1. Some Techniques in the Study of Plant Development Biology Group 3 Tanchuling, Raymund – LEADER Bituin, Dawnn Faustino, Missy Fojas, Juris Gonzaga, Sahara
7. Figure 1. One method of holding a specimen for free hand sectioning; Adapted from http://www.ableweb.org/volumes/vol-19/9-yeung.pdf Do not use the “chopping” action!
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.
11. Figure 2. A V-shaped notched is removed from the papaya block to accommodate a specimen for longitudinal sections; Adapted from http://www.ableweb.org/volumes/vol-19/9-yeung.pdf
29. Advantages and Disadvantages Technique Advatages Disadvantages Smear/Squash rapid and simple excessive pressure lead to cell rupture and a non-diagnostic preparation.
37. Discussion EPIDERMAL PEEL Mounting – done to preserve and support a stained section for light microscopy - used to adhere conversion to slide
38.
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
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 http://protocolsonline.com/histology/paraffin-processing-of-tissue/
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 http://histologycourse.com/Tissue%20Processing.pdf
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
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
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.
http://accessscience.com/content/Microtechnique/424010#S6 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.)
http://microscopy.berkeley.edu/Resources/instruction/acid_fixatives.htm 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.
http://microscopy.berkeley.edu/Resources/instruction/acid_fixatives.htm 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.
http://microscopy.berkeley.edu/Resources/instruction/acid_fixatives.htm 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.
http://accessscience.com/content/Microtechnique/424010#S6 Orcein, safranin, methylene blue, and crystal violet are typically direct nuclear dyes carmine, hematoxylin, and celestin blue B are commonly used with mordants.
http://accessscience.com/content/Microtechnique/424010#S6 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. http://www.archive.org/stream/handbookofbasicm00gray/handbookofbasicm00gray_djvu.txt mordants commonly used are aluminum, ferric and chromiiun salts, and alums (potassium alum, ammonium ahnn, iron alum and chrome alum).
http://www.qml.com.au/Files/Vet_NL_Mar10.pdf
http://www.biology-online.org/dictionary/Toluidine_blue_o 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
