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  • The act or process of cutting or dividing parts from a fixed mass of tissue Free hand is a type of sectioning where it is executed by hand without guiding instruments, or other aids Simple method for preparing specimens for microscopic viewing
  • Most plant parts are too thick to be mounted intact and viewed with a microscope. In order to study the structural organization of the plant body, sections have to be made so that enough light can be transmitted through the specimen to resolve cell structure for microscopic viewing
  • This technique can be used for plant parts that are firm and can be supported by bare hands
  • 1. Use a double edge razor blade. – to minimize the risk of cutting oneself, cover one edge with masking tape 2. Rinse the blade with warm tap water to remove traces of grease from the surface 3. Hold the plant material firmly.
  • The plant material should be held against the side of the firstfinger of the left hand/ right by means of the thumb. 4. In cutting, use a sliding action so that the blade moves through the tissue at an angle *Contol is usually better if the cut is made toward the body
  • 5. Remove sections from the blade by floading them off into the petridish with water. The edge of the blade will be dulled if you rub it on the edge of the watch glass. You can also use a small spatula * Do not use forceps because it can squash or destroy the cells therefore can affect the view of the specimen in the microscope
  • additional support, such as support matrix, can be used to facilitate free hand sectioning
  • To make a support matrix… Obtain a green papaya or cassava pith and cut into longitudinal strips (0.6 inch to 1 inch long) Divide the strips into equal halves Make a groove in each, make sure the groove is enough to put in place the specimen.
  • 4. Collect leaves from various plants and cut along the leaf midrib. 5. Place the leaf midrib into the papaya and tie the halves securely using a strand of thread 6.Using sharp razor blades, cut the specimens
  • Put the sections in petridish with a fixing soltuion – Farmer’s - Fixing specimens are used for making permanent slides. -It kills the specimen -It also preserve the structures - for example in farmer’s - 3 parts ethanol and 1 part glacial acetic acid. This combination denatures proteins. The alcohol also removes some lipids. This is probably the preferred fixing agent because it is less toxic than some other fixatives.
  • Mount the material on a slide in a drop of 50% glycerol and cover with cover slip. Glycerol - This viscous liquid that provides a suitable mounting media that lasts longer.
  • otherwise digests or damages   Fixatives are toxic to most common microorganisms ( bacteria  in particular) that might exist in a tissue sample or which might otherwise colonise the fixed tissue. many fixatives chemically alter the fixed material to make it less palatable (either indigestible or toxic) to opportunistic microorganisms. on a molecular level Generate chemical bonds between proteins and other substances: ↑ their rigidity,mechanical strength or stability
  • autolysis , more commonly known as self-digestion, refers to the destruction of a  cell  through the action of its own  enzymes . It may also refer to the digestion of an enzyme by another molecule of the same enzyme. The term derives from the  Greek  words  αυτό  ("self") and  λύσις  ("splitting"). Putrefaction  is one of seven stages in the  decomposition  of the body of a dead animal. It can be viewed, in broad terms, as the decomposition of  proteins , in a process that results in the eventual breakdown of cohesion between tissues and the liquefaction of most organs.
  • immobilization of highly contractile creatures requires instant denaturing of the proteins, while the preservation of protoplasmic structure is best retained by reasonably slow tanning Actual fixative solutions, which by convention bear the name of the technician who first published them, are compromises
  • The result of stabilizing tissues with these types of fixatives produces the so-called  acid fixation image  (cf.  Basic fixatives , this chapter). These fixatives are good for preserving chromatin, nucleoli, and spindles. The cytoplasm is preserved as a stringy, coagulated mass, but some organelles are dissolved ( e.g.,  mitochondria.)
  • The fixative is stable and does not induce hardening , so tissues may be stored in these solutions indefinitely . Johansen (1940) and Brooks  et al . (1966) consider FPA to be an excellent fixative for anatomical and morphological studies. Johansen (1940) and Bruni and Tosi (1980) specifically recommended FPA for preserving laticifers.
  • It penetrates tissues extremely rapidly and can fix small tissue pieces in minutes rather than the hours required for other fixatives (Chamberlain, 1932; Sass, 1958). Delicate tissues can be damaged when transferred from aqueous solutions to this fixative, due to the extreme hydrophobicity of chloroform and resultant rapid tissue dehydration.
  • As with Carnoy’s fixative, the rapid exchange of tissue water for fixative can cause extreme cellular disruption. However, these two fixatives are excellent for cytological investigations (Sass, 1958; Golubovskaya, 1994).
  • . Some dyes require the use of a mordant, which is a chemical compound that reacts with the stain to form an insoluble, colored precipitate. The mordanted stain will remain on/in the sample when excess dye solution is washed away.
  • Orcein, safranin, methylene blue, and crystal violet are typically direct nuclear dyes carmine, hematoxylin, and celestin blue B are commonly used with mordants.
  • mordant - intermediary which is either applied before, or in the same solution as, the dye . Some dyes require the use of a mordant, which is a chemical compound that reacts with the stain to form an insoluble, colored precipitate. The mordanted stain will remain on/in the sample when excess dye solution is washed away. mordants commonly used are aluminum, ferric and chromiiun salts, and alums (potassium alum, ammonium ahnn, iron alum and chrome alum).
  • C15H16N3SCl, metachromatically certain  structures  (e.g., the  granules  in  mast cells  which are believed to contain  heparin  and  cartilage matrix  which is  rich  in  chondroitin   sulfate ), also antagonises the anticoagulant   action  of heparin. AO-When bound to DNA, it is very similar spectrally to fluorescein which is useful as a non-specific stain for backlighting conventionally stained cells on the surface of a solid sample of tissue (fluorescence backlighted staining).
  •   final stages of  apoptosis  - such cells have much more permeable  membranes
  • (haematoxylin and eosin) staining—one of the most common procedures in  histology .
  • 1. Collect leaf samples designated by your teacher. 2. Wipe the leaves with clean piece of cloth to remove dust particles on it. 3. Thinly apply colorless nail polish on leaf surface. Allow to dry and detach the leaves from the plant 4. Cut a small square of the leaf with nail polish and peel the nail polish. Place the peeled nail polish on the slide and cover with slip. 5. Examine the slides under the microscope and store in a cool dry place.
  • Since plant dev bio studies ontogeny, plant microtechnique allows students to keep track of ontogeny itself (e.g. From a single cell  plant from meristematic tissues  organs (via differential staining, etc. )
  • 2. AFTER substance MUST be fluid when hot, solid when cold Solution - used in lieu of waxes; which solidifies upon evaporation of the solvent , providing a firm mass suitable for sectioning Dehydration displaces h2o in cells, for wax to enter (after infl- embedded into paraffin wax) Inflt- preserves structural integrity of cells; absence of infl will flatten, crumple, tear plant tissue during embedding
  • Embedding and infiltr go hand in hand (during embed, infl also occurs) DE- tissues embedded and infiltrated with supporting medium such as agar or nitrocellulose, then infiltrated a second time with wax in which they are also embedded Plant placed in liquid mold/wax/agar =  solidifies  leave in H20  block is ready for sectioning Vacuum infiltr: minimizing heat-induced tissue hardening, reduces solvent contam (longer life); req’s a vacuum infiltrator Paraffin wax--- tissue wax adhesion is good; melting point: 39 to 68 degrees
  • Miscible- able to mix; become homogenous Transcluscent- indication of effectiveness or completeness of clearing process
  • Transcript

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