Prepare as directed under Microbial Enumeration Tests—Nutritional and Dietary Supplements 2021.

Mix, then heat with frequent agitation, and boil for 1 minute to effect solution.

pH after sterilization: 7.4 ± 0.2.

Heat to boiling. Do not autoclave; use the same day. Immediately before use, add a solution prepared by dissolving 5 g of potassium iodide and 6 g of iodine in 20 mL of water. Then add 10 mL of a solution of brilliant green (1 in 1000), and mix. Do not heat after adding the brilliant green solution.

Boil for 1 minute. Sterilize just prior to use, melt, pour into Petri dishes, and allow to cool.

pH after sterilization: 6.9 ± 0.2

Heat, with swirling, just to the boiling point. Do not overheat or sterilize. Transfer at once to a water bath maintained at about 50, and pour into Petri plates as soon as the Medium has cooled.

Final pH: 7.4 ± 0.2.

Mix, and allow to stand for 10 minutes. Heat gently, and allow to boil for a few seconds to dissolve the agar. Do not sterilize. Cool to 60, and pour into Petri dishes.

Final pH: 7.5 ± 0.2.

pH after sterilization: 7.3 ± 0.2.

Boil for 1 minute to effect solution.

pH after sterilization: 7.1 ± 0.2.

Dissolve pancreatic digest of gelatin, dibasic potassium phosphate, and agar in water, with warming, and allow to cool. Just prior to use, liquefy the gelled agar solution, and add the remaining ingredients, as solutions, in the following amounts: for each 100 mL of the liquefied agar solution, add 5 mL of lactose solution (1 in 5), 2 mL of the eosin Y solution (1 in 50), and 2 mL of methylene blue solution (1 in 300). Mix. The finished Medium may not be clear.

pH after sterilization: 7.1 ± 0.2.

Heat with frequent agitation, and boil for 1 minute. Sterilize, cool to between 45 and 50, and add 10 mL of sterile potassium tellurite solution (1 in 100) and 50 mL of egg yolk emulsion prepared as follows. Disinfect the surface of whole-shell eggs, aseptically crack the eggs, transfer intact yolks to a sterile graduated cylinder, add sterile saline TS to obtain a 3 to 7 ratio of egg yolk to saline, add to a sterile blender cup, and mix at high speed for 5 seconds. Mix all ingredients well but gently, and pour into plates.

pH after sterilization: 6.8 ± 0.2.

Boil for 1 minute. Sterilize, cool to between 45 and 50, and add 20 mL of sterile potassium tellurite solution (1 in 100).

pH after sterilization: 7.2 ± 0.2.

Mix, and heat to effect solution. Then heat in flowing stream for 15 minutes. Do not sterilize.

Final pH: 7.0 ± 0.2.

Dissolve agar in water by heating to boiling, while stirring continuously. Adjust the pH if necessary, and sterilize.

pH after sterilization: 6.8 ± 0.2.

Dissolve agar in water by heating to boiling and with continuous stirring. If necessary, adjust the pH. Sterilize, and allow to cool to 45 to 50. Add, when necessary, gentamicin sulfate, equivalent to about 20 mg of gentamicin base, and pour into Petri dishes.

Pre-reduction of the medium is recommended.

pH after sterilization: 7.3 ± 0.2.

Dissolve, warming slightly. Sterilize in an autoclave using a validated cycle, at a temperature not exceeding 115.

The pH is 5.2 ± 0.2 at 25 after heating and autoclaving.

Incubate at 30 to 35 for 18 to 24 hours. From FSCD, pipet a 1-mL aliquot into 10 mL of Rappaport Vassiliadis Salmonella Enrichment Broth, mix, and incubate at 30 to 35 for 18 to 24 hours. Streak a loopful from both incubated media onto individual surfaces of one or more of following media: Brilliant Green Agar Medium (BG-Agar), Xylose–Lysine–Desoxycholate–Agar Medium (XLDC-Agar), and Hektoen Enteric Agar Medium (HE Agar). Cover, invert the plates, and incubate at 30 to 35 for 24 to 48 hours. Examine the inoculated plates of BG-Agar, XLDC-Agar, and/or HE Agar, and interpret the results with reference to Table 2: if no colonies having the characteristics described are observed, the test specimen meets the requirement for the absence of Salmonella species. If colonies with characteristics described in Table 2 are present, the suspect colonies are transferred to a slant of Triple Sugar–Iron–Agar Medium (TSI) using an inoculating wire, by first streaking the surface of the slant, and then stabbing the wire well beneath the surface. Incubate at 30 to 35 for 24 to 48 hours. If the tubes do not have red alkaline slants and yellow acid butts, with or without concomitant blackening of the butts from hydrogen sulfide production, the test specimen meets the requirement for the absence of Salmonella species.

Table 2. Characteristics of Salmonella Species on Specified Agar Media

Incubate at 30 to 35 for 24 to 48 hours. From FSCD, pipet a 1-mL aliquot into a container containing 10 mL of MacConkey Broth, mix, and incubate at 42 to 44 for 24 to 48 hours. Streak a loopful from both incubated media onto individual surfaces of MacConkey Agar Medium (MC Agar), and incubate at 30 to 35 for 18 to 24 hours. Examine the inoculated MC Agar plate, and interpret the results with reference to Table 3: if no colonies having the characteristics described are observed, the test specimen meets the requirement for the absence of Escherichia coli. Suspect colonies showing the characteristics described in Table 3 are transferred individually, using an inoculating loop, to the surface of a plate with Levine Eosin–Methylene Blue–Agar Medium (LEMB-Agar). If a large number of suspect colonies are to be transferred, divide the surface of each plate into quadrants, each quadrant being inoculated with a different colony. Cover the plates, invert, and incubate at 30 to 35 for 24 to 48 hours. If none of the colonies exhibit a characteristic metallic sheen under reflected light, and if none exhibit a blue-black appearance under transmitted light, the test specimen meets the requirement for the absence of Escherichia coli.

Table 3. Characteristics of Escherichia coli on MacConkey Agar Medium

Medium	Gram Stain	Catalase
Reinforced Medium for Clostridia	(+), rods
Columbia Agar	(+), rods	Negative

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