Fluorescent vital staining and rRNA-targeted in situ hybridization were used to investigate the physiological activity and microbial population structures of two types of activated sludge: fresh returned sludge from a municipal wastewater treatment plant and MITI-standard sludge (a microbial source in a biodegradation test, Modified MITI Test). The ratio of esterase-active bacteria, as determined by double-staining with 4', 6-diamidino-2-phenylindole (DAPI) and 6-carboxyfluorescein diacetate (6CFDA), was 60-80% to total bacterial number in both types of activated sludge; both ratios being much higher than those of the colony forming bacteria. Microbial population structures in both types of activated sludge were determined by 2-hydroxy-3-naphthoic acid-2'-phenylanilide phosphate (HNPP) and Fast Red TR in situ hybridization (HNPP-FISH). Approximately 50% of the DAPI-stained bacteria was detected by HNPP-FISH in wastewater treatment plant sludge and 40% in the MITI-standard sludge. The bacterial community structure in wastewater treatment plant sludge was dominated by the Flavobacterium-Cytophaga group. Characteristic coccoid cells, which fluoresced bright yellow under UV excitation after DAPI-staining, were dominant in the MITI-standard sludge.
This study was carried out to clarify the properties of Arbuscular Mycorrhizal Fungi (AMF) from different types of land used at Sunbarjaya in Southern Sumatra, Indonesia. The soil samples were collected from primary forest (P. F.: Hopea mengarawan, H. sangal, and Dryobalanops spp.), secondary forest (S. F.: Daemonorops oblongata, H. ficus and H. calamus), coffee field (C. F.: Coffea robusta) and native grassland (N. G.: Imperata cylindrica) where were about 1, 200, 100, 800 and 800m above sea level, respectively. Sampling sites for C. F. were selected from three locations along the hillslope. There were four genera and ten species of AM fungal spores identified from the P. F., S. F., C. F., and bush soils of the Sunbarjaya areas. Glomus etunicatum, G. constrictum and G. aggregatum were found in all land uses. The total spore numbers of G. constrictum and G. aggregatum were much greater in the C. F. soils compared to other soils. But, the number of genera and species in the C. F. soils was low compared to the P. F. and S. F. soils. G. constrictum predominated in all soils and G. aggregatum only predominated in the C. F. soils.
Acridine orange direct count (AODC) of bacterial community was examined with two types of epifluorescence microscope, BX-60 (OLYMPUS OPTICAL CO., LTD.) and BHS-RFK (OLYMPUS OPTICAL CO., LTD.). When observed under the naked eye, AO-stained particles appeared clearer under the BX60 than under the BHS-RFK. This difference in the image was considered to influence the AODC containing particles of various size and shape. AODC of a bacterial pure culture did not provide significant difference when determined under the naked eye with these two epifluorescence microscopes. However, the AODC of complex communities from river water and epilithon showed difference in counting among 4 investigators. To clarify the reason for these differences, the particle image of each scope was compared with an image analyzer. Mean AO-stained particle area of BHS-RFK was larger than that of BX60, probably caused by the existence of a dim margin surrounding particles for the former system. As a result, it is not necessary to correct AODC when determined under the naked eye. However, when an image analyzer is employed to determine AODC of natural bacterial community, procedure for application should be checked carefully for newly developed microscope.
Increasing 16S rRNA gene sequence analyses have greatly advanced our knowledge of the phylogeny and diversity of bacterial species. Bacterial taxonomic systems are being reconstructed along the phylogenetic lines based on 16S rRNA sequence data. Ribosomal RNA approaches such as rRNA-targeted fluorescent in situ hybridization and PCR cloning of environmental 16S rRNA genes are now widely used to evaluate phylogenetic structures of bacterial communities and to identify bacterial species in situ without any cultivation and isolation. The current knowledge of bacterial diversity based on the molecular data and the rRNA and biomarker approaches to in situ species identification are reviewed.