Release of microorganisms from soil with respect to transmission electron microscopy viewing and plate counts

Microorganisms were detached and washed from soil by various procedures involving blending and sonication of the soil in water or pyrophosphate solution, followed by successive low-speed, centrifugal-washing separations of the suspended cells from the soil debris. Some of these procedures were previously used for separating and concentrating cells from soil for transmission electron microscopy viewing. Exhaustive applications of these procedures separated up to 27% of the platable cells from the soil. Based on filterability, these cells either were no longer attached to soil particles, or were attached to very small particles. The cells fractionating with the soil debris, however, seemed to be strongly attached to it or to other cells so that they were not filterable. Laboratory-grown cultures added to sterile and non-sterile soil did not attach to the soil materials and were easily recovered from the soil even though low-speed centrifugations were being used. Electron microscopy evidence for cells released and concentrated from non-inoculated natural soil, and for cells remaining with the soil debris, suggests that the very small, probably non-platable, cells tend to release more easily than do cells in the size range of 0.3 to 0.5 μm in diameter, and that cells larger than this, including bacterial and fungal spores, are more difficult to separate from soil. Plating data for heated preparations are in agreement with this for bacterial spores. The results are considered in relation to the validity of plate counts and direct soil transmission electron microscopy for evaluating the microbial flora of soil. This research was authorized for publication as paper no. 5131 in the journal series of the Pennsylvania Agricultural Experiment Station on 7/20/76.     

Persistence of Nosema locustae Spores in Soil as Determined by Fluorescence Microscopy

Nosema locustae, a protozoan parasite of grasshoppers, is used as a bioinsecticide. In the present study, the persistence of N. locustae spores in soil and the interaction of these spores with the indigenous soil microflora were examined with various forms of microscopy and staining. Fluorescence microscopy was found to be better than phase-contrast or bright-field microscopy for detecting and viewing spores in soil. Fluorescein isothiocyanate was a better fluorescent stain than acridine orange or fluorescein diacetate; water-soluble aniline blue did not stain spores. The eight bright-field microscopy stains tested (phenolic erythrosin, phenolic rose bengal, malachite green, crystal violet, safranin, Congo red, methyl red, and eosin B) were not satisfactory, as spore staining characteristics were either poor or masked by overstained soil debris. A procedure was developed which allowed spores to be extracted from soil with a peptone-phosphate buffer, recovered on a membrane filter, and stained with fluorescein isothiocyanate for microscopic counting. This procedure was used to assess the persistence of N. locustae spores in field and laboratory soils. The number of N. locustae spores in a laboratory model soil system persisted at a high level for over 8 weeks when the soil was incubated at 5°C but exhibited a 1,000-fold decrease after 1 week of incubation at 27°C. Persistence was related to the temperature-dependent activity of the indigenous soil microflora, which, on the basis of microscopic observations, appeared to prey on N. locustae spores. N. locustae spores were detected in an N. locustae-treated field soil at a low level consistent with the level for laboratory soil incubated at 27°C, and they persisted at this level for over 2 months. No spores were detected on vegetation from this field or in the soil from an adjacent, nontreated control field. N. locustae-like spores were also detected in soil from nontreated fields supporting large grasshopper populations.     

A method for the extraction and enumeration of resting spores of Plasmodiophora brassicae from infested soil

A technique is described for the extraction of the resting spores of Plasmodiophora brassicae from soil. After deflocculation, coarse mineral matter was filtered from soil samples and the remainder mixed with 40% sucrose. The mixture was allowed to stand for 2–5 days; this enabled most mineral particles to settle out leaving the spores in suspension. The spores were counted optically on a microscope using Nomarski interference contrast optics, although preliminary studies had indicated that electronic image analysis was a more satisfactory method. Good recovery of spores was achieved from artificially infested soils containing 10⁶ spores/g but the technique can be satisfactorily applied to less heavily infested soils. Clubroot symptoms developed on seedlings dipped in suspensions of spores recovered in this way.     

乌毛蕨配子体发育的研究

采用混合土培养乌毛蕨（Blechnum orientale）孢子,显微镜下观察记录其孢子萌发及配子体发育过程。结果表明：孢子黑褐色,赤道而豆形,极而观椭圆形,单裂缝。播种1周左右孢子萌发,萌发类型为书带蕨型,配子体发育为叉蕨型。丝状体5—10细胞时开始发育为片状体。播种2周后发育形成幼原叶体,成熟原叶体呈心脏形。原叶体边缘及表面均可产生毛状体,数量丰富,为单细胞。播种后1个月左右开始有颈卵器出现,成熟颈卵器颈部由4列细胞组成,3—5层细胞高。精子器产,扛时间较颈卵器早10d左右,精子器近圆球形,由3细胞组成。精卵受精后2周左右即可观察到从原叶体上生成的幼胚。     

日本蹄盖蕨配子体发育的研究

采用混和土培养日本蹄盖蕨(Athyrium niponicum)孢子,显微镜下观察记录其孢子萌发及配子体发育过程。结果表明:孢子黑褐色,赤道面豆形,极面观椭圆形,单裂缝。播种7 d左右孢子萌发,萌发类型为向心型,配子体发育为铁线蕨型。丝状体7~11细胞时开始发育为片状体。播种14 d后发育形成幼原叶体,成熟原叶体呈心脏形。原叶体边缘可产生单细胞毛状体。播种后20 d左右精子器出现,精子器近圆球形,由3细胞组成。7 d后颈卵器出现,成熟颈卵器3~5层细胞高。精卵受精后14 d左右即可观察到从原叶体上生成的幼胚。     

三角鳞毛蕨配子体发育的研究

采用混合土培养三角鳞毛蕨(Dryopteris subtriangularis)孢子,显微镜下观察记录其孢子萌发及配子体发育过程。结果表明：孢子深褐色,赤道面豆形,极面观椭圆形,单裂缝。播种1周左右孢子萌发,萌发类型为书带蕨型,配子体发育为三叉蕨型。播种2周左右发育为片状体。播种4周左右发育形成幼原叶体,成熟原叶体呈心脏形。原叶体边缘及腹面均可产生毛状体,数量丰富,为单细胞。播种6周左右开始有性器官出现,精子器近圆球形,由3细胞组成,成熟颈卵器颈部由4列细胞组成,3～5层细胞高。原叶体受精后1个月内即可观察到从原叶体上生成的幼胚。     

Responses of Indigenous Microorganisms to Soil Incubation as Viewed by Transmission Electron Microscopy of Cell Thin Sections

Air-dried soils were adjusted to 50% moisture-holding capacity and incubated for 2 weeks at 30 C. Samples were removed at intervals, and their total microbial populations were physically separated and concentrated from the soil debris for sectioning and ultrastructure examination. Although the total numbers of cell sections in these preparations remained relatively constant during the soil incubations, the percentages of dwarf cells (相似文献   

普通针毛蕨配子体发育的研究

采用混合土培养普通针毛蕨（Macrothelypteris torresiana (Gaud.) Ching）孢子,显微镜下观察记录其孢子萌发及配子体发育过程。结果表明：孢子褐色,赤道面观为半圆形,极面观椭圆形,单裂缝。播种3 d左右孢子萌发,萌发类型为书带蕨型,配子体发育为槲蕨型。播种7 d左右发育为片状体,丝状体与片状体两阶段的界限不明显。原叶体边缘及背腹面均可产生毛状体。播种21 d左右发育形成幼原叶体,成熟原叶体呈心脏形。播种35 d左右开始有性器官出现,精子器近圆球形,由3个细胞组成,成熟颈卵器颈部由4列细胞组成,4～5层细胞高。原叶体受精后1个月内即可观察到从原叶体上生成的幼胚。     

槲蕨配子体形态发育的研究

用无机培养基和土壤培养基分别培养槲蕨(Drynaria roosii Nakaike)孢子,显微镜下观察孢子萌发及配子体形态发育过程.结果表明:孢子黄色,具单裂缝,赤道面观豆形,极面观椭圆形,不具周壁,孢子外壁局部具大小不一的颗粒状纹饰.接种后10～12 d孢子萌发,萌发类型为书带蕨型,原叶体发育为槲蕨型.接种后20 d左右发育为片状体,片状体形成顶端细胞的时间较晚.毛状体出现在片状体形成之后,数量丰富,多为单细胞,分布于原叶体背腹面及边缘.接种后60 d左右发育形成幼原叶体,成熟原叶体呈心脏形.接种后65 d左右开始有性器官出现,精子器的出现较颈卵器早10 d左右.颈卵器成熟后,颈部常向原叶体基部倾斜或弯曲.原叶体受精后幼胚突破颈卵器生长.     

龙头节肢蕨配子体发育的研究

采用原生境土、Knorp’s营养液和MS培养基的培养方法对龙头节肢蕨(Arthromeris lungtauensis Ching)的孢子进行对照培养,观察其孢子萌发和配子体发育过程。结果：孢子黄色,呈二面体型,赤道面观为肾形,极面观椭圆形,周壁瘤状,有刺,单裂缝;接种5 d后孢子开始萌发,萌发为书带蕨型（Vittaria-Type),原叶体的发育为槲蕨型(Drynaria-Type）;接种7 d后向丝状体发育,为3~8个细胞长;接种2周后向片状体发育,片状体呈长阔匙形,达6~8纵列细胞;接种6周后向原叶体发育,成熟的原叶体呈对称心形,毛状体二型（单细胞乳头状的毛状体和4~5个细胞分枝的毛状体）和假根分叉;接种9周后出现精子器,早于颈卵器1周出现和成熟;精子器具宽而短的柄,颈卵器的颈沟部较短而且通常弯曲。研究表明龙头节肢蕨的配子体发育过程中出现毛状体,假根分叉,颈卵器弯曲等进化特征,并对其分类学意义初步讨论。     

Celery leaf spot: sources of inoculum

The relative importance of infected celery seed, infected leaf debris in the soil, and infected wild celery, in the incidence of Septoria leaf spot in cultivated celery has been investigated. Infection can be caused when the sole source of inoculum is viable spores on the seed surface; such spores are considered to be the main cause of disease outbreaks. Of all the seed samples examined, 93% were infected by Septoria spp. In untreated seed samples, 40% carried viable spores which survived for up to 15 months on seed stored in the laboratory, and for longer periods on seed stored at -20d? C. However, ageing of seed is not recommended as a commercial control measure. The fungus was not found in seed embryos or endosperms but mycelium was present in pericarps and testas. Unconfirmed evidence suggests that in favourable circumstances new spores might be produced in old seed-borne pycnidia.     

粗齿黔蕨配子体发育的研究

用原生境腐殖土对粗齿黔蕨(Phanerophlebiopsis blinii(Lévl.)Ching)的孢子进行培养,显微镜下观察孢子萌发和配子体发育过程.结果表明:粗齿黔蕨的孢子深棕色,单裂缝,极面观椭圆形,赤道面观为半圆形.播种1周后孢子萌发,萌发类型为书带蕨型(Vittaria-type),配子体发育为叉蕨型(Aspidium-type).丝状体由4～12个细胞组成,片状体宽达9个细胞,斜向一侧生长,边缘具毛状体.播种约2个月后形成原叶体,成熟原叶体成对称心形.在粗齿黔蕨的配子体发育过程中,成熟原叶体的背腹面和边缘均被毛状体,假根有分叉且尖部常膨大,并含有较大的颗粒状贮藏物,精子器有3个壁细胞等特征较为进化,而颈卵器粗短且直立的特征较为原始.粗齿黔蕨的精子器和颈卵器发育不同步,精子器的出现和成熟均早于颈卵器的发育.从配子体发育的角度,初步探讨了粗齿黔蕨野外种群数量较少的成因.     

A New Fluorescence Staining Assay for Visualizing Living Microorganisms in Soil

5- (and 6-)Sulfofluorescein diacetate (SFDA), which is converted to a fluorescent product by intracellular esterase activity, was used to stain living microorganisms, including bacteria, Saccharomyces cerevisiae, and fungi, in soil. SFDA (1 mM) dissolved in ethyl alcohol was added to an intact soil sample, and the preparation was examined with an epifluorescence microscope. Bright single cells and colonies of live bacteria were observed without interference from the autofluorescence of soil minerals and detritus. Cultured Escherichia coli was killed through heat treatment; thus, SFDA was concluded to stain only living cells. Microbial colonies obtained from natural soils and various cultured strains were tested. It was found that 151 of 154 colonies from natural soils were stained and that hyphae and spores from 1 of 28 cultured microbial strains were not stained. The SFDA method was successfully used to visualize and count bacteria in soil samples from Mount Shiga in Japan.     

Spore Formation and Toxin Production in Clostridium difficile Biofilms

The ability to grow as a biofilm can facilitate survival of bacteria in the environment and promote infection. To better characterize biofilm formation in the pathogen Clostridium difficile, we established a colony biofilm culture method for this organism on a polycarbonate filter, and analyzed the matrix and the cells in biofilms from a variety of clinical isolates over several days of biofilm culture. We found that biofilms readily formed in all strains analyzed, and that spores were abundant within about 6 days. We also found that extracellular DNA (eDNA), polysaccharide and protein was readily detected in the matrix of all strains, including the major toxins A and/or B, in toxigenic strains. All the strains we analyzed formed spores. Apart from strains 630 and VPI10463, which sporulated in the biofilm at relatively low frequencies, the frequencies of biofilm sporulation varied between 46 and 65%, suggesting that variations in sporulation levels among strains is unlikely to be a major factor in variation in the severity of disease. Spores in biofilms also had reduced germination efficiency compared to spores obtained by a conventional sporulation protocol. Transmission electron microscopy revealed that in 3 day-old biofilms, the outermost structure of the spore is a lightly staining coat. However, after 6 days, material that resembles cell debris in the matrix surrounds the spore, and darkly staining granules are closely associated with the spores surface. In 14 day-old biofilms, relatively few spores are surrounded by the apparent cell debris, and the surface-associated granules are present at higher density at the coat surface. Finally, we showed that biofilm cells possess 100-fold greater resistance to the antibiotic metronidazole then do cells cultured in liquid media. Taken together, our data suggest that C. difficile cells and spores in biofilms have specialized properties that may facilitate infection.     

扇蕨配子体发育与濒危机制的探讨

采用改良Knop’s固体培养基、原生境腐殖质土和红壤分别培养扇蕨（Neocheiropteris palmatopedate）孢子,光学显微镜及解剖镜下观察记录其孢子萌发及配子体发育过程,比较了3种培养方法对其配子体发育和有性繁殖的影响,并在此基础上探讨了扇蕨的濒危原因。结果表明：成熟孢子黄褐色,赤道面观为豆形,极面观椭圆形,单裂缝。孢子萌发类型为书带蕨型,原叶体发育为槲蕨型。成熟原叶体呈心脏形。毛状体在原叶体阶段出现。有性生殖周期长及配子体发育成幼孢子体的百分率低是扇蕨在配子体世代的主要濒危原因。此外,红壤固有的理化性质导致扇蕨配子体发育极其缓慢、精子器和颈卵器发生的时间间隔过长使其不能受精产生孢子体。原生植被遭受破坏引起的林下腐殖质土消失、红壤裸露,加剧了扇蕨的濒危。     

Simplified procedures for releasing and concentrating microorganisms from soil for transmission electron microscopy viewing as thin-sectioned and frozen-etched preparations.

A simplified procedure is presented for releasing and concentrating indigenous microbial cells from soil for viewing by transmission electron microscopy as thin sections or replicas of frozen-etched preparations. This procedure is compared with two others reported earlier, and their relative merits are discussed as concerns the choice of procedure for the cellular information desired from the soil. Freeze-etching showed that the cell types and size distributions for cells which have been released and concentrated from soil are in general agreement with those for cells in a crude soil slurry in which no attempt to release and concentrate cells was made. Microcolonies were present both in the crude slurry and in the discard soil debris centrifugation pellets from the cell release and concentration procedures. In contrast to the historic assumptions, these microcolonies, as well as some individual cells embedded in soil debris could not be broken up and (or) dislodged so that they would be washed from the soil. The relative numbers of these cells remaining with the soil debris, however, could not be quantitated in the present study.     

Survival of Bacillus thuringiensis Spores in Soil

Bacillus thuringiensis spores and parasporal crystals were incubated in natural soil, both in the laboratory and in nature. During the first 2 weeks, the spore count decreased by approximately 1 log. Thereafter, the number of spore CFU remained constant for at least 8 months. B. thuringiensis did not lose its ability to make the parasporal crystals during its residence in soil. Spore survival was similar for a commercial spore-crystal preparation (the insecticide) and for laboratory-grown spores. In contrast to these results, spores that were produced in situ in soil through multiplication of added vegetative cells survived for only a short time. For spore additions to soil, variations in soil pH had little effect on survival for those spores that survived the first 2 weeks of incubation. Also without effect were various pretreatments of the spores before incubation in soil or nutritional amendment or desiccation of the soil. Remoistening of a desiccated soil, however, caused a decrease in spore numbers. Spores incubated in soil in the field did not show this, but the degree of soil desiccation in nature probably never reached that for the laboratory samples. The good survival of B. thuringiensis spores after the first 2 weeks in soil seemed to be a result of their inability to germinate in soil. We found no evidence for the hypothesis that rapid germination ability for spores in soil conferred a survival advantage.     

Stored Bacillus popilliae spores and their infectivity against Popillia japonica larvae

Bacillus popilliae spores were stored for about 7 years under three separate conditions: frozen in sterile distilled water, smeared on glass microscope slides, and stored in loam soil at room temperature. In separate experiments, each of the 7-year-old preparations was fed to Popilla japonica larvae at concentrations of 10³, 10⁵, 10⁷, and 10⁹ spores/g of soil. A significant decrease in the percentage of larvae infected occurred in all of the aged spore tests. B. popilliae spores stored in soil, for the extended period, produced 3% larval infection only at the 10⁹ spores concentration; similar results were obtained from frozen spores. When P. japonica larvae were fed spores stored dried on slides, about 20% of the larvae developed milky disease. When aged frozen spores were artificially injected into larvae, 12% became infected at concentrations of 1 × 10⁶ spores/larvae; dried spores at the same concentration infected about 38% of the insect larvae. We conclude from these data that aged B. popilliae spores are significantly less infective against P. japonica larvae than young spores.     

Detection of Bacillus thuringiensis in soil by immunofluorescence

Persistence of viable and heat-killed vegetative cells, parasporal crystals, and spores of Bacillus thuringiensis in soil was monitored by immunofluorescence. The rates of disappearance of the different bacterial components decreased in the following order: viable cells, heat-killed cells, parasporal crystals, and spores. Vegetative cells disappeared at rapid, exponential rates; viable cells autolysed, whereas heat-killed cells were digested by an actinomycete-like, soil microorganism. Parasporal crystals disappeared at a slower, nonexponential rate. Numbers of spores remained unaltered throughout 91 days incubation at 25°C and no germination was detected in this period.     

Artificial wind-gust liberation of microbial bioaerosols previously deposited on plants

Summary A bioaerosol research chamber was constructed and used to evaluate wind-gust release of previously depositedPseudomonas syringae, spores ofBacillus subtilis var.niger, and fluorescent microspheres (FM) on oat plants, and the airborne survival of the releasedP. syringae. Observations of wind gusts on the releasedBacillus spores and FM showed they had similar particle size distributions and therefore FM could act as bacterial sized surrogates in particle dispersion. Microscopic examination of the released FM containing particles revealed that 84% were associated with either fungal spores and hyphae were epiphytic on the plant, or with plant and soil debris. The release rate ofBacillus spores decreased as the number of gusts experienced by the plants increased, with a greater proportion of larger particles removed. The larger the particle associated with the releasedP. syringae, the longer the bacteria survived.