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Population determination of biological indicators

1. Aseptically extract 10 unexposed spore strips from their glassine envelopes and place them within 100 ml sterile chilled water in a sterile high speed blender. Homogenise 3-5 min until no large particles are visible. Homogenisation with a glass-bed may result in a lower CFU-count, due to insufficient homogenisation. Available spore suspensions can be used directly after shaking well. If the spores were exposed to formaldehyde, they have to be pre-treated to inactivate resident formaldehyde particles. The formaldehyde residues would hamper the outgrowth of the spores. Therefore, the sterilized spore strips are aseptically transferred to Na2SO3 solution (2 %, 10 ml, 10 min) and heat activated (90-93°C, 60 min). Then, the spores are homogenized as described above. A further heat shock treatment is not necessary (see below). For further information please pay attention to the standard DIN EN ISO 11138-5 or the publication of Gömann et al.: Reaction Kinetics of the Low-Temperature-Steam-Formaldehyde (LTSF) Sterilization Process, Zentr. Steril. 2000, 8 (5): 290-293).

2. Make appropriate dilutions with sterile distilled water to reach a titter of approximately 30 to 300 CFU (Colony Forming Units)/ml. Preferred dilutions are 1:10 using 1 ml test suspension and 9 ml sterile distilled water. Please take dilution samples only from homogenous suspension! We recommend to make two separate dilutions from the original suspension and incubate at least three plates each. If the population of the examinated biological indicator is unknown, several dilution steps have to be made. The dilution that contained 30-300 CFU/ml will be used for the further population calculation.

3. Following USP (US Pharmacopoeia) heat shock half of the suspension with the following temperatures and times and leave the rest untreated. • Bacillus pumilus and atrophaeus 80-85°C for 10 min • Geobacillus stearothermophilus 95-100°C for 15 min Rapidly cool down the suspensions in ice bath (0 – 4°C).

4. Fill 1 ml of the heat shocked and untreated suspension on 3 (6) TSA (Tryptic Soja Agar) plates each and poor 15-20 ml liquid TSA (50°C) over the culture. Mix the liquids on the agar plate by carefully rotating.

5. For safety purposes plate out 1 ml of the used sterile water to check contamination free operation.

6. Incubate the plates at least 2 days under following temperature: gke – Technical Information TI 730-067-EN

  • • Bacillus pumilus and atrophaeus 30 – 35°C
  • • Geobacillus stearothermophilus 55 – 60°C

Attention: If the wrong incubation temperature is used, reduced growth occurs.

7. Following incubation count the bacterial colonies on plates containing 30 to 300 colonies and average the population of 3 plates. Do not mix heat shocked and untreated samples. Disregard plates with less than 10 colonies due to
inaccuracies caused by absorption effects.

8. Finally, the population of the examinated biological indicator is calculated by multiplying the measured CFU/plate value with all concentration or dilution steps carried out in the population assay.

9. Compare the population values of untreated and heat shocked results. Usually you will find that the heat shocked plates show a higher population. The population values of gke-certificates use heat shock values using the USP
procedures. EN ISO 11138-1 requires an average recovery rate of -50 + 300% referring to the population in the certificate of that batch.