A Method for the Staining of Bacterial Flagella

A modification of the Loeffler method of staining bacterial flagella is proposed. The chief points of the modification are: The cultures are inoculated into distilled water after two successive daily transfers on agar slants, and the distilled water cultures are incubated at optimum temperature for from 48 to 72 hours. The mordant (tannic acid, ferrous sulphate, basic fuchsin) is allowed to stand 18 to 24 hours before use, and then cleared by centrifuging or filtering. An anilin water fuchsin is used as a stain. No heat is used for either mordanting or staining; but both mordant and stain are allowed to act on the preparation for 15 minutes. The writer finds the method admirably adapted for use in class work, where nearly 100 per cent success has been obtained except in the case of some three or four species of bacteria that are especially difficult to stain.     

A Simple, Rapid Method for Demonstrating Bacterial Flagella

We developed a simple, rapid method for demonstrating flagellation of bacteria using the fluorescent protein stain NanoOrange (Molecular Probes, Eugene, Oreg.). The NanoOrange reagent binds to hydrophobic regions of proteins, which results in substantial enhancement of fluorescence. Unbound reagent is essentially nonfluorescent. NanoOrange fluorescently stained bacterial cell bodies, as well as flagella and other appendages, which could be directly observed by epifluorescence microscopy. Detection of flagella was further improved by using a charge-coupled device camera for image capture and processing. The reliability of the method was tested by using 37 pure cultures of marine bacteria. Detection of flagella on the isolates by NanoOrange staining was compared to detection by transmission electron microscopy (TEM). For 36 of 37 cultures, the two methods yielded the same results. In one case, flagella were detected by TEM but not by NanoOrange, although the difference may be attributable to differences between the culture preparations. NanoOrange staining is rapid (10 to 15 min) and does not require fixation or dehydration, so live samples can be stained. Since NanoOrange is a general protein stain and works directly in seawater, it may also prove to be useful for staining other proteinaceous material that is of interest to aquatic microbial ecologists.     

Enhanced Staining of Bacterial Flagella Using Aged Mordant in the Silver Stain

Intensity of bacterial flagella staining using a modified silver stain was increased by aging the mordant for one week at room temperature. The use of aged mordant increased the apparent diameters of stained flagella and resulted in a darker stain. The mordant remained stable for at least four months at room temperature. The staining protocol presented allows application to liquid or solid cultures.     

Basal Organelles of Bacterial Flagella

Liberated by enzymatic lysis of the cells, the flagella of Rhodospirillum rubrum, R. molischianum, and R. fulvum all have a similar structure. The hook at the base of the flagellum is connected by a short, narrow collar to a paired disc in the basal organelle. This paired disc is in turn connected to a second paired disc. The disposition of flagella to which fragments of the cell membrane still adhere suggests that the narrow collar at the base of the hook traverses both the wall and the membrane, and that the upper pair of discs in the basal organelle lies just beneath the surface of the membrane.     

Visualization of Flagella during Bacterial Swarming

When cells of Escherichia coli are grown in broth and suspended at low density in a motility medium, they swim independently, exploring a homogeneous, isotropic environment. Cell trajectories and the way in which these trajectories are determined by flagellar dynamics are well understood. When cells are grown in a rich medium on agar instead, they elongate, produce more flagella, and swarm. They move in coordinated packs within a thin film of fluid, in intimate contact with one another and with two fixed surfaces, a surfactant monolayer above and an agar matrix below: they move in an inhomogeneous, anisotropic environment. Here we examine swarm-cell trajectories and ways in which these trajectories are determined by flagellar motion, visualizing the cell bodies by phase-contrast microscopy and the flagellar filaments by fluorescence microscopy. We distinguish four kinds of tracks, defining stalls, reversals, lateral movement, and forward movement. When cells are stalled at the edge of a colony, they extend their flagellar filaments outwards, moving fluid over the virgin agar; when cells reverse, changes in filament chirality play a crucial role; when cells move laterally, they are pushed sideways by adjacent cells; and when cells move forward, they are pushed by flagellar bundles in the same way as when they are swimming in bulk aqueous media. These maneuvers are described in this report.Swarming is a common yet specialized form of surface translocation exhibited by flagellated bacteria and is distinct from swimming (23). When cells are grown on a moist nutrient-rich surface, they differentiate from a vegetative to a swarm state: they elongate, make more flagella, secrete wetting agents, and move across the surface in coordinated packs. Here we focus on the mechanics of bacterial swarming, as exhibited by the model organism Escherichia coli. Others have worked on swarm-cell differentiation in a variety of organisms, including Proteus, Salmonella, Pseudomonas, Serratia, Bacillus, and Vibrio. For example, screens for genes required for swarming in E. coli or Salmonella have been made by Inoue et al. (25) and Wang et al. (40, 41). Vibrio is a special case, because a single polar flagellum enables cells to swim while multiple lateral flagella promote swarming (32). For general reviews, see the work of Allison and Hughes (1), Shapiro (37), Fraser and Hughes (17), and Fraser et al. (16). Also see the work of Eberl et al. (15), Sharma and Anand (38), Harshey (18), Daniels et al. (11), Kaiser (26), O''Toole (33), and Copeland and Weibel (10).Swarming was first observed with Proteus mirabilis by Hauser (22), who named the genus for a sea god able to change his own form. Proteus is distinctive because cells switch periodically from the vegetative to the swarming state, building terraced colonies (36, 42). This is not observed with E. coli under the conditions used here, where swarms expand at a constant rate propelled by cells swimming vigorously in a monolayer behind a smooth outer boundary.Swarming in E. coli was discovered by Harshey, who found that K-12 strains, which lack the lipopolysaccharide O antigen, swarmed on Eiken agar (from Japan) but not on Difco agar (from the United States), presumably because the former is more wettable (19, 20). Chemotaxis is not required: cells lacking the chemotaxis response regulator CheY swarm perfectly well, provided that mutations in the motor protein FliM enable transitions between clockwise (CW) and counterclockwise (CCW) rotational states (31). It was suggested that these reversals promote wetness by causing cells to shed lipopolysaccharide.How do cells in E. coli swarms move across an agar surface? What are their flagella doing? We sought to answer such questions by performing a global analysis of videotaped data (of phase-contrast images) collected from 5 regions of 2 swarms, plotting body lengths, speeds, propulsion angles, local track curvatures, and temporal and spatial correlations, and we found that cells reorient on a time scale of a few tenths of a second, primarily by colliding with one another (13). Our previous report did not describe analyses of individual tracks or visualization of flagella. This aspect of the work is presented here.Most of the time, cells are driven forwards by a flagellar bundle in the usual way. Flagellar filaments from different cells can intertwine and form common bundles, but this is rare. However, cells in swarms do something not ordinarily seen with swimming cells: they back up. They do this without changing the orientation of the cell body by moving back through the middle of the flagellar bundle. This involves changes in filament shape (in polymorphic form), from normal to curly and back to normal. Polymorphic forms were classified by Calladine (7), on the basis of earlier work by Asakura (3), in terms of the relative lengths of 11 protofilaments, longitudinal arrays of protein subunits that comprise the filament. All polymorphic forms are helical, with some being left-handed (e.g., the normal form) and some being right-handed (e.g., the semicoiled and curly forms, which have half the pitch of the normal filament and half the pitch and half the amplitude, respectively). Transformations from one shape to another can be caused in various ways, e.g., by changes in pH, salinity, or temperature (21, 27, 28) or by application of torque (24). The changes observed with swarm cells are driven by the latter mechanism, when motors switch from CCW to CW rotation. When swimming cells tumble, polymorphic transformations also occur, in the order normal to semicoiled to curly and back to normal (14, 39). But we rarely see the semicoiled form with cells in swarms, and when it appears, it is quite transient. We wonder whether polymorphic transformations evolved to enable cells to escape confined environments when the only way out is to back up, keeping the filaments close to the sides of the cell body.     

Effect of Proteolytic Enzymes on Bacterial Flagella

Sheared flagella of Salmonella typhimurium strains SL 870 (Nml(+)Fla(+)) and SL 871 (Nml(-)Fla(+)) were found to be susceptible to proteolytic digestion by trypsin, chymotrypsin, and Pronase. The rate of tryptic digestion was similar for the epsilon-N-methyllysine-containing and the nonmethylated flagella. Thin fibers, which appeared to originate from only one end of the flagellar filament, were formed upon trypsin digestion. The fibers were not dissociated at extremes of pH or upon heating. The amino acid composition of the purified fibers was very different from that of intact Salmonella flagellin, and the fibers did not cross-react with antiflagellin or antiflagellar antiserum. The possible significance of these findings is discussed in relation to the flagellar structure.     

Development of a Vital Fluorescent Staining Method for Monitoring Bacterial Transport in Subsurface Environments

Previous bacterial transport studies have utilized fluorophores which have been shown to adversely affect the physiology of stained cells. This research was undertaken to identify alternative fluorescent stains that do not adversely affect the transport or viability of bacteria. Initial work was performed with a groundwater isolate, Comamonas sp. strain DA001. Potential compounds were first screened to determine staining efficiencies and adverse side effects. 5-(And 6-)-carboxyfluorescein diacetate, succinimidyl ester (CFDA/SE) efficiently stained DA001 without causing undesirable effects on cell adhesion or viability. Members of many other gram-negative and gram-positive bacterial genera were also effectively stained with CFDA/SE. More than 95% of CFDA/SE-stained Comamonas sp. strain DA001 cells incubated in artificial groundwater (under no-growth conditions) remained fluorescent for at least 28 days as determined by epifluorescent microscopy and flow cytometry. No differences in the survival and culturability of CFDA/SE-stained and unstained DA001 cells in groundwater or saturated sediment microcosms were detected. The bright, yellow-green cells were readily distinguished from autofluorescing sediment particles by epifluorescence microscopy. A high throughput method using microplate spectrofluorometry was developed, which had a detection limit of mid-10⁵ CFDA-stained cells/ml; the detection limit for flow cytometry was on the order of 1,000 cells/ml. The results of laboratory-scale bacterial transport experiments performed with intact sediment cores and nondividing DA001 cells revealed good agreement between the aqueous cell concentrations determined by the microplate assay and those determined by other enumeration methods. This research indicates that CFDA/SE is very efficient for labeling cells for bacterial transport experiments and that it may be useful for other microbial ecology research as well.     

An Efficient Staining Method for Ultrathin Sections

A staining method to handle simultaneously as many as 20 electron microscope grids is described. The devices used are easily constructed of readily obtained inexpensive materials. The volumes of stain and wash water required are very small and drying grids is simplified.     

Rapid Nuclear Staining Method for Saccharomyces cerevisiae

Mithramycin was used to stain nuclei in mitotically dividing and sporulating yeast.     

The Benzidine Staining Method for Blood Vessels

Two modifications of the method are described: A. Living specimens of sabellid and serpluid polychaetes, earthworms, small tadpoles, or fish larvae are immersed in an approximately saturated solution of benzidine for 30 minutes and then 3% hydrogen peroxide is added until bubbles of gas appear. When the blood vessels appear dark blue, the specimens are fixed in acidified 70% alcohol, dehydrated, cleared and either mounted in Canada balsam as whole mounts, or embedded in paraffin, sectioned at 100 to 250µ and mounted. B. Material fixed in 10% formalin in sea-water, or in formalin hypertonic saline, is incubated at 37°C. for one hour in an aqueous mixture containing sodium nitroprusside, 0.1%; benzidine, acetic acid 0.5%, followed by a weak (0.01–0.02%) hydrogen peroxide solution for a further hour, embedded in paraffin, cut into thick sections and mounted.     

Bacterial Flagella: Twist and Stick,or Dodge across the Kingdoms

The flagellum organelle is an intricate multiprotein assembly best known for its rotational propulsion of bacteria. However, recent studies have expanded our knowledge of other functions in pathogenic contexts, particularly adherence and immune modulation, e.g., for Salmonella enterica, Campylobacter jejuni, Pseudomonas aeruginosa, and Escherichia coli. Flagella-mediated adherence is important in host colonisation for several plant and animal pathogens, but the specific interactions that promote flagella binding to such diverse host tissues has remained elusive. Recent work has shown that the organelles act like probes that find favourable surface topologies to initiate binding. An emerging theme is that more general properties, such as ionic charge of repetitive binding epitopes and rotational force, allow interactions with plasma membrane components. At the same time, flagellin monomers are important inducers of plant and animal innate immunity: variation in their recognition impacts the course and outcome of infections in hosts from both kingdoms. Bacteria have evolved different strategies to evade or even promote this specific recognition, with some important differences shown for phytopathogens. These studies have provided a wider appreciation of the functions of bacterial flagella in the context of both plant and animal reservoirs.