The use of anaerobic jars for the enrichment, isolation and enumeration of anaerobic organisms, though very convenient for larger laboratories, is cumbersome and expensive for smaller ones. We have therefore examined a number of methods that do not require anaerobic jars, and give details of one which we have found satisfactory. Preliminary experimentsMethods involving the use of pumps were considered to be as inconvenient as jar methods. The technique of Fortner (1929, 1957) using Serratiu marcescens as the aerobe is rather uneconomical, and as growth of anaerobes is sometimes slow and not very luxuriant, strict anaerobes may be overgrown by facultative anaerobes. Absorption of oxygen with solutions of pyrogallol and sodium carbonate or copper-spotted steel wool (Parker, 1955) failed to reduce the oxygen tension sufficiently to give rapid anaerobic growth, no doubt because the oxygen-absorbers had become partly saturated before use. We therefore developed a method, originally suggested by Koch (1934) and recommended by Terpstra and Akkermans (1955), involving the use of a dry mixture of pyrogallol, potassium carbonate and diatomaceous earth in double plates sealed with paraffin wax. This mixture does not absorb much oxygen until it is adequately moistened by water evaporated from the medium. Materials and methodsStrains. We used Clostridiunz bifermentwns (1 strain), CE. botulinum (3), Cl. butyricum (4), Cl. welchii (4), Cl. sporogenes ( 7 ) , Cl. pasteurianum ( l ) , a stenothermophilic sulphite-reducing clostridmm isolated from a spoiled meat pack, one strain each of Cl. chauvai, GI. histolyticum, Cl. adenlatiens, Cl. septicum and Cl. tetani, and fiwe strains of Rijidibacte~ium bijidum isolated from infant or adult human faxes.The oxygen-absorbent was a freshly prepared mixture of pyrogallol, 3 g., K,CO,, 3 g. and diatomaceous earth, 15 g., packed in 2 g. quantities in filter-paper envelopes 7 x 4 cm. These were found to reduce the Eh in Koch plates in 3 hr a t 37" C. or in 4 hr a t 32" C. to the level a t which resazurin incorporated in thioglycollate agar is reduced, i.e. to -0.04 volt a t p H 7 . Reagent.Media. The following media were used : Thioglycollate-resazurin broth (Pittman, 1946) plus 1.5 per cent. agar, with or without 10 per cent. by volume defibrinated shecp blood ; sulphite-iron agar (Mossel et al., 1956), containing only 0.05 per cent. sulphite, since we have confirmed Beerens' (1958) observation that some strains of Cl. sporogenes will not tolerate 0.1 per cent. sulphite.For general subculture work thioglycollate-resazurin agar was preferrcd, since failure of the normal colour change from pink to greenish yellow to develop within 3 hr was good evidence that the plate was leaking.Unfortunately resazurin cannot be used as indicator for snlphite-agal* (equilibrium Eh -0.03 at pH 7) or for blood-agar. If these media are used,
Packer's crystal violet sodium azide blood agar (Packer, 1943) used in poured plates at 36*1", gave satisfactory recovery of pure cultures of Lancefield group D streptococci and completely inhibited the growth of 11 other species of aerobic and anaerobic food bacteria, including Strep. lactis ( 5 strains). Later, however. one group N streptococcus was obtained which did grow in Packer's agar a t 3651'. To eliminate this organism the incubation temperature had to be increased to 39.5", using agar strips (Stirling et al. 1950) incubated in a water bath to secure strict temperature control. Under these conditions the recovery of typical group D streptococci was never consistently below 50% of the count in tryptone dextrose yeast extract agar at 31&l".
In the course of 1949 interest was aroused by a series of patent applications on the use of dehydroacetic acid (an unsaturated cyclic diketone: 3-acetyl,6-methylpyrandione-2,4, synthesized for purpose of research on this class of compounds) as a preservative for foods (I).Since it was claimed that this compound and its sodium salt were non-toxic, sufficiently soluble and did not impart any special colour, flavour or taste to foods, they looked attractive and therefore were tested in the techniques, in current use in our laboratory.The experiments were carried out with the sodium salt of dehydroacetic acid as it was found in a preliminary experiment that this was slightly less potent than the free acid. The sample used was kindly procured by The Dow Chemical Company, Midland U.S.A. under the trade name: DHA-S. It was a white, odourless powder, pH in 0.2 % aqueous solution = 7.0. FUNGISTATIC ACTION.Antifungal action was tested in the new technique, recently published by the senior author (4), wherein rye flour is used as a spoiling substrate. Spoilage is favoured by exposing the sample to a relative humidity of 90 % (sat. aq. solution of Na2CO3. 10 H20 ) at 25 ° C., which however inhibits any development of bacteria (3). In order to eliminate any non-microbiological limiting factors, the samples are uniformly moistened before incubation to an equilibrium humidity of 0.90.While controls were completely covered with mycelia in 7--9
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