2022 MICROBIOLOGICAL PROCEDURES FOR ABSENCE OF SPECIFIED MICROORGANISMSNUTRITIONAL AND DIETARY SUPPLEMENTS
Good manufacturing practices require that objectionable organisms be absent from nonsterile nutritional and dietary products. A microorganism can be considered objectionable if it represents a potential health hazard to the user who is using the product as directed, or if it is capable of growing in the product. Objectionable microorganisms are defined as contaminants that, depending on the microbial species, number of organisms, dosage form, intended use, and patient population, would adversely affect product safety. Additionally, microorganisms may be deemed objectionable if they adversely affect product stability or if they may damage the integrity of the container closure system.
This chapter describes the testing of nutritional and dietary articles for specified microorganisms, which are specified in the individual monographs or whose absence is recommended by the guidance under Microbiological Attributes of Nonsterile Nutritional and Dietary Supplements 2023. When objectionable microorganisms are not specified in the individual monograph, it is the manufacturers' responsibility to determine which microorganisms in their products are objectionable. It is not intended that all nonsterile nutritional and dietary articles be tested for the absence of all of the microorganisms mentioned in this chapter, nor is the testing of relevant microorganisms restricted to those presented in this chapter.
Alternative microbiological, physicochemical, and biotechnological methods, including automated methods, may be substituted for these tests, provided they have been validated as being equivalent in their suitability for determining compliance.
BUFFER AND MEDIA
See Buffer Solution and Media under Microbial Enumeration TestsNutritional and Dietary Supplements 2021. The appropriateness of each medium for the intended purpose is to be assessed. Control sets of Fluid SoybeanCasein Digest Medium for Preparatory Testing are also used to assess the appropriateness of these media in the growth promotion of the specified microorganisms. For other media, streak agar plates to obtain isolated colonies of appropriate microorganisms, and inoculate the fluid media with the appropriate microorganisms at a final concentration of less than 100 cfu per mL. Observe the growth to establish the appropriateness of the media.
Buffer Stock Solution and pH 7.2 Phosphate Buffer Proceed as directed under Microbial Enumeration TestsNutritional and Dietary Supplements 2021.
fluid soybeancasein digest medium
Prepare as directed under Microbial Enumeration TestsNutritional 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.
fluid tetrathionate medium
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.
brilliant greenagar medium
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.
hektoen enteric agar medium
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.
triple sugarironagar medium
pH after sterilization: 7.3 ± 0.2.
macconkey agar medium
Boil for 1 minute to effect solution.
pH after sterilization: 7.1 ± 0.2.
levine eosinmethylene blueagar medium
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.
bairdparker agar medium
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.
vogeljohnson agar medium
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.
fluid selenitecystine medium
Mix, and heat to effect solution. Then heat in flowing stream for 15 minutes. Do not sterilize.
Final pH: 7.0 ± 0.2.
reinforced medium for clostridia
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.
rappaport vassiliadis salmonella enrichment broth
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.
Proceed as directed for Preparatory Testing under Microbial Enumeration TestsNutritional and Dietary Supplements 2021.
For enrichment broth, selective media, and differential media use an inoculating loop to transfer the inoculum of each test organism to the plated or liquid media being tested. If a plated medium is being tested, streak the surface of plate with the loop in four directions to obtain a pattern of isolated colonies. Incubate the media, and examine the plated or liquid media for the characteristic growth of the inocula (See Tables 1, 2, 3, and 4).
Proceed as directed for Sampling under Microbial Enumeration TestsNutritional and Dietary Supplements 2021.
Test Preparation Prepare as directed for Sampling. Transfer to a suitable container with 100 mL of Fluid SoybeanCasein Digest Medium (FSCD). Mix by shaking gently. [NoteOn the basis of results for Preparatory Testing, modify the Test Preparation as appropriate. ]
Test for Absence of Staphylococcus aureus
Incubate at 30 to 35 for 18 to 24 hours. Streak a loopful from FSCD onto the surface of one or more of the following media: VogelJohnson Agar Medium (VJ Agar), MannitolSaltAgar Medium (MS-Agar), and Baird-Parker Agar Medium (BP Agar). Cover the Petri plates, invert them, and incubate at 30 to 35 for 24 to 48 hours.
Examine the plates of VJ Agar, MS-Agar, and/or BP Agar, and interpret the results with reference to Table 1: if no plate contains colonies having the characteristics described, the test specimen meets the requirement for the absence of Staphylococcus aureus. If characteristic colonies are present, perform coagulase test as follows. Transfer representative colonies to separate tubes containing 0.5 mL of rabbit plasma, horse plasma, or any other mammalian plasma. Incubate in a water bath at 37. Examine for coagulation after 3 hours of incubation and at suitable intervals up to 24 hours. Comparing with positive and negative controls, the absence of a coagulase reaction indicates the absence of Staphylococcus aureus in the tested article.
Table 1. Characteristics of Staphylococcus aureus on Specified Agar Media
Test for Absence of Salmonella Species
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), XyloseLysineDesoxycholateAgar 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 SugarIronAgar 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
Test for Absence of Escherichia coli
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 EosinMethylene BlueAgar 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
Test for Absence of Clostridium Species
Test Preparation Prepare as directed for Sampling. [NoteOn the basis of results for Preparatory Testing, modify the Test Preparation as appropriate. ]
Procedure Take two equal portions of the Test Preparation, heat one to 80 for 10 minutes, and cool rapidly. Transfer 10 mL of each portion to separate containers, each containing 100 mL of Reinforced Medium for Clostridia, and incubate under anaerobic conditions at 35 to 37 for 48 hours. After incubation, subculture each specimen on Columbia Agar Medium to which gentamicin has been added, and incubate under anaerobic conditions at 35 to 37 for 48 hours. Examine the plates, and interpret with reference to Table 4: if no growth of microorganisms is detected, the test specimen meets the requirement for the absence of Clostridium species.
Table 4. Characteristics of Clostridium Species on Specified Media
If growth occurs, subculture each distinct colony on Columbia Agar Medium, and separately incubate in aerobic and in anaerobic conditions at 35 to 37 for 48 hours. The occurrence of only anaerobic growth of gram-positive bacilli, giving a negative catalase reaction, indicates the presence of Clostridium sporogenes. To perform the catalase test, transfer discrete colonies to glass slides, and apply a drop of dilute hydrogen peroxide solution: the reaction is negative if no gas bubbles evolve. If the test specimen exhibits none of these characteristics, it meets the requirement for the absence of Clostridium species.
For the purpose of confirming a doubtful result by any of the procedures outlined in the foregoing tests following their application to a 10 g specimen, a retest on a 25 g specimen of the nutritional or dietary supplement may be conducted. Proceed as directed under Procedure, but make allowances for the larger specimen size.
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