Method for studying grass leaf epidermis and stomata



Convenient method for studying grass leaf epidermis

by Hilu K. W., Randall J. L. (1984)

Khidir Hilu,, Virginia Polytechnic Institute and State University, Blacksburg, United States


in Taxon 33(3): 413- 415 – DOI: 10.2307/1220980 –


Leaf epidermis of grasses is structurally elaborate and quite important in modern systematics of the Poaceae.
Available techniques used in preparing leaf epidermis for microscopic studies are time-consuming and do not always produce adequate preparations.
A simple and convenient technique is proposed in this paper which requires the application of a film of clear nail polish directly to the leaf surface.
The impressions left on the polish film after drying produce an excellent detailed image of the epidermis. This technique is also applicable to other plant families.

Functionally isolated guard cells (stomata) in epidermal strips



A simple technique of obtaining functionally isolated guard cells in epidermal strips of Vicia faba

by Durbin R. D., Graniti A. (1975)

Richard D. Durbin, Antonio Graniti,

Centro di studio del CNR su le tossine e i parassiti sistemici dei vegetali Istituto di Patologia vegetale, Università:Bari, Italy


in Planta 126: 285-288 – doi: 10.1007/BF00388970 –


Ultrasonic irradiation of Vicia faba L. epidermal strips for 2 min preferentially disrupts the epidermal cells but does not impair guard cell movements.

Maximal opening induced by fusicoccin requires that K(+) be provided to the guard cells from external sources. A mobile organic anion is not required.


Procedures for obtaining epidermal strips with only the guard cells alive have been developed but they are either tedious, require considerable experience or may damage the guard cells (Meidner and Mansfield, 1968; Squire and Mansfield, 1972b; Allaway and Hsiao, 1973). Such material is, however, very useful for studies on some aspects of the mechanism of guard cell movements. In this paper a simple, rapid and reproducible technique using ultrasonic irradiation to preferentially disrupt epidermal cells is reported which largely overcomes these drawbacks. ll[aterials

Preparation of leaf epidermis for experiments on stomatal physiology



Selection and preparation of leaf epidermis for experiments on stomatal physiology

by Weyers J. D. B., Travis A. J. (1981)

Screen Shot 2018-07-17 at 20.51.10


in J. Exp. Bot. 32, 837–850 – doi: 10.1093/jxb/32.4.837 –


A simple method for reproducibly peeling leaf epidermis tissue is described. Epidermis was obtained from eight species using the method, and its properties of value in research on stomatal physiology evaluated.
Effects of the method of peeling on cell viability and mesophyll contamination were quantified, and a comparison was made between the effects of peeling and subsequent treatments on a plant with morphologically distinct subsidiary cells, Commelina communis, and one without, Vicia faba.
The results indicate that artefacts in experiments on stomatal physiology involving leaf epidermis could arise not only from the peeling method, but also the plant species chosen.

Stomata and epidermal cells of herbaria specimens



Non-invasive method for looking at stomata / epidermal cells of herbaria specimens

by Wyatt A. (2018)

Amy Wyatt, Cardiff University, UK


in Project DOI: 10.13140/RG.2.2.15999.30884 –

Earlier on in the year I perfected a non-invasive method to look at stomatal density and guard cell size of herbarium specimens. After preliminary tests using clear nail varnish proved unsuccessful, I found a method online ( that involved using Germolene liquid plaster on the herbaria specimens to make an imprint of the leaf surface. For each individual, a photo was taken marking leaves 1-5 with a number so later on I know which leaves have been analysed, and I can account for leaf area, length and width when determining the stomatal density / guard cell size of individuals.


1.) Spread germolene, liquid skin over surface of leaf in a 1cm2 patch and allow to dry for 5-10 minutes so it is no longer tacky on the leaf.

2.) The germolene will dry transparent, and you will need to gently pry the surface of the germolene until it goes opaque. I was using dental tools for this, the ‘dental explorer’ tool (pictured below) with a right-angled head was perfect for this. Run the outside edge of right angle (not the sharp end) gently over the surface of the germolene, you will start to see the germolene turning opaque as it detached from the leaf surface.

3.) Once the whole surface has turned opaque, use the sharp end of the tool to gently pry at the edge of the germolene until it starts to peel off, use fine tweezers to remove gently without damaging the leaf.

4.) Once you have your germolene imprint, mount onto a microscope slide and place a cover slip over the top of the sample.

*** for the species I was working with Impatiens glandulifera I took one measurement from the left hand side of the abaxial surface 1cm from the leaf vein.

Stomata in leaf epidermis images for robust identification

Screen Shot 2018-04-11 at 14.39.35
Figure 1. Samples of epidermal images with low variation in images of the same species (row). From top to bottom: Ilex affinis, Myrsine guianensis, Handroanthus impetiginosus and Xylopia sericea.


Leaf epidermis images for robust identification of plants

by da Silva N. R. , daSilva Oliveira M. W., Antunes deAlmeida Filho H.  , Souza Pinheiro L. F., Rossatto D. R., Kolb R. M., Martinez Bruno O. (2015)

Núbia Rosa da Silva1,2 , Marcos William da Silva Oliveira1,2 , Humberto Antunes de Almeida Filho2 , Luiz Felipe Souza Pinheiro3, Davi Rodrigo Rossatto4, Rosana Marta Kolb3 & Odemir Martinez Bruno1,2

1 Institute of Mathematics and Computer Science, University of São Paulo, USP, Avenida Trabalhador são-carlense, 400, 13566-590 São Carlos, São Paulo, Brazil.

2 Scientific Computing Group, São Carlos Institute of Physics, University of São Paulo, PO Box 369, 13560-970, São Carlos, SP, Brazil.

3 Department of Biological Sciences, Faculty of Sciences and Languages, Univ Estadual Paulista, UNESP. Av. Dom Antônio, 2100, 19806-900, Assis, São Paulo, Brazil.

4 Department of Applied Biology, Faculty of Agriculture and Veterinary Sciences, Univ Estadual Paulista, UNESP, Via de Acesso Prof. Paulo Donatto Castellane S/N. 14884-900, Jaboticabal, São Paulo, Brazil.


in Scientific Reports  6:25994 – DOI: 10.1038/srep25994 –

Screen Shot 2018-04-11 at 14.41.36
Figure 2. Samples of epidermal images with wide variations in images of the same species (row). From top to bottom: Miconia cuspidata, Tapirira guianensis, Symplocos mosenii and Guapira noxia.

This paper proposes a methodology for plant analysis and identification based on extracting texture features from microscopic images of leaf epidermis. All the experiments were carried out using 32 plant species with 309 epidermal samples captured by an optical microscope coupled to a digital camera.

The results of the computational methods using texture features were compared to the conventional approach, where quantitative measurements of stomatal traits (density, length and width) were manually obtained. Epidermis image classification using texture has achieved a success rate of over 96%, while success rate was around 60% for quantitative measurements taken manually. Furthermore, we verified the robustness of our method accounting for natural phenotypic plasticity of stomata, analysing samples from the same species grown in different environments.

Texture methods were robust even when considering phenotypic plasticity of stomatal traits with a decrease of 20% in the success rate, as quantitative measurements proved to be fully sensitive with a decrease of 77%.

Results from the comparison between the computational approach and the conventional quantitative measurements lead us to discover how computational systems are advantageous and promising in terms of solving problems related to Botany, such as species identification.

Methods to measure stomatal and morphological features.

(a) Input image I. [6] Fig 1 (b) Result after binarization [6]


A Review: Methods of Automatic Stomata Detection and Counting Through Microscopic Images of a Leaf

by Bhaiswar N., Dixit V. V. (2016)

Nitin Bhaiswar 1 ,P.G. Student, Department of E&TC, Sinhgad College of Engineering, Vadgaon (BK), Maharashtra, India

Dr.V. V. Dixit 2 , Professor, Department of E&TC, Sinhgad College of Engineering, Vadgaon (BK), Maharashtra, India



in International Journal of Innovative Research in Science, Engineering and Technology  5(6): 10612-10617 –

Screen Shot 2018-02-07 at 21.07.50

Fig 2 (a) Response map [6] Fig 2 (b) Regions of maximum responses after Thresholding [6]


Stomata are the small pores in leaf epidermis of a plant which are important for the intake of carbondioxide and release of oxygen for the growth of plant.

Our aim is to discuss methods to design a tool which can automatically detect number of stomata present on an epidermis of a leaf and count them.

First method is by using morphological operation and another by using the template matching algorithm.

These methods also measure their stomatal and morphological features.

Screen Shot 2018-02-07 at 21.10.12

Fig.3 a) original image of species 1 [7] Fig 3b) Image after stomata segmentation of species1[7]

Measuring Stomatal Density



Measuring Stomatal Density

by Meatyard B., MacDonald M. (xxxx)

Barry Meatyard and Mary MacDonald.

in SAPS –

Science & Plants for Schools:  HYPERLINK “”

Introduction and context

Estimation of stomatal density is often done when studying photosynthesis (at GCSE and higher levels), and can offer a way of illustrating use of the graticule with post-16 students. There are a number of ways to measure stomatal density. Because of the size of stomata, you will need a reasonably good microscope for this. Your choice of magnification will depend on the leaf material that you are using, and the size of the stomata.

One popular method has been to use clear nail varnish to make an impression of the epidermis. Making the impression and viewing it under a microscope can be completed in one lesson. However, some leaves are prone to damage from the solvent in the nail varnish. The leaves absorb it, turn brown, and fail to produce any impression. Pupils lose interest and get frustrated because their leaves ‘aren’t working’. Also, for a GCSE class, several pots of nail varnish are needed so that no one is left waiting, thus adding to expense. Other methods include using Germolene New Skin and using a water-based varnish from DIY shops.


Selecting your plants

One of the best plants for doing epidermal peels is the red hot poker plant Kniphofia. Being a monocot its stomata are highly ordered in rows, but they are big and great for stomatal opening and closing using solutions of different concentrations.

Almost as good is the Elephants Ear Saxifrage Bergenia. This also peels very easily, but the stomata are smaller although clearly visible at x100 magnification. This is a dicot so the distribution is more random.

Many labs have a Pelargonium, and these can also be used for leaf peels.

Spider plants (Chlorophytum comosum variegatum) make excellent leaf peels, with particularly interesting and regular patterns of stomata along the green leaf areas only.