The Infection of Clover Root Hairs by Nodule Bacteria Studied by a Simple Glass Slide Technique

Abstract

SUMMARY: A simple glass slide technique has been devised for the continuous microscopical observation of growth and infection of root hairs of clover seedlings. The method involves an aseptic cultivation of seedlings on microscope slides which are partly immersed in a mineral salts medium. The roots are protected by a coverslip.By this procedure, the root hairs of white clover inoculated with nodule bacteria were studied. The earliest infection was observed to take place within 48 hr. of inoculation, on 4-day-old seedlings. In branched hairs the growth of the thread from a lateral branch towards the hair tip is tentatively explained as an effect of the position of the hair nucleus relative to the site of infection.The infection of leguminous plants by nodule bacteria has, as a rule, been studied with fixed and sectioned material. The first stages of this process may also be studied in vivo, but no suitable technique has yet been described for a continuous microscopical examination of these stages.A method for the study of growing root hairs (of wheat) was devised by Lundegkrdh (1946) and modified by Ekdahl(1953). In their work, a chamber containing flowing salt solution and resting on a microscope stage was employed. However, difficulties arise with this method if aseptic conditions are also necessary. The present author has therefore used a technique for the aseptic cultivation of young seedlings on microscope slides in a manner which permits periodic observations under the microscope of the growth and infection of individual root hairs. The arrangement also offers good conditions for photomicrography .The details of the method are as follows. METHODA nitrogen-free mineral solution of the following composition was prepared :CaCl,, 0.1 g.; MgSO4.7H,O, 0.12 g.; KH,PO,, 0.1 g.; Na,HPO,.ZH,O, 0.15 g.;Fe citrate, 0.005 g.; Mn, Cu, Zn, B, Mo traces; dist. water, 1000 ml.; pH 6.5 (after autoclaving).Portions (25 ml.) of this solution were distributed into large glass tubes (39 x 125 mm.). The tubes were plugged with cotton wool or covered with closely fitting glass caps (45 x 60 mm.) and were sterilized in the autoclave for 20 min. at 120" (15 lb./sq.in.). Microscope slides (26 x 75 mm.) and cover-slips (24 x 40 mm.) were sterilized by heating dry in Petri dishes, preferably only one slide and one cover-slip in each dish.

Analysis of Pseudomonas fluorescens F113 genes implicated in flagellar filament synthesis and their role in competitive root colonization

“…Seeds were germinated at 4 uC for 16 h followed by incubation in darkness, at 28 uC for 1 day. Germinated alfalfa seeds were sown in Leonard jar gnotobiotic systems using Perlite as the solid substrate and 8 mM KNO 3 -supplemented FP (Fahraeus, 1957) as the mineral solution.…”

“…Seeds were germinated at 4 uC for 16 h followed by incubation in darkness, at 28 uC for 1 day. Germinated alfalfa seeds were sown in Leonard jar gnotobiotic systems using Perlite as the solid substrate and 8 mM KNO 3 -supplemented FP (Fahraeus, 1957) as the mineral solution.After 2 days, alfalfa seedlings were inoculated with~10 8 cells of the appropriate strain. For the competitive colonization experiments, the tested strain and the competitor were inoculated at a ratio of 1 : 1.…”

A search for peas (Pisum sativum L.) showing strain specificity for symbiotic Rhizobium leguminosarum

“…(iii) Cultures and assays Peas were sterilized in sodium hypochiorite solution (12 per cent w/v available chlorine) for 30 mm, and placed singly in 250 ml conical flasks closed with foam bungs and containing 200 ml of sterile nutrient agar. This contained nitrogen-free mineral salts (FAhraeus, 1957) and trace elements (Gibson, 1963) adjusted to pH 65, solidified with 5 gF1 Oxoid No. 3 agar.…”

Phase variation has a role in Burkholderia ambifaria niche adaptation

“…After 3 days, germinated seeds were sown in tubes containing Perlite as the solid substrate and 8 mM KNO 3 À -supplemented Fahraeus liquid medium (Fahraeus, 1957). Bacterial isolates were inoculated in a 1:1 ratio (10 5 CFU of wild-type and variant).…”