Microwaves in the laboratory: effective decontamination.

OBJECTIVE: We hypothesize that microwave irradiation of certain contaminated materials typically used in a clinical laboratory or a home healthcare setting could produce efficient and effective sterilization when compared to standard autoclave methods. DESIGN: A standard household carousel microwave oven unit used at the High setting was employed to expose certain materials that had been contaminated with either bacteria or yeast to microwaves for specific intervals of time. Following each time interval, materials were checked for effectiveness of decontamination using standard culture techniques and colony counting. Additionally, powdered media was prepared and microwave irradiated for specific times. The media was then poured into plates and checked for microbial contamination; another set of plates was examined to determine the ability of the irradiated media to support bacterial growth. SETTING: This study was carried out at Texas Tech University Health Sciences Center in Lubbock TX. MAIN OUTCOME MEASURE: Standard culture and colony counting techniques were used to determine the efficacy of microwave sterilization. RESULTS: The study indicated that microwave irradiation provided effective and efficient sterilization of all materials tested. Of the bacteria studied, only E. coli survived beyond 30 seconds of microwave exposure. Yeast did not survive beyond 15 seconds of microwave exposure. Swabs and gauze contaminated with bacteria or yeast were completely sterilized after 30 seconds. After three minutes in the microwave oven, powdered, prepared media was free of contamination while able to support growth when inoculated with S. aureus. CONCLUSION: We conclude that a household carousel microwave oven unit can provide fast, effective sterilization of certain contaminated materials typically used in a clinical laboratory, student laboratory, or home healthcare setting.

Clinical laboratory radioimmunoassay usage.

OBJECTIVES: To determine the extent of radioimmunoassay utilization in clinical laboratories in the state of Texas; to ascertain what methods have replaced it as an analytical tool; to identify trends and elicit comments regarding attitudes toward radioimmunoassay; and to ascertain the extent of instruction of radioimmunoassay principles required in clinical laboratory science curricula. DESIGN: Mailed, written survey designed by the authors. PARTICIPANTS: Laboratory managers or directors of 203 clinical pathology laboratories in Texas. MAIN OUTCOME MEASURE: Responses to seven forced-choice items, prompting information regarding laboratory type and size, extent of radioimmunoassay use, benefits or radioimmunoassay, and replacement technologies; a single item that elicited responses and opinions regarding general attitudes about radioimmunoassay and its place in clinical laboratory science curriculum. DATA SOURCE: Clinical laboratory managers or directors in the state of Texas. RESULTS: A total of 203 surveys were mailed within 127 respondents, yielding a response rate of 63%. The majority (77%) of clinical laboratories surveyed no longer use radioimmunoassay as a diagnostic tool with the predominant reason being the availability and affordability of automated enzyme immunoassays. Time-consuming recordkeeping was another common reason for abandoning the technique. Enzyme immunoassays were by far the most common method replacing radioimmunoassay. Comments reflected the general attitude that radioimmunoassay is a technique of the past in the clinical laboratory. A variety of views were elicited regarding the education of the principles of RIA to clinical laboratory science students. CONCLUSION: Radioimmunoassay, although a viable assay in some situations, has been abandoned as an analytical tool in most clinical laboratories in Texas. Current users are unhappy with the amount of paperwork that accompanies use of the technology, while non-users consider non-isotopic assays equivalent in sensitivity to RIA. In regard to information presented to clinical laboratory science students, the advent of molecular diagnostic techniques requires continued instruction in the principles of radioactivity, although not radioimmunoassay.

Alterations in actin-binding beta-thymosin expression accompany neuronal differentiation and migration in rat cerebellum.

The beta 4- and beta 10-thymosins, recently identified as actin monomer-sequestering proteins, are developmentally regulated in brain. Using specific mRNA and protein probes, we have used in situ hybridization and immunohistochemical techniques to investigate the distribution of the beta-thymosin mRNAs and their proteins in developing rat cerebellum. Early in postnatal development, both beta-thymosin mRNAs were expressed at highest levels in the postmitotic, premigratory granule cells of the external granular layer; expression diminished as granule cells migrated to and differentiated within the developing internal granular layer. In addition, both beta-thymosin proteins were present in bundles of cerebellar afferent fibers in the white matter at this time. Throughout the maturation period, both proteins were present in elongating parallel fibers in the upper portion of the molecular layer. Later in cerebellar development, thymosin beta 4, but not thymosin beta 10, was expressed in Golgi epithelial cells and Bergmann processes. Thymosin beta 4 was expressed in a small population of cells with microglial morphology scattered throughout the gray and white matter. Thymosin beta 10 was detected in an even smaller population of glia. Expression of thymosin beta 4 and thymosin beta 10 in premigratory granule cells and in growing neuronal processes is consistent with the possibility that both beta-thymosins are involved in the dynamics of actin polymerization during migration and process extension of neurons.

Glutamate immunoreactivity in the rat basilar pons: light and electron microscopy reveals labeled boutons and cells of origin of afferent projections.

Immunohistochemical methods that employed a polyclonal antiserum directed against a glutamate-hemocyanin conjugate were utilized to examine the rat basilar pontine nuclei at both light and electron microscopic levels in order to identify putative glutamatergic neural elements. A large number of cells ranging in size from 11 to 32 microns in diameter and present in all subdivisions and at all rostrocaudal levels of the basilar pons exhibited intense glutamate immunoreactivity. Immunoreactive punctate structures, confirmed by electron microscopy to be axon terminals, were homogeneously distributed throughout the pontine neuropil, although a somewhat greater accumulation was apparent medially at mid-levels of the basilar pons and laterally at more caudal levels. Immunolabeled axons were also present throughout the pontine nuclei. In order to demonstrate possible extrinsic sources of glutamate-immunoreactive axon terminals within the pontine gray, injections of wheat germ agglutinin-horseradish peroxidase were made directly into the basilar pons. Tissue was then evaluated for the presence of retrogradely transported wheat germ agglutinin-horseradish peroxidase and the same tissue sections processed for glutamate immunocytochemistry. Following this combined protocol, neuronal somata exhibiting both wheat germ agglutinin-horseradish peroxidase and glutamate immunoperoxidase reaction products were observed within layer Vb of the cerebral cortex, zona incerta, the dentate nucleus of the cerebellum, nucleus paragigantocellularis of the medullary reticular formation, and the dorsal column nuclei. Such double-labeled cells were considered to represent glutamatergic neurons that provide axonal projections to the basilar pons. Ultrastructural studies of the pontine nuclei confirmed the presence of glutamate immunogold labeling in dendrites, neuronal somata, axons, and axon terminals. Immunoreactive boutons contained round vesicles and primarily formed asymmetric synapses at various postsynaptic loci which included glutamate-immunolabeled dendritic profiles and somata. These results suggest that glutamatergic basilar pontine neurons form one segment of a multisynaptic pathway involving glutamatergic afferents to the basilar pons, glutamatergic pontocerebellar projection neurons, and the glutamatergic granule cells of the cerebellar cortex.

Evidence for the presence of presynaptic dendrites and GABA-immunogold labeled synaptic boutons in the monkey basilar pontine nuclei.

Two general categories of GABA-immunoreactive (GABA-Ir) boutons are present in the monkey basilar pontine nuclei (BPN). One type characterized by a pale or lucent appearance is involved both in glomerular arrangements that include serial, triadic synapses, and in non-glomerular synapses. The second type of GABA-Ir bouton exhibits a wide variety in size and shape and contains a greater complement of synaptic vesicles than the first type, giving it a darker appearance in comparison to the pale GABA-Ir boutons. Such boutons participate only in non-glomerular synapses. It is suggested that the pale GABA-Ir boutons arise from the intrinsic population of GABA neurons, while the darker appearing boutons might take origin from one of the GABA-ergic afferent systems that reach the BPN.

GABAergic neural elements in the rat basilar pons: electron microscopic immunochemistry.

Previous light microscopic immunoperoxidase studies of glutamic acid decarboxylase (GAD)-immunoreactive neural elements in the rat basilar pontine nuclei revealed immunocytochemical reaction product in neuronal somata and axon terminals. In the present study, pre-embedding immunoperoxidase labeling of GAD or gamma-aminobutyric acid (GABA) and postembedding immunogold labeling of GABA allowed the ultrastructural visualization of these neural elements in the basilar pontine nuclei of colchicine-treated animals. At the electron microscopic level, immunolabeled neuronal somata exhibited smoothly contoured nuclei, whereas some dendrites also contained reaction product after immunocytochemical treatment and were postsynaptic to both immunoreactive and nonimmunoreactive axon terminals. Synaptic boutons immunoreactive for GAD or GABA exhibited cross-sectional areas that ranged from 0.1 to 3.8 microns 2 and generally appeared round or elongated in most sections. The majority (95%) of immunolabeled boutons contained pleomorphic synaptic vesicles and formed symmetric synapses at their postsynaptic loci; however, boutons exhibiting round vesicles and boutons forming asymmetric synapses (5%) were also immunopositive. Small (less than 1.5 microns 2) GAD- or GABA-labeled axon terminals formed synaptic contact mainly with small dendritic profiles, dendritic spines, and neuronal somata, whereas large labeled boutons (greater than 1.5 microns 2) formed synapses with all sizes of dendritic profiles. Occasionally, a single immunolabeled bouton formed synaptic contact with two separate postsynaptic dendrites. It is suggested that the immunolabeled neuronal somata and dendrites observed in the rat basilar pontine nuclei represent a population of pontine local circuit neurons; however, it is known that GABAergic cell groups extrinsic to the pontine gray provide afferent projections to the basilar pons, and therefore at least some immunoreactive axon terminals present in the pontine nuclei are derived from these extrinsic sources. The ultrastructural observation of GABAergic neural elements in the rat basilar pontine nuclei confirms previous light microscopic findings and provides an anatomical substrate through which GABAergic neurons, whether arising from an intrinsic or extrinsic source, might exert an inhibitory influence on target cells within the pontine nuclei.

Survey of noncortical afferent projections to the basilar pontine nuclei: a retrograde tracing study in the rat.

The retrograde transport of the conjugate wheat germ agglutinin-horseradish peroxidase (WGA-HRP) was used in the rat to identify the cell bodies of origin for all subcortical projections to the basilar pontine nuclei (BPN). A parapharyngeal surgical approach was used to allow the injection micropipette to enter the BPN from the ventral aspect of the brainstem and thus avoid any potential for false-positive labeling due to transection and injury-filling of axonal systems located dorsal to the basilar pontine gray. A surprisingly large number of BPN afferent cell groups were identified in the present study. Included were labeled somata in the lumbar spinal cord and a large variety of nuclei in the medulla, pons, and midbrain, as well as labeled cells in diencephalic and telencephalic nuclei such as the zona incerta, ventral lateral geniculate, hypothalamus, amygdala, nucleus basalis of Meynert, and the horizontal nucleus of the diagonal band of Broca. Quite a number of cell groups known to project directly to the cerebellum also exhibited labeled somata in the present study. To explore the possibility that such neurons were labeled because their axons were transected and injury-filled as they coursed through the BPN injection site to enter the cerebellum via the brachium pontis, a series of rats received complete, bilateral lesions of the brachium pontis followed 30-60 minutes later with multiple, diffuse injections of WGA-HRP (12-16 placements per animal) throughout the cerebellar cortex. In another series of animals, the massive cerebellar WGA-HRP injections were not preceded by brachium pontis lesions. In the latter cases, each of the cell groups in question that were known to project directly to the cerebellum exhibited labeled somata. However, when the cerebellar HRP injections were preceded by brachium pontis lesions, each of the cell groups in question continued to exhibit labeled somata in numbers comparable to that observed in the nonlesion cases. This implies that such neurons project to the BPN and the cerebellar cortex and that the axons of these particular neurons do not project to the cerebellum via the brachium pontis.

A review of recent observations concerning the synaptic organization of the basilar pontine nuclei.

Ultrastructural studies are described that have identified in the basilar pontine nuclei (BPN), the synaptic boutons formed by the corticopontine, cerebellopontine, tectopontine, and dorsal column nuclei-pontine afferent projection systems. In addition, immunocytochemical studies visualized neuronal somata, dendrites, and synaptic boutons that contain immunoreactivity for GABA or the synthesizing enzyme glutamic acid decarboxylase (GAD). Based upon differences in the mode of degeneration and postsynaptic locus of degenerative synaptic boutons in the BPN, it is suggested that two types of cortical neurons and three classes of deep cerebellar nuclear cells project to the BPN. For similar reasons, it appears that two types of neurons in the dorsal column nuclei project to the BPN while only one type of afferent synaptic bouton takes origin from the superior colliculus. Furthermore it appears that the population of BPN neurons projecting to the paramedian lobule receives convergent inputs from the cutaneous periphery and the corresponding region of sensorimotor cortex. Studies employing GAD immunohistochemistry indicate that GABA-ergic neurons and axon terminals are present in the BPN and thus support the suggestion that a local inhibitory interneuron is present within the BPN. Taken together these observations suggest that basilar pontine neurons might play a more active role in the integration of various types of information destined for the cerebellar cortex than has previously been recognized.

Origin and ultrastructural identification of dorsal column nuclear synaptic terminals in the basilar pontine gray of rats.

The ultrastructural characteristics of HRP-WGA-labeled or degenerating axon terminals arising from neurons in the dorsal column nuclei (DCN) were identified within the contralateral basilar pontine nuclei (BPN) following unilateral HRP-WGA injections or ablations of the DCN. The cells of origin of these projections were also identified through the application of the retrograde tracer HRP-WGA. Two groups of degenerating DCN-pontine terminals were identified. Both formed asymmetrical synaptic contacts with dendritic shafts and/or dendritic appendages of pontine neurons. One group of degenerating terminals contained small, round synaptic vesicles, while the other exhibited a mixture of dense core and pleomorphic vesicles. The former group, which clearly represented the majority of degenerating terminals observed, was interpreted to progress from an early filamentous form of degeneration to a later electron-dense variety and to originate from dorsally located DCN cells distributed primarily at the level of and just caudal to the area postrema. Other DCN-labeled neurons were more ventrally located and were postulated to give rise to those degenerative boutons that contained a mixture of dense core and pleomorphic-shaped vesicles. The present study also identified the cells of origin of two additional projections to the basilar pons: one from cells in the external cuneate nucleus and another from neurons of the medullary reticular formation.

Certain basilar pontine afferent systems are GABA-ergic: combined HRP and immunocytochemical studies in the rat.

Injection of the tracer substance wheat germ agglutinin-horseradish peroxidase (WGA-HRP) directly into the basilar pontine nuclei using a ventral surgical approach resulted in the labeling of somata in many areas both rostral and caudal to the basilar pons. Certain of the sections that had been reacted for HRP were also incubated in antiserum prepared against glutamic acid decarboxylase (GAD) and processed according to routine peroxidase anti-peroxidase immunocytochemical procedures. Neuronal somata exhibiting both HRP and GAD reaction products were considered to represent GABA-ergic neurons that provide axonal projections to the basilar pontine nuclei. Such double-labeled neurons were observed within the zona incerta, anterior pretectal nucleus, lateral cerebellar nucleus, perirubral area, and the pontine and medullary reticular formation.

GAD-immunoreactive neural elements in the basilar pontine nuclei and nucleus reticularis tegmenti pontis of the rat. I. Light microscopic studies.

Immunocytochemical procedures employing the unlabeled antibody enzyme (PAP) method were used to visualize those neuronal elements in the basilar pontine nuclei (BPN) and nucleus reticularis tegmenti pontis (NRTP) of the rat which contain glutamic acid decarboxylase (GAD), a key enzyme involved in the synthesis of the neurotransmitter gamma-aminobutyric acid (GABA). Neuronal somata, axons, and axon terminals in the BPN and NRTP exhibited GAD-immunoreactivity. Both thick and thin varieties of labeled axons and terminals were distributed in varying densities throughout the BPN and NRTP. The greatest accumulation of labeled terminals was noted in the ventrolateral and lateral border regions of the BPN while a slightly less dense aggregation was observed along the ventral, ventromedial and midline regions of the pontine gray. Labeled fibers and terminals were also observed in the dorsal and ventral peduncular regions as well as central portions of the lateral, ventral, and medial pontine areas. Axonal and terminal labeling was present throughout the NRTP but no focal increases in density similar to those in the BPN were apparent. No obvious GAD-labeled fiber bundles could be observed to enter the BPN or NRTP. However, small fascicles of labeled axons were seen to course ventrally around the dorsolateral aspect of the cerebral peduncle to reach lateral pontine areas while other labeled axons descended through clefts in the mid-portion of the cerebral peduncle or passed through the medial lemniscus and around the medial portion of the cerebral peduncle to enter the pontine gray.