2.1.1 Lot: A batch or production unit which may be identified by a code. When there is no code identification, see Table II

2.1.2 Sample: The sample units (subsamples) taken per lot for analysis.

2.1.3 Sample Unit: A minimum of 25 g of whole froglegs. A sample unit is often referred to as a subsample.

2.1.4 Analytical Unit: That amount of product withdrawn from the sample unit for analysis. Here, it may be equal to the sample unit.

2.2.1 A sample, consisting of five sample units drawn at random from a lot, shall be taken. (Table I)

2.2.2 Each sample unit shall consist of at least 25 g of whole froglegs.

2.2.3 Employ aseptic techniques in collecting the sample units.

2.2.4 Place each collected sample unit into a separate sterile container.

2.2.5 Keep sample units frozen during transport.

3.1.1 Keep sample units frozen in the laboratory prior to analyzing them.

3.1.2 Analyze sample units as soon as possible after receipt at the laboratory.

3.3.1 Thaw frozen sample units. Do not allow the temperature of any sample unit to exceed 45°C.

3.3.2 Place a minimum of 25g of whole froglegs (analytical unit) from each of the sample units into a separate sterile container.

3.3.3 Alternatively, composite the analytical units and place each composite into a separate sterile container.

Since compositing presents difficulties in preparation and disposal of the material, exercise care in handling bulk preparations.

3.3.4 Add sufficient NB to cover the froglegs.

3.3.5 Shake the container(s) to unite the contents.

3.3.6 Check the pH of each suspended analytical or composite unit. If the pH is outside the range of 6.0 - 7.0, adjust to 7.0 with either sterile NaOH or HCl.

3.3.7 Inoculate NB with a known culture of Salmonella and subsequently make transfers to all other media used in the analysis. This is the positive media control. Set up a negative control by incubating appropriate uninoculated media during each step of the analysis.

3.3.8 Incubate the inoculated pre-enrichment broth(s) and the controls at 35°C ± 0.5^o for 18-24 hr. In no circumstances shall the incubation be prolonged for more than 24 hr.

3.4.1 Transfer 1 ml of the incubated pre-enrichment broth into each of 9 ml of SC and TBG broths using a sterile pipette.

3.4.2 Incubate the SC broth and TBG broth for 24 ± 2 hr at 35°C ± 0.5^o and at 43°C ± 0.5^o, respectively.

3.5.1 After the incubation period, streak a loopful of each of the selective enrichment broths onto BS agar and BGS agar plates to obtain well-isolated colonies. The broths may also be streaked onto a third commercially available plating medium.

3.5.2 Incubate the plates at 35°C ± 0.5^o for 24 ± 2 hr. It may be necessary to incubate the BS agar plates for 48 ± 2 hr.

3.5.3 Examine the plates after the incubation period for colonies indicative of Salmonella. On BGS agar, such colonies are pink to fuchsia surrounded by red medium. On BS agar, they are usually black, with or without a metallic sheen; with increasing time of incubation the surrounding medium is gradually blackened. It should be Noted, however, that lactose and/or sucrose-fermenting strains (e.g. S. arizonae) may develop a coliform-like (greenish) appearance on BGS agar. A heavy growth of coliforms may mask the appearance of the Salmonella colonies. On BS agar, some Salmonella types may form dark brown rather than black colonies.

3.5.4 If there are no colonies indicative of Salmonella on the plates, bacteria of the genus Salmonella are considered to be absent from the analytical or composite unit from which the colonies originated.

3.6.1 Streak suspect colonies onto MA plates for purification.

3.6.2 Incubate the agar plates at 35°C ± 0.5^o for 24 ± 2 hr.

3.6.3 Observe MA plates after the incubation period. Typical Salmonella colonies are lactose-negative and will appear colourless. However, lactose-positive biotypes are known to occur and may appear pink.

3.7.1 Using an inoculating needle, pick colonies indicative of Salmonella from the MA plates and inoculate the biochemical media listed in Table 1. Incubate these media at 35°C ± 0.5^o for 18 - 24 hr. Cap the tubes loosely to avoid the occurrence of erroneous results.

3.7.2 Other media may be used to observe the reactions listed in Table II. If additional biochemical information is desired, other reaction-media or commercially available diagnostic kits may be used.

3.7.3 If none of the isolates from a particular analytical or composite unit show biochemical reactions indicative of Salmonella, then bacteria of the genus Salmonella are considered to be absent from the analytical or composite unit from which the isolates originated. If Salmonella are suspected, proceed to serological testing.

3.7.4 Use TSI or LI agar cultures less than 72 hr old for the serological identification but store the cultures at 2-8°C if the serological identification is not carried out within 12 hr after the incubation of the TSI or LI agar cultures.

3.7.5 If the serological identification is not performed within 72 hr after the incubation period, streak suspect cultures onto NA slants.

3.7.6 Incubate the inoculated NA slants at 35°C ± 0.5^o for 24 ± 2 hr.

3.7.7 Use NA slant cultures less than 48 hr old for the serological identification but store the cultures at 2-8°C if the serological identification is not carried out within 12 hr after the incubation of the NA slant cultures.

3.8.1 Testing with somatic polyvalent antiserum (Groups A to I + Vi)

3.8.2 Testing with Somatic Grouping Antisera

It is preferable to test the culture against somatic grouping antisera whenever possible; however, the culture may be sent to a typing centre for identification. The majority of Salmonella isolated from foods belong to groups B, C, D, and E.

It is important to recognize that unless a complete set of grouping sera is available some Salmonella may be missed. In such cases any culture possessing the biochemical reactions indicative of Salmonella should be sent to a typing centre for identification.

3.8.3 If all of the serological tests performed on an isolate are negative but the original culture gave biochemical reactions indicative of Salmonella (see Table 1), that culture shall be sent to a typing centre for verification.