á2021ñMICROBIAL ENUMERATION TESTS—NUTRITIONAL AND DIETARY SUPPLEMENTS
INTRODUCTION
This chapter provides tests for the estimation of the number of viable aerobic microorganisms present in nutritional supplements of all kinds,from raw materials to the finished forms.Alternative methods may be substituted for the tests,provided that they have been properly validated as giving equivalent or better results.In preparing for and in applying the tests,observe aseptic precautions in handling the specimens.The term “growth”is used in a special sense herein,i.e.,to designate the presence and presumed proliferation of viable microorganisms.
PREPARATORY TESTING
The validity of the results of the tests set forth in this chapter rests largely upon the adequacy of a demonstration that the test specimens to which they are applied do not,of themselves,inhibit the multiplication,under the test conditions,of microorganisms that may be present.Therefore,preparatory to conducting the tests on a regular basis and as circumstances require subsequently,inoculate diluted specimens of the material to be tested with separate viable cultures of the challenge microorganisms.
For theSoybean–Casein Digest Agar used forTotal Aerobic Microbial Counts,inoculate duplicate plates with 25to 250cfu of Staphylococcus aureus (ATCC1No.6538),Escherichia coli (ATCC No.8739),andBacillus subtilis (ATCC No.6633)to demonstrate a greater than 70%bioburden recovery in comparison to a control medium.For theSabouraud Dextrose Agar used forTotal Combined Yeast and Mold Counts,inoculate duplicate plates with 25to 250cfu ofCandida albicans (ATCC No.10231)andAspergillus niger (ATCC No.16404)to demonstrate a greater than 70%bioburden recovery in comparison to a control medium.ForEnterobacterial Probable Number Determinations (Bile-Tolerant Gram-Negative Bacteria),appropriate dilutions ofEscherichia coli (ATCC No.8739)andSalmonella typhimurium (ATCC No.13311)are used.Failure of the organism(s)to grow in the relevant medium invalidates that portion of the examination and necessitates a modification of the procedure by (1)an increase in the volume of diluent,the quantity of test material remaining the same,or by (2)the incorporation of a sufficient quantity of suitable inactivating agent(s)in the diluents,or by (3)an appropriate combination of modifications to (1)and (2)so as to permit growth of the inoculum.
The following are examples of ingredients and their concentrations that may be added to the culture medium to neutralize inhibitory substances present in the sample:soy lecithin,0.5%;and polysorbate 20,4.0%.Alternatively,repeat the test as described in the preceding paragraph,usingFluid Casein Digest–Soy Lecithin–Polysorbate 20Medium to demonstrate neutralization of preservatives or other antimicrobial agents in the test material.Where inhibitory substances are contained in the product and the latter is soluble,a suitable,validated adaptation of a procedure set forth underProcedures using the Membrane Filtration Methodmay be used.
If,in spite of the incorporation of suitable inactivating agents and a substantial increase in the volume of diluent,it is still not possible to recover the viable cultures described above,and where the article is not suitable for the employment of membrane filtration,it can be assumed that the failure to isolate the inoculated organism is attributable to the bactericidal or bacteriostatic activity of such magnitude that treatments are not able to remove the activity.This information serves to indicate that the article is not likely to allow proliferation or contamination with the given species of microorganism.Monitoring should be continued in order to determine the inhibitory range and bactericidal activity of the article.
BUFFER SOLUTION AND MEDIA
Culture media may be prepared as follows,or dehydrated culture media may be used provided that,when reconstituted as directed by the manufacturer or distributor,they have similar ingredients and/or yield media comparable to those obtained from the formulas given herein.
In preparing media by the formulas set forth herein,dissolve the soluble solids in the water,using heat if necessary to effect complete solution,and add solutions of hydrochloric acid or sodium hydroxide in quantities sufficient to yield the desired pHin the medium when it is ready for use.Determine the pHat 25±2
.
.Where agar is called for in a formula,use agar that has a moisture content of not more than 15%.Where water is called for in a formula,use Purified Water.
pH7.2Phosphate Buffer
Prepare a stock solution by dissolving 34g of monobasic potassium phosphate in about 500mLof water contained in a 1000-mLvolumetric flask.Adjust to a pHof 7.2±0.1by the addition of sodium hydroxide TS(about 175mL),add water to volume,and mix.Dispense and sterilize.Store under refrigeration.For use,dilute the stock solution with water in the ratio of 1to 800,dispense as desired,and sterilize.
Media
Prepare media for the tests as described below.Alternatively,dehydrated formulations may be used provided that,when reconstituted as directed by the manufacturer or distributor,they meet the requirements of theGrowth Promotion Testing.Unless otherwise indicated elsewhere in this chapter,media are sterilized in autoclaves using a validated process.The exposure time within the autoclave at 121will depend on the volume of media to be sterilized.Thus,for example,a 500-mLvolume would need to be autoclaved using a temperature and time relationship that will ensure that the medium has attained at least anF0of 12–15in the sterilization process.However,the appropriate time and temperature duration for sterilizing prepared media at any given volume should be confirmed by a thermal penetration study using a thermocouple or thermoprobe placed within the liquid medium.
will depend on the volume of media to be sterilized.Thus,for example,a 500-mLvolume would need to be autoclaved using a temperature and time relationship that will ensure that the medium has attained at least anF0of 12–15in the sterilization process.However,the appropriate time and temperature duration for sterilizing prepared media at any given volume should be confirmed by a thermal penetration study using a thermocouple or thermoprobe placed within the liquid medium.
FLUID CASEIN DIGEST–SOY LECITHIN–POLYSORBATE20MEDIUM
| Pancreatic Digest of Casein | 20g |
| Soy Lecithin | 5g |
| Polysorbate 20 | 40mL |
| Water | 960mL |
Dissolve pancreatic digest of casein and soy lecithin in 960mLof water,heating in a water bath at 48to 50for about 30minutes to effect solution.Add 40mLof polysorbate 20.Mix,dispense as desired,and sterilize.
to 50for about 30minutes to effect solution.Add 40mLof polysorbate 20.Mix,dispense as desired,and sterilize.
SOYBEAN–CASEIN DIGEST–AGAR MEDIUM
| Pancreatic Digest of Casein | 15.0g |
| Papaic Digest of Soybean Meal | 5.0g |
| Sodium Chloride | 5.0g |
| Agar | 15.0g |
| Water | 1000mL |
pHafter sterilization:7.3±0.2.
FLUID SOYBEAN–CASEIN DIGEST MEDIUM
| Pancreatic Digest of Casein | 17.0g |
| Papaic Digest of Soybean Meal | 3.0g |
| Sodium Chloride | 5.0g |
| Dibasic Potassium Phosphate | 2.5g |
| Dextrose | 2.5g |
| Purified Water | 1000mL |
Dissolve the solids in the water,heating slightly to effect a solution.Cool the solution to room temperature,and adjust the pHwith 1Nsodium hydroxide so that after sterilization it will have a pHof 7.3±0.2.Filter,if necessary,and dispense into suitable containers.Sterilize at a temperature and time relationship that will ensure that the medium has attained at least an F0of 12–15in the sterilization process,or by a validated filtration process.
SABOURAUD DEXTROSE–AGAR MEDIUM
| Dextrose | 40.0g |
| Mixture of Peptic Digest of Animal Tissue and Pancreatic Digest of Casein (1:1) |
10.0g |
| Agar | 15.0g |
| Water | 1000mL |
Mix,and boil to effect solution.
pHafter sterilization:5.6±0.2.
VIOLET-RED BILE AGAR WITH GLUCOSE AND LACTOSE
| Yeast Extract | 3.0g |
| Pancreatic Digest of Gelatin | 7.0g |
| Bile Salts | 1.5g |
| Lactose | 10.0g |
| Sodium Chloride | 5.0g |
| D-Glucose Monohydrate | 10.0g |
| Agar | 15.0g |
| Neutral Red | 30mg |
| Crystal Violet | 2mg |
| Water | 1000mL |
Adjust the pHso that it is 7.4±0.2after heating.Heat to boiling,but do not heat in an autoclave.Pour onto plates.
MOSSEL–ENTEROBACTERIACEAE ENRICHMENT BROTH
| Pancreatic Digest of Gelatin | 10.0g |
| D-Glucose Monohydrate | 5.0g |
| Dehydrated Ox Bile | 20.0g |
| Monobasic Potassium Phosphate | 2.0g |
| Dibasic Potassium Phosphate | 8.0g |
| Brilliant Green | 15mg |
| Water | 1000mL |
Suspend the solids in water,and heat to boiling for 1to 2minutes.Transfer 120-mLportions to 250-mLvolumetric flasks or 9-mLportions to test tubes,all being capped with cotton plugs or loose-fitting caps.Heat on a steam bath for 30minutes.Adjust the pHso that it is 7.2±0.2after heating.
GROWTH PROMOTION TESTING
Each lot of dehydrated medium bearing the manufacturer’s identifying number or each lot of medium prepared from basic ingredients must be tested for its growth-promoting qualities.Cultures ofStaphylococcus aureus (ATCC No.6538),Escherichia coli (ATCC No.8739),Bacillus subtilis (ATCC No.6633),Candida albicans (ATCC No.10231),andAspergillus niger (ATCC No.16404)are used.A10-3dilution of a 24-hour broth culture of the microorganism to the first dilution (inpH7.2Phosphate BufferorFluid Soybean–Casein Digest Medium)may be used as the inocula.Serially streak plates of the media with the appropriate inocula to obtain isolated colonies to demonstrate the growth-promotion qualities of theSoybean–Casein Digest andSabouraud Dextrose Agar media.Inocula theFluid Soybean–Casein Digest Medium andMossel–Enterobacteriaceae Enrichment Broth with 10to 100cfu of the appropriate challenge organisms to demonstrate their growth-promotion qualities.
SAMPLING
Provide 10-mLor 10-g specimens for the tests called for in the individual monograph.
PROCEDURE
Prepare the specimen to be tested by a treatment that is appropriate to its physical characteristics and that does not alter the number and kind of microorganisms originally present,in order to obtain a solution or suspension of all or part of it in a form suitable for the test procedure(s)to be carried out.
For a solid that dissolves to an appreciable extent but not completely,reduce the substance to a moderately fine powder,suspend it in the vehicle specified,and proceed as directed underTotal Aerobic Microbial Count.
For a fluid specimen that consists of a true solution,or a suspension in water or a hydroalcoholic vehicle containing less than 30%of alcohol,and for a solid that dissolves readily and practically completely in 90mLofpH7.2Phosphate Buffer or the media specified,proceed as directed underTotal Aerobic Microbial Count.
For water-immiscible products,prepare a suspension with the aid of a minimal quantity of a suitable,sterile emulsifying agent (such as one of the polysorbates),using a mechanical blender and warming to a temperature not exceeding 45,if necessary,and proceed with the suspension as directed underTotal Aerobic Microbial Count.
,if necessary,and proceed with the suspension as directed underTotal Aerobic Microbial Count.
Total Aerobic Microbial Count
For specimens that are freely soluble,use theMembrane Filtration Method orPlate Method.For specimens that are sufficiently soluble or translucent to permit use of thePlate Method,use that method;otherwise,use theMultiple-Tube Method.With either method,first dissolve or suspend 10.0g of the specimen if it is a solid,or 10mL,accurately measured,if the specimen is a liquid,inpH7.2Phosphate Buffer,Fluid Soybean–Casein Digest Medium,orFluid Casein Digest–Soy Lecithin–Polysorbate 20Medium to make 100mL.For viscous specimens that cannot be pipeted at this initial 1:10dilution,dilute the specimen until a suspension is obtained,i.e.,1:50or 1:100,etc.,that can be pipeted.Perform the test for absence of inhibitory (antimicrobial)properties as described underPreparatory Testing before the determination of Total Aerobic Microbial Count.Add the specimen to the medium not more than 1hour after preparing the appropriate dilutions for inoculation.
Membrane Filtration Method
Dilute the fluid further,if necessary,so that 1mLwill be expected to yield between 30and 300colonies.Pipet 1mLof the final dilution into 5to 10mLofpH7.2Phosphate Buffer,Fluid Soybean–Casein Digest Medium,orFluid Casein Digest–Soy Lecithin–Polysorbate 20Medium.Wash each membrane with an appropriate amount of one of the above diluents.Transfer each membrane to a Petri dish containingSoybean–Casein Digest Agar Medium,previously solidified at room temperature.Incubate the plates at a temperature between 30and 35for 48to 72hours.Following incubation,examine the plates for growth,count the number of colonies,and express the average for the two plates in terms of the number of microorganisms per g or per mLof specimen.If no microbial colonies are recovered from the dishes representing the initial 1:10dilution of the specimen,express the results as “less than 10microorganisms per g or per mLof specimen”.
and 35for 48to 72hours.Following incubation,examine the plates for growth,count the number of colonies,and express the average for the two plates in terms of the number of microorganisms per g or per mLof specimen.If no microbial colonies are recovered from the dishes representing the initial 1:10dilution of the specimen,express the results as “less than 10microorganisms per g or per mLof specimen”.
Plate Method
Dilute the fluid further,if necessary,so that 1mLwill be expected to yield between 30and 300colonies.Pipet 1mLof the final dilution onto each of two sterile Petri dishes.Promptly add to each dish 15to 20mLof Soybean–Casein Digest–Agar Medium,previously melted and cooled to about 45.Cover the Petri dishes,mix the sample with agar by gently tilting or rotating the dishes,and allow the contents to solidify at room temperature.Invert the Petri dishes and incubate for 48to 72hours.Following incubation,examine the plates for growth,count the number of colonies,and express the average for the two plates in terms of the number of microorganisms per g or per mLof specimen.If no microbial colonies are recovered from the dishes representing the initial 1:10dilution of the specimen,express the results as “less than 10microorganisms per g or per mLof specimen”.
.Cover the Petri dishes,mix the sample with agar by gently tilting or rotating the dishes,and allow the contents to solidify at room temperature.Invert the Petri dishes and incubate for 48to 72hours.Following incubation,examine the plates for growth,count the number of colonies,and express the average for the two plates in terms of the number of microorganisms per g or per mLof specimen.If no microbial colonies are recovered from the dishes representing the initial 1:10dilution of the specimen,express the results as “less than 10microorganisms per g or per mLof specimen”.
Multiple-Tube Method
Into each of 14test tubes of similar size,place 9.0mLof sterile Fluid Soybean–Casein Digest Medium.Arrange 12of the tubes in four sets of three tubes each.Put aside one set of three tubes to serve as the controls.Into each of three tubes of one set (“100”)and into a fourth tube (A)pipet 1mLof the solution or suspension of the specimen,and mix.Pipet 1mLfrom tube Ainto the one remaining tube (B),not included in a set,and mix.These two tubes contain 100mg or 100µLand 10mg or 10µLof the specimen,respectively.Into each of the second set (“10”)of three tubes pipet 1mLfrom tube A,and into each tube of the third set (“1”)pipet 1mLfrom tube B.Discard the unused contents of tubes Aand B.Close well,and incubate all of the tubes.Following incubation,examine the tubes for growth:the three control tubes remain clear,and the observations in the tubes containing the specimen,when interpreted by reference to Table 1,indicate the most probable number of microorganisms per g or per mL.
Table 1.Most Probable Count by Multiple-Tube Method
| Observed Combinations of Numbers of Tubes Showing Growth in Each Set |
Most Probable Number of Microorganisms per g or per mL | ||
| Number of mg or µLof specimen per tube | |||
| 100 | 10 | 1 | |
| 3 | 3 | 3 | more than 1100 |
| 3 | 3 | 2 | 1100 |
| 3 | 3 | 1 | 500 |
| 3 | 3 | 0 | 200 |
| 3 | 2 | 3 | 290 |
| 3 | 2 | 2 | 210 |
| 3 | 2 | 1 | 150 |
| 3 | 2 | 0 | 90 |
| 3 | 1 | 3 | 160 |
| 3 | 1 | 2 | 120 |
| 3 | 1 | 1 | 70 |
| 3 | 1 | 0 | 40 |
| 3 | 0 | 3 | 95 |
| 3 | 0 | 2 | 60 |
| 3 | 0 | 1 | 40 |
| 3 | 0 | 0 | 23 |
| 2 | 2 | 0 | 21 |
| 2 | 1 | 1 | 20 |
| 2 | 1 | 0 | 15 |
| 2 | 0 | 1 | 14 |
| 2 | 0 | 0 | 9 |
| 1 | 2 | 0 | 11 |
| 1 | 1 | 0 | 7 |
| 1 | 0 | 0 | 4 |
| 0 | 1 | 0 | 3 |
| 0 | 0 | 0 | <3 |
Total Combined Molds and Yeasts Count
Procedure—
Proceed as directed for Membrane Filtration MethodorPlate Methodunder Total Aerobic Microbial Count,except to use the same amount of Sabouraud Dextrose–Agar Mediuminstead of Soybean–Casein Digest–Agar Mediumand to incubate the plates for 5to 7days at 20to 25.
to 25.
Retest—
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 an additional 10-g specimen from the original sample and a 10-g specimen from the new sample of the nutritional supplement may be conducted.Proceed as directed underProcedure.
Enterobacterial Count (Bile-Tolerant Gram-Negative Bacteria)
Dissolve or suspend the sample in a sufficient volume ofpH7.2Phosphate Buffer orFluid Soybean–Casein–Digest Medium and dilute withFluid Soybean–Casein–Digest Medium to 100mL.Pre-incubate for 2to 5hours at 20–25inSoybean–Casein Digest Brothdiluent;inoculate suitable quantities ofMossel–Enterobacteriaceae Enrichment Broth to contain 0.1,0.01,or 0.001g or mLof the product.Incubate at 30–35for 24to 48hours.Subculture onto a plate ofViolet-Red Bile Agar with Glucoseand Lactose,and incubate at 30–35for 18to 24hours.Growth of well developed,generally red or reddish,colonies of Gram-Negative bacterial reveals the presence of enterobacteria.Determine the most probable number of microorganisms per g or per mLby reference to Table 2.
–25inSoybean–Casein Digest Brothdiluent;inoculate suitable quantities ofMossel–Enterobacteriaceae Enrichment Broth to contain 0.1,0.01,or 0.001g or mLof the product.Incubate at 30–35for 24to 48hours.Subculture onto a plate ofViolet-Red Bile Agar with Glucoseand Lactose,and incubate at 30–35for 18to 24hours.Growth of well developed,generally red or reddish,colonies of Gram-Negative bacterial reveals the presence of enterobacteria.Determine the most probable number of microorganisms per g or per mLby reference to Table 2.
Table 2.The Most Probable Enterobacterial Count
| Observed Presence of Enterobacteria | Most Probable Number of Enterobacteria per g | ||
| Number of g of specimen per tube | |||
| 0.1 | 0.01 | 0.001 | |
| + | + | + | more than 100 |
| + | + | – | fewer than 100but more than 10 |
| + | – | – | fewer than 10but more than 1 |
| – | – | – | fewer than 1 |
1
Available from ATCC,10801University Boulevard,Manassas,VA20110-2209.Equivalent microorganisms,provided that they are from a national collection repository,can be used in lieu of ATCCstrains.However,the viable microorganisms used in the test must not be more than five passages removed from the original ATCCor national collection culture.