CSM News Electronic Edition Volume 3, number 2 July 9, 1994 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to CSM-News@worms.cmsbio.nwu.edu. Back issues of CSM-News, the CSM Reference database and other useful information is available by anonymous ftp from worms.cmsbio.nwu.edu [165.124.233.50], via Gopher at the same address, or by World Wide Web through www.nwu.edu. =========== Abstracts =========== Detection of specific microorganisms in environmental samples using flow cytometry. Graham Vesey, Joe Narai,Nicholas Ashbolt, Keith Williams & Duncan Veal School of Biological Sciences, Macquarie University, Sydney, NSW 2109, Australia Methods in Cell Biology, in press. Summary Flow cytometers are technologically advanced instruments which combine laser interogation of a fluid stream with sophisticated data handling technology for obtaining information about, and potentially isolating, particles that pass through the laser beam. Traditionally they are amongst the most expensive of laboratory instruments and require highly skilled personnel to operate them. This combined with the fact that samples must be particulate and of reasonably uniform size has limited their application in biology to well funded laboratories in biomedical research where they are used to analyse blood cell sub populations (immunology, AIDS, cancer) or separate chromosomes. The reduced cost and ease of operation of analytical flow cytometers (which collect data but do not sort particles) means that applications in other areas of biology are now envisaged. The applications of flow cytometry to clinical microbiology have been described by Shapiro (1990). In this chapter we discuss the use of flow cytometry within the environmental microbiology laboratory. In particular we focus on flow cytometric methods for the detection of low numbers of, and even single, specific microorganisms within environmental samples. Flow cytometric analysis performed in environmental microbiology laboratories is often more stringent than that required for the analysis of mammalian cells and can push sensitivities close to limits of operation. This is because the volume, nucleic acid and protein content of bacteria are approximately 1000x less than in mammalian cells Since detection involves identification of light scatter, specific proteins or DNA the signals produced by bacteria are generally several orders of magnitude lower than those from eukaryotic cells. However, recently flow cytometers have been used to great effect for microbiological diagnosis and even more recently they have been applied in environmental microbiology. Developments in both biological techniques and instrumentation, described in this chapter, will considerably increase the range of applications of flow cytometry within environmental microbiology laboratories. Furthermore, these developments result in greatly simplified protocols allowing not only research laboratories but also routine environmental testing laboratories to perform these analyses. We envisage that within the foreseeable future small, robust, relatively cheap and simple to operate flow cytometers will be available for the detection of a vast range of microorganisms in environmental samples. NOTE TO: Dictyologists - In this paper we show a novel use of Dictyostelium in setting up assays for detecting very low numbers of samples in environmental waters. Using two monoclonal antibody tags to spores, we detected a single spore in a water sample! ------------------------------------------------------------------ Mitosis in amoebae of the cellular slime mold (mycetozoan) Acytostelium leptosomum. Bruno Guhl and Urs-Peter Roos Institute of Plant Biology, University of Zurich, Zollikerstr. 107, 8008-Zurich, Switzerland European J. Protozol., in press SUMMARY We investigated mitosis in amoebae of Acytostelium leptosomum, grown in liquid culture, by video microscopy of live cells, by indirect immunofluorescence with antibodies against tubulins, and by transmission electron microscopy of ultrathin sections. Amoebae in interphase contain a single microtubule-organizing center (MTOC) at each nucleus, from which microtubules (MTs) radiate into the cytoplasm. These disappear as the intranuclear spindle forms. Concomitantly, the nucleolus disperses, and the chromosomes that are visible in phase contrast congress to the spindle equator. The spindle is closed except for polar fenestrae occupied by broad, amorphous spindle pole bodies (SPBs). The chromosomes at metaphase are joined to form several blocks, each attached to several kinetochore MTs. Anaphase was accomplished within 2.2 min (s.d.=0.5 min, n=11). Anaphase A was virtually absent, but anaphase B contributed substantially to chromosome segregation. The mean velocity of pole separation was 3.2 !m/ min (s.d.=0.8 !m/min) and the mean elongation factor was 2.8 (range) 1.9 to 3.4). The telophase spindle was a shaft consisting of a few MTs traversing each incipient daughter nucleus and joining in the interzone. The amorphous SPBs were reconverted to compact interphase MTOCs as the chromosomes decondensed and the nucleolus re-formed during cytokinesis. Duration of mitosis and velocities of its movements are within values typical for lower eucaryotes. In most aspects of mitosis A. leptosomum is very similar to two other dictyostelid cellular slime molds, Dictyostelium discoideum and Polysphondylium violaceum, and the three lower eucaryotes are clearly distinct from other myceotozoans. ------------------------------------------------------------------ Mitosis in amoebae of the cellular slim mold (mycetozoan) Protostelium mycophaga Bruno Guhl and Urs-Peter Roos Institute of Plant Biology, University of Zurich, Zollikerstr. 107, 8008-Zurich, Switzerland Eur. J. Protozol., in press SUMMARY We investigated mitosis in amoebae of Protostelium mycophaga by video microscopy of live cells, by indirect immunofluorescence with antibodies against tubulins, and by transmission electron microscopy of ultrathin sections. Amoebae in interphase usually contain two microtubule centers (MCs) on opposite sides of the nucleus, from which microtubules (MTs) radiate into the cytoplasm. During prophase these MTs shorten to form two asters between which the mitotic spindle develops during prometaphase. Concomitantly, the nucleolus fragments, the numerous small chromosomes orient amphitelically in the spindle and congress to the spindle equator, and the asters diminish further until metaphase. The spindle is open and acentric, but with complex spindle pole bodies. Each sister-chromatid is attached to a single MT by a tiny, layered kinetochore. During anaphase and telophase, asters develop anew and enlarge to become the elaborate MT cytoskeletons of the daughter cells. Anaphase lasted 2 min on average (s.d.= 0.6 min, n=4), during which the chromosomes moved poleward with a mean velocity of 4.0 !m/min (s.d.=0.8 !m/min, n=5). The intermingling of kinetochore MTs and the numerous non-kinetochore MTs allows for a sliding interaction between them, but depolymerisation-driven chromosome movement is also possible. The spindle elongated at a mean rate of 5.9 !m/min (s.d.= 2.2 !m/min, n=5), and the mean elongation factor was 2.4. in live cells. In immunofluorescence preparations the longest spindles were 3.5. times longer than the average metaphase spindle. Spindle elongation thus requires the growth of interzonal MTs, that assemble as several bundles, from an ample pool of tubulin. At the end of telophase the nuclear envelope is reconstructed from membrane vesicles and flattended cisternae that appose to the masses of decondensed chromosomes and nucleolar material. ----------------------------------------------------------------------- PROGRESSION OF AN INDUCTIVE SIGNAL ACTIVATES SPORULATION IN DICTYOSTELIUM DISCOIDEUM. Delwood L. Richardson 1, William F. Loomis 2, and Alan R. Kimmel 1 1 Laboratory of Cellular and Developmental Biology, NIDDK, NIH, Bethesda, MD 20892 2 Dept. of Biology, UCSD, La Jolla, CA 92093-0322. DEVELOPMENT, in press. Summary spiA, a marker for sporulation, is expressed during the culmination stage of Dictyostelium development, when the mass of prespore cells has moved partly up the newly formed stalk. Strains containing a full- length spiA promoter/lacZ fusion were stained for beta-galactosidase activity at intervals during development. The results indicate that expression of spiA initiates in prespore cells at the prestalk/prespore boundary (near the apex) and extends downward into the prespore mass as culmination continues. A spatial gradient of staining expands from the top of the prespore mass and intensifies until the front of activation reaches the bottom, whereupon the entire region stains darkly. The spiA promoter can be deleted to within 301 bp of the transcriptional start site with no effect on the relative strength, timing, or spatial localization of expression. Further 5' deletions from -301 to -175 reduce promoter strength incrementally, although timing and spatial expression are not affected. Deletions to -159 and beyond result in inactive promoters. Treatment of early developmental structures with 8- Br-cAMP in situ activates the intracellular cAMP-dependent protein kinase (PKA) and precociously induces spiA expression and sporulation. The absence of an apparent gradient of staining in these structures suggests that PKA is equivalently activatable throughout the prespore region and that all prespore cells are competent to express spiA. Thus, we postulate that the pattern of expression of spiA reveals the progression of an inductive signal for sporulation and suggest that this signal may originate from the prestalk cells at the apex. ----------------------------------------------------------------------- [End CSM-News, volume 3, number 2]