limit of detection

For the listeria experiment, what is the theoretical limit of detection for the enrichment procedure and what are the advantages of using two selective enrichment procedure than just one?

For this question, the solution depends on which type of enrichment methods used to detect the Listeria monocytogenes found in foods. Listeria monocytogenes is a foodborne pathogen commonly present in foods, such as dairy products, meats, vegetables, and seafood. (1 ) Listeria monocytogenes can cause listeriosis, and the most common symptoms are meningitis, encephalitis, septicaemia, and flu-like symptoms. (1) Pregnant women that are infected with Listeria monocytogenes can result in premature birth. (1) In the US, the tolerance level for the presence of L. monocytogenes allow in food is zero. (1) This means that the detection level for listeria should be very low. (1)
The are several methods for the detection of listeria and each of these culture-based methods have a different theoretical limit of detections.

In the method developed by the FDA for the detection of L. monocytogenes in dairy, fruit, vegetable, and seafood; the theoretical limit of detection is < 0.7 cfu/ml. (1) The theoretical limit of detection is the lowest concentration of Listeria cells that can be detected by the procedure. In this case, it is less than 0.7 colony forming units/ ml.

The steps in the enrichment for this method required the growth in FDA enrichment broth (FDA-EB) containing naladixic acid, acriflavine, cycloheximide for 24 hour at 30 degree Celsius. (1) Then, the culture is further subcultured and enriched by selectively using FDA-EB media and incubate for 48 hour at 30 degree Celsius. (1) After this, the subculture is plated onto Oxford and LPM agars for the further isolation of Listeria colonies. (1) To further validate the Listeria colonies, biochemical and metabolic assays must be performed in order to confirm their identification. (1) After the enrichment steps, additional tests must be performed to confirm the identity of the Listeria species. (1) The physiology of the Listeria should be determined using gram-staining and catalase activity testing. (1) If the culture is positive for Listeria, the cells should be gram positive and positive for catalase activity. (1) The culture should also be tested for the ability to induce haemolysis on blood agar, and for mannitol degradation. (1)

In contrast, another method approved by the International Organization for Standardization (ISO) required that the theoretical limit of detection for the enrichment of Listeria to be between 5-100 cfu/25g from all foods. (1) This means that the lowest level of Listeria cells that can be detected by this method is between 5-100 colony forming units/ 25 g of food.

This method utilized a different media for the enrichment of the Listeria. The Listeria is cultured in Fraser broth for 24 h at 30 degree Celsius, and then subcultured in Fraser broth containing ammmonium iron (III) citrate, acriflavine, and nalidixic acid for 48 hour at 37 Celsius. (1) In addition, the culture is further enriched with PALCAM or Agar Listeria Ottavani and Agosti (ALOA) plates. (1) The purpose of the ALOA plates is to distinguish one Listeria species from another. In this case, ALOA plate is selective for L. monocytogenes. (1)

Another method of enrichment developed by the Netherlands Government Food Inspection Service (NGFIS) has a theoretical limit of detection for the enrichment procedure to be about < 10 cfu/g in all foods, especially meat, fermented sausage, mushroom, and cheese. (1) This means that the lowest level of Listeria cells that can be detected in food is < 10 colony forming units/ gram of food.

In this method, the Listeria is grown in Listeria enrichment broth (LEB) at 30 Celsius for 48 hours, and subcultured onto PALCAM agar. (1) PALCAM agar is used to distingush between L. monocytogenes and other species in the culture. (1)

The advantages in using two or more selective enrichment methods rather than just one is that the success of the culturing of Listeria depends on how selective the media tend to become. One media may be more selective than another. So using additional selective media to subculture the Listeria can be very beneficial in the purification of the Listeria, and help in the identification of the optimum detection limit. (1) The requirement for more than one enrichment methods can selectively distinguished between target organisms and others non-target competitors in the same culture. (1) Ideally, the enrichment and detection method should be sensitive enough to detect organisms as low as 1 cell/g of food material, and be specific only to the target pathogen within the genus. (1) Using more than one methods can accurately detect the Listeria, and prevent false positives.

The theoretical limit of detection is the lowest level of concentration that can be detected by the listeria experiment. For example, if the lowest detection concentration of Listeria cells in the culture is 5 cfu/ml. That means that it only takes 5 colony forming units per 1ml of culture for the culture to be positive for Listeria.