See also: Visikol Peer Reviewed Article
Summary: Visikol™ is a new formula designed to replace chloral hydrate in a wide variety of microscopic applications. Here, the superior clearing action of Visikol on Arabidopsis thaliana can be easily observed when directly compared to acidified chloral hydrate solution.
Microscopic observation has been long used by scientists conducting biological research. In plant science, botanical pathology, and anatomy, some unique challenges are encountered when attempting to observe cells under the microscope. Most tissues contain pigments or other opaque structures, and so they require a clearing procedure to improve visualization. Also when light passes through optically clear tissues and organelles, the different refractive indices of the materials can scatter the light and severely reduce the clarity of the image, obscuring the internal structures of interest. So in order to get the best image possible, the tissue is treated with a clearing agent to both remove the internal pigmentation and improve the refractive indices of the different components of the tissue.
Throughout the scientific literature, one of the most frequently used clearing agents used is acidified chloral hydrate (Lersten, 1967). Chloral hydrate is used as an aqueous solution, often along with glycerol to prevent crystallization when used in a temporary mount to examine a wider variety of plant structures (see page 4). With advances in optical imaging, the utilization of clearing agents has allowed scientists to capture incredibly detailed high-resolution images (Haseloff, 2003).
Chloral hydrate is ideal because of its high refractive index (around 1.4280) and its ability to penetrate and clear a wide variety of tissues, which allows for light to pass through the medium and on to the microscope observer without refraction between the boundary of the specimen and the cover glass. An additional benefit of the refractive index is an increased depth of field; meaning that more vertical depth is in focus and can be observed in the same image without adjusting the focus controls of the microscope (Rost and Oldfield, 2000).
Pharmacopeias like the US Pharmacopeia, American Herbal Pharmacopoeia, and the WHO have published procedures for microscopic authentication of herbal preparations using acidified chloral hydrate as clearing agent (also known as Hertwig’s solution). And consequently, chloral hydrate has become the industry standard used daily in laboratories focused on the quality assessment of herbal products.
However not everyone can obtain chloral hydrate. Since, it also has powerful narcotic effects and its use as a date rate drug, access to chloral hydrate is strictly regulated by governmental authorities. In the U.S., chloral hydrate is a Schedule IV substance, controlled by the Drug Enforcement Administration. In addition to high yearly permit fees, the DEA requires detailed documentation of every transfer and use to ensure no material is illegally diverted. The compliance cost of these regulations makes chloral hydrate impractical for the large majority of users in the field, and as such, their ability to perform most accepted standard techniques is limited. This restriction leads to substandard quality control in industry, and constricts both research and educational opportunities.
Visikol™ has been reported in the scientific literature as a suitable, non-regulated substitute for chloral hydrate in microscopic applications for botanical and agricultural quality assessment, pathology and histology, research as well as for teaching (Villani, 2013).
Materials and Methods
The control solution of acidified chloral hydrate-glycerol solution was prepared by dissolving 45g chloral hydrate into a solution consisting of 25mL 4.2% HCl (1:8,38% HCl to H2O) (Fisher Scientific, Pittsburgh, PA, cat. no A508-4) and 10mL glycerol (Fisher Scientific, Pittsburgh, PA, cat. no G33-1) as in standard methods.
The refractive index for each chemical was determined using a temperature controlled refractometer at 23°C (Fisher Scientific Model #: 334620). The refraction index of Visikol™ (1.4450) was higher than acidified chloral hydrate in glycerol, lactic acid, ethanol and water (Table 1).
Seven day old, dried Arabidopsis thaliana (L.) Heynh. (Brassicaceae) seedlings were submerged in Visikol™, acidified chloral hydrate solution, or water for 30 minutes. Specimens were placed on a microscope slide (Fisher Scientific, Cat No. 12-544-1, 3”x1”x1mm) and mounted either with two drops of Visikol, acidified chloral hydrate solution (control), or water and a cover slip (Fisher Scientific, Cat. No. 12-548-B, 22x22-1, 0.17 mm thickness) was put over each. Slides were then heated on a hot plate (60-80°C) for 30-60 seconds until just before boiling, when the air bubbles moved out to the edges of the slide. Each sample was replicated three or more times. All the microscopic image analyses were taken on a Nikon eclipse 80i microscope, with NIS D 3.00 SP7 imaging software (Nikon, Tokyo, Japan).
Results are shown below in Figure 1.
The images presented here show that Visikol can be effectively used as a direct replacement of chloral hydrate in botanical microscopy. Visikol yields high quality microscopic images and can be used to clear herbal products for research, quality assessment and botanical authentication. Treatment with Visikol clears tissues and, due to the increased depth of field over chloral hydrate, it allows different layers of internal structures as well as surface details of the specimen to be simultaneously identified, without the need for sectioning or remounting. As these results show, Visikol is the superior, non-regulated alternative to chloral hydrate for use in research, education and quality control.
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