23 replies to this topic

[AJL]

Hi, 

We have an internal lab and test for yeast and mould amongst other tests. On petrifilm, it is not always easy to identify the colonies as yeast or mould, and with grain based products we have observed that occiasonally food particles cause discolouration or cause a phosphotase reaction which can interfere with the count.

Lately we had some 'high' mould results. We retested on petrifilm and DG18 agar. The differences were huge, as in a log difference between the 2 test types. 

Which one should I rely on? Furthermore, who has observed this phenomenom and has an explanation?

[Charles.C]

Hi, 

We have an internal lab and test for yeast and mould amongst other tests. On petrifilm, it is not always easy to identify the colonies as yeast or mould, and with grain based products we have observed that occiasonally food particles cause discolouration or cause a phosphotase reaction which can interfere with the count.

Lately we had some 'high' mould results. We retested on petrifilm and DG18 agar. The differences were huge, as in a log difference between the 2 test types. 

Which one should I rely on? Furthermore, who has observed this phenomenom and has an explanation?

Hi AJL,

 

Y&M not my area of microbial expertise but yr type of problem is not so unusual IMEX of other counting systems, eg for coliforms.

 

Some obvious initial queries are  -

 

(1a) Same growth media ?

(1b) Same sample ?

(2) Same operational setups, eg incubation temps/times ?

(3) Duplicate measurements agree within "usual" microbial accuracy ?

(4) Either/both agars/methods are officially approved for use with your specific product ? (IMEX the 2nd "red" can be important).

(5) Confidence that counted colonies are being correctly identified.

 

Above are all micro 101 but IMEX often ignored in practice until a discrepancy occurs.

[AJL]

(1) Assume you mean dilution broth? Yes. Used exactly the same sample and diluted sample to plate on both mediums.
(2) yes all the same
(3) yes
(4) yes: DG18 us actually the preferred Medium for low water activity foods (I tested a grain Product).
Petrifilm is approved but studies have shown that there can be phosphate interference or food particles from Grain interfering. However this does not seem to be the case. Phosphate causes a green colour and food particles a blue colour. These 'colonies' are black. Unfortunately do not have a microscope for further investigation.
(5) I have streaked colonies from the Petrifilm on DG18 to see if it is actual growth.
Other than that I am not sure?
I had considered sending to an external lab for a second opinion, but can see they use DG18 as a standard test so...

Edited by AJL, 08 April 2023 - 09:17 PM.

[Charles.C]

No. 1a,  I meant are the agars which generate the counted colonies the same ?

 

By "huge" differences do you mean like 2 :20 or 10:100 or 100:1000 ?

[AJL]

No, one is Petrifilm and the other is DG18. They are different agar types.

[AJL]

On the Petrifilm we got counts in the 1000's
On the DG18 we got counts in the 100s

Edited by AJL, 08 April 2023 - 09:28 PM.

[Charles.C]

Hi AJL,

 

From quick look, seems -

 

BAM        25degC - 5days

Petrifilm   25/28degC - 2-3days

 

Petrifilm agar appears anonymous.

Maybe apples and oranges.

 

PS - sorry, posts overlapped.

Edited by Charles.C, 08 April 2023 - 09:58 PM.
edited

[Charles.C]

Looks like you will need to initially validate yr own DG18 data as per yr post 3

 

+ Maybe discuss with Petrifilm supplier if suitable for yr specific product. I'm sure they will be interested if their product seems to overestimate by x10.

 

PS -  Assuming that 2 or 3 dilutions were utilised, I would also do one more check regarding confidence in the results - ensure that the (hopefully) duplicate plates on the (hopefully) 2 dilutions used to calculate the average result are in reasonable compliance with respect to the ratio of results for individual dilutions. Data presented in several previous APC queries here has immediately revealed that the dilution/plating/evaluation  was simply not (somewhere) performed properly.

 

PPS - Hopefully other members here may have direct experience with the 2 methods under discussion.

Edited by Charles.C, 09 April 2023 - 01:18 AM.
edit

[AJL]

Yes that's what I am looking for, someone with experience with these 2 methods for this particular (grains) product.
No issue with dilutions here, they were checked at (-1) (-2) (-3)
On Petrifilm (-1) and (-2) were overgrown and only (-3) was a statistically valid plate.

On DG18 (-1) and (-2) were readable

[Charles.C]

Yes that's what I am looking for, someone with experience with these 2 methods for this particular (grains) product.
No issue with dilutions here, they were checked at (-1) (-2) (-3)
On Petrifilm (-1) and (-2) were overgrown and only (-3) was a statistically valid plate.

On DG18 (-1) and (-2) were readable

Hi AJL,

 

I get the impression that no duplicate plates per dilution were done. If so, TBH, this is flawed technique.

 

The difficulty with above data is that it's impossible to evaluate the reliability of the Petrifilm data. Even more so if no duplicate plate.

 

For DG18 the average (assuming duplicate plates per dilution) result for -1 dilution should be "approx" 10x the average result for -2 dilution assuming same plated sample sizes.

 

An additional requirement nominally applies to the ratio of the 2 individual results for each dilution (assuming duplicate plates).

 

Inasmuch as this was a "test exercise" it would have been preferable to take at least 2 samples for comparison assuming the source permitted such.

[AJL]

Hi Charles,
More than one replicate for each sample and dilutions across -1,-2,-3 made sense on both agar types.
Everything married up nicely and there was enough data to rule out errors with dilution, sampling errors and such.

Only key difference was agar type (dilution temp and time was the same for both).
So I can hear what you are saying but it seems as if either
A) there was mould, and it grew on Petrifilm but not DG18. Why?
B) DG18 results are most reliable and there is a type of bacteria growing on the Petrilm which is not mould? Or an interference?
Hence reaching out, hoping someone else had experienced this and found out why.

[Charles.C]

Hi AJL,

 

Actually BAM recommends spread plate procedure but it's a very old methodology (2001), eg -

 

https://www.fda.gov/...-and-mycotoxins

[AJL]

Yes looks familiar. I used spread plate for DG18. 🤗

[Charles.C]

Hi AJL,

 

I was unable to locate any specific AOAC approval for the Petrifilm YM product for grain related items although I noticed  several (apparently satisfactory) independent references/valdations for this application. No doubt you have already well researched this aspect with more success than I.

 

I deduce from OP that previously mould results from the 2 methods under discussion were "similar".

 

Looks like you have a conundrum which may require a microscope et al.

 

Quickest solution (in addition to waiting for responses here) is probably to ring 3M and ask why their (proprietary) product appears sometimes far more "productive" than DM18 in your experience for certain case(s) of grain.

No doubt 3M will be absolutely thrilled with your News.

 

PS - Its OT but JFI I noticed this abstract in passing -

 

A total of 260 samples from six food groups (grains and grain products, tree nuts, dried fruits, fresh produce, fruit juice, and dairy products) were tested for levels of fungal contamination using the SimPlate Yeast and Mould Color Indicator (YM-CI) and the FDA official (BAM) method. Results showed that the SimPlate, in most cases, gave higher yeast and mould (YM) counts than the FDA (reference) method. Statistical analysis of the data (paired t-test) revealed that there were significant differences (α = 0.05) between the two methods for several foods tested. The SimPlate was easy to use, saved time during sample preparation and inoculation and gave results faster than the reference method. Some difficulties were encountered when spreading moulds were present

(Tournas et al,2011)

[AJL]

So 3Ms explanation was simply that some strains are happier on Petrifilm.
Also that I should try and test again on agar using 0,1ml rather than 1ml, or spreading 1ml over 3 plates, and also to make sure that the agar is fresh
🙂
So that's where I am at

Edited by AJL, 14 April 2023 - 05:22 PM.

[Charles.C]

So 3Ms explanation was simply that some strains are happier on Petrifilm.
Also that I should try and test again on agar using 0,1ml rather than 1ml, or spreading 1ml over 3 plates, and also to make sure that the agar is fresh

So that's where I am at

Hi AJL,

 

Petrifilm appear (surprise! ) to be implying that their system may be better than DM18 for grain ( although not afaik AOAC officially approved). However this suggestion is contrary to various publications which afaik validate no significant difference. As apparently did your own previous results (> 1 year's data?).

 

I don't get the 0.1ml suggestion since (a) as I understand you are getting higher counts on Petrifilm albeit within the correct numbers for reliable statistical counting and with acceptable ratios between dilutions, (b) smaller samples decrease the accuracy, other things being equal.

 

As I understand you are doing DM18 using spread plates/0.1ml so I half wondered if someone had forgotten to allow for the 0.1ml as compared to the 1ml on Petrifilm but such an error should be disallowed by the reported good previous agreement between 2 methods.

 

Afai can see, the critical question is why you have a history of agreement between the 2 methods but no longer. Indicates something has changed, either with respect to the agar/materials/equipment etc, operational factors, eg diluting/counting, samples themselves.

 

I would still send a sample(s) out to compare DM18 results. (And Petrifilm if you can find a suitable lab).

 

Good Luck !!

 

PS - you also still need to confirm that there are no false positives on Petrifilm Plate (or DM18).

[AJL]

Ok to anyone who was following this post.
I had used 1 ml and spread on DG18 which is not recommended. It's best practice to use 0,1ml or 1ml spread across 2 plates.
Once I did this, and read results after 7 days rather than 5, I got consistent results with Petrifilm. At least they were in the same log
The colonies were small and were not visible at 5 days, but were distinct at 7 days.

I will be sending in to an external lab as well.

Charles: the 0,1 ml is because sometimes 1ml can be too 'wet' and the colonies then do not stick properly to the agar. 🙂
Also if you invert plates when they are wet the liquid just run down the side.

But from what it seems like, the colonies were stressed so required 7 days of incubation to ensure enough time to be visible.

Edited by AJL, 20 April 2023 - 08:46 PM.

[Charles.C]

Ok to anyone who was following this post.
I had used 1 ml and spread on DG18 which is not recommended. It's best practice to use 0,1ml or 1ml spread across 2 plates.
Once I did this, and read results after 7 days rather than 5, I got consistent results with Petrifilm. At least they were in the same log
The colonies were small and were not visible at 5 days, but were distinct at 7 days.

I will be sending in to an external lab as well.

Charles: the 0,1 ml is because sometimes 1ml can be too 'wet' and the colonies then do not stick properly to the agar.
Also if you invert plates when they are wet the liquid just run down the side.

But from what it seems like, the colonies were stressed so required 7 days of incubation to ensure enough time to be visible.

Hi AJL,

 

TBH I thought using 0.1ml for spread plates was "Microbiology 101". (For reasons similar to which you have stated).

 

from BAM -

Count plates after 5 days of incubation. If there is no growth at 5 days, re-incubate for another 48 h.

 

But, as I understood, you did not get zero growth after 5 days, rather a lower count as compared to Petrifilm.

 

I don't think you are "allowed" to adjust BAM's Procedure in order to get agreement with Petrifilm.

[AJL]

Yes but Charles, I write my own procedures 😅
Not but in all serious, noted.
Let's see what the external lab says. 👍

[Charles.C]

Hi AJL,

 

JFI I noticed this adaption of BAM's method which relaxes the comment in Post 18 and whose dilution comment may/may not relate to yr system.

 

 y-m count.pdf   79.31KB   3 downloads

 

Other variations can also be seen, eg -

 

 y-m(2).PNG   16.92KB   0 downloads

(Rossana,2019)

[AJL]

Yes I used 1ml over 2 plates which is nearly the same as 1ml over 3 plates. Due to the counts I wish I had used 3 plates. They were crawling 😅
I have had a lot of experience with the pour plate method but to be honest I was kind of making up my own procedure for DG18 spread.
I think there is more moisture available on the Petrifilm medium because the lid is closed, and the strain was just happier there.
I am happy I solved the conundrum. ! Just goes to show that methods do need to be adapted occasionally. 😉
Even though it isn't my role I do fancy myself as an applications specialist (is that what you call it?) or in other words 'micro detective '. In my next life maybe. 😅

[Charles.C]

Yes I used 1ml over 2 plates which is nearly the same as 1ml over 3 plates. Due to the counts I wish I had used 3 plates. They were crawling
I have had a lot of experience with the pour plate method but to be honest I was kind of making up my own procedure for DG18 spread.
I think there is more moisture available on the Petrifilm medium because the lid is closed, and the strain was just happier there.
I am happy I solved the conundrum. ! Just goes to show that methods do need to be adapted occasionally.
Even though it isn't my role I do fancy myself as an applications specialist (is that what you call it?) or in other words 'micro detective '. In my next life maybe.

Hi AJL,

 

With all due respect you are perhaps a little over- optimistic.

(1) My own conclusion based on yr comments is that, for the samples under present discussion, the preferable methodology (if any) to achieve "reliable" results for Y&M or the individual components remains unknown/unproven (See 3).

(2) I do agree that the variability of results indicates that interpreting any interlaboratory data will demand knowledge of the specific, hopefully approved (for your Product), Procedure involved.  (I speculate this may exclude Petrifilm data).

(3) Further conclusions IMO will need Proficiency data. And maybe a Microscope.

 

I have (multiply) experienced similar confusion to the Y&M results under current discussion when attempting to resolve Customer differences in, particularly, APC and Coliform results for exported seafood. Most of the causes after exhaustive (and exhausting!) discussions between labs have been due to variations in Sampling, Procedures and interpretations of results. Some of the disagreements have been irreconcilable, eg the Customer is compelled to apply Local Standards which are themselves intrinsically flawed or technically incomplete. As I understand, the Y&M data under discussion is primarily of internal interest. Hopefully it stays that way !

[AJL]

No it is not just of internal interest, hence the need for better data
(as in agreement between the two methods) and sending to an external lab.
As we don't have a microscope available I isolated and identified many colonies (they were all of similar appearance) from Petrifilm to confirm it was indeed mould, and we saw growth in every case.
So we can rule out false positives on Petrifilm which I had initially suspected.
Due to the small colony size after 7 days on DG18 I can conclude that it was a slow growing mould, and there must be in some point developed a method for extra incubation time when there is suspected a slow growing mould. ☺️
I would agree with you that descrepancies between inter laboratory results are often due to sampling and sample preparation. Sometimes even due to reading/interpretation errors (I have encountered particles of food being counted as colonies for example).
Usually after a long enough discussion the other lab has concluded that our interpretation was correct. 😉

Edited by AJL, 22 April 2023 - 06:50 AM.

[Charles.C]

Hi AJL,

 

I guess the commercial interest depends on your Product specifications and any (hopefully) agreed sampling/analytical methodology if product acceptance is involved.

 

If ALL the mould results or YM values from both methods are far below acceptance limits (are they?), the commercial interest becomes somewhat, pragmatically, moot.

 

From a purely Research  POV, If results from 2 methods do show a significant, repeatable, discrepancy then  the preferred  validated method  (eg samples sent out to a trusted lab [or 2 labs] give reasonably matching data) is presumably the one which gives higher results. (and particularly if you are the receiver).

 

Hopefully answers yr OP.

 

PS - I just noticed that BAM-2001 do not give an absolute counting method for mould on DG18 agar (only for YM)  although APHA-2001 uses a simillar procedure to BAM and does include a typical direct  mould counting procedure option.

 

It is worth noting that your data for these particular Product samples appears to prove that the DG18  methodology of 5 days incubation may sometimes give low results as compared to 7 days and particularly when compared to Petrifilm. (Although yr older mould data afaik did not show this characteristic).

 

Since both yeast and mould represent a wide group of species it is hardly surprising IMO that count variations may occur when comparing agars, incubation times etc.between Procedures etc. Frankly I'm more surprised that Petrifilm/reduced incubation time is able to closely match results from DG18 (unless the agars are actually similar [?]). (Also see the incubation times in .png in post 20)