Growth Promotion Test Guide for EZ‐CFU™, EZ‐CFU™ One Step, and EZ‐Accu Shot™  

Introduction
The Growth Promotion Test (GPT) is an important function in the United States (USP), European (Ph. Eur.) and Japanese
(JP) Pharmacopeias. Its purpose is to determine the suitability of media used in pharmaceutical tests. This Technical
Information Bulletin (TIB) discusses the basic requirements of the GPT and explains how to perform the test using EZ-
CFU™ and EZ-CFU™ One Step, and EZ-Accu Shot™ lyophilized microorganisms. The GPT should be performed on
each batch of purchased ready-prepared medium, each batch of dehydrated medium or medium prepared from
components in the laboratory (henceforth, any new, untested batch of medium will be called the “new batch of medium”).

The basic requirements for the GPT are as follows:

1. The new batch of medium must be inoculated with a small number of microorganisms.
2. The laboratory should test the medium with the microorganisms required by the pharmacopeias.
3. The microorganisms must not be more than five passages removed from Reference Culture (also called the
original master seed lot).
In order for the new batch of medium to be approved for use, growth on the new batch of medium must be comparable to
growth obtained on a batch of medium previously approved by the laboratory.

The EZ-CFU™, EZ-CFU™ One Step, and EZ-Accu Shot™ are standardized, lyophilized microorganism preparations
designed to deliver an inoculum of less than 100 CFU when used as directed. The microorganisms are no more than four
passages from internationally recognized Reference Culture Collections. Documentation records a chain-of-evidence to
ensure that each product is traceable to a Reference Culture. See Table 4 for a list of EZ-CFU™, EZ-CFU™ One Step,
and EZ-Accu Shot™ microorganisms.

The difference between EZ-CFU™, EZ-CFU™ One Step, and EZ-Accu Shot™ is that the EZ-CFU™ has a greater
microorganism concentration and therefore requires a 1:10 dilution in order to deliver less than 100 CFU per 0.1 mL. EZ-
CFU™ One Step and EZ-Accu Shot™ do not require the dilution step.

MicroBioLogics indicates the average number of CFU per mL on the Certificate of Assay included with every EZ-CFU™,
EZ-CFU™ One Step, and EZ-Accu Shot™ product. The value on the Certificate of Assay is obtained using a statistically
valid method. The laboratory using the product may obtain a different CFU value because different methods can yield
different results. MicroBioLogics guarantees that an EZ-CFU™, EZ-CFU™ One Step, or EZ-Accu Shot™ inoculum will
deliver less than 100 CFU per 0.1 mL when used as directed. The customer should establish the CFU count in the
inoculum by plating it on nonselective agar.

The USP, Ph. Eur. and JP have harmonized the Microbial Enumeration Tests and the Tests for Specified Microorganisms.
The Sterility Test is scheduled to be fully harmonized by the pharmacopeias soon. The GPT varies slightly between the
Microbial Enumeration Tests, the Tests for Specified Microorganisms, and the Sterility Test. For this reason, the GPT
used for each type of test is described in this TIB, in addition to other important considerations for all three tests. This TIB
is a guide; please refer to the pharmacopeias for official text.

Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests

Purpose
The purpose of the Growth Promotion Test (GPT) for Microbial Enumeration Tests is to assure the nutritive properties of a
new batch of medium by challenging it with a small number of microorganisms.

Requirements
1. Inoculate agar plates or broth with only a small number of microorganisms (not more than 100 CFU according to
the USP, Ph. Eur. and JP).
2. Use the microorganisms recommended by the pharmacopeias. The microorganisms should be traceable to a
Reference Culture Collection, and should be no more than five passages from Reference Culture.
3. Follow USP, Ph. Eur., or JP instructions. The USP, Ph. Eur. and JP are harmonized for this test.
4. Test the new batch of medium and the previously approved batch of medium in parallel.
5. Test all media in duplicate.
6. Test each agar plate or media tube with only one microorganism at a time.

Preparation
1. Determine which EZ-CFU™, EZ-CFU™ One Step, and EZ-Accu Shot™ microorganisms are needed to test the
medium (See Table 1).
2. For each microorganism to be tested, label two plates or tubes from the new batch of medium and two plates or
tubes from the previously approved batch of medium.

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3. For each batch of new medium, test a negative control to ensure the new medium and the diluent (if one is used)
are sterile.
If using EZ-CFU™ One Step or EZ-Accu Shot™, the negative control is a non-inoculated plate or tube from the
new batch of medium. The medium is not inoculated because the microorganism suspension is not diluted. If
using EZ-CFU™, the negative control is the diluent used to dilute the microorganism suspension 1:10. Inoculate
a tube or agar plate from the new batch of medium with 0.1 mL of the diluent.
Incubate the negative control at the same temperature as the microorganisms tested. It may be necessary to
incubate the negative control at 20-25°C and at 30-35˚C if both bacteria and yeast or fungi are being tested. The
negative control must have no growth after incubation, in order for the new batch of medium to be approved. If the
new medium is broth and the broth is clear after incubation, it may be subbed to nonselective agar in order to
ensure that the broth is sterile.

Test Procedure
1. Prepare an inoculum of each of the test microorganisms by following the directions in the EZ-CFU™,
EZ-CFU™ One Step, or EZ-Accu Shot™ product inserts.
2. Dispense 0.1 mL of the inoculum per plate of agar or tube of broth. 0.1 mL will deliver less than 100 CFU. Use the
same microorganism preparation to inoculate two plates or tubes from the new batch of medium and two plates or
tubes from the previously approved batch of medium.
3. If the new medium is broth, also test each microorganism individually on two nonselective control agar plates.
Inoculate the nonselective agar plates with 0.1 mL of the same inoculum suspension used to inoculate the new
and previously approved batches of medium. The purpose of testing the microorganism on nonselective agar is to
verify that 0.1 mL of inoculum contains less than 100 CFU. It is possible to determine the CFU concentration in
the inoculum by counting the colonies on the agar plates whereas this is not possible after incubating broth.
4. If using solid media, use the Surface-Spread Method. Use a spreader to disperse the inoculum and distribute it
over the entire agar plate.
5. Follow pharmacopeia directions for incubation temperature and length of incubation for each microorganism
tested (See Table 1). The negative control and the inoculated media should be subjected to the same conditions.
6. Determine if the new medium is suitable for use by using the acceptance criteria listed below.

Acceptance Criteria
1. SOLID MEDIA
Average the number of colonies on the two plates from the new batch of medium and average the number of
colonies on the two plates from the previously approved batch of medium. In order for the new batch of medium to
be approved, the following acceptance criteria must be met for each microorganism tested:
A. There must be growth on the agar plates.
B. There must be no more than 100 colonies on the agar plates.
C. The average number of colonies on the plates from the new batch of medium must be within a factor of 2 of
the average number of colonies from the previously approved batch of medium. For example, if the average
number of colonies on the previously approved media is 40, then the average number of colonies on the
plates from the new batch of medium must be between 20 and 80.
2. BROTH
Visually compare growth in the two tubes from the new batch of liquid medium to growth in the two tubes from the
previously approved batch of medium. In order for the new batch of medium to be approved, the following
acceptance criteria must be met for each microorganism tested:
A. There should be growth in the tubes from both the new and previously approved batches of medium and on
the control agar plates.
B. The control (nonselective) plates should have less than 100 colonies.
C. The amount of turbidity in the tubes from the new batch of liquid medium should be comparable to amount of
turbidity in the tubes from the previously approved batch of liquid medium. A quantitative definition of
“comparable” is not established by the USP, Ph. Eur. or JP; visual comparability is sufficient.

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Growth Promotion Test for Solid Medium Used in Microbial Enumeration Test
Step 1
Prepare inoculum.

Step 2
Inoculate new and previously approved media in duplicate.

Step 3
Spread inocula & incubate.

Step 4
Count colonies on previously approved media.

Step 5
Count colonies on new media.

Step 6
Compare results. Counts should be within a factor of 2.

Growth Promotion Test for Liquid Medium Used in Microbial Enumeration Test
Step 1
Prepare inoculum.

Step 2
Inoculate new and previously approved media.

Step 3
Inoculate nonselective agar.

Step 4
Spread inocula & incubate all media.

Step 5
Compare new to previously approved media. Amount of turbidity should be similar.

Step 6
Count colonies on nonselective media to confirm count is <100.

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Table 1: Growth Promotion Test Requirements for Microbial Enumeration Tests
Microorganism 1,2 Type of Medium Temperature Incubation Period
Staphylococcus aureus
Soybean-Casein Digest Agar
EZ-CFU™ One Step 0485Z or 0579Z ≤3 days (growth can be seen in
and Soybean-Casein Digest 30-35° C
EZ-CFU™ 0485C 18-24 hrs.)
Broth
EZ–Accu Shot™ 0485A
Pseudomonas aeruginosa Soybean-Casein Digest Agar
≤3 days (growth can be seen in
EZ-CFU™ One Step 0484Z or 0576Z and Soybean-Casein Digest 30-35° C
18-24 hrs.)
EZ-Accu Shot™ 0484A Broth
2
Bacillus subtilis subsp. spizizenii
Soybean-Casein Digest Agar
EZ-CFU™ One Step 0486Z or 0582Z ≤3 days (growth can be seen in
and Soybean-Casein Digest 30-35° C
EZ-CFU™ 0486C 18-24 hrs.)
Broth
EZ-Accu Shot™ 0486A
Candida albicans
Soybean-Casein Digest Agar ≤5 days (growth can be seen at
EZ-CFU™ One Step 0443Z Soybean-Casein Digest Agar
30-35°C 48 hrs. at 30-35 °C and at 72 hrs.
EZ-CFU™ 0443C and Sabouraud Dextrose Agar
Sabouraud Dextrose Agar 20-25˚C at 20-25°C )
EZ-Accu Shot™ 0443A
3
Aspergillus brasiliensis
Soybean-Casein Digest Agar ≤5 days (growth can be seen at
EZ-CFU™ One Step 0392Z Soybean-Casein Digest Agar
30-35°C 48 hrs. at 30-35 °C and at 72 hrs.
EZ-CFU™ 0392C and Sabouraud Dextrose Agar
Sabouraud Dextrose Agar 20-25˚C at 20-25°C )
EZ-Accu Shot™ 0392A
1
See Table 4 for Cross Reference of MicroBioLogics Products with recommended Reference Cultures
2
Bacillus subtilis subsp. spizizenii is referred to Bacillus subtilis in the pharmacopeias. More information about the name can be found in Table 4.
3
Aspergillus brasiliensis has replaced the name Aspergillus niger in legacy pharmacopeias. More information can be found in Table 4 footnotes.

Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms

Purpose
The purpose of this growth promotion test is to ensure that the new batch of medium is performing correctly with respect
to growth promotion, inhibitory and indicative properties by challenging the medium with a small number of
microorganisms.

Requirements
1. Inoculate the agar or broth with the number of microorganisms required by the type of test. The types of tests
required by the USP, Ph. Eur. and JP are as follows:
A. Test for Growth-Promoting Properties, Solid Media – Inoculate medium with no more than 100 CFU of the
appropriate microorganism.
B. Test for Growth-Promoting Properties, Liquid Media – Inoculate medium with no more than 100 CFU of
the appropriate microorganism.
C. Test for Inhibitory Properties, Liquid or Solid Media – Inoculate medium with at least 100 CFU of the
appropriate microorganism.
D. Test for Indicative Properties – Inoculate medium with no more than 100 CFU of the appropriate
microorganism.
2. Use the microorganisms recommended in the pharmacopeias. The microorganisms should be traceable to a
Reference Culture Collection, and should be no more than five passages from Reference Culture (original master
seed lot).
3. Follow USP, Ph. Eur., or JP instructions. The USP, Ph. Eur. and JP are harmonized for this test.
4. The new batch of medium may require more than one type of test. All the test results must meet the criteria
described in the procedures in order for the new batch to be approved.
5. Test the new batch of medium and the previously approved batch of medium in parallel.
6. Test all media in duplicate.
7. Test such medium with only one microorganism at a time.

Test for Growth-Promoting Properties, Solid Media

Test for Growth-Promoting Properties, Liquid Media
Test for Indicative Properties

Preparation
1. Determine which EZ-CFU™, EZ-CFU™ One Step, or EZ-Accu Shot™ microorganisms are needed to test the
medium (See Table 2).
2. For each microorganism to be tested, label two plates or tubes from the new batch of medium and two plates or
tubes from the previously approved batch of medium.
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3. For each batch of new medium, test a negative control to ensure the new medium and the diluent (if one is used)
are sterile.
If using EZ-CFU™ One Step or EZ-Accu Shot™, the negative control is a non-inoculated plate or tube from the
new batch of medium. The medium is not inoculated because the microorganism suspension is not diluted. If
using an EZ-CFU™ product, the negative control is the diluent used to dilute the microorganism suspension 1:10.
Inoculate a tube or agar plate with 0.1 mL of the diluent.
Incubate the negative control under the same conditions as the microorganisms being tested. The negative
control must have no growth after incubation, in order for the new batch of medium to be approved. If the new
medium is broth and the broth is clear after incubation, it may be subbed to nonselective agar to ensure the broth
is sterile.

Procedure
1. Prepare an inoculum of each of the required microorganisms by following the directions in the EZ-CFU™, EZ-
CFU™ One Step, or EZ-Accu Shot™ product insert.
2. Dispense 0.1 mL of the inoculum per plate of agar or tube of broth. 0.1 mL will deliver less than 100 CFU. Use
the same microorganism preparation to inoculate two plates or tubes from the new batch and two plates or tubes
from the previously approved batch of medium.
3. If the new medium is selective or liquid, also test each microorganism individually on two nonselective control
agar plates. Inoculate the plates or tubes with 0.1 ml of the same inoculum suspension that was used to inoculate
the new and previously approved batches of medium. This is done because:
• If testing a solid, selective medium, it is necessary to determine how many microorganisms are in the
inoculum since growth-promoting medium can be inhibitory even to microorganisms that should grow on it.
• If testing a liquid medium, it is necessary to verify that it was inoculated with less than 100 CFU since there
are no colonies to count after the liquid medium has been incubated. Inoculate the plates with the same
inoculum used on the new and the previously approved batches of medium.
4. If using solid media, use the Surface-Spread Method. Use a spreader to disperse the inoculum and distribute it
over the entire agar plate.
5. Follow pharmacopeia directions for incubation temperature and length of incubation for each microorganism
tested (See Table 2). The negative control should be subjected to the same conditions as the inoculated media.
6. Determine if the new medium is suitable for use by using the acceptance criteria listed below.

Acceptance Criteria
1. SOLID MEDIA
Average the number of colonies on the two plates from the new batch of medium and average the number of
colonies on the two plates from the previously accepted batch of medium. In order for the results to be
acceptable, the following acceptance criteria must be met for each microorganism tested:
A. There must be growth on the control (nonselective) agar plates and the growth-promoting agar plates.
B. There must be less than 100 colonies on the control agar plates or the growth-promoting agar plates.
C. The average number of colonies on the new batch of medium must be “comparable” to the average number
of colonies on the batch of previously approved medium. No quantitative definition of “comparable”, such as
50% or 70%, has been established in the USP, Ph. Eur. or JP.
2. LIQUID MEDIA
Visually compare growth in the two tubes from the new batch of liquid medium to growth in the two tubes from the
previously approved batch of medium. In order for the results to be acceptable, the following acceptance criteria
must be met for each microorganism tested:
A. There should be growth in the tubes from both the new and previously approved batches of liquid medium
and on the control agar plates.
B. There must be no more than 100 colonies on the control agar plates.
C. The amount of turbidity in the new batch of liquid medium should be “comparable” to that in the previously
approved batch of medium. No quantitative definition of “comparable” has been established by the USP, Ph.
Eur. or JP.
3. INDICATIVE REACTIONS
Visually compare the colonies on the agar plates to the colonies on the previously approved batch of medium.
The colonies should be similar in appearance. Expected indicative reactions are described in Table 2.

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Growth Promotion Test for Solid Media Used in Tests for Specified Microorganisms – Growth Promoting and
Indicative Properties
Step 1
Prepare inoculum.

Step 2
Inoculate new and previously approved media in duplicate.

Step 3
Inoculate nonselective agar. Spread inoculum &
incubate all media.

Step 4
Count colonies on previously approved media.

Step 5
Count colonies on new media. Colony count and indicative reactions on new media should be comparable to colony
count and indicative reactions on previously approved media.

Step 6
Count colonies on nonselective media to confirm count is <100.

Growth Promotion Test for Liquid Media Used in Tests for Specified Microorganisms – Growth Promoting
Properties
Step 1
Prepare inoculum.

Step 2
Inoculate new and previously approved media in duplicate.

Step 3
Inoculate nonselective agar.

Step 4
Spread inocula and incubate all media.

Step 5
Compare new to previously approved media. Turbidity should be comparable.

Step 6
Count colonies on nonselective media to confirm count is <100.

Test for Inhibitory Properties, Liquid or Solid Media

Procedure
This test should be performed at the same time as the Tests for Growth-Promotion and Indicative Properties.
Preparation
1. Determine which EZ-CFU™ microorganism is needed to test the medium (See Table 2).
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2. Label two plates or tubes from the new batch of medium and two plates or tubes from the previously approved
batch of medium.
3. For each batch of new medium, test a negative control to ensure the new medium is sterile. (This may have
already been done during the Tests for Growth Promoting and/or Indicative Properties). The negative control is a
non-inoculated plate or tube from the new batch of medium.
Incubate the negative control at the same temperature as the microorganisms being tested.
If the new medium is broth and the broth is clear after incubation, it may be subbed to nonselective agar in order
to ensure that the broth is sterile.
Procedure
1. Prepare an inoculum of each of the required microorganism. The inoculum must contain at least 100 CFU per 0.1
mL. To prepare the inoculum do the following:
• Use an EZ-CFU™ product.
• Rehydrate 2 pellets in 2 mL hydration fluid as described in the EZ-CFU™ product insert.
• Do not dilute further. The pellet-hydration fluid suspension contains 100 to 999 CFU per 0.1 mL
2. Dispense 0.1 mL of the inoculum per plate or agar of tube of broth. Use the same microorganism preparation to
inoculate two plates or tubes from the new batch of medium and two plates or tubes from the previously approved
batch of medium.
3. Also, test two nonselective control agar plates with the test microorganism. This is done in order to verify the agar
or broth being tested for inhibitory properties was inoculated with at least 100 CFU since there will be no growth
or very little growth on the media that has inhibitory properties. Inoculate the control plates with 0.1 mL of the
same inoculum used on the two plates or tubes from the new media and the previously approved media.
4. If using solid media, use the Surface-Spread Method. Use a spreader to disperse the inoculum and distribute it
over the entire agar plate.
5. Follow pharmacopeia directions for incubation temperature and length of incubation for each microorganism
tested (See Table 2). The negative and positive control should be subjected to the same conditions.
6. Determine if the new medium is suitable for use by using the acceptance criteria listed below.
Acceptance Criteria
1. The test microorganism should be inhibited on the plates or tubes from both the new and previously approved
batches of inhibitory medium.
2. There should be at least 100 colonies on the nonselective control agar plate.

Growth Promotion Test for Solid and Liquid Media Used in Tests for Specified Microorganisms – Inhibitory
Properties
Step 1
Prepare inoculum.

Step 2
Inoculate new and previously approved media.

Step 3
Inoculate nonselective agar. Spread inoculum & incubate all media.

Step 4
Examine previously approved media. Growth should be inhibited.

Step 5
Examine new media. Growth should be inhibited.

Step 6
Count colonies on nonselective media to confirm count is ≥100.

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Table 2: Growth Promotion Test Requirements for Tests for Specified Microorganisms
Type of 1 Incubation
Microorganisms Product Numbers Properties Temp
Medium Period
Enterobacteria EZ-CFU™ One Step 0483Z or 0581Z
E. coli
Enrichment EZ-CFU™ 0483C Growth Promoting 30-35°C 24 hrs.
Broth Mossel EZ-Accu Shot™ 0483A
P. aeruginosa EZ-CFU™ One Step 0484Z or 0576Z
Growth Promoting 30-35°C 24 hrs.
EZ-Accu Shot™ 0484A
S. aureus EZ-CFU™ 0485C
Inhibitory 30-35°C 48 hrs.
EZ-CFU™ One Step 0483Z or 0581Z Growth: 18 hrs.
Violet Red Bile E. coli Growth Promoting & Indicative
EZ-CFU™ 0483C 30-35°C Indicative: 18-24
Glucose Agar (purplish-red colonies)
EZ-Accu Shot™ 0483A hrs.
Growth: 18 hrs.
P. aeruginosa EZ-CFU™ One Step 0484Z or 0576Z Growth promoting & Indicative
30-35°C Indicative: 18-24
EZ-Accu Shot™ 0484A (colorless colonies)
hrs.
EZ-CFU™ One Step 0483Z or 0581Z
MacConkey E. coli
EZ-CFU™ 0483C Growth Promoting 42-44°C 24 hrs.
Broth
EZ-Accu Shot™ 0483A
S. aureus
EZ-CFU™ 0485C Inhibitory 42-44°C 48 hrs.
EZ-CFU™ One Step 0483Z or 0581Z Growth: 18 hrs.
MacConkey E. coli Growth Promoting & Indicative
EZ-CFU™ 0483C 30-35°C Indicative: 18-72
Agar (pink colonies)
EZ-Accu Shot™ 0483A hrs.
S. enterica subsp.
EZ-CFU™ One Step 0363Z
Rappaport enterica serovar
EZ-CFU™ 0363C Growth Promoting 30-35°C 18 hrs.
Vassiliadis Typhimurium
OR
Salmonella S. enterica
EZ-CFU™ One Step 0890Z
Enrichment subsp.enterica Growth Promoting 30-35°C 18 hrs.
EZ-CFU™ 0890C
Broth serovar Abony
S. aureus EZ-CFU™ 0485C Inhibitory 30-35°C 24 hrs.
S. enterica subsp.
Growth Promoting & Indicative Growth:18 hrs.
enterica serovar EZ-CFU™ One Step 0363Z
Xylose Lysine (red colonies with or without 30-35°C Indicative: 18-48
Typhimurium EZ-CFU™ 0363C
black centers) hrs.
OR
S. enterica Growth Promoting & Indicative Growth:18 hrs.
Deoxycholate EZ-CFU™ One Step 0890Z
subsp.enterica (red colonies with or without 30-35°C Indicative: 18-48
Agar EZ-CFU™ 0890C
serovar Abony black centers) hrs.
P. aeruginosa EZ-CFU™ One Step 0484Z or 0576Z
Cetrimide Agar Growth Promoting 30-35°C 18 hrs.
EZ-Accu Shot™ 0484A
E. coli
EZ-CFU™ 0483C Inhibitory 30-35°C 72 hrs.
EZ-CFU™ One Step 0485Z or 0579Z Growth Promoting & Indicative Growth: 18 hrs.
Mannitol Salt S. aureus
EZ-CFU™ 0485C (yellow or white colonies with 30-35°C Indicative: 18-72
Agar
EZ-Accu Shot™ 0485A yellow zone) hrs.
E. coli
EZ-CFU™ 0483C Inhibitory 30-35°C 72 hrs.
EZ-CFU™ One Step 0487Z or 0580Z
48 hrs.
Reinforced C. sporogenes EZ-CFU™ 0487C
Growth Promoting 30-35°C Anaerobic
Medium for EZ-Accu Shot™ 0487A
conditions
OR
EZ-CFU™ One Step 0317Z or 48 hrs.
Clostridia EZ-CFU™ 0317C Growth Promoting 30-35°C Anaerobic
EZ-Accu Shot™ 0317A conditions
EZ-CFU™ One Step 0487Z or 0580Z
48 hrs.
C. sporogenes EZ-CFU™ 0487C
Columbia agar Growth Promoting 30-35°C Anaerobic
EZ-Accu Shot™ 0487A
conditions
OR
EZ-CFU™ One Step 0317 48 hrs.
EZ-CFU™ 0317C Growth Promoting 30-35°C Anaerobic
EZ-Accu Shot™ 0317A conditions
EZ-CFU™ One Step 0443Z
Sabouraud C. albicans Growth Promoting & Indicative Growth: 24 hrs.
EZ-CFU™ 0443C 30-35°C
Dextrose Agar (white colonies) Indicative: 48 hrs.
EZ-Accu Shot™ 0443A
EZ-CFU™ One Step 0443Z
Sabouraud C. albicans
EZ-CFU™ 0443C Growth Promoting 30-35°C 3 days.
Dextrose Broth
EZ-Accu Shot™ 0443A
1
See Table 4 for Cross Reference of MicroBioLogics Products with recommended Reference Cultures

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Sterility Tests
Purpose
The purpose of the growth promotion test for the Sterility Test is to check the nutritive properties of the new batch of
medium by challenging the medium with a small number of microorganisms. The medium being tested is always liquid.

Requirements
1. Inoculate the broth with only a small number of microorganisms (not more than 100 CFU according to the USP,
Ph/ Eur. and JP).
2. Use the microorganisms recommended by the USP, Ph. Eur. or JP. The microorganisms should be traceable to
a Reference Culture Collection and should be no more than five passages from Reference Culture.
3. Follow USP, Ph. Eur. or JP instructions.
4. Test the new batch of medium and the previously approved batch of medium in parallel.
5. Test all media in duplicate.
6. Test each medium with only one microorganism at a time.

Preparation
1. Determine which EZ-CFU™, EZ-CFU™ One Step, or EZ-Accu Shot™ microorganisms are needed to test
medium (See Table 3).
2. For each microorganism to be tested, label two tubes from the new batch of medium and two tubes from the
previously approved batch of medium.
3. For each batch of new medium, test a negative control to ensure, the new medium and diluent (if one is used) are
sterile.
If using the EZ-CFU™ One Step or EZ-Accu Shot™, the negative control is a non-inoculated tube from the new
batch of medium. The medium is not inoculated because the microorganism suspension is not diluted. If using an
EZ-CFU™ product, the negative control is the diluent used to dilute the microorganism suspension 1:10.
Inoculate a tube from the batch of new medium with 0.1 mL of the diluent. Incubate the negative control tube for
14 days under the same conditions as the microorganisms being tested. If the broth is clear after incubation, it
may be subbed to nonselective agar plates in order to ensure that the broth is sterile. The negative control must
have no growth after incubation, in order for the new batch of medium to be approved.

Procedure
1. Prepare an inoculum of each of the required microorganisms by following the directions in the EZ-CFU™, EZ-
CFU™ One Step, or EZ-Accu Shot™ product insert.
2. Dispense 0.1 mL of the inoculum in two tubes of the batch of new media and two tubes of the batch of previously
approved media. 0.1 mL will deliver less than 100 CFU. Use the same microorganism preparation to inoculate
the new batch and the previously approved batch of medium.
3. Also, test each microorganism individually on two nonselective control agar plates. Inoculate the plates with 0.1
mL of the same inoculum that was used to inoculate the new and previously approved batches of medium. This is
done in order to verify 0.1 mL contains less than 100 CFU. It is possible to determine the CFU concentration in
the inoculum by counting the colonies on the agar plates whereas this is not possible after incubating broth.
4. Use a spreader to disperse the inoculum and distribute it over the entire plate of nonselective medium.
5. Follow pharmacopeia directions for incubation temperature and length of incubation for each microorganism
tested (See Table 3). The negative control should be incubated at the same temperature as the inoculated media.
6. Determine if the new medium is suitable for use by using the acceptance criteria listed below.

Acceptance Criteria
After incubation, visually compare the turbidity in the two tubes from the new batch of medium to the turbidity in the two
tubes from the previously approved batch of medium. In order for the new batch of medium to be approved the following
acceptance criteria must be met for each microorganism tested:
1. There should be growth in the tubes from both the new and previously approved batches of liquid medium and on
the control agar plates.
2. The control plate should have less than 100 colonies
3. The amount of turbidity in the tubes from the new batch of liquid medium should be “comparable” to that in the
tubes from the previously approved batch of medium. No quantitative definition of “comparable” has been
established by the USP, Ph. Eur. or JP.

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Growth Promotion Test for Media Used in Sterility Tests

Step 1
Prepare inoculum.

Step 2
Inoculate new and previously approved media.

Step 3
Inoculate nonselective agar.

Step 4
Spread inoculum and incubate all media.

Step 5
Compare new to previously approved media. Amount of turbidity should be similar.

Step 6
Count colonies on nonselective media to confirm there are <100.

Table 3: Growth Promotion Test Requirements for the Sterility Test

1 Incubation
Microorganism Product Type of Medium Temp.
Period
EZ-CFU™ One Step 0317Z
Clostridium sporogenes Fluid Thioglycollate
EZ-CFU™ 0317C 30-35° C ≤3 days
Medium
EZ-Accu Shot™ 0317A OR
EZ-CFU™ One Step 0487Z
Fluid Thioglycollate
EZ-CFU™ 0487C 30-35° C ≤3 days
Medium
EZ-Accu Shot™ 0487A
Pseudomonas EZ-CFU™ One Step 0484Z or 0576Z Fluid Thioglycollate
2 30-35° C ≤3 days
aeruginosa EZ-Accu Shot™ 0484A Medium
EZ-CFU™ One Step 0485Z or 0579Z
Staphylococcus aureus Fluid Thioglycollate
EZ-CFU™ 0485C 30-35° C ≤3 days
Medium
EZ-Accu Shot™ 0485A
EZ-CFU™ One Step 0317Z
Alternative Fluid
EZ-CFU™ 0317C 30-35° C ≤3 days
Thioglycollate Medium
EZ-Accu Shot™ 0317A OR
3 EZ-CFU™ One Step 0487Z
Clostridium sporogenes Alternative Fluid
EZ-CFU™ 0487C 30-35° C ≤3 days
Thioglycollate Medium
EZ-Accu Shot™ 0487A
4 EZ-CFU™ One Step 0392Z
Aspergillus brasiliensis Soybean-Casein Digest
EZ-CFU™ 0392C 5 20-25°C ≤5 days
Medium
EZ-Accu Shot™ 0392A
6 EZ-CFU™ One Step 0486Z or 0582Z
Bacillus subtilis Soybean-Casein Digest
EZ-CFU™ 0486C 5 20-25°C ≤ 3 days
Medium
EZ-Accu Shot™ 0486A
EZ-CFU™ One Step 0443Z
Soybean-Casein Digest
Candida albicans EZ-CFU™ 0443C 5 20-25°C ≤5 days
Medium
EZ-Accu Shot™ 0443A
1
See Table 4 for Cross Reference of MicroBioLogics Products with Reference Cultures.
2
Kocuria rhizophila, MicroBioLogics EZ-CFU™ One Step, 0688Z, or EZ-CFU™, 0688C may be substituted for Pseudomonas aeruginosa (USP).
3
Bacteroides vulgatus, MicroBioLogics EZ-CFU™, 0445C, may be substituted for Clostridium sporogenes when a nonspore-forming microorganism is desired.
See relevant Pharmacopeia.
4
Aspergillus brasiliensis has replaced the name Aspergillus niger in the legacy pharmacopeias. More information can be found in Table 4 footnotes.
5
Fluid Thioglycollate medium may be used when testing a product containing a preservative that cannot be tested by the membrane filtration method provided the
method has been validated.
6
Bacillus subtilis subsp. spizizenii is referred to Bacillus subtilis in the pharmacopeias. More information about the name can be found in Table 4.

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Important Considerations for all Growth Promotion Tests

In order to get consistent results, do the following:
• Use the microorganism cultures recommended by the pharmacopeia. The cultures should be traceable to a
Reference Culture Collection, and should be no more than five passages from Reference Culture (original master
seed lot).
• If testing selective media, run a control on nonselective media such as Tryptic Soy Agar at the same time
because recovery on selective media is not as high as on nonselective media.
• If performing a Growth Promotion Test on liquid medium, run a control on nonselective agar at the same time in
order to verify that the correct number of microorganisms was added for the test.
• If using EZ-CFU™ or EZ-CFU™ One Step, don’t exchange the rubber stoppers on the Hydrating Fluid for the
rubber stoppers on the pellet vials. The moisture on the Hydrating Fluid stopper may harm the pellets.
• Allow the media and the vial of pellets to equilibrate to room temperature before use.
• If using EZ-CFU™ or EZ-CFU™ One Step, the Hydrating Fluid and any dilution fluids should warm for 30 minutes
at 35˚ before use. Use only the Hydrating Fluid that was received with the kit.
• Use a pipettor and calibrate it routinely.
• Vortex suspensions until they are homogenous.
• Use a spreader to disperse the inoculum over the entire plate.
• Follow pharmacopeia directions for temperature, incubation time, and atmosphere.
• After testing EZ-CFU™ One Step or EZ-Accu Shot™ suspension, refrigerate it if planning to use it again. With the
exception of 0320Z and 0318Z, the refrigerated suspension can be used for up to eight hours.
• Calibrate thermometers and validate incubators yearly.
• Some microorganisms such as Bacillus and Aspergillus produce colonies that spread. These colonies can be
hard to count if they become too mature.
• If using the pour plate method, add 0.1 mL of inoculum containing less than 100 CFU to an empty agar plate.
Pour molten agar over the inoculum and mix well by swirling the contents in the plate. Since recovery may not be
as high as when using the spread plate method, run a spread plate as a control. The molten media must be
cooled to 45° Celsius. Invert and incubate the agar plate after it has solidified.
• For optimum results, many experts recommend using parallel testing (side by side) instead of historical data when
comparing new media to previously approved media. When parallel testing is used, the new media and the
previously approved media are inoculated with the same inoculum by the same technician and are subjected to
identical incubation conditions. The only variable is the media.
• A Verification Study is sufficient when using EZ-CFU™, EZ-CFU One Step™, or EZ-Accu Shot™ product for the
first time. MicroBioLogics has already validated the procedures and, per the USP, all the customer needs to do is
verify that the product works for them using their procedures, equipment etc. MicroBioLogics recommends five
repetitive tests, using the same inoculum suspension, should be performed for the verification study. Results
should meet the specifications outlined in this TIB. Verification Protocol Guide, TIB.281, is available for the EZ-
Accu Shot™. It may be downloaded from the MicroBioLogics website, www.microbiologics.com.
• In order to ensure the safety of the consumer, a pharmaceutical product may need to be tested for
microorganisms other than those mentioned in the Tests for Specified Microorganisms. If microorganism controls
other than those mentioned in this TIB are needed, go to www.microbiologics.com to find a complete list of EZ-
CFU™, EZ-CFU™ One Step, and EZ-Accu Shot™ microorganisms that can be used in Growth Promotion
Testing.

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Table 4: Cross Reference of MicroBioLogics Products with Reference Cultures
Microorganism Name Reference Culture # EZ-CFU™ One Step, EZ-CFU™, and
1
EZ-Accu Shot™ Product Numbers
2
Aspergillus brasiliensis ATCC® 16404™* 0392Z, 0392C, 0392A
3
Bacillus subtilis subsp. spizizenii ATCC® 6633™* 0486Z, 0486C, 0486A
3
Bacillus subtilis subsp. spizizenii NCIMB 8054 0582Z
Bacteroides vulgatus ATCC® 8482™* 0445C
Candida albicans ATCC® 10231™* 0443Z, 0443C, 0443A
Clostridium sporogenes ATCC® 19404™* 0317Z, 0317C, 0317A
Clostridium sporogenes ATCC® 11437™* 0487Z, 0487C, 0487A
Clostridium sporogenes NCIMB 12343 0580Z
Escherichia coli ATCC® 8739™* 0483Z, 0483C, 0483A
Escherichia coli NCIMB 8545 0581Z
Kocuria rhizophila ATCC® 9341™* 0688Z, 0688C
Pseudomonas aeruginosa ATCC® 9027™* 0484Z, 0484A
Pseudomonas aeruginosa NCIMB 8626 0576Z
Salmonella enterica subsp.enterica NCTC 6017 0890C, 0890Z
Salmonella enterica subsp.enterica serovar Typhimurium ATCC® 14028™* 0363C, 0363Z

Staphylococcus aureus subsp. aureus ATCC® 6538™* 0485C, 0485Z, 0485A

Staphylococcus aureus subsp. aureus NCIMB 9518 0579Z
1
The code Z indicates an EZ-CFU™ One Step product. The code C indicates an EZ-CFU™ product. The code A indicates an EZ-Accu Shot™ product.
2
Aspergillus brasiliensis is sometimes referred to as Aspergillus niger in the pharmacopeias. ATCC® changed the name of ATCC® 16404™ in 2008 because
ATCC® determined that this strain belonged to the species Aspergillus brasiliensi after performing a polyphasic study in which molecular data was combined with
physiological characteristics. More information can be found at the following link. http://www.microbiologics.com/docs/LIT.256.pdf
3
Bacillus subtilis subsp. spizizenii is referred to Bacillus subtilis in the pharmacopeias. A taxonomic update in 1999 reclassified ATCC® 6633™ as Bacillus subtilis
subspecies spizizenii. The species did not change. Rather, a new subspecies was added. More information can be found in the following reference. Nakamura
LK, et al. Relationship of Bacillus subtilis clades associated with strains 168 and W23: A proposal for Bacillus subtilis subsp. subtilis subsp. nov. and Bacillus
subtilis subsp. spizizenii subsp. nov. International Journal of Systematic and Evolutionary Bacteriology 49: 1211-1215, 1999.

Abbreviations and acronyms

* The ATCC Licensed Derivative Emblem, the ATCC Licensed Derivative word mark and the ATCC
catalog marks are trademarks of ATCC. MicroBioLogics, Inc. is licensed to use these trademarks and to sell products
derived from ATCC® cultures. ATCC is the American Type Culture Collection.

NCIMB is the National Collection of Industrial and Marine Bacteria Ltd. It is located in Aberdeen, Scotland.
MicroBioLogics, Inc. is licensed to use these trademarks and to sell products derived from NCIMB cultures.

NCTC™ is the National Collection of Type Cultures. It is registered to PHLS Central Public Health Laboratory in the
United Kingdom. MicroBioLogics, Inc. is licensed to use these trademarks and to sell products derived from NCTC™
cultures.

References
The United States Pharmacopeia-National Formulary. 2009. <61> Microbiological Examination of Nonsterile Products:
Microbial Enumeration Tests, First Supplement to USP32-NF27.

The United States Pharmacopeia-National Formulary. 2009. <62> Microbiological Examination of Nonsterile Products:
Tests for Specified Microorganisms, First Supplement to USP32-NF27.

The United States Pharmacopeia-National Formulary. 2009. <71> Sterility Tests, First Supplement to USP32-NF27.

The United States Pharmacopeia-National Formulary. 2009. <1226> Verification of Compendial Procedures.

The United States Pharmacopeia-National Formulary. 2009. <1111> Microbiological Examination of Nonsterile Products:
Acceptance Criteria for Pharmaceutical Preparations and Substances for Pharmaceutical Use.
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European Pharmacopoeia. 2009. 2.6.12, Microbiological Examination of Nonsterile Products: Microbial Enumeration
Tests.

European Pharmacopoeia. 2009. 2.6.13, Microbiological Examination of Nonsterile Products: Tests for Specified
Microorganisms.

European Pharmacopoeia. 2009. 2.6.1, Sterility Tests.

Japanese Pharmacopoeia. May.19. 2009. 4.05, Microbiological Examination of Nonsterile Products: Microbial
Enumeration Tests and Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms. 15th
Partial Revision (English version).

Japanese Pharmacopoeia. May. 19. 2009. 4.06, Sterility Tests. 15th Partial Revision (English version).

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