Environmental Engineering Reference
In-Depth Information
2.3.2 Ice-Affinity Selection
Another new method of microbe selection takes advantage of the principle that
when ice grows slowly it excludes solutes. Charles Knight devised methodology
some years ago to examine the adsorption of proteins on the faces of ice [37] and
this was subsequently tailored to allow ice-affinity purification of AFPs [38].
A modification of this procedure has now been adopted for the selection
of microbes that are included into a growing elongated hemisphere of poly-
crystalline ice. The apparatus consists of an 'ice finger' or brass cylinder in
which temperature-controlled ethylene glycol solution is circulated (Fig. 2.3).
The sample beaker contains dilute cultures initiated from sites of interest.
The sample is maintained just at the freezing point and frost is allowed to
accumulate on the cold finger by water condensed from the air. After lowering
the cold finger into the sample solution, an ice ball or 'Popsicle' is formed. It is
important to grow the ice slowly (about 0.01 ml/min) over a 24-48 h period.
After approximately 50% of the culture is frozen, the ice is rinsed and then
melted to obtain the microorganisms that partition into the ice phase. Indivi-
dual colonies can be obtained after limited dilution or by culturing on nutrient
plates.
In experiments where cultures were initiated with single isolates, E. coli
numbers were reduced four orders of magnitude in the melted ice fraction
compared to the original sample [39]. In contrast, when a Chryseobacterium
isolate, originally discovered after cryocycler selection, was subjected to ice
affinity, the recovery from the ice represented a reduction of only two orders
of magnitude in cell number. Although this strain of Chryseobacterium is 5-6
fold more freeze-tolerant than E. coli after a single freeze period, the observed
differential recovery encouraged us to use ice affinity to fractionate cultured
communities in order to reduce both the complexity of the original population
Fig. 2.3 Diagram of the ice finger apparatus fabricated to select microbial cells with an
affinity for ice. Small arrows show the flow pattern of chilled ethylene glycol through the
ice finger. The sample in the beaker is stirred with a magnetic stir bar while ice forms on the
finger (modified from [38])
