Comparative Study on Preparation of Botanical Permanent Slides by - PDF document

Universities Research Journal 2013, Vol.6 1 Comparative Study on Preparation of Botanical Permanent Slides by Different Methods Bay Dar 1 , Moe Moe Lwin 2 , Ohnmar Than 2 , Aye Aye Myint 3 Abstract In the study of biology and medicine, historical permanent slides are widely demanded for effective teaching and learning. Thus the main aim of this paper is to prepare botanical permanent slides by different methods and to study their quality. Sandoricum koetjape Merr. (Thit-to) and Eupatorium odoratum L. (Bi-zat) growing in University of Yangon campus were selected. The selected samples can mainly support the biological study (in Basic Education High School (BEHS) level and undergraduate level. It may be useful for school and college students to know the technique of preparing histological slides as a part of their study of biology. In this paper, permanent slides for histological study in plants were prepared by tertiary butyl alcohol (TBA), xylene-alcohol method and free hand technique. The quality of 537 permanent slides out of 1100 slides were classified as class A. TBA method was found to be the best and cost effective in this work. Key words : Sandoricum koetjape Merr., Eupatorium odoratum L., TBA, xylene-alcohol, free hand Introduction In the biology of high schools and undergraduate courses, the permanent tissue slides are used for learning the histology of plant parts, tissue and microorganisms. Normally, they are imported and they are very expensive, so the main purpose of this research is to make cost effective teaching aid materials (Tissue slides) in the country instead of importing them. Medicinal plants still play an important role in Myanmar people for curing many diseases. This study will provide histological information on two plant species in University of Yangon campus. 1 Associate Professor (Botany), Universities' Research Centre (URC), University of Yangon 2 Lecturer, Department of Botany, University of Yangon 3 Professor (chemistry), Universities' Research Centre, University of Yangon

2 Universities' Research Journal 2012, Vol.4 The aim of the present study is to find out various medicinal plants which could be classified and identified using their histological characters based on the prepared permanent slides. The objective of this research was to evaluate botanical permanent slides using three methods: Tertiary Butyl alcohol (TBA), Xylene-alcohol and Free hand method. Materials and Methods The plant samples were collected from University of Yangon campus and verified at the Department of Botany, University of Yangon. Preparation of permanent slides was conducted at Universities’ Research Centre, University of Yangon. (A) (B) Figure 1. Dehydration and cleaning the tissue in the tissue processor (A) Tissue processor (Citadel TM Shandon, USA) set up at URC (B) Placing the tissue cassette into the cassette hanger The samples of Dicot: the lamina, midrib, petiole and stem of Sandoricum koetjape Merr. (Thit-to), Family-Meliaceae and lamina, midrib, petiole, stem and root, of Eupatorium odoratum L. (Bi-zat), Family- Asteraceae were cut in transverse section (15 - 25 µm). Plant tissues were divided into soft tissues and hard tissues. CRAF III solution was used for soft tissues and Formalin Aceto-Alcohol (FAA) solution was used for hard tissues to carry out fixation of plant tissue. There are five steps in the

Universities Research Journal 2013, Vol.6 3 histological process including: 1. Fixation, 2. Dehydration and Clearing, 3. Embedding, 4. Slicing by Microtome, 5. Staining and Mounting. In this study, the tissue processors were programmed for fixation, dehydration, cleaning (Figure. 1), and infiltration into paraffin (Figure. 2). Figure 2. Tissue embedding by paraffin dispenser The embedded paraffin was then poured into a mold and cooled on the Shandon Histocentre TM 3 cold plate. When paraffin block was frozen, they were kept in the refrigerator (Figure. 3). Figure 3. Chilling the mould on the Shandon Histocentre TM 3 cold plate

4 Universities' Research Journal 2012, Vol.4 The cooled wax block with the tissue inside was sliced into very thin ribbons that have the thickness of 5 μm, using a microtome (Leica, RM 2155) (Figure. 4). The tissue ribbon was transferred to a tissue floating bath, not exceeding 40  C. The section was then quickly picked up on the slide and dried on the slider warmer for 24 hours. Figure 4. Microtome for tissue slicing For the examination of histological tissues, the staining reagent for the specific tissue was systematically conducted. After 3 days or 5 days fixing, tissue samples were processed using with TBA or Xylene-alcohol method and then blocked with paraffin. Then lamina and midrib sections of 10-15  m thickness and stem, petiole and root sections of 20-30  m thickness were cut by microtome. Then the sliced tissue sections were placed on glass slides using warm water (35-40  C) and they were dried in incubator (37-40  C) overnight. And then tissue samples were stained stepwise by the procedure of staining method using Saffranin (Avilla, 2000). Dehydration and clearing (TBA) series for plant tissues were carried out as listed in Table (1). Xylene-alcohol series for plant tissues were prepared as shown in Table (2) (Donald, 1940 and Mya Mya, 2003). The staining procedure for plant tissue was summarized in Table (3) (Avilla, 2000). Finally, the stained tissue slides were mounted with Canada balsam and dried overnight. The permanent slides were labeled and kept in slide boxes for microscopic studies.

Universities Research Journal 2013, Vol.6 5 Table 1. Dehydration and clearing series for plant tissue by TBA method 100 % Step 95 % Tertiary Butyl- Distilled Absolute Time (hr) No. Alcohol (mL) Alcohol (mL) water (mL) alcohol (mL) 1 5 95 2-4 2 10 90 2-4 3 20 80 2-4 4 30 70 2-4 5 40 60 2-4 6 50 50 2-4 7 50 10 40 2-4 8 50 20 30 2-4 9 50 35 15 2-4 10 50 50 2-4 11 25 75 2-4 12 100 + erythrosin 2-4 13 100 12 14 100 12 15 Soft Paraffin 2 16 Hard Paraffin 2

6 Universities' Research Journal 2012, Vol.4 Table 2. Dehydration and clearing series for plant tissue by Xylene-alcohol method Step No. 98% Alcohol Xylene (mL) Distilled Time (mL) water (mL) (hr) 1 5 95 2-4 2 10 90 2-4 3 20 80 2-4 4 30 70 2-4 5 40 60 2-4 6 50 50 2-4 7 60 40 2-4 8 70 30 2-4 9 85 15 2-4 10 95 5 2-4 11 100 - 4-12 12 100 - 4-12 13 95 5 - 2-3 14 90 10 2-3 15 85 15 2-3 16 75 25 2-3 17 50 50 2-3 18 25 75 2-3 19 100 12 20 100 12 21 Soft Paraffin 2 22 Hard Paraffin 2

Universities Research Journal 2013, Vol.6 7 Table 3. Staining procedure for sliceable plant tissue Step No. Chemical Reagents Time (min) 1 Xylene I (pure xylene) 10 2 Xylene II (pure xylene) 10 3 3:1 (xylene: aniline) 10 4 2:1 (xylene: aniline) 10 5 1:1:1 (xylene: aniline: 95 % ethanol) 10 6 97% ethanol 10 7 85% ethanol 10 8 70% ethanol 10 9 50 % ethanol 10 10 Distilled water 10 11 1% aqueous water Saffranin (staining) 6-24 hrs 12 50% ethanol 3 13 70% ethanol 3 14 85% ethanol 3 15 95% ethanol 3 16 0.5% Fast green in 95% ethanol (counterstains) 3 17 1:1:1 (xylene: aniline: 95 % alcohol) 3 18 2:1 (xylene: aniline) 3 19 3:1 (xylene: aniline) 3 20 Xylene III (pure xylene ) 3 21 Xylene IV (pure xylene) 3 Results In this process, a total of 1100 permanent tissue slides were obtained by TBA and Xylene-alcohol methods. The high quality permanent slides (537) were recorded as class A. Some samples (Class A slides) were shown in Figures 5-27. The taxonomy and structure of laminar, midrib, root, various types of stems, trichomes and calcium oxalate crystals were clearly

8 Universities' Research Journal 2012, Vol.4 observed. About 300 slides were damaged due to the thinness or thickness of cell or imperfection including loss of tissue orientation, teared section and round holes while sectioning and they were classified as B. During staining, 263 out of 800 slides were damaged and classified as class C. Some High Quality Tissue Slides Samples (Class A) by Different Methods Upper epidermis Palisade parenchyma cell Intercellular space Spongy mesophyll cell Lower epidermis Figure 5. Classs A - T.S* of Lamina of Sandoricum koetjape ( × 10) by TBA method Palisade parenchyma cell Intercellular space Parenchyma cell Vascular bundle Lower collenchyma cell Lower epidermis Figure 6. Class A - T.S* of Midrib of Sandoricum koetjape ( × 10) by TBA method T.S *= Transverse Section

Universities Research Journal 2013, Vol.6 9 Epiblema Xylem Phloem Figure 7. Class A - T.S of Root of Eupatorium odoratum L. ( × 4) by TBA method Xylem Phloem Cortex Epiblema Figure 8. Class A - T.S of Root of Eupatorium odoratum L. ( × 20) by TBA method Class A - Quality tissue slides by Xylene-alcohol method Upper epidermis Palisade parenchyma cell Intercellular space Spongy mesophyll cell Lower epidermis Figure 9. Class A-T.S of Lamina of Sandoricum koetjape Merr. ( × 10) by Xylene- alcohol method