213 Bot Alanoud Alfagham
Histologic plant tissue preparation techniques.
(1-4 steps)
To prepare micro specimen slide would observed under light microscope and kept for long time. It should passes through several steps. This steps depend in times and concentration of chemicals.
Steps of Permanent preparation slides:
1 - القتل والتثبيت Fixation 5- الطمر Embedding
2 - نزع الماء Dehydration 6- التشذيب Trimming
3- الترويق Clearing 7- التقطيع Sectioning
4- التشريب Infiltration 8- الصبغ والتحميل Stainig
1. Fixation step
Fixation is the most important step in performing histologic specimen preparation
techniques. Fixation is a complex series of chemical events that differ for the different groups of substance found in tissues. The best fixation solution which can kill and fix the tissue very fast than any potential change in plant tissue.
A. The aim of fixation:
· To prevent autolysis and bacterial attack.
· To fix the tissues so they will not change their volume and shape during processing.
· To prepare tissue and leave it in a condition which allow clear staining of sections.
· To leave tissue as close as their living state as possible, and no small molecules should be lost.
· Fixation is coming by reaction between the fixative and protein which form a gel, so keeping everything as their in vivo relation to each other.
B. Factors affect fixation:
· PH.
· Temperature.
· Penetration of fixative.
· Volume of tissue.
· According to previous factors we can determine the concentration of fixative and fixation time.
C. Types of fixative:
Acetic acid, Formaldehyde, Ethanol, Glutaraldehyde, Methanol and Picric acid.
D. Fixation Protocol:
Meterial:
FAA solution, Plant tissue (Stems and Leaves), Razors, Glass tube with lid, Ruler.
Prepar the fixation solution: (FAA solution)
90 ml 70 % Ethanol alcohol
5 ml 40 % Formalin
5 ml Glacial acetic acid
Method:
1. For each grope 5 pieces from leaves and stems, 1 cm long stem, 2 cm long lef.
2. Put separately the specimen (leaves in tube and stems in another tube) in glass tube covers with FAA solution and close it with the lid.
3. Write your information grope on each tube and leave it 24 h for woody tissue ,12 h for flowers tissue.
2. Dehydration step
The aim of this step is to remove fixative and water from the tissue and replace them with dehydrating fluid. There are a variety of compounds many of which are alcohols. Several are hydrophilic so attract water from tissue.
In the paraffin wax method, following any necessary post fixation treatment, dehydration from aqueous fixatives is usually initiated in 60%-70% ethanol, progressing through 90%-95% ethanol, then two or three changes of absolute ethanol before proceeding to the clearing stage.
Types of dehydrating agents: Ethanol, Methanol, Acetone.
Duration of dehydration: should be kept to the minimum consistent with the tissues being processed. Tissue blocks 1 mm thick should receive up to 30 minutes in each alcohol, blocks 5 mm thick require up to 90 minutes or longer in each change. Tissues may be held and stored indefinitely in 70% ethanol without harm.
Protocol:
1. Replace the FAA solution to 80% ethanol for 2 h.
2. Then replace the 80% ethanol to 90% ethanol for 2 h.
3. Then replace the 90% ethanol to 100 % ethanol for 2 h.
4. Replace the 100 % to another 100% ethanol for 24 h.
3. Clearing step
Replacing the dehydrating fluid with a fluid that is totally miscible with both the dehydrating fluid and the embedding medium.
Choice of a clearing agent depends upon the following:
1. The type of tissues to be processed, and the type of processing to be undertaken.
2. The processor system to be used.
3. Intended processing conditions such as temperature, vacuum and pressure.
4. Safety factors.
5. Cost and convenience.
6. Speedy removal of dehydrating agent .
7. Ease of removal by molten paraffin wax .
8. Minimal tissue damage .
Some clearing agents: Xylene, Toluene, Chloroform, Benzene, Petrol.
Protocol
Using gradual concentration xylene and Ethanol:
1. Replace the 100% ethanol to Ethanol: Xylene 1:3 for 2 h then replace this solution carefully to,
2. Ethanol: Xylene 1: 1 for 2 h then replace this solution carefully to,
3. Ethanol: Xylene 3:1 2 h then replace this solution carefully to xylene absolute for 24 h.
4. Infiltration step
This step aim to adding the paraffin wax gradually and reduce the clearing solution. To prepare the tissue for the embedding step. To do this: add the crystals of paraffin wax to the tissue gradually inlet the tissue infiltrate completely. For example add one small piece to the xylene from previous step then don’t add another pieces inlet the first one is full dissolved. Make sure that the lid of the tube is removed while you are adding the wax. And incubate the tube in 50 c. this step take approximately 24-48 h.