3/8/24, 2:25 PM USP-NF 〈1119.

1〉 Bioburden Test
Printed on: Fri Mar 08 2024, 14:25:41 pm
Printed by: Ramkishan Rao Gourineni
Commenting open for 23 more days
Published On: 02-Jan-2024
Open for Commenting as of: 08-Mar-2024
Document Type: GENERAL CHAPTER
DocId: GUID-E2174C65-C3B2-4D5B-8127-0D830104D0FD_10101_en-US
DOI: https://doi.org/10.31003/USPNF_M18395_10101_01
DOI Ref: mk0tz
Printed from: https://online.uspnf.com/uspnf/document/2_GUID-E2174C65-C3B2-4D5B-8127-0D830104D0FD_10101_en-US
Do not distribute
© 2024 USPC

BRIEFING

T
〈 1119 .1〉 Bioburden Test. The General Chapters—Microbiology Expert Committee proposes this new chapter for the testing of bioburden.
Historically, there has been an erroneous assumption that Microbial Enumeration Tests 〈61〉 could be applied as a generic test for bioburden for a

N
wide variety of sample types. This interpretation had been supported in Monitoring of Bioburden 〈1229.3〉, which stated that qualification of a
modified version of 〈61〉 might be appropriate for the testing of bioburden yet failed to provide specific details. Chapter 〈61〉 is a test specifically
for the release of nonsterile finished drug products and associated substances for pharmaceutical use as stipulated in Microbiological

TE
Acceptance Criteria for Nonsterile Pharmaceutical Preparations and Substances for Pharmaceutical Use 〈1111〉. In contrast, this chapter provides a
proposed method that may be used as a generic bioburden test for purposes other than release testing of nonsterile products. An informational
chapter, Bioburden Monitoring 〈 1119 〉, contains additional information regarding scope, sampling, and acceptance limits, which also appears in
this issue of PF.
Additional proposals in this issue of PF include:
1. Addition of a new chapter, Bioburden Monitoring 〈 1119 〉.

N
2. Omission of an existing chapter, Monitoring of Bioburden 〈1229.3〉.
(GCM: L. Furr)
Correspondence Number—C326398

Add the following:

CO
▲
〈 1119 .1〉 BIOBURDEN TEST
INTRODUCTION
L

The tests described hereafter will allow for quantitative enumeration of mesophilic bacteria and fungi that may grow under aerobic
conditions. The tests are designed to determine whether a sample complies with an established limit for bioburden (see Bioburden Monitoring
〈 1119 〉). The term sample refers to the item subject to testing, which might be a unit item (e.g., vial stopper) or a quantity (e.g., weight or
IA

volume). Justified changes to the instructions below may be used, as described in 〈 1119 〉.

GENERAL PROCEDURES
Carry out the test under conditions designed to avoid extrinsic microbial contamination of the product to be examined (see Microbiological
IC

Best Laboratory Practices 〈1117〉). The precautions taken to avoid contamination should not affect microbial recovery in the test.

ENUMERATION METHODS
The method and parameters chosen should be included in a bioburden test user requirements document with an accompanying rationale as
per 〈 1119 〉. The suitability of the chosen method (membrane filtration, pour-plate, or surface-spread method) and stated method parameters
FF

must be established.

GROWTH PROMOTION TEST, NEGATIVE CONTROLS, AND SUITABILITY OF THE COUNTING METHOD
General Considerations
O

The ability of the test to detect microorganisms in the sample must be established. Method suitability must be confirmed if there is a change
in testing performance or sample that may affect the test outcome.

Preparation of Test Strains

N

Use standardized, stable suspensions of test strains or prepare as stated below. Ensure that the viable microorganisms used for inoculation
are not more than 5 passages removed from the original master seed lot. Grow each bacterial and fungal test strain separately, as described in
Table 1.
U

Table 1. Preparation and Use of Test Microorganisms

https://online.uspnf.com/uspnf/document/2_GUID-E2174C65-C3B2-4D5B-8127-0D830104D0FD_10101_en-US?source=Quick Search&highlight… 1/4

3/8/24, 2:25 PM USP-NF 〈1119.1〉 Bioburden Test

Suitability of Counting Method

in the Test Matrix and
Microorganism Preparation of Test Strain Growth Promotion Bioburden Testing

Staphylococcus aureus such as

ATCCa 6538, NCIMBb 9518, CIPc
4.83, or NBRCd 13276

Pseudomonas paraeruginosa
(formerly P. aeruginosa) such as

T
ATCC 9027, NCIMB 8626, CIP
82.118, or NBRC 13275 soybean–casein digest
soybean–casein digest agar and soybean–casein soybean–casein digest

N
Bacillus spizizenii (formerly B. agar or soybean–casein digest broth agar
subtilis) such as ATCC 6633, digest broth ≤100 CFU ≤100 CFU
NCIMB 8054, CIP 52.62, or 30°–35° 30°–35° 30°–35°

TE
NBRC 3134 18–24 h ≤3 days ≤3 days

soybean–casein digest
agar or soybean–casein
Candida albicans such as ATCC digest broth

N
10231, NCPFe 3179, CIP 48.72, 30°–35°
or NBRC 1594 ≤7 days

soybean–casein digest soybean–casein digest

Aspergillus brasiliensis (formerly

A. niger) such as ATCC 16404,
agar or soybean–casein
digest broth
30°–35°
CO agar and soybean–casein
digest broth
≤100 CFU
soybean–casein digest
agar
≤100 CFU
IMIf 149007, CIP 1431.83, or ≤7 days, or until good 30°–35° 30°–35°
NBRC 9455 sporulation is achieved ≤5 days ≤5 days
a American Type Culture Collection, https://www.atcc.org.
b National Collection of Industrial, Food and Marine Bacteria, https://www.ncimb.com/.
L
c
Collection of the Institut Pasteur, https://www.pasteur.fr.
d
NITE Biological Resource Center, https://www.nite.go.jp/en/nbrc/index.html.
e National Collection of Pathogenic Fungi, https://www.phe-culturecollections.org.uk/collections/ncpf.aspx.
IA

f CABI Bioscience, https://www.cabi.org/products-and-services/bioscience-services/.

Use buffered sodium chloride–peptone solution pH 7.0 or phosphate buffer solution pH 7.2 to make fresh test suspensions. To suspend A.
brasiliensis spores, 0.05% of polysorbate 80 may be added to the buffer. Use the suspensions within 2 h, or within 24 h if stored at 2°–8°.
Alternatively, a stable spore suspension for A. brasiliensis and B. spizizenii may be prepared. The stable spore suspension may be maintained at
IC

2°–8° for a validated period.

Negative Control

To verify testing conditions, include a negative control using the chosen diluent in place of the test preparation. There must be no growth of
microorganisms. A negative control is also performed when testing samples described under Testing for Bioburden. A failed negative control
FF

requires an investigation.

Growth Promotion of the Nutrient Culture Media

Test each batch of ready-prepared nutrient culture medium. Likewise, test each batch of nutrient culture prepared from dehydrated medium or
from the described ingredients.
O

Inoculate portions or plates of the nutrient culture media with a small number [not more than 100 colony-forming units (CFU)] of the
microorganisms indicated in Table 1, using a separate portion or plate of nutrient culture medium for each. Incubate according to the conditions
described in Table 1.
N

For solid nutrient culture media, growth obtained must not differ by a factor greater than 2 from the calculated value for a standardized
inoculum. For freshly prepared inoculum, microbial growth and quantity must be comparable to growth obtained with a previously qualified lot
of nutrient culture medium. Liquid nutrient culture media are deemed suitable when clearly visible microbial growth is comparable to that from a
U

previously qualified batch of nutrient culture medium.

Suitability of the Counting Method in the Presence of Product

Test Sample Preparation

Test sample preparation depends upon the test sample, acceptance criteria, and test method as defined in 〈 1119 〉. For a fluid test sample,
no preparation is required. For raw materials and excipients, the sample should be dissolved or diluted in buffered sodium chloride–peptone

https://online.uspnf.com/uspnf/document/2_GUID-E2174C65-C3B2-4D5B-8127-0D830104D0FD_10101_en-US?source=Quick Search&highlight… 2/4

3/8/24, 2:25 PM USP-NF 〈1119.1〉 Bioburden Test

solution pH 7.0, phosphate buffer solution pH 7.2, or soybean–casein digest broth. If necessary, adjust to a pH of 6–8. Dissolution (or further
dilution) must ensure the appropriate sensitivity of the test, allowing conformance to specification. For primary container closures and ready-
to-sterilize components, bioburden must be recovered by a validated method into an appropriate buffer solution.

Inoculation and Dilution

Add a sufficient volume of microbial suspension (not more than 100 CFU) to the prepared sample and a control (with no test material
included). The inoculum volume should not exceed 1% of the volume of the prepared sample.
To demonstrate acceptable microbial recovery, the test must use the lowest possible dilution of the prepared sample. If the sample causes
growth inhibition due to factors like antimicrobial activity or poor solubility, add the microbial inoculum after neutralization, dilution, or filtration
using a validated method.

T
Neutralization
If the test sample to be examined has antimicrobial activity, neutralization methods described in Validation of Microbial Recovery from

N
Pharmacopeial Articles 〈1227〉 should be applied. Antimicrobial activity may be negated by membrane filtration and rinsing, dilution, use of a
specific neutralizing agent, or a combination of these methods. If no suitable neutralizing method can be found, the failure to isolate the
inoculated organism may be attributable to the microbicidal activity of the test sample. This information indicates that the sample is unlikely to

TE
be contaminated with the given microorganism. However, it is possible that the product may only partially inhibit certain specified
microorganisms and may not inhibit other strains that are not listed in Table 1. In this case, perform the test at the lowest dilution compatible
with microbial growth and the specific acceptance criterion.

Recovery of Microorganisms in the Presence of Product

Prepare a test sample as described under Test Sample Preparation, Inoculation and Dilution, and Neutralization. For each of the

N
microorganisms listed, separate tests should be performed in duplicate. Only colonies of the added test strain should be counted.
Membrane filtration: Use membrane filters with a nominal pore size not greater than 0.45 µm. Choose the appropriate filter material such that
the microbial-retaining efficiency is not affected by the test sample. Use one membrane filter for each microorganism listed in Table 1.

CO
Transfer a suitable quantity (ensuring adequate test method sensitivity) of the test sample to the membrane filter, filter immediately, and
rinse the membrane filter with an appropriate volume of diluent. Transfer the membrane filter to the surface of the soybean–casein digest agar
and incubate the plates as indicated in Table 1. Perform the counting.
Pour-plate method: For Petri dishes 9 cm in diameter, add 1 mL of the prepared sample to the dish and 15–20 mL of soybean–casein digest
agar maintained at not more than 45°. If larger Petri dishes are used, increase the agar medium accordingly. Use two Petri dishes for each of
the microorganisms listed in Table 1. Incubate the Petri dishes as indicated in Table 1. Perform the counting.
Surface-spread method: For Petri dishes 9 cm in diameter, add 15–20 mL of soybean–casein digest agar held at not more than 45° and allow
to solidify. If larger Petri dishes are used, increase the volume of the agar accordingly. Dry the plates, for example, in a laminar-airflow cabinet
AL

or an incubator. Spread a measured volume of not less than 0.1 mL of the sample over the surface of the medium. Incubate the Petri dishes as
indicated in Table 1. Perform the counting.

Results and Interpretation

Take the arithmetic mean of the counts per nutrient culture medium and calculate the number of CFU in the original inoculum. The mean
CI

count of the test organisms must not differ by a factor greater than 2 compared with the control. If the above criteria cannot be met for one or
more organisms tested using the described methods, the method and test conditions closest to the criteria should be used to test the sample.

TESTING FOR BIOBURDEN

The amount of sample tested must be sufficient to determine compliance with the established specification for bioburden.
I

Membrane Filtration
FF

Use a filtration apparatus designed to allow the transfer of the filter to the nutrient culture medium. Prepare the sample using a method that
has been shown to be suitable, as described in Growth Promotion Test, Negative Controls, and Suitability of the Counting Method. Transfer the
appropriate amount to a membrane filter, filter immediately, and wash the filter. Transfer the membrane filters to the surface of soybean–casein
digest agar and incubate at 30°–35° for 3–5 days. Calculate the number of CFU per quantity (e.g., CFU/mL, CFU/g, or CFU/unit) of sample.

Pour-Plate Method and Surface-Spread Method

O

Prepare the sample using a method that has been shown to be suitable as described in Growth Promotion Test, Negative Controls, and
Suitability of the Counting Method. Prepare duplicate Petri dishes using soybean–casein digest agar for each sample and incubate the plates at
N

30°–35° for 3–5 days. Take the arithmetic mean of the counts and calculate the number of CFU per quantity (e.g., CFU/mL, CFU/g, or CFU/unit)
of sample.▲ (USP 1-Aug-2025)
U

Auxiliary Information - Please check for your question in the FAQs before contacting USP.

https://online.uspnf.com/uspnf/document/2_GUID-E2174C65-C3B2-4D5B-8127-0D830104D0FD_10101_en-US?source=Quick Search&highlight… 3/4

3/8/24, 2:25 PM USP-NF 〈1119.1〉 Bioburden Test

Topic/Question Contact Expert Committee

< 1119 .1> BIOBURDEN TEST Leslie Furr GCM2022 General Chapters - Microbiology
Associate Scientific Liaison 2022
Page Information:

Not Applicable

DocID: GUID-E2174C65-C3B2-4D5B-8127-0D830104D0FD_10101_en-US
DOI: https://doi.org/10.31003/USPNF_M18395_10101_01
DOI ref: mk0tz

T
N
TE
N
CO
AL
I CI
FF
O
N
U

https://online.uspnf.com/uspnf/document/2_GUID-E2174C65-C3B2-4D5B-8127-0D830104D0FD_10101_en-US?source=Quick Search&highlight… 4/4