Production of Fluorescence on Packaged Chicken ' , 2

KRAFT, A. A. (Iowa State University, Ames), AND J. C. AYRES. Production of fluorescence on packaged chicken. Appl. Microbiol. 9:549-553. 1961.-Development of fluorescence caused by pseudomonads proliferating on packaged chicken was determined by examination of the poultry under ultraviolet light and by measurements of absorption spectra. Asparagine broth inoculated with organisms from chicken showed absorption maxima at 270 m,u and 410 m,; these peaks are characteristic of the fluorescent pigment, pyoverdine. Absorbance calculated as the ratio (A270m, + A410m,)/A35Om, provided a convenient measure of amount of pigment produced; this ratio was related to numbers of fluorescing organisms recovered from chicken. Absorption peaks generally increased during the first few days the poultry was stored at 5 C and then declined during the latter part of the 7-day holding period. Production of fluorescence was influenced by packaging materials. Fluorescence was not visible on poultry until counts of fluorescing bacteria were as great as 100,000 to 1,000,000 per cm2. Growth of fluorescent pigment-producing pseudomonads on chicken was stimulated during storage after the poultry was dipped in solutions containing iron. Fluorescent spoilage of shell eggs has been recognized for many years. The pigment complex produced by pseudomonads has been referred to as "fluorescein," but Elliott (1958) indicated that the term "pyoverdine," as used by Turfreijer (1941), more correctly described the green, water-soluble pigment. In contrast to reports concerning eggs, fluorescence as a manifestation of spoilage of chicken has received relatively little attention. Examination of poultry under ultraviolet light was considered by Cotterill (1956) as a means of demonstrating growth of fluorescing organisms and as a method for determining shelflife. The reliability of this technique was questioned by 1 Journal paper no. J-4099 of the Iowa Agricultural and Home Economics Experiment Station, Ames, Iowa. Project no. 1392, Center for Agricultural and Economic Adjustment cooperating. 2 This investigation was supported in part by research grant E-1430 (C5) from the National Institutes of Health, U. S. Public Health Service. Barnes and Shrimpton (1958), who reported a poor correlation between development of off-odors and the appearance of fluorescence on chicken. In an extensive study characterizing Pseudomonas fluorescens and other species of pseudomonads, Rhodes (1959) indicated that pigment production was not a constant property of these organisms. Earlier work (Sullivan, 1905; Georgia and Poe, 1931; Jamieson, 1942; King, Campbell, and Eagles, 1948) stressed the importance of various minerals and salts for pigment formation and indicated that composition of the medium had a marked influence on ability of pseudomonads to elaborate the water-soluble, fluorescent pigment. Similarly, formation of pyocyanine, the chloroform-soluble pigment, was shown by Haynes (1951) to be dependent upon components of the medium in which Pseudomonas aeruginosa was grown. In view of observations indicating that fluorescence may become apparent on poultry undergoing microbiological spoilage, the present study was initiated in an attempt to evaluate development of fluorescent pigment on poultry meat in relation to growth of pseudomonads. In addition, a limited investigation was conducted concerning the effects of iron on spoilage of poultry, since a recent report by Garibaldi and Bayne (1960) indicated that iron had an appreciable effect on spoilage of shell eggs by pseudomonads. MATERIALS AND METHODS Freshly cut chicken wings were purchased at a retail store and packaged with various films having different rates of transmission of moisture vapor and gases. These materials included 300 MSAD-803 cellophane, 300 LSAD3 cellophane, 50 ga HS Mylar,3 and 75 ga Cryovac.4 Except for one trial, Cryovac packages were not heat-shrunk or evacuated so that procedures were consistent with those employed with other films. When the effect of iron was tested, the chicken was immersed momentarily in a sterile solution of demineralized water containing 10 ,ug FeCl3.6H20 per ml, drained, and packaged. Samples similarly dipped in demineralized water served as controls. The packaged poultry was stored in a display case at approximately 5 C and examined at intervals for numbers of fluorescing organisms and for total microflora. Surfaces of wings 3E. I. du Pont de Nemours & Co., Inc., Wilmington, Del. 4W. R. Grace & Co., Cryovac Division, Cambridge, Mass. 549 on O cber 7, 2017 by gest ht://aem .sm .rg/ D ow nladed fom A. A. KRAFT AND J. C. AYRES were sampled by firmly rolling moist absorbent cotton swabs over two 2-cm2 areas outlined by sterile metal guides (Ayres et al., 1956). Fluorescing organisms were enumerated on the fluorescin medium of King, Ward, and Raney (1954). Plating was performed using the surface technique described by Silliker, Shank, and Andrews (1958), since greater numbers of fluorescing bacteria were recovered from plates prepared by surface inoculation than from pour plates. Total counts were made from pour plates of Trypticase soy agar (BBL).5 All plates were incubated at 15 C for 3 or 4 days. The chicken was examined periodically for fluorescence under ultraviolet light.6 Determinations were also made of absorption spectra of broths inoculated with organisms obtained from the poultry. For these measurements, chicken was sampled as indicated previously and the moist cotton swab placed in a flask containing the broth described by King et al. (1954) or the asparagine medium of Georgia and Poe (1931). The flask was shaken at 30 C for 18 to 20 hr and incubated without shaking for an additional 18 to 20 hr. This procedure was accepted after testing several combinations of time and temperature with and without shaking the flask. The broth was then centrifuged and the absorption spectrum of the supernatant liquid was measured with a Beckman model DU spectrophotometer.7 Absorption spectra of stock cultures of Pseudomonas were determined in a similar manner. RESULTS AND DIsCUSSION Prior to tests with organisms sampled from chicken, measurements were made of absorption spectra of known pseudomonads cultured in asparagine broth. Curves for P. aeruginosa (2F41)5 and Pseudomonas fragi (2F36)8 are presented in Fig. 1. Absorption I Baltimore Biological Laboratory, Inc., Baltimore, Md. 6 Vogelite Egg Candler, model 205, distributed by Alf McNamee and Sons, Oakland, Calif. 7Beckman Instruments, Inc., Fullerton, Calif. 8 Department of Bacteriology, Iowa State University, Ames, Iowa.