Vol. 51, No. 5
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1986, p. 956-962
Copyright C 1986, American Society for Microbiology
Methodology for Enumeration of Coliphages in Foodst
J. E. KENNEDY, JR.,t4 C. I. WEI,'* AND J. L. OBLINGER2
Food Science and Human Nutrition Department, University ofFlorida, Gainesville, Florida 32611,' and College of
Agriculture, University ofMissouri, Columbia, Missouri652112
Received 14 November 1985/Accepted 11 February 1986
The effects of eluent composition, pH, and chaotropic agents on the recovery of T2, MS2, and indigenous
coliphages from various foods were investigated. Additionally, methods of sample suspension and clarification
were evaluated for coliphage recovery and application to various foods. Clarified sample suspensions were
assayed for coliphages with a modified agar layer technique and appropriate Escherichia coli hosts.
Centrifugation and polypropylene mesh filtration were more rapid and effective than glass wool filtration for
clarification of sample suspensions and subsequent recovery of coliphages. Blending, stomaching, and shaking
procedures were generally comparable for sample liquefaction and release of coliphages from foods. Complex
basal eluents, EC medium and 1%S casein, were generally more effective than a less complex eluent, phosphate
buffer, for elution of coliphages from foods. For most foods, incorporation of sodium chloride or chaotropic
agents, i.e,, sodium trichloroacetate, urea, Tween 80, Triton X-100, and sodium nitrate, into basal eluents did
not enhance recovery of coliphages. Indigenous coliphage recovery was not affected by sample suspension pH
over a range of 6.0 to 9.0. With an optimal procedure, i.e., EC medium eluent, blending, and centrifugation,
the recovery of T2 and MS2 ranged from 48 to 81% and from 58 to 100%, respectively, depending on the food
Coliphages have received increased attention and support
in recent years as rapid and inexpensive indicators of fecal
pollution and enteric pathogens in water and wastewater (15,
19, 26, 30, 31, 35, 37). Likewise, methods for quantitative
recovery of coliphages and other viruses from the environ-
ment have been extensively investigated over the past sev-
eral years (2-4, 26, 33). Although numerous methods for
recovering animal viruses from various foods have been
developed (5, 6), few attempts have been made to enumerate
indigenous bacteriophages in foods, and the procedures
reported were not evaluated or refined in relation to recov-
ery efficiency or applicability to various types of food (17,
18, 36). Development of coliphage enumeration methods
which are rapid, simple, economical, and efficient as well as
broadly applicable to different foods is necessary to investi-
gate the distribution of coliphages and their possible role as
indicator organisms in foods.
Techniques used for elution or desorption of viruses and
phages from solid materials, e.g., membrane filters, soil,
sludge, and aquatic sediments, generally involve lowering
the ionic strength of the eluent or sample suspension, adding
soluble proteins or other organic compounds to the suspen-
sion, raising the pH of the suspension, adding chaotropic
agents to the suspension, or various combinations of these
techniques (2-4, 7-12, 27, 29, 33, 37). Similar approaches
have been used for desorption and recovery of animal
viruses from foods with eluents or suspension media con-
taining various concentrations of salts, acids or bases,
buffers, proteins, and various combinations of these compo-
nents (5, 6). Little information is available on the effects of
different eluent compositions and suspension medium pHs
on the elution of viruses from foods (13), and no information
is available on the effects of chaotropic agents.
t FloridaAgricultural Experiment Stationjournal series no. 6899.
t Present address: American Home Foods, Central Research
Laboratory, Milton, PA 17847.
A number of methods, including shaking, stirring and
blending, and homogenization, have been used for physical
suspension or liquefaction of food samples to release animal
viruses (5, 6, 22), but these methods have not been standard-
ized or extensively evaluated for broad application to vari-
ous foods. Several techniques for clarifying food sample
suspensions prior to virological assay, e.g., centrifugation,
filtration, chemical phase separation, and adsorption-
elution, have been reported (5, 6). Tierney et al. (32) found
the glass wool or woven fiber glass filtration technique to be
more efficient than other techniques, including centrifuga-
tion, for clarification of ground beef suspensions and subse-
quent recovery of polioviruses. A modification of the glass
wool filtration technique (19, 32) was used to clarify food
suspensions for subsequent assay of coliphages, but the
technique was time-consuming for most samples (17).
The objective of this study was to develop and evaluate
broadly applicable procedures for efficient recovery of
coliphages from various types offoods. The effects of eluent
composition, eluent and sample suspension pH, and
chaotropic agents on the recovery of T2, MS2, and indige-
nous coliphages from various foods was investigated. Meth-
ods of sample suspension and clarification were also evalu-
ated for recovery efficiency and ease of use.
MATERIALS AND METHODS
Media and chemicals. The chemicals used in these studies
and their sources were as follows: sodium trichloroacetate
(NTCA), sodium chloride, sodium nitrate, sodium hydroxide,
potassium phosphate monobasic, hydrochloric acid, and
Tween 80 were obtained from Fisher
Springfield, N.J.; lysine, casein, and urea were obtained from
Sigma Chemical Co., St. Louis, Mo.; and Triton X-100 was
obtained from Eastman Kodak Co., Rochester, N.Y. All
bacteriological media and peptone and bile salts mixture
number 3 were obtained from Difco Laboratories, Detroit,
Samples. Samples of fresh chicken breasts, fresh ground
KENNEDY ET AL.
APPL. ENVIRON. MICROBIOL.
method and test strategy .for recovery of enteric viruses from
shellfish. Appl. Environ. Microbiol. 39:141-152.
23. Montgomery, D. C. 1984. Design and analysis of experiments,
2nd ed. John Wiley & Sons, Inc., New York.
24. Peterkin, P. L., and A. N. Sharpe. 1981. Filtering out food debris
before microbiological analysis. Appl. Environ. Microbiol.
25. Sabatino, C. M., and S. Maier. 1980. Differential inactivation of
three bacteriophages by acid and alkaline pH used in the
membrane adsorption-elution method of virus recovery. Can. J.
26. Scarpino, P. V. 1978. Bacteriophage indicators, p. 201-227. In
G. Berg (ed.), Indicators of viruses in water and food. Univer-
sity of Michigan, Ann Arbor.
27. Seeley, N. D., and S. B. Primrose. 1982. The isolation of
bacteriophages from the environment.
28. Sharpe, A. N., and G. C. Harshman. 1976. Recovery of Clos-
tridium perfringens, Staphylococcus aureus and molds from
foods by the stomacher: effect of fat content, surfactant con-
centration, and blending time. Can. Inst. Food Sci. Technol. J.
29. Shields, P. A., and S. R. Farrah. 1983. Influence of salts on
electrostatic interactions between poliovirus and membrane
J. Appl. Bacteriol.
filters. Appl. Environ. Microbiol. 45:526-531.
30. Simkova, A., and J. Cervenka. 1981. Coliphages as ecological
indicators of enteroviruses in various water systems. Bull.
31. Stetler, R. E. 1984. Coliphages as indicators of enteroviruses.
Appl. Environ. Microbiol. 48:668-670.
32. Tierney, J. T., R. Sullivan, E. P. Larkin, and J. T. Peeler. 1973.
Comparison ofmethods for the recovery ofvirus inoculated into
ground beef. Appl. Microbiol. 26:497-501.
33. Wait, D. A., and M. D. Sobsey. 1983. Method for recovery of
enteric viruses from estuarine sediments with chaotropic
agents. Appl. Environ. Microbiol. 46:379-385.
34. Wellings, F. M., A. L. Lewis, and C. W. Mountain. 1976.
Demonstration of solids-associated virus in wastewater and
sludge. Appl. Environ. Microbiol. 43:430-434.
35. Wentsel, R. S., P. E. O'Neil, and J. F. Kitchens. 1982. Evalua-
tion of coliphage detection as a rapid indicator of water quality.
Appl. Environ. Microbiol. 43:430-434.
36. Whitman, P. A., and R. T. Marshall. 1971. Isolation of
psychrophilic bacteriophage-host systems from refrigerated
food products. Appl. Microbiol. 22:220-223.
37. Zaiss, U. 1981. Dispersal and fate of coliphages in the River
Saar. Zentralbl. Bakteriol. Hyg. Abt. 1 Orig. Reihe B 1974: