fatty acid extraction

Hemant Chikarmane hchikarmane at mediaone.net
Wed Jan 13 03:11:52 EST 1999


On Jan 12, 1999

Jaime Finguerut wrote:

> ... Coincidentally I asked a question in another list (Lactic Acid) about
> the possibility of using fatty acid profiles for direct ID or
> detection of bacteria in natural mixtures of bacteria and yeast that
> occurs in our industrial alcoholic fermentation.
> I mean, is it possible to extract the fatty acids directly from a
> sample taken from the fermenters and have a positive ID or detecting
> the presence of a bacteria even when there is a lot of yeast
> together?
> One researcher of the LAB list say that it is possible and he pointed
> out a firm (MIDI?). Do you know it?...

A colleague of mine and I spend a good bit of time with MIDI procedures and
software for bacterial identifications.  Our interest was marine bacteria,
but based on our results, here are some observations:


1.  Mixtures of organisms.  If you examine Fatty Acid (FA) profiles, there
are (sometimes) diagnostic peaks which are indicative of a particular
species.  You may be able to use these to determine the presence of that
species in the mixture.  More often, ratios of FA are indicative of specific
organisms, but in a mixture, you would be hard pressed to tell the
difference.

2.  Remember that the FA profiles are generated by growing cells under
defined conditions (medium, temp, time) before FA extraction.  However, the
FA profile that you will get will likely be slightly different than when
cells are grown under MIDI recommended conditions.  You could pellet the
cells, arnd then extract.

3.  Reliability:    In one epidemiology project I worked on, major bacterial
isolates were obtained from diseased lobsters over a period of 4 months in a
mariculture facility, and keyed as Pseudomonas.  The four isolates were
grown, fatty acids (FA) extracted, and FA profiles were obtained by GC.
      The FA profiles of the isolates were identified by the software as
three isolates of  P. putida, and one as P. fluorescens.  The "P. putida"
identification scores were between 0.6 and 0.8, with a P. fluorescens score
which was close enough to cast doubts.  The "P. fluorescens" isolate had
score of 0.8, with P. putida as second possibility.

When RAPD-PCR fingerprinting was performed with four different primers, it
turned out that the 4 isolates were in fact IDENTICAL, never mind the MIDI
id's, and confirming our suspicion that a specific strain of the pathogen
was established in the mariculture facility.

So with a view to identifying STRAINS, you will have a lot of trouble.  I
feel that MIDI  id's are better for genus level, than species or strains.


4.  MIDI profiles may be OK with known bacterial genera/species.  If you are
dealing with natural strains, you may have new species/strains which the
software will likely gag on.

5.  Analysis - A great problem with the MIDI profile database is that each
entry is an "averaged" profile for the species, i.e. a number of isolates
for that species are run through, and an average profile is generated.  This
ensures (a) that strain identifications are impossible, and (b) that you,
the user, cannot try other pattern matching or statistics to confirm MIDI's
determination of the id.


These are just the major gripes, so I'll stop here.  If I were you, I would
use PCR with species-specific primers, and dump MIDI.

Regards,



Hemant Chikarmane

hchikarmane at mediaone.net
hemant at mbl.edu



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