D-715

Microbial Limit Testing using the Neogen Petrifilm System

Section D — Laboratory Operations and Specifications Revision 14 43 pages

Original Document

Scanned document (image-only PDF)

Extracted Text

Searchable text extracted from PDF

1.0 Purpose

The purpose of this procedure is to define the test methods for selective and non-selective
finished product and raw material microbial limit testing using Petrifilm. This procedure

defines processes for sample preparation, incubation conditions, and interpretation of the
results. The methods defined in this procedure, for enumeration testing, are not applicable to

products containing viable microorganisms as active ingredients. Recovery studies for all
Petrifilm methods were performed under protocols MV-LAB-13-002.

2.0 Scope

This procedure applies to all finished product and raw materials with D-715 listed as the test
method.

3.0 Responsibility

a1 It is the responsibility of QC Analysts to follow this procedure.

3.2 ‘It is the responsibility of QC Laboratory Management to implement this procedure and
to ensure that the procedure is being followed.

3.3. It is the responsibility of QC Laboratory Management to keep this procedure aligned
with current practices and to oversee validations and recovery studies.

4.0 Definitions

4.1 Diluent — A sterile solution used to dissolve and dilute samples (e.g., Butterfield’s

Phosphate Buffer)

[SOP

Standard Operating Procedure
SOP No | Rev
Microbial Limit Testing using the D-715 14 Page 2 of 43
Neogen® Petrifilm® System

4.2. Inoculum — The prepared sample that is placed on the Petrifilm® plate via pipet

4.3 IPA — Isopropyl Alcohol

44 QC -— Quality Control

45 OOS - Out of Specification

4.6 | TNTC-— Too Numerous to Count

4.7 Est — Estimation

4.8 TAPC — Total Aerobic Plate Count

49 | AC- Aerobic Plate Count

410 EC-E. coli/ Coliform Count Plates

4.11 EB-—Enterobacteriaceae Count

4.12 STX-—Staph Express Count

4.13 RYM-—Rapid Yeast and Mold Count

414 PQV-—Process Quality Verification

4.15 eGMP — Current Good Manufacturing Practices

5.0 References

5.1 6475/6477, Neogen® Petrifilm® Rapid Yeast and Mold Count Plate Product Instructions

5.2 6400/6406/6442/6403, Neogen® Petrifilm® Aerobic Count Plate Product Instructions

5.3 6404/6414/6444, Neogen® Petrifilm® E. Coli/Coliform Count Plate Product Instructions

5.4 6420/6421, Neogen® Petrifilm® Enterobacteriaceae Count Plate Product Instructions

[SOP

Standard Operating Procedure
SOP No | Rev
Microbial Limit Testing using the D-715 14 Page 3 of 43
Neogen® Petrifilm® System

fe 6446/6490/6491/6492/6493, Neogen® Petrifilm® Staph Express Count System Product
Instructions

5.6 2003.07, AOAC Official Method, Staphylococcus aureus Count in processed foods

PY| 990.12, AOAC Official Method, Aerobic Plate Count in Foods

5.8 991.14, AOAC Official Method, Coliform and Escherichia coli Counts in Foods

a 2003.01, AOAC Official Method, Enumeration of Enterobacteriaceae in Selected

Foods

5.10 997.02, AOAC Official Method, Yeast and Mold Counts in Foods

5.11 Neogen® Petrifilm® Plate Guide to Dilution Preparations

NSF/ANSI 173 -2012- Standards for Dietary Supplements, August 7", 2012
S12

Dl USP <2023>, Monograph, Microbiological Attributes of Nonsterile Nutritional and
Dietary Supplements

5.14 U.S. Food and Drug Administration (2001) Bacteriological Analytical Manual, Chapter
3, Edition 8, Revision A, 1998

5.15 D-715-F1, Form, Microbial Limit Test Ticket

5.16 D-715-F2, Microbial Sample Log

5.17 D-715-F3, Conductivity and pH Test Ticket

5.18 D-809, SOP, Use and Calibration of Conductivity Meter

3.19 D-706, SOP, Use and Calibration of pH Meters

5.20 D-715.0, SOP, Microbial Limit Testing using Agar Plates

[SOP

Standard Operating Procedure
SOP No | Rev
Microbial Limit Testing using the Page 4 of 43
D-715 14
Neogen® Petrifilm® System

5.21 D-902, SOP, Establishment of Specifications

5.22 D-125, SOP, Microbiological Method Suitability

5.23 C-201, SOP, Deviation and Investigation Procedure

5.24 QS-108, SOP, Corrective and Preventative Action (CAPA)

5.25 D-824, SOP, Operation and Cleaning of the Autoclaves in the QC Laboratory

5.26 D-101, SOP, Laboratory Housekeeping

5.2/ A-106, SOP, Documentation Guidelines for cGMP Records

5.28 C-501, SOP, Document Control Procedure

5.29 C-502, SOP, Record Storage, Retention, and Destruction

5.30 RPT-21-0028, Report, D-715 Estimation of Uncertainty

6.0 Required Supplies, Media and Equipment

6.1 Butterfield’s Phospate Buffer-99mL or 90mL

6.2 0.1% Peptone Salt-90mL

6.3 Sterile Millipore Water

6.4 Isopropyl Alcohol

6.5 Sterile 120mL containers with lid

6.6 200ul and 1mL variable pipette with sterile tips

6.7 100, 250, 500 and 1L wide mouth storage bottles with screw cap top.

6.8 Balance

[SOP

Standard Operating Procedure
SOP No | Rev
Microbial Limit Testing using the D-715 14 Page 5 of 43
Neogen® Petrifilm® System

6.9 pH meter

6.10 Sterile IN NaOH solution

6.11 Sterile IN HCl solution

6.12 Neogen® Petrifilm® Rapid Yeast and Mold Count Plates

6.13 Neogen® Petrifilm® Aerobic Count Plates

6.14 Neogen® Petrifilm® Enterobacteriaceae Count Plates

6.15 Neogen® Petrifilm® E. coli / Coliform Count Plates

6.16 Neogen® Petrifilm® Staph Express Count Plates

6.17 Neogen® Petrifilm® Staph Express Disks

Neogen® Petrifilm® spreaders for Aerobic, Rapid Yeast and Mold, E. coli /Coliform,
6.18 Enterobacteriaceae and Staph Express Count plates.

6.19 Compound Microscope with LCD Display

6.20 Biological Safety Cabinet

6.21 20 °C to 25°C and 30 °C to 35°C Incubators (adjustable)

6.22 Autoclave

6.23 Darkfield Colony Counter

7.0 Procedure

When a sample is received into the QC Laboratory that requires Microbial Testing,
7.1 check the final product profile specification to ascertain if Petrifilm is an allowed

method.

[SOP

Standard Operating Procedure
SOP No | Rev
Microbial Limit Testing using the D-715 14 Page 6 of 43
Neogen® Petrifilm® System

7.2 Before initiating testing, record sample information onto form D-715-F2 Microbial

Sample Log as follow:

7.2.1 Product Name

7.2.2 Batch#

7.2.3 Sample preparation number

7.2.3.1 Sample preparation number will be assigned to the sample based on the

next consecutive number listed

7.2.4 Automatic Pipette#

7.2.5 Date testing started

7.2.6 Analyst initial

7.3 Alternatively, any use log associated with the micro lab may be used as the sample

logbook. All samples tested must be captured in a logbook at time of testing.

7.4. Diluent Preparation

7.4.1. Butterfield’s Phosphate Buffer. First, prepare a Potassium Dihydrogen
Phosphate Stock Solution by mixing 34.0 g of Monobasic Potassium Phosphate

in 500 ml of Millipore water. Mix to dissolve. Calibrate the pH meter at pH 7.00

and 10.01 then adjust pH of stock solution to 7.2 + 0.1 using 1N Sodium
Hydroxide. Complete stock solution to 1 L with Millipore water. Prepare bottle

and autoclave at 121°C for 15 minutes. Store in refrigerator after sterilization.
Prepare Butterfield’s Phosphate dilution blanks by adding 1.25 ml of the

Potassium Dihydrogen Phosphate Stock Solution to 1 L of Millipore water.
Prepare bottle and autoclave at 121°C for 15 minutes. Butterfields can also be

bought thru a commercial vendor at 90mL or 99mL.

[SOP

Standard Operating Procedure
SOP No | Rev
Microbial Limit Testing using the D-715 14 Page 7 of 43
Neogen® Petrifilm® System

7.4.2 0.1% Peptone Salt (85% Sodium Chloride and 0.1% Peptone). Add 8.5 g of

Sodium Chloride and 1.0 g of Peptone with 1L of Millipore water. Mix and

heat to dissolve. Autoclave at 121°C for 15 minutes. Refrigerate until use. 0.1
% Peptone Salt can also be bought through commercial vendor at 90mL.

7.5. Sample labeling and Preparation

7.5.1 Sterilize all sample preparation equipment and media/diluents prior to use as per

SOP D-824 Operation and Cleaning of the Autoclaves in the QC Laboratory.
Single use, sterile disposables may also be used.

7.5.2 Label sample containers with a minimum of sample preparation number, and
any extra testing over the following: TAPC, Y&M, EC, SAL

7.5.3. Create a parent sample of no less than 10g by transferring NLT 10g to a
sterile/sanitized mortar (sanitize by wiping thoroughly with 70% IPA and allow

the mortar and pestle time to dry before use). Aseptically pulverize tablets using
the mortar and pestle or open capsules to create a blend of fill and capsules.

Alternatively, parent sample may also be allowed to sit and disintegrate in

diluent for approximately 15 minutes.

7.5.4. Typical sample dilutions are 1:10 and 1:100. 1:10 samples can be prepared in

120mL sterile capped cups using approximately 10g of sample and 90 mL of
diluent or 11g of sample in 99mL of diluent. 1:100 samples are typically

prepared by adding approximately 1g of material to a 120mL sterile capped cup
and diluting up to 100mL with diluent. Other dilution ratios can be used if they

have been validated or are higher than the validated dilution (i.e. validate
dilution is 1/10, plate either 1:10. 1:100 etc). Document volume of diluent used

for dilution ratios that require a diluent volume different from 90 or 99 ml (e.g.
1:20, 1:50). Refer to Finished Product Summary for Validated Dilution and

Diluent Attachment 6.

[SOP

Standard Operating Procedure
SOP No | Rev
Microbial Limit Testing using the D-715 14 Page 8 of 43
Neogen® Petrifilm® System

7.5.5 If the sample has not been validated for petrifilm method, prepare a sample

dilution as described above, using Butterfields’s Buffer or Peptone Salt as the

diluent. Reserve the original sample for validation testing (if applicable).
Alternatively, agar method (D-715.0) can be used as reference test method at

any time, even if it is not listed as the preferred method. Refer to SOP D-715.0
Microbial Limit Testing using Agar Plates.

7.5.6 Before inoculation, sample pH should be adjusted to neutral pH with IN NaOH
for acids products and 1N HC] for alkaline products per Neogen instructions. If

pH indicator strips are used, sample pH should be adjusted to 7.0.

7.5.7 The sampling of certain raw materials such as RMS001583 can be challenging.

To ensure an appropriate aseptic technique, the QC analyst must adhere to the

steps defined below:

7.5.7.1 Inspect the 5-Gallon Water Jug submitted for testing to look for cracks,
perforations or broken seal. Reject the 5-Gallon Water Jug if has visible

damage or seal integrity has being compromised.

7.5.7.2 Spray with 70% IPA to saturate all the exterior surface of the water jug,

before opening.

7.5.7.3 Allow to air dry for couple of minutes.

7.5.7.4 Use new gloves to release the seal and prepare materials for sample

collection.

7.5.7.5 Carefully take off the lid with pre-saturated 70% IPA wipes to avoid

contamination of the exposed ridge.

7.5.7.6 To prevent prolonged exposure, water sample should be immediately

collected after opening.

[SOP

Standard Operating Procedure
SOP No | Rev
Microbial Limit Testing using the D-715 14 Page 9 of 43
Neogen® Petrifilm® System

7.5.8 Document the sample and test information required on form D-715-F1

Microbial Limit Test Ticket before start plating. For raw materials such as

RMS001583 that require testing other than microbial, document additional
testing on D-715-F3 Conductivity and pH Test Ticket. Refer to SOP D-706 and

SOP D-809 for pH and conductivity measurements.

7.6 Inoculation of Petrifilm® Plates- General

7.6.1 Sanitize the interior of the biological safety cabinet with IPA as per D-101
Laboratory Housekeeping before plating. Sterile sleeve covers and gloves

should be used from plate preparation to sample plating.

7.6.2 Obtain the Petrifilm® plates and confirm that they are not expired. Label the

Petrifilm with a minimum of sample preparation number and dilution plated (if
multiple dilutions are being plated)

7.6.3 For routine screening, inoculate two plates per testing required as follow:

7.6.3.1 AC petrifilm for TAPC Test

7.6.3.2 RYM petrifilm for Yeast and Mold Test

7.6.3.3 EC petrifilm for E.coli and Coliforms Test

7.6.3.4 EB petrifilm for Salmonella and Enterobacteriacea Test

7.6.3.5 STX petrifilm for Staphylococcus aureus Test

7.7. Plating sample on Neogen® Petrifilm® Aerobic Count (AC) Plates:

7.7.1. Place AC Petrifilm® plate on a level surface in a biological safety cabinet. Lift

top film.

7.7.2 With pipette perpendicular to Petrifilm® plate, place 1 mL of sample onto

[SOP

Standard Operating Procedure
SOP No | Rev
Microbial Limit Testing using the D-715 14 Page 10 of 43
Neogen® Petrifilm® System

center of bottom film.

7.7.3 Release top film and allow it to drop. Do not roll top film down.

7.7.4 With ridge side down, place spreader on top film over inoculum.

7.7.5 Gently apply pressure on spreader to distribute inoculum over circular area. Do

not twist or slide the spreader.

7.7.6 Lift spreader. Wait at least one minute for gel to form.

7.8 Plating sample on Neogen® Petrifilm® Rapid Yeast and Mold Count Plates:

7.8.1 Place Petrifilm® plate on a level surface in a biological safety cabinet. Lift top
film.

7.8.2 With pipette perpendicular to Petrifilm® plate, place 1 mL of sample onto

center of bottom film.

7.8.3 Release top film and allow it to drop.

7.8.4 Place the Petrifilm Rapid Yeast and Mold spreader on the center of the plate.

7.8.5 Distribute the sample with a gentle downward pressure on the center of the
spreader. Do not twist or slide the spreader.

7.8.6 Lift spreader. Wait at least one minute for gel to form.

7.9 Plating sample on Neogen® Petrifilm® Enterobacteriaceae Count Plates (EB) and E.

coli / Coliform Count (EC) Plates:

7.9.1 Place Petrifilm® plate on a level surface in a biological safety cabinet. Lift top

film.

7.9.2 With pipette perpendicular to Petrifilm® plate, place 1 mL of sample to onto

[SOP

Standard Operating Procedure
SOP No | Rev
Microbial Limit Testing using the D-715 14 Page 11 of 43
Neogen® Petrifilm® System

center of bottom film.

7.9.3. Carefully roll top film down to avoid entrapping air bubbles. Do not let top film

drop.

7.9.4 Wait at least one minute for gel to form.

7.10 Plating sample on Neogen® Petrifilm® Staph Express Count Plates:

7.10.1 Place Petrifilm® plate on a level surface in a biological safety cabinet. Lift top

film.

7.10.2 With pipette perpendicular to Petrifilm® plate, place 1 mL of sample to onto

center of bottom film.

7.10.3 Carefully roll top film down to avoid entrapping air bubbles. Do not let top film

drop.

7.10.4 Spread the inoculant using a Staph Express spreader.

7.10.5 Gently apply pressure on spreader to distribute inoculum over circular area. Do

not twist or slide the spreader.

7.10.6 Lift spreader. Wait at least one minute for gel to form.

7.11. Remove all materials from the biological safety cabinet and sanitize per D-101

Laboratory Housekeeping.

7.12 Incubation of Petrifilm® (AC, EB, EC, and STX) Plates:

7.12.1 Place AC, EB, EC, and STX plates in the incubator (typically 33°C + 2°C) with

the clear side up in stacks of no more than 20 plates. Document the sample start

date on form D-715-F1 Microbial Limit Test Ticket.

7.12.2 Incubate the Aerobic Count plates for 48 + 2 hours to 72 hours before reading.

[SOP

Standard Operating Procedure
SOP No | Rev
Microbial Limit Testing using the D-715 14 Page 12 of 43
Neogen® Petrifilm® System

The smallest colonies should be at least 1mm diameter before reading.

Document the results and length of incubation on form D-715-F1 Microbial

Limit Test Ticket. Justification is required for reads less than 46 hours.

7.12.3 Incubate the Enterobacteriaceae Count plates for 24 + 2 hours to 72 hours before

reading. Document the results and length of incubation on form D-715-F1
Microbial Limit Test Ticket. Justification is required for reads less than 22

hours.

7.12.4 Incubate the E. coli / Coliform Count plates for 48 + 2 hours to 72 hours before

reading. Document the results and length of incubation on form D-715-F1
Microbial Limit Test Ticket. Justification is required for reads less than 46

hours.

7.12.5 Incubate the Staph Express Count plates for 24 + 2 hours to 72 hours. For plates

that have colonies other than red-violet at 24 hours, insert a Staph Express disk

between the plate and cover and incubate at 37°C + 2°C for an additional 60 to
180 minutes (blue-green colonies do not require this step). If colonies exist that,

do not have pink zones around them the plate must be incubated for the full 180
minutes before reading (Reference Attachment 5). Document the results and

length of incubation on form D-715-Fl Microbial Limit Test Ticket.
Justification is required for reads less than 22 hours.

7.13 Incubation of Petrifilm® Rapid Yeast and Mold Count Plates:

7.13.1 Place the plates in the incubator (typically 23°C + 2°C) with the clear side up in

stacks of no more than 20 plates. Document the sample start date on form D-

715-F1 Microbial Limit Test Ticket.

7.13.2 Rapid Yeast and Mold Count Plates are incubated for 72 hours before reading.
Document the results and length of incubation on form D-715-F1 Microbial

Limit Test Ticket.

[SOP

Standard Operating Procedure
SOP No | Rev
Microbial Limit Testing using the D-715 14 Page 13 of 43
Neogen® Petrifilm® System

7.14 In the event of plates drying in the incubator ovens, place a 500mL beaker of sterile

water in the incubator to achieve approximately 30 — 70% RH to minimize moisture

loss and ensure the plates do not dry out.

7.15 Interpretation of Results

7.15.1 Visually determine the raw count or if the colonies are difficult to see, use the

Darkfield Colony Counter to determine the raw count. Document and calculate

the final result on form D-715-F1 Microbial Limit Test Ticket. Refer to the
excerpts from the Neogen Interpretation Guides (Attachments 1 — 4) for

counting instructions. The raw count is totaled as follows:

7.15.1.1 The raw count is the actual number of colonies on the plate.

7.15.1.2 Calculate the cfu / (g or mL) and document the result on form D-715-
Fl Microbial Limit Test Ticket. The reported count is calculated as

follows:

(CFUs (plate 1 + plate 2) / 2) (x Total DF)

divided by the volume plated (mL)

Dilution factor (DF) = Final Volume (mL)

divided by the sample (g) or (mL)

Example: DF = (90 mL + 10.30 g)/ 10.30 g

Note: If multiple dilutions are plated, multiply
each dilution factor up to the readable dilution.

7.15.2 For accurate calculations, raw counts should be between 25 to 250 cfu per plate.

7.15.3 If no colonies appear on the plate, the result should be reported as less than 1

times the corresponding lowest dilution used. Calculated count from raw counts
outside the acceptable range should be reported as an estimated result (est).

[SOP

Standard Operating Procedure
SOP No | Rev
Microbial Limit Testing using the D-715 14 Page 14 of 43
Neogen® Petrifilm® System

7.15.4 For AC plates, all red colonies are counted. Petrifilm AC Plates containing

greater than 250 colonies can either be estimated or recorded as TNTC. If the

colonies are high in density but separated enough to count, estimation can be
done by counting the number of colonies in one or more representative squares

and determining the average number per square. The average number can be
multiplied by 20 to determine the estimated count per plate. If a more accurate

count is required, the sample will need to be retested at higher dilutions.

7.15.4.1 If spreaders or liquefiers are present (i.e., bacteria species than can

liquefy the gel in the AC petrifilm), count only colonies in a few
unaffected squares. Determine average and estimate counts per plate

(See Attachment 1).

7.15.5 For RYM plates, all colonies are counted. Petrifilm RYM Plates containing

greater than 250 colonies can either be estimated or recorded as TNIC.

Estimation can be done by counting the number of colonies in one or more
representative squares and determining the average number per square. The

average number can be multiplied by 30 to determine the estimated count per
plate. If a more accurate count is required, the sample will need to be retested at

higher dilutions.

7.15.5.1 Yeast are small, defined edges, pink-tan to blue-green in color, usually

raised, and uniform color.

7.15.5.2 Mold are large colonies, diffuse edged, variable in color, appear flat,

look fuzzy, may have dark centers.

7.15.6 The EB plates detect salmonella as well as other Enterobacteriaceae.

Enterobacteriaceae can be identified on the plate by red colonies with gas
bubbles with yellow zones or yellow colored environment around the colony.

These colonies are recorded as total Enterobacteriaceae count. Red colonies

[SOP

Standard Operating Procedure
SOP No | Rev
Microbial Limit Testing using the D-715 14 Page 15 of 43
Neogen® Petrifilm® System

without gas are not to be counted. Upon the presence of presumptive salmonella

growth, selective and differential media can be used as supporting media for

confirmatory test. Follow section 7.15.10 to transfer suspected salmonella
colonies to a selective and differential agar. Refer to SOP D-715.0 Microbial

Limit Testing using Agar Plates. Additionally, the excerpts from
HardyCHROMTM instructions guides (Attachment #6) can be used as reference

for differential agar.

7.15.7 The EC plates detect E. coli as well as other coliforms colonies. E. coli can be

identified on the plate by gas bubbles surrounding blue to blue-red colonies.
Non-E. coli Coliforms can be identified on the plate by gas bubbles surrounding

red and blue colonies and are recorded as total coliforms. E. Coli specific

colonies are blue colonies with gas. Upon the presence of presumptive E. coli
growth, selective and differential media can be used as supporting media for

confirmatory test. Follow section 7.15.10 to transfer suspected E. coli colonies
to a selective and differential agar. Refer to SOP D-715.0 Microbial Limit

Testing using Agar Plates. Additionally, the excerpts from HardyCHROMTM
instructions guides (Attachment #6) can be used as reference for differential

agar.

7.15.8 The STX plates detect Staphylococcus aureus as well as other Staphylococcus
species. Staphylococcus aureus can be identified by red-violet colonies and

colonies with pink zones when Staph Express Disks are used (Reference
Attachment 5 for interpretation of positive results). If no colonies or only red-

violet colonies are present after 24 + 2 hours, red-violet colonies are recorded
and the test is completed. If confirmation step is required, count only pink

highlighted colonies. Alternatively, selective and differential media can be used
as supporting media to confirm the presence Staphylococcus aureus growth.

Follow section 7.15.10 to transfer suspected Staphylococcus aureus colonies to a
selective and differential agar. Refer to SOP D-715.0 Microbial Limit Testing

[SOP

Standard Operating Procedure
SOP No | Rev
Microbial Limit Testing using the D-715 14 Page 16 of 43
Neogen® Petrifilm® System

using Agar Plates. Additionally, the excerpts from HardyCHROMTM

instructions guides (Attachment #6) can be used as reference for differential

agar.

7.15.9 For selective plates such as EC, EB, and STX showing no growth, the result is

reported as “Negative” or “Absent” per product profile specifications.

7.15.10 Where necessary, colonies may be isolated for further identification. Lift the

top film using proper aseptic technique; pick the colony from the gel and
subculture on agar. Reference SOP D-1016 Microbial Identification via Biolog

Microstation for further steps.

7.15.11 In the event of inconclusive readings, noted results as “Inconclusive” and

proceed with retest. Serial dilutions of the original sample (if available) should
be performed. New sample can be requested for testing and D-715.0 method can

be use as reference method if considered most suitable for sample testing.

7.15.12 If any material does not meet acceptance criteria for any microbial testing, then

the original sample may be immediately re-plated for confirmation. If the value

observed after the re-plate is still outside of acceptance criteria then a Deviation
should be initiated as detailed in C-201 Deviation and Investigation Procedure.

Alternatively, initiation of a CAPA following SOP QS-108 Corrective and
Preventative Action (CAPA) may be performed.

8.0 Specifications

8.1 The maximum specification thresholds for microbial activity in dietary supplements are

described NSF/ANSI 173- August 7, 2012. Raw material and finished product
specifications for use in HBI Ion Labs dietary supplements comply with the specifications

set forth in NSF/ANSI 173. Specifications for a raw material are listed on the
corresponding raw material test ticket. Specifications for a finished product are listed in the

product profile. Additional specification details are located in SOP D-902 Establishment of

[SOP

Standard Operating Procedure
SOP No Rev
Microbial Limit Testing using the Page 17 of 43
D-715 14
Neogen® Petrifilm® System
Specifications. Unless a deviation in testing is required, the method listed on Product

Profile is the method to use even if it has been validated on other methods. If both methods

are on the Product Profile, D-715 Microbial Limit Testing using the Neogen® Petrifilm®
System is preferred.

8.2 Raw Material Categories and Definitions

8.2.1 Vitamin and/or mineral ingredient — Purified chemical or mineral.

8.2.2 Botanical ingredient (non-extract) — Crude botanical material (whole, cut or
powdered herb.)

8.2.3. Botanical ingredient (Extract / other dietary supplement ingredient) — The
complex, multicomponent mixture obtained after using a solvent to dissolve

components of the biomass. Extracts may be in dry, liquid, or semi-solid form.
Excipients may be added to extracts to adjust the concentration, enhance

stability, limit microbial growth, and to improve drying, flow, or other
manufacturing characteristics.

8.3 Acceptable limits for microbial contaminants in raw materials.

Table 1 *Specifications listed as colony forming units per gram (CFU/g)
Raw Material
Ingredient Aerobic Yeast/Mold | Enterobacteriaceae | Salmonella spp. E. coli® S. aureus
Vitamin and/or 1 X 10° 1 X 10? 1X 10° None Detected None Detected None Detected
mineral ingredient
Botamcal ingredient 1X 10’ 1 X 10° 1 X 10° None Detected 1 X 10? None Detected
— non-extract
Botanical Ingredient-
a fuer 1 X 10° 1 X 10° 1 X 10° None Detected None Detected None Detected
dietary supplement
Ingredient
(a) No guidance is available on acceptable limits for non-E. Coli coliforms.
(b) Upon the presence of E. coli type testing must be performed for enterovirulent E. coli. There is a zero
tolerance for the presence of enterovirulent E. coli.

8.4 Finished product Categories and definitions

[SOP

Standard Operating Procedure
SOP No Rev
Microbial Limit Testing using the Page 18 of 43
D-715 14
Neogen® Petrifilm® System
8.4.1 Category I — Finished products containing only vitamin and minerals.

8.4.2 Category II — Finished products containing Botanical ingredient — extract /

other dietary supplement ingredient.

8.4.3 Category III — Finished products containing botanical ingredients — non extract.

8.5 Maximum acceptable limits for microbial contaminants in Finished Products.

Table 2
* Specifications listed as colony forming units per gram (CFU/g)
Finished
Product Aerobic | Yeast/Mold | Enterobacteriaceae | Salmonella spp. E. coli! S. aureus
Category I 1 X 103 1 X 10? 1 X 10? None Detected None Detected None Detected
Category II 1 X 104 1X 10° 1X 10? None Detected None Detected | None Detected
Category III 1X 107 | 1X10° 1X 104 None Detected ()1 X 10? None Detected

8.6 Upon the presence of E. coli type testing must be performed for enterovirulent E. coll.
There is a zero tolerance for the presence of enterovirulent E. coli USP Microbial

Limits per <2023>.

Table 3 *Specifications listed as colony forming units per gram (CFU/g)

Material Aerobic | Yeast/Mold E. coli / Salmonella
Powdered Botanicals 1 X 10° 1X 10° None Detected
Powdered Botanical Extracts 1 X 104 1 X 10° None Detected
Nutritional supplements with botanicals | 1 X 10° 1X 10° None Detected

Nutritional supplements with highly 1X 10° 1X 102 None Detected
refined ingredients

9.0 Reporting

9.1 Reporting results for TAPC

[SOP

Standard Operating Procedure
SOP No Rev
Microbial Limit Testing using the Page 19 of 43
D-715 14
Neogen® Petrifilm® System
9.1.1 The expanded uncertainty for the method when quantifying TAPC is 28% with a

coverage factor of 2

a4 Reporting results for Yeast and Mold

9.2.1 The expanded uncertainty for the method when quantifying Yeast and Mold is 7%
with a coverage factor of 2

10.0 Documentation Requirements

A PQV check must be performed for each completed page of form D-715-F2 Microbial
10.1 Sample Log as outlined in SOP A-106 Documentation Guidelines for cGMP Records.

10.2 All documentation will be distributed and maintained as outlined in SOP C-501

Document Control and SOP C-502 Record Storage, Retention, and Destruction.

11.0 Revision History

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 01/23/12 | New - - Changed the SOP format, revised and updated SOP to be consistent with 1 (5/09/13 validated test method, updated D-715-F2 Test Ticket to reflect new format, - - added D-715-F 1 microbial sample log. | - | - |
| 2 | 11/11/13 | Added rapid yeast and mold count plates to Section 5.6.2.2 | 13-1027 | B. Johns |
| 3 | 11/06/14 | Added Staph Express System Instructions New title. Added NSF/ANSI 173-2012 microbial limit specifications and 4 OizeHS r s e e f c e t r i e o n n c s e . 7 . A 0 d S d p e ec d i f U i S ca P t i < o 2 ns 0 2 a 3 n > d S 8 p . e 0 c i S f a i f c e a t t y i o T n e s s t a i n n d g. r e A f d e d re e n d c e D . - A 1 d 0 d 1 e L d a b n o e r w atory buh ie B. Jonnie Housekeeping reference. New Title. New D-715-F2 format to include use of agar plates. Reference | 14-0888 | B. Johns |
| 5 | 10/11/16 | recovery study protocols. Expanded incubation times based on subvalidations. Expanded incubation temperature ranges to reflect current practices. | 16-0914 | R. Casabianca |
| 6 | 03/08/17 | . Update test tickets for clarity and to better define start / stop incubation points. Edited D-715-F1 sample log to include pH data. Removed P. aeruginosa : 04/11/18 tresetintg ,fr Momo vteesdt tDic-k7e1t;5 -teFs2tibng f o iDn-c7l1u5d.e0d- iFn2 ,D -A7d15d.e0d. DaUrpkdfaiteeldd Canodl ocnlyar iCfoiuend tDe-r beuhee B, Juinson to equipment list. Changed growth recovery calculation for clarity. Edited form D-715.0 to | 17-0257 | B. Johns |
| 8 | 03/04/20 | combine testing methods D-715 and D-715.0. Added diluent prep according to 3M recommendations. Added what colonies are to look like for all petri-film plates. Added | 19-0733 | L. McWade |
| 9 | 05/19/21 | Butterfield’s Buffer required media. Corrected final result calculation. CC- Corrected all dilutions for each of use 10 LLi23/2! M of o d u i n f ce i r e t d a i S nt O y P . t C o h a a c n t g u e al d p f r o o r c m e s n s u u m s b e e d r . s A f d o d r e t d es t I S ti O c k r e e t q a u n i d r e l m o e g. n ts/measurement caeeaniaddall ©. bie | 21-0204 | G. Shaw |
| 11 | 07/12/23 | Added information related to Validated dilution and diluents for Final CC- Products. Updated format. Added documentation requirements. | 23-0339 | G. Shaw |

[SOP

Standard Operating Procedure
SOP No | Rev
Microbial Limit Testing using the Page 20 of 43
D-715 14
Neogen® Petrifilm® System
Added documentation requirements. Added additional SOP references.
12 11/04/23 | Revised test ticket and log form to include logbook number. Added alternative CC-23-0545 K. Burris
options for sample logbook.
Updated incubation times, reporting terminology, addition of form D-715-F3.
13 09/23/24 | Clarified sample preparation, growth recovery calculation and interpretation. CC-24-0470 A. Perez
Added sampling procedure for 5 Gallon Water Jug. Updated SOP to reflect
14 05/27/25 current practices, including the use and interpretation of alternative methods. CC-25-0230 A Pp
Added information related to Validated dilution and diluents for Final 2 —
Products. Edited brand name for petrifilm plates.
12.0 Attachments

10.1 Attachment 1 — Aerobic Count Plate Interpretation Guide Excerpt

10.2 Attachment 2 — Yeast and Mold Count Plate Interpretation Guide Excerpt

10.3. Attachment 3 — Enterobacteriaceae Count Plate Interpretation Guide Excerpt

10.4 Attachment 4 -E. coli / Coliform Count Plate Interpretation Guide Excerpt

10.5 Attachment 5 — Staph Express Count Plate Preparation and Interpretation Guide

10.6 Attachment 6 — Chromogenic Medium Interpretation Guide Excerpt

10.7 Attachment 7 — Finished Product Summary for Validated Dilution and Diluent

[SOP

Standard Operating Procedure
SOP No | Rev
Microbial Limit Testing using the D-715 14 Page 21 of 43
Neogen® Petrifilm® System

Attachment I — Aerobic Count Plate Interpretation Guide Excerpt

The Petrifilm Aerobic Count (AC) plate is a ready-made culture medium system that contains Standard Methods
nutrients, a cold-water-soluble gelling agent, and an indicator that facilitates colony enumeration. Petrifilm AC
plates are used for the enumeration of aerobic bacteria.

Aerobic Bacteria Count = 152
: A red indicator dye in the plate colors the colonies.
’ : Count all red colonies regardless of their size or
: : a color intensity.

Count = 0 Count = 16

It is easy to interpret the Petrifilm AC plate. Figure 2 Figure 3 shows a Petrifilm AC plate with a few
shows a Petrifilm AC plate without colonics, bacterial colonics.

[SOP

Standard Operating Procedure eating. ie
Microbial Limit Testing using the D-71 s “nd Page 22 of 43

Neogen® Petrifilm® System

Attachment 1 — Aerobic Count Plate Interpretation Guide Excerpt (Cont’d)

Count = TNTC (Estimated count = 10°) Count = TNTC {Estimated count = 10°)
Figure 6 shows a Petrifilm AC plate with colonies thal are With very high counts, the entire growth area may turn
too numerous to count (TNTC). pink, as shown in figure 7. You might observe individual
colonies only at the edge of the growth area. Record this
as a TNTC result.

: BOE ” % Hs #
potot ice OE age ‘ : ‘4

' : e — . : :

| ee ~~ sila : " : . ‘ye |

Count = TNTC (Estimated count = 10°) Count = TNTC {Estimated count = 10°)
Occasionally, distribution of colonies appears uneven. The colonies on the Petrifilm AC plate in tigure 9 appear
as shown in figure 8. This is also an indication countable at first glance. However, when you look closely
of a TNTC result. at the edge of the growth area, you can see a high
concentration of colonies, Record this as a TNTC result.

[SOP

Standard Operating Procedure
SOP No Rev
Microbial Limit Testing using the Page 23 of 43
D-715 14
Neogen® Petrifilm® System
Attachment 1 — Aerobic Count Plate Interpretation Guide Excerpt (Cont’d)

— a Te Estimated Count = 160
oo i | : A few species of bacteria liquify the gel in the
: fs, Petrifilm AC plate, as shown in figure 10. When
l, + ees 8 * tuhniasf ofceccutresd. s dqeutaerremsi anned t theh eanv meurlatgiep cloy uint tb yin 2 a0 fteow
| | obtain the estimated count. Do not count red spots
ete F ‘ within the liquified area.

# P . "4 . : # * ie

Count = 83
Because colonies on Petrifilm AC plates are red. you
can distinguish them from opaque, irregularly shaped
food particles (see circles | and 2).

[SOP

Standard Operating Procedure
SOP No Rev
Microbial Limit Testing using the Page 24 of 43
D-715 14
Neogen® Petrifilm® System

Attachment 2 — Yeast and Mold Count Plate Interpretation Guide Excerpt

The PetrifilmTM Yeast and Mold (YM) Count Plate is a ready-made culture medium that contains
a cold-water soluble gelling agent, nutrients and an indicator dye to provide contrast and
facilitate counting.

Total Count = 20
Yeast Count = 16
Mold Count = 4
This Petrifilm YM Plate contains both yeast colonies and mold colonies.

[SOP

Standard Operating Procedure
SOP No | Rev
Microbial Limit Testing using the D-715 14 Page 25 of 43
Neogen® Petrifilm® System

Attachment 2 — Yeast and Mold Count Plate Interpretation Guide Excerpt
(Cont'd)

Yeast and Mold Count = 0 Estimated Total Count ~ 500
Figure 2 shows a Petrifilm YM Plate without yeast Estimated Yeast Count ~ 480
Mold Count = 24
or molds.
When colonies number more than 150, estimate the count.
Determine the average number of colonies in one square
(1 cm) and multiply it by 30 to obtain the total count per
plate. The inoculated area is approximately 30 cm’. Yeast
colonies may range in color from tan (as in this example)
io pink to blue-green.

Estimated Mold Count ~ 64
The Petrifilm YM Plate in figure 4 contains yeast colonies The mold colonies in figure 5 are beginning to crowd and
too numerous to count (TNTC). The small, blue colonies overlap cach other on the plate. Count cach colony margin
at the edge of the plate (highlighted in the box) are present or focus. The plate can be divided into sections bo assist in
throughout the entire plate although less visible. counting. In this example, approximately 1/4 of the plate
was counted, then the number of colonics counted was
multiplied by 4 to pet the estimated count on the plate.
The section shown has 16 molds,

[SOP

Standard Operating Procedure on
Microbial Limit Testing using the D-71 My ve Page 26 of 43

Neogen® Petrifilm® System

tachment 2 — Yeast and Mold Count Plate Interpretation Guide Excerpt

Cont’d)

Plates in figures 6a and 6b are the same sample. Figure 6a is a 1:10
dilution and has colonies that are small, faint and numerous, making
it difficult to count. Figure 6b is a 1:100 dilution and shows how
diluting product to obtain a colony count of less than 150 colonies
makes counting easier. As with most growth media. in a highly
competitive environment (such as figure 6a), typical colony growth
will be inhibited. For heavily contaminated samples such as these,
higher dilutions are recommended for a more accurate count and
more typical colony groreth (as in figure 6b).
4

Yeast and Mold Count = 0 Yeast and Mold Count = 0

Petrifilm YM Plates utilize a phosphatase indicator dye. Therefore, same food products that contain phosphatase may cause a blue
color reaction to occur on the Petrifilm YM Plate. Two types of color reactions are sometimes ca uniform blue background
coler or intense, blue spots. Figure 7 shows uniform blue background color and figure 8 shows intense blue spots which are often
seen with spices or granulated products. Figure 8 also shows food particles that yielded phosphatase
Ta reduce a phosphatase reaction, follow one or more of these techniques:
1. Dilute Sample: Further sample dilution will minimize blue backgsound color or reduce the number of intense blue spots.
2. Sample Preparation: Mix sample and fet settle for 3-5 minutes before plating. Draw sample from center portion of sample
container or use filtered homogenizer bag to avoid plating large particles.
3. Check and Note: Observe plates within 24-36 hours of incubation and make note of any color change to aid in
final interpretation.

[SOP

Standard Operating Procedure
SOP No | Rev
Microbial Limit Testing using the D-715 14 Page 27 of 43
Neogen® Petrifilm® System

Attachment 3 — Enterobacteriaceae Count Plate Interpretation Guide Excerpt

Enterobacteriaceae count = 13 Enterobacteriaceae count = 9
plate colors all colonies red. The top ff ilm traps: gas wFiitghu rae f2e sw hoEwnste raa Pbeatcrtiefriilma cEeanet ceolronoibesa acndt ea rhCiioguahn ctn euplmaabteeer

soe seen wo soil cxdlinien senecmnasded toy yellow nome. a
Bacteria producing gas and/or acid are considered removed from the selective influence of the medium.
to be presumptive Enterobactearnid awiclle haavee
one of the following characteonr tihes Ptetiricfislm
EnterobacterCoiuantc pelaatee: colonies associated
with gas bubbles and no acid zones (see figure |, circle 1),
colonies with yellow acid zones but no gas
(see fig1, ucirrcle e2), or colomes producboithn ggas
and acid (see figure |. circle 3).
Figure | also illustrates how babble patterns can vary.
Sometimes gas disrupts the colony so that the colony
“outlinetsh”e gas bubble as in figure |, circle 3.

[SOP

Standard Operating Procedure
SOP No | Rev
Microbial Limit Testing using the Page 28 of 43
D-715 14
Neogen® Petrifilm® System
Attachment 3 — Enterobacteriaceae Count Plate Interpretation Guide Excerpt

(Cont'd)

Enterobacteriaceae count = 0 Enterobacteriaceae count = 35

Notice the change in gel color in figures 3 through 8. As
the Enterobacteriaceae count increases, the color of the
ge! lightens from purple to yellow or cream colored.

onaguininnnenipunalangeall
bactonaeae Coun platesa 15-100 cobonies sSanpes
suarsiche wien Grsaientedl The ciecelar groeth

arch is approDonmei. Esmtimaatest caen bel mayde
by counting the munber af colesties it one oF npn
roypecsentative squares and determuning the average murmber
per square. Moltply the average number of coolomes per
sparbey 20 to determine the estimated count per phate.

[SOP

Standard Operating Procedure
SOP No | Rev
Microbial Limit Testing using the D-715 14 | Page 29 of 43
Neogen® Petrifilm® System

tachment 3 — Enterobacteriaceae Count Plate Interpretation Guide Excerpt
(Cont'd)

Enterobacteriaceae count = TNTC Enterobacteriaceae count = TNTC
Petrifilm Enterobacteriaceac Count plates with more than In figure 7, the count is so high that acid zonaned gsas
100 colonies are considered too numerous to count (TNTC) bubbles are not easily seen. A lightening of the gel color
and have a light backgrcooloru anlondg with at least one indicates that the result is TNTC
of the following characteristics: many small colonies or
many gas bubbles. See figure 6.

Enterobacteriaceae count = TNTC
The Petrifilm Enterobacteriaceae Count plate in figure 8 has
two characteristics indicating TNTC colonies: lightening of
the gel color and many small colonies.

[SOP

Standard Operating Procedure mrp Miaal ae
Microbial Limit Testing using the D-71 - i Page 30 of 43

Neogen® Petrifilm® System

Attachment 3 — Enterobacteriaceae Count Plate Interpretation Guide Excerpt
(Cont'd)

Enterobacteriaceae count = 44 Enterobacteriaceae count = 2

of the Petrifilm Enterobacteriaceae Count plate. They and are not associated with gas bubbles or acid zones.
are irregularly shaped and not associated with a red colony. See figur10e.

Enterobacteriaceae count = 29

associated with gas bubbles or acid zones. See figure 11.

[SOP

Standard Operating Procedure
SOP No | Rev
Microbial Limit Testing using the D-715 14 Page 31 of 43
Neogen® Petrifilm® System

Attachment 4 — E. coli / Coliform Count Plate Interpretation Guide Excerpt

Petrifilm E. coli/Coliform Count (EC) plates contain Violet Red Bile (VRB) nutrients, a cold-water-soluble gellmg agent. an
err glucuronidase activity. and an indicator that facilitates colony enumeration. Most£. coli (about 97%) produce
beta-glucuronidase which produces a blue precipitate associated with the colony. The top film traps gas produced by the
lactose fermenting colifoarndm Es£. coli. About 95% of E. coli produce gas, indicated by blue to red-blue colonies associated
with entrapped gas on the Petrifilm EC plate (within approximately one colony diameter).
AOAC INTERNATIONAL and U.S. FDA Bactenological Analytical Manual (BAM) define coliforms as gram-negative rods which
produce acid and gas from lactose during metabolic fermentation. Coliform colomes growmg on the Petrifilm EC plate
produce acid which causes the pH imdicator to make the gel color darker red. Gas trapped around red coliform colomes
ond alia call
‘we
The identification of E. coli may vary by country
(see Reminders for Use section for incubation times
and temperatures):
E. coli = 49 (blue colomes with gas)
Total coliform = 87 (red and blue colonies with gas)

Do not use this plate alone for the detection of E. coii 0157. Like most other £. coliicoliform media. this plate will not
specifically mdicate whether any O157 strain is present.

[SOP

Standard Operating Procedure
icrobial Limit Testing using the “~ag i Page 32 of 43

Neogen® Petrifilm® System

Attachment 4 -— E. coli / Coliform Count Plate Interpretation Guide Excer

Cont’d)

E. coli count = 1
Notice the changes im gel color in figures 2 through 8. Total coliform count = 28
A of s t t he h g e e E l c t ou lmis o rt oc odlairfko rremd c ooru nptu rmpclree-absleuse.. the color The counting range for the to-ta l population on| Petnifilm
EC plates 1s 15-150.
B i ackgr e o n u a o n t d a bubb r e l su e lt s e o a f r E e . a c c o e h li a racter l i i e s f t ic of t o h w e t h ge . l D be o c a n u o s t e c t o h u e n y t a c r o e l o r n e i — m e o s v t e h d a t f r a o p m p e t a h r e o n se l t e h c e t i f v o e a m m f b i a v r e n n .e c r e
of the medaum. See circle 1.

E. coli count = 3 E. coli count = 1
Any blue in a colony (bine to red-blue) indicates the Estimated total coliform count = 156
p de r te e ct s i e on n o o fc f Ee b . l u c e oi p l r . ec F i r p o i n t t a t h e g h f t o i r n m g e w d i b ll y e a n h c a o n lo c n e y . t he The — ci rcular growth area is approxi o mate e ly 2 0 cm- i .
Estimates can be made on plates containing greater than
Circle 1 shows a red-blue colony counted using back 150 colonies by counting the number of colonies in one
lighting. Circle 2 shows the same colony with front of more representative squares and determing the average
lighting. The blue precipitate is more evident m circle 2. number per square. Multiply the average number by 20 to
determine the estimated count per plate.

[SOP

Standard Operating Procedure
SOP No ev
Microbial Limit Testing using the 14 Page 33 of 43
D-715
Neogen® Petrifilm® System
tachment 4 — E. coli / Coliform Count Plate Interpretation Guide Excerpt

Cont’

Actual count ~ 10° Actual count ~ 10°
High concentratioofn £s. coli may causthee growth
have one or more of the ing characteristics:
many small colonies. many gas bubbles, anda
deepening of the gel color from red to purple-blue.

Presumptive E. coli count ~ 8 Actual count ~ 10°
Esttiotalm colaifotrm ceoundt ~ 10° When high numbers of non-coliform orgamsms such as
Pseudomonas axe presenotn PetrEiC pflatiesl. tmhe gel
When high levels of coliforms are present (10% may tum yellow.
some strains of E. coli may proledss ugasc aend
colonies without gas andor blue zones as presumptive
if mecessary.

[SOP

Standard Operating Procedure |
Microbial Limit Testing using the =eee er Page 34 of 43

Neogen® Petrifilm® System

Attachment 4 — E. coli / Coliform Count Plate Interpretation Guide Excerpt
(Cont’d)

Total coliform count = 3 Total coliform count = 78

associated with gas bubbles. colony so that the colony “outlines” the bubble.
See circles 1 and 2.
moculation os from trapped air within the sample.

with a coloSene cyirc.le 3.

Qo
‘s) OF
oe a wie 2 ®
i oO 4

Examples 1-16 show varbuibbloe pauttesrns
assowcithi gaas ptrodeucidng coloAmlle shsou.ld
be enumerated.

[SOP

Standard Operating Procedure
SOP No | Rev
Microbial Limit Testing using the D-715 14 Page 35 of 43
Neogen® Petrifilm® System

Attachment 5 — Staph Express Count Plate Preparation and Interpretation Guide

Excerpt

A) Inoculate Plate B) Stamp plate

C) Incubate Plate nen D) Add Staph Express Disk

E) Read Plate- Staphylococcus aureus

distinguished from other aerobic
cultures by pink highlighted colonies.

[SOP

Standard Operating Procedure cain
Microbial Limit Testing using the D-71 . i Page 36 of 43

Neogen® Petrifilm® System

Attachment 6 — Chromogenic Medium Interpretation Guide Excerpt

Method of Use: Allow the plates to warm to room temperature. The agar surface should be dry prior
to inoculating. Streak for isolation with a sterile loop. Incubate plates in an inverted position,
protected from the light, aerobically at 35-37 degrees C. for 24 hours. Examine plates for colonies
showing typical morphology and color.

HardyCHROMTM Salmonella

Salmonella spp., including S. typhi and S. paratyphi A, produce magenta colored colonies. Other
members of the Enterobacteriaceae (if present) produce blue, blue-green, white, or colorless colonies.
Gram-positive bacteria and nonglucose fermenting bacteria will be inhibited.

Salmonella enterica( 14028) colonies growing | Escherichia coli (ATCC 25922) colonics growing on
on HardyCHROMTM Salmonella (Cat. no. G309). HardyCHROMTM Salmonella (Cat. no. G39).
incubated acrobically for 24 hours at 35 deg. C. Incubated aerobically for 24 hours at 35 deg. C.
HardyCHROMTM ECC

Pink to violet colored colonies are a positive test for the presence of E. coli. Turquoise colonies are a
positive test for the presence of coliform bacteria other than E. coli. Other gram-negative bacteria
appear as white or colorless colonies. Gram-positive bacteria are inhibited

Escherichia coli (ATCC&re6g; 23922) oolomes. growing on Miebyiella preumoniae {ATCC® 135883) cobunmes growing on
Hardy HROMTM BCC (Cat. mo. G303). incubated acrobbcautly for Hardy CHROM'TM BCC (Cat. no. 6303). incubated acrofacally for
24 hours at 39°C. 24 hors wt 359°C".

[SOP

Standard Operating Procedure
Microbial Limit Testing using the ply Rev
Page 37 of 43
14
Neogen® Petrifilm® System
Attachment 6 —- Chromogenic Medium Preparation and Interpretation Guide

Excerpt (Cont'd)

HardyCHROMTM Staphylococcus aureus

Staphylococcus aureus will appear as smooth, deep pink to fuchsia colored colonies. Most other
organisms, including Staphylococcus epidermidis will be partially to completely inhibit. Other
organisms that may grow on HardyCHROMTM Staph aureus may appear as cream, blue, or colorless

colonies. Staphylococcus saprophyticus will appear as turquoise colored colonies. Some gram-
positive organisms other than S. aureus may appear as blue colonies.
Some non-S. aureus colonies may develop a light pink color after 48 hours. Do not incubate plates
more than 24 to 28 hours.

Staphylococcus aureus (aTcc® 25923) colonies Staphylococcus saprophyticus (ATC 15305)
growing on HardyCHROMTM Staph aureus (Cat. colonies growing on HardyCHROMTM Staph
no. G311). incubated aerobically for 24 hours at aureus (Cat. no. G311). Incubated aerobically for
36°C. 24 hours at 35°C.

[SOP

Standard Operating Procedure
SOP No | Rev
Microbial Limit Testing using the D-715 14 Page 38 of 43
Neogen® Petrifilm® System

Attachment 7 — Finished Product Summary for Petrifilm Validated
Dilution and Diluent

Product Profile Number Product (g) | Dilution Diluent
PGM00021 10 g 1:10 Peptone Salt-90 mL

PGM00022 10 g 1:10 Butterfields buffer- 90 mL
PLS00007 10 g 1:10 Peptone Salt-90 mL
PPW00005 10g 1:10 Butterfields buffer- 90 mL
PPWO00011 10 g 1:10 Peptone Salt- 90 mL
PPW00020 10 g 1:10 Butterfields buffer- 90 mL
PTBOOOO1 10 g 1:10 Butterfields buffer-90 mL
PTBO0012 10g 1:10 Butterfields buffer-90 mL
PTBO00014 10 g 1:10 Butterfields buffer-90 mL
PTBOO0019 10 g 1:10 Butterfields buffer-90 mL
PTB00020 10g 1:10 Butterfields buffer-90 mL
PTBO00023 10g 1:10 Butterfields buffer-90 mL
PTB00026 10 g 1:10 Butterfields buffer-90 mL
SCT00004 10 g 1:10 Butterfields buffer-90 mL

SCT00024 10g 1:10 Peptone Salt-90 mL
SCT00058 10g 1:10 Peptone Salt-90 mL
SCT00059 10g 1:10 Peptone Salt-90 mL
SCT00085 10g 1:10 Peptone Salt-90 mL
SCT00113 10g 1:10 Peptone Salt-90 mL
SCT00128 10 g 1:10 Peptone Salt-90 mL
SCT00179 10 g 1:10 Butterfields buffer- 90 mL
SCT00257 10g 1:10 Peptone Salt-90 mL
SCT00297 10 g 1:10 Butterfields buffer- 90 mL
SCT00361 10 g 1:10 Butterfields buffer- 90 mL
SCT00364 10g 1:10 Peptone Salt-90 mL
SCT00384 10g 1:10 Peptone Salt-90 mL

SCT00392 10g LO Peptone Salt-90 mL
SCT00393 10 g 1:10 Butterfields buffer- 90 mL
SCT00439 10 g 1:50 Butterfields buffer
SCT00452 10g 1:10 Peptone Salt — 90 mL
SCT00467 10 g 1:10 Butterfields buffer- 90 mL
SECO001 1 10g 1:10 Butterfields buffer- 90 mL
SEC00013 10g 1:10 Butterfields buffer- 90 mL
SECO0025 10 g 1:10 Butterfields buffer- 90 mL
SEC00030 10g 1:10 Butterfields buffer- 90 mL
SEC00037 10g 1:10 Butterfields buffer- 90 mL
SEC00038 10g 1:10 Butterfields buffer- 90 mL
SECO0040 10g 1:10 Butterfields buffer- 90 mL

[SOP

Standard Operating Procedure
SOP No | Rev
Microbial Limit Testing using the D-715 14 Page 39 of 43
Neogen® Petrifilm® System

Attachment 7 — Finished Product Summary for Petrifilm Validated Dilution and
Diluent (continued)

Product Profile Number | Product (g) | Dilution Diluent
SECO00052 10g 1:10 Butterfields buffer- 90 mL
SEC00079 10g 1:10 Peptone Salt-90 mL
SECO00133 10g 1:10 Peptone Salt-90 mL

SEC00139 10g 1:10 Butterfields buffer- 90 mL
SECO00144 10g 1:10 Butterfields buffer- 90 mL
SEC00176 10g 1:10 Peptone Salt-90 mL
SEC00199 10 g 1:10 Butterfields buffer-90 mL
SEC00200 10g 1:10 Peptone Salt-90 mL
SEC00205 10g 1:10 Peptone Salt-90 mL
SEC00208 10g 1:10 Peptone Salt-90 mL
SEC00220 10 g 1:10 Peptone Salt-90 mL
SEC00237 10g 1:10 Peptone Salt-90 mL
SEC00239 10 g 1:10 Butterfields buffer-90 mL
SEC00243 10 g 1:10 Peptone Salt-90 mL
SEC00264 10 g 1:10 Peptone Salt-90 mL
SEC00269 10g 1:10 Butterfields buffer-90 mL

SEC00270 10g 1:10 Peptone Salt-90 mL
SEC00290 10 g 1:10 Peptone Salt-90 mL
SEC00291 10 g 1:10 Peptone Salt-90 mL
SEC00295 10 g 1:10 Peptone Salt-90 mL
SEC00308 10 g 1:10 Butterfields buffer-90 mL
SEC00320 10 g 1:10 Butterfields buffer-90 mL
SEC00325 10g 1:10 Butterfields buffer-90 mL
SEC00327 10 g 1:10 Peptone Salt-90 mL
SEC00332 10g 1:10 Butterfields buffer-90 mL
SEC00333 10 g 1:10 Butterfields buffer-90 mL
SEC00341 10 g 1:10 Butterfields buffer-90 mL
SEC00362 10 g 1:10 Butterfields buffer- 90 mL

SEC00368 10 ¢ 1:10 Butterfields buffer-90 mL
SEC00372 10 g 1:10 Butterfields buffer- 90 mL
SEC00373 10 g 1:10 Butterfields buffer- 90 mL
SEC00398 10 g 1:10 Peptone Salt-90 mL
SEC00401 10g 1:10 Butterfields buffer-90 mL
SEC00403 10 g 1:10 Peptone Salt-90 mL
SEC00421* 10g 1:10 Butterfields buffer-90 mL
SEC00423 10 g 1:10 Peptone Salt- 90 mL
SEC00426 10 g 1:10 Peptone Salt-90 mL
* Refer Method Suitability test details

[SOP

Standard Operating Procedure
SOP No Rev
Microbial Limit Testing using the Page 40 of 43
D-715 14
Neogen® Petrifilm® System
Attachment 7 — Finished Product Summary for Petrifilm Validated Dilution and

Diluent (continued)

Product Profile Number Product (g) | Dilution Diluent
SEC00433 10 ¢ 1:10 Peptone Salt-90 mL
SEC00438 10 g 1:10 Peptone Salt- 90 mL
SEC00442 10g 1:10 Peptone Salt- 90 mL
SEC00498 10g 1:50 Butterfields buffer + 14 mL of 1N HCl
SEC00593* 10g 1:10 Butterfields buffer-90 mL

SEC00601 10g 1:50 Butterfields buffer
SEC00602 10 g 1:100 Butterfields buffer + 17 mL of IN NaOH
SEC00609 10g 1:10 Butterfields buffer-90 mL
SEC00610 10g 1:50 Butterfields buffer + 3 mL of 1N HCl
SEC00617 10 g 1:50 Butterfields buffer + 25 mL of 1N HCl
SGMO00281 10 g 1:10 Peptone Salt-90 mL
SGM00331 10g 1:10 Peptone Salt-90 mL
SGM00336 10g 1:10 Peptone Salt-90 mL
SGM00337 10g 1:10 Peptone Salt-90 mL
SGM00349 10g 1:10 Peptone Salt-90 mL
SGM00351 10 g 1:10 Peptone Salt-90 mL
SGM00357 10g 1:10 Butterfields buffer-90 mL + 5 mL of IN NaOH
SGM00358 10 g 1:10 Peptone Salt-90 mL
SGM00359 10 g 1:10 Peptone Salt-90 mL
SGM00360 10g 1:10 Peptone Salt-90 mL
SGM00379 10 g 1:10 Peptone Salt-90 mL
SGM00391 10 ¢ 1:10 Peptone Salt-90 mL
SGM00418 10 g 1:10 Peptone Salt- 90 mL
SGM00428 10 ¢ 1:10 Peptone Salt-90 mL
SGM00475 10 g 1:10 Butterfields buffer-90 mL
SGM00507 10 ¢ 1:20 Butterfields buffer + 4 mL of IN NaOH
SGM00549 10 ¢ 1:20 Butterfields buffer + 5 mL of IN NaOH
SGM00550* 10g 1:10 Butterfields buffer-90 mL
SGM00552 10 ¢ 1:10 Butterfields buffer-90 mL
SGM00553 CA 10 ¢g 1:10 Butterfields buffer-90 mL + 1 mL of IN NaOH
SGM00558 10g 1:10 Butterfields buffer-90 mL
SGM00562 10 g 1:10 Butterfields buffer-90 mL
SGM00567 10¢ 1:20 Butterfields buffer
SGM00585 10 g 1:10 Butterfields buffer-90 mL + 2 mL of IN NaOH
SGM00598 10g 1:10 Butterfields buffer-90 mL + 2 mL of IN NaOH
SLC00039 10 g 1:10 Peptone Salt-90 mL
SLC00048 10 ¢ 1:10 Peptone Salt-90 mL
SLC00049 10g 1:10 Butterfields buffer-90 mL
SLC00056 10 g 1:10 Butterfields buffer-90 mL
* Refer Method Suitability test details

[SOP

Standard Operating Procedure
SOP No | Rev
Microbial Limit Testing using the D-715 14 Page 41 of 43
Neogen® Petrifilm® System

Attachment 7 — Finished Product Summary for Petrifilm Validated Dilution and

Diluent (continued)

Product Profile Number | Product (g) Dilution Diluent
SLC00084 10g 1:10 Butterfields buffer-90 mL
SLC00173 10g 1:10 Butterfields buffer-90 mL

SLC00174 10g 1:10 Butterfields buffer-90 mL
SLC00183 l0g 1:10 Peptone Salt-90 mL
SLC00215 10g 1:10 Butterfields buffer-90 mL
SLC00218 10g 1:10 Butterfields buffer-90 mL
SLC00232 10g 1:10 Butterfields buffer-90 mL
SLC00234 10g 1:10 Butterfields buffer-90 mL
SLC00321 10g 1:10 Butterfields buffer-90 mL
SLC00334 10g 1:10 Butterfields buffer-90 mL
SLC00343 10g 1:10 Butterfields buffer-90 mL
SLC00356 10 g 1:10 Peptone Salt-90 mL
SLC00375 10g 1:10 Butterfields buffer-90 mL
SLC00381 10 g 1:10 Peptone Salt- 90 mL
SLC00385 10g 1:10 Butterfields buffer-90 mL
SLC00387 10g 1:10 Butterfields buffer-90 mL
SLC00389 10g 1:10 Butterfields buffer-90 mL
SLC00396 10g 1:10 Peptone Salt-90 mL
SLC00408 10 g 1:10 Butterfields buffer-90 mL
SLC00415 10 ¢ 1:10 Butterfields buffer-90 mL
SLC00425 10g 1:10 Peptone Salt-90 mL
SLC00508 10g 1:10 Butterfields buffer-90 mL
SLC00575 10g 1:10 Butterfields buffer-90 mL
SLS00226 10g 1:10 Butterfields buffer-90 mL
SLS00305 10g 1:10 Butterfields buffer-90 mL
SLS00306 10g 1:10 Peptone Salt-90 mL

SLS00307 10g 1:10 Peptone Salt-90 mL
SLS00348 10 g 1:10 Butterfields buffer-90 mL
SLS00355 10 g 1:10 Butterfields buffer-90 mL
SLS00370 10 g 1:10 Butterfields buffer-90 mL
SLS00394 10 g 1:10 Butterfields buffer-90 mL
SLS00395 10g 1:10 Butterfields buffer-90 mL
SLS00405 10 g 1:10 Butterfields buffer-90 mL
SLS00427 10g 1:10 Peptone Salt-90 mL
SPW00003 10 g 1:10 Butterfields buffer-90 mL
SPW00008 10g 1:10 Butterfields buffer-90 mL
SPW00080 10 g 1:10 Butterfields buffer-90 mL
SPW00149 10g 1:10 Butterfields buffer-90 mL
SPW00157 10 g 1:10 Butterfields buffer-90 mL

[SOP

Standard Operating Procedure
SOP No Rev
Microbial Limit Testing using the Page 42 of 43
D-715 14
Neogen® Petrifilm® System
Attachment 7 — Finished Product Summary for Petrifilm Validated Dilution and

Diluent (continued)

Product Profile Number | Product (g) Dilution Diluent
SPW00193 10g 1:10 Peptone Salt-90 mL
SPW00278 CA 10g 1:10 Butterfields buffer-90 mL
SPW00278 US 10g 1:10 Butterfields buffer-90 mL
SPW00339 10 g 1:10 Peptone Salt-90 mL
SPW00342 10 g 1:10 Peptone Salt-90 mL
SPW00352 10g 1:10 Butterfields buffer-90 mL
SPW00353 10 g 1:10 Butterfields buffer-90 mL
SPW00354 10g 1:10 Butterfields buffer-90 mL
SPW00365 10g 1:10 Butterfields buffer-90 mL
SPW00366 10 g 1:10 Butterfields buffer-90 mL
SPW00367 10g 1:10 Butterfields buffer-90 mL
SPW00376 10 g 1:10 Peptone Salt-90 mL
SPW00383 10g 1:10 Butterfields buffer-90 mL
SPW00400 10g 1:10 Butterfields buffer-90 mL
SPW00413 10g 1:10 Butterfields buffer-90 mL

SPW00417 10g 1:10 Peptone Salt-90 mL
SPW00429 10g 1:10 Peptone Salt-90 mL + 25 mL of IN NaOH
SPW00430 l0g 1:10 Peptone Salt-90 mL + 25 mL of IN NaOH
SPW0043 | l0g 1:10 Peptone Salt-90 mL + 25 mL of IN NaOH
SPW00432 10g 1:10 Peptone Salt-90 mL + 25 mL of IN NaOH
SPW00434 10g 1:10 Peptone Salt-90 mL + 25 mL of IN NaOH
SPW00435 10 g 1:10 Peptone Salt-90 mL + 25 mL of IN NaOH
SPW00436 10g 1:10 Peptone Salt-90 mL
SPW00437 10 g 1:10 Peptone Salt-90 mL
SPW00445 10g 1:10 Butterfields buffer-90 mL
SPW00458 10g 1:10 Butterfields buffer-90 mL
SPW00460 10g 1:10 Butterfields buffer-90 mL
SPW00468 10g 1:50 Butterfields buffer + 8 mL of IN NaOH
SPW00469 10g 1:50 Butterfields buffer + 7 mL of IN NaOH
SPW00488 10g 1:100 Butterfields buffer-900 mL
SPW00489 10g 1:50 Butterfields buffer + 4 mL of IN NaOH

SPW00490 10g 1:100 Butterfields buffer-900 mL
SPW00523 10g 1:50 Peptone Salt + 3 mL of IN NaOH
SPW00530 10g 1:50 Butterfields buffer + 6 mL of IN NaOH
SPW00565 10g 1:50 Butterfields buffer + 4 mL of IN NaOH
SSG00087 10g 1:10 Peptone Salt-90 mL
STB00134 10g 1:10 Butterfields buffer-90 mL
STB00177 10g 1:10 Peptone Salt-90 mL
NFIGO0001 10g 1:10 Butterfields buffer-90 mL

[SOP

Standard Operating Procedure
SOP No | Rev
Microbial Limit Testing using the D-715 14 Page 43 of 43
Neogen® Petrifilm® System

Attachment 7 — Finished Product Summary for Petrifilm Validated Dilution and
Diluent (continued)

Product Profile Number | Product (g) Dilution Diluent
NFIG00007 10g 1:10 Butterfields buffer-90 mL
NFYO00003 10g 1:10 Butterfields buffer-90 mL