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Basic Histological Techniques for Planarians

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Planarian Regeneration

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1774))

Abstract

The chapter provides a concise account of collecting techniques, and basic histological techniques for investigating planarians, intended for use by practicing scientists and postgraduate students new to the field. Selected hands-on methods are described that will lead to well-stained preparations suitable for light microscopic examination for subsequent taxonomic identification or general investigations into planarian functional cytology and histology. Major equipment items that are minimal essential requirements of a functional histological laboratory are indicated. The chapter includes sectioning and staining troubleshooting guides that will be very helpful for those who must work in the laboratory without the opportunity to consult qualified and experienced technicians or microscopists.

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Notes

  1. 1.

    Chloral hydrate acts as a preservative and can be omitted. Add a few crystals of thymol as a preservative.

  2. 2.

    Synonym: Crystal Ponceau 6R, available through SIGMA chemicals.

  3. 3.

    Synonym: Aniline Blue WS. Methyl Blue (C.I. 42780) may be substituted for the Soluble Blue.

  4. 4.

    The original method stated 10 min staining in Soluble Blue. This was found to be generally too long for land planarians.

References

  1. Sluys R (2001) Towards a phylogenetic classification and characterization of dugesiid genera (Platyhelminthes, Tricladida, Dugesiidae): a morphological perspective. In: Littlewood DTJ, Bray RA (eds) Interrelationships of the Platyhelminthes, Systematics association special volume series, vol 60. Taylor & Francis, London, pp 57–73

    Google Scholar 

  2. Sluys R, Kawakatsu M (2006) Towards a phylogenetic classification of dendrocoelid freshwater planarians (Platyhelminthes): a morphological and eclectic approach. J Zool Syst Evol Res 44:274–284

    Article  Google Scholar 

  3. Baguñà J, Carranza S, Paps J et al (2001) Molecular taxonomy and phylogeny of the Tricladida. In: Littlewood DT, Bray RA (eds) Interrelationships of the Platyhelminthes, Systematics association special volume series, vol 60. Taylor & Francis, London, pp 49–56

    Google Scholar 

  4. Álvarez-Presas M, Baguñà J, Ruitort M (2008) Molecular phylogeny of land and freshwater planarians (Tricladida, Platyhelminthes): from freshwater to land and back. Mol Phyl Evol 47:555–568

    Article  Google Scholar 

  5. Sluys R, Solà E, Gritzalis K et al (2013) Integrative delineation of species of Mediterranean freshwater planarians (Platyhelminthes: Tricladida: Dugesiidae). Zool J Linnean Soc 169:523–547

    Article  Google Scholar 

  6. Carbayo F, Álvarez-Presas M, Olivares T et al (2013) Molecular phylogeny of Geoplaninae (Platyhelminthes) challenges current classification: proposal of taxonomic actions. Zool Scr 42:508–528

    Article  Google Scholar 

  7. Von Gelei J (1929) Untersuchungsmethoden für Turbellarien. Z wiss Mikrosk mikrosk Techn 46:45–88

    Google Scholar 

  8. Gabe M (1976) Histological techniques. Masson, Paris

    Book  Google Scholar 

  9. Gray P (1964) Handbook of basic microtechnique, 3rd edn. McGraw-Hill, New York

    Google Scholar 

  10. Humason GL (1967) Animal tissue techniques, 2nd edn. Freeman, San Francisco

    Google Scholar 

  11. McClung-Jones R (1966) Basic microscopic technics. University of Chicago Press, Chicago

    Google Scholar 

  12. Romeis B (1989) Mikroskopische Technik, 17th edn. Urban & Schwarzenberg, Munich

    Google Scholar 

  13. Sanderson JB (1994) Royal microscopical handbooks 28: biological microtechnique. BIOS Scientific Publishers Ltd., Oxford

    Google Scholar 

  14. Subarna SK, Layton C, Bancroft JD (2013) Bancroft’s theory and practice of histological techniques, 7th edn. London, Churchill Livingstone-Elsevier

    Google Scholar 

  15. Renier-DiCiaula LL, Foley GL, Schaeffer DJ (1995) Fixation and staining of planaria for histological study. Biotech Histochem 70:121–123

    Article  Google Scholar 

  16. Buesa RJ, Peshkov MV (2009) Histology without xylene. Ann Diagn Pathol 13:246–256

    Article  Google Scholar 

  17. Lillie RD, Fullmer HM (1976) Histopathologic technic and practical histochemistry, 4th edn. McGraw-Hill, New York

    Google Scholar 

  18. Cason JE (1950) A rapid one-step Mallory Heidenhain stain for connective tissue. Stain Technol 25:225–226

    Article  CAS  Google Scholar 

  19. Galigher AE, Kozloff EN (1971) Essentials of practical microtechnique. Lea and Febiger, Philadelphia

    Google Scholar 

  20. Neuhaus B, Schmidt T, Riedel J (2017) Collection management and study of microscope slides: storage, profiling, deterioration, restoration procedures, and general recommendations. Zootaxa 4322(1):173

    Article  Google Scholar 

  21. Sluys R (1989) A monograph of the marine triclads. Balkema, Rotterdam

    Google Scholar 

  22. Bouin P (1897) Etudes sur l’evolution normal et l’involution du tube séminifère. Arch d’Anat Microsc 1:229

    Google Scholar 

  23. Lillie RD (1997) H.J. Conn’s Biological Stains, 9th edn. Williams & Wilkins, Baltimore

    Google Scholar 

  24. Kenk R (1976) Freshwater planarians (Turbellaria) of North America. 2nd printing. U. S. Environmental Protection Agency, Water Pollution Control Research Series 18050 ELDO2/72

    Google Scholar 

  25. Kawakatsu M, Takai M, Hori H et al (1985) Some collecting methods of freshwater planarians. Occ Publ Biol Lab Fuji Women’s College 14:2–8

    Google Scholar 

  26. Wilhelmi J (1909) Tricladen. Fauna und Flora des Golfes von Neapel, vol 32. Friedländer und Sohn, Berlin

    Google Scholar 

  27. Ohbayashi T, Okochi I, Sato H et al (2005) Food habit of Platydemus manokwari De Beauchamp, 1962 (Tricladida: Terricola: Rhynchodemidae), known as a predatory flatworm of land snails in the Ogasawara Islands, Japan. Japan J Appl Entomol Zool 40:609–614

    Article  Google Scholar 

  28. Ogren RE (1955) Ecological observations on the occurrence of Rhynchodemus, a terrestrial turbellarian. Trans Am Microsc Soc 74:54–60

    Article  Google Scholar 

  29. Blackshaw RP (1990) Studies on Artioposthia triangulata (Dendy) (Tricladida: Terricola), a predator of earthworms. Ann Appl Biol 116:169–176

    Article  Google Scholar 

  30. Yeates GW, Boag B, Johns PM (1998) Field and laboratory observations on terrestrial planarians from modified habitats in New Zealand. Pedobiologia 42:554–562

    Google Scholar 

  31. Southwood TRE (1996) Ecological methods. Chapman & Hall, London

    Google Scholar 

  32. Raw F (1959) Estimating earthworm populations by using formalin. Nature 184:257

    Google Scholar 

  33. Blackshaw RP, Stewart VI (1992) Artioposthia triangulata (Dendy, 1894), a predatory terrestrial planarian and its potential impact on lumbricid earthworms. Agricult Zool Rev 5:201–219

    Google Scholar 

  34. Lee AB (1921) The microtomist’s vade-mecum, 8th edn. J. & A Churchill, London

    Google Scholar 

  35. Leong AS-Y (1994) Fixation and fixatives. In: Woods AE, Ellis RC (eds) Laboratory Histopathology—a complete reference, vol 1. Churchill Livingstone, Edinburgh

    Google Scholar 

  36. Churukian CJ (1993) Zinc facts. Histo-Logic 1:13

    Google Scholar 

  37. Kawakatsu M, Miyazaki T (1972) Effect of different fixatives on common Japanese freshwater planarian Dugesia japonica Ichikawa et Kawakatsu. Bull Fuji Women’s Coll 10:81–117. Series II

    Google Scholar 

  38. Ball IR, Reynoldson TB (1981) British planarians. (Synopses of British fauna 19). Linnean Society of London/Cambridge University Press, Cambridge

    Google Scholar 

  39. Young JZ (1935) Osmotic pressure of fixing fluids. Nature 135:823–824

    Article  CAS  Google Scholar 

  40. Justine J-L, Briand MJ, Bray RA (2012) A quick and simple method, useable in the field, for collecting parasites in suitable condition for both morphological and molecular studies. Parasitol Res 111:341–351

    Article  Google Scholar 

  41. Dawar BL (1973) A combined relaxing agent and fixative for Triclads (Planarians). Stain Technol 48:93–94

    Article  CAS  Google Scholar 

  42. Winsor L (1991) Methods for taxonomic and distributional studies of terrestrial flatworms (Tricladida: Terricola). Hydrobiologia 227:349–352

    Article  Google Scholar 

  43. Winsor L (1998) Collection, handling, fixation, histological and storage procedures for taxonomic studies of terrestrial flatworms (Tricladida: Terricola). Pedobiologia 42:405–411

    Google Scholar 

  44. von Graff L (1899) Monographie der Turbellarien. II. Tricladida Terricola (Landplanarien). Wilhelm Engelmann, Leipzig

    Google Scholar 

  45. Huber JT (1998) The importance of voucher specimens, with practical guidelines for preserving specimens of the major invertebrate phyla for identification. J Nat Hist 32:367–385

    Article  Google Scholar 

  46. Mesibov R (2012) Known unknowns, Google Earth, plate tectonics and Mt Bellenden Ker: some thoughts on locality data. Zookeys 247:61–67. http://www.pensoft.net/journals/zookeys/article/4195/

    Article  Google Scholar 

  47. Steigerwald M, Laframboise S (1996) Tape application: a jar sealing method for reduced ethanol evaporation in fluid-preserved collections. Collection Forum 12:45–54

    Google Scholar 

  48. Gillette JW (2004) Jar sealing tape for use with fluid-preserved collections: a summary of recommendations and sources. SPNHC Newsletter 18:15

    Google Scholar 

  49. Moore S (1989) Conservation of spirit specimens. In: Howie CV (ed) Conservation of natural history specimens spirit collections. University of Manchester, Manchester, Manchester Museum and Department of Environmental Biology

    Google Scholar 

  50. Simmons JE (2014) Fluid preservation—a comprehensive reference. Rowman & Littlefield, Maryland

    Google Scholar 

  51. Winsor L (1994) Tissue processing. In: Woods AE, Ellis RC (eds) Laboratory histopathology—a complete reference, vol 1. Churchill Livingstone, Edinburgh. (also at https://www.researchgate.net/publication/260272795_Tissue_Processing)

    Google Scholar 

  52. Doxtader EK (1948) Isopropyl alcohol in the paraffin infiltration technic. Stain Technol 23:1–2

    Article  CAS  Google Scholar 

  53. Hauser J (1952) Ausschaltung des Xylols in der histologischen Technik. Microscopie 7:208–211

    CAS  Google Scholar 

  54. da Vara DC, Leal-Zanchet AM, Lizardo-Daudt HM (2001) Histological processing techniques for the study of Dugesiidae development (Platyhelminthes, Tricladida, Paludicola). Rev Bras Biol 61:341–345

    Article  Google Scholar 

  55. Leica Biosystems Microtomy and paraffin sectioning preparation http://www.leicabiosystems.com/pathologyleaders/topics/histology-sample-preparation/more/topic/Topic////page_t/2/ (this site includes a valuable library of histological methods and trouble-shooting guides including for difficult tissues)

  56. Lindekens Y (2007) Making your own paraffin sections @ home. Part 2. http://www.microscopy-uk.org.uk/mag/indexmag.html?http://www.microscopy-uk.org.uk/mag/artfeb07/yl-para2.html

  57. Steedman HF (1960) Section cutting in microscopy. Blackwell Scientific Publications, Oxford

    Google Scholar 

  58. Cooper DW (1988) The preparation of serial sections of platyhelminth parasites, with details of the materials and facilities required. Syst Parasitol 12:211–229

    Article  Google Scholar 

  59. Richards OW (1959) The effective use and proper care of the microtome. American Optical, Buffalo, NY

    Google Scholar 

  60. Frandsen JC (1964) Serial sections; simplified handling of paraffin ribbons on a floating-out bath. Stain Technol 39:279–282

    Article  CAS  Google Scholar 

  61. Drury RAB, Wallington EA (1980) Carleton’s histological technique, 5th edn. Oxford University Press, Oxford

    Google Scholar 

  62. Pantin CFA (1969) Notes on microscopical technique for zoologists. Cambridge University Press, Cambridge

    Google Scholar 

  63. Hauser J (1956) Beiträge zur Turbellarienhis-tologie: untersuchungen über die histologische struktur des Tricladenpharynx. Rev Bras Biol 16:37–58

    Google Scholar 

  64. Froehlich EM, Leal-Zanchet AM (2003) A new species of terrestrial planarian of the genus Notogynaphallia Ogren & Kawakatsu (Platyhelminthes, Tricladida, Terricola) from south Brazil and some comments on the genus. Rev Brasil Zool 20:745–753

    Article  Google Scholar 

  65. Heidenhain M (1915) Über die Mallorysche Bindegewebsfärbung mit Karmin und Azokarmin als Vorfarben. Z wiss Mikrosk mikrosk Techn 32:361–372

    Google Scholar 

  66. Lendrum AC, Fraser DS, Slidders W et al (1962) Studies on the character and staining of fibrin. J Clin Pathol 15:401–413

    Article  CAS  Google Scholar 

  67. Culling CFA (1974) Handbook of histopathological and histochemical techniques, 3rd edn. Butterworths, England

    Google Scholar 

  68. Kiernan JA (2008) Histological and histochemical methods. Theory and practice, 4th edn. Scion Publishing, Oxfordshire

    Google Scholar 

  69. Thompson SW, Luna LG (1978) An atlas of artifacts encountered in the preparation of microscopic tissue sections. Thomas, Springfield

    Google Scholar 

  70. Schoobridge MPK (1983) A new principle in polychrome staining: a system of automated staining, complementary to haematoxylin and eosin, and useable as a research tool. Stain Technol 58:245–258

    Article  Google Scholar 

  71. Horobin RW, Flemming L (1988) One-bath trichrome staining: investigation of a general mechanism based on a structure-staining correlation analysis. Histochem J 20:29–34

    Article  CAS  Google Scholar 

  72. Horobin RW, Bancroft JD (1998) Troubleshooting histology stains. Churchill Livingstone—Harcourt Publishers Ltd, Edinburgh

    Google Scholar 

  73. Gurr E (1973) Biological staining methods, 8th edn. Searle Diagnostic Gurr Products, High Wycombe

    Google Scholar 

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Acknowledgments

The authors thank Eduard Solà for his fine rendition of Fig. 9. N. Korenhof and M. Hermsen (Naturalis Biodiversity Center) are thanked for the digital rendering of the figures, and Laurie and Sue Reilly, and Karen Reeks (James Cook University) are thanked for access to histological facilities and the photomicroscope.

Author information

Authors and Affiliations

  1. Zoology & Ecology, College of Science and Engineering, James Cook University, Townsville, QLD, Australia

    Leigh Winsor

  2. Naturalis Biodiversity Center, Leiden, The Netherlands

    Ronald Sluys

Authors
  1. Leigh Winsor
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  2. Ronald Sluys
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Corresponding author

Correspondence to Ronald Sluys .

Editor information

Editors and Affiliations

  1. Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany

    Jochen C. Rink

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Winsor, L., Sluys, R. (2018). Basic Histological Techniques for Planarians. In: Rink, J. (eds) Planarian Regeneration. Methods in Molecular Biology, vol 1774. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7802-1_9

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  • DOI: https://doi.org/10.1007/978-1-4939-7802-1_9

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  • Publisher Name: Humana Press, New York, NY

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