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Research Note: Two Processing Methods for the Isolation of Salmonella from Naturally Contaminated Alfalfa Seeds

Journal of Food Protection: Vol. 64, No. 8, pp. 1240–1243.
Gregory B. Inami, Sue Ming C. Lee, Robin W. Hogue, and Rita A. Brenden
California Department of Health Services, Microbial Diseases Laboratory, 2151 Berkeley Way, Berkeley, California 94704, USA

Abstract—Two processing methods were examined for the recovery of Salmonella from naturally contaminated alfalfa seed. Seed samples, from each of three investigations, were processed by sprouting and shredding before preenrichment and culture. In lot A, Salmonella serotype Newport was isolated from 3 of 30 sample units with the sprouting method and 2 of 30 with the shredding method. In lot B, three serotypes in various combinations were isolated from 10 of 30 sample units with the sprouting method and 9 of 30 with the shredding method. In lot C, Salmonella group C₁ was isolated from 27 of 30 sample units with the sprouting method and 24 of 30 with the shredding method. Additionally, serotype Newport was found in one lot C sample unit. Using shredded seed data, a most probable number (MPN) for Salmonella contamination per lot was calculated. Serotype Newport was estimated at 0.07 MPN/100 g in lot A; the concentration for three serotypes was estimated to be 0.36 MPN/100 g in lot B; Salmonella group C₁ was estimated at 1.8 MPN/100 g in lot C. Our success in isolating Salmonella from alfalfa seeds was likely attributed to the volume of material tested and the quick acquisition of the seeds after the outbreak was identified. Shredding the seeds was easier and yielded definitive results more quickly than sprouting.

Bob Rust’s (ISS) Comments on the Above Research

You only need to find one pathogen in a seed lot to determine it is unfit for human consumption.  In this study, the researchers were able to determine pathogens 100% of the time.  For instance, in lot A, they found pathogens in 3 of the 30 samples.  This may sound like 10% of the time, but because they took 30 samples, they found it not just once, which would have rejected it, but three times.  They used two different methods for each lot, making the total samples 60.  If you pull one sample out of a lot and test it, odds are, you won’t find it.  Pull 60 samples and the odds increase dramatically.

Lot A,   5 of 60 = REJECTED
Lot B, 19 of 60 = REJECTED
Lot C, 51 of 60 = REJECTED 

Using the formula, Probability = 1-(C/T)^N, I calculate the probability of finding a pathogen in each of these lots is:

Lot A (.07 MPN/100g) = 98.5%
Lot B (.36 MPN/100g) = 99.9999999%
Lot C (1.8 MPN/100g) = 99.9^30+%

With such probabilities it is no wonder the researchers were able to find a pathogen in each lot more than once.

(Note:  (C/T) = assumed ratio of clean seeds to total.  N = the number of seeds sampled.)

Lot A is the least contaminated with a most probable number of 0.7 cells per kilogram.  A seed trier (at least the one we use), draws 25 grams.  When we pull samples from every bag of a truckload we are pulling 880 samples.  Statistically, the odds of capturing a pathogen in this lightly contaminated seed are in excess of 99.9999%.   In the heavily contaminated seed the probabilities are astronomical.

If you would like a copy of the entire article please give your ISS or Sungarden salesperson a call.

Bob Sanderson's Comments on the Same Article

Dear Bob (Rust)

"Congratulations! Not only for your courage and successful outcome in this lawsuit, but wait a minute- since when are you getting listed in JFP article references?

I am quite impressed with the Inami et al research note in the August JFP. For several reasons. One of course being that you are deservedly mentioned.

If I read Greg Unami's report right, he seemed to find that taking a 3Kg sample from each lot, they had no trouble finding contamination in the three lots they looked at, even in one seed which was only contaminated at .7cfu/Kg, which is much lower than the 4 cfu/kg mentioned as being very low (and by implication very hard to detect) in the White Paper.

It looks as though they got a positive from this seed (Lot A; going on 5 years old) 3 times out of 30 using the sprouting method. To me, that means that any 100 g sample of that seed, grown out and tested, would have a 10% likelihood of being positive. Therefore, sampling and testing 30: 100 g samples, the likelihood of detection becomes as close to 100% as death and taxes.

Using the formula P = 1-(C/T)^N, the probability of getting a positive from a 100g sample of seed contaminated at .7cfu/kg would be: P = 1-(9993/10000)^100, which solves to P = 6.7%, rather than the 10% which they got using the Thomas formula. So the two are close, but my formula is a little more conservative. Well, I'm a conservative kind of person.

The contamination levels in the other two seed lots were estimated at 3.6 cfu/Kg and 18 cfu/Kg. Using my P formula, a 100 g sample of the first would have a P = 1-(9964/10000)^100, or 30% likelihood of being positive. Inami got 10 out of 30, for 33%. The third seed lot probability would be P = 1-(982/1000)^100, or 84%. Inami got 27 out of 30, or 90%.

It seems that the whole emphasis of the paper is in terms of researchers being able to determine contamination levels in seed, rather than as a method for suppliers or growers to determine if seed is contaminated.

You know what gets my goat (along with about a thousand other things): If people get sick because a simple procedure is not being used because the FDA says it isn't any good, and meanwhile the grower failed to use another procedure which the FDA says they should use, chlorine, which is horrible and dangerous and doesn't work very well, and someone's ass ends up in the fire, and the FDA issues another press release about how dangerous the product is and what a bunch of slobs the sprout growers are... that gets my goat. How's your goat?

I would think that if there is another outbreak, and it can be determined that it could have been averted if Inami's sampling and testing had been done, that the victims might consider a class action lawsuit against the FDA, if such things are possible."

Bob Sanderson