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Guidelines for sterility testing of therapeutic goods

13 September 2006

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Annex I. Guidance on obtaining small numbers of vegetative organisms and spores

All procedures for the preparation, maintenance and cultivation of challenge organisms should be documented. At the time of use, cultures maintained by seed lot culture techniques should be no more than 5 passages from the original type culture strain that has been obtained from a recognised reference culture supplier. The identity (morphological and physiological properties) should be checked periodically.

The methods described below are for preparing suspensions of low numbers of the challenge organisms from the major groups (aerobes, anaerobes and fungi) in Table 5. They are provided as examples of acceptable procedures and are for guidance only. Some incubation times and temperatures differ from those in the sterility test procedures; they are suggested because they have been found to provide, for the particular strains used, conditions under which useful numbers of viable cells can be cultured within 24 hours.

The methods detailed may be modified for other organisms by changing the media, diluents or culture conditions. Other methodology (such as the use of suspensions on glass beads or suspensions stored in a 10% glycerol preparation at -70°C) may be equally satisfactory. Irrespective of the method used, it is advisable for any laboratory to carry out viable counts on the selected working dilution of each challenge organism on a daily basis for a whole week to determine the viability (stability) of their particular strains of organisms.

Method for preparation of cell suspension of Staphylococcus aureus or Pseudomonas aeruginosa

Every 4 months open a new ampoule and subculture to Soy Bean Casein Digest (SCD) broth. The incubation conditions for these organisms are 24 hours at 37°C for S. aureus and 30°C for P. aeruginosa. Subculture from the SCD broth to Soy Bean Casein Digest agar (SCDA) slopes (stock slopes) and concurrently plate on to a SCDA plate to check for purity.

Every month subculture from the stock slope to a fresh SCDA slope.

Every week subculture from the monthly slope to a SCDA plate. If the culture is pure, subculture from the slope into 10 mL of SCD and incubate for 24 hours at 37°C for S. aureus and 30°C for P. aeruginosa.

This culture is used to prepare the working dilution as follows:

  • assuming that the 24 hour culture contains 1 x 109  CFU/mL, carry out sufficient serial dilutions in 0.1% peptone saline to arrive at approximately 100 CFU/mL;
  • prepare about 100 mL of this dilution, which will be the working dilution.

Carry out a viable count on the working dilution on SCDA plates.

From the results of the viable count calculate the volume of the working dilution which contains not more than 100 CFU and use this for validation, growth promotion and stasis testing. A concurrent viable count should be carried out when performing any of these tests, as a check that the working dilution has been correctly prepared and calculated.

Store the 100 mL of the working dilution at 2-8°C. Use as needed but do not keep longer than a week.

Method for preparation of Cell Suspension of Candida albicans

Every 4 months open a new ampoule and subculture to Sabouraud Dextrose Broth (SDB). Unless otherwise stated the incubation conditions for this organism are 24-48 hours at 30°C. Subculture from the SDB to Sabouraud Dextrose Agar (SDA) slopes (stock slopes) and concurrently plate on to an SDA plate to check for purity. Prepare sufficient stock slopes to last 4 months.

Every month subculture from the stock slope to a fresh SDA slope.

Every week subculture from the monthly slope to a SDA plate. If the culture is pure, subculture from the slope into 10 mL of SCD and incubate for 24 hours at 30°C.
This culture is used to prepare the working dilution, as follows:

  • assuming that the 24 hour culture contains 1 x 108  CFU/mL, carry out sufficient serial dilutions in 0.1% peptone saline to arrive at approximately 100 CFU/mL;
  • prepare about 100 mL of this dilution, which will be the working dilution.

Carry out a viable count on the working dilution on SDA plates and incubate at 30°C for 24 hours.

From the results of the viable count calculate the volume of the working dilution that contains not more than 100 CFU and use this for validation, growth promotion and stasis testing. A concurrent viable count should be carried out when performing any of these tests, as a check that the working dilution has been correctly prepared and calculated.

Store the 100 mL of the working dilution at 2-8°C. Use as needed but do not keep longer than a week.

Preparation of spore suspensions of Bacillus subtilis

Stock suspension

Preparation of the stock suspension may be carried out every 12 months. Open ampoule and subculture into SCD and incubate at 37°C for 24 hours.
Inoculate five 45 mL Sporulation Agar slopes (in 100 mL medical flats) with approximately 1.0 mL of the 24 hour broth culture and incubate at 37°C for 5 days. Concurrently, plate the 24 hour broth culture onto SCDA to check for purity. Incubate at 37°C overnight. Next day, if pure, discard; otherwise purify.

Check spore production after 5 days by spore stain. If the percentage of cells sporing is less than 70-80% continue incubation. When a 70-80% spore yield is achieved, wash off the growth from the flats with 20 mL of sterile normal saline and dispense into sterile McCartney bottles or centrifuge tubes.

Centrifuge at 1500 rpm for 20 minutes. Decant (and discard) the supernatant liquid.

Resuspend the sediment in 10 mL of fresh sterile normal saline and spin again; repeat this process three times.

After the third wash decant off the supernatant liquid except for approximately 1mL. Resuspend the spores in 2 mL of normal saline.

Heat the spore suspension at 56°C for 30 minutes to kill the vegetative cells.

Carry out a viable count on SCDA using peptone saline as diluent, incubating at 37°C for 24 hours. The final preparation should contain approximately 108 spores/mL.

Working dilution suspension

To prepare a working dilution, dilute the spore suspension in peptone saline to contain approximately 100 spores/mL. Prepare sufficient to last a week for validation, growth promotion and stasis testing. Keep refrigerated at 2-8°C.

Carry out a viable count on the working dilution on SCDA plates; incubate the plates at 37°C for 24 hours.

From the results of the viable count calculate the volume of the working dilution that contains not more than 100 CFU and use this for validation, growth promotion and stasis testing. A concurrent viable count should be carried out when performing any of these tests, as a check that the working dilution has been correctly prepared and calculated.

Preparation of spore suspension of Clostridium sporogenes

Stock suspension

Preparation of the stock suspension may be carried out every 12 months.
Open ampoule and inoculate into Reinforced Clostridial Medium (RCM). Incubate under anaerobic conditions at 32°C for 48 hours.

Subculture about 3-5 mL of the broth onto each of five 45 mL solid RCM agar slopes (in 100 mL medical flats) and incubate anaerobically at 32°C for until 70-80% of the population is sporing (approximately 2 weeks). Spore production should be checked every few days by spore stain.

When a 70-80% spore yield is achieved, wash off the growth from the flats with 20 mL of sterile normal saline and dispense into sterile McCartney bottles or centrifuge tubes.

Centrifuge at 1500 rpm for 20 minutes. Decant (and discard) the supernatant liquid.

Resuspend the sediment in 10 mL of fresh sterile normal saline and spin again; repeat this process three times.

After the third wash decant off the supernatant liquid except for approximately 1mL. Resuspend the spores in 2 mL of normal saline.

Heat the spore suspension at 56°C for 30 minutes to kill the vegetative cells.

Carry out a viable count on SCDA using peptone saline as diluent, incubating anaerobically at 32-37°C for 24-48 hours. The final preparation should contain approximately 108 spores/mL.

Working dilution suspension

To prepare a working dilution, dilute the spore suspension in peptone saline to contain approximately 100 spores/mL. Prepare sufficient to last a week for validation, growth promotion and stasis testing. Keep refrigerated at 2-8°C.

Carry out a viable count on the working dilution on SCDA plates; incubate the plates anaerobically at 32-37°C for 24-48 hours.

From the results of the viable count calculate the volume of the working dilution that contains not more than 100 CFU and use this for validation, growth promotion and stasis testing. A concurrent viable count should be carried out when performing any of these tests, as a check that the working dilution has been correctly prepared and calculated.

Preparation of spore suspensions of Aspergillus niger

Stock suspension

Preparation of the stock suspension may be carried out every 12 months.

Open ampoule and subculture into SDB and incubate at 25°C for 3 days.

Inoculate five 45 mL SDA slopes (in 100 mL medical flats) with approximately 1.0 mL of the broth culture and incubate at 25°C for 5 days. Concurrently, plate the broth culture onto SDA to check for purity. Incubate at 25°C for 3 days. Next day, if pure, discard; otherwise purify.

Check spore production after 5 days by the appearance of black spores on the surface. When the entire surface of the culture is black, wash off the growth from the flats with 20 mL of sterile normal saline and dispense into sterile McCartney bottles or centrifuge tubes.

Centrifuge at 1500 rpm for 20 minutes. Decant (and discard) the supernatant liquid.

Resuspend the sediment in 10 mL of fresh sterile normal saline and spin again; repeat this process three times.

After the third wash decant off the supernatant liquid except for approximately 1mL. Resuspend the spores in 2 mL of normal saline.

Carry out a viable count on SDA using peptone saline as diluent, incubating at 25°C for 3 days. The final preparation should contain approximately 108 spores/mL.

Working dilution suspension

To prepare a working dilution, dilute the spore suspension in peptone saline to contain approximately 100 spores/mL. Prepare sufficient to last a week for validation, growth promotion and stasis testing. Keep refrigerated at 2-8°C.

Carry out a viable count on the working dilution on SDA plates; incubate the plates at 25°C for 3 days.

From the results of the viable count calculate the volume of the working dilution that contains not more than 100 CFU and use this for validation, growth promotion and stasis testing. A concurrent viable count should be carried out when performing any of these tests, as a check that the working dilution has been correctly prepared and calculated.

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