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  1. 1. Some Techniques in the Study of Plant Development Biology Group 3 Tanchuling, Raymund – LEADER Bituin, Dawnn Faustino, Missy Fojas, Juris Gonzaga, Sahara
  2. 2. Free Hand Sectioning
  3. 3. <ul><li>Terms: </li></ul><ul><ul><li>Sectioning - The act or process of cutting or dividing parts from a fixed mass of tissue </li></ul></ul><ul><li>Simple method </li></ul><ul><li>Used for specimens that are able to withstand the impact of the sectioning knife or blade </li></ul>
  4. 4. Why do we use free hand sectioning? <ul><li>In order to study the structural organization of the plant body. </li></ul>
  5. 5. The General technique <ul><li>Soft herbaceous stems and small woody twigs </li></ul><ul><li>Generally, tissue is cut without a supporting matrix </li></ul>
  6. 6. Double razor blade Water Stem +
  7. 7. Figure 1. One method of holding a specimen for free hand sectioning; Adapted from Do not use the “chopping” action! 
  8. 8. Petridish with water *Do not use forceps! With sectiongs
  9. 9. Select and transfer the thinnest sections onto the glass slide, stain and cover with cover slip View in the microscope **The general histological stain for free hand sections is Toluidine Blue stain
  10. 10. Special technique for delicate and hard to hold specimens <ul><li>For thin leaves and tiny roots </li></ul><ul><li>Support matrix is used. </li></ul>
  11. 11. Figure 2. A V-shaped notched is removed from the papaya block to accommodate a specimen for longitudinal sections; Adapted from
  12. 12. Leaf Midrib Leaf midrib inserted into the papaya
  13. 13. Petridish with a fixing solution <ul><li>Farmer’s Solution </li></ul><ul><li>It kills the specimen </li></ul><ul><li>It also preserve the structures </li></ul>
  14. 14. Mount the material on a slide in a drop of 50% glycerol and cover with cover slip.
  15. 15. Definition of terms <ul><li>Fixing </li></ul><ul><ul><li>Disable intrinsic biomolecules (proteolytic enzymes) which otherwise digests sample </li></ul></ul><ul><ul><li>protects a sample from extrinsic damage </li></ul></ul><ul><ul><li>alter cells/tissues to increase their mechanical strength/stability </li></ul></ul><ul><li>Smear or squash </li></ul><ul><ul><li>spread cells evenly in a single layer to facilitate killing of the cells instantly; evenly fix cells without the formation of artifacts; staining and dehydration processes with the loss of only a few cells </li></ul></ul><ul><li>Staining </li></ul><ul><ul><li>application of stain to a sample to color cells, tissues, components, or metabolic processes </li></ul></ul>
  16. 16. Squash or Smear Technique <ul><li>Useful in: </li></ul><ul><ul><li>counting monoploid an diploid chromosomes </li></ul></ul><ul><ul><li>studying the chromosomes themselves </li></ul></ul><ul><ul><li>critical cytological studies </li></ul></ul>
  17. 17. Squash or Smear Technique <ul><li>Methodolgy </li></ul><ul><ul><li>FAA prep in hood </li></ul></ul><ul><ul><ul><li>FAA: 66ml 95% ethanol </li></ul></ul></ul><ul><ul><ul><li>21ml water </li></ul></ul></ul><ul><ul><ul><li>8ml commercial formalin (37%) </li></ul></ul></ul><ul><ul><ul><li>5ml glacial acetic acid </li></ul></ul></ul><ul><ul><li>Dissect open anthers (diff dev’l stages) </li></ul></ul><ul><ul><li>↓ </li></ul></ul><ul><ul><li>Fix (FAA (formalin+acetic acid+alcohol) </li></ul></ul><ul><ul><li>for 10-15 mins (or 1-2 days) </li></ul></ul><ul><ul><li>↓ </li></ul></ul><ul><ul><li>Stain (toluidine blue /acetocarmine) </li></ul></ul><ul><ul><li>↓ </li></ul></ul><ul><ul><li>Mount (glass slide+cover slip) </li></ul></ul><ul><ul><li>↓ </li></ul></ul><ul><ul><li>Squash (eraser of pencil) </li></ul></ul>
  18. 18. Fixation <ul><li>Kill tissue so that decay is prevented </li></ul><ul><li>preserves tissue/cells as close to its natural state as possible in preparation for examination. </li></ul>
  19. 19. Fixation <ul><li>Noncoagulating tanning agents </li></ul><ul><ul><li>Less violent denaturants </li></ul></ul><ul><ul><li>Potassium dichromate, osmium tetroxide (osmic acid), acetic acid, and formaldehyde </li></ul></ul><ul><li>Coagulating agents </li></ul><ul><ul><li>Violent denaturants </li></ul></ul><ul><ul><li>Coagualate proteins </li></ul></ul><ul><ul><li>Heat, trinitrophenol (picric acid), chromium trioxide (chromic acid), mercuric chloride, and ethanol </li></ul></ul>
  20. 20. <ul><li>Coagulating fixatives </li></ul><ul><ul><li>Acid fixatives </li></ul></ul><ul><ul><ul><li>good for preserving chromatin, nucleoli, and spindles </li></ul></ul></ul><ul><ul><ul><li>cytoplasm is preserved as a stringy, coagulated mass, but some organelles are dissolved ( e.g.,  mitochondria.) </li></ul></ul></ul>
  21. 21. Acid fixatives <ul><li>Formalin-Acid-Alcohol </li></ul><ul><ul><li>Formalin-Acetic acid-Alcohol (FAA); Formalin-Propionic acid-Alcohol (FPA) </li></ul></ul><ul><ul><li>Penetration not fast due to alcohols (shrinkage may occur) </li></ul></ul><ul><ul><li>↑ conc’n of acetic/propionic acid </li></ul></ul><ul><ul><ul><li>Induce ↑ tissue swelling </li></ul></ul></ul><ul><ul><ul><li>Couneract alcohol shrinkage </li></ul></ul></ul><ul><ul><li>Tissues killed and hardened within 18-24 h </li></ul></ul><ul><ul><li>Stable, does not induce hardening </li></ul></ul><ul><ul><li>FPA>FAA (preservation) </li></ul></ul><ul><ul><li>FPA </li></ul></ul><ul><ul><ul><li>for anatomical and morphological studies </li></ul></ul></ul><ul><ul><ul><li>Preserve laticifers </li></ul></ul></ul><ul><ul><li>FAA </li></ul></ul><ul><ul><ul><li>Loses effectiveness with storage </li></ul></ul></ul>
  22. 22. Acid fixatives
  23. 23. Acid fixatives <ul><li>Carnoy’s fixative </li></ul><ul><ul><li>Chloroform-containing fixative </li></ul></ul><ul><ul><li>Rapidly penetrates and fix small tissue </li></ul></ul><ul><li>Damage delicate tissues transferred from aqueous solutions </li></ul><ul><ul><li>Due to </li></ul></ul><ul><ul><ul><li>↑ hydophobicity of chloroform </li></ul></ul></ul><ul><ul><ul><li>Rapid tissue dehydration </li></ul></ul></ul><ul><li>*(1)Fix for1 h(2)wash several times in absolute ethanol(3)infiltrate(4)embed immediately. </li></ul>
  24. 24. Acid fixatives <ul><li>Farmer’s fixative </li></ul><ul><ul><li>Anhydrous solution: causes rapid dehydration and fixation </li></ul></ul>
  25. 25. Staining <ul><ul><li>better visualize cells and cell components under a microscope  </li></ul></ul><ul><ul><li>can preferentially stain certain cell components </li></ul></ul>
  26. 26. Acid dyes <ul><ul><li>Colors the nucleus </li></ul></ul><ul><ul><li>attach to bacteria and cellulose </li></ul></ul>Source:
  27. 27. Basic dyes <ul><ul><li>color other cellular components </li></ul></ul><ul><ul><li>eosins, orange G, ponceau 2R, light green SF, and methyl blue </li></ul></ul><ul><ul><li>background stain when used as direct dyes </li></ul></ul><ul><ul><li>some specific to special cytoplasmic substances when used with mordants </li></ul></ul>
  28. 28. Guide Questions
  29. 29. Advantages and Disadvantages Technique Advatages Disadvantages Smear/Squash rapid and simple excessive pressure lead to cell rupture and a non-diagnostic preparation.
  30. 30. List and describe characteristics of some stains used in different techniques <ul><li>Toluidine blue </li></ul><ul><ul><li>Blue, basic dye </li></ul></ul><ul><ul><li>Stain: </li></ul></ul><ul><ul><ul><li>nucleus </li></ul></ul></ul><ul><ul><ul><li>metachromatically certain structures </li></ul></ul></ul><ul><ul><ul><li>rNa, rNase, mucopolysaccharides in electrophoresis </li></ul></ul></ul><ul><li>Carmine </li></ul><ul><ul><li>intensely red dye </li></ul></ul><ul><ul><li>stain  glycogen </li></ul></ul><ul><ul><li>Carmine alum - nuclear stain </li></ul></ul><ul><ul><li>requires a mordant </li></ul></ul><ul><li>Acridine orange  (AO) </li></ul><ul><ul><li>nucleic acid selective fluorescent cationic dye useful for cell cycle determination </li></ul></ul><ul><ul><li>cell-permeable, and interacts with DNA and RNA by intercalation or electrostatic attractions </li></ul></ul>
  31. 31. <ul><li>Coomassie blue (brilliant blue) </li></ul><ul><ul><li>nonspecifically stains proteins a strong blue color </li></ul></ul><ul><ul><li>often used in gel electrophoresis. </li></ul></ul><ul><li>Crystal violet </li></ul><ul><ul><li>when combined with a suitable mordant, stains  cell walls  purple. </li></ul></ul><ul><ul><li>important component in Gram staining. </li></ul></ul><ul><li>DAPI </li></ul><ul><ul><li>fluorescent  nuclear stain </li></ul></ul><ul><ul><li>excited by UV light </li></ul></ul><ul><ul><li>blue fluorescence when bound to  DNA </li></ul></ul><ul><ul><li>binds with A=T rich repeats of chromosomes </li></ul></ul><ul><ul><li>not visible with regular transmission microscopy </li></ul></ul><ul><ul><li>for cell counting. </li></ul></ul>
  32. 32. <ul><li>Ethidium bromide </li></ul><ul><ul><li>stains DNA </li></ul></ul><ul><ul><li>fluorescent red-orange stain </li></ul></ul><ul><ul><li>marker for apoptosis in cells populations </li></ul></ul><ul><ul><li>locate bands of DNA in  gel electrophoresis </li></ul></ul><ul><ul><li>EB/AO combined stain </li></ul></ul><ul><ul><ul><li>live cells to fluoresce green while apoptotic cells retain the distinctive red-orange fluorescence. </li></ul></ul></ul><ul><li>Iodine </li></ul><ul><ul><li>indicator for  starch </li></ul></ul><ul><ul><li>dark blue colour (+starch) - starch/iodine complex. </li></ul></ul><ul><ul><li>Lugol's solution /Lugol's iodine (IKI) </li></ul></ul><ul><ul><ul><li>brown solution- turns black (+starch) </li></ul></ul></ul><ul><ul><ul><li>cell stain </li></ul></ul></ul><ul><ul><li>as a mordant </li></ul></ul>
  33. 33. <ul><li>Osmium tetroxide (formal name: osmium tetraoxide) </li></ul><ul><ul><li>stain  lipids </li></ul></ul><ul><ul><li>dissolves in fats, </li></ul></ul><ul><ul><li>Is reduced by organic materials to elemental osmium, an easily visible black substance. </li></ul></ul><ul><li>Safranin </li></ul><ul><ul><li>nuclear stain </li></ul></ul><ul><ul><li>red stain </li></ul></ul><ul><ul><li>a counterstain </li></ul></ul><ul><ul><li>give a yellow colour to collagen </li></ul></ul>
  35. 35. Methodology EPIDERMAL PEEL
  36. 36. Results EPIDERMAL PEEL
  37. 37. Discussion EPIDERMAL PEEL Mounting – done to preserve and support a stained section for light microscopy - used to adhere conversion to slide
  38. 38. Discussion EPIDERMAL PEEL <ul><li>Advantage </li></ul><ul><li>study of shape of epidermal cells, their arrangement and distribution. </li></ul><ul><li>study the structure of stomata, as well as their distribution </li></ul><ul><li>Approximation of stomata frequency </li></ul><ul><li>Easy to do (especially in plants with larger leaf) </li></ul><ul><li>Can be mounted even without the use of water </li></ul><ul><li>Disadvantage </li></ul><ul><li>Difficult to perform in plants with smaller leaves </li></ul><ul><li>Only the structures found at the surface of the epidermis can be observed </li></ul>
  39. 39. Microtechnique- preparation of animal materials for microscopic study - important in developmental biology since the field requires cytological and anatomical studies that can only be performed upon tissue preparation by microtechniques
  40. 40. fix- dehydrate-clear-infiltrate-embed-section-stain-mount
  41. 41. Embedding is the process by which tissues are surrounded by a medium such as agar, gelatin, or wax which when solidified will provide sufficient external support during sectioning. Figure 1. tissue and paraffin in block form, ready for sectioning; Adapted from
  42. 42. Infiltration -also, interpenetration -saturation of tissue cavities and cells by a supporting substance which is the medium in which they are finally embedded 1. infiltration by wax 2. infiltration by solution Usual procedure (embedding and infiltration) Figure 2. a sample infiltration procedure; Adapted from
  43. 43. Modifications/ related techniques: Double embedding- infiltration and embedding done twice (e.g. 1 st : agar/ nitrocellulose; 2 nd : paraffin wax); - provides improved tissue support and sectioning qualities Investment- embeds wax- infiltrated tissues in another wax (e.g. Piccolyte- paraffin wax) - improved tissue support and sectioning qualities Vacuum infiltration- impregnates tissues under reduced pressure
  44. 44. Clarification- follows dehydration - replaces dehydrant with substance that will be miscible with the embedding medium -“clear”- clearing agents often have the same refractive indices as proteins - result: transluscent tissue - e.g. Xylene, toluene , chloroform, methyl salicylate, Histo- Clear
  45. 45. Johansen’s Safranin and Fast Green method – method involving additions to the stain (e.g. dehydrating and clearing agents) to enhance and differentate tissue structure Safranin O- brilliant red in chromosomes, nuclei, lignified, suberized, or cutinized cell walls Fast Green- brilliant green in cytoplasm and cellulosic cell walls; blue to bluish-green in the stems and leaves of aquatic plants and most gymnosperms Sass’s Safranin and Fast Green method- fewer additions than Johansen’s method Sharman Staining Series- meristematic tissues; cell walls stain blue-black, nuclei stain yellow to orange, starch grains appear black, and lignified cell walls stain brilliant red 
  47. 47. INTRODUCTION <ul><li>Maceration – treatment of plant tissue with chemicals to allow cells to become separated </li></ul><ul><ul><li>To examine the nature of cells from different parts of the plant (viewed under a microscope) </li></ul></ul><ul><ul><li>To extract plant essences </li></ul></ul><ul><li>Killing – termination of the organism’s (or part of the organism) life processes abruptly and permanently </li></ul><ul><ul><li>Dead: no longer capable of reproducing itself and stopped receiving nourishment </li></ul></ul>
  48. 48. INTRODUCTION <ul><li>Fixation – process in which aims to preserve the natural living condition of cellular and structural elements as much as possible </li></ul><ul><ul><li>“ A good fixative is one that changes the cell chemistry the least and preserves the cell structure the best. ” (Schiller, 1930) </li></ul></ul><ul><li>Staining – addition of stains or dyes to highlight and make tissue structures visible when viewed under a microscope </li></ul><ul><ul><li>Safranin: stains lignified, cutinized, suberized and chitinized structures as well as chromosomes, nucleoli and centrosomes </li></ul></ul>
  49. 49. INTRODUCTION <ul><li>Dehydration – remove water from tissues through the addition of a dehydrating agent </li></ul><ul><ul><li>Hygrobutol (tert-butyl alcohol) </li></ul></ul><ul><li>Mounting – placing of plant tissue on a slide to be viewed under a microscope </li></ul><ul><ul><li>Mounting media: used to adhere the cover slip to the slide with the plant tissue sample </li></ul></ul><ul><ul><ul><li>Canada balsam: amorphous when dried (forms transparent non-crystalline solid) </li></ul></ul></ul>
  50. 50. METHODS w/ DISCUSSION “ macerate” the tissue; dissolve the middle lamella T o allow the sample to sink into the acid mixture (air bubbles may cause them to float)
  51. 51. METHODS w/ DISCUSSION Remove the acids (may interfere with the staining process) Further separate the tissues
  52. 52. METHODS w/ DISCUSSION Remove excess stain (irremovable precipitates may form) Remove water from the tissue
  53. 53. RESULTS Figure 1. Tilia sp. vessel elements. Source:
  54. 54. RESULTS Figure 2. Tilia sp. macerated wood. Source:
  55. 55. CONCLUSION <ul><li>Advantages </li></ul><ul><ul><li>Show the real nature of cells as much as possible </li></ul></ul><ul><ul><li>Explicitly show cell’s distinguishing characteristics such as thickenings and pores </li></ul></ul><ul><li>Disadvantages </li></ul><ul><ul><li>Cells cannot be viewed in their proper arrangement patterns </li></ul></ul><ul><ul><li>Time-consuming </li></ul></ul>
  56. 56. REFERENCES <ul><li>Johansen, D. A. 1968. Plant Microtechnique . United States of America: McGraw-Hill Book Company, Inc. pp. 23, 27-28 &104 </li></ul><ul><li>Schiller, W. 1930. Gewebesfixierung unter Erhaltung der basischen Kernfärbung. Zeitschr . Zellforsh . mikr . Anat . 11:63 </li></ul>