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Measurement of Motility of Helicobacter Pylori, Campylobacter

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Measurement of Motility of Helicobacter Pylori, Campylobacter J Clin Pathol 1998;51:623–628 623 Measurement of motility of Helicobacter pylori, J Clin Pathol: first published as 10.1136/jcp.51.8.623 on 1 August 1998. Downloaded from Campylobacter jejuni, and Escherichia coli by real time computer tracking using the Hobson BacTracker Q N Karim, R P H Logan, J Puels, A Karnholz, M L Worku Abstract organisms, and the slightly slower growth of Aims—(1) To make precise measurements motile flagellate cells would ensure the loss of and comparisons of various aspects of the motile phenotype if it did not confer some motility of three gastrointestinal patho- substantial benefit to the cell, as explained by gens, Helicobacter pylori, Campylobacter Armitage.1 Microbial motility can be assessed jejuni, and Escherichia coli, in log phase microscopically by hanging drop, by phase growth; (2) to provide background infor- contrast and dark ground illumination, or mation on motility data to study the influ- macroscopically, by growth in semisolid agar, ence of pH, viscosity, and chemotactic swarming on surface of solid agar, or by the factors, thereby gaining a better under- crossing of a filter paper bridge over a trench standing of bacterial pathogenesis. cut in solid agar. Methods—Computer image processing These methods distinguish motile from non- technology and phase contrast micros- motile bacteria and assess motility only subjec- copy (Hobson BacTracker) were used to tively, though giving the impression that some measure several indices of bacterial mo- bacteria move faster than others. The methods tility in real time. Ten clinical isolates of are not precise enough to follow transient or each species in log phase liquid culture minor changes in motility. Video recording of were studied. motile bacteria has provided a means of quan- Results—C jejuni moved fastest, with a tifying motility. The video recorded tapes have median curvilinear velocity (CLV) of 38.76 been replayed, and motility measured manually µm/s (range 29.08 to 52.82). Next was H on the screen by planimeter and stop watch, as pylori, median CLV 25.02 µm/s (range described by Ferrero and Lee.2 Such manual 12.07 to 29.07). E coli was the slowest, measurements are cumbersome and diYcult to median CLV 12.73 µm/s (range 8.20 to do on large numbers of bacteria. http://jcp.bmj.com/ 18.04). The straight line velocities showed These problems can be overcome by the similar trends. Measurement of track lin- Hobson BacTracker (Hobson Tracking Sys- earity (TL) showed that C jejuni moved tem, SheYeld, UK), which allows precise and the straightest (TL 60.3%), H pylori objective measurements of motility. It is a moved in wide circles (TL 28.7%), and E measuring system that incorporates unique coli showed spinning movement without “blob and track” image processing technology, Department of much linear displacement (TL 18.3%). analysing bacterial movement in real time. The Microbiology, Imperial There were significant diVerences in these tracker comprises a phase contrast microscope on September 30, 2021 by guest. Protected copyright. College of Science, three variables between the species stud- connected to a video camera. The camera is Technology and ied, but no significant diVerences in linked to a video recorder, which is connected Medicine at St Mary’s measurements of time and frequency of to a computer that tracks 120 moving bacteria Hospital, London W2, halts between movement runs. on screen simultaneously and continuously. UK Q N Karim Conclusions—The BacTracker provides a The results are displayed as histograms or trail J Puels useful technical aid for measuring several draws. This system records several indices of A Karnholz indices of bacterial motility objectively, motility including direction, curvature rates, M L Worku reproducibly, and precisely, which is diY- curvilinear velocity, and straight line velocity, cult to achieve without computer assist- which can be measured accurately, objectively, Division of ance. Accurate quantification of motility and reproducibly so that comparisons can be Gastroenterology, Queens Medical provides a basis for studying the factors made under diVerent experimental conditions. Centre, Nottingham, which influence bacterial motility. It can Recordings of movement can be stored in the UK provide phenotypic measurements of the computer and can be downloaded for more R P H Logan eVect of flagellar gene depletion. detailed statistical analysis. (J Clin Pathol 1998;51:623–628) Real time computer tracking has been used Correspondence to: to assess the motility of Rhodobacter sphaeroides, Dr Q N Karim, Senior Keywords: bacterial motility measurement; computer Lecturer, Department of image processing technology; bacterial tracking Rhodospirullum rubrum, and Salmonella Microbiology, Imperial typhimurium.3 The aim of our present study was College of Science Technology and Medicine at to assess the motility of Helicobacter pylori (H St Mary’s Hospital Medical For many bacteria, motility confers a survival pylori), Campylobacter jejuni (C jejuni), and School, Norfolk Place, advantage by permitting migration towards a Escherichia coli (E coli). These are major patho- London W2 1PG, UK. favourable microenvironment, or away from an gens infecting the upper and lower gastrointes- Accepted for publication unfavourable one. The operation of motility tinal tract, in which motility is acknowledged to 16 April 1998 machinery is very energy expensive for micro- be a virulence factor.2 4–6 Motility measure- 624 Karim, Logan, Puels, et al pears from view or moves out of the analysis window (fig 1). J Clin Pathol: first published as 10.1136/jcp.51.8.623 on 1 August 1998. Downloaded from Stop—A stop occurs when the speed of the bacterial cell falls below the stop speed defined in the setting screen. This was set at the speed shown by Brownian movement of dead bacte- ria. Run—A run is the track between two stops (fig 1). Curvilinear velocity (CLV)—This is the length of a track divided by the time taken to travel it. It is calculated by summing the incre- mental distances moved in each frame along the sampled path and divided by the total time for the track. It is measured for tracks (total path length) and for runs (incremental path lengths between two stops) in µm/s (fig 2). Straight line velocity (SLV)—This is calcu- lated by measuring the straight line distance between the start and end point of the track and dividing by the time taken to travel it. It is measured in µm/s (fig 2). Track linearity percentage (TL%)—This is the ratio of the straight line velocity to curvilinear velocity × 100 (SLV/CLV(100)). For a bacte- Figure 1 Quantities analysed by the BacTracker. rium that runs straight this value is 100%. The ments of these bacteria in log phase growth can more curved the route taken by the bacterium form the basis for studying and comparing the greater will be the curvilinear velocity, and changes that take place because of diVerent the lower the straight line velocity and the value environmental factors, such as phases of of track linearity percentage. For a bacterium growth, pH, temperature, and the presence of that spins around a point the straight line antimicrobial agents. These measurements can velocity may be so small compared with the be used to study chemotaxis and help us to curvilinear velocity that the value may ap- understand this aspect of bacterial physiology proach 0%. Thus, depending on the straight- and pathogenesis. ness or curvature of the path of the bacterium, the value of track linearity will be between 100% and 0%. This is shown diagrammatically Methods in fig 2. http://jcp.bmj.com/ THE HOBSON BACTRACKER Curvature rate (CVRT+/s)—This is meas- The BacTracker separates the moving parts of ured using the incremental sum of change in the image from the static parts. The moving angle as the bacterium changes direction for object is then filtered and a threshold imposed the length of the track. It includes the sign to to produce coloured “blobs” which are super- reflect the direction of change. For each track imposed on the moving objects on the black point the signed change in angle from the pre- and white video image. Once the moving vious track point is measured. The sum of the objects have been identified the BacTracker has signed change of angle is accumulated along on September 30, 2021 by guest. Protected copyright. the coordinates of every moving object in the the track. At the end of the track the sum of the frame and with this information various meas- signed angle change is divided by the time for urements can be made. The BacTracker is a the track to give a value for the curvature rate in real time image processing system where degrees per second. For bacteria with net anti- motility measurements are carried out as they clockwise movement the value is positive, and are occurring—the computer measures con- for a bacteria with net clockwise the value is tinuously and does not have to stop, calculate, negative. This is shown as direction change and measure as in snapshot measurements (DRCH) (fig 1). made over a short period of time. Various Stop time (STTM)—This is the time of a measurements are displayed on the computer defined stop between two adjacent runs. The screen, such as summary graphs and histo- average of all the stop time is displayed in sec- grams, while the tracking screen shows the cells onds (fig 1). as they are tracked. Thus the system can be Stop frequency (STFRQ)—This is a measure validated while watching in real time. The of how often the cell stops. The time is video images are processed at 25 Hz in PAL measured from the start of a run through to the format, or 30 Hz or 6 Hz in NTSC North end of the following stop or the start of a new American format.
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