á61ñMICROBIAL LIMIT TESTS
This chapter provides tests for the estimation of the number of viable aerobic microorganisms present and for freedom from designated microbial species in pharmaceutical articles of all kinds,from raw materials to the finished forms.An automated method may be substituted for the tests presented here,provided it has been properly validated as giving equivalent or better results.In preparing for and in applying the tests,observe aseptic precautions in handling the specimens.Unless otherwise directed,where the procedure specifies simply “incubate,”hold the container in air that is thermostatically controlled at a temperature between 30
and 35,for a period of 24to 48hours.The term “growth”is used in a special sense herein,i.e.,to designate the presence and presumed proliferation of viable microorganisms.
and 35,for a period of 24to 48hours.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 Staphylococcus aureus,Escherichia coli,Pseudomonas aeruginosa,and Salmonella.This can be done by adding 1mLof not less than 10-3dilution of a 24-hour broth culture of the microorganism to the first dilution (in pH7.2Phosphate Buffer,Fluid Soybean–Casein Digest Medium,or Fluid Lactose Medium)of the test material and following the test procedure.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 (1)and (2)so as to permit growth of the inocula.
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,using Fluid Casein Digest–Soy Lecithin–Polysorbate 20Mediumto 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 in the section Membrane Filtrationunder Test for Sterility of the Product to be Examinedunder Sterility Tests á71ñ,may 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 employment of membrane filtration,it can be assumed that the failure to isolate the inoculated organism is attributable to the bactericidal activity of the product.This information serves to indicate that the article is not likely to be contaminated with the given species of microorganism.Monitoring should be continued in order to establish the spectrum of inhibition 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
Stock Solution—
Dissolve 34g of monobasic potassium phosphate in about 500mLof water contained in a 1000-mLvolumetric flask.Adjust to pH7.2±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 Solutionwith water in the ratio of 1to 800,and sterilize.
Media
Unless otherwise indicated,the media should be sterilized by heating in an autoclave (see Steam Sterilizationunder Sterilization á1211ñ),the exposure time depending on the volume to be sterilized.
I.Fluid Casein Digest–Soy Lecithin–Polysorbate 20Medium
| Pancreatic Digest of Casein | 20g |
| Soy Lecithin | 5g |
| Polysorbate 20 | 40mL |
| Water | 960mL |
Dissolve the 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,and dispense as desired.
to 50for about 30minutes to effect solution.Add 40mLof polysorbate 20.Mix,and dispense as desired.
II.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.
III.Fluid Soybean–Casein Digest Medium
| Prepare as directed for Soybean–Casein Digest Mediumunder Sterility Tests á71ñ. |
IV.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.
V.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.Mix intimately but gently,and pour into plates.(Prepare the egg-yolk emulsion by disinfecting the surface of whole shell eggs,aseptically cracking the eggs,and separating out intact yolks into 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.)
and 50,and add 10mLof sterile potassium tellurite solution (1in 100)and 50mLof egg-yolk emulsion.Mix intimately but gently,and pour into plates.(Prepare the egg-yolk emulsion by disinfecting the surface of whole shell eggs,aseptically cracking the eggs,and separating out intact yolks into 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.)pHafter sterilization:6.8±0.2.
VI.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 the solution of solids 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.
VII.Cetrimide Agar Medium
| Pancreatic Digest of Gelatin | 20.0g |
| Magnesium Chloride | 1.4g |
| Potassium Sulfate | 10.0g |
| Agar | 13.6g |
| Cetyl Trimethylammonium Bromide (Cetrimide) | 0.3g |
| Glycerin | 10.0mL |
| Water | 1000mL |
Dissolve all solid components in the water,and add the glycerin.Heat,with frequent agitation,and boil for 1minute to effect solution.
pHafter sterilization:7.2±0.2.
VIII.Pseudomonas Agar Medium for Detection of Fluorescin
| Pancreatic Digest of Casein | 10.0g |
| Peptic Digest of Animal Tissue | 10.0g |
| Anhydrous Dibasic Potassium Phosphate | 1.5g |
| Magnesium Sulfate (MgSO4·7H2O) | 1.5g |
| Glycerin | 10.0mL |
| Agar | 15.0g |
| Water | 1000mL |
Dissolve the solid components in the water before adding the glycerin.Heat,with frequent agitation,and boil for 1minute to effect solution.
pHafter sterilization:7.2±0.2.
IX.Pseudomonas Agar Medium for Detection of Pyocyanin
| Pancreatic Digest of Gelatin | 20.0g |
| Anhydrous Magnesium Chloride | 1.4g |
| Anhydrous Potassium Sulfate | 10.0g |
| Agar | 15.0g |
| Glycerin | 10.0mL |
| Water | 1000mL |
Dissolve the solid components in the water before adding the glycerin.Heat,with frequent agitation,and boil for 1minute to effect solution.
pHafter sterilization:7.2±0.2.
X.Fluid Lactose Medium
| Beef Extract | 3.0g |
| Pancreatic Digest of Gelatin | 5.0g |
| Lactose | 5.0g |
| Water | 1000mL |
Cool as quickly as possible after sterilization.
pHafter sterilization:6.9±0.2.
XI.Fluid Selenite–Cystine Medium
| Pancreatic Digest of Casein | 5.0g |
| Lactose | 4.0g |
| Sodium Phosphate | 10.0g |
| Sodium Acid Selenite | 4.0g |
| L-Cystine | 10.0mg |
| Water | 1000mL |
Final pH:7.0±0.2.
Mix,and heat to effect solution.Heat in flowing steam for 15minutes.Do not sterilize.
XII.Fluid Tetrathionate Medium
| Pancreatic Digest of Casein | 2.5g |
| Peptic Digest of Animal Tissue | 2.5g |
| Bile Salts | 1.0g |
| Calcium Carbonate | 10.0g |
| Sodium Thiosulfate | 30.0g |
| Water | 1000mL |
Heat the solution of solids to boiling.On the day of 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 the medium after adding the brilliant green solution.
XIII.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 | 80mg |
| Agar | 20.0g |
| Brilliant Green | 12.5mg |
| Water | 1000mL |
Boil the solution of solids for 1minute.Sterilize just prior to use,melt the medium,pour into petri dishes,and allow to cool.
pHafter sterilization:6.9±0.2.
XIV.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 | 2.5g |
| Sodium Thiosulfate | 6.8g |
| Ferric Ammonium Citrate | 800mg |
| Water | 1000mL |
Final pH:7.4±0.2.
Heat the mixture of solids and water,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 plates as soon as the medium has cooled.
,and pour into plates as soon as the medium has cooled.
XV.Bismuth Sulfite Agar Medium
| Beef Extract | 5.0g |
| Pancreatic Digest of Casein | 5.0g |
| Peptic Digest of Animal Tissue | 5.0g |
| Dextrose | 5.0g |
| Sodium Phosphate | 4.0g |
| Ferrous Sulfate | 300mg |
| Bismuth Sulfite Indicator | 8.0g |
| Agar | 20.0g |
| Brilliant Green | 25mg |
| Water | 1000mL |
Final pH:7.6±0.2.
Heat the mixture of solids and water,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 plates as soon as the medium has cooled.
,and pour into plates as soon as the medium has cooled.
XVI.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.
XVII.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 Chloride | 5.0g |
| Agar | 13.5g |
| Neutral Red | 30mg |
| Crystal Violet | 1.0mg |
| Water | 1000mL |
Boil the mixture of solids and water for 1minute to effect solution.
pHafter sterilization:7.1±0.2.
XVIII.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 the pancreatic digest of gelatin,the dibasic potassium phosphate,and the agar in the water,with warming,and allow to cool.Just prior to use,liquefy the gelled agar solution,add the remaining ingredients,as solutions,in the following amounts,and mix:for each 100mLof the liquefied agar solution—5mLof lactose solution (1in 5),2mLof the eosin Ysolution (1in 50),and 2mLof methylene blue solution (1in 300).The finished medium may not be clear.
pHafter sterilization:7.1±0.2.
XIX.Sabouraud Dextrose Agar Medium
| Dextrose | 40g |
| Mixture of equal parts of Peptic Digest of Animal Tissue and Pancreatic Digest of Casein | 10g |
| Agar | 15g |
| Water | 1000mL |
Mix,and boil to effect solution.
pHafter sterilization:5.6±0.2.
XX.Potato Dextrose Agar Medium
| Cook 300g of peeled and diced potatoes in 500mLof water prepared by distillation,filter through cheesecloth,add water prepared by distillation to make 1000mL,and add the following: | |
| Agar | 15g |
| Glucose | 20g |
Dissolve by heating,and sterilize.
pHafter sterilization:5.6±0.2.
For use,just prior to pouring the plates,adjust the melted and cooled to 45medium with sterile tartaric acid solution (1in 10)to a pHof 3.5±0.1.Do not reheat the pH3.5medium.
medium with sterile tartaric acid solution (1in 10)to a pHof 3.5±0.1.Do not reheat the pH3.5medium.Sampling
Provide separate 10-mLor 10-g specimens for each of the tests called for in the individual monograph.
Procedure
Prepare the specimen to be tested by 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 under Total Aerobic Microbial Count,and under Test for Staphylococcus aureus and Pseudomonas aeruginosaand Test for Salmonella species and Escherichia coli.
For a fluid specimen that consists of a true solution,or a suspension in water or a hydroalcoholic vehicle containing less than 30percent of alcohol,and for a solid that dissolves readily and practically completely in 90mLof pH7.2Phosphate Bufferor the media specified,proceed as directed under Total Aerobic Microbial Count,and under Test for Staphylococcus aureus and Pseudomonas aeruginosaand Test for Salmonella species and Escherichia coli.
For water-immiscible fluids,ointments,creams,and waxes,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 under Total Aerobic Microbial Count,and under Test for Staphylococcus aureus and Pseudomonas aeruginosaand Test for Salmonella species and Escherichia coli.
,if necessary,and proceed with the suspension as directed under Total Aerobic Microbial Count,and under Test for Staphylococcus aureus and Pseudomonas aeruginosaand Test for Salmonella species and Escherichia coli.For a fluid specimen in aerosol form,chill the container in an alcohol-dry ice mixture for approximately 1hour,cut open the container,allow it to reach room temperature,permit the propellant to escape,or warm to drive off the propellant if feasible,and transfer the quantity of test material required for the procedures specified in one of the two preceding paragraphs,as appropriate.Where 10.0g or 10.0mLof the specimen,whichever is applicable,cannot be obtained from 10containers in aerosol form,transfer the entire contents from 10chilled containers to the culture medium,permit the propellant to escape,and proceed with the test on the residues.If the results of the test are inconclusive or doubtful,repeat the test with a specimen from 20more containers.
Total Aerobic Microbial Count
For specimens that are sufficiently soluble or translucent to permit use of the Plate Method,use that method;otherwise,use the Multiple-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,in pH7.2Phosphate Buffer,Fluid Soybean–Casein Digest Medium,or Fluid Casein Digest–Soy Lecithin-Polysorbate 20Mediumto 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 under Preparatory Testingbefore the determination of Total Aerobic Microbial Count.Add the specimen to the medium not more than 1hour after preparing the appropriate dilutions for inoculation.
PLATE METHOD
Dilute further,if necessary,the fluid 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 Mediumthat previously has been melted and cooled to approximately 45.Cover the petri dishes,mix the sample with the agar by 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 the agar by 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 fourteen test tubes of similar size place 9.0mLof sterile Fluid Soybean–Casein Digest Medium.Arrange twelve of 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.From tube A,pipet 1mLof its contents into the one remaining tube (B)not included in a set,and mix.These two tubes contain 100mg (or 100µL)and 10mg (or 10µL)of 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 the incubation period,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 mLof specimen.
Table 1.Most Probable Total 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 |
||
| No.of mg (or mL)of Specimen per Tube | |||
| 100 (100µL) |
10 (10µL) |
1 (1µL) |
|
| 3 | 3 | 3 | >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 |
Test for Staphylococcus aureusand Pseudomonas aeruginosa
To the specimen add Fluid Soybean–Casein Digest Mediumto make 100mL,mix,and incubate.Examine the medium for growth,and if growth is present,use an inoculating loop to streak a portion of the medium on the surface of Vogel–Johnson Agar Medium(or Baird–Parker Agar Medium,or Mannitol–Salt Agar Medium)and of Cetrimide Agar Medium,each plated on petri dishes.Cover and invert the dishes,and incubate.If,upon examination,none of the plates contains colonies having the characteristics listed in Tables 2and 3for the media used,the test specimen meets the requirements for freedom from Staphylococcus aureusand Pseudomonas aeruginosa.
Table 2.Morphologic Characteristics of Staphylococcus aureus on Selective Agar Media
| Selective Medium | Characteristic Colonial Morphology | Gram Stain |
| Vogel-Johnson Agar Medium |
Black Surrounded by yellow zone | Positive cocci (in clusters) |
| Mannital-Salt Agar Medium |
Yellow colonies with yellow zones | Positive cocci (in clusters) |
| Baird-Parker Agar Medium |
Black,shiny,surrounded by clear zones 2to 5mm | Positive cocci (in clusters) |
Table 3.Morphologic Characteristics of Pseudomonas aeruginosaon Selective and Diagnostic Agar Media
| Selective Medium | Characteristic Colonial Morphology |
Fluorescence in UV Light |
Oxidase Test | Gram Stain |
| Centrimide Agar Medium | Generally greenish | Greenish | Positive | Negative rods |
| Pseudomonas Agar Medium for Detection of Fluorescin |
Generally colorless to yellowish | Yellowish | Positive | Negative rods |
| Pseudomonas Agar Medium for Detection of Pyocyanin |
Generally greenish | Blue | Positive | Negative rods |
Coagulase Test (for Staphylococcus aureus)—
With the aid of an inoculating loop,transfer representative suspect colonies from the agar surfaces of the Vogel–Johnson Agar Medium (or Baird–Parker Agar Medium,or Mannitol–Salt Agar Medium)to individual tubes,each containing 0.5mLof mammalian,preferably rabbit or horse,plasma with or without suitable additives.Incubate in a water bath at 37,examining the tubes at 3hours and subsequently at suitable intervals up to 24hours.Test positive and negative controls simultaneously with the unknown specimens.If no coagulation in any degree is observed,the specimen meets the requirements of the test for absence of Staphylococcus aureus.
,examining the tubes at 3hours and subsequently at suitable intervals up to 24hours.Test positive and negative controls simultaneously with the unknown specimens.If no coagulation in any degree is observed,the specimen meets the requirements of the test for absence of Staphylococcus aureus.
Oxidase and Pigment Tests (for Pseudomonas aeruginosa)—
With the aid of an inoculating loop,streak representative suspect colonies from the agar surface of Cetrimide Agar Medium on the agar surfaces of Pseudomonas Agar Medium for Detection of Fluorescin and Pseudomonas Agar Medium for Detection of Pyocyanin contained in petri dishes.If numerous colonies are to be transferred,divide the surface of each plate into quadrants,each of which may be inoculated from a separate colony.Cover and invert the inoculated media,and incubate at 35±2for not less than three days.Examine the streaked surfaces under UVlight.Examine the plates to determine whether colonies having the characteristics listed in Table 3are present.
for not less than three days.Examine the streaked surfaces under UVlight.Examine the plates to determine whether colonies having the characteristics listed in Table 3are present.
Confirm any suspect colonial growth on one or more of the media as Pseudomonas aeruginosaby means of the oxidase test.Upon the colonial growth place or transfer colonies to strips or disks of filter paper that previously has been impregnated with N,N-dimethyl-p-phenylenediamine dihydrochloride:if there is no development of a pink color,changing to purple,the specimen meets the requirements of the test for the absence of Pseudomonas aeruginosa.The presence of Pseudomonas aeruginosamay be confirmed by other suitable cultural and biochemical tests,if necessary.
Test for Salmonellaspecies and Escherichia coli
To the specimen,contained in a suitable vessel,add a volume of Fluid Lactose Medium to make 100mL,and incubate.Examine the medium for growth,and if growth is present,mix by gently shaking.Pipet 1-mLportions into vessels containing,respectively,10mLof Fluid Selenite–Cystine Medium and Fluid Tetrathionate Medium,mix,and incubate for 12to 24hours.(Retain the remainder of the Fluid Lactose Medium.)
Test for Salmonella Species—
By means of an inoculating loop,streak portions from both the selenite-cystine and tetrathionate media on the surface of Brilliant Green Agar Medium,Xylose–Lysine–Desoxycholate Agar Medium,and Bismuth Sulfite Agar Medium contained in petri dishes.Cover and invert the dishes,and incubate.Upon examination,if none of the colonies conforms to the description given in Table 4,the specimen meets the requirements of the test for absence of the genus Salmonella.
Table 4.Morphologic Characteristics of SalmonellaSpecies on Selective Agar Media
| Selective Medium | Characteristic Colonial Morphology |
| Brilliant Green Agar Medium |
Small,transparent,colorless or pink to white opaque (frequently surrounded by pink to red zone) |
| Xylose-Lysine- Desoxycholate Agar Medium |
Red,with or without black centers |
| Bismuth Sulfite Agar Medium |
Black or green |
If colonies of Gram-negative rods matching the description in Table 4are found,proceed with further identification by transferring representative suspect colonies individually,by means of an inoculating wire,to a butt-slant tube of Triple Sugar–Iron–Agar Medium by first streaking the surface of the slant and then stabbing the wire well beneath the surface.Incubate.If examination discloses no evidence of tubes having alkaline (red)slants and acid (yellow)butts (with or without concomitant blackening of the butt from hydrogen sulfide production),the specimen meets the requirements of the test for the absence of the genus Salmonella.*
Test forEscherichia coli—
By means of an inoculating loop,streak a portion from the remaining Fluid Lactose Medium on the surface of MacConkey Agar Medium.Cover and invert the dishes,and incubate.Upon examination,if none of the colonies conforms to the description given in Table 5for this medium,the specimen meets the requirements of the test for absence of Escherichia coli.
Table 5.Morphologic Characteristics of Escherichia colion MacConkey Agar Medium
| Gram Stain | Characteristic Colonial Morphology |
| Negative rods (cocco-bacilli) |
Brick-red;may have surrounding zone of precipitated bile |
If colonies matching the description in Table 5are found,proceed with further identification by transferring the suspect colonies individually,by means of an inoculating loop,to the surface of Levine Eosin–Methylene Blue Agar Medium,plated on petri dishes.If numerous colonies are to be transferred,divide the surface of each plate into quadrants,each of which may be seeded from a separate colony.Cover and invert the plates,and incubate.Upon examination,if none of the colonies exhibits both a characteristic metallic sheen under reflected light and a blue-black appearance under transmitted light,the specimen meets the requirements of the test for the absence of Escherichia coli.The presence of Escherichia colimay be confirmed by further suitable cultural and biochemical tests.
Total Combined Molds and Yeasts Count
Proceed as for the Plate Methodunder Total Aerobic Microbial Count,except for using the same amount of Sabouraud Dextrose Agar Medium or Potato Dextrose Agar Medium,instead of Soybean Casein Digest Medium,and except for incubating the inverted petri dishes 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 10.0-g specimen,a retest on a 25-g specimen of the product may be conducted.Proceed as directed for Procedure,but make allowance for the larger specimen size.
*
Additional,confirmatory evidence may be obtained by use of procedures set forth in Official Methods of Analysis of the AOAC,12th ed.(1975),sections 46.013-46.026.