Conventional tube and microplate Limulus amoebocyte lysate procedures for determination of gram-negative bacteria in milk.

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 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 degrees 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 less than .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 10(3) to 10(4)/ml.

Heat Inactivation and Reactivation of Alpha Toxin from Clostridium perfringens 1.

Crude and purified preparations of commercial alpha toxin from Clostridium peifringens were heated in physiological saline and in fractions separated from proteose peptone (Difco, 0120). Heating temperatures ranged from 55-95 C for 6-18 min. Alpha toxin remained active at temperatures ⩽ 85 C when heated in the presence of a fraction of proteose peptone having a MW ⩾ 50,000. At 95 C, some destruction was observed with a D-value of 444 min. In saline or a proteose peptone fraction having a MW < 50,000, heat inactivation of alpha toxin occurred at 65 C. Gel filtration revealed that heat protection was associated with complex formation of the alpha toxin with proteose peptone. Alpha toxin heat-treated in saline was reactivated when proteose peptone was added after heat treatment and the toxin was subsequently incubated in hemolysin indicator plates at 37 C for 24 h. Reactivation of alpha toxin was not observed when saline was added after the toxin was heat-treated and then incubated in hemolysin indicator plates at 37 C for up to 48 h.

Simple Technique to Determine Heat Resistance of Bacillus stearothermophilus Spores in Fluid Systems 1.

A simple, rapid, highly reproducible procedure was developed to determine heat resistance of Bacillus stearothermophilus spores in milk and soy protein-based formulas at temperatures > 100 C. Plating efficiencies on different media and heat activation temperatures were also studied. The procedure involved use of a serum bottle to which was added formula. The bottle was closed with a rubber septum and sealed air-tight with a crimped aluminum cap. The formula was agitated during heating in a thermostatically controlled oil bath, using a wrist action shaker. When the formula attained the desired temperature, a spore suspension was injected through the rubber septum, using a high-pressure GLC syringe. At selected time intervals, a portion was withdrawn from the bottle, using a sterile GLC syringe. The number of surviving spores was determined by plating on Trypticase Soy agar, which yielded significantly higher spore recovery count than did Trypticase Soy broth fortified with 1.5% agar with and without starch, or Dextrose Tryptone agar. The serum bottle procedure yielded higher D values than did the capillary tube procedure. The difference was significant where p = 0.05 but not where p = 0.01. With the serum bottle procedure, D values for spores in the milk protein base formula were 18.46, 3.56 and 1.13 min at 115, 121 and 125 C, respectively. In the soy protein base formula, D values were 26.1, 3.64 and 1.26 min, respectively. The z values were 7.7 and 7.6 Centigrade degrees (13.86 and 13.68 Fahrenheit degrees). Maximum heat activation of the spore was at 95 C for 10 min in milk protein base formula and at 100 C for 5 min in the soy product.

Effect of Temperature on Growth and Alpha Toxin Production by Clostridium perfringens 1.

Growth and alpha toxin production by a strain of Clostridium perfringens was determined in Thioglycollate medium, beef broth with ground beef, and beef broth with ground beef and soy protein. Incubation temperatures ranged from 15 to 50 C. In Thioglycollate medium, maximum alpha toxin production occurred at 35 C and was 40 times greater than that observed at 45 C. However, generation time and maximum population were approximately the same at 35 and 45 C. At 15 C, a two log cycle reduction in viable counts occurred within 6 h. Irrespective of incubation temperature, alpha toxin levels in Thioglycollate medium declined as the incubation period was extended beyond the stationary growth phase. In the beef broth with ground beef system which was studied at 35 C only, the organism grew slower and produced less toxin than in Thioglycollate medium. The amount of alpha toxin detected was influenced to a greater extent by the incubation time and temperature, the holding time beyond the stationary growth phase, and the growth medium than by the population level of C. perfringens .

Influence of heat treatment on antigenicity of bovine serum albumin in milk and model systems.

Electroimmunodiffusion on a cellulose acetate support medium was used to study the effect of heat treatment on the antigenic activity of bovine serum albumin suspended in water, simulated milk ultrafiltrate (J & K buffer), or saline (.85%) and of bovine serum albumin in milk per se. Heat treatments were at 70, 73, and 75 C for 0 to 30 min. The suspending medium influenced the loss of antigenicity of bovine serum albumin upon heat treatment. The loss in increasing order was J & K buffer, saline, and water. In milk, the loss of antigenicity of bovine serum albumin was 70 C for 30 min, none; 73 C for 30 min, 24.6%; and 75 C for 20 min, almost complete with no characteristic cone formation upon electroimmunodiffusion.

Heat Resistant Psychrotrophic Bacteria in Raw Milk and Their Growth at 7 C.

Microbiological data are presented for 109 raw milk samples, and for the same samples after heat treatment at 80 C for 12 min, and upon subsequent storage at 7 C for 7, 14, and 28 days. The Standard Plate Count of the raw milk averaged 110,000/ml with 65% of these organisms being penicillin-resistant. Immediately after heat treatment, 87% of the samples had psychrotrophic spore counts< 10/ml. After 14 and 28 days of storage, 50 and 83% of the samples had psychrotrophic counts ≥ 100,000/ml. It was concluded that growth of heat-resistant psychrotrophic organisms may cause spoilage of heated milk. No relationship could be demonstrated between gram-negative counts of raw milk, or initial mesophilic spore counts of heated milk, and bacterial numbers in heated, stored milk.

Factors Affecting Quantification of Clostridium perfringens Alpha Toxin by the Hemolysin Indicator Plate Test and Other Procedures 1.

Application of the following methods, electroimmunodiffusion on cellulose acetate, radial immunodiffusion, lecithovitellin agar plate, and hemolysin indicator plate, for quantification of alpha toxin of Clostridium perfringens revealed that the hemolysin indicator plate method was the most sensitive detecting 0.0003 unit/ml. Sensitivity of the hemolysin indicator plate method was enhanced 67-fold when alpha toxin was diluted with Brewer Thioglycollate medium (Difco, 0236) rather than physiological saline solution. The proteose-peptone ingredient of Thioglycollate medium was the major contributor to increased sensitivity. Less refined agar yielded a larger hemolytic zone. This was attributed, in part, to its higher calcium content. The reducing agent, sodium thioglycollate, diminished the hemolytic zone. In terms of clarity of hemolytic zone, the optimum human red blood cell concentration was 2.1 × 106/ml of base agar. Incubation of hemolytic indicator plates at 35 rather than 21 or 45 C yielded greater hemolytic zones. When incubation time was extended, hemolytic zones also increased in size.

Purification and Activity of Clostridium perfringens Alpha Toxin 1.

Clostridium perfringens a-toxin obtained from a commercial source contained at least seven protein bands when examined by SDS polyacrylamide disc gel electrophoresis. The fraction with an Rm of 0.29-0.56 contained 95.6% of the total hemolytic activity and all of the phospholipolytic acitivity. The a-toxin when passed through a Sephadex G-100 column yielded several peaks but only one active fraction. This fraction upon electrophoresis in a SDS polyacrylamide disc gel system contained two protein bands. Only one protein with a Rm of 0.64 possessed hemolytic and phospholipolytic activites. A second passage of the active fraction through Sephadex G-100 yielded only one protein band. This protein exhibited both hemolytic and phospholipolytic activities. For C. perfringens , it is apparent that both of these activities reside with the same protein.