A Simple Medium for Isolation of Coagulase-Positive Staphylococci in a Single Step

218

Journal ofFood Protection, Vol. 45. No.3. Pages 218-222I.February 1982) Copyright International Association of Milk, Food, and Environmental Sanitarians

LEONIE MINTZER-MORGENSTERN and ELIY AHU KATZENELSON"

The A. Felix Public Health Laboratory, Ministry ofHealth. P.O.B. 8255, Tel Aviv, Israel

(Received for publication April1, 1980) Downloaded from http://meridian.allenpress.com/jfp/article-pdf/45/3/218/1650405/0362-028x-45_3_218.pdf by guest on 28 September 2021

ABSTRACT which makes quantitative determination a complicated An agar medium containing NaCl, egg yolk and tellurite for and cumbersome technique. selective quantitative isolation of coagulase-positive staphy This communication describes a selective agar lococci from food was developed. Isolation and identification of medium which enables isolation and identification of the staphylococci was achieved in a single step. A properly coagulase-positive staphylococci in a single step pro diluted food sample was spread over the medium and cedure, thus providing an easy and simple method for the incubated for 24 h at 42 C. Coagulase-positive staphylococci quantitative determination of these bacteria in food. appeared as small grey to dark-grey colonies surrounded by a dense white opacity. Coagulase-negative bacteria which, at MATERIALS AND METHODS times, grow on this medium, did not produce this reaction. The identification on this selective medium of isolates from 683 Single-step-sU!phylococcus-selective agar (4-S agar) different food samples as coagulase-positive staphylococci was Basal agar was prepared by successively dissolving the following subsequently confirmed by the coagulase test. Comparative ingredients in 900 ml of distilled water: Bactotryptone (Difco), 4 g; titrations of 29 various coagulase-positive staphylococcus yeast extract (DifcoJ. 3 g; dextrose, 10 g; NaCl, SO g; Bacto agar (Difco), 13 g. The suspension was brought to a boil and then sterilized strains on both the selective medium and nutrient agar yielded at 121 C for 15 min. Before preparing the 4-S agar, the basal agar was nearly identical titers. The growth of heat-stressed staphylo lignified by boiling and cooled to 55 C. Thirty ml of egg yolk solution cocci was inhibited by the selective medium. Complete reversal (prepared by mixing egg yolk with an equal volume of sterile saline of the inhibition was achieved by a 3-h pre-incubation in brain solution, followed by homogenization for a few seconds in a sterile heart infusion at 37 C. blender) and 3 ml of sterile 1 o/o potassium tellurite solution [prepared by dissolving 1 g of potassium tellurite (BDH Chemicals, Poole. England) in 100 ml of distilled water and subsequent filtration through a Seitz filter] were then added to the liquified basal agar. In a number of experiments, 10 g of sodium pyruvate (Merck) were introduced. The Isolation and enumeration of coagulase-positive suspension was thoroughly mixed, poured onto sterile plastic plates and staphylococci from clinical specimens or food samples left to solidify for several minutes at room temperature. During implicated in food poisoning outbreaks, when these solidification, the turbid medium clarified almost completely. This microorganisms are present in large numbers, is selective agar can be stored at 4 C for several weeks. relatively simple. In routine food samples, on the other Quantitative enumeration of coagulase positive staphylococci in food hand, these bacteria are usually found in small samples quantities, if at all, compared with other microorganisms Portions (0.1 ml) of properly diluted food sample suspensions were spread with a Drigalski spatula on plates containing the 4-S agar and present in food. Selective media which prevent the incubated at 42 C for 20-24 h. Typical colonies were counted and the staphylococci from being overgrown are therefore used. number of coagulase·positive staphylococci in the samples was These media, however, may also be inhibitory to the calculated. In some experiments, the samples were incubated at 35 C staphylococci to such a degree as to make their instead of 42 C. quantitative enumeration almost impossible. Despite Coagulase test these limitations, a number of these media are in use for Suspicious colonies were transferred to a nutrient agar slant and determination of staphylococci in food, a very popular incubated for an additional 24 h. A loopful was then seeded in one being that of Baird-Parker (2). The latter exhibits approximately 0.5 ml of a I :4 dilution of citrated rabbit plasma and incubated at 37 C. Results were read after 2 and 24 h. and were certain drawbacks. such as complexity, lability in storage considered coagulase-positive when clotting of the plasma occurred and interference by Proteus vulgaris. In addition, within 24 h. occasional staphylococcus strains do not give the typical Heat-stressed staphylococci egg yolk reaction produced by coagulase-positive An isolated colony of a Staphylococcus strain was seeded in 10 ml of staphylococci on this medium (J). Moreover, each sterile brain heart infusion (BHI) (Difco) and incubated for 24 h at suspicious colony has to be confirmed separately, 37 C. after which the culture was diluted in phosphate buffer (0.15 M,

JOURNAL OF FOOD PROTECTION. VOL. 45. FEBRUARY 1982 MEDIUM FORSTAPHYLOCOCCI 219

pH 7.0) to 10·4 • To obtain heat-stressed microorganisms, a test tube containing about 10 ml of the diluted culture was placed in a water bath at 52 C for IS min. Before titration, the latter suspension was further diluted.

RESULTS Growth of coagulase-positive staphylococci on 4-S agar After incubation for 24 h at 35 C, colonies of coagulase-positive staphylococci appeared as small (1 - 1.5 mm) grey or dark-grey dots, surrounded by a narrow zone of white opacity (Fig. 1). At incubation of 42 C, however, this zone became much wide and denser and also manifested itself beneath the colony (Fig. 2).

The diameter of the opaque zone varied with the type of Downloaded from http://meridian.allenpress.com/jfp/article-pdf/45/3/218/1650405/0362-028x-45_3_218.pdf by guest on 28 September 2021 Staphylococcus strain and time of incubation; the longer the incubation period, the broader the opaque zone. The colonies that produced these opacities were very distinct Figure 1. Colonies of coagulase positive staphylococci on 4-S and were easily distinguished, even in the presence of medium after incubation at 35 C for 24 h. heavy growth of other bacteria (Fig. 3). Identification of the opacity reaction-producing micro organisms During the course of this investigation, 683 food sample isolates displaying the characteristic opacity were examined microscopically and by the coagulase test. All proved to be coagulase-positive staphylococci. In fact, no other aerobic microorganisms produced this type of reaction on the 4-S agar at 42 C incubation; strains of Proteus vulgaris, for example, did not grow at all on this medium. Quantitative determination of coagulase-positive staphy lococci with 4-S agar To assess the suitability of the 4-S agar for quantitative determination of coagulase-positive staphylococci, comparative titrations of 29 strains of this bacterium isolated from different food and clinical sources were carried out on 4-S and nutrient agar. The plates Figure 2. Colonies of coagulase positive staphylococci on 4·$ containing 4-S agar were incubated at 42 C and those medium after i:zcubation at 42 C for 24 h. with nutrient agar at 35 C. (fhe initial isolation of the 29 strains was carried out on Baird-Parker medium or on blood agar.) The titers obtained with the 4-S agar do not differ significantly from those produced on the nutrient agar (fable 1). Furthermore, on the 4-S agar, all strains yielded typical colonies surrounded by the characteristic opacity zone. Quantitative determination of heat-stressed staphy lococci with 4-S agar and 4-S agar supplemented with pyruvate The adequacy of 4-S agar for quantitative determina tion of heat-stressed staphylococci was evaluated by means of comparative titrations of six Staphylococcus strains treated at 52 C. The titrations were carried out on nutrient agar, 4-S agar and 4-S agar supplemented with pyruvate. The plates containing the 4-S agar and 4-S agar-supplemented with pyruvate were incubated at 42 C, and those containing nutrient agar at 35 C. The number of staphylococcus colonies which appeared on the 4-S agar Figure 3. Bacterial growth on 4-S medium from a food was considerably lower than that which developed on the sample containing four coagulase positive staphylococci.

JOURNAL OF FOOD PROTEC110N, VOL. 45, FEBRUARY 1982 220 MINTZER-MORGENSTERN AND KATZENELSON

TABLE 1. Comparative titration of 29 coagulase-positive was investigated as follows: 1 ml of a suspension staphylococcus strains on nutrient agar and on 4-S agar. containing heat-stressed staphylococci was added to 9 ml of pre-incubation medium (see below}. As control served Titer (x 1 ml of the heat-stressed microorganisms suspension Strain Source Nutrient 4-S agar added to 9 ml of phosphate buffer (0.15 M, pH 7.0). No. (a) (b) bia After thorough stirring, the mixtures were placed in a 1 Meat 1.0 2.7 2.70 water bath at 37 C. Samples were taken at 0, 1, 2, 3, 4 2 Meat 1.6 1.4 0.86 and 5 h of pre-incubation and titrated on 4-S and 3 Meat 0.8 0.4 0.50 nutrient agar. Incubation temperatures were 42 C for the 4 Meat 5.0 3.8 0.76 4-S agar and 35 C for the nutrient agar. The results of a 5 Meat 3.9 3.2 0.82 6 Vegetables 4.1 2.9 0.71 typical experiment with BHI as pre-incubation medium, 7 Vegetables 1.5 3.7 2.47 are presented in Table 3. In the control, the number of 8 Vegetables 1.7 2.3 1.35 bacteria, as measured on nutrient agar, did not change

9 Cake 4.3 5.0 1.16 significantly throughout the pre-incubation period. When Downloaded from http://meridian.allenpress.com/jfp/article-pdf/45/3/218/1650405/0362-028x-45_3_218.pdf by guest on 28 September 2021 10 Cake 4.8 2.9 0.60 measured on 4-S agar, however, the initial number of 11 Cake 3.6 2.9 0.81 bacteria appeared to be much lower (about 6o/o of that 12 Cake 1.0 1.1 1.10 manifested on nutrient agar), and even after a pre 13 Cream 2.8 4.9 1.75 incubation of 5 h, recovery did not exceed 35o/o. When 14 Cream 1.1 2.3 2.09 employing BHI, on the other hand, the number of 15 Ice cream 7.2 4.2 0.58 bacteria, as measured on nutrient agar, remained 16 Ice cream 2.1 1.8 0.86 constant for the first 3 h of pre-incubation, after which it 17 Ice cream 4.6 4.8 1.04 18 Ice cream 1.3 1.3 1.00 rose sharply. Assaying on 4-S agar resulted in an initially 19 Ice cream 4.1 3.1 0.76 low recovery, similar to that of the control. Recovery 20 Nose 3.4 3.7 1.09 increased rapidly thereafter, until, after 3 h of pre-incu 21 Nose 3.6 2.2 0.61 bation, it reached 100o/o. Prolongation of pre-incubation 22 Nose 4.5 3.6 0.80 beyond 3 h resulted, here too, in a sharp increase in the 23 Nose 3.4 2.6 0.76 number of bacteria. 24 Nose 2.4 2.7 1.13 The above results provide evidence that pre-incubation 25 Pharynx 2.7 3.9 1.44 in BHI for 3 h should facilitate complete recovery of 26 Pharynx 4.3 3.4 0.79 stressed staphylococci. And indeed, pre-incubation for 27 Pharynx 4.0 3.5 0.86 3 h in BHI of 22 different strains of heat-stressed 28 Pharynx 3.8 2.8 0.74 staphylococci resulted in their full recovery on 4-S agar 29 Vagina 2.5 2.4 0.96 (Table 4}. Mean 1.07 Replacing BHI with nutrient broth or lactose broth S.D. ±0.54 and altering the pre-incubation temperatures to 4 C or 22 C (instead of 37 C), did not provide satisfactory nutrient agar (Table 2). Addition of pyruvate to the 4-S agar did not improve its performance. recovery. The e.fJect of pre-incubation on recovery of heat-stressed DISCUSSION staph_vlococci An ideal method should afford isolation and In an attempt to increase the recovery of heat-stressed identitication of a given type of bacterium from among a microorganisms on 4-S agar, the effect of pre-incubation collection of microorganisms in a single step, even when

TABLE 2. Comparative titration ofsix heat-stressed staphylococcus strains on nutrient agar. 4-S agar and 4-S agar supplemented with

Titer x 102 Strain Nutrient 4-S agar 4-S agar no. agar + pyruvate (al (b) (c) b/a c/a 1 196 8 6 0.04 0.03 2 81 5 2 0.06 0.02 3 75 3 7 0.06 0.09 4 113 4 5 0.04 0.04 5 300 15 18 0.05 0.06 6 20 0 0 0 0 Mean 0.04 0.04 S.D . ± O.o2 0.03

.JOURNAL OF FOOD PROTECTION. VOL. 45. FEBRUARY 1982 MEDIUM FORSTAPHYLOCOCCI 221

TABLE 3. Titers ofstressed staphylococci obtained on nutrient agar and on 4·S agar after various times of pre· incubation in brain heart infusion (JJH/) and in buffer.

Titer x 102 Pre- Pre-incubation Pre-incubation incubation inBHI in buffer time (h) Nutrient 4-S Nutrient 4-S agar agar agar agar (a) (b) (c) (d) b/a d/c 0 92 2 88 5 0.02 0.06 1 82 20 99 15 0.24 0.15 2 86 39 62 26 0.45 0.42 3 85 87 83 21 1.02 0.25 4 178 156 91 23 0.88 0.25

5 >600 >600 77 29 1.00 0.38 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/45/3/218/1650405/0362-028x-45_3_218.pdf by guest on 28 September 2021

TABLE 4. Titers ofstressed staphylococci obtained on nutrient agar and on 4-S agar before and after pre-incubation in brain heart 1nnus1tm at 37 h.

Titer x 102 Strain Before pre-incubation After pre-incubation no. Nutrient 4-S Nutrient 4-S agar agar agar agar (a) b/a d/c 1 101 7 95 68 0.07 0.72 2 177 32 190 181 0.18 0.95 3 149 5 182 143 0.03 0.79 4 570 22 524 540 0.04 1.03 5 16 <1 19 18 0 0.95 6 7 1 9 11 0.14 1.22 7 510 53 563 537 0.10 0.95 8 45 1< 51 37 0 0.73 9 558 35 529 517 0.06 0.98 10 340 166 307 319 0.49 1.04 11 31 2 34 33 0.06 0.97 12 196 1 155 206 0.01 1.33 13 163 2 134 143 O.ot 1.07 14 286 10 242 282 0.03 1.17 15 101 3 73 77 0.03 1.05 16 34 2 43 39 0.06 0.91 17 367 45 286 302 0.12 1.06 18 9 2 16 16 0.22 1.00 19 545 40 482 562 O.G7 1.17 20 197 19 155 217 0.10 1.40 21 134 7 126 112 0.05 0.89 22 4 <1 6 6 0 1.00 Mean 0.09 1.02 S.D. ±0.10 0.17 it is present in small quantities. the main problem when using the Baird-Parker medium, Isolation of the microorganisms is usually achieved by did not grow on 4-S agar. inhibiting growth of the immaterial microorganisms The coagulase-positive staphylococci were identified while, at the same time, the development of the desired easily by their typical opaque zone. This effect is similar bacterium is selectively supported. Identification of to the one seen with the "egg yolk" reaction, first microorganisms is attained by incorporating into the observed by Gillespie and Adler (6), and which was later system reagents that give rise to a biochemical reaction shown to be an amalgamate of a number of reactions typical of the specific type of bacterium. associated with both lecitinase and lipolytic enzyme In our method for isolation and identification of activity (8, 9). coagulase-postivie staphylococci, selectivity was achieved Owens and John (9) demonstrated that lipolytic by combining a high concentration of sodium chloride activity is suppressed by C02• Our results suggest that (5%) with tellurite and incubating at 42 C. P. vulgaris, incubation at 42 C also inhibits lipolytic activity. This

JOURNAL OF FOOD PROTECTION. VOL.4S,FEBRUARY 1982 222 MINTZER-MORGENSTERN AND KATZENELSON assumption is supported by the enlarged zones of opacity coagulase-positive staphylococci, it is recommended to appearing on the 4-S agar when incubated at 42 C. It suspend the examined food in BHI, incubate the seems that at 35 C both enzymes are engendered by suspension for 3 h at 37 C and, finally, assay it on 4-S coagulase-positive staphylococci, and since their activ agar. When the presence of stressed staphylococci is not ities are antagonistic (one enzyme producing opacity and suspected, pre-incubation can, of course, be omitted. the other clarification of the egg yolk material), the result is a small or even complete absence of the opaque zone. At 42 C, on the other hand, the antagonistic activity to lecitinase is obliterated and, hence, the widened zone of REFERENCES opacity. l. All wood, M. C., and A. D. Russell. 1970. Mechanisms of thermal The combination of 4-S agar and incubation at 42 C injury in nonsporulating bacteria. Adv. Appl. Microbiol. 12:89. showed to be a quantitative and selective method. All 2. Baird-Parker, A. C. 1962. An improved diagnostic and selective medium for isolating coagulase-positive staphylococci. J. Appl. isolates from 683 food samples producing the opacity Bacteriol. 25:12. reaction with our method were subsequently identified as 3. Baird-Parker, A. C.1962. The performance of an egg yolk-tellurite Downloaded from http://meridian.allenpress.com/jfp/article-pdf/45/3/218/1650405/0362-028x-45_3_218.pdf by guest on 28 September 2021 coagulase-positive staphylococci. Other bacteria able to medium in practical use. J. Appl. Bacteriol. 25:441. produce the egg yolk reaction on various media (8), either 4. Brewer, D. G., S. E. Martin, and Z. J. Ordal. 1977. Beneficial did not grow on the 4-S agar or did not display the typical effect of catalase or pyruvate in a most-probable-number technique opacity. for the detection of Staphylococcus au reus. Appl. Environ. Microbiol. 34:797. As the 4-S agar contains a high sodium chloride 5. Busta, F. F.1976. Practical implications of injured microorganisms concentration (5%), this would be expected to inhibit in food. J. Milk Food Techno!. 39:138. growth of damaged staphylococci (7) occurring in 6. Gillespie, W. A .. and V. G. Adler. 1952. Production of opacity heat-processed or dried foods (1,5,10). Heat-stressed in egg-yolk media by coagulase-positive staphylococci. J. Pathol. Bacteriol. 64:187. staphylococci assayed on 4-S agar indeed yielded a poor 7. Iandolo, J. J., and Z. J. Ordal. 1966. Repair of thermal injury of recovery (4%- 7%). Addition of pyruvate, which is Staphylococcus aureus. J. Bacteriol. 91:134. supposed to enhance the recovery of injured staphy 8. Owens, J. J. 1974. The egg-yolk reaction produced by several lococci (4), proved unsatisfactory. Pre-incubation of 3 h species of bacteria. J. Appl. Bacteriol. 37:137. at 37 C in brain heart infusion, on the other hand, was 9. Owens, J. J.. and P. C. L. John. 1975. The egg yolk and lipolytic reactions of coagulase positive staphylococci. J. Appl. Bacteriol. beneficial. Pre-incubation in different media, or at 39:23. temperatures other than 37 C, resulted in low recoveries. 10. Ray. B., and M. Speck. 1973. Freeze-injury in bacteria. CRC For routine quantitative isolation and identification of Crit. Rev. Clin. Sci. 4:161.

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JOURNAL OF FOOD PROTECTION, VOL. 45, FEBRUARY 1982