9 replies to this topic

[lilii]

Hi everyone,

 

I have a question about the presence of coliform bacteria.
Safety data sheet lauryl sulphate bulion with durham tubes says about the presence of coliform when gas appears.
Sometimes the gas present in the durham tube, after inverting the sample, escapes. Is this a positive result?

[Charles.C]

Hi everyone,

 

I have a question about the presence of coliform bacteria.
Safety data sheet lauryl sulphate bulion with durham tubes says about the presence of coliform when gas appears.
Sometimes the gas present in the durham tube, after inverting the sample, escapes. Is this a positive result?

 

Hi Lilii,

 

The text is slightly unclear.

 

I guess you mean use of lauryl sulphate tryptose broth (LST).

 

This (evidence of gas) is a presumptive test for coliforms using a standard method.

 

Presumptives require confirmation.

 

eg see this FDA procedure -

 

https://www.fda.gov/...tm#conventional

[lilii]

hi,

We have a standard in which we add 1 ml of the initial suspension to three tubes of lauryl sulphate, incubate at 30 ° C and then read the results for the presence of coliforms.
A positive result is turbidity and / or gas. Then we confirm it on the ground with brilliant green.
Only sometimes the gas appearing in the tube of Durham escapes and I do not know if this is a positive result.
When there is cloudiness and the gas that is embedded in the tube is a positive result.

[Charles.C]

hi,

We have a standard in which we add 1 ml of the initial suspension to three tubes of lauryl sulphate, incubate at 30 ° C and then read the results for the presence of coliforms.
A positive result is turbidity and / or gas. Then we confirm it on the ground with brilliant green.
Only sometimes the gas appearing in the tube of Durham escapes and I do not know if this is a positive result.
When there is cloudiness and the gas that is embedded in the tube is a positive result.

 

Hi Lilii,

 

Yr procedure seems unusual. Usual incubation temperature is 35 - 37 degC ?

 

Is there a reason for yr lower temperature ?

 

eg -

Coliform bacteria are defined as rod-shaped Gram-negative non-spore forming and motile or non-motile bacteria which can ferment lactose with the production of acid and gas when incubated at 35–37°C. They are a commonly used indicator of sanitary quality of foods and water.

 

I am also guessing yr incubation time is 24 hrs ?  If so, i suggest continuation to 48 hrs and check again (see my previous link).

 

Personally i routinely use 48 hrs since IMEX it helps to give more definite results.

 

Maybe the "decision" also depends on the Product type,  specification, and absolute level of Coliform ?

[lilii]

Thanks for the answer,

In our procedure, the incubation takes 24 h /30^oC. If there is cloudiness and/or gas, then sift to the ground with brilliant green.
If there is no turbidity or gas, we incubate another 24 hours, which is a total of 48 hours.
Next time I will try to incubate at 37^oC.

[liberator]

Incubation temp i.e. 30°C or 37°C will depend on what organism you're interested in.

 

i.e. coliforms (30°C), E.coli (37°C).  If you're interested in both organisms, then incubate at 37°C, just coli, then 30°C, and continue onto confirmation step according to the organism you're interested in after 24/48 hours.

 

The standard method for coliform/E.coli/bacterial test, pres/abs  is, for Australia, AS 1766.2.3. Not too sure what the ISO standard number is. Tubes can be incubated at 30°C for up to 48 hours (E. coli 37°C). Tubes can checked at 24 hours and then if gas present in the Durham tube, onto confirmation step. If not then continue to incubate up to 48 hours and confirmation step where gas is present in the Durham tubes.

 

Presence of gas = presumptive coliforms. (typically 2/3 tubes with gas = presumptive +ve, 1/3 not considered to be presumptive. It's not always easy to see gas due to size of gas bubbles and turbidity of tubes. If time is not a factor then incubate to 48 hours and leave the 24 hour check - unless results need to be known sooner rather than later.

[Charles.C]

Incubation temp i.e. 30°C or 37°C will depend on what organism you're interested in.

 

i.e. coliforms (30°C), E.coli (37°C).  If you're interested in both organisms, then incubate at 37°C, just coli, then 30°C, and continue onto confirmation step according to the organism you're interested in after 24/48 hours.

 

The standard method for coliform/E.coli/bacterial test, pres/abs  is, for Australia, AS 1766.2.3. Not too sure what the ISO standard number is. Tubes can be incubated at 30°C for up to 48 hours (E. coli 37°C). Tubes can checked at 24 hours and then if gas present in the Durham tube, onto confirmation step. If not then continue to incubate up to 48 hours and confirmation step where gas is present in the Durham tubes.

 

Presence of gas = presumptive coliforms. (typically 2/3 tubes with gas = presumptive +ve, 1/3 not considered to be presumptive. It's not always easy to see gas due to size of gas bubbles and turbidity of tubes. If time is not a factor then incubate to 48 hours and leave the 24 hour check - unless results need to be known sooner rather than later.

 

Hi Liberator,

 

I agree yr initial comment. As you probably know there is a (ongoing) historical encyclopedia on the subject of "Coliforms". Started around 100 years ago.

 

To illustrate the reason for the (probably) infinite discussion here are 2 quotes from Compendium of Methods for Microbiological examination of Foods, 4th ed,2001 (there is, I think, a later edition but i anticipate that situation not significantly different) -

 

The coliform group is defined on the basis of biochemical reactions, not genetic relationships, and thus the term "coliforms" has no taxonomic validity. Coliforms are aerobic and facultatively anaerobic, gram negative, non-sporeforming rods that ferment lactose, forming acid and gas within 48 hours at 35 degC. An incubation temperature of 32 degC is usually used for dairy products.

 

 

"Coliforms"  and "Faecal Coliforms," practically speaking, are those microorganisms that are detected by the "coliform test" and the "faecal coliform test," respectively.

 

Dairy not my area of expertise but the reason for the differences in incubation temperature are probably, primarily, that it optimizes  "bacterial" growth. For example see -

https://www.ncbi.nlm...les/PMC1558775/

(eg pg 3)

also can see this dairy article -

https://www.ncbi.nlm...les/PMC5043024/

 

The general result is that Globally procedural variations do undoubtedly occur.

 

I have unfortunately no idea as to Slovenian Standard Procedures (if any) for the (unknown) product.

 

Regarding Australia, I have Coliform Standard AS 1766.2.3 - 1992 (latest ?) which has the following text -

 

Where the test is to estimate the level of coliforms only as an index of potential spoilage of the product and of hygiene, an incubation temperature of 30degC is specified. However, where the test is to be extended to include the estimation or detection of E.coli, incubation at 37degC is specified. Cultures which have been incubated initially at 30degC cannot be used for further tests for E.coli.

(I daresay the justification for this general use of 30degC is available somewhere (?) but so far I have only seen it (generally) applied in Australia. But it's a big World and this measurement as illustrated above is, in Principle, self-definable. I have personally had numerous data arguments with customers who used their own shortcut modifications rather than an official Procedure).

 

PS - afaik ISO used to use 37degC but currently appear to (ingeniously) sit on the fence, ie -

 

 

ISO 4831:2006(en)

Microbiology of food and animal feeding stuffs — Horizontal method for the detection and enumeration of coliforms — Most probable number technique

 

Enumeration is carried out by calculation of the most probable number (MPN) after incubation in a liquid medium at 30 °C or 37 °C.

NOTE The temperature is subject to agreement between the parties concerned. In the case of milk and milk products, the temperature of incubation is 30 °C.

 

3   Terms and definitions

For the purposes of this document, the following terms and definitions apply.

3.1

coliforms

bacteria which, at the specified temperature (i.e. 30 °C or 37 °C, as agreed) cause fermentation of lactose with the production of gas under the test conditions specified in this International Standard

 

 

[liberator]

Thanks Charles,

I agree too may variables with this test and it all comes down to what you or the customer is requesting. I've always argued the PRES/ABS test really is not too helpful when you're trying to make decision about what to do with product.

 

I think if you're doing a coliform test then you should always incubate at 37°C. Then when/if you get a presumptive positive test you can confirm for both coliforms and E.coli. I think we used 30°C as we were more interested in a coliform count and not too worried about E.coli, but again this comes down to our customer requirements.

 

With the pres/abs test yes, you have coliforms/E.coli shown as being present but it doesn't really tell you how many. We can go to the MPN test, either 9 or 15 tube - which gives you an "estimated" statistical count based on the number of tubes showing or not showing gas and you can then determine the statistical number of presumptive coliforms present in the sample but if you get a result of <0.3 coliforms cfu/g or ml what does that really mean? (in reality zero, as your tubes are not present for gas - but the result can't be recorded as zero.) You will still need to complete confirmation testing using this test as well.

 

My preference for coliform testing has always been plate count methods, you get actual counts.  Petri film has certainly advanced that method with is chromogenic agar and overlay to show gas bubble production. Actual counts to me are a more robust  result than the pres/abs methodology inc. MPN.

 

Thanks for the links - interesting reading and one to quote is also so very true of the Australian Dairy Industry:

 

"However, coliform testing remains a cornerstone of microbial testing in the U.S. dairy industry, from raw milk testing to processed dairy product testing. Recent studies provide evidence that coliform testing should be reconsidered as a marker for unsanitary conditions in the dairy industry as further understanding of this diverse group of microbes is achieved."

 

 

Things can move quite slowly in the dairy industry so I wonder if I'll see a move away from coliform testing for assessing food hygiene? I don't think I'll see a change in testing in what's left of my working career.

[Charles.C]

Hi Liberator,

 

Thanks yr comments.

 

Regarding plate count methods, I imagine the VRB 24 hr procedure(s) is still the most popular plate method however IMEX this can give significantly high results for certain food matrices if no colony confirmation step is carried out.. The latter adds time of course.

 

To stir the pot a little more, I just noticed this intriguing summary (> some support for Australia) in the 3M interpretation document for their Coliform/VRB Petrifilm method -

 

 Coliform Incubation temperatures-times.PNG   93.94KB   5 downloads

 

And this additional comment in same document -

The definition of coliforms may vary with country.

[lilii]

Hi,

 

I tried to incubate at 37^oC. Sometimes I incubate medium as a control ang gas bubbles appear in Durham tube. For example now I have turbidity and a small gas bubble that escapes after inverting the test tube (incubated 72h). It is unambiguous when there is turbidity and gas bubbles visible in the tube that doesn't escapae after inverting tube.