Enhancement of the Sensitivity of the Limulus Assay for the Detection of Gram Negative Bacteria

Abstract

In the Limulus pyrogen assay a linear relationship has been demonstrated between optical density and viable cell number of the strains of Gram negative bacteria studied. When the bacteria are suspended in a solution of 0.9% saline and 10‐5 M‐ethylenediamine‐tetra‐acetic acid (EDTA) the sensitivity of the assay is markedly improved and the variation between different species or strains is greatly reduced. The Limulus assay is able to detect fewer than 103 of the least reactive Gram negative bacteria studied within 1 h. It is being developed as a rapid method for detecting Gram negative contamination in raw materials and products.

Potential value of the Limulus lysate assay for the measurement of meat spoilage

“…The results presented here showed no enhancing effect of EDTA treatment. However, the end point detection was probably less sensitive than that used by Coates (1977) and only treatments causing a change in sensitivity of at least a factor of ten would be apparent. An increase of this nature was apparent after sonication which improved the sensitivity of the assay to cells of P. putida and P. fragi by an order of magnitude.…”

“…However, Jay (1977) did note that the values he obtained for the minimum number of cells required to give a positive titre were approximately ten-fold higher than those reported by other workers. Coates (1977) using a spectrophotometric method found that treatment with EDTA/Tris/HCl increased the sensitivity of the Limu1u.s assay to LPS. The results presented here showed no enhancing effect of EDTA treatment.…”

Determination of the Gram Type Using the Reaction Between Polymyxin B and Lipopolysaccharides of the Outer Cell Wall of Whole Bacteria

“…Maeda & Taga, 1979;Flowers, 1979;Coates, 1977;Janda & Work, 1971). This extremely sensitive test is used mainly to detect bacteria in medical fluids.…”

Changing perspectives in food microbiology: Implementation of rapid microbiological analyses in modern food processing

“…It should further be realized that the LPS content depends on the growth phase (111) and that correction factors must be introduced when heat-treated foods (UHT milk, dried milk) are being examined. Because the evaluation of gel strength is subjective, a modification was developed in which the coagulation was determined turbidimetrically (112) or colorimetrically with the help of a chromogenic substrate (113)(114)(115), which after activation of the proteolytic system liberates a colored product quantifiable by measuring at 405 nm. Although at present not commercially available, the LAL test with chromogenic substrate is suitable for automation (116,117).…”