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Section IV. Guidance Documents
Chapter II. Growing Areas
Guide Contents
.10 Approved National Shellfish Sanitation Program Laboratory Tests: Microbiological and Biotoxin Analytical Methods
1. Microbiological Methods
Application |
Sample Type |
Total Coliform1 |
Fecal Coliform2 |
Standard Plate Count3 |
A-1M |
APHA |
ETCP |
APHA
Decimal Dilution
MPN |
12 tube single dilution
MPN |
Other |
Decimal dilution
MPN |
12 tube single dilution
MPN |
Decimal dilution
MPN |
12 tube single dilution
MPN |
mTEC |
Growing Area Survey & Classification |
Seawater |
X |
X |
|
X4 |
X4 |
X |
X |
X |
|
|
Controlled Relaying |
Seawater |
X |
X |
|
X4 |
X4 |
X |
X |
X |
|
|
Shellfish |
|
|
|
|
|
X |
|
|
|
|
Wet Storage |
Seawater |
|
|
X |
|
|
|
|
X |
|
|
Shellfish |
|
|
|
|
|
X |
|
|
|
|
Controlled Purification |
UV Effluent |
|
|
X |
|
|
|
|
X |
|
|
Shellfish |
|
|
|
|
|
X |
X |
|
X |
|
Market Shellfish |
Shellstock |
|
|
|
|
|
X |
|
|
|
X |
Shucked |
|
|
|
|
|
X |
|
|
|
X |
PHP |
Shucked |
|
|
|
|
|
|
|
|
|
|
In Shell |
|
|
|
|
|
|
|
|
|
|
References
- Total Coliform Methods
- American Public Health Association. 1970. Recommended
Procedures for the Examination of Sea Water and Shellfish, 4th
Edition, APHA, New York, N. Y. [Decimal Dilution MPN test]
- American Public Health Association, American Water Works
Association, and Water Environmental Federation. 1992. Section 9221.
Examination of a 100 ml aliquot by the Multiple Tube Fermentation
Method (MTF). Standards Methods for the Examination of Water and
Wastewater, 18th Edition, APHA/AWWA/WEF. Washington,
D.C. [Decimal Dilution MPN test]
- Redman, J. H. 1974. A simpler multiple fermentation tube test for
monitoring the bacteriological quality of shellfish harvest waters; the
examination of twelve 1.0 ml sample portions, p.123-124. In
Wilt, D. S. (ed.), Proceedings 8th National Shellfish
Sanitation Workshop,. U.S. Food and Drug Administration,
Washington, D.C. [12-tube, Single Dilution MPN test]
- Springer, J. A. 1974. Statistical considerations in using the
twelve-tube MPN test for routine monitoring of shellfish waters,
p.125-126. In Wilt, D. S. (ed.), Proceedings 8th
National Shellfish Sanitation Workshop. U.S. Food and Drug
Administration, Washington, D.C. [12-tube, Single Dilution MPN test]
- Fecal Coliform Methods
- 40 CFR 136.3 Table I-A. List of Approved Microbiological Methods
- A-1M, 1990 AOAC International - Official
Methods of Analysis, 15th Edition. Association of
Official Analytical Chemists. Washington, D.C. [A-1 Modified MPN test]
- APHA. 1998. Standard Methods for the Examination of Water and Wastewater APHA, 20th Edition,
APHA, Washington, DC
- American Public Health Association. 1970. Recommended
Procedures for the Examination of Sea Water and Shellfish, 4th
Edition, APHA, New York, N.Y.
- Rippy, Scott, et. al, Enumeration of Fecal Coliforms and E. coli in marine
and estuarine waters: an alternative to the APHA-MPN approach. Journal Water
Pollution Control Federation. August 1987, pg. 795-798.
- U.S. Food and Drug Administration. 2001. (Revised 12-8-02). NSSP
Interpretation, 03-III-@.02-100, Options for the use of the 12-tube,
single dilution MPN test. [12-tube, Single Dilution MPN test for
seawater]
- U.S. Food and Drug Administration. 2001 (Revised 12-8-02). NSSP
Interpretation 03-XV-.03-100, Method for determining fecal coliform
levels in end product depurated shellfish. [12-tube, single dilution
MPN test for the controlled purification of shellfish]
- Cabelli, V. J. and W. P. Heffernan. 1970. Accumulation of Escherichia
coli by the northern quahog. Appl. Microbiol. 19:239-244. [ETCP
for the controlled purification of hard- and soft-shelled clams]
- Standard Plate Count Method
- American Public Health Association. 1970. Recommended
Procedures for the Examination of Sea Water and Shellfish, 4th
Edition, APHA, New York, N. Y.
Footnote:
4 The use of A-1 Medium Minus Salicin is optional with the following condition. Any State that eliminates Salicin must show equivalency with a minimum of 30 samples seasonally (4 sets of 30 samples). The samples must generate results and the results must not be significantly different as shown by using a T test. The data generated to support the elimination of Salicin must be submitted to FDA for review and concurrence prior to State action to eliminate the use of Salicin.
2. Marine Biotoxin Method
Application |
Sample Type |
Paralytic Shellfish Poison (PSP) |
Neurotoxic Shellfish Poison (NSP) |
Growing Area Survey & Classification |
Shellfish |
X |
X |
Controlled Relaying |
Shellfish |
X |
X |
References:
- Paralytic Shellfish Poison (PSP) and Neurotoxic Shellfish Poison (NSP) Methods
American Public Health Association. 1970. Recommended Procedures for the Examination of Sea Water and Shellfish, 4th
Edition, APHA, New York, N.Y.
3. Type III and Type IV Microbiological Test Methods
Application |
Sample Type |
Total Coliform |
Fecal Coliform |
Other |
Growing Area Survey & Classification |
Seawater |
|
|
|
Controlled Relaying |
Seawater |
|
|
|
Shellfish |
|
|
|
Wet Storage |
Seawater |
|
|
|
Shellfish |
|
|
|
Controlled Purification |
UV Effluent |
Type III1 |
|
|
Shellfish |
|
|
|
Market Shellfish |
Shellstock |
|
|
|
Shucked |
|
|
|
Footnotes:
- 1. Single step direct mEndo-LES Membrane Filter Technique used in the Newburyport Depuration Plant, Massachusetts.
4. Type III and Type IV Marine Biotoxin Test Method
Application | Sample Type |
NSP |
PSP |
DSP |
AS |
Other |
Growing Area Survey & Classification |
Shellfish |
|
Type III2 |
|
Type III1 |
|
Controlled Relaying |
Shellfish |
|
|
|
Type III1 |
|
Footnotes:
- Peer recognized HPLC Methods with or without clean up.
- Jellett Rapid Test for PSP
- Method can be used to determine when to perform a mouse bioassay in a previously closed area.
- A negative result can be substituted for a mouse bioassay to maintain an area in the open status.
- A positive result shall be used for a precautionary closure.
5. Interim Approval by ISSC Executive Board August 2007
Application |
Sample Type |
Vv |
Vv |
Vp |
Vp |
PHP |
Shucked |
EIA1 |
MPN2 |
MPN3 |
PCR4 |
In-Shell |
EIA1 |
MPN2 |
MPN3 |
PCR4 |
Footnotes:
1 EIA procedure of Tamplin, et al, as described in Chapter 9 of the FDA Bacteriological Analytical Manual, 7th Edition, 1992,
2 MPN method in Chapter 9 of the FDA Bacteriological Analytical Manual, 7th Edition, May 2004 revision, followed by confirmation using biochemical analyses or by the DNA -alkaline phosphatase labeled gene probe (vvhA).
3 MPN format with confirmation by biochemical analysis, gene probe methodology.
4 PCR methods as they are listed in Chapter 9 of the FDA Bacteriological Analytical Manual, 7th Edition, May 2004 revision, or a method that a State can demonstrate is equivalent.