13 replies to this topic

[kumar7]

Hello everyone! Here's a scenario: 1mL sample transferred to Petri dish in duplicates. Agar were poured, upon solidified were incubated. After incubation period, one agar plate got count, the other agar plate had no count. 1)How does the colony counting rule works when one agar plate has count but the other agar plate has NO count? 2)Does this result consider valid in the first place? I couldn't find any standard/reference that could explain on this. Appreciate your input on this. Do share if there's any standard/official method reference. Thank you. Have a great day! Kumar

[Evans X.]

Greetings Kumar,

 

There is no definitive answer to this. The simple way would be to discard this analysis and perform a new one. Since you deal with living organisms many things could go wrong, like one petri having air bubbles so the strictly anaerobic microorganisms (guessed that from your process method, so correct me if I'm wrong) you found in one couldn't grow on the other. If by some chance you were looking for any microbes then the opposite could happen.

If you do want to move ahead and give an explanation, then you must consider the result of the petri with the plate count. If the result is within limits you or legislation have set then you could pass it as safe. If however the count is off limits then you must repeat the analysis and I would also suggest you quadruple the petri dishes you will incubate for a safer statistical result (it could possibly reveal analysis handling mistakes).

 

Regards!

[kumar7]

Hi Evans,

 

Thank you for the input.

I guess repeating the test again is a feasible solution.

 

Most of the sample tested (liquid) doesn't involved dilution since low count is expected. 

Just that encountering this scenario is quite frequent. No discrepancy is the media prepared or analyst competency.

 

FYI, the microbes I work on is aerobic.

 

Regards

[Brothbro]

For samples expecting a low count, would an MPN method be more suitable? My understanding is that traditional agar plate counts are less precise outside a 25-250 colony count range. You could repeat the test, but it's still likely that you will get disagreements between the plates.

[juanolea1]

Evans,

 

It seems to me that it was more of a technique error. Ensure that the sample is thoroughly mixed when drawing the aliquot. In liquids it’s easy to draw a bad sample if not taken right after mixing the solution because solids that may settle to the bottom can carry a greater bacterial load. Also, solids may at times appear like bacteria when in fact they are just contaminating particles that may be interpreted as a colony by the inexperienced eye.

I hope it helps.

Juan

 

[Kara S.]

I think you should first review your process and conduct retraining with the staff and testing methods. Some common things to look out for include:

1. Sample Preparation - if you are adding the sample into a dilution blank, is it mixed well prior to transferring to agar? If your sample is liquid already, maybe still invert or mix prior to taking the 1mL to ensure homogeneous. 
2. Sample Collection - watch how everyone is collecting their 1mL samples. Are they using aseptic techniques? Are they collecting air bubbles and skewing the 1mL amount?
3. Temperature of Agar - the agar may be too hot and killing off some of the microbes - maybe by the time you reach the second petri dish the agar has cooled that it is not causing that issue. 

[Charles.C]

Hello everyone! Here's a scenario: 1mL sample transferred to Petri dish in duplicates. Agar were poured, upon solidified were incubated. After incubation period, one agar plate got count, the other agar plate had no count. 1)How does the colony counting rule works when one agar plate has count but the other agar plate has NO count? 2)Does this result consider valid in the first place? I couldn't find any standard/reference that could explain on this. Appreciate your input on this. Do share if there's any standard/official method reference. Thank you. Have a great day! Kumar

Hi kumar,

 

Results on one sample are often meaningless.

 

IIRC, ISO's APC Standard offers some statistical advice where very low counts do occur.

 

It's difficult to give meaningful recommendations without more context, eg

 

What is product ?

What is specification ? (+ defined procedure ?)

What was the actual Count which did occur ?

 

Offhand, Post 4 looks most likely to quantitatively apply (eg as typically used for water analysis) but elsewhere exceptions also exist,eg as shortcuts.

[kumar7]

Evans,

 

It seems to me that it was more of a technique error. Ensure that the sample is thoroughly mixed when drawing the aliquot. In liquids it’s easy to draw a bad sample if not taken right after mixing the solution because solids that may settle to the bottom can carry a greater bacterial load. Also, solids may at times appear like bacteria when in fact they are just contaminating particles that may be interpreted as a colony by the inexperienced eye.

I hope it helps.

Juan

 

 

I think you should first review your process and conduct retraining with the staff and testing methods. Some common things to look out for include:

1. Sample Preparation - if you are adding the sample into a dilution blank, is it mixed well prior to transferring to agar? If your sample is liquid already, maybe still invert or mix prior to taking the 1mL to ensure homogeneous. 
2. Sample Collection - watch how everyone is collecting their 1mL samples. Are they using aseptic techniques? Are they collecting air bubbles and skewing the 1mL amount?
3. Temperature of Agar - the agar may be too hot and killing off some of the microbes - maybe by the time you reach the second petri dish the agar has cooled that it is not causing that issue. 

 

Hi Kara,

 

Thank you for the suggestion.

Will take into account for improvement.

 

Kumar

[kumar7]

Hi kumar,

 

Results on one sample are often meaningless.

 

IIRC, ISO's APC Standard offers some statistical advice where very low counts do occur.

 

It's difficult to give meaningful recommendations without more context, eg

 

What is product ?

What is specification ? (+ defined procedure ?)

What was the actual Count which did occur ?

 

Offhand, Post 4 looks most likely to quantitatively apply (eg as typically used for water analysis) but elsewhere exceptions also exist,eg as shortcuts.

 

Hi Charles,

 

Thank you for your input.

 

The samples being tested here are dairy products (UHT & pasteurized milk, condensed milk).

Our count limit is <1 CFU/mL (for UHT), <10 000 CFU/mL (for pasteurized & condensed milk).

Method is pour plate using Plate Count Agar (added with skimmed milk).

One of the result obtained was ~ 30 CFU/mL in 1st plate, <1 CFU/mL in 2nd plate. No dilution involved.

 

Kumar 

[Brothbro]

Hi Charles,

 

Thank you for your input.

 

The samples being tested here are dairy products (UHT & pasteurized milk, condensed milk).

Our count limit is <1 CFU/mL (for UHT), <10 000 CFU/mL (for pasteurized & condensed milk).

Method is pour plate using Plate Count Agar (added with skimmed milk).

One of the result obtained was ~ 30 CFU/mL in 1st plate, <1 CFU/mL in 2nd plate. No dilution involved.

 

Kumar 

 

Hi Kumar,

 

For your UHT milk, I'm assuming it's commercially sterile? If that's the case aerobic plate count testing is not an appropriate method for verifying sterility. Typically, complete packages are incubated at ~35C for a couple days before streak plating onto an appropriate agar type. Unless you give bacteria time to grow within the package, they will be difficult to spot. I think it could be argued that your current method may be missing defects.

[kumar7]

Hi Kumar,

 

For your UHT milk, I'm assuming it's commercially sterile? If that's the case aerobic plate count testing is not an appropriate method for verifying sterility. Typically, complete packages are incubated at ~35C for a couple days before streak plating onto an appropriate agar type. Unless you give bacteria time to grow within the package, they will be difficult to spot. I think it could be argued that your current method may be missing defects.

 

Hi Brothbro,

 

Thank you for your comment.

Yes, it's commercially sterile. 

Noted on the method.

 

We don't usually see the trend in UHT product but in condensed milk/pasteurized milk.

 

Regards,

Kumar

[Charles.C]

Hello everyone! Here's a scenario: 1mL sample transferred to Petri dish in duplicates. Agar were poured, upon solidified were incubated. After incubation period, one agar plate got count, the other agar plate had no count. 1)How does the colony counting rule works when one agar plate has count but the other agar plate has NO count? 2)Does this result consider valid in the first place? I couldn't find any standard/reference that could explain on this. Appreciate your input on this. Do share if there's any standard/official method reference. Thank you. Have a great day! Kumar

 

 

Hi Charles,

 

Thank you for your input.

 

The samples being tested here are dairy products (UHT & pasteurized milk, condensed milk).

Our count limit is <1 CFU/mL (for UHT), <10 000 CFU/mL (for pasteurized & condensed milk).

Method is pour plate using Plate Count Agar (added with skimmed milk).

One of the result obtained was ~ 30 CFU/mL in 1st plate, <1 CFU/mL in 2nd plate. No dilution involved.

 

Kumar 

Hi Kumar,

 

Re ^^(red) - I assume this was sterilized milk.

This pour plate method is presumably a "rapid" test to (hopefully) result in no colonies on the plates. 

IMEX 0/30 would be typically, politely, interpreted as a "Laboratory Accident" (ie somewhere a gross error). > Need to repeat.

 

ISO 7218, 2001 is afaik unique in proposing that in case of 2 plates (test sample or first dilution) containing less than 15 colonies, calculate the estimated number N_e of microorganisms present in test sample as arithmetic mean of colonies on 2 plates via -

 

N_e = S/(V x n x d)

S=sum of colonies on 2 plates, V= volume of inoculum applied to each plate in mls, n=number of plates, d= dilution factor of initial suspension [=1 when undiluted liquid is used]

eg at a first dilution (10^-2), 12 and 13 colonies counted so -

 

N_e = (12+13)/(1 x 2 x 10^-2) = 25/0.02 = 1250 > 1300 cfu/ml

In almost all cases, this calculation would obviously fail your limit.

Most (other) APC Standards afaik would consider the result of 0/30 colonies unacceptably divergent and recommend to repeat.

 

MPN methodology using 3 x 5 tubes can also achieve < 1 cfu/ml in a more precise way than Pour Plate if so desired.

 

Milk not my area of expertise  but I noticed that yr APC limit for "condensed milk" is 10x that in US-58.938 (sweetened condensed milk).

[kumar7]

Hi Kumar,

 

Re ^^(red) - I assume this was sterilized milk.

This pour plate method is presumably a "rapid" test to (hopefully) result in no colonies on the plates. 

IMEX 0/30 would be typically, politely, interpreted as a "Laboratory Accident" (ie somewhere a gross error). > Need to repeat.

 

ISO 7218, 2001 is afaik unique in proposing that in case of 2 plates (test sample or first dilution) containing less than 15 colonies, calculate the estimated number N_e of microorganisms present in test sample as arithmetic mean of colonies on 2 plates via -

 

N_e = S/(V x n x d)

S=sum of colonies on 2 plates, V= volume of inoculum applied to each plate in mls, n=number of plates, d= dilution factor of initial suspension [=1 when undiluted liquid is used]

eg at a first dilution (10^-2), 12 and 13 colonies counted so -

 

N_e = (12+13)/(1 x 2 x 10^-2) = 25/0.02 = 1250 > 1300 cfu/ml

In almost all cases, this calculation would obviously fail your limit.

Most (other) APC Standards afaik would consider the result of 0/30 colonies unacceptably divergent and recommend to repeat.

 

MPN methodology using 3 x 5 tubes can also achieve < 1 cfu/ml in a more precise way than Pour Plate if so desired.

 

Milk not my area of expertise  but I noticed that yr APC limit for "condensed milk" is 10x that in US-58.938 (sweetened condensed milk).

 

Hi Charles,

 

Thank you for the detailed explanation. Much appreciated.

Noted on the points you have highlighted.

 

I believe that repeating the test if such result is obtained is the safer option here.

 

Regards,

Kumar

[fahimabbasreza]

https://ileanafilio....doanh-viet-anh/ https://ileanafilio.com/tu-dong-viet-hoa-dau-dong-trong-excel-2007/ https://ileanafilio.com/tai-bai-hat-yeu-lai-tu-dau/ https://ileanafilio.com/phan-ky-dau-tu-du-an/ https://ileanafilio.com/su-lua-chon-so-phan-tap-62/ https://ileanafilio.com/cong-ty-co-phan-thuong-mai-dau-tu-va-phat-trien-cong-nghe-sctt/ https://ileanafilio.com/karate-co-nguon-goc-tu-dau/ https://ileanafilio.com/tap-doan-co-khi-xay-dung-thuong-mai-dai-dung/ https://ileanafilio.com/cach-tinh-dau-tu-tai-san-co-dinh/