Methodology

6.2.1 Handling Flocs for Microscopic Examination

Due to their size, location and relative fragility, river, lake, or marine flocs are difficult to examine under in situ conditions. Lotic aggregates are often sampled in bottles with, for example, one or two liter volume. Similarly, lake or marine flocs maybe sampled directly into special containers by scuba divers.19 However, within 30 min, these sampling procedures will result in settling and co-aggregation of smaller flocs into larger loosely associated aggregates of several 100 ^m diameter thus analyses of these specimens are extremely time sensitive. Leppard20 reported the occurrence of artifactual aggregation where small aggregates combine to yield a few large aggregates. In addition, it was noted that rough handling (high flow, centrifugation) storage longer than 24 h, and most concentration steps will all result in coagulation of the flocs.

In order to maintain structural integrity of the sample some care must also be exercised in the preparation for microscopic examination. In general, biofilm and floc samples are exposed to physical stress in the real-world environment, therefore in most instances they are resilient enough to be manipulated and mounted for staining and observation. However, laboratory treatments such as drying, freezing, washing, dehydration etc. will all perturb the native structure of the floc. Leppard,20,21 Leppard et al.,22 and Droppo et al.23 provide useful instruction on the handling of flocs for microscopic examination and preservation of their native state and properties. Staining may be carried out by careful addition of the stain and its withdrawal using tissues or small sponges, with subsequent replacement and washing with sterile medium (variously 3x to 5x) or environmental water (river, lake, pond, etc.). In some instances removal of excess stain must be carried out by centrifugation of the floc and resuspension in stain/probe free water. Only careful evaluation can determine at what point these treatments will alter the floc under investigation and this should be assessed for each type of floc examined. Conventional wet mounts and other slide preparations may also be usefully performed to examine flocs.24 Floc or aggregate samples may be fixed to the bottom using flowable silicon adhesives or allowed to settle to the bottom of a small petri dish (diameter 5 cm). In these cases an upright microscope may be used to examine the preparation. In the case of flocs an inverted microscope in combination with a settling chamber having a cover slip bottom such as those provided by NalgeNunc International, Denmark, may be a preferred method of preparation for 1-photon laser scanning microscopy (1P-LSM), 2-photon laser scanning microscopy (2P-LSM), or fluorescence microscopy.1 Although if an inverted microscope is used, access to the sample is limited and the working distance of the objective lens may further limit examination of the material. It is also possible that lotic aggregates be collected directly in the LabTek coverslip chambers (NalgeNunc International). By this sampling procedure the settling and co-aggregation of small flocs is kept to a minimum. Subsequently, the aggregates can be microscopically examined using LSM for reflection signals and autofluorescence (general, algal, cyanobacterial). In addition, flocs may be stained inside the chamber using nucleic acid specific stains to record bacterial distribution and fluorescently labeled lectins to record glycoconjugate distribution.

In the case of synchrotron based imaging such as scanning transmission x-ray microscopy (STXM) the sample must be prepared on an x-ray transparent holder. STXM measurements must be performed with the sample in a wet cell constructed with a silicon nitride window (Silson Inc, Northampton, U.K.) by placing the sample onto one half of the silicon nitride cell and sealing it with the other half. Figure 6.1 shows a typical completed wet cell with enclosed biofilm material. The wet cell is then placed directly in the beamline for imaging.25,26

6.2.2 Epifluorescence Microscopy

Conventional widefield epifluorescence microscopy provides simple effective means to examine the results of most staining of the exopolymers of microbial cells, flocs, and biofilms provided a suitable range of optical filters are available. Optical sectioning may be achieved using epifluorescence, a stepper motor, and a digital video imaging device. The major limitation of the image series collected is poor axial resolution, however, this may be improved by computing intensive restoration procedures or deconvolution (see Section 6.2.3).

6.2.3 CLSM and 2P-LSM

Confocal laser scanning microscopy (CLSM or 1P-LSM) has become an indispensable technique for the study of interfacial microbial communities.27 This is particularly due to the increasing number of fluorescent stains and reporter

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