

1. Entrance/Emergency Exit2. Fire Extinguisher3. Smoke Detector4. Fire Alarm Strobe Light5. Emergency Break Bar Exit6. LN2 In-Line Piping System

7. Phone Access8. Incoming Page Speaker9. Acid Storage Cabinet10. Humidity & Oxygen Sensor11. Solvent Storage Cabinet12. Window13. Eyewash Station14. Emergency Shower15. Teflon Fume Hood

7

8

8

LN2

Freezer

LN2

Freezer

-80 °C

Freezer Cabinet Balance Bio Hood

Teflon

Fume 

Hood

15 Small

Disk

Mill

ANTE ROOM

ROOM 227BCLASS 10000SPECIMEN BANK

ROOM 227ACLASS 100CLEAN ROOM

14

2

5

Figure 14. NBSB-Charleston Safety Features

Large

Disk

Mill

-80 °C

Freezer

Manual Smasher

1

Acid 

Storage

9

Laminar

Flow

Hood

11

10

34

3

4 

13

12

9

LN2

Freezer

LN2

Freezer

LN2

Freezer

LN2

Freezer

LN2

Freezer

3

CompressedAirSmasher

Cabinet

6

6

6

6

6

1. Entrance/Emergency Exit2. Fire Extinguisher3. Smoke Detector4. Fire Alarm Strobe Light5. Emergency Break Bar Exit6. LN2 In-Line Piping System

7. Phone Access8. Incoming Page Speaker9. Acid Storage Cabinet10. Humidity & Oxygen Sensor11. Solvent Storage Cabinet12. Window13. Eyewash Station14. Emergency Shower15. Teflon Fume Hood

7

8

8
32

DATATRON 

The Datatron System 3001: Monitoring, Alarming, and Data Management 

System, created by R & D Scientific Corporation, has been installed to continuously 

monitor the temperatures of all freezers located in NBSB-Charleston as well as monitor 

room conditions (i.e., temperature and humidity). Three probes (channels) have been 

placed in each of the liquid nitrogen vapor freezers to record temperatures at the top, 

center and bottom of each freezer and one probe has been placed at the top of each of the 

-80 °C ultra-cold upright freezers. An alarm will sound if pre-set condition ranges are 

exceeded and a message providing the alarm number and location of the alarm will 

display across the LCD display message board, located in the NIST Office Space, Room 

225. Also, the phone alarm notification system is designed to notify remote users of any 

alarms that are triggered either by channels out of their alarm limits or rebooting of the 

computer system (e.g. due to a power failure). The system uses one of two lists of phone 

numbers and users, List 1 (0800-1659) and List 2 (1700-0759), and begins calling the 

entries on the list. The system will continue calling the entries in order until an 

acknowledgment has been made by one of the users. The system ensures that trained 

personnel will be able to correct the problem or error within the NBSB before any 

damage has occurred. The Datatron System will also store all information that is 

provided by the trained user (i.e., the cause of the alarm event). The computer, LCD 

message board, and printer for the Datatron 3001 system are located in the NIST Office 

space in Room 225. A manual has been designed by R & D Scientific Corporation for 

this system and is stored next to the computer as a reference. 
33

MAINTENANCE 

In general, cleaning should begin in areas that require the most critical level of 

cleanliness and proceed toward areas of less critical requirements (Fig. 15). The 

following cleaning methods are to be followed to ensure that each room maintains the 

Class 100 and 10,000 levels. Basically, the preferred clean room cleaning protocol is to 

vacuum an area, wipe that area with a cleanser and lint-free cloths and vacuum the area 

again. 

Vacuum ceiling panels using a dry vacuum with a soft brush attachment. To 

protect the HEPA filters, only the grid surrounding the filters should be cleaned, using 

overlapping strokes following the grid pattern in one direction only. Remove spots with 

a commercial cleanser, deionized (DI) water and lint-free cloth and then vacuum again to 

remove any loosened particles. 

 Vacuum lighting units with a soft brush attachment. Open the unit and wipe the 

bulbs with a lint-free cloth dampened with DI water, vacuum horizontal surfaces, and 

close unit. Wipe all trim pieces. 

 Vacuum walls first, starting at the ceiling and working in vertical lines toward the 

floor. Next, use a sticky roller to wipe the wall, using overlapping movements from the 

ceiling towards the floor. Vacuum again using a soft brush attachment. 

 Clean doors, frames, and components using a vacuum, followed by wiping with a 

lint-free cloth that has been moistened with Deconahol and DI water. Doors should be 

cleaned while they are ajar. 

 Wipe glass surfaces and windows with a lint-free cloth that has been dampened 

with Deconahol. With a dry wiper or squeegee, dry the window starting at the top and 

move vertically towards the bottom. 

 Use a lint-free cloth dampened with Deconahol and DI water to clean piping 

systems. Wipe from top to bottom, using overlapping strokes. Vacuum pipes with a 

curved pipe attachment and then rewipe the pipe, if necessary, to remove stubborn grime 

and spots. 

 Vacuum freezers with a soft brush attachment. Use a lint-free cloth dampened 

with DI water to clean the top and sides of the freezer, use overlapping strokes to wipe 

the top of the freezer and then from top to bottom for the sides of the unit. Revacuum 

with a soft brush attachment to pick up any loose particles. 

 Wipe work stations/bench tops with a lint-free cloth moistened with Deconahol 

and DI water. Start at the rear of the work surface and use overlapping strokes in one 

direction only. Use a new area of the wiper for each stroke. 

 Vacuum the surface of the floor first, place the sticky mop or vacuum on the floor 

and pull toward you, lift and move the vacuum so that the next stroke starts adjacent to 

and slightly overlaps the first stroke. Next, wash the floor with a solution of DI water 

and floor cleanser using a clean mop head. Rinse with DI water and a new mop head, 

leaving the rinse water on the floor long enough to completely saturate any film build-up. 

Mop up the rinse water, making sure to change the water after mopping every 10 to 15 

square feet of floor surface. Because the floor is the dirtiest part of the clean room, it is 

important to keep the wash water and rinse water clean by changing it after 10 to 15 

square feet of surface has been mopped. After the floor is dry, vacuum again, using the 

same movement as described above. 
34

 

LN2

Freezer

LN2

Freezer

-80 °C

Freezer Cabinet Balance Bio Hood

Teflon

Fume 

Hood

Small

Disk

Mill

ANTE ROOM

ROOM 227BCLASS 10000SPECIMEN BANK

ROOM 227ACLASS 100CLEAN ROOM

Figure 15. NBSB-Charleston Cleanliness Levels

Large

Disk

Mill

-80 °C

Freezer

ManualSmasher

Acid 

Storage

Laminar

Flow

Hood

LN2

Freezer

LN2

Freezer

LN2

Freezer

LN2

Freezer

LN2

Freezer

CompressedAirSmasher

Cabinet

Clean Area

Cleanest Area

Clean Area

Cleanest Area

Sticky Mat

Sticky Mat

Sticky Mat

LN2

Freezer

LN2

Freezer

-80 °C

Freezer Cabinet Balance Bio Hood

Teflon

Fume 

Hood

Small

Disk

Mill

ANTE ROOM

ROOM 227BCLASS 10000SPECIMEN BANK

ROOM 227ACLASS 100CLEAN ROOM

Figure 15. NBSB-Charleston Cleanliness Levels

Large

Disk

Mill

-80 °C

Freezer

ManualSmasher

Acid 

Storage

Laminar

Flow

Hood

LN2

Freezer

LN2

Freezer

LN2

Freezer

LN2

Freezer

LN2

Freezer

CompressedAirSmasher

Cabinet

Clean Area

Cleanest Area

Clean Area

Cleanest Area

Sticky Mat

Sticky Mat

Sticky Mat

LN2

Freezer

LN2

Freezer

-80 °C

Freezer Cabinet Balance Bio Hood

Teflon

Fume 

Hood

Small

Disk

Mill

ANTE ROOM

ROOM 227BCLASS 10000SPECIMEN BANK

ROOM 227ACLASS 100CLEAN ROOM

Figure 15. NBSB-Charleston Cleanliness Levels

Large

Disk

Mill

-80 °C

Freezer

ManualSmasher

Acid 

Storage

Laminar

Flow

Hood

LN2

Freezer

LN2

Freezer

LN2

Freezer

LN2

Freezer

LN2

Freezer

CompressedAirSmasher

Cabinet

Clean Area

Cleanest Area

Clean Area

Cleanest Area

Sticky Mat

Sticky Mat

Sticky Mat
35

The cleaning schedule for the NBSB will depend upon the amount of work being 

conducted in the rooms and the number of particles that are detected on the particle 

counter. After using the clean room for cleaning supplies or homogenizing samples, the 

above procedure should be followed precisely, starting with the ceiling and working 

down to the floors. A weekly particle count will be taken to ensure the rooms are staying 

at Class 100 and 10,000 levels. If the particle counts are above the class levels required, 

a thorough cleaning will be conducted. Because the Clean Room is two class levels 

higher than the Specimen Bank, entry from one room to the next should be kept to a 

minimum. To help prevent particle exchange between rooms, a 1.4 m x 2.5 m vinyl 

curtain made of 0.2 m wide strips has been mounted above the door between the 

Specimen Bank and the Clean Room. A sticky mat has also been adhered to the floor in 

front of that door to trap any particles that are on the bottom of the shoe covers. These 

measures will help to minimize the number of particle exchanges between the two rooms. 
36

REFERENCES 

Ayal, N. 1998, Archival Specimen Tracking Retrieval Operation User’s Guide. Center 

for Disease Control and Prevention, Atlanta, GA. 

Becker, P.R., S.A. Wise, B.J. Koster, and R. Zeisler, 1991, Alaska Marine Mammal 

Tissue Archival Project: Revised Collection Protocol, NISTIR 4529, USDOC, National 

Institute of Standards and Technology, Gaithersburg, MD. 

Becker, P.R., B.J. Koster, S.A. Wise, and R. Zeisler, 1993, Biological specimen banking 

in Arctic research: an Alaska perspective. Sci. Total Environ. 139/140: 69-95. 

Becker, P.R., D. Wilkinson, and T.I. Lillestolen, 1994, Marine Mammal Health and 

Stranding Response Program: Program Development Plan. USDOC, NOAA Tech. 

Memo. NMFS-OPR-94-2, Silver Spring, MD. 

Becker, P.R., S.A. Wise, L. Thorsteinson, B.J. Koster, and T. Rowles, 1997, Specimen 

banking of marine organisms in the United States: current status and long-term 

prospective. Chemosphere, 34(9/10): 1889-1906. 

Institute of Environmental Sciences and Technology, 1997, IEST-RP-CC006.2: Testing 

Cleanrooms. Contamination Control Division, Institute of Environmental Sciences and 

Technology, 35 pp. 

Lillestolen, T.I., P.R. Becker, and D. Wilkinson, 1993, The National Marine Mammal 

Tissue Bank and Stranding Network Program. Coastal Zone 93: Proceedings of the 8th 

Symposium on Coastal and Ocean Management (New Orleans, LA), O.T. Magoon, W.S. 

Wilson, H. Converse, and L.T. Tobin, eds., ASCE, New York, NY, 3: 2494-2504. 

National Bureau of Standards, 1986, Certificate of Analysis, Standard Reference Material 

101g, 18 Cr-10 Ni Steel (AISI 304L). Analytical Chemistry Division, National Bureau of 

Standards, Gaithersburg, MD, 2 pp. 

U.S. General Services Administration, 1992, Federal Standard 209E: Airborne Particulate 

Cleanliness Classes in Cleanrooms and Clean Zones. Institute of Environmental Sciences 

and Technology. 

Wise, S.A., and R. Zeisler, 1985, The U.S. Pilot Environmental Specimen Bank Program. 

In: International Review of Environmental Specimen Banking. National Bureau of 

Standards Spec. Pub. 706, Wise, S.A., and R. Zeisler, eds., Gaithersburg, MD. 34-45. 

Wise, S.A., B.J. Koster, J.L. Langland, and R. Zeisler, 1993, Current activities within the 

National Biomonitoring Specimen Bank. Sci. Total Environ. 139/140:1-12. 
37

Wise, S. A. and B.J. Koster, 1995, Considerations in the Design of an Environmental 

Specimen Bank: Experiences of the National Biomonitoring Specimen Bank Program. 

Environ. Health Perspect. 103 (Suppl. 3): 61-67. 

York, G.W., B.J. Porter, R.S. Pugh, D.G. Roseneau, K. Simac, P.R. Becker, and S.A. 

Wise. 2001, Seabird Tissue Archival and Monitoring Program: Protocols for Collecting 

and Banking Seabird Eggs, NISTIR 6735, National Institute of Standards and 

Technology, Gaithersburg, MD. 

Zeisler, R., B.J. Koster, and S.A. Wise, 1992, Specimen Banking at the National Institute 

of Standards and Technology. In: Analytical Approaches as Related to Specimen 

Banking. Rossbach, M., and J.D. Schladot, eds., Stuttgart: Springer Verlag, 37-49.