NVIRONMENTAL
February 1965
     1964
  PROCEEDINGS
  Shellfish Sanitation
      Research
Planning Conference
          U.S. DEPARTMENT OF
       HEALTH, EDUCATION, AND WELFARE
          Public Health Service

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              7964
            PROCEEDINGS
        Northwest
 Shellfish Sanitation
         Research
Planning Conference
      W.J. Beck, J.C. Hoff and T.H. Ericksen

      Northwest Shellfish Sanitation Laboratory
          Gig Harbor, Washington
     U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE
            Public Health Service
      Division of Environmental Engineering and Food Protection
           Shellfish Sanitation Branch

              1965

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                         Disclaimer Clause
   The use of trade names of products  in this  publication  are  for
example only and do not imply endorsement by the Public Health
Service or the U.  S, Department of Health,  Education and Welfare.
          Public Stealth Service Publication No.

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                           PREFACE
     In 1959 the first Annual Northwest Shellfish Sanitation Research
Planning Conference was held at Purdy, Washington.  The principal ob-
jectives of research in problems relating to the sanitary control of
shellfish in the Pacific Northwest were outlined at this time.

     Many portions of the original objectives have been accomplished.
However, as one facet of research has been completed other projects
in the same realm remain to be completed.  Thus many of the basic ob-
jectives of the original conference are appropriate six years later.
New problems have emerged in the ensuing years that have required
expanding the scope of our research activities beyond that outlined
in the plans developed in 1959.  Publication of proceedings of the
1964 conference will inform participants in the cooperative program
of progress being made in attacking these more recent problems.

     We are deeply grateful to the conferees for their frank discus-
sions of the problems through the past years.  Participation by
personnel of State and Federal agencies, both in this country and
Canada, universities and industry has made these conferences success-
ful.  We pledge this laboratory to continue in the spirit of coopera-
tion that has prevailed in all phases of sanitary control of shellfish
on the international level.
                                           W.  J.  Beck

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                            CONTENTS

                                                                    Page

Summarization of Activities, W. J. Beck	    1

PART I.  RESEARCH PROGRESS REPORTS

     A Study of the Applicability of Several Indices as Sanitary
     Quality Indicators in Commercially Packed Pacific Oysters
     (Crassostrea gieas)
        J. C. Hoff, W. J. Beck, and M.  W.  Presnell	    3

     Effect of Antifoaming Agents and Evacuation on Shellfish
     Homogenization
        T. H. Ericksen	15

     Activities of Public Health Service Research Centers
        C. B. Kelly    	21

     Brief Hydrographic Survey of Burley Lagoon,  Washington
        P. S. Kelley	24

     Bacteriological Study of Stored Pacific Oyster Shellstock
        J. C. Hoff, W. J. Beck, and T.  H.  Ericksen	46

     Storage Studies on Manila Clams (Tapes japonica)  and Native
     Littleneck Clams (Protothaca staminea) shellstock
        T. H. Ericksen, J. C. Hoff, and W.  J. Beck	55

     Studies on Depuration-induced Changes  in the Composition
     of the Pacific Oyster (Crassostrea gigas)
        G. Wedemeyer, J.  R, Chung, B. J. Kemp, and A.  M.  Dollar .  .   66

     Studies on the Behavior of a Bacteriophage in the Pacific
     Oyster (Crassostrea gigas)
        J. C. Hoff and W. J. Beck	69


PART II.  PROPOSED RESEARCH ACTIVITIES  FOR FISCAL YEAR 1965

     Overall Plan for 1965	    84

     Accumulation and Elimination of Enteroviruses in West
     Coast Shellfish   	    85

     Survival, Outgrowth, and Toxin Production from Gram-
     positive Spore Forming Bacteria 	    86
                                 ii

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                                                                Page




     Enteropathogenie E.  coll 	  .........*.    88




     Oceanographic Methods  	    38




     Pilot Plant Study on Depuration   .....  	    89






PART III.  APPENDIX






     Agenda	   92




     Attendance Roster 	   93
                                 iii

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                   Summarization of Activities

                           W. J. Beck
     During the past few years several parts of the original proposed
program of research activities of 1959 have been completed.   The ini-
tial stage of research dealing with methods of analysis and fate of
indicator organisms as shown in the ecological study is ready for pub-
lication.  Various studies on storage of shellfish are reported in
these proceedings.  The laboratory phase of accumulation-elimination
of bacteria by shellfish has been completed.

     With the phasing out of certain research projects others have
been initiated as proposed by the conferees at the Annual Planning
Conference.  One set of data in oceanography of a local estuary has
been obtained.  This study was initiated to complement the sanitary
and bacteriological surveys made in the past.  Accumulation-elimina-
tion studies of viruses by shellfish have also been initiated.

     Through the cooperation of Washington State Departments of Health
and Fisheries, samples of Pacific oysters and overlying water were
analyzed for DDT-DDE during the Hemlock Looper spraying program on
Willapa Basin.

     Technical assistance has been given to State and Regional offices.
The laboratory evaluation of the West Coast States has been placed on
an annual schedule.  A paper on fecal coliform in shellfish growing
areas has been prepared for presentation at the forthcoming National
Shellfish Sanitation Workshop.  Several short-term training courses
for in-service and State Laboratory personnel have been conducted on
various aspects of shellfish sanitation bacteriology.

     The year has been one of change and expansion.  As one research
project was completed other aspects of the overall problem in the
sanitary control of shellfish remained to be solved.   With addition
of new personnel and more space we hope to delve more deeply into
the many complex problems associated with shellfish sanitation re-
search.
                              - 1 -

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Research Progress Reports
           - 2 -

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A Study of the Applicability of Several Indices as Sanitary Quality In-
  dicators in Commercially Packed Pacific Oysters (Crassostrea gigas)


             J. C. Hoff, W. J. Beck and M. W. Presnell
                           INTRODUCTION

     Various tests used in other areas of sanitary bacteriology have
been used to determine the microbiological quality of fresh packed
oysters in commercial channels.  The most important objective of such
a test is to determine the disease transmitting potential of the
product.  Therefore, such a test, or tests, should indicate whether
or not the shellfish have been exposed to fecal pollution from man
and other warm blooded animals and the degree of such exposure if
present.  Such a test or index should remain stable during proper
marketing operations subsequent to harvesting, thereby indicating
the sanitary quality of the product as harvested and reflecting po-
tentially hazardous mishandling during processing.

     Another objective, also related to public health, is to determine
the freshness, i.e. presence or absence of spoilage of the product.
Since temperature and duration of holding time are two primary factors
influencing spoilage, such a test, or tests,  should reflect the quality
of handling from these two aspects.  It is obvious that the same test
could not satisfy both objectives, since in the first instance the in-
dex should remain stable and in the second the index should change in
response to temperature and time.

     This study was undertaken to investigate the value of various in-
dices relative to the above objectivea and also to determine whether
or not modification of the present maximum temperature recommended for
handling of shucked shellfish would be advisable.  The investigation
was initiated in January,  1961 and continued through June, 1963.   Modi-
fications in storage temperatures and bacterial indices applied were
made when predicated by results obtained.
                       MATERIALS AKD METHODS

Description of Study:

     In the 1961 studies, the behavior of five groups of indicator
organisms in Pacific oysters (Crassostrea gigas)  during storage at 3 C
and -21 C was determined.  Individual experiments were conducted over
a period of several months to evaluate possible seasonal effects on
the index groups.  The index groups studied included the coliform,
fecal coliform, fecal streptococcus, 35 C standard plate count,  and
20 C plate count in sea water agar.  The pH of samples was also de-
termined.  The results of this phase of the study indicated that the
fecal coliform test and the 35 C standard plate count gave the best
indication of quality.  Little change in any of the indices occurred
at -21 C.

                                 - 3 -

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     Accordingly, in the 1962-63 studies, the -21 C storage temperature
was discontinued.  In these studies, oysters were stored in crushed ice
and at 3 C and 10 C dry storage.  The latter temperature is the upper
limit recommended for storage of shucked shellfish (Jensen, 1962).
Studies of the coliform, fecal coliform, and 35 C plate count index
groups and pH determinations were continued in the 1962-63 studies.
Commercial sources were broadened to include some Oregon plants and
lots were collected through several seasons as before.
Oysters;

     The oysters used in this study were obtained from commercial sources
in Washington and Oregon.  Each lot consisted of 36 commercially packed
and sealed twelve-ounce containers.  The containers were collected immed-
iately after shucking, washing and packing.  They were then placed in refrig-
erator cases in crushed ice, and transported to the laboratory.  Transport
time varied from a few minutes to eight hours, depending on the proximity
of the commercial source.  On arrival at the laboratory each lot was sub-
divided for storage at various temperatures.

     Iced samples were stored in insulated chests and kept constantly and
completely surrounded by crushed ice.  Standard refrigerators equipped with
special thermoregulators and facilities for air circulation were used for
dry storage at 3 C and 10 C.  One container from each lot, sampled immed-
iately on arrival at the laboratory, provided the 0 hour sample for all
three temperatures.


Samples and Sampling Schedule;

     The samples consisted of the entire contents of one twelve-ounce con-
tainer after one oyster had been removed aseptically and chopped for the
pH determination.  The sampling schedules for the three storage tempera-
tures were as follows:

           (1)  Iced Storage:  0 hour, 1,2,A,7,10,15,20 and 25 days.

           (2)  3 C Storage:   Same as (1) except sampling terminated
                               on 20th day.

           (3)  10 C Storage:  0 hour, 1,2,3,4,5 and 6 days.

     These periods were sufficiently long so that definite signs of spoil-
age, indicated by off-odor and cell lysis, were apparent at or before the
last sampling time at each temperature.

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Examination;

     The bacteriological indices determined on samples consisted of five
tube coliform and fecal coliform MPN's and standard plate counts at 35 C
performed according to recommended procedures (1962).  The pH was de-
termined electrometrically.  Results of examination for fecal streptococci
and 20 C plate counts in sea water agar and the results of -21 C storage
are not included because these tests were discontinued in the latter part
of the study.

     In some of the experiments, DfViC tests on cultures isolated from EC
positive tubes were performed to determine whether or not a differential
die-off of Escherichia coll occurred during storage.
.Storage schedule;

     Storage experiments were planned so that at least three lots were
examined in each of three seasons:  summer, winter, and spring.  In this
way, possible effects of seasonal variations in the oysters and their
bacterial flora on changes in bacterial densities in the shucked product
could be evaluated.
                              RESULTS

     The chronology of the storage experiments and the classification of
the data for analysis are shown in Table 1.  In the 1961 studies changes
in some of the indices in the summer lots differed from those observed
in the winter and spring lots.  Therefore, these lots were separated
into two groups.  Since analysis of the 1962-63 results indicated that
similar bacteriological changes occurred in all lots, these were placed
in one group.  Table 1 shows that the summer lots generally were col-
lected following period of much less rainfall than the winter and spring
lots.  This difference was more pronounced in 1961 than In 1962-63,

     Geometric mean values for coliform MPN's and 35 C plate counts in
each group were calculated.  Because some fecal coliform MPN's were inde-
terminate (<18), mediati values were calculated for this index.  Arithmetic
mean values were used for the pH data.  In most of the 1961 studies, dupli-
cate determinations were performed on each sample.  In the 1962-63 studies,
determinations were done singly.  In a few instances, due to laboratory
accidents or indeterminate results, data were not available for a particu-
lar sampling time.  In some lots, storage was terminated earlier than in
others because of definite signs of spoilage.  In these instances, the
results are based on fewer than the number of lots indicated.  The results
are shown in Figures 1-5.
                                   - 5 -

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     A comparison of the behavior of coliform MPN's and 35 C plate counts
 in  1961 winter-spring and summer lots stored at 3 C is shown in Fig. 1.
 The summer  lots contained lower initial levels of both indices.  Also, the
 initiation  of rapid multiplication of both groups was delayed in the summer
 lots.  Coliform MPN's and 35 C plate counts in the summer lots remained at
 their initial levels for 3 days and 5 days longer, respectively, than in
 the winter-spring lots.  Coliform MPN's in the 1961 lots increased slowly
 and in one  case appeared to decline after the tenth day of storage.  This
 may not have been an actual reduction since there was evidence of inhibi-
 tion in the lower KPN dilutions.  In some cases gas was not produced in
 the lower dilutions in the presumptive medium but was produced in higher
 dilutions.  In other instances, inocula transferred from positive lower
 dilution tubes failed to produce gas in Brilliant Green Bile Broth while
 growth transferred from positive higher dilution tubes did produce gas.
 However, the patterns of gas positive tubes were so erratic that it was
 not possible to interpret the data on the basis of definite inhibition.
 Results similar to these were encountered in a few individual samples
 from several subsequent lots.  Plate count densities were not noticeably
 affected by the inhibition.

     The changes in coliform MPN's of the 1962-63 lots stored in ice, 3 C
 and 10 C are shown in Fig. 2.  The behavior of coliform MPN's in these
 lots stored at 3 C differed from those observed in the 1961 lots.  Rapid
multiplication of coliforms in the 1962-63 lots began immediately and
 numbers became progressively higher.  Coliform MPN's in iced samples
 showed little initial stability but increased less rapidly than at 3 C.
At  10 C, coliform MPN's began to increase immediately and rapidly, reach-
 ing very high levels by the fourth day of storage.

     The changes in 35 C plate counts in the 1962-63 lots stored in ice,
 3 C and 10 C are shown in Fig. 3.  At 3 C, the 35 C plate counts in the
 1962-63 lots began to increase sooner than in the 1961 lots (see Fig.1).
The 35 C plate counts in the 1963 summer lots stored at 3 C increased
much more rapidly than in the 1961 summer lots.  In iced samples, the
 35  C plate count remained relatively stable for 7 days, then increased
 rather rapidly.   At 10 C the 35 C plate counts increased very rapidly,
 reaching high levels by the fourth day of storage.

     Changes in fecal coliform MPN's during storage are shown in Fig. 4
 (1961 lots) and Fig. 5 (1962-63 lots).   As indicated above these results
are based on median values rather than geometric means.  At 3 C and in
 ice, fecal coliform MPN's remained quite stable but decreased somewhat
 during the storage period.  At 10 C (Fig.  5) fecal coliform MPN's did
 increase about thirtyfold during the storage period of six days.  This
was much lower than the increases in coliform MPN's and 35 C plate
 counts.
                                 - 6 -

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     The results of IMViC tests on isolates from EC positive tubes are
shown  in Table  2.  The number of isolates is limited since IMViC tests
were performed  only at selected intervals.  No evidence of either se-
lective survival or dieoff of E. coli compared with other IMViC types
is evident.

     The results of pH determinations are shown in Table 3.  Since differ-
ences  in pH changes between the winter-spring lots and the summer lots
collected in 1962-63 were apparent, these results are presented separately.
The largest pH  decline in the 1961 lots occurred in the winter-spring lots
during the first day of storage.  In the 1963 summer lots the greatest de-
cline  occurred  during the first day of storage at all three temperatures.
In the case of  3 C and ice storage, these sudden drops were not correlated
with increases  in coliform, fecal coliform or plate count densities.  In
addition, in most cases, similar pH values at all three temperatures were
shown  after two and four days storage.  However, coliform and plate count
densities were  much higher in oysters stored at 10 C than at the two lower
temperatures at these time intervals (Figs. 2 and 3).


                              DISCUSSION

     The data presented above confirm and extend the observation of
Presnell, (1962) regarding the fecal coliform group enumerated by the EC
test.  That is, that this group ... "showed the most consistent patterns
of change and was influenced least by duration or condition of storage or
by season."  A  similar observation was made as a result of storage studies
on the Eastern  oyster (Crassostrea virginica) on the Gulf Coast by Presnell
and Kelly (1961).  Their storage studies were carried out at 2-12 C for as
long as 23 days.

     Fecal coliform densities did increase somewhat during storage at 10 C.
However, the rapid increases in coliform and 35 C plate count densities
at this temperature and rapid deterioration of the oysters indicated that
10 C is not an  acceptable storage temperature for shucked oysters.  Fecal
coliform densities decreased slightly during storage at 3 C and in ice.
However, in view of the great increases in coliform and 35 C plate count
densities with  consequent production of substances potentially toxic to
fecal  coliforms, the stability of the group of these temperatures was re-
markable.

     Since fecal coliform densities increased at 10 C,  it would be reason-
able to assume  that enteric pathogens might also multiply during storage
at this temperature.   Presnell and Kelly (1961)  showed that E.  coli and
a mixed suspension of SaImone1la derby,  Salmonella infantis. and
and Salmonella newport showed similar patterns of increase in Eastern
oysters stored  at 20 C.  Similarly, one might postulate that numbers of
                               - 7 -

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enteric pathogens might be reduced during storage at 3 C and in ice.
In neither case is there direct evidence from the current studies to
support these conjectures.  Kelly and Arcisz (1954) found that IE. coli
and Salmonella schottmueLleri showed similar changes in numbers in
shell oysters (Crassostrea virginiea) and soft clams (Mya arenaria)
stored at 5 C and 19 C.  At both temperatures in both shellfish species
similar reductions in numbers occurred.  However, in shell oysters stored
at 5 C the rate of reduction of S. s chpttmue11 e r i was less than that of
E. coli in the initial period of storage.

     Evidence for the biological soundness of the use of fecal coliforms
as an indicator of fecal pollution by man and other warm blooded animals
has been given by Kelly (I960), Presnell, (1961), Kelly et aj., (1962),
Beck jet al (1963) and Kabler ^t al (1964).  Because of this, and because
of the stability of this index during storage indicated above, it appears
that the organisms enumerated by the EC test would be of value as an indi-
cator of the sanitary quality of shucked oysters during the marketable
life of the product.

     The 35 C plate count, because of its marked response to storage
temperature differences would be useful as an indicator of temperatures
maintained and length of time involved in transport and holding shucked
oysters in commercial channels.  While differences in initial densities
of microorganisms enumerated by this test and different patterns of in-
crease were shown by different lots of oysters,  the changes observed
correlated overall with time and temperature of storage.

     The coliform group has disadvantages as an indicator of either sani-
tary quality or state of freshness of the product.  Large increases in
density of this index occurred at all three temperatures, making it un-
suitable as a stable indicator of the sanitary quality of shucked oysters
when harvested.  Changes in numbers in response to storage time and temp-
erature were much less consistent than shown by the 35 C plate count in-
dex,  making the coliform index less desirable as an index of shucked oyster
spoilage.

     The results of this study indicate that pH determinations were of
little value in determining the quality of shucked Pacific oysters.  A
definite change toward lower pH levels was observed but the changes in
many cases were not correlated with changes in any of the microbial in-
dices used in this study.  From some of the results above,  it would ap-
pear that factors other than bacterial multiplication are major causes of
postmortem pH changes in Pacific oysters.

     It is evident from the results presented above that 10 C is not a
satisfactory temperature for holding shucked Pacific oysters because of
the rapid increase in microorganisms observed at this temperature.  It
is also evident that as one approaches 0 C, the rate of multiplication
of microorganisms present in the oysters becomes much lower.  The rates
of enzymatic reactions causing autolysis are also temperature dependent.
Therefore, it is obvious that storage temperatures which approach 0 C as
nearly as possible will result in much longer shelf life for shucked
Pacific oysters.

                                   -  8  -

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                              SUMMARY
     A  study  of  the applicability of coliform MPN, fecal coliform MPN, 35 C
plate count,  and pH as  indicators of the quality of commercially packed
Pacific oysters  stored  in crushed ice, and at 3 C and 10 C dry storage was
made over a period of nearly three years.

     The results  indicated that the fecal coliform MPN, because of its
stability during storage and its value as an indicator of fecal pollution,
would be of value as an indicator of sanitary quality of the oysters when
packed.  The  35 C plate count, because of its response to storage condi-
tions,  i.e. time  and temperature, appeared to be a satisfactory indicator
of spoilage.  Coliform MPN's were not stable and did not show consistent
changes  in all lots of oysters.  The pH gave little information as to the
bacteriological quality of the oysters.

     As  storage  temperatures approached 0 C, rates of bacterial multipli-
cation  became slower and the lag before multiplication began became longer.
Storage  either in ice or at 3 C greatly extended the marketable life of
the product beyond that observed during storage at 10 C.
                     COMMENTS BY PARTICIPANTS

     Mr. Sam Reed opened the discussion by asking for comments.  Mr. Neufeld
discussed the relationship of indicator organisms.  Dr. Hoff quoted earlier
work by Kelly and Arcisz on the relationship of indicator organisms to patho-
genic organisms in light of the results from the present paper.  Mr. Kelly
discussed the methods used and the relationship of Salmonellae to the indi-
cator organisms used in the Gulf Coast study.

     Dr. Listen commented on the relative fate of fecal coliform, coliform
and total plate count at the various storage temperatures.   Mr. Neufeld
stated that somewhat similar results had been observed in British Columbia.
                                -  9  -

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                            REFERENCES

American Public Health Association. 1962.  Recommended procedures for the
  bacteriological examination of sea water and shellfish, 3rd ed.

Beck, W. J., M. W. Presnell, and J. C. Hoff. 1963.  Ecological study of
  bacterial indices of pollution.  1963 Shellfish Sanitation Research
  Conference, Purdy, Washington.

Jensen, E. T. 1962.  Sanitation of shellfish growing areas.  Part II.
  Sanitation of the harvesting and processing of shellfish.  Public Health
  Service Publ. No. 33, revised 1962.

Kabler, P. W., M. A. Clark, and E. E. Geldreich, 1964.  Sanitary signifi-
  cance of coliform and fecal coliform organisms in surface water.   Publ.
  Hlth. Repts. 79: 58-60.

Kelly, C. B. 1960.  Bacteriological criteria for market oysters.  Techni-
  cal Report F60-2.  Robert A. Taft Sanitary Engineering Center, Cincinnati,
  Ohio 15p.

Kelly, C. B. and W. Arcisz, 1954.  Survival of enteric organisms in shell-
  fish.  Pub. Hlth. Repts. .69: 1205-1210.

Kelly, C. B., W. J. Beck, M. W. Presnell, and K. J. Zobel.  1962.  Ecologi-
  cal study of bacterial indices of pollution.   1962 Shellfish Sanitation
  Research Conference, Purdy, Washington.

Presnell, M. W., 1961.  Sanitary significance of "fecal coliform organisms"
  in a shellfish growing area - sanitary survey of Burley Lagoon.  1961
  Shellfish Sanitation Research Conference, Purdy, Washington.

Presnell, M. W. 1962.   Studies on stored Pacific oysters.  1962 Shellfish
  Sanitation Research Conference, Purdy, Washington.

Presnell, M. W. and C. B. Kelly, 1961.  Bacteriological studies of commer-
  cial shellfish operations on the Gulf Coast.   Sanitary Engineering Center
  Technical Report No. F61-9 U. S. Public Health Service.
                                 - 10 -

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                                                                        nra KPN/lOOk

                                                                        Plate Count If,	

                                                                        on. MPN/lOOn

                                                                        Plutc Count/1
                                                                                                                                 10              Ts~
                                                                                                                                .'toragc time (day*)
                                                                                                  Flj. 2,  Change  In  collforniMPN In ahucked Pacific oyttera atored  In  let   4nd
                                                                                                           at  1 C ond  10 C  ( 1962-liJ lota)
                                     10—            F5~
                                  .. lor.R. tln» (d.iyi)
        1. ChnnK«l  In  colKorm MPN nnd )5 C pUie count In ihucked Pacific  oyatet
            lored at 1 C  < 19M lota)
                    I
                                                                      I
10'
                                     Storag* t(Tn« 
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Table 1.  Grouping of shucked Pacific oyster storage for data analysis
Storage Storage Lot
Temperatures Classification No.
3 C 8 lots 1
Winter-Spring 2
3
4
5
6
7
8
8 lots 9
Summer, 1961 10
11
12
13
14
15
16
Ice, 3 C and 15 lots 17
10 C Winter, Spring 18
and Summer 19
1962-63 20
21
22
23
24
25
26
27
28
29
30
31
Date Storage
Initiated
1-11-61
1-12-61
1-17-61
1-17-61
2-10-61
2-10-61
4-21-61
4-21-61
6-26-61
6-27-61
6-28-61
8-15-61
9-18-61
9-18-61
9-18-61
9-18-61
11-26-62
11-26-62
11-26-62
3- 4-63
3- 4-63
3- 4-63
4- 1-63
4- 1-63
4- 1-63
4- 1-63
6-10-63
6-10-63
6-10-63
6-10-63
6-10-63
Inches Rainfall3
in preceding
7 days 14 days
3.48
3.50
5.25
5.25
4.07
4.07
1.35
1.35
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
7.96
7.96
6.96
•1.84
1.84
0.29
5.29
5.29
2.41
2.83
0.64
0.64
0.64
0.64
0.44
3.54
3.56
8.75
8.75
5.13
5.13
2.51
2.51
0.0
0.0
0.0
0.0
0.03
0.03
0.03
0.03
11.33
11.33
11.68
3.32
3.32
0.79
5.93
5.93
2.51
3.74
0.84
0.84
1.27
1.27
0.56
     a
      Data from:   Climatological Data, Washington and Oregon.
                  Volumes  65,  66 and  67  (1961,62 and 63),
                  U.  S.  Dept.  of Commerce, National Weather
                  Records  Center, Asheville, N. C.
                                  -12-

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Table 2.  IMViC analysis of EC positive cultures isolated during
                storage of shucked Pacific oysters
Storage Temperature
Storage
(Davs)
0
1
2
3
4
5
6
7
10
15
20
25
30
Iced
1962-63
No. of
Isolates
43
11


33


8
31
24
28
5
24

lots
%
E.coli
74
82


80


63
93
96
90
100
100

1961
No. of
Isolates
39
46
108

63


60
60
28
25
5

3 C
lots3 1962-63 lots
% No. of %
E.coli Isolates E.coli
92
72 6 33
75

84 33 80


82 8 88
83 29 93
100 6 83
64 4 100
100

10 C
1962-63 lots
No. of %
Isolates E.coli

15 87

16 100
74 88
13 92
16 100
18 78





S1961 data from Presnell, M. WT 1962.   Studies on stored Pacific
      oysters (presented at Shellfish Sanitation Research Conference*
      Purdy, Washington).
                            - 13 -

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Table 3.  Changes in pH of shucked Pacific oysters stored at 10 C,  3 C
                             and in ice
Time
(Days)
0
1
2
3
4
5
6
7
10
15
20
25
Ice
1962-63
Winter-
Spring S
6.4a
6.4
6.4

6.4


6.2
6.1
6.0
6.0
5.9

lots
unnner
6.5
6.1
6.0

6.1


5.8
5.9
5.9
5.9
5.9

1961
Winter-
Spring
6.5
6.2
6.1

6.0


5.9
5.7
5.5
5.7

3
lots
Summer
6.6
6.4
6.2

6.1


5.8
5.6
5.5
5.6

C

1962-63 lots
Winter-
Spring Summer
6.4
6.4
6.3

6.2


6.1
6.0
6.0
5.9

6.5
6.2
6.1

6.1


5.8
5.8
5.8
6.0
5.8
10
C
1962-63 lots
Winter-
Spring Summer
6.4
6.3
6.2
6.1
6.1
5.9
5.9





6.5
6.0
6.0
6.0
5.9
5.7
5.6





      Arithmetic means of individual determinations.
                                 - 14 -

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Effect of Antifoaming Agents and Evacuation on Shellfish Homogenization

                           T. H. Erlcksen


                            INTRODUCTION

     It has been observed that during the blending process of the bacteri-
ological examination of shellfish foaming occurred.  Since the indices of
bacterial density as described in APHA Recommended Procedures for the Bac-
teriological Examination of Sea Water and Shellfish (Third Edition,  1962),
are based on a weight-to-volume relationship,  any property which would
interfere with this relationship may affect the number of bacteria indi-
cated by these indices.

     Preliminary data were obtained on the weights of 10 ml aliquots of
the 1:2 dilution of shellfish homogenates.  The ideal weight-to-volume
relationship would be lOg/lOml.  The results indicated this relationship
was less than maximum.  The average weight per 10 ml of these samples
was 7.9g.  It was believed that this deficiency was due to foaming dur-
ing the blending process.   Therefore, a number of companies were contacted
and 17 different defoaming compounds were obtained.  These compounds were
examined and evaluated on the basis of toxicity,  ability to reduce foaming
and practicality.  Also included was a method of blending under reduced
atmospheric pressure.

                         MATERIALS AND METHODS

     An aluminum Waring blendor jar was modified by laboratory personnel
for attachment to a vacuum pump.  It was also modified by the addition of
aluminum baffling fins to insure homogeneous blending.  Pacific oysters
(Crassostrea gigas), Manila clams (Tapes japonica) and Native Littleneck
clams (Protothaca staminea) were obtained from various sources to supply
different bacterial populations for testing.

     Each shellfish sample was shucked in the prescribed manner,  then
chopped and divided into lOOg portions.  These portions were treated in
the following manner:

     1.   The control sample was blended with the shellfish meats  and
         liquor only for 15-20 seconds, after which a 20 ml aliquot
         was removed and the pH determined.  An equal weight of
         phosphate buffer was added to the sample and blending con-
         tinued.   After blending for the prescribed period, dupli-
         cate 10 ml aliquots were pipetted to  tared beakers.   A
         20 ml aliquot was obtained for the pH determination and
         the bacteriological examination then  proceeded as outlined
         in recommended procedures.
                               - 15 -

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    2.  A minimum sample of lOOg of shellfish meats and liquor was
        weighed and placed in a sterile modified blendor jar.  An
        equal weight of phosphate buffer was added to the sample to
        be evacuated.  The blendor jar was placed on the motor and
        attached to the vacuum pump.  The container was evacuated to
        a vacuum gauge reading of 15 pounds.  This mixture was blended
        for the prescribed time (90 seconds for oysters and 100 seconds
        for clams),  After blending the atmosphere within the blendor
        container was allowed to reach equilibrium with that of the
        surrounding atmosphere very rapidly.  Duplicate 10 ml aliquots
        were immediately pipetted to tared beakers.  A 20 ml aliquot
        was obtained for the electrometric pH determination.  Bacter-
        iological examination then followed the outline in recommended
        procedures.

    3.  The antifoam compound was added to the phosphaae buffer and
        sterilized.  A shellfish sample was weighed and placed in a
        blending container.  An equal weight of the phosphate buffer
        with the antifoam agent added was placed in the blending con-
        tainer.  After blending for the prescribed period two 10 ml
        duplicate aliquots were pipetted to tared beakers,  after which
        a 20 ml aliquot was obtained for the pH determination.  The
        bacterial examination then followed as outlined in recommended
        procedures.
     The pH determinations were obtained on the schedule as described
above to determine the effects of evacuation or the antifoam agent on
the pH of the shellfish homogenates.
                                 RESULTS

     Weights of twenty-one 10 ml preliminary samples of random shellfish
homogenates diluted 1:2 with an equal weight of phosphate buffer and
blended for 90 to 100 seconds ranged from 5.8g to 9.0g with an average
of 7.9g.  This data indicated that all dilutions contained approximately
20 percent less shellfish meats than the ideal amount.

     All compounds investigated to date have been abandoned in favor of
the evacuation method because of either their inability to be sterilized
or to reduce foaming.  Ten of these compounds were eliminated because of
undesirable solubility and emulsifying properties.  The remaining seven
compounds were eliminated on the basis of inability to reduce foaming.
However, one compound which showed some promise was tested further.

     The weight-to-volume relationships of the control, evacuation method
and antifoam agent are shown in Table 1.  These values are based on  18
Pacific oyster samples.   These data indicated that the evacuation method
gave the most consistent maximum values.
                                 - 16 -

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      Data on the effect of evacuation and  the  antifoara agent  on  the
 coliform MPN,  fecal coliform MPN  and  35 C  plate  count are  given  in
 Table 2.   The  data were analyzed  by three  different methods;  median,
 geometric mean and probability  plot.

      The control values of the  coliform MPN  for  the three  methods of
 analysis ranged from 2200to 2400.  The values  for the evacuation
 method ranged  from 1800 to 2700 and the values for the antifoam  agent
 ranged from 1500 to 1900.   Fecal  coliform MPN values ranged from 94
  to  96 for the control;  73 to 97  for  the evacuation method, and  120
 to 130 for the antifoam agent.  The 35 C plate count values ranged
 from 3900 to 5000 for the control; 4300 to 5200  for the evacuation
 method,  and 2900 to 3600 for the  antifoam agent.

      The  most  distinct differences are found in  the 35 C plate count
 values for the three methods  of analysis.

      Electrometric pH determinations  on the homogenates of the evacu-
 ated samples,  antifoam agent  samples  and control samples indicated no
 effect on pH because of  the method employed.

      In addition to the  above a few samples of Pacific Coast  hardshell
 clam species have been examined.  However, sufficient data were not
 available for  analysis  at  the time.


                                DISCUSSION

      From the  limited data  presented  it appears  that a source of error
 in bacteriological  examination  of shellfish may  exist.  The use of anti-
 foam agents  has  not proven  practical  to date.  The method of  choice may
 be that of evacuation technics.

      The  possibility  of  obtaining and  using glass jars for evacuation
 has  been  discussed.   However, recent  correspondence with Dr,  Thomas Hosty,
 Director,  Bureau of Laboratories,  Dept. of Public Health,  State of Alabama,
 has  indicated  that  suitable glass containers may not be available.   Further
 inquiries  with glass  container manufacturers will be made as to possible
 availability of  appropriate containers.

      This  investigation will  be continued.  Emphasis will be placed on
 obtaining readily available,  inexpensive evacuation blending containers
 and  determining  the effects of  lower vacuum pressures on foam reduction
 and  bacterial densities.

                        COMMENTS BY PARTICIPANTS

     Dr. Dollar  discussed the use of other antifoaming agents that  might
 be available as well as other methods  of homogenization.   Dr.  Listen
questioned the limits that should be placed on present methods of
                                   - 17 -

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homogenization of shellfish samples.  Dr. Listen questioned whether or
not the margin of error shown would have significant bearing on the
final results due to other inherent fluctuations in technics used.  It
was generally agreed that further investigation must be made before
this could be resolved.
                               - 18 -

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Table 1.  Weights of duplicate 10 ml aliquots  of Pacific
  oyster homogenates with two foam control  technics
                 Weight in grams  of 10 ml  aliquots	
                rol	Evacuation	Antifoam Agent
	Control

Range              6.7 - 9.1       8.0 - 10         7.0 - 9.9

Average               8.3            9.5               8.9
                        19

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                   Table 2.  Bacterial densities of Pacific oysters with two foam control technics
N>
o
Bacterial Densities

Method
of
Analysis
Median
Geometric
Mean

Coliform
MPN
Fecal Coliform MPN
Foam Control

None
2400

2200

Evacuation
2700

1800
Ant i foam
Agent
1700

1900
Foam Control
35 C Plate Count
Foam Control
Ant i foam
None
94

98
Evacuation
73

97
Agent
130

120
None
5000

3900
Ant i foam
Evacuation
5200

4300
Agent
3500

3600
Probability

  Percentila



     10          34



     50        2300



     90       16000
21
2300
23000
120
1500
18000
20
96
450
16
97
590
40
120
340
250
3400
45000
                                                                                                  410
430
                                                                                                 5000     2900



                                                                                                61000    20000

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      Activities of Public Health  Service  Research Centers

                            C.  B.  Kelly1
      In 1962,  Congress  appropriated more  than $1,500,000 for con-
 structing and  equipping two  shellfish sanitation research centers.
 The Gulf Coast Shellfish Sanitation Research Center,  located at
 Dauphin Island, Alabama,  completed July 1963, has a proposed staff
 of some 32 scientists and supporting staff, in a facility of approxi-
 mately 10,000  square feet in area.  The Northeast Center, some 20,000
 square feet in area located  at Narragansett, Rhode Island was occu-
 pied in May 1964.  Both laboratories are  similar in design and are
 equally complete  in facilities, including laboratories for chemistry,
 biochemistry,  microbiology,  marine microbiology, virology, and marine
 biology.   Each has an experimental laboratory with flowing sea water
 where _in vivo  experiments with shellfish  can be conducted.

      A unique  feature of each Center is a Field Investigations Unit
 that serves two purposes;  it gives technical assistance to the Regions,
 States,  and Industry in problems of unusual nature and it augments the
 staff of the Regional Offices, assisting  them in the  evaluation of
 State shellfish sanitation control programs.  The Field Investigations
 Unit provides  engineers  and  other scientists acting as a buffer to the
 research scientists, allowing an uninterrupted program of research to
 continue.   Technical assistance activities of the Northeast Shellfish
 Sanitation Research Center included collaboration with Maine, Rhode
 Island and Massachusetts  in  the design, development, and evaluation of
 pilot facilities  for depuration of shellfish, a cooperative study with
 Maryland,  New York, and New York City in  commercial handling of soft
 clams,  assistance to Maine in the development of sanitary survey of
 two  shellfish  areas, investigations in Connecticut and New Jersey in
 the  incidents  of hepatitis due to clams.  At Dauphin Island, the pro-
 jects  included a  cooperative study with Alabama to evaluate the pro-
 posed criteria  for shellfish areas and shellfish at the market, assist-
 ance to Florida in shellfish toxicity in  the Sarasota-Tampa Bay area,
 and  assistance  to Louisiana  in pesticides in shellfish areas.

      Of paramount importance in this day  of rapid urbanization of
 coastal  areas  is the need  for knowledge and development of technology
 for  depuration.  In spite  of an active program in pollution control,
 shellfish  control authorities have progressively found it necessary
 to prohibit harvesting of  shellfish from many productive areas to


 IChief, Research and Investigations Section, Shellfish Sanitation
 Branch, Division of Environmental Engineering and Food Protection,
 Public Health Service,  Department of Health, Education,  and Welfare,
Washington,  D.  C. 20201

                                - 21 -

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protect the public from dangers of chance contamination due to
mechanical failures in waste treatment systems.  A system of de-
contamination of shellfish comparable in principle to the pasteur-
ization of milk would afford the needed public health protection
to utilize shellfish from these marginal areas.  That shellfish
will cleanse themselves is a well-recognized biological principle.
The need is for the development of technology that would transcribe
research knowledge to commercial application.  A three phase program
of research in depuration is in progress.  Research knowledge is now
available to demonstrate the effectiveness of the feeding-cleansing
process in all species of shellfish.  Highly efficient systems have
been developed for the sterilization of sea water.  The second phase,
now initiated, will develop a practical process for depuration, first
in pilot plants, then in commercial size facilities.  Long term re-
search needs will gather basic knowledge of the physiological processes
that govern the accumulation, retention, and elimination of bacteria,
viruses and toxic chemicals.  Ultimate goals are to develop a system
effective for all noxious materials and at an economically realistic
level.

     The Centers are fully equipped for research in virology.  The
importance of such research came to focus in 1961 when some 80 cases
of infectious hepatitis occurred in Mississippi and Alabama traced to
the consumption of raw oysters, the source being a localized area at
the mouth of a heavily contaminated river.  Later, hepatitis was as-
sociated with the consumption of raw clams in New Jersey and
Pennsylvania.  Although the exact source was not determined, the
polluted areas in Raritan Bay were strongly incriminated.  While
the epidemiology of these outbreaks does not present evidence  of
failure of the current technical standards, that they occurred
certainly demonstrated the need for basic investigations in the fate
of viruses in the marine environment and their behavior in shellfish
and the development of effective means for decontamination.

     Following the massive fish kill in the Mississippi River last
March, a united effort was exerted to determine the potential hazard
of such agricultural chemicals to consumers of shellfish.  The Public
Health Service laboratories at Cincinnati and Dauphin Island collabo-
rated in the determination of the cause of the mortalities and the
Dauphin Island Laboratory conducted surveys in oyster growing areas.
These studies are still in progress.  A program of monitoring shell-
fish areas for toxic materials is in progress in representative shell-
fish growing areas in the Gulf Coast and South Atlantic areas.

     The scientific literature for many years has mentioned the
presence of certain substances in marine flora and fauna that inhibit
the growth of viruses and bacteria.  Of particular significance is
the recent discovery of antimicrobial, antiviral and most recently
tumor inhibiting agents from certain species of edible mollusks.
                                 - 22 -

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 Such findings  indicate the need for  investigation of  the presence of
 these substances  in sea water and  the  flora and  fauna of the marine
 environment.   They are important as  they  relate  to an understanding
 of  the biological forces  in the sea  responsible  for maintaining the
 biological  balance and the destruction of organisms of terrestrial
 origin,  but these biologically  active  materials  might be of clinical
 significance.

      Recent outbreaks  of  botulinum food poisoning from smoked fish
 brings to focus the need  for investigations of toxigenie spore form-
 ing bacteria in the marine envirotnaent.  While adequate preparation,
 primarily canning,  of  the commercially prepared  product will prevent
 such occurrences,  it is important  to determine the distribution of
 such bacteria  in  the marine environment,  the conditions under which
 the organisms  can multiply and/or  produce toxin  in shellfish.

      The need  for studies of marine  toxins has been recognized for
 many years.  The  Northeast Center  will embark on studies of the
 ecology  of  the paralytic  shellfish poison.  Research will be con-
 ducted on the  development of methods for  the cultivation and growth
 of  the causative  organisms,  the isolation and purification of the
 toxin, ultimately  to gain a better understanding of the physiology
 and biochemistry  of the paralytic  shellfish poison.  The Gulf Coast
 Shellfish Sanitation Research Center is conducting studies in the
 shellfish toxin found  in  the Tampa-Sarasota Bay  area.  Research and
 field investigations includes the  monitoring of  areas in the Gulf
 Coast for the  presence of the toxin, isolation of toxins from shell-
 fish and studies  on the ecology o£ the organisms involved.

      Shellfish sanitation research facilities in New England and the
 Gulf Coast  cannot  contribute research  information completely of value
 to  the West Coast  industry and  control agencies, because of different
 climatic and environmental characteristics and the difference in
 species  of  shellfish under commercial  exploitation.  To correct this
 research weakness,  the Public Health Service is  planning for the con-
 struction of a Northwest  Shellfish Research Center, similar in design
 to  the two  existing facilities  which will provide for a multi-disci-
 plinary  effort, much broader in scope  than now possible at Purdy, to
 work on  problems unique to the West Coast industry.  Facilities for
 virology, chemistry, biochemistry, microbiology, biology and ocean-
 ography  will provide for  a team approach  in fundamental research.
An  engineering and  technical assistance staff will assist, consult
 and advise  control  components in the Regions and States and to indus-
 try in shellfish sanitation  problems.  We look toward the construction
 of  this  facility within the  next five years.  We look forward to this
 as  a  further accomplishment  of  our program responsibilities.
                                 - 23 -

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        Brief Hydrographic Survey of Burley Lagoon, Washington

                           Philip S. Kelleyl


                             INTRODUCTION

     In many of its shellfish sanitation projects, the Shellfish
Sanitation Branch has a need for oceanographic data to supplement
the bacteriological and sanitary surveys usually taken.  Since much,
if not all, of the work presently contemplated will be confined to
estuarine waters where shellfish are found, it seemed desirable to
evaluate available methods of oceanographic sampling in such estu-
arine waters.  To pursue this goal, it was decided to undertake a
limited hydrographic survey of a small tidal estuary.

     The estuary studied was Burley Lagoon, upon the shore of which
is located the Northwest Shellfish Sanitation Laboratory.  Burley
Lagoon (Fig. 1) is located at the head of Carr Inlet, a large arm
of southern Puget Sound in the State of Washington.  It is a narrow,
nearly flat-bottomed estuary, covering about 370 acres, and is ori-
ented with its long axis almost exactly north-south.  The lagoon is
protected by a long spit across its southern end.  There are dwell-
ings on the shore almost everywhere except the spit.  Oysters grow
well in the lagoon, and a commercial harvesting operation based in
part upon these oysters is quite active.  Two streams contribute
fresh water to the lagoon:  Burley Creek at the head and Purdy Creek
near the mouth of the lagoon.  Both creeks flow year round and both
have stream gages near their mouths which are read regularly.  In
addition, there are a few small seasonal streams which drain from
the ridge to the west of the lagoon.  These flow in the late fall and
winter when precipitation is high.

     The study of this lagoon was restricted to simple observations
of temperature, salinity, currents and bottom topography.  Limitations
on personnel, equipment and time precluded any attempt to study the
biological, chemical and geological conditions in the lagoon.  Equip-
ment had to be of such size and tractability as to allow its being
handled without undue difficulty over the side of a sixteen-foot out-
board motorboat,  and it was desirable that the gear be simple enough
that untrained personnel would have no difficulty using it in the
future.  A current meter and portable salinometer were obtained;
these will be described in more detail below.  Other necessary items
were made in the laboratory as needed.  The topography, the currents,
and the temperature and salinity observations will be discussed sep-
arately in the paragraphs following.


 •••Present address:   Northeast Shellfish Sanitation Research Center,
  Narragansett, Rhode Island.

                               -  24 -

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                             BATHYMETRY
 Method:

      Soundings  were taken at times  when  there was  sufficient water  in
 the lagoon to float the boat.   The  87  soundings  recorded  are listed in
 Table 1.   The locations of the  soundings, with the exceptions of numbers
 81  and 82,  are  shown in Fig.  2.  The two missing stations  lie in Henderson
 Bay,  just  off the  border of this chart south of  station 80.  Most of the
 soundings  were  taken with the boat  moving slowly.   The sounder consisted
 of  a  light line, marked at one  foot intervals and  weighted with a three-
 pound cast-iron sash weight.  One observer cast  the sounder, while  the
 other guided  the boat and recorded  depths as the first called them  out.

      The soundings  obtained were corrected for the stand of the tide at
 the time of observation and reduced to depths below mean higher high
 water at Wauna, Washington,  which is on  Henderson  Bay at the west end
 of  the spit across  the  lower end of Burley Lagoon.  Tidal data and  cor-
 rections for Wauna  to be applied to Seattle predictions as published
 by  the U.  S. Coast  and  Geodetic Survey (1962) are  shown in Table 2.
 The contour map  based on the corrected soundings is shown in Fig. 3.
 The shapes  of the contours  have been checked and corrected somewhat  to
 conform generally with  the  forms seen  on stereoscopically viewed aerial
 photographs of  the  lagoon.
Discussion:

     From Fig. 3 it can be seen that Burley Lagoon is a fairly shallow
estuary, generally flat-bottomed except at the southern end.  The area
north of the island is covered with soft mud to a depth of several feet,
whereas south of the island the bottom is fairly firm, being sandy or
gravelly in places.  The depths are such that on a moderately low tide
(about a foot above mean lower low water) most of the lagoon above a
line somewhere between the 12 and 15 foot contours shown will be dry
and exposed.  The deepest place found,  a depth of 31 feet, was in the
hole shown near the center of the lower half of the lagoon.  Local
residents claim that a fresh-water spring exists at the bottom of this
hole, but evidence of this was not found, as will be shown later.  The
banks of the lagoon are quite steep.  Because of this, the 3 and 6 foot
contours are not shown on Fig. 3,  but will be approximately equally
spaced between the shoreline and the 9  foot contour.

     A polar planimeter was used to measure the areas enclosed by each
of the contour lines.   These areas were substituted into the formula
                                - 25 -

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 given  by Raisz  (1948, p. 112)  V - i       *2  + A3 +	+ An>
 where  V is  the  volume of  the body of water, i is the  contour interval,
 AI  is  the surface area, and A2, A3 and so forth are the areas enclosed
 by  consecutively lower contour lines.  The approximate volume of Burley
 Lagoon estimated by  this  method is 1.9 x 108 cu. ft.  or about 4,300 acre-
 ft., approximately 687» of which lies above the 9 foot contour.  In making
 this calculation, the areas enclosed by the 3 and 6 foot contours were
 approximated by linear interpolation between the 0 and 9 foot contour
 areas.  The volume of 4,300 acre-ft. is the amount of water the lagoon
 will hold at mean higher  high water, or at a tide of  13.1 feet above
 mean lower  low  water.

     The contour lines shown in Fig. 3 represent with reasonable accuracy
 the relative shape of the lagoon floor.  However, there is evidence that
 the contours may be  somewhat shallower than shown by perhaps a foot or so.
 The aerial  photographs mentioned above were taken on 3 September 1963 at
 about  1:30  P.M. when the  stand of the tide was almost exactly one foot
 above  mean  lower low water.  These photographs show somewhat less water
 in  the lagoon than would  be predicted from the contour map and the stand
 of  the tide at  the time.  Whether this discrepancy is the result of an
 actual systematic error in measurements and data reduction, or the result
 of a lag between the time of a given tide height at Wauna on Henderson
 Bay and the time of  the same height inside the lagoon cannot be determined
 without further observations.  Until the source of the discrepancy can be
 definitely  located,  the absolute values of the depths in, as well as the
 volume of,  the  lagoon must be considered as only rough approximations.

                              CURRENTS
Methods and Instruments:

     Currents were measured with the boat anchored in position; stations
where currents were measured are shown on Fig. 2.  Except for stations D
and E, it was found desirable to anchor the boat bow and stern to prevent
undesirable swinging caused by shifting winds.  At Stations D and E the
current was strong enough during the time observations were made to
obviate the necessity for a stern anchor.  The boat was anchored from
the bow by a standard Navy-type steel small-boat anchor and from the
stern by two concrete building blocks totalling about thirty pounds in
the water.  The boat was anchored bow into the current.

     The current meter used was the Model 622-SW-l Price current meter,
serial number 610356, manufactured by W. and L. E. Gurley of Troy, N.Y.
This meter, together with its battery-operated electric counter, is shown
in Fig. 4.  A description of this type of meter, together with useful
suggestions on handling and maintenance, is found in a publication by
the U. S. Coast and Geodetic Survey:   Manual of Current Observations.
Special Publication No. 215 (1950, pp. 19-24).  This meter is simple in
construction and mode of operation.  An electric circuit, consisting of
a 6-volt dry cell battery in series with an electric counter and twin-
wire rubber-insulated cable which also serves  as a suspension cable  for
                                  -  26  -

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 the meter,  is made  and  broken by a cam mounted on  the upper end of the
 bucket wheel shaft.  The  cam closes  the circuit momentarily once for
 each  revolution of  the  bucket wheel, causing the counter to register
 each  revolution of  the  wheel.  The number of revolutions per minute
 of the wheel may  be easily  converted to current velocity in meters per
 second,  feet per  second or  knots by means of rating tables, two of which
 are supplied by the manufacturer.

      The meter was  rigged off the port side of the boat, as pictured in
 Figs. 5  and 6.  The suspension cable of the meter was market at one-foot
 intervals with paint, enabling easy positioning of the instrument at any
 desired  depth, down to  about 25 feet.  A small boom, fashioned from two-
 by-four  lumber and  a clothesline pulley, was mounted on the port side.
 A cable  clamp, made from  an ordinary hasp and an eye-bolt and lined with
 thick rubber to prevent damage to the cable, was fastened to the boom.
 The boom was fastened by  one bolt through the seat back, and could be
 either swung up and over  or completely removed when the boat was being
 carried  on a trailer.

      The observer must  be able to see the meter in order to know the di-
 rection  of the current, as  the Price design has no other means of indi-
 cating direction.   It was found, using the two-conductor rubber-covered
 cable for suspension of the meter, that the stiffness of the suspension
 may be sufficient to hold the meter in an orientation not consistent with
 the direction of  the current, especially if the meter was only a few feet
 down  and/or the current was quite weak.  The direction of the current was
 indicated by pieces of  white string, several inches long, fastened to the
 cable just above  the meter.  After the meter was stabilized in the water
 it was possible by  twisting the cable slightly where it passed over the
 pulley to cause the meter to orient itself into the current.  Where the
 current  was strong  enough to exert sufficient force on the stabilizing
 vanes to orient the meter properly this procedure was unnecessary.   It
 was also found essential  to keep the contact chamber, which contains the
 cam and  electrical  contact, filled with light oil to exclude sea water.
Any sea  water in this chamber will create a short circuit, jamming the
 counter  and causing corrosion in the contact chamber.

     Located on the deck  (to the right of the wheel, just above the igni-
 tion  switch in Fig. 6)  was  a box containing a Navy Mark I four-inch boat
 compass, mounted in gimbals.  The box may be rotated horizontally through
 360°, and has two slots on  opposite sides through which sightings for
 bearings may be made.   The  box is held to the boat by a single bolt and
wing-nut, and may be easily removed.  The compass itself may also be re-
moved from the box.  The  true direction of the current was obtained by
 noting the orientation  of the meter, lining up the marks on the compass
 frame approximately parallel to the meter,  and adding 22°  for variation
 to the reading.

     Data was gathered  at eight stations in Burley Lagoon and Henderson
Bay during September and October,  1963.  With two exceptions,  the meter
was suspended about halfway between surface and bottom.   At station D,
 the current was strong  enough to cause considerable vibration of the
meter suspension cable,  hence the meter was kept near the surface to re-
duce  the vibration.  Observations at station F on 15 October were made

                                  - 27 -

-------
both at the surface and midway down, because the velocities observed at
the two depths differed substantially.  This was the only time this effect
was noticed.  By "surface" is meant a depth of between one and two feet,
deep enough to keep the meter below the level of the keel of the boat.
Discussion;

     The currents measured generally showed little change in direction
during the course of the ebb or flood on which they were measured.  The
notable exception was at station B on the ebb tide, where a complete re-
versal in direction occurred because of an eddy which developed as the
water level fell.  Figs. 7 and 8 show the direction and maximum veloci-
ties observed on the ebb and flood tides.  The tidal range was not the
same in all cases, varying from 12.2 to 5.1 feet, so the velocities are
not always strictly comparable one to another.

     Figs. 7 and 8 also show the general current pattern in the lagoon
as observed qualitatively.  One point is particularly worthy of note.
This is the large, strong eddy, centered northwest of the power line
towers standing in the southern end of the lagoon, which develops on the
flood tide.  As can be seen from Fig. 7 the current also flows south on
the ebb tide.  Hence this section of shore, along with the north shore
of the spit, is affected by a unidirectional current, whereas the rest
of the shoreline of the lagoon is influenced by a current which reverses
itself every six hours.  One effect of this current pattern is to cause
some of any pollution introduced along the southwest shore the lagoon
during flood tide to end up on the east shore, rather than the west
shore, further up the lagoon.  The current pattern also tends to ensure
the stability of the sand spit as a natural feature.

     With the exception of those flowing through the narrow mouth, the
measured currents in the lagoon itself were fairly weak, always less
than a knot, averaging overall about 0.25 knot.  The maximum velocity
observed at the mouth was 1.89 knots on a tidal change (flood tide) of
12.2 feet.  Currents in excess of two knots are probably not uncommon
in this vicinity.  As noted above, at station F what appeared to be
a definite velocity gradient between the surface and a depth of about
6 feet was observed.  This was the only time and place that such an
effect was pronounced enough to be obvious before lowering the meter.
There was sufficient plankton  in the water at most stations to enable
the observer to compare velocities at the surface and at a depth of
several feet before lowering the meter.  In all cases except this one,
there was no discernible difference, and hence the current meter was
usually left at depth, with only occasional adjustments to correct for
the changing level of the tide.  The reason for the  gradient observed
at station F is not known.
                                  -  28 -

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                       TEMPERATURE AND SALINITY.

Methods and Instruments:

     Salinity and temperature were measured at the same time and place
that currents were.  Observations were recorded about every fifteen
minutes; data were generally taken from the surface as well as at the
depth of the current meter.  Some observations were recorded for the
bottom, but these did not differ significantly from those halfway to
the bottom.

     The instrument used for these observations was a Model RS5-2
Electrodeless Induction Salinometer, serial number 124, manufactured
by Industrial Instruments, Inc., of Cedar Grove, New Jersey.  This in-
strument, together with its probe, is shown in Fig. 9.  The device
measures salinity, temperature and conductivity in the ranges and
accuracies shown:

         Salinity:     0-40 o/oo ±0.3 o/oo (temp, range 0-27 C)

         Temperature:  0-40 C ± 0.5 C

         Conductivity: 0-60 millimhos/cm ±0.5 millimhos cm.

According to the manufacturer, accuracies may be improved by approxi-
mately one order of magnitude by the use of error curves.

     The control box of the instrument contains an oscillator with an
output of 5 volts at 2.3 kilocycles/second, a four-stage amplifier de-
veloping a gain of about 1000, a discriminator circuit with a gain of
about 4, and a D. C. Wheatstone bridge circuit.  The electronics are
all solid-state and power is uppplied by nine 1.3 volt mercury cells.
The transducer contains a thermistor head and two toroidal coils ar-
ranged about a hollow epoxy tube.  The coils themselves are potted in
epoxy and hence insulated from the salt water, but the hollow tube,
open at both ends, allows sea water to form a core common to both
toroids.  The oscillator drives the first coil, which generates a
closed alternating current loop in the sea water.  The magnitude of
this current is a function of the electrical conductivity of the water
forming the core of the coil.  The current loop in turn induces a
current in the second toroid; the output of this coil is boosted by
the amplifier.  The discriminator compares the amplifier output with
the oscillator signal, and causes the meter to deflect in the proper
direction as dictated by the phase relationship of the two signals.
The meter is then balanced to null by means of a manually adjusted
slide-wire and two auxiliary coils, one of which is in the transducer.
These coils together with the slide-wire are used to induce a flux in
the second transducer coil opposite to that induced by the sea-water
couple; when the fluxes, are equal but opposite, the meter reads null.
A linkage attached to the slide-wire shaft allows the conductivity
to be read directly in the lower window of the control box.


                                   - 29 -

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     The thermistor in the transducer is wired as one leg of the D. C.
Wheatstone bridge circuit, and when the instrument is used to read temp-
erature this circuit is all that is in use.  When salinity is read, the
resistance of the conductivity circuit is switched into another leg of
the Wheatstone bridge, and a self-contained computer circuit integrates
the two into a salinity value.  Hence, to read salinity, conductivity
must be measured first and the reading so obtained left undisturbed
while salinity is being read.  A four-position switch allows selection
of the three described modes of operation of the instrument (conductivity,
salinity and temperature) as well as a battery check.

      The transducer is located at one end of 100 feet of five-conductor
cable.  The manufacturer cautions against subjecting the transducer to
rough treatment such as dropping and further warns against using in cur-
rents in excess of two knots unless some strain relief is provided for
the cable.  Readings should not be taken when the transducer is less
than six inches in any direction from any solid object, as such object
will distort the induced current loop which in turn may lead to an
erroneous reading.  It was presumed that this restriction also applied
to the air-water interface.

     The salinometer cable was aigged off the starboard side of the boat
using a boom arrangement identical to that used for the current meter.
The cable was weighted with a three-pound weight, which was suspended
about two feet below the transducer.  The cable was marked with paint at
one-foot intervals, allowing the transducer to be positioned at any depth
down to about 95 feet.
Data and Discussion:

     Readings were taken at the same time that current measurements were
made.  Readings were taken both at the surface and at various depths,
with emphasis on the surface.  Table 3 shows the average values of tempera-
ture and salinity by station for surface and subsurface observations.

     The variations in salinity and temperature during the period of ob-
servation were slight.  The weather during this time was generally clear
and warm, with no rainfall and low runoff.  The exceptions to this are
reflected in the data for stations F and H.  The effect of rainfall pre-
ceding the taking of data on the second occupation of these stations is
seen in the somewhat lower surface salinities.  Overall, however, there
was little variation from the average temperature and salinity of about
13.7 C and 29.5 o/oo respectively.

     These conditions of low runoff and rainfall do not persist through
the fall and winter, and consequently no inferences about the effects of
the runoff on the lagoon may be drawn for times other than those covered
by the period of observations.  As mentioned above, two creeks contribute
runoff to the lagoon on a year-round basis.  Of these, Burley Creek con-
tributes the larger proportion.  Table 4 shows approximate values for the


                                 -JO -

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 discharges of Burley and Purdy Creeks during September and October of
1963.  These values were obtained from graphs relating stream gage height
in feet to stream discharge in second-feet.  The graphs were based on
reduced Burley and Purdy Creek data for 1962 made available by the  U.S.
Geological Survey; the data for 1963 had not been processed by the Survey
at the time of this writing.  Since it is known that the relationships
between gage height and discharge volume may very from year to year be-
cause of changes in the stream bed near the gage, the values given for
runoff in Table 4 are only approximate.  The months of November, December
and January generally have considerably more precipitation than the
months preceding, e.g., in December 1962 the average discharge from both
Purdy and Burley Creeks was some 99 acre-feet per day as compared with
27.5 acre-feet per day for October, 1963.  It is therefore quite apparent
that further observations will be needed to examine the effects of runoff.

      It was mentioned above that there might be another source of fresh
water for the lagoon:  a spring in the bottom of the hole in the center
of the southern half of the lagoon.  Certain local residents claim to
have seen fresh water boiling to the surface or even geysering into the
air from this position, especially following the last severe earthquake,
presumably in 1949.  The hole was investigated thoroughly with the sal-
inometer but no significant variation from the average salinity was ob-
served.  The area surrounding Burley  Lagoon is served by many drilled
artesian wells, but no natural artesian water is known.  Drilled wells
in the vicinity reach artesian water below hard-rock at about 130 feet
below sea level; water pressure at a sea-level wellhead is reported to
be between ten and twenty pounds per square inch.  Knowing this the
possibility that the hole may be associated with artesian fresh water
cannot be excluded.  However, if such activity does exist, it must
occur at irregular intervals, or some evidence of fresh water would
have been found in the form of lower salinity or a slick on the surface
near the hole.
                         GENERAL DISCUSSION
Methods:
     The equipment and techniques described above satisfactory in
many ways, but some limitations should be noted.  The rig described and
shown in Figs. 5 and 6 is quite obviously a fair weather arrangement.
On Puget Sound there are sufficient winds during the late fall, winter,
and spring that the Coast Guard posts small-craft warnings a large part
of the time; working in these winds on the open sound with a sixteen-
foot boat is out of the question.  Even protected areas such as Burley
Lagoon may become quite rough in a short period of time when the wind
comes up quickly; during the seasons mentioned one cannot count on being
able to spend much time anchored on a station.
                                 - 31 -

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     Having reasonably calm weather is a necessity for making current
measurements with the equipment described above.  As can be readily
seen from Fig. 4 the shape of the buckets on the bucket wheel of the
Price meter is such that vertical motion, such as that imparted to the
meter by pitching or rolling of the boat, will decelerate the bucket
wheel, causing low readings.  Similarly, excessive swinging of the
boat, as may be caused by stiff breezes when no stern anchor is used,
will lead to erroneously high readings.  Consequently the Price meter
can be used from a small boat only under good weather conditions.  The
salinometer is not bothered by the problems mentioned above.  The con-
trol unit and transducer themselves are not affected by motion and can
be used in rough weather as long as due care is taken to prevent damage
to the transducer by its hitting the bottom or the side of the boat.
Data:

     Except as noted earlier in the case of the bathymetry, the data
shown are believed to be representative of conditions in the lagoon
during the months of September and October, 1963.  It should be noted,
however, that neither the current meter nor the salinometer have been
calibrated by the user.  Rough checks on the current meter were made
by noting the length of time required for surface detritus to drift
the length of the boat and calculating velocity from this; the veloci-
ties so obtained did not differ significantly from those registered
by the current meter.  One field check on the salinometer was made
with hydrometers and a mercury thermometer, on surface water sampled
with a Frautschy bottle held horizontally .just under the surface.  A
measurement was made with the salinometer immediately following the
taking of the sample.  The salinometer recorded 29.9 o/oo and 13.3 C
while the hydrometer indicated 30.1 o/oo and 13.6 C, which values are
within the tolerances stated by the manufacturer of the instrument.

     A second method of checking salinity and temperature was available.
The Shellfish Sanitation Laboratory has a Foxboro recording salinometer
and a Tag recording thermometer which continuously record the salinity
and temperature of the sea water furnished to several salt-water aquaria.
The intake of this system lies on the floor of the mouth of the lagoon,
about a hundred feet north of station D.  During the morning of 11
October, when station D was occupied, the laboratory instruments recorded
temperatures and salinities in the ranges 14.5-15.0 C and 29.5-29.7 o/oo
respectively, while, as shown in Table 3, the portable instrument recorded
average temperatures and salinities of 13.5 C and 29.6 o/oo.   The salini-
ties compare very well; the temperatures less so, perhaps because of the
heating effect of the pumping system which is located in a room usually
considerably warmer than the sea water (the thermograph probe is located
on the outlet side of the pump several feet from the pump itself).  Bear-
ing the above in mind,  there does not seem to be any reason to doubt that
the values shown in the appended tables are not reasonably accurate.


                                  -  32 -

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Future Work:

     Several things have been mentioned in this report which deserve
further observation.  First among these is the bathymetry.  In order
to resolve the doubts expressed above about the data, it would be de-
sirable to have a portable tide gage somewhere in the lagoon, perhaps
in the vicinity of the power towers in the southern end.  Data from
such an instrument covering a month or two would settle the question
of time lag between the lagoon and Wauna.  If a tide gage were not
available, a tide staff might be used, fixed to the power line towers
or a bridge pier at the mouth of the lagoon.  If a staff were used,
regular observations several times a day would be desirable.

     Second, the current data should be completed so that a set of
observations based on the same tidal difference for both ebb and flood
tides would be available.  A considerable amount of the observation
recorded herein was based on a tidal range of about ten feet.  If a
full set of data based on this range were available, any generaliza-
tions about the current patterns in the lagoon would be on a firmer
base.  It would be desirable to have several more stations for cur-
rent data to supplement those present in the southern end of the
lagoon.

     Third, there should be a full year's data on surface and mid-depth
or bottom salinity and temperature, for the data presented here show
nothing of the effect of precipitation and runoff on the lagoon.  That
such effect exists is shown by the fact that at the time of this writ-
ing (late November, 1963) the Foxboro recording salinometer mentioned
above is showing salinities as low as 20 o/oo on the very low tides
which occur at this time of year.   But it has been raining almost con-
tinually during the month of November, and very little rain was recorded
during the time the data were taken in September and October.  Data on
salinity and temperature taken at several points in both Burley Lagoon
and Henderson Bay would show any stratification present, the extent of
mixing at various locations,  and the duration of such effects through
the seasons of the year.

     Finally, some investigations should be carried out which time and
facilities did not permit at this time.  Among these are a survey of
the water chemistry of the lagoon,  including dissolved oxygen,  nitrate
and phosphate;  and an examination of the mixing process occurring as
the water from Burley Lagoon spills into Henderson Bay.   It is suspected
that mixing is quite complete in this area, as the flow is noticeably
turbulent, but more concrete evidence of this would be desirable.
                                - 33 -

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                              CONCLUSIONS
Ins trumentat ion:

     For the work carried out, the instruments used proved serviceable
and easy to use, and the use of these instruments is recommended for
work of the sort described in this report.  The limitations of the Price
current meter,  i.e. sensitivity to vertical motion and lack of a direc-
tion sensor which would allow its use in murky water, are not critical
so long as one  chooses the times and conditions of observation with
these limitations in mind.  The inconveniences involved in operation of
meters of the Ekman type from a small boat, and the relatively great
cost involved in such remote-reading meters as the Kelvin-Hughes and
Hytech instruments are also factors which favor the Price meter for
work such as that described.

     The RS 5-2 salinometer is well suited for this work.  Its simplic-
ity and the rapidity with which readings may be made with it especially
commend its use.  The model described in this report is not limited to
the depths at which it was used.  The instrument is available with up
to 300 feet of cable, which means the RS 5-2 can be used in almost any
estuarine situation where high precision is not required.  The manu-
facturer of this instrument now has available a Model RS-6 which measures
conductivity,  temperature, and depth; and a Model RS-7A,  which is a high
accuracy instrument apparently similar to the RS 5-2, but neither of
these have been investigated by the author.
Burley Lagoon;

     During the course of the study, the conditions in Burley Lagoon were
much as would be expected.  The lagoon flushes almost completely on each
tidal cycle, and the quality of the water in the lagoon is dependent al-
most exclusively on the conditions in Henderson Bay, which is the source
for the salt water in Burley Lagoon.  The effect of runoff from Burley
and Purdy Creeks was nonobservable, so such fresh water as was contributed
by these streams must have been well mixed with the salt water not far
from the mouths of the streams.  These conditions prevail during the
summer and early fall, when evaporation dominates over precipitation and
runoff; the effects prevailing during those seasons when precipitation and
runoff dominate will of course be quite different.

     Finally, as suggested earlier in this report, further work is needed.
Until such future work on currents and runoff as well as water chemistry
is undertaken, the knowledge of the hydrography of Burley Lagoon cannot
be considered complete.
                                   - 34 -

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                             ACKNOWLEDGEMENTS

     The author wishes to express his gratitude to the members of the
Northwest Shellfish Sanitation Research Laboratory for their continued
interest during the course of this project, and for their many questions
and suggestions.  Thanks are especially due to Leroy C. Myers for his
assistance in the gathering of data and for his instruction in small-boat
handling.

     Thanks are due also to Dr. Clifford A. Barnes, of the Department of
Oceanography of the University of Washington, for reading the final draft
and offering helpful comments about the presentation of the data.


                          COMMENTS BY PARTICIPANTS

     Mr. Sam Reed commented on what emphasis could be placed on the study.
How would this or similar studies be utilized in the shellfish certification
program:  What information could the various states use to implement their
individual programs in shellfish sanitation?  Mr.  Beck replied that the
initial program would be collated with other studies on Burley Lagoon such
as the bacteriological and sanitary surveys performed as cooperative effort
between Washington State Department of Health and the Shellfish Laboratory.
Further investigations in oceanography would be necessary before the entire
relationship could be completed.
                                      - 35 -

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                                References
U. S. Coast and Geodetic Survey, 1950. Manual of Current Observations.
     Special Publication No. 215, Revised!~"
     U. S. Government Printing Office, Washington,  87 pp.

U. S. Coast and Geodetic Survey, 1962.  Tide Tables.  West Coast.  North  and
     South America. 1963.  U. S. Government Printing Office,  Washington
     224 pp.

Raisz, Erwin,  1948.  General Cartography.   McGraw-Hill Book Company,  Inc.,
     354 pp.
                                    -  36  -

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Table 1.  Soundings in Burley Lagoon
Sounding
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
Depth re
mhhw* feet
9
10
9
9
10
12
10
10
13
12
12
11
12
13
9
11
12
11
12
11
11
13
13
12
13
13
14
14
14
Sounding
No.
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
Depth re.
mhhw* feet
14
15
15
16
15
15
14
16
11
21
15
12
12
14
15
16
18
13
15
17
21
31
25
19
15
18
18
18
16
Sounding
No,
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
Depth re.
mhhw* feet
14
13
14
12
10
12
14
15
23
16
17
20
23
26
23
27
24
21
22
13
14
15
19
21
19
18
15
19
19
     *mhhw = mean higher high water
                       - 37 -

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         Table 2.  Tidal data for Wauna,  Washington
High water    +0h 20m       +1.8 feet   add to Seattle  prediction




Low water     + Oh 36m      +0.0 feet    "    "    "          "






Mean higher water            13.1 feet




Mean high water              12.2




Mean low water               02.8




Mean lower low water         00.0




Mean range                   09,4




Diurnal range                13.1




Mean tide level              07.5
                             - 38 -

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Table 3.  Average surface and mid-depth temperatures and salinities for
                  Burley Lagoon and Henderson Bay
Surface
Station
A
B
C
D
£
F
G
H
Mean
Av. S o/oo
29.4
29.5
29.5
29.6
29.8
29.2
29.5
29.3
29.5
Av. T° C
13.7
14.0
14.4
13.5
13.5
13.7
13.2
13.3
13.7
Mid- depth
Av. S o/oo
29.7
29.5
29.6
-
30.0
29.7
29.9
29.6
29.7
Av. T* C
14.1
13.8
13.5
-
13.3
13.4
13.0
13.0
13.4
Table 4.  Gage height and stream discharge for Burley and Purdy Creeks
                        September-October, 1963
Burley Creek
Sent ember



Max.
Min.
Avg.
Gage
Height
ft.
1.18
1.00
1.05
Discharge
Acre ft.
/day
23.0
11.5
15.0
October
Gage
Height
ft.
1.90
1.06
1.21
Discharge
Acre-ft.
/day
67.0
15.5
25.0
Purdv
September
Gage
Height
ft.
0.48
0.36
0.39
Discharge
Acre-ft.
/day
03.5
01.0
01.5
Creek

October
Gage
Height
ft.
0.68
0.38
0.44
Discharge
Acre-ft.
/day
12.5
01.5
02.5
                                   - 39  -

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        /ii "3t\ JO'
w7:
,37') 30"
-30'
   BURLEY  LAGOON

         WASHINGTON

2J'
                                            FRET
                                                1000   2000
                                                 Projection
                                            Source
                                     U.S.G.S.. 7.5' Quadrangle
                                          Burley, Wash.
                                          -<
    Fig. 1  Base Map of Burley Lagoon, on Henderson Bay at the Head of Carr
                     Inlet, Southern Puget Sound
                            - 40  -

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          \-2.L 31 30"
38
                               37|'30'
   47°
   24'
   3§URLEY  LAGOON

          WASHINGTON
	24'
  23'
  •30"
   47'
   23'
           SAMPLING STATIONS

           Sounding      ^
           Salinity, Temperature '
           and Current    „
                       B
                                              VAR,fo6l)
          122°38'30
I
       122°| 37
                                               47°
                                               24'—I
                                               30"
                                                               2F-
                                                        1
                          1000      0     1000     2000
                          •.  POLYCONIC  PROJECTION
                                                 23'
                                     Source       30'I
                            U. S.G.S.   7.5' QUADRANGLE '
                                 BUKLEY, WASH.
                                                              23'
1963
P.S.K.
                                                          122
                                Ill
        Fig. 2  Data Stations Occupied For Bathymetry,  Temperature,
          Salinity, and Currents on Burley Lagoon and Henderson Bay
                               - 41 -

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iV
30"
BURLEY  LAGOON


      WASHINGTON -   :


         BATHYMETRY

        Depths in Feet

     at Mean Higher High Water
                         .
      Contour Interval 3 Feet '.

      3 and 6 Foot Contours
      Omitted

      Low-Tide Stream
      Channels Shown by
      Broken Lines
AX'
30'
1J'
          ° yt'\3o'
                                       	      FEET

                                      1000     0     1000
                                                        2000
                                   ' POUYCONIC       PROJECTION
                                             Source
                                   - -U.S.G.S. 7.5' Quadrangle
                                          Burley, Wash.
                 Fig. 3 Contour Chart of Burley Lagoon
                              - 42 -

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                                              -
               Fig. 4
Price Current Meter, Salt-Water Modi-
fication, Made by W. and L. E.  Gurley.
Battery and Electric Counter Shown at
Right, Separated from Meter by 30 feet
of Cable.
             Fig. 5
View from Bow of 16-foot Outboard
Motorboat, Showing Detail of Boom
and Cable Clamp for Current Meter.
            Fig. 6
View from Stern of Boat, Showing
General Layout of Equipment.   Pole
in Center Holds Cables for Current
Meter and Salinometer.  Meter Count-
er and Salinometer Control are at
Left.  Box to Right of Steering Wheel
Holds 4-inch Boat Compass .
            Fig.  9
Industrial Instruments Model RS5-2
Portable Salinometer.   Transducer
is at Right, Separated from Control
by 100 feet of Cable.
                                      - 43  -

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   BURLEY  LAGOON

         WA SHI NGTON
  CURRENTS:  EBB TIDE

Maximum Observed Velocities
 General Circulation Shown By ..
       Large Arrows:
                                      1000     0     1000    2000
                                       Pclyconic     Projection
                                               Source
                                       ''U.S.G.S. -7.5' Quadrangle
                                       '       Burley, Wash.
           Fig. 7 Ebb Tide Current Patterns in Burley Lagoon
                              - 44 -

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  BURLEY  LAGOON
        WASHINGTON
  CURRENTS:  FLOOD TIDE
Maximum Observed Velocities

          KNOTS
 General Circulation Shown by
       Large Arrows
OQO      0
•Polyconic
                                                 1000    2000
                                                Projection
                                  ..  -U.S.G.S. 7.5' Quadrangle
                                           .Bur ley, Wash.
          Fig. 8  Flood Tide Current Patterns in Burley Lagoon
                              - 45 -

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     Bacteriological Study of Stored Pacific Oyster Shellstock

            J. C. Hoff, W. J. Beck and T. H. Ericksen

                           INTRODUCTION

     Several indices used in other areas of sanitary bacteriology have
been used to determine the microbiological quality of fresh, packed
oysters in commercial channels.  The changes which occur in these in-
dices during storage of shucked Pacific oysters have been extensively
studied in this laboratory.  However, the effects of storage of shell-
stock on these indices have not been investigated.

     In commercial operations, dry storage of oyster shellstock for two
or more days at temperatures of 10 C or above is not unusual.  Under
these conditions, shucked oysters show rapid changes in bacterial popu-
lations and deteriorate rapidly.  Therefore, it is of interest to de-
termine whether or not similar changes occur in the living oyster stored
under these conditions.

     This study was undertaken to investigate the behavior of several bac-
terial indices during dry storage of Pacific oyster shellstock.  The study
was initiated in September 1963 and continued through August 1964.


                      MATERIALS AND METHODS

Oysters.  The oysters used in this study were obtained through commercial
sources in Washington and Oregon.  Each lot consisted of approximately 370
oysters, 10-15cm long.  The oysters were scraped to remove barnacles and
other fouling organisms and washed in fresh water to remove mud on arrival
at the laboratory.  Ten lots were collected locally directly from the beds
by laboratory personnel.  These lots were placed in storage within four
hours after harvesting.  Two lots, collected in Oregon,  had been collected
some time previous to arrival of laboratory personnel.   The time elapsed
between harvest and storage on these two lots is uncertain.

Storage.  Approximately 120 oysters were stored at 10 C, 20 C and 27.5 C
respectively.  Storage facilities consisted of refrigerators equipped with
refrigeration or heating units and thermoregulators capable of controlling
temperatures to ±0.5 C.  The oysters were placed one layer deep on racks
in these units.
                                - 46 -

-------
Sampling.  Samples consisted of 10 tightly closed oysters.  One sample
tested on arrival at the laboratory constituted the 0 hr. sample for all
three temperatures.  Sampling schedules following the initial sample were
as follows:

              10 C - 1,2,4,7,10,15,20, and 25 days.

              20 C - 1/2, 1,1-1/2,2,3,4,5,6, and 7 days.

              27.5 C - 1/2,1,1-1/2,2,3, and 4 days.

"Gaping" oysters were also examined if more than five were found.  Excess
"gapers" were discarded at each sampling time.


Examination. The bacteriological indices determined on each sample con-
sisted of five tube coliform and fecal coliform MFN's, and standard plate
counts at 35 C performed according to recommended procedures (1962).  The
pH was determined electrometrically on a portion of the blended sample.
At selected intervals IMViC tests on cultures isolated from EC positive
tubes were performed to determine whether or not a differential dieoff of
Escherichia coli occurred during storage.


Storage Schedule.  Storage experiments were planned so that several lots
were examined in each season.  In this way, possible effects of seasonal
variations in the oysters and their bacterial flora on the behavior of
the bacterial populations during storage could be evaluated.


                                RESULTS

     The chronology of the storage experiments, sources, and initial group-
ings for data analysis are shown in Table 1.  Geometric mean values for
each bacterial index in each seasonal group were calculated and the results
were plotted.  The changes shown by the indices in all four groups were
similar.  Therefore, geometric means derived from all 12 lots were calcu-
lated and plotted.  In a few instances, due to laboratory accidents or in-
determinate results, data were not available for a particular sampling time.
In some lots, storage was terminated earlier than in others because no
tightly closed oysters remained.  In these instances the results are based
on fewer than 12 lots.

     The results of storage of the 12 lots at 10 C are shown in Fig. 1.
The fecal coliform MPN remained quite stable but showed a slight overall
decline during the 20-day storage period.  Coliform MPN's increased about
twofold during the first 2 days after which they remained relatively stable.
Plate counts increased about fourfold during the first day and continued to
increase at a slower rate during the remainder of the storage period.


                                   - 47 -

-------
     At 20 C (Fig. 2), the storage time was considerably shortened be-
cause the animals were usually all "gapers" by the seventh day.  Fecal
coliform MPN's increased about twofold during the first two days and
remained stable at that level for the remaining period.  Coliform MPN's
increased approximately ninefold during the first two days then remained
relatively stable.  Plate counts increased twenty-eightfold during the
first day of storage, then continued to increase at a slower rate.

     The results of storage at 27.5 C are shown in Fig. 3.  Oysters sur-
vived for only 3 days at this temperature.  Numbers of all 3 indices in-
creased rapidly during the first day and continued to increase at a rapid
rate.

     The results of IMViC tests on isolates from EC positive tubes are
shown in Table 2.  At 10 C, no change in percent £.. col ft found is evident.
At 20 C and 27.5 C E. coli percentages appeared to drop slightly as stor-
age time progressed.  However, the change if any was slight.

     The results of pH determination are shown in Table 3.  It is obvious
that pH in the live Pacific oyster was not affected by changes in bacterial
populations within the animal.

     "Gapers" were not always found in sufficient numbers to constitute a
sample at each sampling interval in each lot.  When three or more compara-
tive samples of closed and "gaping" oysters were examined, geometric means
and ratios of "gaper" populations to closed oyster populations were calcu-
lated.  The results are shown in Table 4.  The 35 C plate counts showed the
greatest differences and were always as high or higher when the "gapers"
were compared with closed oysters.
                                DISCUSSION

     Previous studies have shown that the fecal coliform group enumerated
by the EC test was influenced least by duration or condition of storage
in shucked Pacific oysters, (Presnell, 1962; Hoff, Beck, and Presnell,
1964) and in the Eastern oyster (Presnell and Kelly, 1961).

     The data presented in this study indicated that the above statement
also applies to dry stored Pacific oyster shellstock.  Although fecal
coliform MPN's did increase at 27.5 C little change occurred at 20 C and
10 C.  Rapid death (gaping) and rapid increases in plate count, coliform
and fecal coliform populations in shellstock stored at 27.5 C indicate
that shellstock should not be held in areas where temperatures in this
range persist.
                                   -  48-

-------
      Evidence  for  the  biological  soundness of  the use  of  the  fecal  coli
 form group  as  an indicator  of  fecal pollution  by man and  other warm
 blooded  animals has  been  given previously (Kelly, 1960; Presnell, 1961;
 Kelly et al, 1962; Beck,  Presnell, and Hoff, 1963; Kabler, Clark and
 Geldreich,  1964).  Because  of  this and because of the  stability of  this
 index during storage indicated above, it appears that  the EC  group  would
 be  of value as an  indicator of the sanitary quality of Pacific oyster
 shellstock  during  the  interim  between harvesting and shucking.

      The 35 C  plate  count showed  the most uniform differential response
 to  storage  time and  temperature.  At 10 C the  time required for a ten-
 fold increase  to occur was  five days while at  20 C a tenfold  increase
 occurred in one-half day.   It  is  apparent that either  low shellstock
 storage  temperatures or prompt shucking of harvested shellstock would
 be  conducive to lower  initial  plate count populations  in  the  product and
 thus to  longer shelf life.

      The response  of the  coliform group to storage time and temperature
was  less consistent  and uniform than that of the 35 C  plate count and
 less stable than that of  the fecal coliform group.  These factors,  in
 combination with its doubtful  validity as an indicator of sanitary
 quality  indicate that the coliform index would be less valuable as  an
 indicator of shellstock bacteriological quality.

      Despite large increases in populations which occurred at the higher
 storage  temperatures, the pH showed little change.  Therefore, it appears
 that this criterion would give no information  as to bacteriological qual-
 ity  of Pacific oyster shellstock.


                            COMMENTS BY PARTICIPANTS

      Dr. Listen discussed the  use of 20 and 27.5 C dry storage.  Mr. Kelly
commented on the lack of  change in fecal coliform make-up at  the three
 storage  temperatures.  Dr. Hoff made further comments which included the
difference  in plate  counts and the possibility of differential control of
microorganisms by the animal.

     Mr. Girard asked about difference between liquors and meats.   Mr. Kelly
commented on other studies on  the Gulf Coast where some multiplication evi-
dently takes place in the animal and liquor.   Drs.  Dollar and Hoff discussed
the  reasons for the  loss  of liquor by some oysters.   Again the relationship
of pathogenic organisms to the indicator organisms were discussed.
Mr.   Kelly and Dr.  Bartsch commented on the difficulties of obtaining samples
with known pathogenic organisms.

      Drs. Sparks and Quayle commented on length of time shellfish can be
held in  dry storage under various conditions.
                                   - 49 -

-------
                            REFERENCES
American Public Health Association. 1962.  Recommended procedures for the
     bacteriological examination of sea water and shellfish, 3rd ed.

Beck, W. J., M. W. Presnell, and J. C. Hoff. 1963.  Ecological study of
     bacterial indices of pollution.  (Paper presented at Shellfish Sanita-
     tion Res. Conf., Purdy, Wash.).

Hoff, J. C., W. J. Beck, and M. W. Presnell. 1964.  A study of the applic-
     ability of several indices as sanitary quality indicators in commer-
     cially packed Pacific oysters (Crassostrea gigas).  (Paper presented
     at Shellfish Sanit. Res. Conf., Purdy, Wash.).

Kabler, P. W., M. A. Clark, and E. E. Geldreich.  1964.  Sanitary signifi-
     cance of coliform and fecal collform organisms in surface water.  Pub.
     Hlth. Rep. 79: 58-60.

Kelly, C. B., 1960.  Bacteriological criteria for market oysters.  Tech.
     Rep. F60-2.  Robert A. Taft Sanit. Engr. Center, Cincinnati, Ohio.

Kelly, C. B., W. J.  Beck, M. W. Presnell, and K. J. Zobel. 1962.  Ecologi-
     cal study of bacterial indices of pollution.  (Paper presented at
     Shellfish Sanit. Res. Conf., Purdy, Wash.).

Presnell, M. W. 1961.  Sanitary significance of "fecal coliform organisms"
     in a shellfish growing area - sanitary survey of Burley Lagoon.
     (Paper presented at Shellfish Sanit. Res.  Conf., Purdy, Wash.).

Presnell, M. W. 1962.  Studies on stored Pacific oysters. (Paper presented
     at Shellfish Sanit. Res. Conf., Purdy, Wash.

Presnell, M. W., and C.  B. Kelly. 1961.  Bacteriological studies of commer-
     cial shellfish operations on the Gulf Coast. Sanit.  Eng. Center Tech.
     Rep. F61-9.  U. S.  Public Health Service.
                                 - 50 -

-------
Table 1.  Chronology of Pacific oyster shellstock storage study
Lot
2
3
4
5
6
7
8
9
10
11
12
13
Date
Stored
9-30-63
10-14-63
10-28-63
1- 6-64
1-27-64
2-10-64
2-24-64
5-11-64
5-18-64
6- 8-64
7-20-64
7-27-64
Source
Washington )
Washington )
Washington )
Washington )
Oregon )
Oregon )
Washington )
Washington )
Washington )
Washington )
Washington )
Washington )
Grouping for
Initial Analysis
3 lots -
4 lots -
3 lots -
2 lots -
Fall
Winter
Spring
Summer
Table 2.  IMViC analysis of E C positive cultures  isolated during
          storage of Pacific oyster shellstock (12 lots)
Storage
time
( days )
0
1/2-1
1-2
1 1/2-2
3-4
4-7
5-6
10
15
20
10 C
Total No.
Isolates
88
-
56
-
-
107
-
74
68
76

E.coli
97
-
91
-
-
91
-
95
100
99
20 C
Total No. %
Isolates E.coli
88 97
114 96
-
121 89
144 88
-
125 87



27.5 C
Total No.
Isolates
88
197
-
198
107






E.coli
97
95
-
92
84





                               -  51  -

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Table 3.  Effect of storage time and temperature
       on pH of Pacific oyster shellstock
Storage Time
(days)
0
1/2
1
1 1/2
2
3
4
5
6
7
10
15
20
10 C
6.5

6.4

6.4

6.5


6.5
6.5
6.5
6.5
20 C 27.5 C
6.5 6.5
6.4 6.4
6.4 6.4
6.4 6.4
6.5 6.3
6.5 6.1
6.5
6.4
6.3




                       - 52 -

-------
                Table 4.  Comparison of  bacterial populations  in  gaping  and  closed Pacific  oysters
v_n
u
Time
(days)
1 1/2
2
3
4
5
6
10
15
20
Bacteria
K.aLio _
Bacteria
27.5 C
Fecal 35 C
Coliform Coliform Plate Coliform
MPN/lOOg MPN/lOOg Count/e MPN/lOOfc
1.1 3.5 1.2
3.0 2.3 1.9
3.9 3.4 13.1 1.1
0.3
0.7
1.2
in "gapers
in closed
20 C
Fecal
Coliform
MPN/lOOe
1.4
0.7
1.1
0.4
ii
oysters

35 C
Plate
Count /R
2.6
2.3
1.1
10.0

10 C
Fecal 35 C
Coliform Coliform Plate
MPN/lOOg MPN/lOOe Count/a
12.5 2.0 16.1
1.3 0.8 9.3
0.9 0.8 2.0

-------
                                                                                                                  1	1	1           I
                                                                                                                                   O  3S°C Tlt
                                                                                                                                   A  Coltfon.
                                                                                                                                   Q  Txtl CoUfon KPVAOOI
: l&. 1.  Cli,inge»  In tnli(.,ni  HP:*,  fecal Ccltri.n. ^•JI^, and  35 C  flat*
    Ci-ui.l in ~atif:c 0>-st«r SlwlUtock Stored ;it In C (U  lots)
I Is. 2.  Chattel  In Cullforii MPK,  Fical Ciillfrrn HPK, and 3S C  Pint.
     Count  In i'aclflc Oyitcr Shdlituck Stored .it 20 C (13  loti)
                                                                                 O IS C Fl«t« Counl./f
                                                                                 A Coll fern NPOAOOf
                                                                                Q FM! Coll fora
                                                                                J	L
                                                                         Staraft TIM
                                              : r.  3.  Hi. i.|.i.» t i, MI IT. sr:.,  I'licni ci'lKc-n Mr., ~"ci  itCil.i
                                                  Ct.«tnl i^' :':!t If U- OVMtl St-t'llKl. ck Sl.Ti-a ; t 'J7. 5 t U * 1' "
                                                                             -  54   -

-------
 Storage Studies on Manila Clams  (Tapes  japonica)  and  Native Littleneck
                  Clams (Protothaca staminea)  shellstock

                T.  H.  Ericksen, J.  C. Hoff,  and W. J.  Beck
                               INTRODUCTION

      This  study was  initiated  to  determine  the effects of a range of
 storage temperatures on the  bacterial quality of  clam shellstock as
 indicated  by  the 35  C plate  count,  coliform MPN and  fecal coliform MPN.
 Recommendations by previous  Shellfish Sanitation  Planning Conferences
 were  made  in  order that aid  in evaluation of present marketing prac-
 tices of Manila (Tapes japonica)  and Native Littleneck (Protothaca
 staminea)  clams could be made.  The study consisted of eleven lots of
 each  species  of clams collected at  seasonal intervals over a period
 of  one year.
                          MATERIALS AND METHODS

     The clam shellstock was collected at various sites in the State
of Washington through the cooperation of the Washington State Depart-
ment of Health.  The first 2 lots were collected by commercial harvest-
ers and the remaining lots by laboratory personnel of the Northwest
Shellfish Sanitation Laboratory.  Transport of samples to the laboratory
and bacteriological examination were carried out as described in APHA
Recommended Procedures for the Bacteriological Examination of Sea
Water and Shellfish (Third Edition, 1962).

     Each lot of clam shellstock consisted of 360 individuals.  Each
lot was divided into three equal groups which were stored at 10 C,
20 C and 27.5 C respectively.  Both species were stored and examined
simultaneously.  Perforated plastic bags were used to store clams to
simulate market storage practices.  Modified refrigerators equipped
with electronic relays,  constant temperature recording devices and
the appropriate thermal regulators were used for storage.   Each sample
for examination consisted of a minimum of five clams and a maximum of
ten clams for each species.

     Clams stored at 10 C were examined at 0,  1,  2,  4, 7,  10,  15 and 20
days.   Clams stored at 20 C  and 27.5 C were examined at 0, 1/2,  1, 1-1/2,
2 days and 1 day intervals thereafter until no clams remained closed.
The inability to close its shell in response to tactile stimuli was an
arbitrary criterion for determining the clam sample to be  examined.
                                 - 55 -

-------
     Samples were selected at each temperature and prepared and shucked
in the prescribed manner.  Each sample of shellfish meats and liquor was
weighed in a sterile tared beaker.  The shellfish sample was placed in a
sterile blending jar and blended for twenty seconds, after which a 20 ml
aliquot of homogenate was removed.  The loss of the 20 ml aliquot was
compensated for and a weight of phosphate buffer equal to that of the
shellfish homogenate remaining in the blender jar was added.  This mix-
ture was blended for an additional eighty seconds.

     Bacteriological examination immediately followed the blending pro-
cedure.  The pH of the sample was determined electrometrically from the
20 ml aliquot.  EC positive tubes at selected intervals were submitted
to the IMViC test.  However, because of the selected intervals the number
of EC positive tubes submitted to the IMViC test was limited.  These data
indicated growth differentials of the various fecal coliform organisms
present throughout the storage period.

     "Gaper" samples, which were determined by the inability of the clam
to close its shell to tactile stimuli, were collected simultaneously with
closed shellstock samples at each of the three respective temperatures
for both clam species.  All "gapers" were removed at each sampling period,
therefore the clam shellstock of each species remaining at each temperature
was closed shellstock.

     Modification of storage temperatures and examination intervals were
made after testing the first 2 lots of each species.  This accounts for
the 27.5 C storage temperature being added on the third lot and thereafter.
                              RESULTS

     The results of storage of Native Littleneck clam shellstock are shown
in Figs. 1, 2 and 3 and Manila clam shellstock in Figs. 4, 5 and 6.

     The coliform MPN and 35 C plate counts were analyzed by the geometric
mean method.  Because of the presence of indeterminate numbers (<18),  med-
ian values were used for fecal coliform MPN's.  Comparison of values for
seasonal series of lots indicated there were no apparent seasonal varia-
tions.  Therefore, lots at each storage temperature were grouped by sampl-
ing time, clam species and specific bacterial index and the geometric
means or medians determined.  The values represented in the figures were
determined from approximately eleven lots each.

     During storage of Native Littleneck clams at 10 C (Fig. 1) only a
slight change in the fecal coliform MPN occurred.  The change was a slight
decrease after ten days of storage.  The coliform MPN showed an immediate
slight decrease for the first two days, which was followed by a leveling-
off period.  The 35 C plate counts indicated an overall continuous increase
as storage progressed.
                               - 56 -

-------
     Native Littleneck  clams  stored at 20 C (Fig. 2) showed little change
 in  the  fecal  coliform MPN.  However, slight fluctuation was evident.  The
 coliform MPN  changed somewhat erratically, but showed a general decrease.
 The 35  C plate count indicated a steady increase throughout storage.

     The 27.5 C  storage of Native Littleneck clams (Fig. 3) showed a
 slight  decrease  in the  fecal coliform MPN.  The coliform MPN indicated a
 continuous increase as  storage progressed.  The 35 C plate counts showed
 a steady sharp increase throughout storage.

     Overall, the fecal coliform MPN's remained relatively stable during
 storage at the three temperatures.  The coliform MPN's at 10 C and 20 C
 showed  slight decreases and at 27.5 C indicated an increase.  The 35 C
 plate counts  showed steady increases at all three temperatures.  However,
 as  the  storage temperature increased this bacterial index increased at a
 faster  rate.

     The Manila  clam shellstock stored at 10 C (Fig. 4) showed little
 change  in the fecal coliform MPN during storage.  The coliform MPN's
 showed  a slight  increase for seven days followed by a decrease for the
 remainder of  the storage period.  The 35 C plate counts indicated an
 initial lag period of two days followed by a steady increase.

     Storage  of Manila clams at 20 C (Fig. 5)  showed little change in
 the fecal coliform MPN.  The coliform MPN indicated a steady increase
 throughout storage.  The 35 C plate counts showed an initial lag period
 of  one-half day  followed by a general increase.

     In Manila clams stored at 27.5 C (Fig. 6) the fecal coliform MPN
 decreased.  The coliform MPN indicated a steady increase throughout
 storage,  the 35 C plate counts indicated a steady sharp increase.

     Overall  in Manila clam shellstock the fecal coliform MPN remained
 relatively stable.  However,  a general decrease was observed at 27.5 C.
The coliform MPN's indicated general increases as storage progressed at
 the three temperatures.  The 35 C plate counts showed general  increases
 at  the  three  temperatures.   However,  lag periods were observed at 10 C
 and 20 C.   Again, as in the Native Littleneck clams,  the 35 C  plate
 counts  increased at a faster rate as the storage temperature increased.

     The data in Fig.  7 showed relatively little change in the pH of
 either species at the three temperatures as storage progressed.   The
values for the Native Littleneck clams were slightly higher than those
 of the Manila clams.
                                    - 57 -

-------
     Bacteriological examination of "gaper" samples at the three tempera-
 tures  for  the  two species of clams indicated predominantly higher bacter-
 ial densities  than samples of closed clams at the same temperature and
 storage  time.  The ratio of geometric means of coliform MPN's, fecal coli-
 form MPN's and 35 C plate counts of "gaping" clams to closed clams are
 shown  in Table 1 for Native Littleneck clams and Table 2 for Manila clams.
 The data indicated generally higher values for "gapers" than closed clams.
 for both species at the three temperatures.  No patterns were apparent.
 Generally  the  ratios were higher for the Native Littleneck clams.  There-
 fore a marked  difference by species was noted.

     Results of 489 IMViC tests indicated the predominant fecal coliform
 was Escherichia coli.  Results of these tests are given in Tables 3 and 4.
 This data  indicated a higher percentage of fecal coliform present as
 El. coli  in Manila clams than in the Native Littleneck clams.  The percent
 of E. coli in  tested samples of Manila clams ranged from 87 to 100.  In
 Native Littleneck clam shellstock, the percentage as E. coli of fecal
 coliform organisms tested ranged from 75 to 88 at 10 C; 79 to 100 at 20 C
 and 65 to 80 at 27.5 C.  At all storage temperatures the E. coli percent-
 age remained relatively stable for both species.

     Several samples of both species of clam shellstock from various lots
 have been stored at -5 C.  However, only a limited number of these have
 been tested.   To date the results appear to be similar to those of the
 zero-hour sample*
                              DISCUSSION

     Data obtained from Manila and Native Littleneck clam shellstock
stored at 10 C, 20 C and 27.5 C indicated 35 C plate count increased as
storage progressed.  The coliform MPN also increased for both species of
clams except for the Native Littleneck clams stored at 10 C and 20 C.
This increase for both indices appeared to be more rapid in the Manila
clams.  Generally it was shown that as the storage temperature increased
the bacterial density increased at a faster rate as indicated by these
two indices.

     The data indicated there was little change in the fecal coliform
MPN's of both clam species at the three temperatures.  However, a de-
crease was observed in Manila clams stored at 27.5 C.

     This data agrees with the previous work on shucked Pacific oysters
(Presnell, 1962; Hoff, Beck, and Presnell, 1964) and in the Eastern oyster
(Presnell and Kelly, 1961).  The coliform MPN and 35 C plate count were re-
lated to temperature and condition of storage whereas the fecal coliform
MPN exhibited no such relationship.  Previous studies have shown that the
fecal coliform group may be used as an indicator of fecal pollution by
warm-blooded animals (Kelly, 1960; Presnell, 1961; Kelly et ill, 1962;
Beck, Presnell and Hoff, 1963; Kabler, Clark and Geldreich, 1964).  There-
fore because the fecal coliform MPN of Manila and Native Littleneck clam
shellstock did not appear to be affected by duration or condition of stor-
age, this index appeared to be a valued indicator of the sanitary quality
of dry stored Manila and Native Littleneck clam shellstock.

                                  - 58 -

-------
     It was also shown that each individual index for each respective
storage temperature exhibited similar growth patterns for both species
of clams.  The general coliform MPN, fecal coliform MPN and 35 C plate
count changes exhibited by both clam species were similar to the changes
exhibited by the same indices for the Pacific oyster shellstock storage
studies.

     The pH data indicated relatively no appreciable change during stor-
age at the three temperatures for both species of clam.

     Data obtained from IMViC tests of selected positive EC tubes indi-
cated the predominant fecal coliform organism present was JE. coli.  From
this data no differential growth patterns of different fecal coliforms
could be discerned during storage at the three temperatures.  However,
there was a higher percentage of fecal coliforms present as E. coli in
Manila clams.  The differences between Manila and Native Littleneck clam
results may be explained by the fact that earlier studies indicated
Manila clams possibly concentrate these organisms to a greater degree.
Also, the Manila and Native Littleneck clams were collected from differ-
ent areas and therefore may have been subject to dissimilar environments.
The changes exhibited by the coliform MPN and 35 C plate counts for both
species of clams during storage also may have influenced this situation.

     The data from the "gaper" study indicated that the inclusion of
"gaping" clam shellstock possibly would increase the bacterial density
of samples.  The ratios for the Native Littleneck clams tended to be
greater than those of Manila clams.  Therefore, a species difference
was apparent.  It was also observed that the Native Littleneck clams
remained closed longer at all temperatures than the Manila clams.

     From this study it may be concluded that of the three storage
temperatures, 10 C would be the better dry storage temperature for
Manila and Native Littleneck clam shellstock.   The coliform MPN and
35 C plate count seemed to be related to duration and condition of
storage.  The fecal coliform did not exhibit this relationship.


                      COMMENTS BY PARTICIPANTS

     Mr. Kelly pointed out the differences between fecal coliform re-
sults as compared with plate counts and coliform results at 27.5 C.
                                  - 59 -

-------
                              REFERENCES
American Public Health Association. 1962.  Recommended procedures for the
     bacteriological examination of sea water and shellfish, 3rd ed.

Beck, W. J., M. W. Presnell, and J. C. Hoff. 1963.  Ecological study of
     bacterial indices of pollution.  .(Paper presented at Shellfish Sani-
     tation Res. Conf., Purdy, Wash.).

Hoff, J. C., W. J. Beck and M. W. Presnell.  1964.  A study of the appli-
     cability of several indices as sanitary quality indicators in commer-
     cially packed Pacific oysters (Crassostrea gigas).   (Paper presented
     at Shellfish Sanitation Res. Conf. Purdy, Wash.).

Kabler, P. W., M. A. Clark and E. E. Geldreich. 1964.  Sanitary significance
     of coliforms and fecal collform organisms in surface water.  Pub. Hlth.
     Rep. 79:58-60.

Kelly, C. B., 1960.  Bacteriological criteria for market oysters.  Tech.
     Rep. F60-2.  Robert A. Taft Sanit. Engr. Center, Cincinnati, Ohio.

Kelly, C. B., W. J. Beck, M. W. Presnell, and K.  J.  Zobel. 1962.  Ecologi-
     cal study of bacterial indices of pollution.  (Paper presented at
     Shellfish Sanitation Res. Conf.,  Purdy, Wash.).

Presnell, M. W., 1961.  Sanitary significance of "fecal coliform organisms"
     in a shellfish growing area - sanitary survey of Burley Lagoon.
     (Paper presented at Shellfish Sanit. Res. Conf., Purdy, Wash.).

Presnell, M. W., 1962.  Studies on stored Pacific oysters.  (Paper pre-
     sented at Shellfish Sanit. Res. Conf., Purdy, Wash.).

Presnell, M. W. and C. B. Kelly.  1961.  Bacteriological studies of com-
     mercial shellfish operations on the Gulf Coast.  Sanit. Eng. Center
     Tech. Rep. F61-9.  U. S. Public Health Service.
                                 - 60 -

-------
 102
                                          	1

                                           O li C Plata count/s

                                           A Coll fora KPN/100,
                                           O Ftcal Col 111.™ !•:>'«/1
     -O-D-
                -o-
  '°'i   ',  J   M   i	1
                             . lorai* tlM <*Uya)
      Fli  1.   Chant.,  in collfon HPN,  ftul colt Tor. MPN. and 15 C Plata
       e«unc In tutu-*  LtttUnccli clan ihclUtock  itorrd at 10 C (tl .loti)
                                                                                     102
                                                                                                                             O  3i c pUtc cuunc/y.

                                                                                                                             A  Col I Com vra/lOOs
                                                                                                                             Q  Frcul Collfon HI'll/
     012         34         i        6         7
                              (.Storaf* tl.u (day!)
        Fit. 2.  ChangM  In  colifom HPN. t*C*l eollfom MPN, and 35 C pl«t«
          count tn Native ltttl«n«ck cla« *htlUcock •Cored  at  20 C (tl loti)
  10*
•io3
                                            0 " * Pl*>*  coynt/1
                                            A Coltfor» HI>H/100§

                                            0 r«.it coll (or. Hm/100K
                                                                                      10"
               I        I        )        •         51.         7
                              toraK* '!•* (days)
       II,   1   Chandra In Col I hint HM),  flteal cplltoni HPN, and DC  plat*
       couiil  In Haltv* l.ltlUn«ck clan ali>tltli«V atond •(  II 1 C  (11 lota)
                                                                                      105
£

*


i
                                                                                      10'
                                                                                                                               O >5 C »lata

                                                                                                                                          HPH/lMi;

                                                                                                                               QF.C.I  eoiirim HPN/IOO«.
                                                                                             I    1  9
                                                                                                                                             li
                                                                                                                                                             >0
                        5       !         10
                                loraK« 11"'* (daya)
         ri>.  «.   chaniiaa  In callforaKI'N. Itcal collfon HFK, and lie plait
               count  In  Manila  CUB ahallatock atorad at IOC Ml lot.)
                                                                            -  61  -

-------
102
                                                  35 C  plaE* counc/g
                                                  Coltfonn NPN/lOOg
                                                  Fecal colifenn HPN/lOOg.
                                                                                      10
                           O  35 C pure counc/g

                           A  Ct>lifon» MPN/lOOt

                           LJ  Fecal  collform MPN/lOOw
                              Stor*g* tl«« (d*yi)
      '!(. S.  Ch»tM  La •olifon MPH.  fecal  collforv KPN, and 35 C
      pUt* count ID fUBilU cl.u  «hellttock  stored  ac  20 C  (11 lot.)
               r.cc,rase time (days)
 Changei In collfom MPN.  fecal collfonu MPN,  and 35 C place
nt in Manila clam shellacock stored at  27.5 C  (11 lots)
                                                                                                             JaU». Uttltnack 01

                                                                                                         O  Hull! Clai
                                g.  7.  Change!  in PH of Eleven Lc*  r
                                                                         lla and Katlve Lltllen.c
                                                                                                k CI™, Stored ,t 10, 20, and 27.5 C
                                                                               -   62   -

-------
Table 1.  Comparison of bacterial populations in "gaping"  Native Littleneck clams with those  in closed
                                            Native Littleneck clams
Storage
Time
(days)
1/2
1
1 1/2
2
I 3
U3
1 4
5
6
7
10
15
20
Patio Bacteria in "aapers
Bacteria in closed
27.5 C 20 C
Fecal 35 C Fecal
Coliform Coliform Plate Coliform Coliform
MPN MPN Count MPN MPN
1.36 1.13 3.57
1.34 0.79 1.67
14.29 52.45 18.57 4.12 14.29
2.0 1.93 3.75
1.75 9.33 10.48 5.93 7.81
5.13 0.67
1.58 3.05
6.25 0.63




H *
clams *

35 C
Plate
Count


7.06

7.78
10.00
6.67
4.86





10 C
Fecal 35 C
Coliform Coliform Plate
MPN MPN Count







4.89 1.47 2.95
1.35 1.16 3.64
2.57 2.00 5.71
0.27 0.92 21.21
    *Geometric means

-------
Table 2.  Comparison of bacterial populations  in "gaping" Manila clams with those in closed Manila clams
Storage
(days)
1/2
1
1 1/2
2
3
4
5
7
10
15
Ratio Bacteria
Bacteria
27.5 C
Fecal 35 C
Coliform Coliform Plate Coliform
MPN MPN Count MPN


1.58 1.35 2.89
0.86 0.60 1.38 0.76
0.46 2.43 1.25 2.29
1.41
1.33



in "gapers
in closed
20 C
Fecal
Coliform
MPN



0.55
2.24
0.85
1.43



n *
clams *

35 C
Plate
Count



10.38
1.74
0.91
1.89




10 C
Fecal 35 C
Coliform Coliform Plate
MPN MPN Count









1.1 2.0 1.42
      ^Geometric means

-------
Table 3.  IMViC analysis of EC positive cultures  isolated  during  storage
                   of Native Littleneck clam shellstock
Storage
time
(days)
0
1/2
1
2
4
7
10
15
Table 4.
10
Total No
Isolates
28
-
8
.
14
4
8
14
C
20
C
% Total No.
E.coli
79
-
88
-
79
75
75
86
IMViC analysis of
Isolates E.coli
28
21
-
15
14



EC positive
of Manila clam
Storage
time

0
1/2
1
2
4
7
10
15
10 C
Total No.
Isolates
64

19

39
16
15
9

%
E.coli
89
-
95
.
90
93
100
100
20
Total No
Isolates
64
46
•
28
43



79
100
-
80
100



cultures
shellstock
C
• At
E.coli
89
87
—
89
88



27.5 C
Total No. %
Isolates E.coli
28 79
20 65
-
15 80




isolated during storage

27.5 C
Total No. 7o
Isolates E.coli
64 89
35 91
• **
25 100




                                 - 65 -

-------
Studies on Depuration*induced Changes in the Composition of the Pacific
                      Oyster (Crassostrea gigas)
       6. Wedemeyer, J. R. Chung, B. J. Kemp, and A. M. Dollar
       (College of Fisheries, University of Washington, Seattle)
                                ABSTRACT

     Oysters are an important fisheries product in coastal areas of the
United States.  One of their principal market forms is as an iced product,
The bacterial flora of the intestinal contents cannot be removed and be-
comes part of the final product.  The number of fecal coliform bacteria
can be reduced by depuration and thus reduce the potential hazard.  This
latter procedure could affect the quality of the final product.  Since
glycogen is important in the commercial value of the oyster, any loss of
carbohydrate would be undesirable.  Thus, a preliminary study on the ef-
fect of depuration on the composition of oysters was undertaken.
                          MATERIALS AND METHODS

     Pacific oysters (Crassostrea gigas) of 7-10 cm, in length were ob-
tained from the depuration unit of the U.S.P.H.S. Shellfish Sanitation
Laboratory at Purdy, Washington.  These oysters had been held for 1/2
to 26 days, while control groups had been held under refrigeration at
4-5 C for 1/2 to 3 days.

     The samples were weighed into blendor jars and two volumes of water
added and then blended at high speed for 1-1/2 minutes.  This procedure
failed to disintegrate the oysters completely.  The procedure was modi-
fied, in that only one-half the water was added, the oysters blended for
1/2 min., and the balance, ±.£. one volume of water, was added and the
blending process continued for an additional 1 min.  One ml. aliquots
were pipetted into tared aluminum dishes and these dried at 105 C to
constant weight.

     The specific gravity of the homogenate was measured, using a pipet
in which the weight of a given volume of oyster homogenate was compared
to a given volume of distilled water at room temperature, approximately
20 C.

     Glycogen was determined, using essentially the method of Good and
Somogyi (1933), as modified by Dubois et al. (1956) and Montgomery(1957).
One ml. of the homogenate was added to 2 ml. of 30% KOH, held in a boil-
ing water bath, and the digestion continued until the samples cleared.
1.1 to 1.2 volumes of 95% ethanol was added to give the final concentra-
tion of 67-70%, in order to precipitate the glycogen.  The samples were
centrifuged and washed two times with additional volumes of 70% alcohol,
                                  - 66 -

-------
 centrifuging and decanting  the supernatant at each washing.  Glycogen
 was determined  colorimetrically, using the phenol-sulfuric acid method
 (Montgomery, 1957), and  the results compared to glycogen standards pre-
 pared  in a similar manner.  The results were calculated on the basis of
 dry matter.


                       RESULTS AND DISCUSSION

     The weight of the homogenate in grams/ml., as shown in Appendix,
 Table  1, reflects the dry matter present in the shellfish.  Those which
 contained a very low proportion of dry matter had a specific gravity of
 about  1.0.  As the dry matter increased, the specific gravity decreased
 within reasonable limits.  The normal oyster would appear to have a
 specific gravity of approximately 0.90 to 0.95.  The glycogen expressed
 as mg/g of dry matter reflected the holding conditions of the oysters.
The controls refrigerated for 1/2 day and those depurated for 1/2 day
had very low glycogen levels.   There is no explanation for these low
values, and the samples held 3 days returned to a normal value, possibly
 due to loss of moisture, as reflected in the dry matter.  Holding oysters
 in the depuration facility did not cause appreciable change in glycogen
 levels during 21 days, except in those oysters which were supplied with
a filtered water.   In these the dry matter declined sharply and the glyco-
 gen increased proportionally.   This change apparently reflected a simple
 increase in the amount of tissue water,  and if the dry matter were cor-
rected to 20%,  the glycogen levels were more consistent.  It is quite
evident that the initial condition of the oyster will vary and that this
condition will reflect the handling methods.
                                 -  67  -

-------
          Table 1.   Relationship of pre-conditioning of oysters to
                                 composition
Days of No.
treat- in
Treatment ment Sample
Controls re-

Weight of Dry
Sample Matter
2 R/100K


Glycoaen
mg/g dry 20% dry
matter basis*


frigerated at
4-5 C

Depurated



1/2 3
3 3
1/2 3
1 3
3 3
21 3
114
120
108
127
65
93
16.2
20.0
15.7
16.3
11.7
18.1
86
162
134
197
297
188
70
162
105
161
174
170
Weight
of homog-
enate g/ml


0.90
0.90
0.91
0.95
0.92
0.88
Starved
26
84
8.3
515
213
1.01
         *Adjusted to 20% dry matter basis,
                                      -.68  -

-------
    Studies on the Behavior of a Bacteriophage  in  the Pacific Oyster
                         (Crassostrea gigas)

                       J.  C. Hoff and W. J. Beck
                             INTRODUCTION

      That shellfish ingest  and  concentrate  bacteria present  in  the water
 during their feeding activities is well known.  The public health impli-
 cations of this  phenomenon  have been  extensively  studied.

      Certain groups of viruses  such as poliomyelitis, Coxsackie, ECHO,
 and infectious hepatitis virus  are present  in the human intestinal tract
 and therefore are possible  pollutants of shellfish growing waters.  It
 is  of interest to know whether  or not these very  small biological part-
 icles (approximately one-fiftieth the size  of Escherichia coli) are in-
 gested,  concentrated, and eliminated by shellfish in the same manner as
 bacteria and also to determine  their ability to survive in shellfish
 under various conditions.

      Because of  a lack of equipment necessary for animal virus work and
 because of the difficulties  in  quantitative analysis of heterogeneous
 mixtures for these  particles, a bacteriophage was used in these studies.
 The bacteriophage used, while larger than the enteroviruses  is much
 smaller than Escherichia coli.  A comparison of the relative sizes is
 given in Table 1.   The shellfish species used in  this study was the
 Pacific oyster (Crassostrea  gigas).


                        MATERIALS AND METHODS

 Bacteriophage.  The phage was isolated from sewage using E. coli C2 as
 the host bacterium.  Electron microscopy1 showed  that the phage was 390 mu
 long and 110 mu wide.  Initial  experiments  showed that the phage was
 stable  in sea water and was  inactivated by ultraviolet irradiation.

Oysters.  The oysters (Crassostrea gigas)  were collected in upper Burley
Lagoon.  Usually oysters 10-15  cm long were used.   Samples consisted of
 from 6  to  10 animals.

Media.  Media and methods described in APHA Recommended Procedures for
 the  Bacteriological Examination of Sea Water and Shellfish (Third Edition,
 1962) were used for determination of E.  .coli 02 in samples except that .the
         Electron microphotographs were made by Mr. Dale Birdsell,
          Dept. of Bacteriology and Public Health,  Washington State
          University, Pullman, Washington.
                                  - 69 -

-------
EC  test was conducted at 46 C rather than 44.5 C.  Media used for phage
production and assay were those described by Groman and Suzuki (1962).
Overlay agar consisted of 1.2% agar (Difco) in distilled water.

Blending procedure for phage assays.  The conventional blending procedure
caused destruction of phage.  A modified procedure in which the samples
were blended for 45 seconds at 8,000 RPM was used in preparing oyster
meats for phage analysis.

Phage assay method.  All samples were assayed in duplicate.  One ml of
the sample dilution to be assayed was placed in sterile cotton plugged
tubes(13mm x 100mm) held in a water bath at 45 C.  One ml of overlay
agar, previously melted and cooled to 45 C was added.  Then 0.1 ml of a
5-6 hour broth culture of E. coli C2 was added and the tubes were shaken
briefly in the water bath.  The contents of the tubes were then poured
over the surface of freshly prepared phage assay agar plates and the
plates were rotated to spread the overlay.  After the overlay had hardened,
the plates were inverted and incubated at 28 C for 48 hours.  Plaques were
counted using a Quebec colony counter.

Accumulation-elimination experiments.  The procedures were similar to those
used previously in bacterial accumulation-elimination experiments (Eresnell,
1963).   Water flow usually was adjusted to 9.6 liters/hr/oyster.  In some
of the experiments the flow rate/animal was reduced.
                                 RESULTS

Development of blending process.  The deleterious effect of the blending
process on recovery of phage from blended suspensions is shown in Table 2.
The results indicate that the loss resulted from the blending process it-
self since blended meats shaken with phage suspension showed no appreciable
loss even after 30 minutes incubation at room temperature.  However, when
blending was used, either with or without oyster meats, losses of approxi-
mately 50 percent occurred.

     In the experiments shown in Tables 3 and 4 sterile 0.1% peptone water
was substituted for oyster meats.  The effects of various blenders and
blending times are shown in Table 2.  Losses became progressively greater
with extended blending time.  Losses were smaller in Waring blendors than
in the Oster blenders but in all cases definite losses occurred.

     In subsequent experiments a rheostat was used to control blending
speeds.  Blender speeds at various rheostat settings were calibrated with
an odometer.  The results of varying blending speeds and times are shown
in Table 4.  In the Oster blender no losses occurred after 45 seconds at
any of the three speeds but definite losses occurred after 90 and 180
seconds blending time.  In the Waring blendor recovery was good at all
                                 - 70 -

-------
 three speeds after 90 seconds blending time.  The results of the same
 type of experiment using oyster meats in place of 0.1% peptone water
 are shown in Table 5.  It was concluded that blending for 45-60 seconds
 at 8,000 RPM would give satisfactory phage recovery.  OBter blenders
 with baffles produced more homogeneous preparations than Waring blenders
 at this speed because of more violent action.

 Accumulation-elimination experiments.  The results of the initial accumu-
 lation-elimination experiment are shown in Table 6.  The phage concentra-
 tion was higher in the oyster shell liquor than in the oyster meats after
 42 hours of pollution.  The presence of feces in the tank and the presence
 of phage in the shell liquor indicated that the oysters had been pumping
 during this period.  This experiment was repeated with results similar to
 those shown in Table 6.  In one of the repeated experiments, oyster feces
 and pseudofeces were examined for phage content.  The results, shown in
 Table 7, indicate that phage particles were not concentrated cither in
 feces or pseudofeces.

     During the initial experiment water samples were collected and centri-
 fuged at 3000 RPM for 40 minutes to determine whether the phages were freely
 suspended in the water or were attached to larger particulate matter which
 would be sedimented at this speed.  The results of these analyses are shown
 in Table 8.  No difference in phage concentration in supernatant and sedi-
 ment fractions was found, indicating that the phages were not attached to
 larger particles.  The centrifugation used is sufficient to sediment E. coli
 from culture media.  When 20 ml samples of the water were filtered, no
 plaques were found.  However, when previously filtered sea water was contami-
 nated with phage and refiltered the phage was also retained by the filter.
 This retention was evidently due to physical factors rather than to attach-
 ment of the phage to larger particulate matter.

     In further attempts to determine whether or not the oysters were filter-
 ing our phage particles, experiments employing a large number of animals
 held in tanks through which phage contaminated water was flowing at a low
 rate were designed.  It was assumed that if the oysters were actively feed-
 ing and ingesting the phages the effluent water from the tank should con-
 tain lower concentrations of phage than the influent water.  The results
 of several of these experiments are shown in Table 9.   Consistent reduc-
 tions in phage concentration in effluent samples compared with influent
 samples were not found.  In experiments 3 and 4, oysters in a control flat
 were exposed under similar flow rate conditions to water polluted with
 E  coli.  In experiment 3, the last three samplings show considerable dif-
ference* between influent and effluent E. coli concentrations.  In both
 experiments 3 and 4, E. coli was present in similar concentrations in both
 oyster meats and shell liquor, while no phage was found in the meats of
 the phage polluted oysters.

     In several of the experiments, the oysters were allowed to pump in
 phage-free flowing sea water following the accumulation phase.  The re*
 sults of three of these experiments are shown in Table 10.  The oysters
 became free of detectable phage in every case by the time the first sample
 was taken.


                                 - 71 -

-------
      In several  instances oysters which had been exposed  to phage pollution
were taken  from  the water at  the end of the polluting period and stored dry
at  5 C.  Table 10  shows  the effect of storage at this temperature on survival
of  the  phage  in  oyster meats  and shell liquor.  The results indicate that
phage was quite  stable in both the meats and shell liquor at this tempera-
ture for at least  12 days.


                                DISCUSSION

      The results of the  initial accumulation experiments were not similar
to  results previously obtained with oysters using E. coli as a pollutant
(Kelly  et al, 1960), (Kelly,  1961).  However, it was possible that the
phage particles were being ingested and subsequently inactivated or held
by  some means that made  them  undetectable.  The mucous of the oyster could
possibly physically bind the  particles or chemically inactivate them since
many virus inhibitors are mucins or mucoproteins (Luria, 1953).

      The series of experiments employing high oyster/water flow ratios were
set  up to test this possibility.  However, the results of these studies are
regarded as inconclusive because EC MPN's of effluent and influent water
samples  from the control tank polluted with E. coli did not differ consist-
ently.  Oyster meats did contain higher concentrations of E. coli than
phage but again this may have been because the phage was held so as to be
undetectable.  The reason for the failure of the oysters to accumulate
E. coli concentrations higher than those of the surrounding water is not
known.  It is possible that lack of oxygen or food because of the low flow
rates caused the oysters to be inactive.

      The results of Hedstrom and Lycke (1964), using poliovirus as a pollut-
ant  in a nonflowing system were similar to those found in this study.  Shell
liquor contained virus concentrations similar to that of the surrounding
water while gill and mantle tissue and the remainder of the oyster body us-
ually contained much lower concentrations.

      Depuration of the phage-infected oysters proceeded rapidly with no
phage being detectable after 18 hours.   The results of Hedstrom and Lycke
(1964) differed from this.   They found that oysters transferred from in-
fected to uninfected water showed little change in virus concentration
after 24 hr. and that oysters transferred five times over a period of 100
hours stij.1 contained poliovirus.   They concluded that the virus was
associated with the oysters in such a way that it was not removed during
these transfers.

     The phage was stable in contaminated oyster shellstock stored at 5 C.
Similarly,  Hedstrom and Lycke (1964), found that poliovirus was stable
for at least 56 hours in either 2 percent or 50 percent oyster tissue
homogenate stored at 23 C.

      It appears that neither phages nor poliovirus freely suspended in
water are accumulated and concentrated to the same extent as E. coli.
However, it is possible that they may be attached to larger particles and
ingested in this way.   The stability of these particles in oyster meats


                                 - 72 -

-------
and shell liquor is of considerable importance from the public health stand-
point.  This is enhanced by dosage response curve data which indicate that
one virus particle is sufficient to initiate infection (Luria, 1953).  On
the other hand, with the exception of several outbreaks of infectious hepa-
titis (Roos, 1956} Mason and McLean, 1962), oysters have not been implicated
in epidemics of other viral diseases.

     The experiments employing large oyster/water flow rate conditions will
be continued.  Attempts will be made to improve conditions, e. g., oxygen
supply, so that the animals will feed more actively.  Oyster meats will be
washed in phage-free water prior to examination in attempts to determine
whether the phage is closely associated with the oyster meats or is found
in the meats because of incomplete separation of meats and shell liquor.


                       COMMENTS BY PARTICIPANTS

     Dr. Liston opened the discussion by commenting on the absorption of
animal viruses as compared to phages.  Mr. Hill and Dr. Hoff discussed
the relative removal of E. coli and the phage used in the study.  Dr. Dollar
and Mr. Hill discussed methodology of isolation of phage.  Dr. Dollar sug-
gested the use of EDTA in connection with filters used.  Dr. Quayle commented
on the low level of accumulation of bacteria by the oysters in the latter
part of the study.  A brief discussion was held between Mr. Kelly and
Dr. Liston on the use of other viruses as a test organism.
                                -73  -

-------
                               REFERENCES

American Public Health Association.  1962.  Recommended procedures for the
      bacteriological examination of  sea water and shellfish, 3rd ed.

Groman, N. B. and G. Suzuki  1962.  Temperature and  lambda phage reproduc-
      tion.  J. Bacteriol. 84: 431-437.

Hedstrom, C. E. and E. Lycke, 1964.  An experimental study on oysters as
      virus carriers.  Am. J. Hyg. 79; 134-142.

Kelly, C. B., 1961.  Accumulation of bacteria by the Pacific and Olympia
      oysters.  (Paper presented at Shellfish Sanit. Res. Conf., Purdy,
     Wash.).

Kelly, C. B., W. Arcisz, and M. W. Presnell. 1960.  Bacterial accumula-
      tion by the oyster, Crassostrea virginica, on  the Gulf Coast.
      Robert A. Taft Sanit. Eng. Center Tech. Rept. F60-4.

Luria, S. E. 1953.  General virology.  John Wiley and Sons, Inc., New York.

Mason, J. 0. and W. R. McLean 1962.  Infectious hepatitis traced to consump-
      tion of raw oysters.  An epidemiological study.  Am. J. Hyg. 75: 90-111,

Presnell, M. W., J. C. Hoff, and W. J. Beck 1963.  Accumulation and elimina-
      tion of bacteria by the Manila clam (Tapes laponica) and the Native
     Littleneck clam (Protothaca staminea).  (Paper presented at Shellfish
      Sanit. Res. Conf., Purdy, Wash.

Roos, B. 1956.  Hepatitis epidemic conveyed by oysters.  Svensk. Lakantidn.
      53: 989-1003.
                                  - 74 -

-------
     Table  1.  Comparative sizes of JS. coll bacteriophage C2 and
                        some enteric viruses
Microorganism
                   SizefMilllmicrons)
Escherlchia coli


Bacteriophage C2


Poliomyelitis virus

Coxsackie

ECHO virus
width
length

width (at head)
length (overall)
  400 -   700
1,000 - 4,000

          110
          390

      27 - 30

      27 - 30

      20 - 90
                             ******
Table 2.  Effect of blending process on recovery of phage from oyster
                                meats
              Treatment
                        Plaque Counts
                        7oLoss or Gain)a
                      Observer  Observer
None (control)

Meats blended then mixed with phage
    suspension and chaken

Above preparation allowed to stand at room
    temperature for 30 minutes, then sampled
    again

Meats blended with phage suspension 90 seconds
    at full speed (15,600RPM) in Oster blender

Same as above except 0.1% peptone water used
    in place of oyster meats
                          1         2
                   295 plaques  286 plaques
                        + 4%
              n
- 6%
-62%
-40%
- 1%
-58%
-39%
       aControl equals 100%.
                               - 75 -

-------
Table 3.  Effects of various blenders and blending times on
      recovery of phage suspended in 0.17o peptone water
Blending Time
Type of Blender Speed(RPM) (Seconds)
None (control)
Pint Oster Blender 15,600

Large Glass
Waring Blendor 15,200

Small Glass
Waring Blendor 15,200

Aluminum Waring
Blendor 15,200


45
90
45
90
45
90
45
90
Plaque Counts
(% Loss or Gain)a
189 plaques
-28%
-70%
-16%
-33%
-17%
-47%
- 7%
-25%
       Control equals 100%.
                              -  76 -

-------
Table 4.  Effects of blending speed and time on recovery of phage suspended
                           in 0.1% peptone water
                    Speed     Blending Time
                                (Seconds)
  Plaque Counts
(%  Loss or Gain)  a
  Observer  Observer
     1         2
None (Control)
Pint Oster Blender 5,600


8,000


12,000


None (Control)
Large Glass
Waring Blendor 5,600

7,500


11,500



45
90
180
45
90
180
45
90
180

45
90
180
45
90
180
45
90
180
174 plaques
+ 5%
-17%
-20%
- 5%
-19%
-20%
+ 2%
-26%
-37%
173 plaques
+21%
+ 9%
+16%
-
-
.
-
-
-
-
-
-
-
.
-
-
_
-
-
171 plaques
+18%
+ 9%
+16%
+16%
+19%
+12%
+ 8%
+ 5%
-11%
           aControl equals  100%,
                                 -  77 -

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Table 5.  Effect oi' blending speed and time on recovery of phage from
                               oyster meats
Plaque Counts
(% Loss or Gain) a
Speed
Type of Blender (RPM)
None (Control)
Half Gallon
Oster Blender 12,000


8,000


Large Glass
Waring Blendor 11,500


7,500


Blending Time
(Seconds)


45
90
180
45
90
180

45
90
180
45
90

Observer
1
110 plaques

+ 13%
-
-
+ 8%
- n
-23%

- 7%
- 9%
-
+ 5%
-15%
-22%
Observer
2
100 plaques

+ 15%
-
-
+ 5%
- 2%
- 20%

- 9%
- 6%
-
+ 6%
+ 11%
- 24%
       Control equals 100%.
                                 - 78 -

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Table 6.  Accumulation and elimination of bacteriophage by Pacific oysters
                       Pollution
                       Aquarium
                                      Plaque Counts/ml  or  g
                                            Flat A
Flat C
                                             Oyster  Oyster      (Control)
Date     Time     Start    Finish   Water   Meats    Liquor  Water  Oysters
3-25-64    2200
           2215    100,000
           2230
                                      50
3-26-64    0900
           0930                        780
           1500              96,000    680       80a
           1530    120,000

3-27-64    1030
           1100                        520
           1500              89,000    430
           1600 -  Pollution stopped             15

3-29-64    1030                          °        0
                                                         190
                                                                        0
                                                                         a
          a
           Liquor not separated from meats.
                                     *****
         Table 7.   Phage concentration  in  oyster  feces and pseudofeces
Duration of Pollution
(days) 	
1
2

Water
460
640
Oyster
Feces
520
740
Oyster
Pseudofeces
620
780
                                    - 79  -

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Table 8.  Effects of centrifugation and filtration on bacteriophage content
                                of sea water
Plaque Count /ml
No
Sample Treatment
Centrifuged3
Supernate Sediment
20 ml
Millipore
Filteredb
Sea Water - Flat A               780
     3-26-64

Sea Water - Flat A
     3-27-64                     520

Phage diluted in Millipore
    filtered sea water         1,000
710
540
780
560
0



0


0
       a
        3,000 RPM for 40 min.

       'filter grids HA 0.45 u
                                     .  80  .

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Table 9.  Accumulation of bacteriophage and Escherichia coli by Pacific oysters under high oyster/water flow

Water
Flow
Exp. No. of Rate
No. Oysters (ml/min.)
1 22 450



2 38 80


3 30 250
(Starved)3



4 33 250



ratio conditions
Duration
of
Pollution
(hours)
6
23
30
47
3
19
20
23
26
44
49
68
14
18
23
35
Phage plaques /ml or R
Water Oysters
Influent
850
550
690
1,300
680
590
610
120
85
87
116
81
126
130
93
111
Effluent Meat Liquor
970
540
730
1,300 35 490
760
530
390 15 290
113
92
122
81
97 0
127
142
101
125 0 33
Escherichia coli MPN/ml or g
Water
Influent







7.9
13
1,300
3,300
330
790
1,300
790
3,300
Effluent







33
33
350
1,300
130
1,300
1,300
1,100
1,700
Oysters
Meat Liquor











230 79



330 490
         a
          Oysters held at flow rate of 250 ml/min for approximately 40 days  prior  to  experiment.

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          Table 10.  Elimination of bacteriophage by Pacific oysters
Depuration
Time (hours)
0
18
0
18
0
26

Water
430
640
111
Phage plaques/ml
Oyster
Heats
15
0
35
0
0
0
or g
Oyster
Liquor
190
0
490
0
33
0
Table 11.  Effect of dry storage at 5 C on bacteriophage concentration  in Pacific
                                 oyster shellstock
Storage
Time (days)
0
2
0
2
5
11
0
11
0
12

Water
430
-
640
-
-
-
Ill
-
300
-
Phage plaques /ml
Oyster
Meats
15
15
35
40
16
6
0
0
3
30
or R
Oyster
Liquor
190
380
490
620
380
300
33
25
220
110
                                       - 82 -

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PART II  PROPOSED RESEARCH ACTIVITIES FOR




           FISCAL YEAR 1965
               - 83 -

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              PROPOSED RESEARCH ACTIVITIES FOR FY  1965

                            W. J. Beck
     The  following proposals were presented at the 1964 conference.  An
outline of  each proposal was presented.  Participants discussed each pro-
posal and offered many excellent suggestions that augmented each proposal.
In addition to formal presentations, several proposals were made from the
floor.

                       OVERALL PLAN FOR 1965

     A brief discussion of previous research, continued research, and
projected research was rendered.  Certain studies have now been completed.
Among these are:  1) storage studies on shucked Pacific oysters; 2) storage
studies on  Pacific oyster and clam shellstock; 3) laboratory phase of
accumulation-elimination of bacteria by oysters and clams and 4) phase 2
of the ecological study.  All of these studies have either been published,
or will be  published within the very near future.

     Studies to be continued through FY 1965 include: 1) accumulation and
elimination of viruses by shellfish; 2) the investigation of oceanography
as applied  to shellfish growing areas.

     New  studies will include:  1) botulism; 2) pilot plant studies on
depuration; 3) antimicrobial agents and 4) interrelationship of commercial
organisms to indicators plus possibly pathogens.

     Thus there has been a certain amount of change in the emphasis placed
on the proposed research program of the Northwest Shellfish Sanitation
Laboratory  for FY 1965.
                        COMMENTS BY PARTICIPANTS

     Mr. Girard asked about former proposals that seemed to have been lost.
He was especially interested in research on wet storage.  Mr. Beck stated
that this study had only been tabled and would be made a part of the FY 1965
program.

     Mr. Foster commented on the need for an evaluation of the relationship
of coliform and fecal coliform in the environment.  Mr. Beck suggested that
part of the question would be forthcoming with the publication of the eco-
logical study.  Other background material may be found in recent publica-
tions.
                                  - 84 -

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     Mr. Bowers described the renewed interest in Olympia oysters.  The
Olympia Oyster Growers Association has indicated a desire for storage
studies similar to that performed on Pacific oysters and clams.  Mr. Beck
stated that the Northwest Shellfish Sanitation Laboratory would be most
happy to cooperate with the Olympia Oyster Growers Association in this
study.


                                PROPOSAL

     Amimulation and elimination of enteroviruses in West Coast shellfish

     The bactericidal efficiency of ultraviolet irradiation for the treat-
ment of sea water used in shellfish depuration has been demonstrated.  That
ultraviolet irradiation inactivates viruses in nonabsorbent media such as
water and salt solutions has also been shown.   However, particulate matter
and other organic compounds found in natural sea water may exert a protective
screening effect.  Therefore, the viricidal efficiency of U. V. irradiation
for sea water under various experimental and natural conditions of turbidity
and color should be determined.

     A small scale U. V. treatment unit will be designed using the principles
incorporated in the "Purdy unit".  Water flow and U. V. intensity will be
variable.  The use of one representative each of the polio, ECHO, and Cox-
sackie virus groups and Escherichia coli bacteriophage C2 is anticipated.
If the assay procedure permits, the pollutant pool will consist of equal
concentrations (plaque forming units) of each virus.  The contaminated sea
water will contain approximately 1000 PFU/ml of each virus.  Assays will be
done by the plaque method.

     After the amount of U. V. irradiation necessary for the destruction of
viruses in deionized water at various flow rates has been determined, similar
determinations will be made using sea water.  The effects of both natural
turbidity and turbidity produced by the addition of diatomaceous earth on
lethality will be determined.

     Studies will be continued on the accumulation, survival and elimina-
tion of viruses in shellfish.  The present studies using a bacteriophage
will be extended wherein the Sabin poliovirus will be used as the test
virus to determine rates of accumulation, survival and elimination.  After
the proper cell lines have been established, assays for the Sabin poliovirus
will be done by the plaque technic.

     Pollution of a similar system with E. coli will be used as a control.
Thus some indication of the activity of the test shellfish can be determined
at the time of the accumulation-elimination of viruses experiments.
                                  - 85 -

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                     COMMENTS BY PARTICIPANTS
     Dr. Bartsch and Mr. Girard requested information on how the test virus
would be removed from the salt water system before discharge into the estu-
ary.  Mr. Beck explained the system of holding tanks and check samples that
would be used to insure the safety of the salt water discharge.

     Drs. Listen, Dollar and Hoff discussed the ramifications of the attach-
ment of viruses to certain particle sizes.  Dr. Berquist suggested the use
of fluorescent microscopy in addition to the other methods for detection of
certain virus.

     Drs. Sparks, Listen and Hoff discussed the reasons why oyster tissues
may or may not be applicable to the research in virology.

     Mr. Kelly summed up the reasons for the need of an intensified study
in the relationship of viruses to shellfish.
                                PROPOSAL

            Survival, outgrowth and toxin production from Gram-positive
                           spore-forming bacteria
     Although in recent years fish products from fresh and salt water
sources have been implicated in outbreaks of botulism caused by Type E
Clostridium botulinum. shellfish have not been associated in any way.
Perhaps in the past the length of time shellfish have been held during
and after processing may have eliminated the danger of toxin production.
However, several factors would seem to make shellfish products a suitable
vehicle for the production of this toxin.  Among the factors are:  a) Type
E C. botulinum has been reported to be endemic in certain marine environ-
ments in the Pacific Northwest; b) with the combination of the feeding
process of shellfish plus the viability of the microorganisms after harvest-
ing potentially hazardous conditions could be established.  It is, there-
fore, proposed that the following investigation be undertaken.

      Attempts will be made to determine survival, outgrowth and toxin pro-
duction of C. botulinum. Type E, in shellfish and other marine fauna from
simulated commercial practices such as harvesting, cold processing, partial
heat processing and frozen storage.  Thus the factors needed to induce sur-
vival, outgrowth and toxin production of Type E C. botulinum in shellfish
may be determined.

     The competence of personnel at this station in working with C. botulinum
has not been ascertained.  In addition, the many ramifications of isolation
and detection from shellfish and related biota must be predetermined.  There-
fore, the initial stage of this research will consist of adding a known spore
suspension of Type E 
-------
     Spores will be added at the rate of 1,000, 100 and 10 per gram of
shucked combined oyster meats and liquors.  Each lot of oysters will be
divided in 17 aliquots of 12 oz. each, placed in commercial plastic con-
tainers and sealed.  The containers will be divided into 4 lots and stored
at -5 C, 3 C, 10 C and 20 C respectively.  Each storage temperature will
be sampled at periodic intervals according to data obtained from previous
experiments on storage of shucked Pacific oysters.

     Each aliquot tested will be homogenized in an evacuated blendor.  The
homogenate will be prepared as outlined in Examination of Foods for Entero-
pathogenic and Indicator Bacteria (PHS Pub. No. 1142) for isolation of
spores and identification of toxin.

     As technics are developed, certain modifications may aid in speeding
the final results.  In addition, as proficiency is increased, studies will
be initiated for the presence or absence of toxin-producing Gram-positive
spore-forming bacteria in terrestrial and marine environments.
                       COMMENTS BY PARTICIPANTS
     Messrs. Bowers, Foster and Beck commented on the relationship of this
research to industry.  It was agreed that emphasis should be placed on the
fact that no problem was present in the shellfish industry.  This type of
research would be used for preventive measures only.  Mr. Beck explained
the system of reporting that would be in effect.

     Mr. Kelly questioned why oysters would be used as the initial animal
in place of clams.  The reason was only one of expediency in developing
technics.  It is hoped that clams would be included during the present
study.

     Dr. Craig explained the type of research that was being performed by
his group and other interested researchers on the West Coast.  Mr. Beck
requested comments on the level of spores to be used.  Dr. Craig considered
these levels somewhat high.  He suggested a level of 3 to 5 spores per gram
of meat as being adequate.  Dr. Craig also indicated that he had found ex-
cellent cooperation with all facets of industry in his studies.
                                - 87 -

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                              PROPOSAL

                       Enteropathogenic E. coll

     Studies will be continued on the relative significance of entero-
pathogenic E. coll as indicators of pollution.  Screening tests using
commercially available fluorescent antiserum will be made at regular
intervals on E. coli isolated from sewage effluents, polluted soils,
animal  fecal material and streams tributary to a shellfish growing
area.   Final identification will  be made by classical serological
methods.

                       COMMENTS BY PARTICIPANTS

     Dr. Berquist remarked on the inadequacies of present commercial
antisera*  Mr. Kelly suggested that perhaps a cooperative effort was
needed  to determine availability of antisera.  Mr. Girard questioned
why this study was limited to a shellfish growing area.  Mr. Kelly
noted that these organisms would only be used as a research tool.
Mr. Michener questioned whether or not they would be found in sufficient
numbers to be significant.  Mr. Beck explained the general outline as
suggested by Dr. Hosty in the State of Alabama.  The general agreement
was that a fairly low priority should be given this project.
                              PROPOSAL

                          Oceanographic methods

     Methods developed by the physical oceanographer in FY64 will be
continued on a seasonal basis to study current patterns, displacement,
retention and other oceanographic factors related to Burley Lagoon.
The data will be gathered by personnel at this station and analyzed
through the cooperation of the physical oceanographer at the Northeast
Shellfish Sanitation Research Center, Kingston, Rhode Island.  Thus an
investigation incorporating seasonal variations will be completed.

                       COMMENTS BY PARTICIPANTS

     Dr. Quayle questioned the use of personnel that were not trained
as oceanographers.  He suggested the possibility of using a student
oceanographer or working with other agencies in order that the proper
interpretation be placed on the data gathered.

     Mr. Foster suggested the use of tagged silt to cover flows etc.
Mr. Girard objected to certain of these items in a commercial shellfish
growing area.  Mr. Kelly commented on the need to continue the study in
order that the entire study on Burley Lagoon as a prototype could be
completed.  There is a need to correlate microbiology with oceanography.
Mr. Beck was directed to contact the University of Washington to explore
possibilities of assistance in this program.


                                 . 88-

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                              PROPOSAL

                       Antimicrobial agents

     Much interest in recent years has been given to certain antimicrobial
agents that may be found in shellfish and other marine biota.  With the
introduction of cell cultures in virology, methods will be available to
determine whether or not these antimicrobial agents are present in western
species of shellfish.  The extraction method of Prescott & Li will be used
for purification of the antimicrobial agents.  These extracts will be tested
against both viruses and bacteria.
                      COMMENTS BY PARTICIPANTS

     Dr. Dollar suggested several possibilities of technics available for
separating materials according to molecular size rather than the simpler
method of dialysis.  Mr. Kelly commented on projects at other research
centers and the increased interest shown in recent years.
                               PROPOSAL
                    Pilot Plant study on depuration

     In the past, research in depuration at this station has been maintained
at the laboratory level.  Interest of certain areas in this country has in-
creased rapidly during the past several years to where the process must now
be evaluated at a pilot plant level.  Among the questions still to be answered
are:   loading capacity of tanks; design of equipment for cleanability, prac-
ticality and economic feasibility; the fate of certain indicator organisms
during the depuration process as related to stacking, moving and water flow
through the tanks; and whether or not recirculation of sea water may be used.
In addition, as technics and laboratory scale depuration operations are com-
pleted in relationship to virology, these microorganisms will have to follow
the research in bacteriology.

     It is therefore proposed that a cooperative effort between a state
agency, industry and the research center be initiated to investigate depura-
tion on a commercial scale.
                      COMMENTS BY PARTICIPANTS

     Dr. Sparks and Mr. Gruble discussed the part that industry might play
in such a proposed study.  Mr. Gruble felt that industry would look ahead
and cooperate to make shellfish available for the study.  Mr.  Beck explained
that the problem was one of disposal of the product rather than construction
of the pilot plant.

     Mr. Girard suggested that the potential problem would be  one of clams
rather than oysters and that clams should have priority over oysters.

-------
     Mr. Stonehouse commented on the problem in British Columbia.  He
then introduced Mr. Timothy,an oysterman from the British Columbia area.
Mr. Timothy commented on the situation from the viewpoint of a grower.

     Mr. Beck again asked whether or not industry, state agencies and
the Northwest Shellfish Sanitation Laboratory could find a common purpose
in planning such a study.  Mr. Gruble stated that industry would look
favorably upon the program if the PHS would initiate action.

     Mr. Kelly outlined what the general concept-as to size and objective
of the pilot plant would be.  Mr. Gruble's only concern was that of sub-
stituting depuration for the continued vigilance of protecting approved
growing areas.

     Mr. Kelly commented that we could not regard depuration for pollution
abatement.  Depuration would have to be regarded as a means of additional
protection of the product.

     Mr. Glude suggested that the study proceed as rapidly as possible.
Ideally, specifications for an approved type of plant for industry use
should be developed immediately.

     Dr. Dollar and Mr. Kelly discussed the potentiality of using radio-
active source as a substitute for ultraviolet treatment of the sea water.
It was suggested that Mr. Beck contact Dr. Dollar in the very near future
to discuss the feasibility of this idea.

     Mr. Kelly concluded the discussion by commenting on various projects
on the East Coast.

     As there were no further comments on this or other proposed projects,
Mr. Beck thanked all the participants for their comments and lively partici-
pation in the entire conference.
                                -  90 -

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APPENDIX
-  91 -

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                                   AGENDA

              Annual Shellfish Sanitation Planning Conference
                          September 14-15, 1964


MONDAY AFTERNOON  Sam Reed, Moderator

   1:00 - 1:30    Opening comment on conference - Dr.  Bernard Bucove

   1:30 - 2:15    Storage studies on shucked Pacific oysters  - J. C. Hoff

   2:15 - 3:00    Effects of antifearning agents and evacuation on
                      bacteriological technics - T. H.  Bricksen

   3:00 - 3:15    COFFEE BREAK

   3:15 - 4:00    Activities at other PHS research centers  -  C. B. Kelly

   4:00 - 4:45    Oceanography report on Burley Lagoon - W. J. Beck


TUESDAY MORNING   Dr.  John Listen,  Moderator

   9:00 - 9:45    Storage studies on Pacific oyster shellstock - J. C. Hoff

   9:45 -10:30    Storage studies on hard-shell clam shellstock -
                                                              T. H. Ericksen

  10:30 -10:45    COFFEE BREAK

  10:45 -11:30    Accumulation and elimination of bacteriophage in Pacific
                      oysters - J.  C.  Hoff

  11:30 - 12:30   Proposed activities for 1965 - W.  J.  Beck

  12:30 -  1:30   NO HOST LUNCH


TUESDAY AFTERNOON C. B.  Kelly,  Moderator

   1:30 -  4:30   Proposed activities for 1965 - W.  J.  Beck
                                     - 92 -

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                         ATTENDANCE ROSTER
Federal Agencies

A. F. Bartsch
P. N. Bardal
W. J. Beck
S. S. Copp
Geo. Dixon Lt. Col.
T. H. Ericksen, Jr.
W. A. Felsing, Jr.
John Glude
R. W. Hill
J. C. Hoff
L. S. Houser
W. Jakubowski
C. B. Kelly
F. S. Kent
W. G. Kupp
L. C. Myers
R. W. Nelson

N. Neufeld
H. M. Risley
R. Speyrer 1st Lt.
G. J. Vasconcelos
D. C., Zeiter 1st Lt,

Industry

Bob Bower
Earl Brenner
Malcolm Edwards
Edward J. Gruble
Edward A. Timothy
Nat Waldrip

State Agencies

K. R. Berquist
T. P. Blair
Bernard Bucove
H. B. Foster, Jr.
John Gerth
John Girard
Clarence V.  Hall
Max G. Hays
Evelyn MacDonald
K. L. Michener
USPHS WS&PC
Dept. of National Health
NW Shellfish Sanitation Lab.
Dept. of National Health
USA Veterinary Corps
NW Shellfish Sanitation Lab.
USPHS Region IX
Bureau of Comm. Fisheries
Food & Drug Directorate
NW Shellfish Sanitation Lab.
USDHEW PHS Shellfish Branch
NW Shellfish Sanitation Lab.
USDHEW PHS Shellfish Branch
USPHS Region IX
U. S. Food & Drug Admin.
NW Shellfish Sanitation Lab.
Bureau of Comm. Fisheries
    Technical Laboratory
Dept. of Fisheries
U. S. Food & Drug Admin.
USA Veterinary Corps
NW Shellfish Sanitation Lab.
USA Veterinary Corps
Ellison Bros. Oyster Co.
J. J. Brenner Oyster Co.
Coast Oyster Company
Hilton Seafoods Co., Inc.
                              Portland, Oregon
                              Vancouver, B. C.
                              Purdy, Washington
                              Vancouver, B. C.
                              Ft. Lewis, Wash.
                              Purdy, Washington
                              San Francisco, Calif.
                              Seattle, Washington
                              Vancouver, B. C.
                              Purdy, Washington
                              Washington, D. C.
                              Purdy, Washington
                              Washington, D. C.
                              San Francisco, Calif.
                              Seattle, Washington
                              Purdy, Washington
                              Seattle, Washington

                              Vancouver, B. C.
                              Seattle, Washington
                              Ft. Lewis, Wash.
                              Purdy, Washington
                              Ft. Lewis, Wash.
                              Olympia, Washington
                              Olympia, Washington
                              So. Bend, Washington
                              Seattle, Washington
                              Ladysmith, B.  C.
Pacific Coast Oyster Growers  Shelton, Washington
Washington State Health Dept. Seattle,
Oregon State Health Dept.     Portland
Washington State Health Dept* Olympia,
California State Health Dept. Berkeley
Washington State Health Dept. Olympia,
Washington State Health Dept. Olympia,
Washington State Health Dept. Seattle,
Washington State Health Dept. Olympia,
Washington State Health Dept. Seattle,
Oregon State Health Dept.     Portland
                                      Washington
                                       Oregon
                                      Washington
                                      , Calif.
                                      Washington
                                      Washington
                                      Washington
                                      Washington
                                      Washington
                                       Oregon
                                  -  93 -

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State Agencies (Cont'd.)
Alfred T. Neale
Sam Reed
V. C. Reierson
C. R. Stonehouse
R. West ley

Universities

James M. Craig
A. M. Dollar
Gary Houghtby
John Liston
Jack Matches
B. M. Slabyj
A. K. Sparks
Wash. Pollution Control Comm.
Washington State Health Dept.
Oregon State Health Dept.
Health Branch, Govt. of B. C.
State Shellfish Laboratory
Oregon State University
College of Fisheries, U of W.
College of Fisheries, U of W.
College of Fisheries, U of W.
College of Fisheries, U of W.
College of Fisheries, U of W.
College of Fisheries, U of W.
Olympia, Washington
Olympia, Washington
Portland, Oregon
Victoria, B: C.
Brinnon, Washington
Corvallis, Oregon
Seattle, Washington
Seattle, Washington
Seattle, Washington
Seattle, Washington
Seattle, Washington
Seattle, Washington
                                -  94 -
                                           * U.S. GOVERNMENT ralNTIM OMICI: IMS O—7*1-107

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