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Conventional Tube and Microplate Limulus Amoebocyte Lysate Procedures for Determination of Gram-Negative Bacteria in Milk¹

Department of Food Science and Nutrition, The Ohio State University, Columbus 43210

ABSTRACT

A comparison was made of the conventional tube and microplate Limulus amoebocyte lysate assay for detection of gram-negative bacterial lipopolysaccharide in milk. Raw whole milk samples were maintained frozen and portions were examined periodically on alternate days during a 13-d storage to evaluate the reproducibility of both Limulus amoebocyte lysate procedures and to determine optimum reaction conditions for the microplate method. One-day-old, raw and locally purchased pasteurized milk samples, held at 7°C, were analyzed during storage to establish the correlation of both procedures with aerobic and modified psychrotrophic plate counts. Vitamin- and mineral-fortified dairy-based products were examined using the microplate Limulus amoebocyte lysate test as a potential indicator of raw material or finished product bacterial quality and possible postprocessing contamination.

Statistical analysis of the data collected comparing the conventional tube and the microplate Limulus amoebocyte lysate assay demonstrated no significant difference exists between the methods when either the modified psychrotrophic bacterial count or the aerobic plate count was used to determine gram-negative bacteria in pasteurized or raw milk (P<.91). The microplate method, which uses half the lysate reagent, was a good indicator of the bacterial quality of milk and fortified dairy products, consistently detecting bacterial levels greater than 10³ to 10⁴/ml.

¹ Salaries and research support provided by state and federal funds appropriated to The Ohio Agricultural Research and Development Center, The Ohio State University, Journal Article Number 202-88. Additional financial support for this study was contributed by the Dairy Farmers Federation Milk Check-Off program and the National Dairy Promotion and Research Board.