Thermal Destruction Curves for Salmonella oranienburg in Egg Products1,2

Thermal Destruction Curves for Salmonella oranienburg in Egg Products1'2 0 . J . COTTERILL, J . Gl-AUERT AND G. F . K R A T J S E 3 Department of Food Science and Nutrition, University of Missouri-Columbia, Columbia, Missouri 65201 (Received for publication June 23, 1972)

POULTRY SCIENCE 52: 568-577, 1973

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HIS paper offers an improved method for constructing thermal destruction curves (T.D.C). T.D.C. indicate heat resistant characteristics of microorganisms. The following generalizations have evolved concerning their construction: (1) T.D.C. are usually linear but extrapolation beyond available data is questioned. (2) T.D.C. for an organism in different media are parallel only if Z is a constant. (3) T.D.C. based on D and F are parallel only if linear portions of survival curves are involved. These suppositions are based on the linearity of survival curves and the assumption that Z is a constant, neither of which is true in all cases. There are many examples of variations within these concepts. Hence, it is usually necessary to experimentally determine the time-temperature requirements for each organism in different products. Non-linear aspects of survival curves are well established, and are accepted as 1 Contribution from the Missouri Agricultural Experiment Station. Journal Series Number 6424. 2 Presented at the 60th Annual meeting of the Poultry Science Association, University of Arkansas, Fayetteville. (see Poultry Science 50:1566, 1971). 3 Agricultural Experiment Station Statistician.

the result of natural causes rather than experimental errors or bias. No further review is necessary. The manner of their use in constructing T.D.C. is important. Moats et al. (1971) and Dabbah el al. (1971a, b) questioned the use of D to express thermal resistance. They suggested that F values as well as a recovery test (e.g., "Salmonella Survival Test") would permit a more complete description of heat resistance. Our experience supports the view that a recovery test is a more valid and useful estimate of thermal resistance and that these data should be used to construct T.D.C. for adequate pasteurization. The following review indicates other deviations within the above generalizations. Differences in Z have been reported, but many authors attributed them to experimental error. For example, Kaplan el al. (1954) summarized the literature and hypothesized that Z could be influenced by media. However, after further work with foods, similar to those used in the various studies, they concluded that the observed variations were due to experimental error. Likewise, Thomas et al. (1966) observed some medium effects on Z but indicated that they were not statis-

568

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ABSTRACT This paper offers an improved method of constructing thermal destruction curves (T.D.C). It summarizes data on the times and temperatures required to kill salmonellae in various egg products. Also, additional data are presented and T.D.C. are constructed for a wide range of conditions. Further evidence is obtained that Z for salmonellae varies in different products. Water activity influences Z. This concept provides a better method of predicting thermal process conditions. The data indicate that T.D.C. for different products intersect near a common point. A point of intersection was determined by minimizing the sum of the squares of the differences between the observed and hypothetical slopes of the T.D.C. This point for several egg products was a t 29,240 minutes and 42.3°C.

Salmonella Oranienburg

The usefulness of T.D.C. for predicting thermal processing time-temperature rela-

tionships has been questioned. Wang et al. (1964) stated that a thermal death time curve (i.e., T.D.C.) is a poor way to correlate data over an extended temperature range. Thomas et al. (1966) recommended that such curves not be extrapolated more that 5.6°C. higher than those actually tested. Baird-Parker et al. (1970) stressed that D and Z values should be determined for each food. The purpose of this paper is to summarize data on egg products so that T.D.C. can be extended over a wider temperature range. Also, a new concept is proposed for the construction of T.D.C. When the curves for various egg products are plotted on one graph, they intersect near a common point. Z is influenced by water activity. MATERIALS AND METHOD

Aseptic samples of egg yolk (E.Y.), egg white (E.W.), and whole egg (W.E.) were prepared according to a method described by Cotterill (1968), except that the separation process for W.E. was eliminated. For adding 10% salt or sugar to a sample, the procedure outlined by Cotterill and Glauert (1969) was followed. Egg samples were inoculated with ca 106 TABLE 1.—F and Z values used to calculate hypothetical TDC shown in Figure 7. F Zi

Product

Egg white—pH 9.0 Whole egg Egg yolk Whole egg + 10% sugar Egg yolk + 10% sugar Whole egg + 10% salt Egg yolk + 10% salt Egg white solids (2.67% moisture)

Xi

Yi

(°C.)

(min.)

56.6a 60.0" 60.0° 57.0b 59.0b 60.0b 61.0e 60.0'

»b Cotterill (1968). This paper." USDA-CMS (1971). d Garibaldi et al. (1969). " Cotterill and Glauert (1969). ' McBee and Cotterill (1971).

3.5 3.5 6.2 45.0 23.0 42.0 100.0 6050.0

(°C.) 4.1b 4.5b 4 . 4 db 4.3 • 5.0b 7.0b 9.1e 32.0f

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tically significant. Angelloti et al. (1961) concluded that their observed differences in Z of 5.1 to 7.8°C. for S. Manhattan in chicken a' la king and custard might be due to error. The parallel T.D.C. based on Z of 4.4°C. for S. typhimurium in various egg products constructed by Garibaldi et al. (1969) and Lineweaver et al. (1969) support the supposition that Z is a constant for a given organism. Mostly the above variations fell within the +1.1°C. range indicated by Schmidt (1957) for vegetative cells. Other authors have reported large differences in Z but did not comment further. For example, Anellis el al, (1954) observed Z values for 5. senftenberg 7 75 W in whole egg (pH 5.5 to 8.5) ranging between 3.9 and 7.9°C. There was some indication that pH affected Z. Lategan and Vaughn (1964) found that the addition of /3-propiolactone had a definite effect on Z but estimates were not made. Recently, decisive evidence that Z is affected by media have been presented. Cotterill and Glauert (1969) reported Z values of 5.0°C. and 9.1°C. for S. oranienburg in 10% sugared egg yolk and 10% salted egg yolk. Also, Baird-Parker et al. (1970) observed that 13.8% salt almost doubled Z for S. senftenberg 775W in heart infusion broth (Z was 13° in the presence of salt but only 6.8°C. in the broth). McBee and Cotterill (1971) summarized the literature on Z values in products with low water activities. Estimates of Z varied between 11.6° and 113°C. They observed a Z value of 32°C. for S. oranienberg in egg white solids. Dega et al. (1972) found that milk solids levels influenced Z for S. typhimurium. They reported values of 4.0°, 4.6°, 6.0°, and 6.8f°C. for 10, 30, 42, and 5 1 % milk solids, respectively.

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Egg White and Whole Egg: T . D . C . for p H 9.0 egg white and p H 7.0 whole egg are shown in Figure 1. The Z values of 4.1 and 4.5°C. (7.4 and 8.3°F.) are in substantial agreement with others in the literature, see Table 2. This indicates that TABLE 2.—Comparison of observed and hypothetical Z values for salmonellae in egg products

58

Observed Zi

Hypothetical Zhi

(°C)

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organisms from a 24 hour Trypticase Soy Broth culture of S. oranienburg. T w o types of experiments were used to arrive at D , F, and Z values (Stumbo, 1965; Cotterill and Glauert, 1969). All D values, and F above 50°C., were determined in thermal death tubes (T.D.T) unless otherwise indicated. Total counts on Trypticase Soy Agar and the Salmonellae Survival Test (S.S.T.) described b y Cotterill and Glauert (1969), were used to determine if salmonellae survived the heat treatment. F was established on the basis of 106 t o 7 10 reduction of viable cells. D u e to the extended period of time needed to determine F below 5 0 ° C , another series of experiments were set up. Ten g. samples of the product were weighed into sterile beakers, which were sealed with sterile aluminum foil and Parafilm. After storage, 90 ml. of sterile lactose b r o t h were added to the sample for the S.S.T.

Egg y o l k + 10% salt

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b

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25.8

This paper. Garibaldi et al. (1969). Corry and Barnes (1968). Lategan and Vaughn (1964). • Anellis e<
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Additional d a t a were obtained so t h a t the range of thermal destruction curves (T.D.C.) for 5 . oranienburg in some egg products could be extended. Also, a series of T D C were constructed which are more applicable t h a n those obtained by extrapolation of a series of parallel lines.

Salmonella Oranienburg

on D and F were parallel. The survival curves were linear.

the basic methods used in this study produced results similar to other workers. Data for F were not obtained. However, since the survival curves in these two systems are linear, F can be estimated for any desired reduction in cells.

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Salted Whole Egg and Yolk: A T.D.C. had not previously been constructed for whole egg containing 10% salt. Hence, all data shown in Figure 4 was obtained specifically for this report. The T.D.C, within ranges observed, based on D and F were parallel. The survival curves resembled those previously reported by Cotterill and Glauert (1969) for salted egg yolk. Also, a curvi-linear function is apparent in the T.D.C. based on F values below 50°C. The value for Z in the temperature range of 50 to 60°C. was 7.0°C. (12.6°F.).

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Sugared Whole Egg and Yolk: The values of 4.3 and 5.0°C. (7.74 and 9.0°F.) for Z obtained in 10% sugared whole egg and yolk are also in close agreement with those previously reported in the literature (see Figures 2 and 3, Table 2). Attempts to destroy S. oranienburg below 50° C. were unsuccessful. These products degraded upon extended storage before the test organism was killed. T.D.C. based :

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The data shown in Figure 5 for egg yolk containing 1 0 % salt above 49°C. and below 40°C. was obtained from Cotterill and Glauert (1969, 1972). T h e other data had not previously been reported. As expected, the thermal resistance in egg yolk was higher t h a n in whole egg containing 1 0 % salt. Otherwise t h e two patterns were similar. T h e Z value of 9.1°C. (16.4°F.) shown in Figure 5 for the temperature range of 50 to 60°C. is t h a t reported previously by Cotterill and Glauert (1969). Since the T . D . C . is curvilinear, Z would be higher for the lower temperatures.

1) from T . D . C . of several egg products were plotted on one graph (Figure 6). Since Z is not constant, the curves are not parallel. T h e solid portions of the curves represent available data. Broken T . D . C . represent ranges where d a t a are not available or pasteurization under these conditions is not recommended within conditions employed. When extrapolated to lower temperatures, they intersect at X , Y coordinates between 300 and 20,000 minutes and 40 and 53°C. This observation suggests t h a t a series of curves intersecting at one point is a better concept t h a n a series of parallel lines established from a constant Z. •

Observed and Hypothetical TDC: Previously observed values of Z and F (Table

T h e point of intersection (P.I.) can be approximated graphically by visually

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6. Observed T.D.C. for several egg products plotted on one graph.

estimating the best point in Figure 6. However, the P . I . shown in Figure 7 was obtained by determining the coordinates with the following equation where the slopes must be changed the least to permit all lines to pass through one point.

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Where: i = ith curve. bi = observed slope of the ith T D C . bhi = hypothetical slope of the ith T D C . bi or bhi = reciprocal of Zi or Zhi. X ; and Yi = coordinates for an observed pasteurization time and temperature on the ith T D C for a specific product. P I = an intersection point in the X , Y grid.

XPI

and Ypi = coordinates for P I where the sum of (bi —bhi)2 is minimized for all lines.

Using the values for X i ; Yi, and bi shown in Table 1 the calculated coordinates for X n , Y P I were 42.3°C. and 29,240 minutes. The hypothetical (Z M ) values are reported in Table 2. With the exception of egg white solids, all Zhi values were within 0.8°C. of an observed value. Published values for S. senftenberg 775W are not included in Table 2 because Z is higher than for the other salmonellae serotypes. The differences between most Zi and Zhi values were within those credited b y Kaplan et al. (1954) and Thomas et al. (1966) to experimental error. The only major descrepancy between Zi and Zhi was in egg white solids. Apparently, this T.D.C. had to be

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SO

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FIG. 7. Hypothetical T.D.C. for several egg products with Z adjusted so all curves pass one point of intersection. changed the most to minimize the sum of (bi — bhi)2 for all lines. DISCUSSION A new concept is recommended for the construction of thermal destruction curves ( T . D . C ) . These data suggest a hypothesis t h a t Z is a function of vapor pressure; such as, water activity, osmotic pressure, etc. As water activity is decreased b y dehydration or by adding solutes, the rate of cell destruction is decreased with increased temperature increments. Z must be increased to produce the same degree of kill within a specified time. Also, a generalization is suggested t h a t T.D.C. for products with widely different water activities tend to converge and intersect at a common point. T h e

significance of this point of intersection (P.I.) is not clear. However, Graham (1971) suggested t h a t it m a y represent a stasis area where the organism does not readily grow or die. The work by Dega et al. (1972) on the influence of milk solids levels on T . D . C . supports the above concepts. Their results showed t h a t Z increases as the solids level is increased (decreased water activity). Also, when extrapolated to lower temperatures they intersect near a common point. Using values shown in Table 3 (obtained from Dega—Fig. 1), calculated coordinates for X P I , Y n were 46.6°C. and 741 minutes. Note t h a t the temperature (Xpi) at P.I. was near t h a t determined from our data. T h e lower value for Y P I would be expected since the d a t a

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100

576

O. J. COTTERILL, J. GLAUERT AND G. F. KRAUSE TABLE 3.—F and Z values used, to calculate Zhi and point of intersection F

Skim milka solids

Xi

Yi

(%)

(°C.)

10 30 42 51

55.2 56.9 58.8 61.9

a b

Zi

Z hi b

(min.)

(°C.)

(°C.)

5.0 5.0 5.0 5.0

4.0 4.6 6.0 6.8

4.0 4.8 5.7 7.1

Data from Dega et al. (1972). Point of intersection at 741 minutes and 46.6°C.

The concept of intersecting lines has limitations. Note the discrepancy in the case of salted whole egg below 55°C. Hence, this technique should be used only to estimate thermal processing conditions. Eventually, these conditions should be confirmed or determined specifically for each product. It is laborious to obtain enough data to

REFERENCES Anellis. A., J. Lubas and M. M. Rayman, 1954. Heat resistance in liquid eggs of some strains of the genus Salmonella. Food Res. 19:377-395. Angelloti, R., M. J. Foter and K. H. Lewis. 1961. Time-temperature effects on salmonellae and staphylococci in foods. Appl. Micro. 9:308-315. Baird-Parker, A. C , M. Boothroyd and E. Jones, 1970. The effect of water activity on the heat resistance of heat sensitive and heat resistant strains of salmonellae. J. Appl. Bact. 33:515-522. Brant, A. W., G. W. Patterson and R. E. Walters, 1968. Batch pasteurization of liquid whole egg. 1. Bacteriological and functional property evaluation. Poultry Sci. 47:878-885. Corry, J. E. L., and E. M. Barnes, 1968. The heat resistance of salmonellae in egg albumen. British Poultry Sci. 9:253-260. Cotterill, O. J., 1968. Equivalent pasteurization temperatures to kill salmonellae in liquid egg white at various pH levels. Poultry Sci. 47:354365. Cotterill, O. J., and J. Glauert, 1969. Thermal resistance of salmonellae in egg yolk products containing sugar or salt. Poultry Sci. 48:1156-1166. Cotterill, O. J., and J. Glauert, 1971. Thermal resistance of salmonellae in egg yolk containing 10% sugar or salt after storage at various temperatures. Poultry Sci. 50:109-115. Cotterill, O. J., and J. Glauert, 1972. Destruction of Salmonella oranienburg in egg yolk containing various concentrations of salt at low temperatures. Poultry Sci. 51:1060-1061.

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were based on D rather than F values. The new Zhi values are also shown in Table 3. The differences between Zi and Zhi were small. The concept of intersecting T.D.C. provides an improved method to predict time-temperature requirements to kill salmonellae (and perhaps other organisms) in foods with widely different moisture or solute levels. These properties determine the water activity. The water activity of egg products varies widely. However, there is not sufficient information available on the water activity of egg products to establish a quantitative relationship with Z. Liquid egg white has a water activity near 1.0 while egg white solids was observed to be 0.008 by McBee and Cotterill (1971). An attempt to measure the water activity of liquid egg products was unsucessful. A liquidvapor water equilibrium technique was attempted but surface denaturation or low surface area interferred.

construct good T.D.C. Also, new product compositions will be formulated or there will be need to heat treat in different time-temperature ranges. With only one value for D or F available, constructing a TDC through P.I. should be helpful in estimating others. Because of the curvilinear nature of survival curves, F values are preferred to extrapolations from D values. These F values should be based on recovery techniques capable of reviving debilitated cells and with a probability of kill sufficient to assure safety. The F values in this paper are based on data obtained by pre-enrichment in lactose broth and capable of destroying about 107 cells per gm. The survival level was less than 0.1/cell/gm.

Salmonella Oranienburg

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Garibaldi and V. F. Kaufman, 1969. Egg pasteurization manual. U.S.D.A., A.R.S. 74-48. McBee, L. E., and 0 . J. Cotterill, 1971. High temperature storage of spray-dried egg white. 3. Thermal resistance of Salmonella oranienburg. Poultry Sci. 50:452^58. Moats, W. A., R. Dabbah and V. M. Edwards, 1971. Interpretation of nonlogarithmic survivor curves of heated bacteria. J. Food Sci. 36:523-526. Schmidt, C. F., 1957. Thermal resistance of microorganisms. p. 831-884 in Reddish, G. F. Ed. Antiseptics, Disinfectants, Fungicides, and Chemical and Physical Sterilization. Lea and Febiger, Philadelphia, Pa. Stumbo, C. R., 1965. Thermobacteriology in Food Processing. Academic Press, New York. Thomas, C. T., J. C. White and K. Longree, 1966. Thermal resistance of salmonellae and staphylococci in foods. Appl. Micro. 14:815-820. U.S.D.A.-C.M.S., 1971. Egg and egg products inspection. Federal Register, 36(104) :9814-9834. Wang, D. I - C , J. Scharer and A. E. Humphrey, 1964. Kinetics of death of bacterial spores at elevated temperatures. Appl. Micro. 12:451-454.

Fellow of the American Society of Animal Science Among those made Fellows of the American Society of Animal Science at the 64th annual meeting at Virginia Polytechnic Institute and State University, Blacksburg, July 29 to August 2, 1972, was Dr. Elmer Roberts. The citation read: "Elmer Roberts, geneticist, pioneer, prophet, philosopher and historian combined all these attributes and abilities in his unusually long and extended career in animal science." "He was born June 29, 1886, at Burnside, Kentucky, and spent his boyhood on the family farm. He received the B.S. degree in General Agriculture from the University of Illinois in 1913, and continued his graduate study receiving the Ph.D. degree in genetics in 1917." "Dr. Roberts was a faculty member of the University of Illinois until his formal retirement in 1954 and he has continued to devote his time and talent to special University assignments. While primarily serving the Department of Animal Science, he elicited the interest and enrollment of students from all areas of the University and continued his interdisciplinary teaching until recently." "He was a pioneer in his understanding of and concern for population control, particularly in

densely inhabited areas such as China. His international involvement in China predated our long lapse in relationships with the Mainland." "His research in genetics covered a wide spectrum of animal life—from laboratory species such as drosophila, mice, rats and rabbits, to chickens, sheep, swine, cattle and horses, and finally, man. He has long been interested in the relation of heredity to disease—specifically resistance and susceptibility to infection as influenced by hereditary factors. This problem was explored in detail in the study of pullorum disease in young chicks. After finding unmistakable evidence of hereditary resistance in strains of chickens kept at the University of Illinois, he spent a year (1929 to 1930) at Tunghsien, China, near Peking, where he worked with staff members of Peking Union Medical College, and found that small-type Chinese chickens had, through natural selection, developed resistance to Salmonella pullorum under conditions in which no disease control methods were in general use." "Other animal studies involved color inheritance in Shorthorn cattle, hairlessness in swine, crossbreeding in swine, twinning in Shropshire sheep, fertility in sheep and swine, anhidrosis in man, and

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Dabbah, R., W. A. Moats and V. M. Edwards, 1971a. Heat curves of food-borne bacteria suspended in commercially sterilized whole milk. 1. Salmonellae. J. Dairy Sci. 54:1583-1588. Dabbah, R., W. A. Moats and V. M. Edwards, 1971b. Survivor curves of selected 5. enteritidis serotypes in liquid whole egg homogenates at 60°C. Poultry Sci. 50:1772-1776. Dega, C. A., J. M. Goepfert and C. H. Amundson, 1972. Heat resistance of salmonellae in concentrated milk. Appl. Micro. 23:415-420. Garibaldi, J. A., R. P. Straka and K. Ijichi, 1969. Heat resistance of Salmonella in various egg products. Appl. Micro. 17:491-496. Graham, D. M., 1971. Personal communication. Kaplan, A. M., H. Reynolds and J. Lichtenstein, 1954. Significance of variations in observed slopes of thermal death curves for putrefactive anaerobes. J. Food Sci. 19:173-181. Lategan, P. M., and R. H. Vaughn, 1964. The influence of chemical additives on the heat resistance of Salmonella typhimurium in liquid whole egg. J. Food Sci. 29:339-344. Lineweaver, H., H. H. Palmer, G. W. Putnam, J. A.

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