Food micro (long-ish)
limbic_lesion at hotmail.com
Wed Dec 15 17:32:22 EST 2004
"The Sceptical Chymist" <polyzoni at otenet.invalid> wrote in message
news:cpq9j4$dp$1 at usenet.otenet.gr...
> Hello everybody.
> I would appreciate any comments on the following:
> We are a company that makes frozen dough products, some of which include
> cured meat in the filling. We're having a problem with one of our
> a major food multinational, and I would like to hear the opinions of
> The situation is as follows:
> Two of our products contain boiled ham and bacon, which are placed in an
> open dough base (they're exposed). The cooking instructions state clearly
> that the product should be baked at 180 deg C for15-20 min. However the
> customer believes that the end-user might let the product thaw rather than
> take it straight from the freezer to the oven and additionally might pick
> and eat the ham or bacon. For that reason they treat those two ingredients
> as ready-to-eat and ask for an "absence of Salmonella spp in 375g"
> We're using a Biomerieux Minividas instrument, and the protocol asks for:
> 1. Homogenising 25g of sample in 225 ml of buffered peptone water (BPP).
> 2. Incubating the resulting 250ml of homogenate at 37 °C for 16-20 hrs.
> 3. Subculturing 0.1 ml of homogenate in 10 ml of Rappaport Vassiliadis
> 4. Incubating the RV broth at 41.5 °C for 6-8 hrs.
> 5. Subculturing of 1 ml from the RV broth in 10 ml of M-broth.
> 6. Incubating the M broth for 16-20 hrs at 41.5 °C.
> 7. Heating 1 ml of M broth at 100 °C for 15 min.
> 8. Adding 0.5 ml of heated M broth to the Vidas SLM test strip and
> the results in 45 minutes.
> Positive results are followed by confirmation by a reference method that
> involves plating the selective broth and running biochemical (API strips)
> and/or serological (agglutination) tests.
> If we were to follow the protocol to the letter we would have to test 15
> samples (the instruments can only run 12 samples at a time) with all the
> reagents, incubator space, staff and time requirements that would entail.
> What we do instead is pool the 15 samples of 25g to 4 samples of 100g
> makes 400 rather than 375g). We dilute those 1:10 with BPP and homogenise,
> ending up with four homogenates of 1000 ml. These we incubate at 37 °C for
> 16-20 hrs and then draw 0.1ml and subculture it in RV broth. From that
> onwards we follow the rest of the protocol to the letter.
> The probability of the 400g sample carrying Salmonella remains the same as
> long as the sample is representative and drawn from different points. What
> does change is the probability that 0.1 ml drawn from 1000 ml of incubated
> homogenate carry Salmonella. There's a better chance of detecting it if
> 0.1ml is drawn from 250 ml as the protocol requires. However we believe
> after incubating the homogenate at 37 °C for 16-20 hrs in BPP, if
> is present it will multiply to such levels that this difference is
> I'm interested in other people's opinion on the matter, so I would like to
> hear from you.
> I will be posting this to more than one food list/newsgroup, so apologies
> anyone who reads it more than once.
> Kostas Polyzonis
> QA Manager
> Michael Arabatzis AVEE - Hellenic Dough
> To reply by private e-mail type replace invalid with gr
I agree that sampling plan and proceedure for bulking samples is fine but
how was the mass of 375gram calculated is it in itself even significant as
a statiscal proportion of your production mass ?. You should know that 375
gram is not a magical figure. The key factor in deciding how much sample to
examine depends on the total mass produced and the limit of sensitivity at
which you decide to test. Working to a even 95 % confidence limit is
relativel trare in the Food Industry .
In my experience the majority of Salmonella sampling plans fall far short
of even the most simple statiscal model. I think you should review the maths
to make certain your sample makes statistical sense otherwise all other
considerations or questions of effciencies are less relevant.
Assuming you are satisfied with sampling plan then please also consider the
A) Large BP volumes may go extremely anaerobic during pre-enrichment
therefore with volumes of 1000 ml it is advised stir or shake.
b) If your dough is "live" you may have extremly high levels of competative
organisms during the temeprature equilibriation stage of the BP pre
enrichment and possibly during some stages of the subsequent incubation.
Addtionally you going to need an understanding the effects of negative pH
swing during preencrichment . Do you know if the buffering capaicty of the
broth is sufficient...check it out I think you might be very supprised !
I would recommed therefore that you conduct validation trials to ensure
that low levels of Salmonella ( 1-5 cells per 25 50 or 75 gram) can be
enriched satisfactorily. I d reccomend working with atleast 5 or 6 strains
taken from each of the most common O antigenic ( B,D,C,E,F,G).
It may be difficult but I recommend you try to make some assesment of
quantative levels of Salmonella obtained in the BP broth after pre
enrichement. In my experience a decent pre enrichment will yeild at least
log 2 cells per ml after the 16- 20 hours incubation. Theoretically
selective enrichment will work on log 1 but more of that latter but if you
do the figures you'll see LOG 2 is attainable post pre enrichment even with
stressed or fragile Salmonella populations.
Quantation at this stage could easily be performed by surface platting on
XLD or BG (etc) and I know this because I ve done it. Given known
pre-enrichment yields from known levels of intial inoculation you will then
be in a position to calculate whether or not your 0.1 ml aliquot fromthe
1000 ml culture would contain Salmonella at what ever CI you choose.
Factually no one can tell you with defendable accuracy that your 0.1 ml
aliquote is sufficient... I suspect it is but I wouldnt put my professional
name to such a statment rather I truelly beleive you need to validate your
method and thus generate your own confidence.
You may care to check yields ( I consider it vital) at the end of selective
enrichment and the M broth stage. You may care to check the yield with M
Borth incubated at 37'c instead of 41.c 'c again you might be suprised.
C) If you do the work and validate your pre enrichment stage I think you
still have a problem and its to do with the RV broth. RV is a superb borth
but single broth selective enrichment for Salmonella species invovles a risk
of producing False negative results ( ie missing true positives) .
In the case of RV broth you have two factors which need consideration.
Firstly it is well documented in literature that numerous common Salmonella
serotypes are Methyleneblue sensitive, temperature sensitive mutatants are
also relatively common. We isolate 3-4 methylene blue Salmonella from food
samples on a weekly basis and perhapes 2 -3 temperature sensitive mutants
per month ( Usually grooup D of C1).
RV contains methylene blue and is a high temperature incubation ( you use
My point is R.V. is " good" but as advised in every international standard I
ve read pertaining to Salmoenlla isolation it is always true that at
least two selective enrichment broths are used to give the highest
statistical chance of recovery and susequent detection.,,,irrespective of
the end confirmation proceedure.
You are obviously interested in obtaining the "correct result " so I mention
these points because no matter what the PR for Vidas system states it is
flawed if implies that a single broth selective enrichment stage is
superior or even equivalent to a dual broth system in the long view... again
I speak from experience on this one .
Best regards Des akaN10
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