Materials Needed For Preparing Samples

First and foremost, all samples should be prepared in a clean area, preferably in a containment hood to avoid any contamination. The slides prepared from the environmental samples are temporary or semipermanent, and may last for only several days to less than 3 months. The rule of thumb is that a freshly prepared sample will always yield a better image and is much easier to examine for minute fungal structures. In addition, the sooner the slides are analyzed, the possibility of deformation or degradation of the fungal structures from water contained within the spore or fungal structures will be lessened. Crystals may also form in the sample from the combination of stain, mounting medium, and/or the sticky compounds used in the commercial sampling devices, such as Air-O-Cell cassettes.

Basic materials needed for preparing spore count samples include a pair of pointed forceps, filter forceps, small stainless-steel scissors, inoculating/dissecting needles, 25 x 75-mm plain or frosted slides, 22 x 22-mm coverslips, bottles of stock solutions of stains and mounting media (e.g., lactofuchsin, aniline blue, cotton blue), a 15-30-mL drop dispenser bottle for each mounting medium and/or stain, or a 1-mL syringe containing mounting medium and/or staining agent, immersion oil type A, 70% ethanol or isopropyl alcohol, clear nail polish, a fine or extrafine permanent marker, alcohol wipe pads, scalpel with disposable blades, and a cardboard slide container or a slide box.

Some environmental laboratories provide prepared or coated slides for use with Burkard or Allergenco air samplers. Some investigators, who conduct on-site investigations, prepare their own slides. Several coating materials are commonly used, including petrolatum or Vaseline-based high-melting-point wax (VHW) mix, mixed cellulose ester (MCE) cleared with triacetin solution, single- or double-sided clear adhesive tape, silicone in xylene 261, silicone grease 60, and Lubriseal stopcock grease 46. More detailed information about coating materials can be found in the AIHA Green Book.31

For samples collected on 25 x 75-mm-coated slides, the samples should be numbered and labeled on the uncoated end or frosted area first. Before a slide is prepared, the spore deposit area (known as a "trace") should be outlined with a permanent marker on the uncoated side of the slide. One or two drops of the mounting medium are gently applied to the trace in a manner that avoids disturbing the fungal spore deposit. A 22 x 22-mm coverslip is then placed over the mounting medium on the trace to avoid creating or trapping air bubbles. Let the coverslip settle down naturally to spread the mounting medium over the whole trace. Do not push the coverslip down with a pen or any other instrument. Pushing down the coverslip may lead to splitting the mounting medium and/or stain or smear the sample trace. It is not necessary to fix fungal spores prior to the staining procedure.

For samples collected with Air-O-Cell cassettes or similar devices, make sure that each sample has a matching labeled slide with the appropriate information obtained from the "chain of custody." Cut the sealing tape around the cassette and pry it open. This can be safely done with a stainless-steel weighing spatula. Mark the spore deposition trace on the underside of the slide with a permanent

4.6. STAINING AND MOUNTING TECHNIQUES 83

marker. If the sample is to be prepared with collection side up, place a drop of clear nail polish on each of the four corners of the sample slide. With a pair of recently alcohol-wiped forceps, carefully remove the slide from the cassette, turn it over, and place it collection side up on a 25 x 75-mm slide. The nail polish on the corners anchors the sample to the slide. One or two drops of mounting agent can be gently applied onto the trace area. Afterward, a 22 x 22-mm coverslip is placed over the mounting agent and the trace. Although it is possible to prepare a slide sample with the collection side down, it is better to place the collection side up to achieve optimal resolution and clarity. Some laboratories use a simplified preparation method and mount the samples with the collection side down. This method does not require the use of nail polish and a coverslip. The coating material on the sample can anchor itself on a slide. The sample slide serves as the coverslip. This preparation and mounting method greatly reduce the working distance between the sample and the objective lens due to the thickness of the coating. Although it is possible to have a reasonable resolution with a 60x objective, it is impossible to use a 100 x lens to observe fine details when such an observation is necessary to properly identify some small spores.

When a sample is collected with a 25-mm mixed cellulose ester (MCE) filter cassette, the filter is removed from the cassette with the filter forceps and mounted on a slide. A clearing agent (such as triacetin) containing a dye is applied to the filter. Again, this process should be conducted in a clean area (e.g., in a hood or a clean Petri dish) to avoid contamination. The clearing process may take approximately 10-30 min. Afterward a mounting agent is applied, a coverslip is gently placed over the sample, and then 25-50% or a fixed area of the filter area is examined. When 37-mm filter cassettes are used for sampling, only a quarter of the filter is gently cut with the alcohol-wiped scalpel blade and mounted on a slide for clearing and staining.

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