Eighty-four samples of ground beef were placed into five half-log cycle groups based upon aerobic plate count (APC) results. Endotoxins were determined by the Limulus amoebocyte lysate test (LAL), and gram-negative viable counts were determined by a violet red bile agar overlay method. Ten samples with a log of APC of less than 5.50 had an APC mean of less than 5.24 and mean endotoxin content by the LAL of 51 ng/g. The 15 samples with APCs between a log of 5.50 and 5.99 had an APC mean of 5.79/g and an endotoxin mean of 103.8 ng/g. Twenty-eight samples had APCs between a log of 6.00 and 6.49 with a mean of 5.28/g and an endotoxin mean of 1106.4 ng/g. The 20 samples with APCs between a log of 6.50 and 7.00 had a mean of 6.77/g and an endotoxin mean of 5067.6 ng/g, while 11 samples had a log of APCs of greater than 7.00 with a mean of 7.53 and an endotoxin mean of 7,472 ng/g. Correlation of half-log cycle mean APC and violet red bile agar counts with mean endotoxin content were both highly significant, indicating that LAL-determined endotoxin content can be used to make a rapid approximation of viable plate counts. Because results can be obtained by LAL in 1 h, the finding of low levels of endotoxins can be taken to indicate low-count meat. The use of additional tests of microbial quality may be necessary when high endotoxin levels are found because the LAL detects both viable and nonviable cells.
Of three methods studied, brisk shaking of samples in dilution blanks by hand and homogenization by a stomacher were compared relative to their capacity to recover both endotoxins and viable bacteria; blending with a Waring blender was compared with these two methods only on the recovery of viable cells. Aerobic plate counts were essentially the same by the three methods for fresh meats, with the stomacher producing slightly higher aerobic plate counts and significantly higher gram-negative counts determined by violet red bile agar. The stomacher produced significantly higher aerobic plate counts and violet red bile agar results on frozen meats than did shaking. Endotoxins were determined by the Limulus amoebocyte lysate test; results by shaking and stomacher on 15 single samples of frozen meat were identical. Of Limulus amoebocyte lysate-negative beef which was spiked with known endotoxin, a higher percentage of recovery was obtained with the stomacher. Although both aerobic plate counts and violet red bile agar counts were found by shaking and stomacher to decrease significantly in frozen meats, endotoxin content was not significantly affected. The stomacher was found to be the better method overall, especially when meats are to be examined for their content of viable gram-negative bacteria, endotoxins, or both.
We developed a new medium, designated peptone bile amphotericin cycloheximide (PBAC) agar, which contains (per liter) 10 g of peptone, 300 mg of bile salts, 1 mg of amphotericin B, 1 g of cycloheximide, and 15 g of agar. When 21 samples of fresh ground beef were studied and plate count agar counts were used as references, we obtained a mean recovery of 28% of total counts with violet red bile agar overlay, whereas we obtained 48% recovery with PBAC agar. With 12 samples of frozen ground beef, recovery on violet red bile agar overlay was 29% of the recovery on plate count agar, whereas the corresponding value on PBAC agar was 45%. PBAC agar allowed the enumeration of 1.4 times as many gram-negative bacteria as violet red bile agar overlay. None of eight strains of gram-positive bacteria and none of eight strains of yeasts grew on PBAC agar. Of 158 colonies randomly selected from pour plates of eight fresh ground meat samples, 95% stained gram negative. In comparison, only 70% of 151 colonies selected from corresponding plate count agar plates were gram negative. The lack of background color, turbidity, and ease of use make PBAC agar easier to handle than other media used for gram-negative bacteria, such as violet red bile agar, violet red bile agar overlay, and crystal violet tetrazolium agar. In the preparation PBAC agar, all ingredients are autoclaved together except amphotericin B, which is filter sterilized and added before the plates are poured.
When the endotoxin content of ground beef was determined by direct serial dilution and by three-tube most-probable-number methods, the results were not significantly different, although the latter method provided more specific values for individual samples.
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