Reading reflection - 4/10/13

A New Method which Gives an Objective Measure of Colonization of Roots by Vesicular-
Arbuscular Mycorrhizal Fungi. Author(s): T. P. McGonigle, M. H. Miller, D. G. Evans, G. L. Fairchild and J. A. Swan


This article argues that previous methods to quantify the proportion of root length colonized by vesicular-arbuscular (VA) mycorrhizal fungi provide only observer-dependent measures of colonization, which is not useful for other researchers to compare roots quantitatively. This study developed a new modified method referred to as the magnified intersection method, which estimates VA mycorrhizal colonization on an objective scale of measurement. The magnified intersection method involves inspection of intersections between the microscope eyepiece crosshair and roots at magnification x 200. The estimate of colonization is the proportion of root length containing arbuscules, called the arbuscular colonization (AC). According to McGonigle (1990), arbuscules (tree like root systems) are the only unique feature of mycorrhizas, and they serve as the principal site of phosphorus transfer from endophyte to plant.

A clover root stained to reveal the arbuscules and vesicles within the root tissues. Jim Deacon 2005

Methods:
Roots were collected by taking a 5cm diameter soil core from a field of Zea mays. The roots were then washed from the soil, and stored in formyl acetic alcohol to be later dispersed in a beaker of water. Root samples were cleared for 20 minutes at 121 degrees Celsius in 10% KOH, and stained in Chlorazol Black E. Roots were mounted in glycerin on microscope slides, covered with coverslips, and observed at magnification x 200. All intersections between roots and the vertical eyepiece crosshair were considered. The AC was calculated by taking the count for the arbuscules divided by the total number of intersections examined.

A compound microscope used to measure the length of arbuscular mycorrhizas (arbuscules, vesicles and internal hyphae) within roots. Mark Brundrett 2008

Discussion:
Individual subjective estimates of colonization are likely to be inaccurate, but can help detect relative colonization levels. Moreover, these data will be observer-dependent and should not be directly compared across experiments conducted by different researchers. The magnified intersections method provides a reliable and objective measure of colonization, AC, which has been shown in this study to vary little from observer to observer. It permits direct comparisons of colonization data from one research to another.