This document proposes using a single general growth medium (TSA) and incubation scheme to monitor cleanrooms for bacterial, yeast, and mold growth. It describes incubating TSA plates at 32.5°C for 3 days followed by 2 days at 22.5°C, aiming to support growth of all microorganisms. A study showed growth of common environmental isolates on TSA was comparable to growth on a selective medium (SDA). This combined approach could reduce the time, cost, resources and space needed for cleanroom environmental monitoring compared to using two media and incubation conditions.

1. The Cost and Time Saving Model
To Monitor Your Cleanroom

2.  Two media, single incubation system.
◦ TSA (bacterial growth), SDA (yeast/fungal)
◦ TSA incubated at 32.5 +/- 2.5oC for 2-3 days
◦ SDA incubated at 22.5 +/- 2.5oC for 3-5 days
 Past Reasoning:
◦ TSA cannot capture fungal growth therefor a
selective media is required.
◦ Selective media allows for yeast/mold growth while
limiting ability of bacteria to grow.

3.  General Use Media (TSA) for all organisms.
 Incubation Scheme:
◦ Incubation at 32.5+/-2.5oC for 3 days followed by
22.5+/-2.5oC for additional 2 days.
 Reasoning:
◦ TSA has ability to capture most mold with correct
conditions.
◦ Method should be qualified against appropriate
environmental isolates (specifically yeast/mold).

4.  USP <1116>
◦ “A general microbiological growth medium such as
soybean–casein digest medium (SCDM) is suitable
for environmental monitoring in most cases
because it supports the growth of a wide range of
bacteria, yeast, and molds.”
◦ “The ability of any media used in environmental
monitoring, including those selected to recover
specific types of organisms, must be evaluated for
their ability to support growth, as indicated in
<71>.”

5.  Study should be performed showing
comparability in media to pick up
environmental isolates.
 Study should target environmental
yeast/mold that show predominance in
trending and/or are slower growing
organisms.
◦ No need to perform bacterial environmental isolates
as they have already been captured on TSA and
therefor do not need to be repeated.

6.  Target Media: TSA with neutralizers
 Control Media: SDA with neutralizers
 Target Organisms
◦ Aspergillus bransiliensis (env. Isolate)
◦ Aspergillus flavus (env. isolate)
◦ Candida albicans (ATCC 10231)
◦ Penicillium chrysogenum (env isolate)
◦ Chaetomium globosum (env isolate)
 Target Growth
◦ Acceptable growth is 0.5 to 1.5 times the growth
seen on control Agar.

7.  Positive Control Media incubated at 22.5+/-
2.5oC for 3 days.
 Test Growth Media incubated at 32.5+/-
2.5oC for 3 days followed by 22.5+/-2.5oC
for additional 2 days.
 Acceptable growth is 0.5 to 1.5 times the growth seen
on Positive Control Agar.
 Negative Control Media to be negative for
growth after 5 days of transferred incubation
alongside samples.

8. Organism SDA 1 SDA 2 SDA 3 Avg
SDA
Acceptance
Range
A. bransiliensis 45 39 48 44 22-66
A. flavus 41 38 43 41 21-62
C. albicans 56 51 46 51 26-77
P. chrysogenum 44 41 48 45 23-68
C. globosum 42 49 52 48 24-72

9. Organism Acceptance
Range
TSA 1 TSA 2 TSA 3 Pass/Fail
A. bransiliensis 22-66 39 48 44 Pass
A. flavus 21-62 38 43 41 Pass
C. albicans 26-77 51 46 51 Pass
P. chrysogenum 23-68 41 48 45 Pass
C. globosum 24-72 49 52 48 Pass

10.  Changes in Growth Promotion Testing.
◦ Modified from USP <71> listed organisms.
◦ Modified to include incubation scheme described in
study.
◦ Incubation time based on worst case scenario.
 Increased sampling scheme based on
decreased resources required and a risk
based approach.

11.  Time
◦ Length of time to sample is typically cut in half.
 Cost
◦ Typical Cost savings range from 30%-50%.
 Resources
◦ Lowered sampling time and plate analysis will
decrease personnel requirements for sampling.
 Space
◦ Decreased refrigeration storage space for sampling
plates (pre-use) and incubation space due to plate
count being cut in half.

12. Questions?
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