Influence of Environmental Sample Pre-Storage on Counting Results

Environmental monitoring using culture media in the form of settle and contact plates is a crucial part of aseptic manufacturing of pharmaceutical products. Culture media sampling is normally carried out in cleanrooms and then transferred to a lab space for incubation and evaluation of the results. The manufacturing process does not always allow for the immediate transfer of samples to the incubator space.

The article discusses interesting data for the workflow of handling environmental samples in a cleanroom. Most lab technicians lack suitable capabilities to carry the samples directly to an incubator. The samples are either pre-stored in a cleanroom or a refrigerator before they start the incubation process. The data presented in this article will demonstrate if pre-storage has any impact on the recovery rates and growth promotion of selected microorganisms.

• Interim Storage Conditions
• Influence of Interim Storage on Selected Microorganisms vs. Direct Inoculation
• in situ Surface Sampling using ICR Plus Contact Plates
• Suitability of Pre-storage Conditions for Environmental Samples
• Related Products

Interim Storage Conditions

The influence of different interim storage conditions is investigated based on the following criteria:

• Growth of selected microorganisms
• in situ surface sampling on naturally contaminated surfaces

For this study, gamma-irradiated casein soya bean digest agar (TSA) with 4 neutralizers was chosen as the test media (TSA w. LTHThio Contact ICR+; Product No. 146783).

Interim storage conditions were chosen based on different lab practices and are listed below:

1. The sample is directly incubated at 30 °C to 35 °C after surface sampling or inoculation.
2. The sample is stored at room temperature for 16 to 18 hours after surface sampling or inoculation and then incubated at 30 °C to 35 °C.
3. The sample is stored at 4 °C to 8 °C for 2 hours after surface sampling or inoculation and then incubated at 30 °C to 35 °C.
4. The sample is stored at 4 °C to 8 °C for 6 hours after surface sampling or inoculation and then incubated at 30 °C to 35 °C.
5. The sample is stored at 4 °C to 8 °C for 16 to 18 hours after surface sampling or inoculation and then incubated at 30 °C to 35 °C.
6. The sample is stored at 4 °C to 8 °C for >55 hours after inoculation and then incubated at 30 °C to 35 °C.

Study of Sample Pre-Storage conditions on the Recovery Rates and Growth Promotion

Part I: Influence of interim storage on selected microorganisms vs. direct inoculation

The test strains were selected based on their general occurrence in cleanrooms,³ their relationship to the human skin microbiome², and US and EU pharmacopeia recommendations for growth promotion tests on casein soya bean digest agar media.^1,4 The list of selected strains and incubation conditions after sampling and direct storage conditions are given in Table 1.

Table 1. Selected test strains and incubation conditions.

Test Strain	ATCC® Number	Direct incubation conditions (excluding interim storage conditions)
Staphylococcus aureus	6538	Aerobic; 24 hours at 30-35 °C
Kocuria rhizophila	9341	Aerobic; 48 hours at 30-35 °C
Staphylococcus epidermidis	14990	Aerobic; 48 hours at 30-35 °C
Propionibacterium acnes	6919	Anaerobic; 10 days at 30-35 °C
Corynebacterium striatum	7094	Aerobic; 48 hours at 30-35 °C
Escherichia coli	25922	Aerobic; 24 hours at 30-35 °C
Penicillium commune	10428	Aerobic; 5 days at 30-35 °C
Surface samples		Aerobic; 3 days at 30-35 °C

An inoculum of fresh overnight cultures was prepared and diluted to achieve approx. 10 to 120 CFU, which is then inoculated on the surface of the test plates. Each test condition was performed in two independent test runs with a five-fold repetition in each run. For the interim storage condition of >55 hours at 4 °C to 8 °C, only one test run was performed. The recovery rates were calculated to the average CFU on the plates without interim storage.

Recovery Rate = (CFU – test) × 100 / (CFU − direct-incubation)

It is observed that recovery rates of the two independent rounds showed the same trends. Hence, figures 1 and 2 only show the results for one test round. The results for the aerobic test strains are shown in Figure 1.

Figure 1. Recovery rates of selected microorganisms after each interim storage condition compared to direct incubation.

In addition, the interim storage for the Propionibacterium acnes is performed both anaerobically and aerobically. The results for this test strain are indicated in Figure 2.

Figure 2. Recovery rates of Propionibacterium acnes after each interim storage condition compared to direct incubation.

Part II: in situ surface sampling using ICR plus Contact plates

Surface samples were taken from a non-classified laboratory environment in 20 rows and 5 positions per row using a TSA w. LTHThio Contact ICR+ contact plate (Product No. 146783). The sampling pattern and various interim storage conditions are shown in Figure 3. These plates were incubated for 3 days at 30 °C to 35 °C following the interim storage conditions. Finally, the colonies were counted, and a comparison was made for each of the interim storage conditions.

Figure 3. Sampling pattern for the first 5 rows (rows 6 to 20 will repeat this pattern every 5 rows).

The average of all test samples is comparable for most interim storage conditions with 10 or 11 CFU. Longer interim storage for 16 to 18 hours at cool temperatures resulted in a lower recovery rate with an average of 6 CFU. This is indicated in Figure 4 for all test results per interim condition. It seems that cool storage conditions might reduce the recovery of some microorganisms, which do not grow properly afterward in incubation.

Figure 4. Recovery of microorganisms at each interim storage temperature.

Suitability of Pre-Storage Conditions for Environmental Samples: Conclusion

This study established that different pre-storage conditions had no influence on the recovery rate of the selected microorganisms (Staphylococcus aureus, Kocuria rhizophila, Staphylococcus epidermidis, Propionibacterium acnes, Corynebacterium striatum, Escherichia coli and Penicillium commune) prior to the standard incubation period. All recovery rates were found to be in the range of 70%-200%.

Also, the different storage conditions did not influence the growth-promoting properties of the samples if they were stored at room temperature for up to 18 hours or placed at a cool temperature for up to 6 hours. A slightly decreased recovery rate was obtained for samples that were pre-stored for longer than 6 hours at cool temperatures before the incubation period. Storage at room temperature after sampling and before official incubation seems to be the preferable choice since this temperature already supports the growth of a variety of microorganisms. On the other hand, the presence of molds might bear the risk of overgrowth when exposed to prolonged storage at room temperature.

The slight difference between the direct inoculation study (Part I) and the in situ study (Part II) to test the pre-storage of samples for 16-18 h at 4-8 °C might be explained by the different compositions of the in situ microflora. In addition, environmental microorganisms may be damaged due to desiccation. Additional studies would be necessary to verify the difference.

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References

1. 2019. 2.6.12. Microbial examination of non-sterile products: Microbial Enumeration Tests. European Pharmacopoeia 9.8;

2. Grice E. A., Segre J. A. . 2011. The Skin Microbiome. Nature reviews Microbiology .(9):244-253.

3. Sandle T. 2011. A Review of Cleanroom Microflora: Types, Trends and Patterns. PDA J Pharm Sci and Tech. 65(4):

4. 2018. United States Pharmacopoeia 41 NF 36: . <61> Microbiological Examination of Non-Sterile Products: Microbial Enumeration Tests.