7 replies to this topic

[Ulysses]

Hi,

 

Im working on on our shelf life test for our fresh meat. My question is on how to determine the "log" or how to calculate it base on my TPC (total platye counts). see below the part of email I received regarding for the requirement that we need to meet for japanese standard.

 

TPC Standards (Japan):

Greater than Log 8 = Spoiled

Greater than Log 7 = initial stage of spoilage

Log 6 or less = General standard for non-processed food (meat, fish etc.) at the point of sale

Log 5 or less = General standard for processed food at the point of sales

 

Thanks,

 

Ulysses

[gagansaini1]

Hi

 

First thing we need to know is the Dilution Factor, or how much the original sample is diluted:

here Initially 5mL in 45mL = Final Volume / Sample volume = 50/5 = 10.

Then two serial dilutions of 1/100.

Total Dilution Factor = 10 * 100 *100 = 10^5 (10 raise to the power 5)

CFU/mL = cfu/ml = (no. of colonies x dilution factor) / volume of culture plate

= (137 * 10^5)/0.1

=1.37*10^8

So Total colony forming units = 1.37*10^8 CFU/mL

Converting CFU/mL to Log value

For example,

Total colony forming units = 1.37*10^8 CFU/mL and you want to convert it into Log value,

Just take Log(CFU/mL)

Here, log (1.37*10^8) = 8.13924922.

 

Hope this help.......

[Charles.C]

Hi,

 

Im working on on our shelf life test for our fresh meat. My question is on how to determine the "log" or how to calculate it base on my TPC (total platye counts). see below the part of email I received regarding for the requirement that we need to meet for japanese standard.

 

TPC Standards (Japan):

Greater than Log 8 = Spoiled

Greater than Log 7 = initial stage of spoilage

Log 6 or less = General standard for non-processed food (meat, fish etc.) at the point of sale

Log 5 or less = General standard for processed food at the point of sales

 

Thanks,

 

Ulysses

 

It's easy although yr email is in Japanese maths.  By "Log 8" the sender actually meant the number 8.00000. Etc.

 

By common convention you will need a result for product "APC"/"TPC"/"TVC"/ whatever in units of cfu/gram (or maybe cfu/ml). Call it X cfu/gram

 

Then you need a scientific calculator with a "log" function/button. (NOT a "ln" button !)

 

Then, as per calculator manual for using log function, enter X and click "log" (sometimes the reverse, follow the manual)

 

Now compare the result to the values in yr email.

 

As a test check, if X = 1000 cfu/gram, the answer in calculator should be 3.000000etc

Similarly, if X = 100,000 cfu/gram, the calculator should show 5.000000etc

 

PS - Unfortunately the procedures/interpretations for measuring X in Canada and Japan may be different so yr data may actually be OK but not necessarily match their opinions.

[Ulysses]

Hi,

 

I just did some calculation base on what weredoing in our lab. Please check if Ididthe right calculation.

 

Formula:

(sample size)+(diluent) = (total solution) then..

(sample size) / (total solution)= (original sample dilution)

then one serial dilution of 1/10...

(original sample dilution) x (diluent + 1ml concentration) = (Dilution Factor)

then one serial dilution of 1/10  again...

(Dilution Factor) x (diluent + 1ml concentration) = (Total Dilution Factor)

For example:

11g + 99ml =110ml

11g / 110ml = 0.1 or 1:10 total dilution factor

then one serial dilution of 1/10, plus 1ml concentration...

0.1 x (9ml +1ml)= 1 or 1:100 total dilution factor

then one serial dilution of 1/10, plus 1ml concentration...

1 x (9ml +1ml)= 10 or 1:1000 total dilution factor

So, now to determine counts, the equation for this is:

 

CFU/mL=  Count on Plate x dilution Factor

                      Volume Plated on Petrifilm

 

So, say with that same example above with the 11g into 99mL. You plated on AC Petri film and saw 5 counts.  

(5 cfu) x (.1) / (0.1 mL) = 5 cfu/ ml

So Total colony forming units = 5 *10¹ cfu/ml

 

If 1 mL of a 1:10 dilution is plated and yields no colonies, 0 CFU/gram for the original sample can never be a reported value because the entire sample was not tested. By convention, the count is reported as < 10 (< 1 x 10) CFU/gram.

Converting cfu/ml to Log value

For example:

Total colony forming units= 5 *10¹ cfu/ml and you want to convert it into Log Value,

Just take Log(CFU/ml)

Here, log (5 *10¹) = 1.7

 

Thanks,

 

Uly

[Charles.C]

Hi Ulysses,

 

I suggest you work with weights.

 

110ml yr solution X contains 11g sample

So 1ml solution X contains 0.1g sample

So 0.1ml solution X contains 0.01g sample

 

you transfer 0.1ml solution X to petrifilm

So 0.01g sample generates 5 cfu

So 1gram sample is equivalent to 500cfu

so TPC = 500cfu/g

 

I wonder why you use 11g sample and 0.1 ml onto Petrifilm ?. These quantities will reduce the accuracy of result compared to normal protocol values.

You should also do duplicate petrifilms.

[Danica]

Hi :)

 

Here are some links that might help you:

 

https://www.research...fu_for_bacteria

 

http://www.microbiol..._.2011.17_3.pdf

 

In my experience, when doing this type of calculation you work with mL since you usually use liquids and pipets to measure the quantity you inoculate on the plate and when you make you dilutions. Then, based on the starting mass of material you used for your first dilution you can bring it to CFU/mg if that is what you are looking for.

 

We also did more than one dilution per sample, for reproducibility and also you ensure that you have at least one dilution with a reasonable plate count and are able to count isolated colonies to have a precise CFU count. Serial dilutions is the best method for dilutions, always change pipet tips between dilutions it DOES make a difference, trust me on that.

 

I have never heard of calculations for this type of test being different between countries as it is an established scientific method used around the world, as far as I know. I have worked in labs for 15 years and we (as well as read publications) all used the same way of calculation for this type of test (same for yeast CFU, viral PFU, etc).

 

What I would do is:

 

1) 'x'g of sample in a final volume of 10ml (homogenize your sample in a volume below 10ml, then once homogenized adjust the volume to 10ml)

2) then serial dilutions in factors of 10 (I would do 4-6 dilutions for the first tests to give you a ball park idea of what your sample's range is)

3) inoculate 100ul per plate (I like to work in factors of 10, less confusing IMO)

4) always do a negative control with only media to ensure that your technique was aseptic and that the colonies you are counting truly come from the meat and not the environment/reagents.

 

Be sure to bring your volume plated to ml if you want CFU/ml when doing your calculation. Also 1:1000 dilution factor as mentioned in your example is not 0.1, it is 1000.

 

If you only have 5 colonies on your plate, I would not use this dilution as this is not a significant/representative count.

 

If you have zero colonies, I would redo the test using different dilutions/sample mass as this seems unlikely to be true.

 

PS-the only way 11g is 11ml is if you have 11g of water. Otherwise 1ml does not equal 1g.

 

Hope this helps!!

Best of luck to you

 

Cheers

DA

[Danica]

Another thing: I do not understand how you do your serial dilutions.

 

what I would do: 1g of meat homogenized in a final volume of 10ml (final volume, not 1g + 10ml. As mentioned above, homogenize in smaller volume and once the meat is homogenized adjust the volume to 10ml).

 

Dilution A: 100ul + 900ul media (1:10)

Dilution B: 100ul of A + 900ul media (1:100)

Dilution C: 100ul of B + 900ul media (1:1000)

and so on until you find your 'optimal dilution range' for this type of sample.

 

In the end you calculate CFU/ml using your dilution factor, then adjust the results with starting mass/volume to get your CFU/g.

 

So if you used 1g of meat in 10ml final volume, multiply your count: CFU/ml * 10ml/1g (meat)=CFU/g

 

Correct me if I'm wrong but this is the method I always used in the past.

 

Good luck :)

[Charles.C]

Hi Danica,

 

Sorry but i have to disagree with you on this one. Maybe different product types have their own individual characteristics/variations.

 

IMEX bacterial data when compared between labs, and particularly in different countries, is often unbelievably variant. And accepted to be so.

I have had endless arguments over methodological variations between micro. labs/countries due to divergences in procedures/interpretations.

Additionally many in-house labs (and some external ones !) generate their own modifications of "standard" methods to suit their specific product types. These operational shortcuts (eg method steps, small samples, small volumes, no duplicates, one dilution only) can often save time/material/effort for internal work but become unreliable when used outside their validated scope.

 

"Standard" methods also display a bewildering variety of calculation methods for plate counts.

 

I suspect that the well-known nmMc decision procedure was invented to handle the typical scatter in microbiological plate counts. Some EC experts even proposed that  the "M" in this menu  is presented with a (+/-)  acceptance range.

 

My recommendation to Ulysses is that he choose one Standard method to follow. Which one depends on the projected usage, and possibly (local?) Regulatory/customer factors.