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Preservative efficacy testing without counting colonies was done by determining growth in dilutions of inoculated product following enrichment in Letheen broth with 0.001% triphenyltetrazolium chloride (TTC) in 96-well microtiter plates. Bacterial growth was indicated by the development of a red/pink color in the enrichment broth. The method was used to determine log reductions of bacteria at specified times after inoculation, and D-values were calculated using the reciprocal of the highest dilution showing growth (pink color) as the log CFU/ml bacteria at each time point. The method using TTC was validated by demonstrating that D-values for Staphylococcus aureus, Pseudomonas aeruginosa, Burkholderia cepacia, and Escherichia coli in 44 aqueous cosmetic and OTC-drug products were virtually identical to those obtained when using Alamar Blue in the miniaturized system. Plotting D-values obtained using TTC as a function of D-values obtained using Alamar Blue gave a line with a slope of 0.98, which shows excellent agreement of results obtained by the two methods. This miniaturized assay system has been used for more than three years for preservative efficacy testing of several hundred cosmetic and OTC-drug product samples in our laboratory. It is recommended for laboratories that conduct large numbers of preservative efficacy tests.

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The objective of these studies was to set up a reliable radioimmunoassay (RIA) for staphylococcal enterotoxins A, B, and C (SEA, SEB, and SEC) in a food system. Significant differences (95% confidence limits) were obtained between the 0- and 1-ng/ml enterotoxin standards, so the sensitivity of the RIAs was 1 ng/ml. Polystyrene tubes coated with anti-SEB and stored at 4 degrees C were unstable. The percentage of iodinated SEB bound to these tubes decreased at a rate of 0.33%/day, in contrast to the rate of 0.07%/day obtained with tubes prepared the day before the analyses. Satisfactory precision and maximum sensitivity were obtained by using six replicates for each sample and freshly coated tubes. The antisera used for coating the tubes were reused four times and were frozen between coatings. The process of drum drying mashed potatoes containing 1 mug of SEB per g of mashed potatoes inactivated 83% (wt/wt) of the SEB. Statistical quality control parameters were used to insure that RIAs were performing reliably with a sensitivity of 1 ng/ml. Over 450 samples of potato flakes and granules, which represented different production lots from 12 different manufacturers, were examined for SEA, SEB, and SEC. No enterotoxins were detected.

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This report describes the conditions that are necessary for iodination of staphylococcal enterotoxin B (SEB) by use of chloramine-T. Makor Chemical Co. SEB and the two major SEB components, which were prepared by isoelectric focusing of partially purified SEB, were used in these studies. The antigenic activity of the SEB preparations was monitored by radioimmunoassay as the oxidation/reduction (O/R) potential was increased by addition of chloramine-T. The SEB preparations lost antigenic activity rapidly at pH 7.5 and room temperature when sufficient chloramine-T was added to raise the O/R potential above 250 mV. Iodinated SEB with satisfactory immunoreactivity was prepared by omitting carrier iodide from the iodination reaction mixture and by using at least 1 mg of SEB/ml, steps which made the O/R potential more stable, and by stopping the reaction before the O/R potential exceeded 250 mV. Comparison of the chloramine-T method with a lactoperoxidase/H2O2 method of iodinating SEB showed the latter to cause a greater loss of immunoreactivity.

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The overall recoveries of spores and of actively growing, heat-stressed, coldshocked, and frozen cells of five strains of Clostridium perfringens were significantly greater (95% confidence limits) on tryptose-sulfite-cycloserine medium without egg yolk than on sulfite-polymyxin-sulfadiazine medium.

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The sera of several animals were examined for suitability in coagulase testing. The assay for coagulase-reacting factor (CRF) activities of the whole sera indicated the following relative concentrations of CRF: human > pig > rabbit > horse > bovine, chicken, and lamb. Human, pig, and rabbit sera had adequate amounts of CRF for coagulase testing. The plasmin activities of the different sera, arranged from the strongest to the weakest, were as follows: rabbit > human > lamb > horse > bovine, chicken, and pig. Fibrinolysis was observed when rabbit, human, lamb, or horse sera were incorporated into coagulase test agars. Pig serum was superior to the other sera for use in the plate test for coagulase since it had adequate amounts of CRF and the plasminogen-plasmin system was not activated by staphylokinase or staphylococcal Müller factor. Heparinized pig plasma was more suitable than citrated pig plasma since citrate interfered with the growth of Staphylococcus aureus, and the use of heparinized plasma prevented false-positive coagulase reactions due to citrate utilization.

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Fibrin halos developed around coagulase-positive Staphylococcus aureus colonies, and deoxyribonuclease production was detected by using a developing solution containing 1 mg of methyl green per ml.

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A selective, differential plating medium was developed for the isolation and identification of coagulase-positive and mannitol-fermenting staphylococci. Coagulase produced by growing Staphylococcus aureus caused an opaque zone of fibrin to form around each colony. Several strains of S. aureus produced a visible coagulase reaction by 8 hr, and all strains gave a positive reaction before 12 hr. Mannitol fermentation was usually observed between 12 and 36 hr. Rabbit serum was filtered through Sephadex G-100 to obtain plasmin- and plasminogen-free coagulase-reacting factor (CRF). False-negative reactions, caused by staphylokinase and staphylococcal Müller factor action on plasminogen, were eliminated when this CRF was used. False-positive reactions by lipolytic, coagulase-negative staphylococci were reduced, since gel filtration removed the serum lipoprotein which served as a primary source of opacity. The addition of 75 mug of polymyxin B per ml selectively retarded the growth of S. epidermidis and minimized false-positive reactions caused by citrate-utilizing gram-negative rods. The preparation, characteristics, and use of the medium are presented.

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Egg white trypsin inhibitor activated coagulase clotting when added to a final concentration between 2 and 60 mg/ml. The greatest increase in clotting rate was observed in reaction mixtures containing the lowest concentrations of serum and plasma. Maximal activation was reached with 40 mg of trypsin inhibitor per ml when either serum or plasma was used as the source of coagulase-reacting factor (CRF). The increased rate of clotting is partly due to inhibition of plasmin. Freezing and thawing reduced plasma clotting inhibition. Soybean trypsin inhibitor also activated the coagulase reaction. The increased rate of clotting was observed with a coagulase preparation from organisms which produced plasminogen activators and with the culture supernatant fraction from organisms which did not activate plasminogen to plasmin. The tube test for coagulase could be made more sensitive for some strains of staphylococci by increasing the concentration of CRF (added as plasma or serum) by adding trypsin inhibitor, or both.

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