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Max micro limits 0, <1 or <10


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#1 earthbornstew

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Posted 11 December 2017 - 11:11 AM

Hi Everyone,

I hope someone can help me with this. I work with a trader in milk powder and help with developing specifications. We have two tests which state a max limit of <1 CFU/g for e.coli ( test method iso 11866-1) and coliforms (iso 4831/4832). Previously these specs stated <1 CFU/0.1g max.

However I don’t believe this is correct, as the testing will usually be carried out by adding 1g milk powder with 9mls diluent to reconstitute the samples, and plated with 1ml. Would I be correct in saying that the lowest reportable result would actually be <10 CFU/g and that reporting <1 for zero detected growth is incorrect?

If not, how would you be able to test for that in milk powder?

My background isn’t very thorough in micro. I’ve tried reading ISO7218 requirements and guidance for micro exams but it doesn’t explain to me clearly my question above.

Thanks for any help on this.


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#2 earthbornstew

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Posted 11 December 2017 - 04:39 PM

Additional question on above, is <10 CFU/g equivalent to <1/0.1g?


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#3 bensmith007

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Posted 11 December 2017 - 06:54 PM

Hi,

 

This is just a simple dilution factor calculation. Often in micro testing you need to use dilutions in order to get a workable number of bacteria growing on the petri dish. If you just try and plate the undiluted plate for something with a lot of bacteria (e.g. a root vegetable) then there are too many bacteria colonies to be able to easily count. In this case you plate a series of dilutions (generally 1 in 10 as you describe above- 1g milk powder plus 9g (or ml) of water is 1 in 10) of decreasing strength until you obtain a plate with a countable number of bacterial colonies on it- between 3 and 50 colonies normally (don't quote me exactly on those numbers). To relate back to the number of bacteria that this represents per gram of milk powder, you need to multiply the number of bacteria by the dilution factor.

 

In the case you describe above, the microbiologist would take the number of colonies grown on the dish and multiply that by 10 to give you CFU/g and thus this will account for the 1 in 10 dilution of the milk powder initially performed.

 

That result will usually be in the units of CFU/g as industry standard, and the actual value will depend on how many bacterial colonies grew on the plate. In order to reach your limit of <1 CFU/g, no bacterial colonies should be found growing on the plate. If one colony was found, then your actual result would be 10 CFU/g after multiplying by the dilution factor.

 

I suggest you speak to the lab conducting the micro testing for you and ask them if they can give you a run down of the process or better yet go visit them and see it done for real. It's not always intuitive.

 

Suffice to say, your COA limit and reporting sounds fine, although I have no idea what lab you are using so I can't vouch for them.

 

I hope that makes a little more sense!

 

Ben



#4 earthbornstew

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Posted 11 December 2017 - 09:51 PM

Hi Ben

Thanks for the answer above. I do have one question on one piece you mentioned.

That result will usually be in the units of CFU/g as industry standard, and the actual value will depend on how many bacterial colonies grew on the plate. In order to reach your limit of <1 CFU/g, no bacterial colonies should be found growing on the plate. If one colony was found, then your actual result would be 10 CFU/g after multiplying by the dilution factor.


I would wonder if you could say <1cfu/g though if no growth was detected but you were using different dilutions, say from one batch to another.

Below is an image from a piece I found online about interpretation of results that would say you should include the dilution factor, even if no detection. So the lowest reportable could be <10 for a 1:10 dilution, <100 for 1:100 ...... etc. it’s from http://www.microchem...ion-of-results/

5bae709c901e2f620f304c3ca29c7c2b.jpg


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#5 Charles.C

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Posted 11 December 2017 - 10:11 PM


Hi Earthbornstew/Ben,

 

ISO are very thorough but always make things so complicated to follow.

 

The simple case is basically arithmetic until you get involved with averages when you hv to examine all the details.

 

Example 1g sample product mixed with 9 ml liquid.This is equivalent to 1g product in 10g solution.

This implies 1ml solution ~ contains 0.1g product.

 

Assume 1ml of solution is transferred to agar plate and after incubation can detect 1 colony.

This implies there is 1cfu in 0.1g product which is equivalent to saying there is 10 cfu in 1g of product, ie 10cfu/g

 

Therefore, zero detection of colonies implies < 10cfu/g product.


Edited by Charles.C, 11 December 2017 - 11:32 PM.
re "formatted" slightly

Kind Regards,

 

Charles.C


#6 Charles.C

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Posted 11 December 2017 - 10:17 PM

addendum

 

to be able to state that <1 cfu/g for a solid, probably need to use a MPN technique involving multiple tubes.

 

I assume the procedure you refer does not include a MPN variation.


Kind Regards,

 

Charles.C


#7 bensmith007

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Posted 11 December 2017 - 10:52 PM

Interesting- I've not come across that before in my education or my (relatively brief) time in industry.

 

Check out slide 26 of this Powerpoint from FSIS: https://www.fsis.usd...pdf?MOD=AJPERES

which makes the point that the portion of test sample will dictate the theoretical limits of detection.

 

Does this explain why a lot of specifications will not say 'less than X per gram' or whatever, but rather 'ABSENCE of pathogen in X grams of food sample'?

 

I am at the limit of my knowledge here, so I suggest finding someone more microbiology oriented on this forum, or just speak to your pathogen testing lab so they can explain!

 

Ben



#8 earthbornstew

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Posted 11 December 2017 - 11:05 PM

Hi Charlie

Thank you for the input above.

What I’m taking from everything so far is that whether we state <10cfu/1g or <1cfu/0.1g , these should be equivalent. It seems that only when the spec states <1cfu/g that they are actually incorrect.

Is there any requirement (Codex, ISO...etc) which would indicate whether <10cfu/1g or <1cfu/0.1g would be the preferred reporting format?


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#9 Charles.C

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Posted 11 December 2017 - 11:54 PM

Interesting- I've not come across that before in my education or my (relatively brief) time in industry.

 

Check out slide 26 of this Powerpoint from FSIS: https://www.fsis.usd...pdf?MOD=AJPERES

which makes the point that the portion of test sample will dictate the theoretical limits of detection.

 

Does this explain why a lot of specifications will not say 'less than X per gram' or whatever, but rather 'ABSENCE of pathogen in X grams of food sample'?

 

I am at the limit of my knowledge here, so I suggest finding someone more microbiology oriented on this forum, or just speak to your pathogen testing lab so they can explain!

 

Ben

 

Hi Ben,

 

Indeed, the sample size is directly related to detection sensitivity, eg some texts use 25g, others are more strict and require 50g which will increase chance of detection.

 

The terminologies used relate as to whether the testing procedure is qualitative or quantitative.

 

ABSENCE in 25g sample means that using a (hopefully) standard qualitative procedure, the pathogen was not detected in 25g. (Absence per se in a product lot is impossible to prove due statistical/analytical sampling errors).

 

As in yr link MPN techniques are quantitative so allow a numeric expression, eg <3 cfu/g where the detection limit can be lower than direct plating techniques but also likely require a lot more work.

 

Since most/all specs have a zero tolerance for pathogens like salmonella the labs normally only use a qualitative procedure for this.

In comparison S.aureus usually has a finite limit in specs so a quantitative procedure is employed, either direct plating or MPN.


Kind Regards,

 

Charles.C


#10 Charles.C

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Posted 12 December 2017 - 12:13 AM

Hi Charlie

Thank you for the input above.

What I’m taking from everything so far is that whether we state <10cfu/1g or <1cfu/0.1g , these should be equivalent. It seems that only when the spec states <1cfu/g that they are actually incorrect.

Is there any requirement (Codex, ISO...etc) which would indicate whether <10cfu/1g or <1cfu/0.1g would be the preferred reporting format?


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Hi earthbornstew,

 

It is more usual to use 1g as a reference. eg <10cfu/g. This format is typically used in Product Specifications.

 

<1cfu/g is a possible Product Specification (obviously more strict than <10cfu/g) but will likely require a MPN technique if product is a solid. (Or perhaps via use of mutiple Plates if direct plating).

 

For a liquid, testing compliance to such a spec. is directly achievable using a 1ml sample (zero diluent) and the Maths in my Post 5 (However sometimes the liquid is too viscous etc so that a diluent must be used)


Kind Regards,

 

Charles.C


#11 earthbornstew

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Posted 21 December 2017 - 10:29 AM

Hi Everyone

Just a quick update. We’ve reviewed this and discussed with some of our suppliers and they’ve agreed going with <1 CFU/0.1g.

Thanks for all the advice.


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