How to do challenge testing on L.Monocytogenes

[redfox]

I need to do a challenge test on L.Monocytogenes in guacamole. I'm to reduce the CFU/g through changing temperature and pH (the primary focus is on pH). Only problem is - I've never done a challenge test before.

Can anyone help me out and point me to a list of steps I need to take to do the tests please? I've searched the net for quite some time but the information isn't terribly relevant.

 

Thanks:)

[CharonY]

You need to establish a protocol to treat your cultures in a standardized way. Mostly you would e.g. dilute your bacteria into a buffer with the respective pH or use the same cell number and heat them and then plate aliquots on a plate to check viability.

[redfox]

Ok, thanks for your reply. I've started to write up the protocol. However I have a few more questions now!

 

How do I know how what inoculum level to use and when do I take the L.m for inoculation - at lag, log or stationary phase?

 

How many repetitions must I use?

 

What should the intervals of testing be for a product of 30 days shelf-life?

 

I'm sure there must be articles with information out there but I've been looking for 3 days now and can't find anything on the lag/log/sta...

and everyone seems to say 'use 3 reps minimum' but I need a little more info than that. Unfortunately I must present the final plan before

I can do any testing so there's not much room for error here!

 

Thanks again:)

[CharonY]

Some short answers:

Growth phase: often (but not always) the log phase is taken as the bacteria show highest viability and activity here.

 

Repetition: as high as possible. You need sufficient for statistical testing. The at least 3 is a lousy rule and it is there as with 3 you can calculate an average and e.g. make a t-test. But I would try as many replicates (ideally biological) as possible. Time constraints should be the limiting factor here.

 

I cannot really comment on the interval, though. At the very least you need the t0 (i.e. time defined as zero point), and at the end of the 30 days. Most likely in the middle and maybe a week after the 30 days would add to the safety margin.