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Spreader Colonies in PCA Plates


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#1 super-tresmarias

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Posted 25 June 2016 - 06:11 AM

Dear All,

 

Good Day!

 

We are having problems with recurring spreader colony growth in Plate count Agar (PCA). We have gone through intense aseptic techniques in performing microbiological testing, and yet, spreader colonies seem to be inevitable.

 

Just recently, we have attended a training regarding food safety and then we asked the speaker as to what kind of microorganism is responsible for the formation of such. According to her, it belongs to the Bacillus group.

 

We want to know further discussion on this problem.

 

Does anybody have relevant inputs that could help us understand the source of the trouble and how to eradicate it?

 

Thank you very much!



#2 Lorbi

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Posted 27 June 2016 - 02:36 AM

Hello,

 

I would like to verify if in intensifying your aseptic techniques, have you considered the sanitation and sterility of the environment, lab apparatus, your incubator, etc.?

 

 

Sincerely,

 

Lorbi



#3 danieljoshua.tayamora

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Posted 27 June 2016 - 07:40 AM

Good day!

 

If you already have confidence in your aseptic techniques (this includes complete sterilization of all equipments) during testing then most probably the contamination comes from the environment as pointed out by Ms. Lorbi.

 

However, if the problem still persists, spreaders may be caused by the microorganisms in the sample itself. Spreaders occur when the dilution factor is not enough (too low) to provide enough space for individual cells to reproduce individually, resulting in a "one big single colony" a.k.a. spreader. The best way to resolve this is to increase your dilution factor (instead of 10^-2, go for 10^-5 or higher) depending on the microbial load of your sample. The higher the microbial load, the higher your dilution should be. 

 

Hope this helps. :)

 

-Dan Tayamora



#4 Charles.C

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Posted 27 June 2016 - 08:48 AM

Good day!

 

If you already have confidence in your aseptic techniques (this includes complete sterilization of all equipments) during testing then most probably the contamination comes from the environment as pointed out by Ms. Lorbi.

 

However, if the problem still persists, spreaders may be caused by the microorganisms in the sample itself. Spreaders occur when the dilution factor is not enough (too low) to provide enough space for individual cells to reproduce individually, resulting in a "one big single colony" a.k.a. spreader. The best way to resolve this is to increase your dilution factor (instead of 10^-2, go for 10^-5 or higher) depending on the microbial load of your sample. The higher the microbial load, the higher your dilution should be. 

 

Hope this helps. :)

 

-Dan Tayamora

 

Hi Dan,

 

A few queries,

 

what kind of food(s) / presentation,eg raw - RTE / storage ?

what incubation temperature ?

what kind of agar plate,eg pour, spread, 3M ?

what level of plate count where spreading is a problem, eg 10,000, 100,000, 1M ?

what frequency of "significant" spreading, eg every plate, 10% total ??

 

eg -

 

http://jid.oxfordjou...8/4/372.extract


Kind Regards,

 

Charles.C


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#5 danieljoshua.tayamora

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Posted 27 June 2016 - 09:28 AM

Hi Dan,

 

A few queries,

 

what kind of food(s) / presentation,eg raw - RTE / storage ?

what incubation temperature ?

what kind of agar plate,eg pour, spread, 3M ?

what level of plate count where spreading is a problem, eg 10,000, 100,000, 1M ?

what frequency of "significant" spreading, eg every plate, 10% total ??

 

eg -

 

http://jid.oxfordjou...8/4/372.extract

 

Hi Charles!

 

Just want to confirm, are you asking for a specific sample scenario? Since the initiator did not clearly state what type of food samples are being tested. But in general, irregardless of the method and incubation time, if very low dilutions were used for testing, spreader will be imminent. 

 

Best regards,

 

Dan



#6 Charles.C

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Posted 27 June 2016 - 10:13 AM

Hi Charles!

 

Just want to confirm, are you asking for a specific sample scenario? Since the initiator did not clearly state what type of food samples are being tested. But in general, irregardless of the method and incubation time, if very low dilutions were used for testing, spreader will be imminent. 

 

Best regards,

 

Dan

 

Hi Dan,

 

Additionally to contamination (investigatable pre-usage), I anticipate that spreaders are likely to be more (or less) common depending on the specific product / microbial profile.

 

(IMEX of  seafood/coated seafood some spreading is inevitable but frequent is not.)

 

I anticipate that also more likely for high bacterial levels on plate, eg NRTE, PHF foods.

 

As per my link, Bacillus group appears common for milk. i suspect for various other product also (haven't looked up book details as yet).

 

Hence my queries. :smile:


Kind Regards,

 

Charles.C


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#7 Charles.C

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Posted 27 June 2016 - 10:29 AM

addendum

 

There are also a variety of "spreaders" of course, eg bacterial, yeasts/moulds, eg -

 

Attached File  spreaders.png   136.73KB   1 downloads

 

A few practical tips -

 

Attached File  plate interpretations.pdf   482.46KB   53 downloads

 

BAM -

 

Spreaders. Spreading colonies are usually of 3 distinct types: 1) a chain of colonies, not too distinctly separated, that appears to be caused by disintegration of a bacterial clump; 2) one that develops in film of water between agar and bottom of dish; and 3) one that forms in film of water at edge or on surface of agar. If plates prepared from sample have excessive spreader growth so that (a) area covered by spreaders, including total area of repressed growth, exceeds 50% of plate area, or (b) area of repressed growth exceeds 25% of plate area, report plates as spreaders. When it is necessary to count plates containing spreaders not eliminated by (a) or (b) above, count each of the 3 distinct spreader types as one source. For the first type, if only one chain exists, count it as a single colony. If one or more chains appear to originate from separate sources, count each source as one colony. Do not count each individual growth in such chains as a separate colony. Types 2 and 3 usually result in distinct colonies and are counted as such. Combine the spreader count and the colony count to compute the APC

Kind Regards,

 

Charles.C


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#8 danieljoshua.tayamora

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Posted 27 June 2016 - 11:00 AM

Hi Dan,

 

Additionally to contamination (investigatable pre-usage), I anticipate that spreaders are likely to be more (or less) common depending on the specific product / microbial profile.

 

(IMEX of  seafood/coated seafood some spreading is inevitable but frequent is not.)

 

I anticipate that also more likely for high bacterial levels on plate, eg NRTE, PHF foods.

 

As per my link, Bacillus group appears common for milk. i suspect for various other product also (haven't looked up book details as yet).

 

Hence my queries. :smile:

 

Hi Charles,

 

Thank you for the clarification! :) I highly agree that high bacterial levels on plate will most likely result in spreader "spr" colony formation as this is the result of using low dilutions when you have high microbial load.

 

And to address some of your questions

 

-food products that are likely to have mold contamination are best candidates for spr formation. This is due to the filamentous characteristic of mold colonies.

 

-As for plating method, when using conventional agar media, it is easier to have spr in pour plating method compared to spread plate. But when using 3M petri plates, it is almost close to impossible to have spr formation except when there is excess liquid plated

 

-lastly, spr colonies may persist in spread plate method if agar plates used during testing were not allowed to dry and age for atleast 24 h 

 

Btw, I would like to clarify that I am referring to spr colonies that are considered as "laboratory accidents" where you can no longer obtain a plate count because of the obstruction because it covers half or more than half of the area of the plate.

 

The difference in colony size as depicted in some of the pictures earlier posted are due to inherent characteristics of a particular organism. It is also a must to have knowledge of the general microflora of the product and its morphology when evaluating plate count tests. This will allow the experimenter to differentiate target microflora from unwanted contamination, in this case, spr colonies

 

Hope I was able to attend to your queries,

 

Many thanks and regards,

 

Dan



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#9 Charles.C

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Posted 27 June 2016 - 11:15 AM

Hi Dan,
 

 

We are having problems with recurring spreader colony growth in Plate count Agar (PCA). We have gone through intense aseptic techniques in performing microbiological testing, and yet, spreader colonies seem to be inevitable.

 

In the context of "laboratory accident" (ie material excluded)  IMEX the condensation factor discussed in attachment is a frequent cause. Not only for PCA plates either.

 

In a general context, one needs to specify the material involved.

 

Personally, if i had a plate to count with 25% spreaders I would usually repeat.


Kind Regards,

 

Charles.C


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#10 super-tresmarias

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Posted 28 June 2016 - 11:16 AM

Hi All!  :spoton:

 

Sirs Charles and Dan, thank you so much for your informative discussions, we learned so much from them.

 

For clarification, the characteristics of the spreaders we usually see are those that occupy almost half of the plate, thick, with rough surface and having a hard-to-scrape texture.  Majority are not transparent although some have thin transparent films with small colonies underneath.  

 

We already have done the relevant aseptic techniques same as those earlier mentioned by both of you and yet, the problem still persists.

 

Anyway, thank you once again for the inputs.

 

All the best! God bless. 






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