SLIDE 1 Universities Research Journal 2013, Vol.6 1
Comparative Study on Preparation of Botanical Permanent Slides by Different Methods
Bay Dar1, Moe Moe Lwin2, Ohnmar Than2, Aye Aye Myint3 Abstract
In the study of biology and medicine, historical permanent slides are widely demanded for effective teaching and learning. Thus the main aim
- f 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
- f 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
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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
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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
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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 40C. 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
- ut 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.
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Table 1. Dehydration and clearing series for plant tissue by TBA method
Step No. 95 % Alcohol (mL) 100 % Absolute alcohol (mL) Tertiary Butyl- Alcohol (mL) Distilled water (mL) Time (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 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
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Table 2. Dehydration and clearing series for plant tissue by Xylene-alcohol method
Step No. 98% Alcohol (mL) Xylene (mL) Distilled water (mL) Time (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
12 100
13 95 5
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
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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
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- bserved. About 300 slides were damaged due to the thinness or thickness
- f 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 T.S*= Transverse Section
Figure 5. Classs A - T.S* of Lamina of Sandoricum koetjape ( × 10) by TBA method Figure 6. Class A - T.S* of Midrib of Sandoricum koetjape ( × 10) by TBA method
Upper epidermis Palisade parenchyma cell Intercellular space Spongy mesophyll cell Lower epidermis Palisade parenchyma cell Parenchyma cell Vascular bundle Intercellular space Lower epidermis Lower collenchyma cell
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Class A - Quality tissue slides by Xylene-alcohol method
Figure 9. Class A-T.S of Lamina of Sandoricum koetjape Merr. ( × 10) by Xylene- alcohol method Figure 7. Class A - T.S of Root of Eupatorium odoratum L. ( × 4) by TBA method Figure 8. Class A - T.S of Root of Eupatorium odoratum L. ( × 20) by TBA method
Cortex Epiblema Xylem Xylem Phloem Epiblema Phloem Upper epidermis Palisade parenchyma cell Intercellular space Spongy mesophyll cell Lower epidermis
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Figure 10. Class A-T.S of Midrib of Sandoricum koetjape Merr. ( × 4) by Xylene– alcohol method
Figure 11. Class A - T.S of Stem of Sandoricum koetjape Merr. ( × 10) by Xylene
–alcohol method Figure 12. Class A - T.S of Stem of Sandoricum koetjape Merr. ( × 20) by Xylene-alcohol method
Upper epidermis Lower epidermis Vascular bundle Lower parenchyma cell Pericyclic fibres Xylem rays Phloem Epidermis Pith Xylem rays
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Figure 13. Class A - T.S of Root of Eupatorium odoratum L. ( × 4) by Xylene- alcohol method Figure 14. Class A - T.S of Root of Eupatorium odoratum L. ( × 20) by Xylene- alcohol method Figure 15. Class A - Unicellular, uniseriate Trichomes of Sandoricum koetjape
- Merr. ( × 20) by Xylene-alcohol method
Epiblema Xylem Phloem Cortex Xylem Unicellular, uniseriate Trichomes
Trichomes
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Figure 16. Class A - Unicellular, uniseriate trichomes, epidermal cells and the cortex of Sandoricum koetjape Merr. ( × 20) by Xylene-alcohol method
Class A Tissue slides by Free hand
Figure 17. Surface view of upper epidermis showing straight anticlinal wall of Sandoricum koetjape Merr.( × 20)
Unicellular, uniseriate Trichomes
Trichomes
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Figure 18. Surface view of lower epidermis paracytic stomata ( × 20) Figure 20. Watery trichomes ( × 20) Figure 19. Calcium oxalate (Raphides) ( × 40)
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Figure 21. T.S of Stem of Sandoricum koetjape Merr. ( × 20) by Free hand Figure 22. T.S of Stem of Eupatorium odoratum L. ( × 4) by Free hand Figure 23. T.S of Stem of Eupatorium odoratum L. ( × 10) by Free hand
Pith Xylem Pericycle Epidermis Pith Xylem Pericycle Epidermis Phloem Collenchyma cell Xylem rays Phloem Pith
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Figure 25. T.S of Root of Eupatorium odoratum L. (× 10) by Free hand Figure 26. Vascular bundles of Root of Eupatorium odoratum L. (× 10) by Free hand Figure 24. T.S of Root of Eupatorium odoratum L. (× 4) by Free hand
Epidermis Xylem Intercellular space Epidermis Phloem Intercellular spaces Xylem rays Xylem rays
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Discussion and Conclusion The sliced tissues prepared by TBA method were the first experience for the researchers in making permanent slides. While making permanent sliced sample tissue with no previous experience and while not very skillful in microtome techniques, many slides were damaged. When Xylene-Alcohol method was used for making slides for the second time, there was less damage. For the process of cutting microtome section, it is better to have three persons at the same time instead of two only. When conducting free hand section, air bubbles were found in tissues during staining although the section was thin and good. In the process of permanent slides by free hand techniques, the cells inside the tissues were found to be damaged. Thus more has to be attempted for better quality. This research was first attempted to prepare permanent slides applying microtome technique in the Botany department. After studying the three methods used systematically TBA method was found to be the best and the most cost effective. Thus, it can be used as teaching aid in biological syllabus of high schools and undergraduate
- courses. This project will be of help in providing information on the
histological characteristics of plant applying different techniques. Microtome techniques were found to be more suitable for Dicot (hard tissue) than Monocot (soft tissue). Based on these findings and experience, quality permanent slides of spore formation of bacteria and fungi that can be used as teaching aids should be prepared for further studies.
Figure 27. Unicellular, Uniseriate trichome and glandular trichome of Eupatorium
- doratum L. ( × 20) by Free hand
Glandular trichome Unicellular, Uniseriate trichome
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Acknowledgements
We would like to express our deepest thanks to Professor Dr. Tin Tun, Rector, University
- f Yangon, for his permission and providing us with essential references. We would also
like to express our gratitude to Professor Dr Pho Kaung, Pro-Rector and Head of Universities' Research Centre (URC), University of Yangon for providing us with research
- facilities. We would like to convey our sincere thanks to all the URC staff who helped in
this project. Finally, we are most indebted to Dr Thet Thet May, Professor, Head of the Department of Botany for her kindness and unstinting support in every way. References Avilla, V. B., 2000. "Manual on the Techniques of Microscopic Slide Processing". Institute of Biological Science, College of Arts and Science, University
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