Environmental Engineering Reference
In-Depth Information
medium
0
P. borealis
-2
E. coli
-4
-6
-8
-10
2000
2500
3000
3500
4000
4500
Time (sec)
Fig. 2.4 Representative thermal profiles showing the heat of fusion in 2 ml samples. A reference
thermistor recording (straight, thin black line) shows the temperature of a control sample containing
ethylene glycol. P. borealis cultures (approximately
1
10
-8
CFU/ml) show freezing beginning at
-2.58C. Culture medium and E. coli cultures (at
1
10
-8
CFU/ml) show freezing at -7 and -88C,
respectively, ([50] reproduced by permission of The Royal Society of Chemistry)
in distinguishing differences between lysates. This protocol has been used to
successfully identify more than a dozen bacteria with putative AFPs, including
those from the genera Idiomarina, Rhodococcus (an Actinobacteria), Pseudomonas,
Bacillus and Marimonas [7]. One of the latter isolates has now been extensively
studied and the Ca
þþ
-dependent AFP has been purified and described [28].
This screening technique is also useful when used as an assay for the char-
acterization of microbes isolated by selective regimes; we have used it to
examine Chryseobacterium isolates identified after cryocycler selection as well
as ice-affinity selection. IR inhibition activity is apparent in these (Fig. 2.5), but
after treatment with protease, activity was destroyed [27]. This suggests that
these particular microbes may have AFP activity.
1
2
3
4
5
6
7
8
9
Fig. 2.5 Inhibition of ice recrystallization. Screens are usually conducted in microtiter plates,
but samples in microcapillaries held at -68C and examined between crossed polarizing filters
more easily demonstrate this principle. From left to right samples include sample buffer controls
(1, 2), serial dilutions of 0.2, 0.02 and 0.002 mg/ml AFP solutions (3-5), Chryseobacterium
sp. C14 cultures (6, 7), and E. coli cultures (8, 9). Note that only the AFP controls and the
Chryseobacterium cultures have crystals too small to be observed at this magnification and the
overlying 'feathery' pattern, seen in rapidly frozen samples, is apparent. Bacterial cultures
were at
2
10
-8
CFU/ml (adapted from [27])
