Preparation of Plant tissues for histological study 213 bot Alanoud Alfagham

Plant histology is the branch of biology concerned with the composition and structure of plant tissues in relation to their specialized functions. Its aim is to determine how tissues are organized at all structural levels, from cells and intercellular substances to organs.

Tissue processing The aim of tissue processing is to embed the tissue in a solid material firm enough to support the tissue and give it sufficient rigidity to enable thin sections to be cut, and yet soft enough not to damage the knife or tissue. In order to keep slide for long time, we should prepare permanent slide of plant tissues. For permanent preparation, there are several steps and plant tissue must pass through all the steps. All steps are depend on time and concentration of chemicals. Steps of Permanent slide preparation of plant tissues: 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 Staining

1-Fixation step: Fixation is the most important step in performing histological specimen preparation technique. Fixation is a complex series of chemical events that differ for the different groups of substance found in tissues. The best fixation solution is that can kill and fix the tissue very fast rather than any potential change in plant tissue.

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.

Types of fixative: Acetic acid, Formaldehyde, Ethanol, Glutaraldehyde, Methanol and Picric acid.   Factors affects fixation: PH. Temperature. Penetration of fixative. Volume of tissue. According to previous factors we can determine the concentration of fixative and fixation time.

Fixation Protocol Material required: FAA solution, Plant tissues (Stems and Leaves), Razors, Glass tube with lid, Ruler. Preparation of fixation solution: (FAA solution) 90 ml 70 % Ethanol alcohol 5 ml 40 % Formalin 5 ml Glacial acetic acid Method: For each group 5 pieces from leaves and stems, 1 cm long stem, 2 cm long leaf. Put separately the specimen (leaves in one tube and stems in another tube) in glass tube covers with FAA solution and close it with the lid. Write information of your group on each tube and leave it 24 h for woody tissue ,12 h for flowering 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: Replace the FAA solution to 80% ethanol for 2 h. Then replace the 80% ethanol to 90% ethanol for 2 h. Then replace the 90% ethanol to 100 % ethanol for 2 h. Replace the 100 % to another 100% ethanol for 24 h.

Clearing step Replace the dehydrating fluid with a fluid that is totally mixable with both the dehydrating fluid and the embedding medium. Choice of a clearing agent depends upon the following: The type of tissues to be processed, and the type of processing to be undertaken. The processor system to be used. Intended processing conditions such as temperature, vacuum and pressure. Safety factors. Cost and convenience. Speedy removal of dehydrating agent . Ease of removal by molten paraffin wax . Minimal tissue damage . Some clearing agents: Xylene, Toluene, Chloroform, Benzene, Petrol.

Protocol Using gradual concentration xylene and Ethanol: Replace the 100% ethanol to Ethanol: Xylene in 1:3 ratio for 2 h then replace this solution carefully to, Ethanol: Xylene in 1: 1 ration for 2 h then replace this solution carefully to, Ethanol: Xylene in 3:1 ratio for 2 h then replace this solution carefully to absolute xylene for 24 h.

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.

date time step الاحد/8-9 24 FAA 1 الاثنين 9-8 2 80 % ethanol الاثنين 11 90 % ethanol 3 الاثنين 1 100 % ethanol 4 الاثنين 3 5 الثلاثاء 9 Ethanol : Xylene 3:1 6 الثلاثاء 11 Ethanol : Xylene 1:1 7 الثلاثاء 1 Ethanol : Xylene 1:3 8 الثلاثاء 3 xylene 9 48 Paraffin 10