á2022ñMICROBIOLOGICAL PROCEDURES FOR ABSENCE OF SPECIFIED MICROORGANISMS—NUTRITIONAL AND DIETARY SUPPLEMENTS
INTRODUCTION
Good manufacturing practices require that objectionable organisms be absent from nonsterile nutritional and dietary products.Amicroorganism 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 of 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 á2023ñ.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 microorganismsm 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
General Considerations
SeeBuffer Solution and Media underMicrobial Enumeration Tests—Nutritional and Dietary Supplements á2021ñ.The appropriateness of each medium for the intended purpose is to be assessed.Control sets ofFluid Soybean–Casein Digest Medium forPreparatory 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 100cfu per mL.Observe the growth to establish the appropriateness of the media.
Buffer
Buffer Stock Solution and pH7.2Phosphate Buffer—
Proceed as directed underMicrobial Enumeration Tests—Nutritional and Dietary Supplements á2021ñ.
Media
FLUID SOYBEAN–CASEIN DIGEST MEDIUM
Prepare as directed under Microbial Enumeration Tests—Nutritional and Dietary Supplements á2021ñ.
MANNITOL–SALT–AGAR MEDIUM
| Pancreatic Digest of Casein | 5.0g |
| Peptic Digest of Animal Tissue | 5.0g |
| Beef Extract | 1.0g |
| D-Mannitol | 10.0g |
| Sodium Chloride | 75.0g |
| Agar | 15.0g |
| Phenol Red | 0.025g |
| Water | 1000mL |
Mix,then heat with frequent agitation,and boil for 1minute to effect solution.
pHafter sterilization:7.4±0.2.
FLUID TETRATHIONATE MEDIUM
| Pancreatic Digest of Casein | 2.5g |
| Peptic Digest of Animal Fat | 2.5g |
| Bile Salts | 1.0g |
| Calcium Carbonate | 10.0g |
| Sodium Thiosulfate | 30.0g |
| Water | 1000mL |
Heat to boiling.Do not autoclave;use the same day.Immediately before use,add a solution prepared by dissolving 5g of potassium iodide and 6g of iodine in 20mLof water.Then add 10mLof a solution of brilliant green (1in 1000),and mix.Do not heat after adding the brilliant green solution.
BRILLIANT GREEN–AGAR MEDIUM
| Yeast Extract | 3.0g |
| Peptic Digest of Animal Tissue | 5.0g |
| Pancreatic Digest of Casein | 5.0g |
| Lactose | 10.0g |
| Sodium Chloride | 5.0g |
| Sucrose | 10.0g |
| Phenol Red | 80.0g |
| Agar | 20.0g |
| Brilliant Green | 12.5mg |
| Water | 1000mL |
Boil for 1minute.Sterilize just prior to use,melt,pour into Petri dishes,and allow to cool.
pHafter sterilization:6.9±0.2
XYLOSE–LYSINE–DESOXYCHOLATE–AGAR MEDIUM
| Xylose | 3.5g |
| L-Lysine | 5.0g |
| Lactose | 7.5g |
| Sucrose | 7.5g |
| Sodium Chloride | 5.0g |
| Yeast Extract | 3.0g |
| Phenol Red | 80mg |
| Agar | 13.5g |
| Sodium Desoxycholate (as Bile Salts) | 2.5g |
| Sodium Thiosulfate | 6.8g |
| Ferric Ammonium Citrate | 800mg |
| Water | 1000mL |
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 Mediumhas cooled.
,and pour into Petri plates as soon as the Mediumhas cooled.Final pH:7.4±0.2.
HEKTOEN ENTERIC AGAR MEDIUM
| Protease Peptone | 12.0g |
| Yeast Extract | 3.0g |
| Lactose | 12.0g |
| Sucrose | 2.0g |
| Salicin | 9.0g |
| Bile Salts No.3 | 9.0g |
| Sodium Chloride | 5.0g |
| Sodium Thiosulfate | 5.0g |
| Ferric Ammonium Citrate | 1.5g |
| Acid Fuchsin | 0.1g |
| Bromothymol Blue | 65mg |
| Agar | 14.0g |
| Water | 1000mL |
Mix,and allow to stand for 10minutes.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.
,and pour into Petri dishes.Final pH:7.5±0.2.
TRIPLE SUGAR–IRON–AGAR MEDIUM
| Pancreatic Digest of Casein | 10.0g |
| Pancreatic Digest of Animal Tissue | 10.0g |
| Lactose | 10.0g |
| Sucrose | 10.0g |
| Dextrose | 1.0g |
| Ferrous Ammonium Sulfate | 200mg |
| Sodium Chloride | 5.0g |
| Sodium Thiosulfate | 200mg |
| Agar | 13.0g |
| Phenol Red | 25mg |
| Water | 1000mL |
pHafter sterilization:7.3±0.2.
MACCONKEY AGAR MEDIUM
| Pancreatic Digest of Gelatin | 17.0g |
| Pancreatic Digest of Casein | 1.5g |
| Peptic Digest of Animal Tissue | 1.5g |
| Lactose | 10.0g |
| Bile Salts Mixture | 1.5g |
| Sodium Salts Mixture | 5.0g |
| Agar | 13.5g |
| Neutral Red | 30mg |
| Crystal Violet | 1.0mg |
| Water | 1000mL |
Boil for 1minute to effect solution.
pHafter sterilization:7.1±0.2.
LEVINE EOSIN–METHYLENE BLUE–AGAR MEDIUM
| Pancreatic Digest of Gelatin | 10.0g |
| Dibasic Potassium Phosphate | 2.0g |
| Agar | 15.0g |
| Lactose | 10.0g |
| Eosin Y | 400mg |
| Methylene Blue | 65mg |
| Water | 1000mL |
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 100mLof the liquefied agar solution,add 5mLof lactose solution (1in 5),2mLof the eosin Ysolution (1in 50),and 2mLof methylene blue solution (1in 300).Mix.The finished Mediummay not be clear.
pHafter sterilization:7.1±0.2.
BAIRD–PARKER AGAR MEDIUM
| Pancreatic Digest of Casein | 10.0g |
| Beef Extract | 5.0g |
| Yeast Extract | 1.0g |
| Lithium Chloride | 5.0g |
| Agar | 20.0g |
| Glycine | 12.0g |
| Sodium Pyruvate | 10.0g |
| Water | 950mL |
Heat with frequent agitation,and boil for 1minute.Sterilize,cool to between 45and 50,and add 10mLof sterile potassium tellurite solution (1in 100)and 50mLof 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 TSto obtain a 3to 7ratio of egg yolk to saline,add to a sterile blender cup,and mix at high speed for 5seconds.Mix all ingredients well but gently,and pour into plates.
and 50,and add 10mLof sterile potassium tellurite solution (1in 100)and 50mLof 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 TSto obtain a 3to 7ratio of egg yolk to saline,add to a sterile blender cup,and mix at high speed for 5seconds.Mix all ingredients well but gently,and pour into plates.pHafter sterilization:6.8±0.2.
VOGEL–JOHNSON AGAR MEDIUM
| Pancreatic Digest of Casein | 10.0g |
| Yeast Extract | 5.0g |
| Mannitol | 10.0g |
| Dibasic Potassium Phosphate | 5.0g |
| Lithium Chloride | 5.0g |
| Glycine | 10.0g |
| Agar | 16.0g |
| Phenol Red | 25.0mg |
| Water | 1000mL |
Boil for 1minute.Sterilize,cool to between 45and 50,and add 20mLof sterile potassium tellurite solution (1in 100).
and 50,and add 20mLof sterile potassium tellurite solution (1in 100).pHafter sterilization:7.2±0.2.
FLUID SELENITE–CYSTINE MEDIUM
| Pancreatic Digest of Casein | 5.0g |
| Lactose | 4.0g |
| Sodium Phosphate | 10.0g |
| Sodium Acid Selenite | 4.0g |
| L-Cystine | 10.0g |
| Water | 1000mL |
Mix,and heat to effect solution.Then heat in flowing stream for 15minutes.Do not sterilize.
Final pH:7.0±0.2.
REINFORCED MEDIUM FOR CLOSTRIDIA
| Beef Extract | 10.0g |
| Peptone | 10.0g |
| Yeast Extract | 3.0g |
| Soluble Starch | 1.0g |
| Glucose Monohydrate | 5.0g |
| Cysteine Hydrochloride | 0.5g |
| Sodium Chloride | 5.0g |
| Sodium Acetate | 3.0g |
| Agar | 0.5g |
| Water | 1000mL |
Dissolve agar in water by heating to boiling,while stirring continuously.Adjust the pHif necessary,and sterilize.
pHafter sterilization:6.8±0.2.
COLUMBIA AGAR
| Pancreatic Digest of Casein | 10.0g |
| Meat Peptic Digest | 5.0g |
| Heart Pancreatic Digest | 3.0g |
| Yeast Extract | 5.0g |
| Cornstarch | 1.0g |
| Sodium Chloride | 5.0g |
| Agar | 15.0g |
| Water | 1000mL |
Dissolve agar in water by heating to boiling and with continuous stirring.If necessary,adjust the pH.Sterilize,and allow to cool to 45to 50.Add,when necessary,gentamicin sulfate,equivalent to about 20mg of gentamicin base,and pour into Petri dishes.
to 50.Add,when necessary,gentamicin sulfate,equivalent to about 20mg of gentamicin base,and pour into Petri dishes.Pre-reduction of the medium is recommended.
pHafter sterilization:7.3±0.2.
Rappaport Vassiliadis Salmonella Enrichment Broth
| Soya Peptone | 4.5g |
| Magnesium Chloride Hexahydrate | 29.0g |
| Sodium Chloride | 8.0g |
| Dipotassium Phosphate | 0.4g |
| Potassium Dihydrogen Phosphate | 0.6g |
| Malachite Green | 0.036g |
| Purified Water | 1000mL |
Dissolve,warming slightly.Sterilize in an autoclave using a validated cycle,at a temperature not exceeding 115.
.The pHis 5.2±0.2at 25after heating and autoclaving.
after heating and autoclaving.PREPARATORY TESTING
Proceed as directed for Preparatory Testingunder Microbial Enumeration Tests—Nutritional and Dietary Supplements á2021ñ.
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,and4).
SAMPLING
Proceed as directed for Sampling under Microbial Enumeration Tests—Nutritional and Dietary Supplements á2021ñ.
TEST PROCEDURES
Test Preparation—
Prepare as directed for Sampling.Transfer to a suitable container with 100mLofFluid Soybean–Casein Digest Medium (FSCD).Mix by shaking gently.[NOTE—On the basis of results forPreparatory Testing,modify theTest Preparation as appropriate.]
Test for Absence of Staphylococcus aureus
Incubate at 30to 35for 18to 24hours.Streak a loopful from FSCDonto the surface of one or more of the following media:Vogel–Johnson Agar Medium (VJ Agar),Mannitol–Salt–Agar Medium (MS-Agar),and Baird-Parker Agar Medium (BP Agar).Cover the Petri plates,invert them,and incubate at 30to 35for 24to 48hours.
to 35for 18to 24hours.Streak a loopful from FSCDonto the surface of one or more of the following media:Vogel–Johnson Agar Medium (VJ Agar),Mannitol–Salt–Agar Medium (MS-Agar),and Baird-Parker Agar Medium (BP Agar).Cover the Petri plates,invert them,and incubate at 30to 35for 24to 48hours.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 0.5mLof rabbit plasma,horse plasma,or any other mammalian plasma.Incubate in a water bath at 37.Examine for coagulation after 3hours of incubation and at suitable intervals up to 24hours.Comparing with positive and negative controls,the absence of a coagulase reaction indicates the absence of Staphylococcus aureusin the tested article.
.Examine for coagulation after 3hours of incubation and at suitable intervals up to 24hours.Comparing with positive and negative controls,the absence of a coagulase reaction indicates the absence of Staphylococcus aureusin the tested article.
Table 1.Characteristics of Staphylococcus aureus on Specified Agar Media
| Agar Medium | Colonial Morphology | Gram Stain |
| Vogel–Johnson | Black surrounded by yellow zone | (+),cocci |
| Mannitol–Salt | Yellow colonies with yellow zone | (+),cocci |
| Baird–Parker | Black,shiny surrounded by 2-5-mm clear zones | (+),cocci |
Test for Absence of SalmonellaSpecies
Incubate at 30to 35for 18to 24hours.FromFSCD,pipet a 1-mLaliquot into 10mLof Rappaport Vassiliadis Salmonella Enrichment Broth,mix,and incubate at 30to 35for 18to 24hours.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),andHektoen Enteric Agar Medium (HE Agar).Cover,invert the plates,and incubate at 30to 35for 24to 48hours.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 Salmonellaspecies.If colonies with characteristics described in Table 2are 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 30to 35for 24to 48hours.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 Salmonellaspecies.
to 35for 18to 24hours.FromFSCD,pipet a 1-mLaliquot into 10mLof Rappaport Vassiliadis Salmonella Enrichment Broth,mix,and incubate at 30to 35for 18to 24hours.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),andHektoen Enteric Agar Medium (HE Agar).Cover,invert the plates,and incubate at 30to 35for 24to 48hours.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 Salmonellaspecies.If colonies with characteristics described in Table 2are 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 30to 35for 24to 48hours.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 Salmonellaspecies.
Table 2.Characteristics of Salmonella Species on Specified Agar Media
| Agar Medium | Colonial Morphology | Gram Stain |
| Brilliant Green | Small,transparent and colorless;or opaque,pink or white (often surrounded by pink to red zone) |
(–),rods |
| Xylose–Lysine–Desoxycholate | Red,with or without black centers | (–),rods |
| Hektoen Enteric | Blue-green,with or without black centers | (–),rods |
Test for Absence of Escherichia coli
Incubate at 30to 35for 24to 48hours.FromFSCD,pipet a 1-mLaliquot into a container containing 10mLofMacConkey Broth,mix,and incubate at 42to 44for 24to 48hours.Streak a loopful from both incubated media onto individual surfaces of MacConkey Agar Medium (MC Agar),and incubate at 30to 35for 18to 24hours.Examine the inoculated MC Agarplate,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 3are 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 30to 35for 24to 48hours.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.
to 35for 24to 48hours.FromFSCD,pipet a 1-mLaliquot into a container containing 10mLofMacConkey Broth,mix,and incubate at 42to 44for 24to 48hours.Streak a loopful from both incubated media onto individual surfaces of MacConkey Agar Medium (MC Agar),and incubate at 30to 35for 18to 24hours.Examine the inoculated MC Agarplate,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 3are 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 30to 35for 24to 48hours.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 onMacConkey Agar Medium
| Colonial Morphology | Gram Stain |
| Brick red,may have surrounding zone of precipitated bile | (-),rods |
Test for Absence of Clostridium Species
Test Preparation—
Prepare as directed for Sampling.[NOTE—On the basis of results for Preparatory Testing,modify the Test Preparationas appropriate.]
Procedure—
Take two equal portions of the Test Preparation,heat one to 80for 10minutes,and cool rapidly.Transfer 10mLof each portion to separate containers,each containing 100mLof Reinforced Medium for Clostridia,and incubate under anaerobic conditions at 35to 37for 48hours.After incubation,subculture each specimen on Columbia Agar Mediumto which gentamicin has been added,and incubate under anaerobic conditions at 35to 37for 48hours.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 Clostridiumspecies.
for 10minutes,and cool rapidly.Transfer 10mLof each portion to separate containers,each containing 100mLof Reinforced Medium for Clostridia,and incubate under anaerobic conditions at 35to 37for 48hours.After incubation,subculture each specimen on Columbia Agar Mediumto which gentamicin has been added,and incubate under anaerobic conditions at 35to 37for 48hours.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 Clostridiumspecies.
Table 4.Characteristics of Clostridium Species on Specified Media
| Medium | Gram Stain | Catalase |
| Reinforced Medium for Clostridia | (+),rods | |
| Columbia Agar | (+),rods | Negative |
If growth occurs,subculture each distinct colony on Columbia Agar Medium,and separately incubate in aerobic and in anaerobic conditions at 35to 37for 48hours.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 Clostridiumspecies.
to 37for 48hours.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 Clostridiumspecies.
Retest
For the purpose of confirming a doubtful result by any of the procedures outlined in the foregoing tests following their application to a 10g specimen,a retest on a 25g specimen of the nutritional or dietary supplement may be conducted.Proceed as directed under Procedure,but make allowances for the larger specimen size.