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TRANSCRIPT
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Some Techniques in the Study of Plant Development Biology
Group 3Tanchuling, Raymund – LEADER
Bituin, DawnnFaustino, Missy
Fojas, JurisGonzaga, Sahara
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Free Hand Sectioning
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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
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Why do we use free hand sectioning? In order to study the structural organization of
the plant body.
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The General technique Soft herbaceous stems and small woody twigs Generally, tissue is cut without a supporting
matrix
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Double razor blade Water Stem
+
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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!
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Petridish with water
*Do not use forceps!
With sectiongs
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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
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Special technique for delicate and hard to hold specimens
For thin leaves and tiny roots Support matrix is used.
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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
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Leaf Midrib
Leaf midrib inserted into the papaya
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Petridish with a fixing solution
Farmer’s Solution
-It kills the specimen
-It also preserve the structures
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Mount the material on a slide in a drop of 50% glycerol and cover with cover slip.
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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
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Squash or Smear Technique Useful in:
counting monoploid an diploid chromosomes studying the chromosomes themselves critical cytological studies
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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)
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Fixation Kill tissue so that decay is prevented preserves tissue/cells as close to its natural
state as possible in preparation for examination.
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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
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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.)
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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
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Acid fixatives
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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.
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Acid fixatives Farmer’s fixative
Anhydrous solution: causes rapid dehydration and fixation
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Staining better visualize cells and cell components under a
microscope can preferentially stain certain cell components
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Acid dyes Colors the nucleus attach to bacteria and cellulose
Source: http://accessscience.com/popup.aspx?id=424010&tableID=424010TB0020&name=table
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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
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Guide Questions
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Advantages and DisadvantagesTechnique Advatages Disadvantages
Smear/Squash rapid and simple excessive pressure lead to cell rupture and a non-diagnostic preparation.
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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
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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.
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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
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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
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EPIDERMAL PEEL
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Methodology
EPIDERMAL PEEL
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Results
EPIDERMAL PEEL
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Discussion
EPIDERMAL PEEL
Mounting – done to preserve and support a stained section for light microscopy
- used to adhere conversion to slide
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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
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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
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fix- dehydrate-clear-infiltrate-embed-section-stain-mount
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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/
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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 wax2. infiltration by solution
Usual procedure (embedding and infiltration)
Figure 2. a sample infiltration procedure; Adapted from http://histologycourse.com/Tissue%20Processing.pdf
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Modifications/ related techniques:
Double embedding- infiltration and embedding done twice (e.g. 1st: agar/ nitrocellulose; 2nd: 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
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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
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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 wallsFast 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
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MACERATION OF TISSUES
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INTRODUCTION 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
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INTRODUCTION 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
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INTRODUCTION 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)
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METHODS w/ DISCUSSION“macerate” the tissue; dissolve the middle lamella
To allow the sample to sink into the acid mixture (air bubbles may cause them to float)
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METHODS w/ DISCUSSION
Remove the acids (may interfere with the staining process)
Further separate the tissues
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METHODS w/ DISCUSSION
Remove excess stain (irremovable precipitates may form)
Remove water from the tissue
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RESULTS
Figure 1. Tilia sp. vessel elements.Source: http://www.lima.ohio-state.edu/biology/images/tiliavessels.jpg
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RESULTS
Figure 2. Tilia sp. macerated wood.Source: http://bio.rutgers.edu/~gb101/lab7_p_evol/p_evol_graphics/tilia_macerated.jpg
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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
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REFERENCESJohansen, 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