Food Microbiology, 1984, 1, 155-158

A simple test to detect staphylococcal thermonuclease using discs of foods

D. M&lain, C. Szymanski and W. H. Lee*

Microbiology Division, Food Safety and Inspection Service, United States Department of Agriculture, 322 ARC, Beltsville, MD 20705, USA

Received 17 November 1983

A method was developed for locating foci of Staphylococcus aureus in suspected foods. Though not rapid, it can detect about 0,015 AzGO units (1 ng) of thermonuclease per food disc (0.2-0.4 g) and is as sensitive as the extraction methods.

Introduction Numerous cases of food poisoning each year are caused by the growth of Staphy- lococcus aureus in improperly prepared or stored foods (Bergdoll 1979, Minor and Marth 1972a, b). The presence of staphy- lococcal thermonuclease enzyme is widely used as an indicator of S. aureus contamination because contaminated foods may show neither organoleptic defects nor high levels of viable bacteria (Todd et al. 1981). Staphylococcus aureus growth in foods may be unevenly distri- buted (Koupal and Deibel 1978, Todd et al. 1981) and the current thermonu- clease test methods (Tatini et al. 1976, Kamman and Tatini 1977, Koupal and Deibel 1978, Park et al. 19791, involving the extraction of 20-30 g of food, make it difficult to pinpoint the foci of S. aureus growth for subsequent enterotoxin tests. A simple thermonuclease test, which uses a small sample, would be a useful adjunct to the more elaborate entero- toxin tests. Methods for the detection of thermonuclease by the direct application of cheese discs (Stadhouders et al. 1980), sausage casings (Emswiler-Rose et al.

Please address requests for reprints to Dr W. H. Lee at the above address.

0740-0020/84/030155 + 04 $02.00/O

1980) and foods in 5 mg amounts to DNA test agar (Lachica et al. 1972) have been published. However, there are many problems arising from this approach, including microbial (Bacillus) growth, dye absorption, and lipid, color and preci- pitation interferences. This paper presents a simple and reliable method that overcomes these problems for the detection of thermonuclease in many kinds of foods.

Materials and Methods Tris-borate (TN) agar was formulated to inhibit microbial growth and also to elimi- nate the problem of ortho-toluidine blue dye absorption by food discs. It has two layers, like an antiobiotic assay plate, and is pur- posely formulated to be calcium-deficient to avoid white precipitation zones. The base layer (10 ml in a 15 x 100 mm Petri dish) of TN agar contains 6 g Tris, 11.5 g sodium borate, 10 g NaCl, 6 mg CaClz and 10 g Difco Noble agar per liter; pH is adjusted to 9. The 4 ml top layer is the base layer media with the addition of 0.45 g DNA and 10 mg ethidium bromide per liter. The dry DNA must be rehydrated separately in 15 ml of cold water for 0.5 h and then boiled for 0.25 h before mixing with the other ingredients. Prepared TN plates are stable for some weeks when refrigerated.

Food discs can be tested in large or small

0 1984 Academic Press Inc. (London) Limited

156 D. M&lain et al.

groups or individually. Discs of solid foods (B-12 x 2 mm) are cut from designated areas with stainless steel cork borers and placed in 20 mm glass screw cap tubes or Petri dishes for heating. Cheese discs are melted onto 12.7 mm antibiotic paper discs in the heating step. Soft and powder foods such as poultry dress- ing and spices can also be tested by applying them to dry or moistened 12.7 mm antibiotic paper discs.

Food discs prepared as above are steamed at 100°C for 15 min to destroy heat-labile nucleases and are then extracted twice, with 2 ml acetone per disc, in a glass tube or flask in a 50°C water bath for 1 h to remove interfering lipids. Five such heated ahd ace- tone extracted food discs are placed on a TN agar plate and rehydrated once or twice with one to two drops of 1 M CaCls. Duplicate sets of plates are incubated with the agar side down in a closed plastic bag at 35 or 45°C for 1 and 2 days. After incubation, the food discs are removed and the TN plates are examined under UV light (302 nm). Thermonuclease zones must extend beyond the diameters of the food discs, and can be clear or very hazy depending on the amount of interfering sub- stances present. Plates that have hazy zones are soaked overnight in tap water in the refrigerator to remove water-soluble interfer- ing substances such as hemes, chlorophylls and tannins. Thermonuclease zones become larger and clearer after soaking.

Food discs known to be positive and nega- tive for thermonuclease should be tested as controls each time. Positive controls can be naturally or artificially contaminated food samples, or food discs with 0.01-0.1 AzCO units (Heins et al. 1966) of thermonuclease added. Staphylococcus aureus thermonu- clease was purchased from Sigma Chemical Co. and assayed by the method of Heins et al. (1966) except that 0.05 M Tris instead of 0.1 M borate buffer was used. Thermonuclease was diluted in thermonuclease-negative sterile evaporated skim milk (Carnation Brand) to give about 0.01-0.1 AzGO units of activity in 25 cl.1 which was then applied to various paper and food discs. Many previous publications express the activity of S. aureus thermonu- clease by weight; 1 pg of commercial S. aureus thermonuclease is said to contain about 8.5-17 A 260 units of activity.

The method was evaluated with various food discs which were naturally contami- nated or cultured with S. aureus, or to which known amounts of thermonuclease were added.

Results and Discussion Results indicated that incubation at 45°C was only slightly superior to 35°C; incubation for 48 h rather than 24 h increased the sensitivity of the assay. Efforts to speed up this test by using 50°C incubation and changing the pH of the TN agar to 10 were not successful (Kam- man and Tatini 1977). The sensitivity of this method was evaluated by adding 0~0075,0~015,0~03,0~06 and 0.12 units of thermonuclease to three kinds of cheese, 10 kinds of meats, and four kinds of vegetable discs before the heating and acetone extraction steps. Repeated test- ing showed that 0.015 units of thermonu- clease could be reliably detected in vari- ous food discs weighing 0.2-0.4 g. However, O-0075 units of thermonu- clease sometimes could not be detected from highly absorbent foods like fried chicken batter and potatoes, or foods like spinach and a dried spicy sausage snack containing large amounts of interfering substances. The current extraction methods for thermonuclease test can detect 10 ng (O-15 units) or more of thermonuclease per gram of foods (Kou- pal and Deibel 1978, Park et al. 1979). Hence this direct method for thermonu- clease in foods is as sensitive as the more cumbersome extraction methods. The reason for this parity in sensitivity is that although 20-30 g of food samples are extracted for thermonuclease test- ing, only a very small aliquot (5-10 ~1 out of l-2 ml of the food extracts) is actually tested. This is equivalent to testing 0.1-0.2 g of foods (Tatini et al. 1976, Kamman and Tatini 1977, Koupal and Diebel 1978, Park et al. 1979).

Tests with inoculated ham and roast beef showed that the method consis- tently detects about lo7 g-l or higher populations of S. aureus, which is the same level of 23. aureus detected in inoculated cheese by another direct method (Stadhouders et al. 1980). It

Direct thermonucleese test in foods 157

Fig. 1. Thermonuclease zones Corn discs of naturally contaminated commercial Genoa salami surface meat after 24 h incubation (left) and Swiss-type cheese after 48 h incubation (right) on TN agar plates (10 cm). Note that the food discs were removed in the washing step and only faint outlines of the discs are visible.

should be noted that there is no exact correlation between thermonuclease and S. aureus population levels, and com- parison of thermonuclease test proce- dures based on 5’. aureus populations is not meaningful.

This method was also evaluated with commercial Swiss cheese (Todd et al. 1981) and Genoa salami (Emswiler-Rose et al. 1980) which were naturally con- taminated with S. aureus and caused widespread staphylococcal enterotoxin food poisonings. Thermonuclease from the above foods was clearly identified as illustrated in Fig. 1 and it was unevenly distributed in the cheese and salami. For these food samples, 24 h incubation was sufficient to detect thermonuclease (Fig. 1). For routine food examinations, it would be prudent to examine one set of food discs after 24 h and another set after 48 h incubation in order to obtain maxi- mum sensitivity. The test is not rapid but it is simple and does not take much time to perform.

The utility of the thermonuclease and enterotoxin tests depends on the circum-

stances, the number of samples, and the availability of enterotoxin assays. No doubt a small number of food samples suspected of causing staphylococcal food poisonings should be analysed for entero- toxins quickly and directly. In the case of huge numbers of suspected or routine food samples, then the probability of finding enterotoxins in thermonuclease positive areas is much greater than in testing for enterotoxins at random. Also, growth of 5‘. aureus in foods can be unevenly distributed (Koupal and Diebel 1978, Todd et al. 1981). The new method uses only 0*2-0.4 g of food to locate thermonuclease positive areas and the same areas can be subsequently tested for enterotoxins. The simplicity and sen- sitivity of this food disc thermonuclease test procedure lends itself to routine quality control operations for the pur- pose of preventing staphylococcal intoxi- cations. Foods with thermonuclease zones like those shown in Fig. 1 indicate heavy S. aureus contamination and a potential health hazard.

158 D. McClain et al.

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