A Rapid Plate Method of Counting Spores of Clostridium Botulinum

Abstract

Making accurate counts of the spores of Clostridium botulinum has been a formidable problem in food bacteriology. A literature survey revealed no standard or widely accepted method. In general, long incubation times have been employed in an effort to count these spores, many of which may remain dormant for weeks or even years. The present paper describes a fast plate method for counting spores of C. botulinum. The method is based on the development of an adequate medium which includes HCO_ and the use of a new plating technique which produces anaerobiosis and permits the plate to be counted or examined at any time. METHOD Medium (VII). Pork infusion, 800 ml; pea infusion, 200 ml; peptone, 5 g; tryptone, 1.6 g; K2HPO4, 1.25 g; soluble starch, 1 g; Na thioglycolate, 0.5 g; N NaOH to pH 7.2; agar, 16 g. The materials are autoclaved 5 minutes at 121 C and clarified while hot by adding 25 g of "celite"2 and filtering through Whatman no. 42 filter paper with suction. The medium is tubed in 9 and 14 ml quantities and can be stored in a closed container in a freezer. As needed the tubes are autoclaved at 15 pounds for 12 minutes. The pork infusion is made as follows: fresh, lean pork is ground, stirred into hot distilled water (1 lb per L), and steamed 1 hour. The meat is filtered out on 4 layers of cheesecloth, and the solidified fat is removed after cooling. The pea infusion is made as follows: fresh or good-quality frozen peas are blended with distilled water (1:1) and steamed 1 hour. The solids are removed by centrifugation and filtration with celite. If not used immediately the pork or pea infusions can be stored at 0 F in enameled cans. The complete medium also contains NaHCO3 which gives best results if added to the dish at the time of pouring. A freshly prepared solution of 5 g of NaHCO3 per 100 ml is sterilized by pressure Seitz filtration and, if not used immediately, is kept in sterile screw-cap tubes in a refrigerator. Plating. The anaerobic petri dish used in this wvork is set up as shown in figure 1. To the petri dish (no. 3162, Corning Glass Works2) 0.4 ml of the NaHCO3 solution, 14 ml of the medium, and the inoculum are added and mixed. After

THE ENUMERATION OF ANAEROBIC BACTERIA, AND OF CLOSTRIDIUM SPECIES IN PARTICULAR, IN FOODS

“…That this occurs with dilution counts is recognized, but it is not generally appreciated that it also applies to anaerobic colony counts. Because of the disadvantages of anaerobic jars, numerous modifications to Petri dishes have been suggested for cultivating and counting anaerobes (Streng, 1903 ;M'Leod, 1913;Huntemuller, 1924;Kadisch, 1924;Brekenfeld, 1924;Spray, 1930;Koch, 1934;Muller, 1037;van Riemsdijk, 1939;Brewer, 1942;Kaesz, 1042;Andersen, 1951;Leistner, 1955). None of the modifications has been found sufficiently convenient and reliable in routine use, except for the Morse-Kopeloff double plate (Tetrault, 1929).…”

Growth of Food Spoilage Bacteria in Banana Purée

“…3679 cell suspension was done according to the method of Reed et al (8) with the modifications that the procedure was scaled down to a final volume of one liter, instead of 5 liters, of pork broth, and that the final incubation period was for one week, instead of two, at 30" C. The centrifuged and filtered cell suspension was stored under refrigeration in a 2-0~ screw cap jar containing glass beads. An aliquot part of the cell suspension was heated at 80" C for 15 min; this and an unheated portion were examined for cell counts on Andersen's medium (1) in anaerobic culture tubes. The colonies were counted as described in the preceding paragraph, with results as shown in Table 1.…”

SPORE FORMATION BY CLOSTRIDIUM SPECIES IN AN ARTIFICIAL MEDIUM

“…Spray, 1930). Anderson (1951) used a standard Petri dish with a glass plate on top of the medium and a final thioglycollate agar seal. Other workers recommended counting in deep agar tubes, such as Prickett tubes (Miller, Garrett & Prickett, 1939).…”

“…A spore may conveniently be described as an organism with a certain high heat resistance, but exact knowledge of the amount of heating necessary to kill vegetative cells but not spores is essential. Many arbitrary heat treatments have been used by different investigators, for example, 75" for 30 min (Wynne t Foster, 1948), 80" for 10 min (Hirsch & Grinsted, 1954), 80" for 15 min or 85" for 10 min (Sugiyama, 1951), 90" for 5 rnin (Halvorson, 1955) and I miri in boiling water (Anderson, 1951). The choice is somewhat arbitrary since clostridial cultures consisting entirely of spores or entirely of vegetative cells have never been used for this purpose and in our experience are very difficult to obtain.…”