Prova de tipográfica (In Press).Aims: To improve bead ingestion counts in Tetrahymena pyriformis by automated image analysis as an alternative to direct-counts.
Methods and Results: Fluorescent latex beads were added to T. pyriformis cultures for ingestion tests. The number of beads ingested by 25 cells was counted directly by epifluorescence microscopy and compared with similar data from image analysis. ANOVA indicated that counts were not significantly different (P < 0.05). The image
analysis particularly provided advantages in terms of speed.
Conclusions: The image analysis is superior to direct beads counting in T. pyriformis particularly in terms of speed of analysis.
Significance and Impact of the Study: The image analysis method is very rapid and will allow many more toxicological analyses to be undertaken with less operator error

Alevisopoulos G.

Blackburn N.

Corkidi G.

Kuehn M.

Nilsson J.R.

Letters in Applied Microbiology

Dias, Nicolina

Amaral, A. L.

Ferreira, Eugénio C.

Lima, Nelson

Universidade do Minho: RepositoriUM

UNCORRECTEDPROOFAutomated image analysis to improve bead ingestion toxicitytest counts in the protozoan Tetrahymena pyriformisN. Dias1, A.L. Amaral1,2, E.C. Ferreira1 and N. Lima11Centro de Engenharia Biolo´gica, Universidade do Minho, Campus de Gualtar, Braga, Portugal, and 2Departamento de TecnologiaQuımica, Escola Superior de Tecnologia e de Gesta~o, Instituto Polite´cnico de Braganc¸a, Campus de Santa Apolo´nia, Braganc¸a,Portugal2003/30: received 14 January 2003, revised 24 March 2003 and accepted 1 June 2003ABSTRACTN. D IAS , A .L . AMARAL, E .C. FERREIRA AND N. L IMA. 2003.Aims: To improve bead ingestion counts in Tetrahymena pyriformis by automated image analysis as an alternative todirect-counts.Methods and Results: Fluorescent latex beads were added to T. pyriformis cultures for ingestion tests. Thenumber of beads ingested by 25 cells was counted directly by epifluorescence microscopy and compared with similardata from image analysis. ANOVA indicated that counts were not significantly different (P < 0Æ05). The imageanalysis particularly provided advantages in terms of speed.Conclusions: The image analysis is superior to direct beads counting in T. pyriformis particularly in terms of speedof analysis.Significance and Impact of the Study: The image analysis method is very rapid and will allow many moretoxicological analyses to be undertaken with less operator error.Keywords: Tetrahymena pyriformis, image analysis, ingestion, toxicity tests, bead counts.INTRODUCTIONThe protozoan Tetrahymena is used to assess the bioactivityof various chemicals because of its ability to ingest modelsubstances and can be used as an alternative to laboratoryanimals for this purpose (Nilsson 1981; Wakatsuki et al.1986; Stefanidou et al. 1990; Alevisopoulos et al. 1997;Stefanidou et al. 1999; Schiess et al. 2001). Nicolau et al.(1999) studied the influence of several toxicants on Tetra-hymena pyriformis grazing activity using fluorescent labelledlatex beads (FLLB), which are considered a valid materialfor these purposes. Recently, green fluorescent protein-expressing Escherichia coli coupled with fluorometry hasbeen used to determine protozoan ingestion rates (Parryet al. 2001). Image analysis techniques provide qualitativeand quantitative analysis of micro-organisms and providesignificant advantages over microscopic visualization usingthe naked eye (e.g. speed, accuracy and objectivity) (Bun˜oet al. 1998). The methodology has been used in the (a)determination of bacterial abundance, biovolume, morphol-ogy and growth (Blackburn et al. 1998); (b) detection ofconfluent microbial colonies for automated counting (Cork-idi et al. 1998); (c) analysis of biofilms (Kuehn et al. 1998)and (d) microbial community characterization (Amaral et al.1999). In the present work, image analysis was comparedwith direct FLLB counting to assess ingestion by T. pyr-iformis.MATERIALS AND METHODSMicro-organism and culture conditionsTetrahymena pyriformis (strain GL, ref. CCAP/1630/1F;Culture Collection of Algae and Protozoa, UK) was grownaxenically to a density of approximately 104 cells ml)1 inthe exponential phase, at room temperature (ca 22C), pHL A M 1 3 8 2 B Dispatch: 1.7.03 Journal: LAM CE: NagarajJournal Name Manuscript No. Author Received: No. of pages: 4 PE: BalajiCorrespondence to: Nelson Lima, Centro de Engenharia Biolo´gica, Universidade doMinho, Campus de Gualtar, 4710-057, Braga, Portugal (e-mail: nelson@iec.uminho.pt).ª 2003 The Society for Applied MicrobiologyLetters in Applied Microbiology 2003, 37, 1–4UNCORRECTEDPROOF7Æ0–7Æ5. Proteose peptone yeast extract medium (PPY),Difco proteose peptone 2% and Oxoid yeast extract 0Æ25%,was used for growth. The medium was filtered twice(0Æ45 lm membrane; Gelman1 ), before autoclave steriliza-tion, to avoid particle background upon microscopicobservation.Ingestion assayThe assay was performed in 2-ml Eppendorf test tubes, byadding the yellow–green FluoSpheres fluorescent micro-spheres (0Æ5 lm diameter, Molecular Probes Europe BV2 )to the cells at a rate of 1Æ05 · 106 bead per ml (Nicolauet al. 1999). The solution was vortexed for a minimum of5 min to avoid bead clustering. After incubation in thedark for 20 min, samples were fixed for 1 h with neutralbuffered formalin [10% (v/v) formalin in phosphate-buffered saline (PBS) pH 7Æ0, at a final concentration of2–5% (v/v)]. Cells were washed and resuspended in fresh0Æ1% (w/v) sodium azide in PBS and stored in the darkfor subsequent analysis. Bead counting was performed withan epifluorescence Laborlux S (Leitz, Wetzlar3 ) microscopeusing UV light (EX 450–490 nm excitation filter). Thenumber of beads ingested by 25 randomly selected cellswas counted at 1000· magnification, with minimum fielddiaphragm aperture in order to minimize excess illumin-ation, control glare and avoid internal reflection. Sam-ples from each independent assay were taken at 0Æ1, 1 and24 h.Image acquisitionThe image acquisition (50 images per sample) was accom-plished through fluorescence microscopy using the sameconditions as described above. All the images were digitizedusing a 3CCD DXC-9100P colour camera (Sony, Tokyo,Japan) and a Matrox Meteor II frame grabber (Matrox,Dorval4 ), with a 768 · 576 pixel size and 24 bits per imageresolution (8 bits per each colour channel, red, green andblue) by the Matrox Intellicam (Matrox) software package.Image processing and analysis was then performed inMATLAB 5Æ1 software package (The Mathworks Inc.,Natick, MA, USA). Graphical analysis of data fromMATLAB was performed with EXCELTM (MicrosoftCorp., Redmond, DC, USA).Image processing and analysisWith the purpose of determining the total area of beadspresent in each image, a program was created in MATLABcomprising the following steps:i Image loading: the images were loaded from a folder andacquired as colour TIFF format.ii Channel selection: the image was then decomposed in itsthree colour channels and the green channel image wassubsequently used in the program.iii Image smoothing: the resulting image was smoothed bya 2 · 2 window Wiener filter5 which performs a two-dimensional adaptive noise-removal filtering using theWiener method based on statistics estimated from thelocal neighbourhood of each pixel. This filter firstestimates the local mean l and the local variance r2 (1),(2):l ¼ 1NMXn1;n22gaðn1; n2Þ ð1Þr2 ¼ 1NMXn1;n22ga2ðn1; n2Þ  l2 ð2Þwhere g is the N · M local neighbourhood of each pixelin the image a. The filter then creates a pixelwiseWiener filter b using these estimates (3):bðn1; n2Þ ¼ lþ r2  m2r2 aðn1; n2Þ  l½ 	 ð3Þwhere m2 is the noise variance, i.e. the average of all thelocal estimated variances.iv Segmentation: the resulting image of the previous stepwas then segmented into a binary image with pixelsvalue of 1 for the objects and 0 for the background.Hence, the pixels above a user chosen threshold (defaultvalue of 0Æ35) were segmented to 1 (beads) and thevalues below that threshold to 0 (background).v Debris removal: in this step, all the objects in the imagewith an area smaller than 10 pixels were considered asdebris and, therefore, removed.vi Parameters determination: the morphological parameterbeads area was then determined from the final binaryimage, as the projected surface of the beads, i.e. the totalnumber of pixels belonging to all beads present in theimage.vii Result saving: the final binary image was saved in aTIFF format and the beads area in an ASCII file.Bead number determinationA previous calibration of 50 beads was performed in order toconvert the pixel area of one bead given by image analysis, inthe numerical determination of fluorescent beads. The meanarea A of a single bead was calculated by:A ¼ pr2 ð4Þwhere r is the radius of the bead.For the 1000· magnification, the area of each pixel was0Æ01918 lm2 and the average bead area was found to be0Æ19625 lm2 corresponding to 10Æ232 pixels.2 N. DIAS ET AL.ª 2003 The Society for Applied Microbiology, Letters in Applied Microbiology, 37, 1–4UNCORRECTEDPROOFStatistical data analysisANOVA was used to test the statistical significance (P < 0Æ05)of differences between automatic and direct count data.RESULTSPrior to the bead number determination, a microsphere sizecalibration must be performed. The area of 50 isolated beadswas determined and a mean area value of 9Æ958 ± 2Æ030 lm2was obtained. After image acquisition, the segmentationwas achieved using a chosen threshold or the default value of0Æ35.At the sampling times 0Æ1, 1 and 24 h good correlations of0Æ974, 0Æ971 and 0Æ983, respectively, were obtained for thetwo methods. The linear regressions between the automaticand direct-count are shown in Fig. 1. Some variation wasobserved in samples where the number of beads ingested byT. pyriformis was greater than 20. No significant differences(P < 0Æ05) were found between the numbers of beadscounted by both methods at any time of incubation (Fig. 2).DISCUSSIONImage analysis is used routinely in cellular biology studies.The present work demonstrates that the automated countmethod was as effective as the conventional method using amicroscope with the naked eye. The computerized methodallowed the acquisition of hundreds of images and subse-quent analysis in 1 h compared with approximately three tofour samples by conventional method. The choice of thethreshold value was of crucial importance in order to resolvethe beads from background and therefore great care isrequired with this step.Some underestimation in the number of beads counted bythe direct-count method occurred if more than 20 beads wereingested because of the intense fluorescence of the beads.This is not the case with the automated method. Pla¨sier et al.(1999) refer to the intrinsic subjectiveness of the direct-countmethod. Accordingly, the counting of a large number ofbeads in this work may differ from person to person or evenwithin different counts by the same operator. In conclusion,the high correlation values and the absence of significantdifferences between each method at all sampling timessuggest the superiority of image analysis to quantify beadsinside protozoa in subsequent toxicological studies.ACKNOWLEDGEMENTSThe authors wish to thank Dr Russell Paterson for hishelpful comments and corrections. N. Dias and A. L.Amaral were supported by PRAXIS-XXI BD/20328/99and BD/20325/99 grants, respectively, from FCT (Fun-dac¸a˜o para a Cieˆncia e Tecnologia), Portugal.REFERENCESAlevisopoulos, G., Stefanidou, M., Pagou, M., Siatra-Papastaikoudi,T. and Koutselinis, A. (1997) The effects of two new amphetamine-like derivatives on the phagocytic ability of the protozoon Tetra-hymena pyriformis. Alternatives to Laboratory Animals 25, 547–553.Amaral, A.L., Baptiste, C., Pons, M.N., Nicolau, A., Lima, N.,Ferreira, E.C., Mota, M. and Vivier, H. (1999) Semi-automated0102030405060100 20 30 40 50 60Number of beads (automatic-count)Number of beads (direct-count)Fig. 1 Regression lines between the number of beads counted bydirect count and automatic count, in 25 protozoa, at different samplingtimes (e) 0Æ1 h, (j) 1 h and (s) 24 h0501001502002503003504004505000·1 1 24Sampling time (h)Number of beadsFig. 2 Enumeration of beads ingested by Tetrahymena pyriformis, in25 protozoa counted by direct count (h) and by automatic count (j) atdifferent sampling timesAUTOMATIC COUNT OF BEAD INGESTION 3ª 2003 The Society for Applied Microbiology, Letters in Applied Microbiology, 37, 1–4UNCORRECTEDPROOFrecognition of protozoa by image analysis. Biotechnology Techniques13, 111–118.Blackburn, N., Hagstrom, A., Wikner, J., Cuadros-Hansson, R. andBjornsen, P.K. (1998) Rapid determination of bacterial abundance,biovolume, morphology, and growth by neural network-based imageanalysis. Applied and Environmental Microbiology 64, 3246–3255.Bun˜o, I., Juarranz, A., Can˜ete, M., Villanueva, A., Gosalvez, J. andStockert, J.C. (1998) Image processing and analysis of fluorescentlabelled cytoskeleton. Micron 29, 445–449.Corkidi, G., Diaz-Uribe, R., Folch-Mallol, J.L. and Nieto-Sotelo, J.(1998) COVASIAM: an image analysis method that allows detectionof confluent microbial colonies and colonies of various sizes forautomated counting. Applied and Environmental Microbiology 64,1400–1404.Kuehn, M., Hausner, M., Bungartz, H.J., Wagner, M., Wilderer, P.A.and Wuertz, S. (1998) Automated confocal laser scanning micro-scopy and semiautomated image processing for analysis of biofilms.Applied and Environmental Microbiology 64, 4115–4127.Nicolau, A., Mota, M. and Lima, N. (1999) Physiological responses ofTetrahymena pyriformis to copper, zinc, cyclohexymide and TritonX-100. FEMS Microbiology Ecology 30, 209–216.Nilsson, J.R. (1981) Effects of copper on phagocytosis in Tetrahymena.Protoplasma 109, 359–370.Parry, J.D., Heaton, K., Drinkall, J. and Jones, H.L.J. (2001)Feasibility of using GFP-expressing Escherichia coli, coupled withfluorometry, to determine protozoan ingestion rates. FEMS Micro-biology Ecology 35, 11–17.Pla¨sier, B., Lloyd, D.R., Paul, G.C., Thomas, C.R. and Al-Rubeai, M.(1999) Automatic image analysis for quantitation of apoptosis inanimal cell culture by annexin V affinity assay. Journal of Immuno-logical Methods 229, 81–95.Schiess, N., Csaba, G. and Kohidai, L. (2001) Chemotactic selectionwith insulin, di-iodotyrosine and histamine alters the phagocyticresponsiveness of Tetrahymena. Comparative Biochemistry and Phys-iology 128, 521–530.Stefanidou, M.E., Georgiou, M., Maravelias, C. and Koutselinis, A.(1990) The effects of morphine, cocaine, amphetamine and hashishon the phagocytosis of the protozoon Tetrahymena pyriformis strainW. Toxicology in Vitro 4, 779–781.Stefanidou, M.E., Alevisopoulos, G., Maravelias, C., Loutsidis, C. andKoutselinis, A. (1999) Phagocytosis of the protozoon Tetrahymenapyriformis as an endpoint in the estimation of cocaine salt and cocainefreebase toxicity. Addiction Biology 4, 449–452.Wakatsuki, T., Tazaki, Y. and Imahara, H. (1986) Respiratoryinhibition by copper in Tetrahymena pyriformis GL. Journal ofFermentation Technology 64, 119–127.4 N. DIAS ET AL.ª 2003 The Society for Applied Microbiology, Letters in Applied Microbiology, 37, 1–4

Automated image analysis to improve bead ingestion toxicity test counts in the protozoan Tetrahymena pyriformis

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Automated image analysis to improve bead ingestion toxicity test counts in the protozoan Tetrahymena pyriformis

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