semi-automated of fully automated sample processing.
The software can be linked to existing laboratory data
systems, take over sample data from them and subsequently
provide the results for further processing.
For the test procedure, the milk samples are pasteurized
at 80 °C in a water bath for 20 min and mixed with the
specific nutrient medium. Subsequently, the individual dilutions
are pipetted into the 96 wells of a microtiter plate and
the plates are incubated in a sufficiently large anaerobic jar
at 37 °C for 48 hours. After incubation sample evaluation
and calculation of the spore count will be performed by the
means of the AmpScan evaluation unit.
Depending on the number of tests, the procedure can
be carried out semi-automatically using the SY-LAB AMP-
6000® APS pipetting robot (up to approx. 180 samples/day)
or fully automatically with a Xantus full robotic system from
Raudszus Electronics (up to 400 samples/day) respectively.
Detection of very low spore
The new method achieves a lower detection limit of 75
spores per litre in the basic version with three different
sample dilutions and 32 replicates per dilution used. In this
configuration the test is already significantly more sensitive
than the conventional MPN methods used for raw milk
testing (lowest detection limit 180 spores/litre, see Tab. 1).
However, the new Mini-MPN application can easily be
scaled up to analyse up to 96 replicates per milk sample. As
a result, the detection limit can be reduced to 14 spores per
litre. In this configuration, even cheese milk can be analysed
with high sensitivity very easily and rapidly.
For users who want to test for cheese spoiling clostridia
only a few samples per day or just occasionally, an instrument
independent but nevertheless very simple manual
pipetting variant of the method is available (Fig.3). Sixteen
replicates with a detection limit of 86 spores/litre are examined
and the colour change is determined visually. The
spore count can be easily retrieved from a simple table
based on the number of tubes with colour change.
Praxis results from Italy
Using the new enumeration method a regular routine monitoring
and rapid diagnosis of the supplier milk for cheese
making dairies is now possible with justifiable effort. The
technique is used since autumn 2017 at the Sennereiverband
Südtirol in Bolzano in the routine testing of the supplier's
milk." Due to the newly possible weekly monitoring
of the Clostrida spore content of the suppliers milk accompanied
by consulting for the farmers, the spore contents of
raw milks from farms with silage feeding could be reduced
since the method was introduced," reports Andreas Österreicher
from Sennereiverband Südtirol.
Regular testing and comprehensive supplier consultation
thus verifiably will lead to a quality improvement of
raw milk in terms of contamination with anaerobic spore
formers. By the new detection method a very efficient tool
is now available for that purpose.
The method is already used in eight European Countries
replacing the conventional MPN method. In 2018 the
method was honoured with the Innovation Award of the
district of Lower Austria as well as with a nomination for
the Econovius 2019 in the context of the Austrian State
Award for Innovation.
Moreover, many additional quantitative microbiological
parameters can be determined using the AMP-6000®
platform. The AMP application for the enumeration of
the total aerobic mesophilic counts in various food matrices
has already been validated by Microval according to
ISO 16140-2 (2016).
1 Brändle, J. et.al.: Relevance and analysis of butyric acid
producing clostridia in milk and cheese. Food Control 67
2 Brändle, J. et al.: A critical assessment of four most
probable number procedures for routine enumeration of
cheese-damaging clostridia in milk. International Dairy
Journal 73 (2017), 109-115
3 Brändle, J. et al.: Novel approach to enumerate clostridial
endospores in milk. Food Control 85 (2018) 318-326