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Microwave Processing of Pneumocystis carinii for Electron Microscopy

Although improvements have been made in processing for transmission electron microscopy (“EM), ultrastructural details of many Pneumocystis features frequently are poorly preserved. Improved TEM resolution could better monitor structures altered by drug exposure, development, and pathogenesis processes. Microwave energy has been used to accelerate a number of types of chemical processes. The principles behind its use are not well understood, and there is dispute as to whether it accelerates biochemical processes such as fixation through simple heat or through other mechanisms (12). The use of microwave energy during fixation, dehydration, and infiltration steps significantly reduces processing time for TEM. However, the control and reliability of microwave-enhanced fixation has been problematic and there are disputes about whether there are other advantages to microwave fixation or processing, including enhanced preservation of some types of tissues (1 2). Recent publications have suggested that improvements in microwave power regulation, sample tube configuration, placement of samples in “cold spots” of the microwave oven and temperature regulation (cooling and maintaining low heat) can improve the reliability of microwave fixation and processing (12). For relatively impermeable cells with high levels of hydrolases like Pneurnocysfis, rapid processing with reduced heat may be expected to improve ultrastructural details, so preliminary comparisons of microwave-coupled and conventional fixation were performed. MATERIALS AND METHODS. After ketamine anesthesia and killing by euthanasia and cervical dislocation, lungs of dexamethasone immunosuppressed transtracheally infected rats 131 were dissected and minced in Hank‘s buffered saline. Duplicate lung fragments were placed into Kamofiky’s fixative, one was processed as previously described (1) in 0. 4 ml in 1.5 ml Eppendorftubes and fixed at 4C for 2.5 to 96 hours. Duplicate pieces of tissue were placed into Karnofsky’s fixative and processed in parallel, except that some samples were placed in either conventional or EM microwave ovens and exposed to 15 to 60 seconds of microwave fmtion at 15-37C, in an area of the microwave with less energy, and in the presence oftwo 1-liter beakers of water as microwave dampeners. Additionally, short-term cultured organisms (4) were processed using microwave treatment after overnight incubation in either Hank’s saline or Karnofsky‘s fixative at 4C as previously described (9). Embedding was done using either Epon or Epon-Spur’s 1:l mixture. RESULTS AND DISCUSSION. Approximately 400 trophozoites and 70 cysts were examined from microwave-coupled and conventionally fixed samples. Full microwave processing was rapid ( 3 4 hrs) compared with conventional processing (2 days). However, because each step was monitored, there was more hands-on time for microwave processing relative to controls which used automated dehydration and embedding steps. In total, several thousand cells were examined. In the distinct areas examined for a given sample, there was significant variability in the apparent quality of fixation as monitored by cytoplasmic and membrane integrity and other features, for both microwave-coupled and conventionally-processed samples. Also, a limited number of independent samples were microwave-processed soon after immersion in fixative. With the available samples, it was not possible to determine whether conventional or microwave samples were better preserved but potential differences if any, were slight. In general, near-equivalent ultrastructural details were observed for both normal and microwaveprocessed samples, similar to those previously reported (5,6). Interestingly, trophozoites seem relatively less well preserved than cysts, although the latter have thicker walls. Cultured organisms, even those mailed overnight on ice, displayed comparable ultrastructural details relative to those fixed immediately from rat lung. Typical trophozoites from conventional and microwave-coupled processing are shown (Fig. 1). More detailed blinded studies may allow more definitive comparisons.