The Recovery Of Clinically Important Anaerobes On Solid Media

Abstract

IT was shown by Collee, Rutter and Watt (1971) that the difficulties encountered in the surface culture of Clostridium oedematiens (CZ. novyi) types B and D can be overcome by the use of modified Moore's medium (Moore, 1968) or by sprinkling sterile iron filings on the surface of a seeded blood agar plate. This approach made it possible to study quantitatively whether the growth was derived from spores or from vegetative cells. It was found for type D of this exacting organism that vegetative cells can account for an appreciable part of the growth obtained when a broth culture is subcultured on solid medium. This finding may be of practical importance also in the case of other anaerobes. It raises the question whether the traditional methods give adequate recovery of anaerobic organisms for clinical purposes or whether more elaborate procedures such as those of Hungate (1950) The following organisms were subcultured in nutrient broth after isolation from clinical material and identification : two strains of Staphylococcus aureus; one strain of Streptococcus pyogenes (group A), one strain of Bacillus anthracoides; one strain of Neisseria gonorrhoeae ; one strain of Veillonella sp.; and two strains of Haernophilus influenzae.Media. Cooked-meat broth was prepared as described by Cruickshank (1968, p. 757), but the infusion broth component was made from Nutrient Broth no. 2 (Oxoid). Oxoid Nutrient Broth was also used for the subculture of some of the test organisms. "Presteamed nutrient broth " was used as a diluent in the quantitative studies; it was heated by steaming at 100°C for 30 min. to remove dissolved air, then cooled rapidly to 37°C.In the initial studies, Watt's modification of Moore's medium (cysteine-dithiothreitol human blood agar) was used (Collee et ~1 . ) . In later studies, a further modification (cysteinedithiothreitol equine blood agar was prepared by substituting 10 per cent. horse blood for 33 per cent. human blood.Blood agar plates were prepared with Oxoid Blood Agar Base no. 2 enriched with equine blood (10 per cent.) or human blood (33 per cent.).Blood. Outdated citrated human blood was obtained from the Blood Transfusion Department, Edinburgh Royal Infirmary. Defibrinated equine blood was supplied by Wellcome Laboratories.

The Isolation of Strict Anaerobes: the Use of an Anaerobic Cabinet Compared with a Conventional Procedure

“…Viable counts were performed by a spread-plate method (Watt, 1972); they were recorded as the mean obtained from six to ten plates seeded from a dilution that gave 100-700 discrete colonies per plate. To minimise errors in comparative studies, dilutions were prepared inside the cabinet with a pre-set constant volume pipette which was then passed out of the cabinet and used for preparation of dilutions at the bench.…”

A Defined Medium for the Growth of Clostridium Tetani and Other Anaerobes of Clinical Interest

“…Although vegetative cells of C. tetani can be recovered on blood-agar media with conventional anaerobic procedures, the yield may still be sub-optimal in terms of the number of cells in the inoculum (Watt, 1972). The development of such studies requires a defined solid medium that can be reliably prepared without the marked variation that is often encountered between batches of blood agar media.…”

Some Variables Affecting the Recovery of Anaerobic Bacteria: a Quantitative Study

“…anaerobic jar can be used with confidence provided that the procedures outlined above are followed. Although previous workers have described experiments with two catalyst sachets in each of their anaerobic jars (Moore, (Watt, 1972 b). Further quantitative studies are clearly needed to define improved methods for the catalytic removal of oxygen from anaerobic jars.…”

“…2 enriched with equine blood (10 %, v/v). Cysteine-dithiothreitol equine blood agar (CDEBA) plates were prepared as described by Watt (1972 a). Blood.…”