Michael B. Howard and Steven W. Hutcheson* 

Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland 20742 

Received 21 June 2002/Accepted 15 October 2002

Evaluation of the ability of germinating alfalfa seeds to support S. enterica growth. Since most of the increase in population levels occurred during the initial 24 h (>104-fold increase), the ability of S. enterica to colonize germinating seeds later in development was evaluated. A seed batch was inoculated as described above, and six germinating seeds from the inoculated seed batch were transferred at 24, 48, 72, and 96 h to batches of previously uncontaminated germinating seeds (approximately 6,000 seeds). Populations developing on germinating seeds and in the waste irrigation water were monitored as before. Seed batches inoculated at 0, 24, and 48 h after the initiation of germination all established roughly equivalent populations of 98E01362SH2 within 48 h after inoculation of the seed batch (Fig. 3). A 10-fold reduction in the size of maximum population was detected in seeds inoculated at 72 h, and little growth was detected in seed lots inoculated at 96 h. Similar results were obtained when waste irrigation water was sampled (data not shown). 

The contamination of alfalfa sprouts by S. enterica strains and other pathogenic bacteria has become a significant public health concern and has resulted in multiple warnings by regulatory agencies (e.g., references 2 and 3). Comparatively little was known about how S. enterica strains grow on germinating alfalfa sprouts and what aspects of that growth can be exploited to reduce the human health risk. We have shown here that the ability of S. enterica strains to grow on germinating alfalfa 

The presence of S. enterica in the waste irrigation water was shown to be a key indicator of S. enterica contamination of germinating alfalfa sprouts. 

Populations in waste irrigation water were strongly correlated with populations present on developing alfalfa sprouts. S. enterica could be detected in waste irrigation water from contaminated seed batches within 12 h after the initiation of the germination process. Each S. enterica strain tested could be detected in the waste irrigation water collected from germinating seeds inoculated with that strain. S. enterica populations in waste irrigation water from inoculated seed batches reached a maximum by 48 h, irrespective of the strain, but germinating alfalfa seeds could support the growth of S. enterica strains when they were inoculated at any time during the initial 72 h of the process. Thereafter, the rate of growth appeared to be significantly lower. Since waste irrigation water still contained comparatively high numbers of S. enterica CFU, the reduced ability of S. enterica to grow may have been related to the diminished release of sugars from the germinating seed, active defenses by the emerging root radicle, or a reduction in the ability of S. enterica strains to adhere to and colonize plant tissue. Interestingly, the relative numbers of CFU of S. enterica strains in waste irrigation water were reduced late in the seed germination process. The reduced levels may have been due to the formation of biofilms on the emerging root radicles, as reported previously by other groups (8, 11). 

Our data lend support to the U.S. Food and Drug Administration’s recommended testing methods to ensure that commercial sprout batches are free of S. enterica contamination (2). The testing of waste irrigation water 48 h after the initiation of seed germination was recommended (1), but supporting evidence was not provided. 

This testing should be done early in the production cycle, as delayed sampling of waste irrigation water could diminish the chances of detecting contaminated seed batches. The early identification of S. enterica contamination of alfalfa sprouts should prevent contaminated products from reaching the consumer 

Alfalfa Sprouts Held at Room Temperature Do Not Support The Growth of Salmonella.  Bin Liu and Donald W. Schaffner, Ph. D.

Food Risk Analysis Initiative, Rutgers University, 65 Dudley Road, New Brunswick, NJ 08901-8520, Tel: (732) 932-9611 ext. 214, Fax: (732) 932-6776, Email: schaffner@aesop.rutgers.edu

Sprouts products has become a popular produce product, which are often consumed freshly. Even very low concentration of pathogen is able to grow exponentially and reach a high level posing a risk for human consumption. Sprouts are regarded as Potentially Hazardous Foods (PHF) which requires critical Time/Temperature control to reduce the risk of foodborne illness in the post-harvesting stage. Previous researches have shown that Salmonella population on contaminated seeds could gain 5-6 log increase during sprouting process. On the other hand, evidences have shown that Salmonella growth could be reduced by nutrient depletion or community level competitive inhibition at the late stage of sprouting.  This research has shown that alfalfa sprouts held at room temperature does not support the growth of Salmonella. Although it is obviously a sanitary or quality issue of sprouts product.

Below are excerpts from two web sites and the conclusions we are drawing from them for a position statement we are pulling together for the International Sprout Growers Assn. (ISGA).

(1.)  “Most Salmonella serotypes can grow over the temperature range 7 – 48 ºC, but growth is slow at temperatures below 10 ºC. (50 ºF.) .Reports suggesting that some serotypes can grow at temperatures as low as 4 ºC are not universally accepted. Nevertheless Salmonella is able to survive for extended periods in chilled and frozen foods.” (See link).


This article suggests that below 7 ºC. (44.6 ºF.) Salmonella is unlikely to grow, and that growth is slow up to 50 ºF.

(2.) “Mesophiles, which include virtually all human pathogens, have an optimum growth range of between 30 °C (86 °F) and 45 °C (113 °F), and a minimum growth temperature ranging from 5 to 10 °C (41 to 50 °F).”


Both articles indicate that probably the maximum temperature for sprouts would be 50 °F. but optimum (according to the Foodsafetywatch article) would be below 44.6°F.

(Because of their normal high bacterial load (8.0 log CFU/g) several studies have shown that sprouts inoculated with Salmonella or Listeria M. after 48 to 72 hours of sprouting, do not allow growth of the pathogen… most likely due to competition from the normal flora. This suggests that storage temperature is probably not a safety issue with sprouts. This is the part that the ISGA is working with researchers and FDA in putting together an official position statement.)