Ex1

Some Techniques in the Study of Plant Development Biology

Group 3Tanchuling, Raymund – LEADER

Bituin, DawnnFaustino, Missy

Fojas, JurisGonzaga, Sahara

Free Hand Sectioning

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

Why do we use free hand sectioning? In order to study the structural organization of

the plant body.

The General technique Soft herbaceous stems and small woody twigs Generally, tissue is cut without a supporting

matrix

Double razor blade Water Stem

+

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!

Petridish with water

*Do not use forceps!

With sectiongs

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

Special technique for delicate and hard to hold specimens

For thin leaves and tiny roots Support matrix is used.

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

Leaf Midrib

Leaf midrib inserted into the papaya

Petridish with a fixing solution

Farmer’s Solution

-It kills the specimen

-It also preserve the structures

Mount the material on a slide in a drop of 50% glycerol and cover with cover slip.

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

Squash or Smear Technique Useful in:

counting monoploid an diploid chromosomes studying the chromosomes themselves critical cytological studies

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)

Fixation Kill tissue so that decay is prevented preserves tissue/cells as close to its natural

state as possible in preparation for examination.

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

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.)

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

Acid fixatives

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.

Acid fixatives Farmer’s fixative

Anhydrous solution: causes rapid dehydration and fixation

Staining better visualize cells and cell components under a

microscope  can preferentially stain certain cell components

Acid dyes Colors the nucleus attach to bacteria and cellulose

Source: http://accessscience.com/popup.aspx?id=424010&tableID=424010TB0020&name=table

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

Guide Questions

Advantages and DisadvantagesTechnique Advatages Disadvantages

Smear/Squash rapid and simple excessive pressure lead to cell rupture and a non-diagnostic preparation.

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

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.

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

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

EPIDERMAL PEEL

Methodology

EPIDERMAL PEEL

Results

EPIDERMAL PEEL

Discussion

EPIDERMAL PEEL

Mounting – done to preserve and support a stained section for light microscopy

- used to adhere conversion to slide

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

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

fix- dehydrate-clear-infiltrate-embed-section-stain-mount

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/

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

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

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

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

MACERATION OF TISSUES

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

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

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)

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)

METHODS w/ DISCUSSION

Remove the acids (may interfere with the staining process)

Further separate the tissues

METHODS w/ DISCUSSION

Remove excess stain (irremovable precipitates may form)

Remove water from the tissue

RESULTS

Figure 1. Tilia sp. vessel elements.Source: http://www.lima.ohio-state.edu/biology/images/tiliavessels.jpg

RESULTS

Figure 2. Tilia sp. macerated wood.Source: http://bio.rutgers.edu/~gb101/lab7_p_evol/p_evol_graphics/tilia_macerated.jpg

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

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