Use of Presumpto Plates to identify anaerobic bacteria.

Identification of anaerobic bacteria requires special media and growth conditions that contribute to a higher cost per identification than that for aerobic isolates. Newer rapid methods streamline the identification process, but confirmation to the species level is often difficult. The Presumpto Plate method for the identification of commonly encountered anaerobes consists of three quadrant plates, each containing four conventional media, that result in the generation of 21 test parameters: growth on Lombard-Dowell medium; production of indole, indole derivative, catalase, lecithinase, and lipase; proteolysis of milk, H2S, and esculin; growth on 20% bile; precipitate on bile; DNase, glucose, casein, starch, and gelatin hydrolysis; and fermentation of lactose, mannitol, and rhamnose. Identification charts were developed by using the results from 2,300 anaerobic isolates. Because conventional media were used, there was a high degree of agreement between the Presumpto Plate method and the reference method when testing commonly encountered anaerobes. The Presumpto Plate method is as accurate as commercially available enzyme systems for the identification of many anaerobic species but is less expensive to perform.

Comparison of media in the Anaerobe-Tek and Presumpto plate systems and evaluation of the Anaerobe-Tek system for identification of commonly encountered anaerobes.

Using a variety of sporeforming and nonsporeforming anaerobic bacteria, we compared 10 differential agar media of the Anaerobe-Tek (A/T) system recently marketed by Flow Laboratories, Inc. (McLean, Va.) with 10 comparable media in Presumpto quadrant plates (Presumpto 1, 2, and 3) developed by the Centers for Disease Control Anaerobic Bacteria Branch. The A/T identification system was evaluated by comparing the species identity of anaerobes determined as recommended by the manufacturer's instruction manual with the identity of the strains obtained by the Centers for Disease Control Anaerobe Reference Laboratory by using conventional procedures. We also compared reactions obtained with the Presumpto plates with a chopped meat glucose broth culture as a source of inoculum with those obtained by using a turbid cell suspension from growth on blood agar as inoculum. The agreement of results for the 16 characteristics compared ranged from 92.8 to 100%. Comparison of test results obtained with 10 media in the Presumpto plate and A/T systems from the examination of 223 strains of anaerobes, representing 54 different taxa, showed the following agreement between A/T and CDC systems: catalase production, esculin hydrolysis, glucose fermentation, and lecithinase production (100%); inhibition of growth by bile agar (99.6%); lipase production (99%); DNase (98.7%); fermentation of lactose and mannitol (98.2%); starch hydrolysis (96.9%); gelatin hydrolysis (96.4%); and casein hydrolysis (94.6%). Of the 204 strains of common anaerobes tested with the A/T system, only 70% were correctly identified to the species level. However, several strains could have been identified correctly with the A/T system if data on certain other characteristics had been included in the A/T data base.

Gelatin agar medium for detecting gelatinase production by anaerobic bacteria.

A new medium, Lombard-Dowell gelatin agar, was developed for detecting gelatinase activity by anaerobic bacteria. The medium contained: Trypticase (BBL Microbiology Systems), 5.0 g; yeast extract (Difco Laboratories), 5 g; sodium chloride, 2.5 g; sodium sulfite, 0.1 g; L-tryptophan, 0.2 g; L-cystine, 0.4 g; hemin, 10.0 mg; vitamin K1, 10.0 mg; agar, 20.0 g; D-glucose, 1.0 g; gelatin, 4.0 g; and distilled water to 1 liter. The pH was adjusted to 7.5. The medium was dispensed in 100- by 15-mm quadrant plastic dishes (5 ml per quadrant). To test for gelatinase activity, we inoculated the medium with a young enriched thioglycolate or chopped meat glucose broth culture or a turbid cell suspension in Lombard-Dowell broth, using a sterile cotton swab, and incubated it under anaerobic conditions for 48 h at 35 degrees C. The quadrants were then flooded with Frazier solution, and clear zones around the bacterial growth were recorded as positive for gelatinase activity. The new medium was tested with a variety of anaerobic bacteria, and the results were compared with data obtained with the conventional technique for detecting gelatinase activity. Overall, there was satisfactory agreement between the two tests in the detection of gelatinase activity, but the Lombard-Dowell gelatin agar tests was more rapid and somewhat more sensitive than the conventional test.

Cultural and physiological characteristics of Clostridium botulinum type G and the susceptibility of certain animals to its toxin.

Strain 89 of Clostridium botulinum type G, isolated by Gimenez and Ciccarelli in 1969, was characterized culturally, biochemically, and toxigenically. It was motile, hemolytic asaccharolytic, weakly proteolytic, lipase and lecithinase negative, and it produced acetic, isobutyric, butyric, and isovaleric acids in peptone-yeast extract-glucose broth. No spores were seen in smears from solid or liquid media. Very low levels of toxin were produced in regular broth cultures, but dialysis cultures yielded 30,000 mouse 50% mean lethal doses (LD50 per kg, orally and subcutaneously, respectively; and for guinea pigs, 10,000 to 20,000 and 100 mouse LD50 per kig, intragastrically and intraperitoneally, respectively.

An inexpensive device for evacuating and gassing anaerobic systems with in-house vacuum.

An inexpensive, compact device to aid in establishing an anaerobic environment for bacterial cultures is described. This device is designed for use with an in-house vacuum system and can replace the vacuum pump, "U" utbe manometer system commonly used for this purpose.

Low-temperature irradiation of beef and methods of evaluation of radappertization process.

An inoculated, irradiated beef pack (1,240 cans) was conducted for the determination of microbiological safety for unrestricted human consumption. Each can contained a mixture of 10(6) spores of each of 10 strains of Clostridium botulinum (5 type A and 5 type B), or a total of 10(7) spores/can. The cans were irradiated to various doses (100 cans/dose) with 60Co gamma rays at -30 +/- 10 C, incubated at 30 +/- 2 C for 6 months, and examined for swelling, toxicity, and recoverable botulinal cells. The minimal experimental sterilizing dose based on nonswollen, nontoxic sterile cans were 2.2 less than experimental sterilizing dose based on nonswollen, nontoxic sterile cans was 2.2 less than experimental sterilizing dose less than or equal to 2.6 Mrad. Using recoverable cells as the most stringent criterion of spoilage, and assuming the conventional simple exponential (without an initial shoulder) rate of spore kill, the "12D" dose was 3.7 Mrad when estimated on the basis of mixture of 10 strains totaling 10(7) spores/can, and 4.3 Mrad if it is assumed that each can of beef contained 10(6) spores of a single most resistant strain and all of these spores were of identical resistances. However, an analysis of the data by extreme value statistics indicated with 90% confidence that the spore death rate was not a simple exponential but might be a shifted exponential (with an initial shoulder), Weibull, lognormal, or normal, with a "12D" equivalent of about 3.0 Mrad regardless of the initial spore density per can. There was an apparent antagonism between the irradiated type A and B strains in the cans. Some of the cans contained type B toxin but did not include type B viable cells. Other cans had a mixture of type A and B toxins, but a large number of these cans did not yield recoverable type B cells. However, type A viable cells could always be demonstrated in those cans containing type A toxin.