Food micro (long-ish)

N10 limbic_lesion at
Wed Dec 15 17:32:22 EST 2004

"The Sceptical Chymist" <polyzoni at otenet.invalid> wrote in message 
news:cpq9j4$dp$1 at
> 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 
> customers,
> a major food multinational, and I would like to hear the opinions of 
> others.
> 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" 
> analysis.
> 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 
> (RV)
> broth.
> 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 
> reading
> 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 
> 25g
> 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 
> (that
> 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 
> point
> 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 
> that
> after incubating the homogenate at 37 °C for 16-20 hrs in BPP, if 
> Salmonella
> is present it will multiply to such levels that this difference is
> insignificant.
> 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 
> to
> anyone who reads it more than once.
> Sincerely
> Kostas Polyzonis
> QA Manager
> Michael Arabatzis AVEE - Hellenic Dough
> To reply by private e-mail type replace invalid with gr

Hello Micheal

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 
following ;

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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