Lactose Pre-enrichment Method for Isolation of Salmonella from Dried Egg Albumen

Transcription

1 Lactose Pre-enrichment Method for Isolation of Salmonella from Dried Egg Albumen Its Use in a Survey of Commercially Produced Albumen WILLIAM R. NORTH, JR. U. S. Food and Drug Administration, Department of Health, Education, and W1'elfare, Washington, D. C. Received for publication July 20, 1960 A review of the literature has revealed a scarcity of attempts to compare directly various methods proposed for the detection of Salmonella organisms in food products. Unfortunately, often it has been assumed that methods useful in clinical or public health laboratories, engaged in identifying cases and carriers, are also suitable for the examination of food products. Furthermore, in many instances new methods have not been adequately proved in effectiveness nor have they been subjected to comparisons with noninhibitory media to determine how well they will support the growth of Salmonella. Productivity, as well as selectivity, is very important where interest lies in quantitative determination or in the detection of small numbers of Salmonella in a food product. The medium selected should not only be noninhibitory for Salmonella, either alone or when combined with the product under examination, but should also be capable of supporting growth of small numbers of organisms especially in higher dilutions where any possible nutritional or inhibitory substances from the food under test would have been diluted out. In a previous paper, North and Bartram (1953) demonstrated the favorable effect of L-cystine on the productivity of selenite F broth. These findings were confirmed by Byrne, Rayman, and Schneider (1955) and more recently by Taylor, Silliker, and Andrews (1958). Among other things, Taylor et al. compared selenite cystine (SC) broth with broths prepared with dulcitol or mannitol instead of lactose. They found no statistical advantage by these substitutions and thereby confirmed Leifson's (1936) claim that there was no improvement when a carbohvdrate other than lactose was used. Also, they found that the selenite brilliant green sulfapyridine enrichment broth of Osborne and Stokes (1955) and Stokes and Osborne (1955) did not produce more isolations of Salmonella and in some instances was markedly inferior to the SC broth. Comparative studies by the author (unpublished) on various enrichment broths showed that these modifications which contain brilliant green, with or without sodium sulfapyridine, were inferior to SC broth in productivity. 188 In another paper, Silliker and Taylor (1958) called attention to "skips" occurring in the most probable numbers (MPN) determinations which involved the testing of 10-, 1. 0-, and 0.1-g portions of dried egg white, using SC and tetrathionate (TT) as enrichment media. They found that, by centrifuging, a soluble inhibitory substance could be decanted without appreciable loss of microorganisms. The sediment cultured in either SC or TT enrichment broth permitted detection of small numbers of Salmonella frequently missed when large amounts of egg white were cultured directly in the enrichment broth. The author has confirmed the superiority of this method over direct enrichment in either SC or TT broth for the examination of egg whites. We have encountered difficulty in the detection of small numbers of Salmonella in other foods where relatively large portions are cultured directly in selective enrichment media. In testing a variety of dried food products, we have found that reconstitution in water or broth and incubation for several hours prior to inoculation of selective enrichment media significantly enhances the recovery of Salmonella, presumably attenuated by prolonged storage in the dry state. The purpose of this paper is to describe (a) a procedure employing a nonselective medium, lactose broth, for pre-enrichment of dried foods prior to inoculation of selective media, which eliminates "skips" in quantitative estimations of Salmonella, and _(b) the results of this procedure in the examination of commercially produced dried egg white. MATERIALS AND METHODS Lactose Broth Pre-enrichment Method 1. Weigh into sterile wide-mouth screw-capped jars of at least 8-oz capacity, one 14-g and two 10-g portions of dried egg. To the one 14-g portion add 126 ml of sterile lactose broth (APHA, 1955). Since it is important in preparing serial dilutions that the egg be perfectly dissolved or suspended without lumps, add only a small amount of the diluent (not more than 30 ml) and stir with a sterile glass rod so that the egg is thoroughly wetted. Now add with constant stirring the remainder

2 196,11 ISOLATION OF SALMONELLA FROM DRIED EGG ALBUMEN 189 of the diluent. Let stand for about 12 hr. After shaking, the egg should be free from lumps. Dissolve or suspend the two 10-g portions by adding to each jar 90 ml of sterile lactose broth in the same manner as described above. Some lots of egg white, particularly those produced by the fermentation process, may be so acid as to lower the ph of the lactose broth below that optimum for growth of Salmonella. If such is the case, the emulsion should be neutralized to a point above ph 6.0, preferably to ph 7.0, by the addition of N NaOH. 2. From the 14-g sample of diluted egg, remove three 10-ml, three 1.0-ml, and three 0.1-ml portions, transferring each portion into a tube containing 10 ml of sterile lactose broth (with inner fermentation tubes for simultaneous coliform determination). This provides: 3 jars containing 10 g of egg (the remainder of the 14-g sample is slightly in excess of 10 g of egg). 3 tubes containing 10 ml = 1 g of egg each. 3 tubes containing 1 ml = 0.1 g of egg each. 3 tubes containing 0.1 ml = 0.01 g of egg each. Further decimal dilutions may be prepared in the usual manner to detect higher levels of contamination. 3. Incubate the inoculated tubes and jars for 24 hr at 35 C. 4. Using a 5-mm loop make transfers from the jars and from those lactose tubes, which show growth, to 1.0 ml of SC broth in small tubes (14 by 100 mm). Tilt the lactose tubes and jars to assure sampling the culture and not egg froth. Incubate 6 to 7 hr at 35 C. 5. Streak each tube of SC broth on Salmonella- Shigella (SS), brilliant green, and bismuth sulfite agar. The SC broth tubes should be reincubated for the full 24-hr period and examined for growth which may not have been apparent during the shorter period. Restreaking at this time may in some cases increase the recovery of Salmonella. The surface of an entire plate may be used but with the multiple tube "set up" it is possible to conserve on the number of plates by marking off the bottom of each plate into three segments with a wax pencil to provide streaking area for the three tubes representing the same dilution. Modification of the streaking technique is necessary to avoid overcrowding and provide good isolations. The material on the loop may, in large measure, be knocked off on the side of the tube above the culture. Puncture the periphery of the agar close to the edge of the plate several times (3 or more) before streaking. It is advisable, in the case of brilliant green medium, to use an entire plate for streaking each tube of a dilution because of the lack of selectivity of this medium for Salmonella over lactose fermenters and some other types. An additional precaution in streaking is to avoid creating aerosols from vibration of the loop which may encroach upon an adjoining segment. As a screening procedure on samples of unknown Salmonella level, the three 10-g, three 1.0-g, three 0.1-g, and three 0.01-g portions are usually sufficient. However, should all these dilutions be positive and information as to total Salmonella content be needed, a repeat examination extending the dilutions will be necessary. 6. Fish suspected colonies and confirm as to genus or species by the usual serological and/or biochemical tests. 7. Calculate the MPN per 100 g from Hoskins Tables' (Hoskins, 1934). The selective media used for comparison of direct enrichment procedures were: (a) Kauffmann's modification of TT broth (TTK)2 (Barnes, 1944) prepared as follows: To 1000 ml of reconstituted tetrathionate dehydrated base medium (BBL or Difco) add 20 g sodium thiosulfate, 40 g CaCO3 (precipitated chalk) and 1.0 ml of 1.0 per cent brilliant green, (1: 100,000). Autoclave at 15 lb (121 C) for 15 min. At time of use cool below 50 C, and add 2.0 ml per 100 of the iodine solution (6.0 g iodine, 5.0 g potassium iodide, and 20 ml distilled water). (b) Modified TT broth (TTH) (Hajna and Damon, 1956). Note: This medium is not autoclaved but heated 30 min in flowing steam. (c) SC broth3 (North and Bartram, 1953) as follows: Polypeptone (BBL),4 5.0 g, or Tryptone (Difco),5 4.0 g; lactose, 4.0 g; sodium acid selenite, 4.0 g; dibasic sodium phosphate (anhydrous), 5.5 g; monobasic potassium phosphate (anhydrous), 4.5 g; water, 1000 ml; L-cystine (1.0 per cent'), 1.0 ml. Steam in Arnold sterilizer 15 to 20 min (do not autoclave). The medium needs no adjustment since the reaction will approximate ph 7.1. The streaking media consisted routinely of SS agar (BBL) and bismuth sulfite agar (BBL). To shorten the task of streaking the many dilutions used in these experiments, brilliant green agar was omitted. ' When three 10-g samples are tested and all are negative the result is recorded as <3.6 per 100 g. When only one 10-g portion is tested and found negative the result is recorded as <10 per 100 g. 2 Complete formulae is given here, since the reference cited does not refer to F. Kauffmann and it is not included in his text, Enterobacteriaceae. 3This simplified formula was not specifically included in the reference cited. 4Baltimore Biological Laboratory, Inc., Baltimore, Maryland. 5 Difco Laboratories, Inc., Detroit, Michigan. 6 L-Cystine was prepared by dissolving 1.0 g in 10 to 20 ml of N NaOH and making up to 100 ml with distilled water. One milliliter per L equals 10 mg per L or 10 ug per ml.

3 190 W. R. NORTH, JR. [VOL. 9 RESULTS Experiment 1. To compare the above enrichment media with the lactose pre-enrichment method a lot of naturally infected dried egg white was chosen because of its fairly low incidence of Salmonella organisms. To increase the precision of the results, 15 tubes each of lactose, SC, and of the two TT broths (TTK and TTH) were prepared for inoculation with 1.0-g and with 0.1-g portions of the dried egg white. Measured volumes of media were used so that the concentration of the egg in all the media would be equal, or 5 per cent and 0.5 per cent, respectively. In addition, one jar containing 10 g of egg white in approximately 100 ml of each of the respective media was prepared and run simultaneously. Table 1 presents the results in MPN values and lists the Salmonella species isolated by the various procedures. The marked increase in the MPN recovery by the lactose pre-enrichment method over direct inoculation in any of the enrichment broths will be noted. The TABLE I Comparison of lactose broth pre-enrichment with direct inoculation of selenite cystine (SC) and tetrathionate (TTK and TTH) broths for recovery of salmonellae from naturally infected dried egg white Tubes Positive for Salmonellae Medium and/or MPN Method per Salmonella Species log, I g, O.l g, loog 1 por- 15 rep 15 reption licates licates hr SC S. montetideo SC S. montevideo and S. anatum TTK S. montevideo and S. reading TTK S. montevideo and S. reading TTH S. montevideo and S. reading TTH S. montevideo and S. reading Lactose 24 SC S. montevideo, S. reading, Lactose 24 SC S. montevideo, S. reading, Lactose 48 SC S. montevideo, S. reading, Lactose 48 SC S. montevtideo, S. reading, Lactose 24 TTH S. montevideo, S. reading, Lactose S. montevideo, S. read- TTH 24 ing, Coliform~MPN = 2400 per 100 g. results in the two TT broths are not conclusive for arriving at an evaluation of their relative merits. To demonstrate statistically the superiority of one tetrathionate modification over the other, a much larger series of experiments would be necessary. Both of these modifications, however, showed higher MPN readings when the cultures were streaked after 48 hr of incubation. This was also true, although not so pronounced, in the SC broth. Productivity tests, by means of comparative MPN determinations, using broth cultures of Salmonella, repeatedly have demonstrated greater recovery in SC broth than in either modification of the TT medium after 24 hr of incubation. It is interesting to observe that the coliform MPN per 100 g (not included in the table) in these eggs was 2400 and the Salmonella MPN per 100 g was 1700 by the lactose pre-enrichment method, whereas the highest Salmonella MPN per 100 g by any of the direct enrichment procedures was 200. After 24 hr in lactose broth followed by loop transfers to SC broth and 7 hr of incubation at 35 C, the Salmonella MPN per 100 g was 490, and was increased to 1300 per 100 g after 24 hr of incubation. A maximal value of 1700 per 100 g occurred after 48 hr in lactose broth and 24 hr in SC or TTH broth. By the lactose pre-enrichment procedure, recovery of the different Salmonella species found in the TABLE 2 Lactose broth pre-enrichment compared with direct inoculation of selenite cystine and tetrathionate broth for the examination of naturally infected egg white powder Comparative MPN per 100 g Sample No. Tetrathionate Selenite cystine Lactose pre-enrich- (TTH),* 48 hr (SC), 24 hr SC, 24 hr X X X X X X 106 * Hajna and Damon (1956) modification. TABLE 3 Comparison of centrifugation method and lactose broth pre-enrichment method Egg White Method 10 g, 1.0 g, 10-1 g, 102 g, 10-3 g, 1 por- 3 repli- 3 repli- 3 repli- 3 repli- MPN per tion, cates, cates, cates, cates, 100 g no. pos- no. pos- no. pos- no. pos- no. positive itive itive itiv e itive Centrifugation ,000* Lactose broth, 24 hr; SC, 7 hr ,000* Lactose broth, 24 hr; SC, 24 hr ,000* * Predominating species isolated was Salmonella montevideo. No attempt was made to determine the number of species present.

4 1961] ISOLATION OF SALMONELLA FROM DRIED EGG ALBUMEN 1.91 product was facilitated. It is apparent that inhibitory enrichment media can be selective of species and tend to reproduce those which are less susceptible to the media. This has been observed in a high count sample where in SC broth, at high concentration of the egg white, the Salmonella colonies on the streak plates were confined to one species, S. montevideo. With lactose broth pre-enrichment no difficulty was encountered in isolating four additional species. Although tetrathionate will give as good recovery following lactose pre-enrichment, it has the disadvantage of requiring longer incubation. Experiment 2. In table 2, the Salmonella content of two highly contaminated but naturally infected samples of dried egg white, as determined by direct enrichment in TTH and SC broths and compared with the lactose pre-enrichment procedures, is shown. By lactose broth pre-enrichment Salmonella recoveries as MPN per 100 g were 46 X 106 and 92 X 106; by TTH 2.4 X 105 and 17 X 106; by SC 2.4 X 106 and 17 X 106. On a percentage basis using recovery by the lactose pre-enrichment TABLE 4 Comparison of centrifugation with selenite cystine (SC) direct enrichment method in the examination of whole eggs with added albumen and artificial flavor Method of Direct Enrichment in SC Broth Centrifugation Method Sam- MPN MPN pie log, 1.0 g, 0.1 g, per log, g '5oi~g, per No. 1 por- 5 repli- 5 repli- INOOg po.tios replicates, 5 repli- 100 g tion, cates, cates, n.portio n, r Pliats- cates, no. pos- no. pos- n.j0 no. pos- ii tv no. p os itive itive itive itive <10 1 M 1 M <10 1 M 2M M* 1 A* M 3 AM R* M R 5AM 2 M <10 1 M 3 M R 1 R MR 3 M R 1 M 110 * A = Salmonella anatum; M = Salmonella montevideo; R = Salmonella reading. TABLE 5 Comparison of lactose broth pre-enrichment with the centrifugation method in the examination of dried whole eggs with added albumen and artificial flavor Method of Lactose Pre-enrich- Centrifugation Method 6 ment 24 hr; SC 24 hrcetigaonmhd - - ~~~~ ~~MPN -MPN 0.1 g, per log,1 g1,-o.i g, per E 10 g, 1 por- 1.0 g, 5 rep- 5 repli- MPN portion, crepli-s repli- 10O g rn tion, no. licates, no. cates, no. cates, cates, positive positive no. pos- pos- no. pos- no. positive it'v itive itive 1 1 M* 5 M 1 M M 4M 3M M 3 M R* 1 M M A* M 5 M 1 R A R 2M 2M A M 1 M 4 M 40 1 M R * A = Salmonella anatum; M = Salmonella montevideo; R = Salmonella reading. procedures as standard, the TTH broth recoveries were 0.52 and 18.3 per cent and by SC broth 5.2 and 18.3 per cent, respectively. Experiment 3. In table 3 the recovery of Salmonella from egg white using the lactose broth pre-enrichment and the centrifugation method is compared. The egg white employed was a mixture of samples of Salmonellafree and naturally infected egg white powder. It will be seen that in this experiment the lactose pre-enrichment method gave a 10-fold greater recovery than the centrifugation method. The 7-hr and 24-hr streak plates, after loop inoculation into SC broth from the 24-hr lactose tubes, yielded in both instances a Salmonella MPN per 100 g recovery of 110,000 as compared with 11,000 by centrifugation. After lactose pre-enrichment for 24 hr and loop transfer to SC broth and streaking after 7 hr of incubation, the numbers of Salmonella per streak plate ranged from few to moderate with little or no interference from coliform or other types. After 24 hr in SC broth the numbers of Salmonella per plate increased without change in the ratio of Salmonella to coliform. Experiment 4. Results of a comparison of the centrifugation method with the direct inoculation in SC broth in the examination of naturally infected mixture of whole dried egg containing added dried egg white are given in table 4. The six samples show by direct enrichment in SC broth, 3 negative (< 10 per 100 g) and 3 positive with MPN counts of 10 to 20 per 100 g. By the centrifugation method all 6 were positive with counts varying from 20 to 470 per 100 g. The species identified were S. montevideo, S. anatum, and S. reading. Attention is called to the effect of the inhibitory factor which may have been incorporated in the whole egg (usually not so inhibitory) by the addition of the extra egg white and possibly by the substance added to impart artificial flavor to the eggs. Experiment 5. Other samples of the same eggs were examined by the lactose broth pre-enrichment and by the centrifugation methods. The results obtained are shown in table 5. It will be observed that in all instances greater recoveries were obtained by the former method which yielded 330, 110, 1300, and 40 Salmonella, whereas recoveries by the centrifugation method were 270, 10, 93, and 10, per 100 g. Examination of Commercial Dried Egg White A survey of dried egg white produced in 11 egg drying plants7 revealed that of 79 samples examined, 30 contained Salmonella (table 6). Six of these samples showed 3.6 per 100 g, an additional 9 were under 50 per 100 g, and in 8 the number of Salmonella ranged from 150 to 930 per 100 g. One sample from plant B2 contained 4600 per 100 g and 3 additional samples 7Plants designated A to I. Letter with subscript represents same company at different locations.

5 192 W. R. NORTH, JR. [VOL. 9 from the same plant showed very high counts of Salmonella, which ranged from 1.1 X 105 to 1.1 X 109 per 100 g with multiple species isolated. Two samples from plant H ranged in MPN per 100 g from 1.0 X 107 TABLE 6 Salmonella examination by the lactose pre-enrichment method of commercial dried egg white Plant Lots Nega- PNso: MPN tive tive Salmon- Salmonella Species Identified A 7 7* 4t 3* 1* 3.6 S. montevideo B, S. tennessee B S. infantis S. infantis and S. montevideo 1 43 S. bareilly and S. infantis 2t S. tennessee 1 110,000 S. oranienburg, S. bareilly, and S. montevideo 2t X 108 S. bareilly, S. tennessee, and S. norwich X 109 S. oranienburg and S. montevideo B3 6 6 C 6t S. oranienburg S. typhimurium D 6t S. montevideo S. montevideo 1 10 S. montevideo E 6t 6 F S. oranienburg and S. montevideo S. infantis S. oranienburg and S. bareilly S. oranienburg S. montevideo G 6 6 H 2t X 107 S. montevideo, S. muenchen, and S. denver X 107 S. bareilly and S. bredeney 2 2 I S. oranienburg 1 46 S. oranienburg S. oranienburg 2 24 S. oranienburg S. oranienburg * Held at 150 F for 1 to 9 days. t Pan-dried egg white. to 5.4 X 107, whereas two from the same plant were negative (< 3.6 per 100 g). All 6 of the samples with high Salmonella content were pan-dried products. In an attempt to determine the possible source of the high levels and incidence of Salmonella found in some of the commercial samples of dried egg, line samples were obtained from the same 11 plants. The samples consisted of the liquid egg, and as far as possiple, samples from the intermediate stages to the final dried product. The dried egg white was examined by the lactose preenrichment method and the liquid eggs by direct inoculation into SC broth.8 In some cases only one 10-g and lesser portions of the dried eggs or one 10-ml and lesser amounts of the liquid eggs were examined and, when negative, the MPN per 100 g was expressed as < 10. When three 10-g portions were included, the negative reading was expressed as < 3.6. The results recorded in table 7 show that the samples from plants A, B1, B3, C, D, and G were negative for Salmonella (MPN per 100 g of < 3.6 or < 10.0). The pan-dried albumen produced in plant A showed 430 organisms per 100 g of S. montevideo, although the liquid fermented egg white was negative. The untreated liquid egg white from plant B2 was also negative (<3.6 per 100 g) but the inoculum used to start the fermentation process contained 240 S. montevideo per 100 ml, and the fermented liquid eggs contained 1500 per 100 ml Salmonella. Species isolated were S. montevideo, S. infantis, and S. bareilly; however, in the finished, dried whites, only S. montevideo (9.1 per 100 g) was found. In liquid untreated egg white at plant E, an MPN of 150 per 100 g of S. pullocum was found in one sample and < 3.6 per 100 g in a duplicate sample. Two samples of dried egg were negative or < 3.6 per 100 g. At plant F the untreated liquid eggs contained 23 per 100 g S. infantis and in the dried product the count was 240 per 100 g. From plant H the untreated liquid eggs showed 2400 per 100 ml of S. montevideo and S. infantis and 2.4 X 105 per 100 ml in the fermented eggs. The pan-dried whites showed an MPN per 100 g of 4.6 X 106 with S. montevideo predominating. The liquid fermented eggs contained four Salmonella species, S. oranienburg, S. montevideo, S. infantis, and S. tennessee. Two prior production spray-dried, enzyme-fermented samples from this plant were negative or <3.6 per 100 g. At plant I, the Salmonella content in the liquid untreated eggs was negative (< 3.6 per 100 ml). The eggs fermented with enzyme showed 2400 per 100 ml S. oranienburg, the same species being found in the dried product (46 per 100 g). It should be noted (table 6 and 7) that some of the samples of dried egg white were taken from lots which 8 Lactose pre-enrichment did not sufficiently increase recovery to justify extra time involved in the test.

6 1961] ISOLATION OF SALMONELLA FROM DRIED EGG ALBUMEN 193 had been held at 150 F for periods of 9 days, or for longer periods at 120 F. The results of the examination of these samples tend to demonstrate the efficiency of ;such procedures since all but one were negative for Salmonella. The exception showed only 3.6 per 100 g S. montevideo in a sample of pan-dried albumen. DISCUSSION The consistently greater recovery of Salmonella, as shown by data presented, demonstrated the superiority of the lactose broth pre-enrichment method over the other procedures tested. This is particularly apparent in the examination of egg albumen, where, in the lower dilutions, the high concentration of the product in the selective medium appears to be inhibitory for Salmonella. This inhibition was not observed when lactose pre-enrichment is employed. The inhibitory effect of the substance has also been noted by Silliker and Taylor (1958) who employed centrifugation to allow the interfering solutions to be decanted off. Our results confirm the superiority of this procedure over direct inoculation into selective media. However, when quantitative TABLE 7 Salmonella content of in-plant sample taken during production of dried egg albumen Plant Description of Material No. MPN Salmonella per Salmonella Species Identified A Untreated and fermented 2 <3.6 Spray-dried 1 <3.6 Untreated and fermented S. montevideo and S. infantis Pan-dried S. montevideo B1 Untreated and fermented 3 <3.6 Dried 2 <3.6 B2 Untreated 2 <3.6 Inoculum* S. montevideo Fermented eggs S. montevideo, S. infantis, and S. bareilly Dried S. montevideo B3 Untreated 2 < 10 Fermented 1 < 10 Fermented 1 10 S. montevideo Dried 1 < 10 C Untreated, fermented, and dried 3 <10 D Untreated and enzyme fermentation 3 <10 Pan-dried 1 < 10 E Untreated S. pullorum Untreated 1 <3.6 Dried 2 <3.6 F Untreated 1 23 S. infantis Fermented 1 <3.6 Ditto after cooling 1 21 S. tennessee Dried S. infantis, S. oranienburg, and S. montevideo Driedt 1 <3.6 G Untreated and enzyme fermentation 2 <10 Dried 1 < 10 H Untreated S. montevideo and S. infantis Fermented 1 240,000 4 species tte Fermented, acidified 1 16,000 3 speciesf Pan-dried X 106 S. montevideo (predominate) I Untreated 1 <3.6 Fermented (enzyme) S. oranienburg Dried 1 46 S. oranienburg * Liquid egg whites used as a starter to initiate fermentation. t Held for 20 days at 120 F.

7 194 W. R. NORTH, JR. [VOL. 9 procedures, using higher dilutions, are to be used, the precipitate must be completely re-emulsified, a process which is frequently difficult to accomplish. There are several other factors which may contribute to this enhanced recovery. The processes employed in drying egg products as well as prolonged freezing serve to attenuate or reduce the viability of most microorganisms and a selective medium does not offer as favorable conditions for growth of these organisms as exist in a noninhibitory medium such as lactose broth. By preenrichment, a larger number of organisms, in a state of active growth, will be transferred to the selective medium, with a correspondingly better chance for multiplication therein. Results employing a selective and nonselective medium (plain broth), have demonstrated that with a 24-hr pure culture of Salmonella, the former will give lower recoveries, indicating an inhibitory effect. With pure cultures the use of a plain broth might prove as satisfactory as one containing lactose; however, in the presence of a mixed flora, the fermentation of the carbohydrate (lactose) results in a lower ph which serves to hold in check other types of microorganisms. This ph change does not appear to be sufficient to affect the growth of or be lethal for Salmonella since recoveries from lactose broth could be made over a period of at least a week without loss. In addition to the greater numerical recovery of Salmonella, the pre-enrichment method results in many instances in the isolation of additional species, particularly in the lower dilutions which contain a higher concentration of the egg albumen. Since the additional species were demonstrated by direct inoculation from the higher dilutions, it would appear possible that the inhibitory effect of the egg white in the selective media, SC and TT, may vary considerably for different species. These observations are illustrated in the results presented in table 1. On direct inoculation, S. montevideo was the predominating species isolated with recovery of S. anatum or S. reading in the highest dilution. However, by the lactose pre-enrichment all three species were isolated from all dilutions. Surveys conducted on dried albumen, produced by a major portion of firms preparing this item, have revealed that the majority of the samples were free of Salmonella. However, the output of a few manufacturers were contaminated to a high degree. Attempts to demonstrate the factors responsible for the high incidence of Salmonella in these samples have not been completely successful. Definite increases were apparent in some instances in the liquid egg during fermentation. The two plants whose products contained the highest numbers of Salmonella and the greatest number of types routinely utilized the fermenting albumen as an inoculum for subsequent batches. Although these plants also employed pan drying, it would appear that the high degree of contamination resulted in part from a build-up from batch to batch. However, additional sampling would be necessary to demonstrate the significance of these observations. In one instance a reduction in Salmonella content was demonstrated in dried product held for 20 days at 120 F. This is in accord with the findings of Banwart and Ayres (1956) who demonstrated marked reduction of Salmonella in egg albumen stored at this temperature, without loss of functional properties of the product. The marked increase in numbers of proved cases of salmonellosis reported by the National Office of Vital Statistics (1959) and the occasional association of egg products with salmonellosis outbreaks, warrants adoption of methods of analysis capable of detecting the presence of Salmonella in small numbers. This is especially true in the case of egg albumen which is frequently employed in food products without adequate cooking to destroy these organisms or which is not held at sufficiently low temperatures to prevent their multiplication. ACKNOWLEDGMENT The author wishes to thank Dr. P. R. Edwards, Communicable Disease Center, Enteric Bacteriology Unit, U. S. Public Health Service, Atlanta, Georgia, for identifying some of the Salmonellae species isolated during the course of these studies. SUMMARY A lactose pre-enrichment method for detecting Salmonella when present in small numbers in dried egg products has been described. Higher most probable number counts have been demonstrated by this method than by direct enrichment methods. The natural inhibitory effect exerted on Salmonella by high concentration of egg white is not operative in lactose broth, and therefore fewer "skips" are encountered by this method than by direct enrichment methods. The results obtained during a survey of commercial production of dried egg white occasionally show very high counts of Salmonella, with some indication that increases may occur during the desugaring process. REFERENCES American Public Health Association 1955 Standard methods for the examination of water, sewage and industrial wastes, 10th ed. New York, New York. BANWART, G. J. AND AYRES, J. C The effect of high temperature storage on the content of Salmonella and on the functional properties of dried egg white. Food Technol., 10, BARNES, L. A U. S. Naval Med. Bull., 43, 713. BYRNE, A. F., RAYMAN, M. M., AND SCHNEIDER, M. D Methods for the detection and estimation of numbers of Salmonella in dried egg and other food products. Appl. Microbiol., 3, HAJNA, A. A. AND DAMON, S. R New enrichment and plating media for the isolation of Salmonella and Shigella organisms. Appl. Microbiol., 4,

8 1961] COAGULASE POSITIVE STAPHYLOCOCCI FROM RAW MILK 195 HOSKINS, J. K Most probable numbers for evaluation of coli-aerogenes test by fermentation tube method. Public Health Repts., 49, LEIFSON, E New selenite enrichment media for the isolation of typhoid and paratyphoid (Salmonella) bacilli. Am. J. Hyg., 24, National Office of Vital Statistics, Morbidity and Mentality Report, 1959, 7 (54), 4. NORTH, W. R. AND BARTRAM, M. T The efficiency of selenite broth of different compositions in the isolation of Salmonella. Appl. Microbiol., 1, OSBORNE, W. W. AND STOKES, J. L A modified selenite brilliant green medium for the isolation of Salmonella from egg products. Appl. Microbiol., 3, SILLIKER, J. H. AND TAYLOR, W. I Centrifugation method for removal of inhibitory substance in soluble material. Appl. Microbiol., 6, STOKES, J. L. AND OSBORNE, W. W A selenite brilliant green medium for the isolation of Salmonella. Appl. Microbiol., 3, TAYLOR, W. I., SILLIKER, J. H., AND ANDREWS, H. P Isolation of Salmonella from food samples. I. Factors affecting the choice of media for the detection and enumeration of Salmonella. Appl. Microbiol., 6, Characterization of Coagulase-positive Staphylococci Isolated from Raw Milk1"2 WARREN S. CLARK, JR.3, T. D. MOORE, AND F. E. NELSON4 Department of Dairy and Food Industry, Iowa Agricultural and Home Economics Experiment Station, Ames, Iowa Raw milk commonly contains staphylococci, and various dairy products have been implicated in staphylococcus food poisoning outbreaks. Increasing interest has been shown in the characterization of staphylococci during recent years. Characteristics such as coagulase and hemolysin production, formation of enterotoxin, mannitol fermentation, gelatinolytic and lipolytic action, bacteriophage type, antibiotic resistance, and pigmentation have been of particular interest. Staphylococci isolated from raw milk were characterized using these various criteria in an attempt to evaluate the significance of raw milk as a reservoir of staphylococci of potential importance from a public health standpoint. Evans and Niven (1950) reported that most enterotoxigenic staphylococci were members of the coagulasepositive group. However, they did not preclude the possibility that some varieties of coagulase-negative staphylococci also might be associated with food poisoning. Evans, Buettner, and Niven (1950) reported that 24 cultures of coagulase-negative staphylococci were fed to 59 monkeys without a single symptom of illness appearing, whereas 14 cultures of coagulasepositive staphylococci produced typical food poisoning 1 Journal Paper No. J-3928 of the Iowa Agricultural and Home Economics Experiment Station, Ames, Iowa. Project No This investigation was supported in part by research grant RG-5502, National Advisory Health Council, U. S. Public Health Service. 3 Dairy Industrial Research Fellow, supported by Dairy Industries Supply Association, Inc. 4 Present address: Department of Dairy Science, University of Arizona, Tucson, Arizona. Received for publication July 25, 1960 symptoms in 11 cases. On the basis of these reports, only coagulase-positive staphylococci were considered potentially enterotoxigenic, and were isolated from raw milk and examined in this study. MATERIALS AND METHODS Nineteen raw milk samples of manufacturing grade were collected from individual Iowa producers, all of whom used bulk tanks for cooling and holding of the milk, during the period from March through May, During June, July, and August, 20 samples of grade A milk were collected from producers in the Iowa State University milkshed. The milk was plated on a modified Tellurite-glycine medium consisting of Trypticase, 5 g; Proteose peptone, 5 g; yeast extract, 5 g; glycine, 8 g; mannitol, 15 g; K2HPO4, 5 g; LiCl, 5 g; and agar, 15 g. A 1 per cent potassium tellurite solution was prepared and sterilized. Twenty milliliters of the solution were added aseptically to 1000 ml of the medium after it had been autoclaved at 121 C for 20 min and cooled to 50 C. A surface plating technique was used. Plates were incubated at 37 C for 48 hr. Isolations for further study and characterization were made from typical coagulasepositive staphylococcus colonies, appearing smooth, convex, glistening, and jet-black in color. The tube coagulase test was performed, using Warner-Chilcott diagnostic plasma,5 on 24-hr Tryptosephosphate broth cultures. Chapman (1944) suggested the use of this medium because it enhances clotting 5 Warner-Chilcott Laboratory Supply Division, Morris Plains. New Jersey.