Re: how to measure TMA in fish tissue by Dyer method

From: Ted Labuza (tplabuza@umn.edu)
Date: Sat Oct 06 2007 - 14:18:58 PDT

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    Sorry Peter

    In a response to a question you stated:
    Dear Shuming

    You are right; do not try to dry the picric acid. I still have a copy of the
    procedure as used at Torry Research Station and the section on preparation
    of reagents states:

    "Approximately 2% picric acid stock solution. Picric acid is normally stored
    under water and is unstable when dry. Remove some crystals from the bottle
    and dry as much as possible and dry as much as possible by pressing between
    filter paper. Weigh out 3g of the damp crystals and add to 100ml of toluene.
    Shake to dissolve."

    The weight of damp crystals to take agrees more or less with that
    recommended by Wekell, and the concentration of picric acid is not critical
    anyway. Be careful how you dispose of any unused crystals and the filter
    paper; down the sink with plenty of water

    This is not acceptable.

    In the US , at least in Universities, such as U Mass where Shuming is at,
    the sink is not a disposal route for
    any hazardous material. All procedures with such materials need prior
    approvals, they might accept this but it needs to be approved.
     Perhaps the industry is more lax but we don't train our students
    to accept that. Worse cases with hazardous microbes, see below;

    The Chronicle of Higher Education Wednesday, September 5, 2007

    Texas A&M Violated Numerous Biosafety Rules, Federal Agency Says

    By JEFFREY BRAINARD <mailto:jeffrey.brainard@chronicle.com>

    Texas A&M University at College Station violated a dozen safety and security
    rules for research on dangerous microbes, federal officials say. The
    government says it will continue its two-month-old suspension of such
    studies at the university until the problems are fixed.

    The findings came in a letter
    <http://chronicle.com/weekly/documents/v54/i02/2007_09_04_09_56_50.pdf> to
    the university on Friday from the federal Centers for Disease Control and
    Prevention, which regulates research on microbes and toxins that could be
    used in biological attacks. The agency's continuing suspension of Texas A&M
    has struck a blow at the university's goal of becoming a national leader in
    research to counteract such attacks.

    The violations alleged by the CDC include the university's inability to
    account for at least three vials of microbes, which the CDC described as
    "missing." In addition, one researcher was working to develop
    antibiotic-resistant strains of a regulated bacterium even though he had not
    received the CDC's permission.

    What's more, laboratory workers did not don proper laboratory clothing or
    face masks to prevent their becoming infected and sometimes wore the
    clothing outside of the lab before it was decontaminated. At least seven
    times, workers were given access to regulated microbes before they gained
    permission under CDC rules, which require each worker to undergo a
    background security check.

    The letter describes the findings of an 18-member CDC inspection team that
    visited Texas A&M for five days in July. In late June, the agency had
    suspended all of the university's research with microbes defined as "select
    agents" under federal rules that took effect in 2002 as a result of the
    anthrax attacks a year earlier. The June suspension, the first of its kind,
    came after the university failed to promptly report two cases of accidental
    exposure of laboratory workers to infectious agents (The Chronicle,
    <http://chronicle.com/weekly/v53/i45/45a00101.htm> July 13).

    The inspector general's office of the U.S. Department of Health and Human
    Services, the CDC's parent agency, will now review the center's findings.
    The office has the power to fine the university up to $500,000 per
    violation. Under the rules, individual researchers involved face fines of up
    to $250,000 and prison terms of five years for each violation.

    In its letter Friday, the Centers for Disease Control did not characterize
    the severity of the alleged violations, and an agency spokesman, Von
    Roebuck, said the CDC would not elaborate.

    A Texas A&M spokeswoman, Sherylon Carroll, said university officials had
    scheduled a news conference for Thursday morning and would not comment
    before then. University officials have said that none of the university's
    regulated research threatened the public.

    But a critic said the university repeatedly disregarded public safety. "It's
    obvious that the program" at Texas A&M "was a disaster," said Edward
    Hammond, director of the American office of the Sunshine Project, an
    arms-control group that first publicized apparent problems at Texas A&M
    after obtaining public documents. "What's really striking" about the
    university, he said, "is the breadth of the violations. It's in all
    different aspects of their program."

    Among the problems cited by the CDC are the following: * The three "missing"
    vials contained Brucella abortus, a bacterium that causes fevers, but rarely
    death, in humans. They were used by a former university researcher. * Texas
    A&M's plans for safety, security, and emergency response involving research
    on the microbes were listed as drafts, and the university had not conducted
    drills at least annually to test the plans' effectiveness. * The university
    failed to produce several types of relevant records. They included training
    records for regulated lab workers, and documentation that deficiencies
    identified by the university's internal oversight board, the Institutional
    Biosafety Committee, had been corrected. * There was no coordinated response
    or assessment after routine blood tests of laboratory workers indicated
    possible exposure to microbes being studied. * Three of four principal
    investigators inspected by the CDC had "poorly organized inventory records"
    of the microbes they were studying, making "inventory reconciliation
    difficult and cumbersome." A laboratory device used in the research lacked a
    required filter to catch dangerous microbes in its exhaust.

    Days after the inspectors' visit, the university's vice president for
    research, Richard E. Ewing, resigned from that position. Texas A&M has since
    hired Claudia A. Mickelson, a biosafety expert from the Massachusetts
    Institute of Technology, to advise the university about how to improve its
    compliance with all select-agent rules.

    All locations should use approved procedures The solution by dilution is no
    longer adequate 

    -- 
Dr. Ted Labuza  
Morse Alumni Distinguished Teaching Professor of Food Science and
Engineering
Department of Food Science and Nutrition Univ. of Minnesota  Rm. 136 A 1354
Eckles Ave. St Paul 55108
email - tplabuza@umn.edu Text Msg  6513072985@mobile.mycingular.com  Cell
651-307-2985
Office ( 612-624-9701            UM 7  fax 612-625-5272          NFNC 7
Fax 661-483-3302
web     http://www.ardilla.umn.edu/Ted_Labuza

    "
> 
> The weight of damp crystals to take agrees more or less with that
> recommended by Wekell, and the concentration of picric acid is not critical
> anyway. Be careful how you dispose of any unused crystals and the filter
> paper; down the sink with plenty of water.
> 
> There are a couple of other points with regard to the picrate procedure
> which are in my opinion not adequately dealt with in descriptions of
> procedure.
> 
> The TMA in extract after making alkaline is extracted into toluene and the
> calibration curve is prepared by similarly extracting standard solutions of
> TMA into toluene. You should note that the extraction of TMA into the
> toluene phase is not complete. The proportion is high, around 90% depending
> on the concentration of alkali used, and is affected by the total solids in
> solution - a salting out effect. The fish extract is prepared in a TCA or
> perchloric acid solution whereas the procedures describe the TMA standards
> being prepared in dilute hydrochloric. I recommend that the working TMA
> standards be diluted from the stock using TCA or perchloric acid of
> approximately the same concentration as in the extract. Similarly extracts
> should be diluted with this solution if they are of high concentration and
> need to be diluted into the working range. This brings me to the other
> consideration.
> 
> Typical demersal fish muscle contains about 80% water. A common extraction
> ratio for TMA determination is 1:3 fish muscle:extractant. Starting with,
> say, 100g of fish the final volume of the aqueous phase is 380ml. In my
> opinion the TMA in the 100g is dissolved in 380 ml and the amount in the
> aliquot taken for analysis should be scaled up to this volume to give the
> concentration in the muscle. This is the procedure always used in the
> laboratory I worked in, but some published procedures, including the AOAC
> procedure, scale up to the total mass of the extraction blend, 400g in the
> case of my example. This latter procedure will overestimate the
> concentration by around 5% compared with the former. The procedure as
> described by Dyer scales up to total mass, (using 200ml of TCA solution in
> this case, i.e scaling up to 300 rather than 280). Most papers on TMA
> contents do not declare the how final concentration are calculated or
> adequately cite the reference for the procedure used. These comments apply
> also to determination of TVB by distillation of extracts.
> 
> Another consideration for reproducible results is to standardise how the
> sample is taken from the fish. The TMA concentration within a fillet as the
> fish spoils and the TMA increases varies throughout the fillet. The highest
> concentration as one might expect are around the belly cavity and lowest
> near the tail. In the case of a small fish perhaps the whole fillet can be
> taken, in a large fish perhaps standardise by taking a section above the
> belly cavity, but not including the belly flaps.
> 
> Peter Howgate
> 
> ----- Original Message -----
> From: "Shuming Ke" <shumingk@foodsci.umass.edu>
> To: "Seafood research and extension information exchange"
> <seafood@ucdavis.edu>
> Sent: Friday, October 05, 2007 4:53 PM
> Subject: how to measure TMA in fish tissue by Dyer method
> 
> 
>> hi,
>> I plan to use picrate method described by Dyer (j fish res bd can,
>> 1945:351-
>> 358) to determine TMA content in marine fish tissue. The basic concept is
>> to
>> react TMA extracted in toluene with 0.02% picric acid and measure
>> absorbance
>> at 410nm. In Dyer' method, dry picric acid is disolved in toluene to
>> prepare
>> 2% picric acid stock solution in toluene. Since dry picric acid is highly
>> explosive and shock sensitive, this raises the question-- how to prepare
>> picric acid solution in toluene? Do I just order dry (moisture free)
>> picric
>> acid solid and disolve in toluene? Any one has practical ideas on how to
>> carry
>> on the analysis? Many thanks.
>> -- 
>> Shuming Ke, Ph.D.
>> Research Associate
>> University of Massachusetts Marine Station
>> P.O.Box 7128
>> 932 Washington Street
>> Gloucester, MA 01930
>> shumingk@foodsci.umass.edu
>> 
>> Office: 978-281-1930 x. 2
>> Fax: 978-281-2618
>> 
>> 
>> 
>> 
>
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