Micromanipulation in the Examination of Rumen Bacteria

Abstract

THE culture of bacteria from single cells isolated by micro-manipulation techniques has been used for some time, but only once (in published reports) has the technique been used in attempts to isolate bacteria from rumen contents¹. Some new results using the technique as an adjunct to conventional investigations of rumen bacteria are reported here. The method used was essentially that of Johnstone², as this is simpler than the oil-chamber method originated by de Fonbrune³, and although most rumen bacteria are non-sporing strict anaerobes it was felt that, provided manipulation was rapid, a brief exposure of the cells to air would not affect their viability. The method of casting the agar block and the design of the manipulation cell were as described by Johnstone² and the manipulation was observed through a × 40 long working-distance phase-contrast objective. A 2 per cent solution of ‘Oxoid Ionagar’ was found to give the clearest gel, and after cooling for 10 min the agar blocks were kept in the refrigerator for 15–20 min to give a surface of the required humidity. A 1-mm loopful of rumen fluid was streaked down one end of the block and single cells of the desired organism drawn away by means of a glass micro-needle and deposited at least 0.5 cm from other organisms. The positions of the separated organisms were marked by an electrically heated needle². About 8 cells were separated on one agar block, which was then cut up with a sterile micro-knife to leave the organisms on separate blocks of agar which were transferred to 2 ml. of a suitable medium and incubated.