Janine Coetzer Integral Labs Agar sterilization time, temperature and expiry dates for prepared media Janine Coetzer Integral Labs

No matter how prepared you are for your next audit, there will always be something you missed, there is always something you can improve on and there are always ways to make your life easier and to work more efficient. Working under conditions which does not allow for the preparation of media every day, it is necessary to prepare bigger batches of media when high volume of samples are expected. Even though well maintained and calibrated equipment are being used, time and temperature limits are sometimes exceeded. This can become very expensive if this media has to be discarded. These questions popped up in an Internal Audit 1. How long can you keep your prepared agar in the fridge. 2. How many degrees can you go above 121°C while sterilizing agar. 3. How much extra time can you allow at the required temperature with no effect to agar. And off course there were no answers. The idea behind this exercise was be to establish expiry dates for prepared agar, Minimum/maximum time and temperatures for sterilization of agar that would not affect the integrity of the agar.

Question 1. How long can we store prepared agar: There is no data available under the manufacturers preparation procedures regarding the time prepared media can be stored. The idea is to prepare a batch, and over a period of time perform: 1. Positive and negative controls 2. pH 3. Sterilities to try and establish a suitable time media can be stored. LAB M Harlequin e.coli/coliform Medium Soak for 10min, swirl to mix. Autoclave at 121°c for 15min. pH7.2 ±0.2 Biolab Plate Count Agar for HPCs 1. Boil until completely dissolved. 2. Autoclave at 121°c for 15min. 3. pH 7.0 ±0.2

Harlequin coliform agar Agar was also poured into 4 sterile containers to allow for pH’s to be performed over a period of 4 weeks. Every week a pH would be run Plates was poured into Petri dishes and stored in a sealed container at 2-8°C these were to be used as Positive and Negative controls. Plate Count Agar for HPCs Agar were stored in separate containers at 2-8°C, allowing it to be melted every week in order to perform positive controls, sterilities and pH

LAB M Harlequin e.coli/coliform Medium Soak for 10min, swirl to mix. Autoclave at 121°C for 15min. pH7.2 ±0.2 Date Prep Date Run Steri Temp Av time at Max temp Steri Time 121 ±5°c Agar/Media pH pH range Positive Negative Blank Logger position 2016/09/14 123°c 12 min at 123°C 26 min Harlequin 7.25 7.0 ± 0.2 Blue colonies Pink colonies No Growth External 2016/09/20 2016/09/27 7.21 2016/10/03 7.23 2016/10/11 7.16

Biolab Plate Count Agar for HPCs 1. Boil until completely dissolved Biolab Plate Count Agar for HPCs 1. Boil until completely dissolved. Autoclave at 121°C for 15min. pH 7.0 ±0.2 Date Prep Date Run Steri Temp Av time at Max temp Steri Time 121 ±5°C Agar/Media pH pH range Pos Blank Logger position 2016/06/02 2016/6/02 123°C 11 min at 123°C 18 min PCA 6.96 7.0 ± 0.2 Pass No Growth External 2016/06/08 6.93 2016/06/15 2016/06/22 2016/06/27 7.00

The next step was to run the plates in duplicate every week with our daily controls. Every week I would prepare a freshly spiked sample using e.coli ATCC 25922 as a positive control and k.pneu ATCC 31488 as negative for e.coli and positive for total coliforms. This spiked sample was then run on freshly prepared agar and the “old” agar, the results were compared and evaluated as per a 95% confidence table.   Total coliform E Coli Date Dupl 1 Dupl 2 QC Indicator Analyst/comments UWL UAL 08-May-17 68 81 ACCEPT 8 12 49.35 75.0 100.0 09-May-17 128 147 3 2 25.00 10-May-17 204 228 200 0.00 11-May-17 88 94 12-May-17 54 79 91 37.84 13-May-17 - 14-May-17 15-May-17 62 78 98 61.24 16-May-17 84 65 17-May-17 18-May-17 120 111 20 25 35.90

Harlequin Duplicates Fresh vs “Old” Prepared: 14/9/2016 Run: 20/9/16 e.Coli ATCC 25922 k.Variicola ATCC 31488 Date Dupl 1 Dupl 2   20-Sep-16 >200 #VALUE! Date Dupl 1 Dupl 2   20-Sep-16 >200 #VALUE!

Prepared: 14/9/2016 Run: 27/9/16 e.Coli ATCC 25922 k.Variicola ATCC 31488 Date Dupl 1 Dupl 2   27-Sep-16 1 2 ACCEPT Date Dupl 1 Dupl 2   27-Sep-16 121 113 ACCEPT

Prepared: 14/9/2016 Run: 3/10/16 e.Coli ATCC 25922 k.Variicola ATCC 31488 Date Dupl 1 Dupl 2   03-Oct-16 9 8 ACCEPT Date Dupl 1 Dupl 2   03-Oct-16 236 203 ACCEPT

Prepared: 14/9/2016 Run: 11/10/16 e.Coli ATCC 25922 k.Variicola ATCC 31488 Date Dupl 1 Dupl 2   11-Oct-16 >200 ACCEPT Date Dupl 1 Dupl 2   11-Oct-16 >200 #VALUE!

PCA Duplicates Fresh vs “Old” Fresh Agar e.Coli ATCC 25922 Prepared: 2/6/2016 Run: 8/6/2016 pH: 6.93 e.Coli ATCC 25922 Date No. Dupl 1 Dupl 2 08-Jun-16 1 24 22

Fresh Agar e.Coli ATCC 25922 Prepared: 2/6/2016 Run: 15/6/2016 pH: 6.96 e.Coli ATCC 25922 Date No. Dupl 1 Dupl 2 08-Jun-16 1 24 22 15-Jun-16 2 1000

Fresh Agar e.Coli ATCC 25922 Prepared: 2/6/2016 Run: 22/06/2016 pH: 6.96 e.Coli ATCC 25922 Date No. Dupl 1 Dupl 2 08-Jun-16 1 24 22 15-Jun-16 2 1000 22-Jun-16 3 253 274

Fresh Agar e.Coli ATCC 25922 Prepared: 2/6/2016 Run: 29/06/2017 pH: 7.00 e.Coli ATCC 25922 Date No. Dupl 1 Dupl 2 08-Jun-16 1 24 22 15-Jun-16 2 1000 22-Jun-16 3 253 274 29-Jun-16 4 18 17

Decision: Looking at the results obtained during the 4 weeks, all checks passed and the agar was still in good condition( no visible deterioration). We’ve settled on a 4 week storage time for prepared plates, but prepared [plates hardly lasts longer than 1 week. At least this means business has picked up a lot.

Question 2. What time is acceptable at 121°C, and what temperature deviation would be tolerated by the media? Sterilization Time and Temperature is still a bit of a Toffee. On all the manufacturers preparation steps it states autocalve at 121°C for 15 minutes, no +/- this way or that. I’ve never seen a run this specific. With a SANAS audit it was noted that the temperature logger was not submerged in the media during the sterilization process, and that different volumes were sterilized at the same time. Subsequently all autoclaves have been re-calibrated with Loaded and Unloaded runs, and sterilizing of media and glassware etc. has been separated. The temperatures on most of our runs are fairly regular and hardly ever exceeding 124°C. On the odd occasion where the temperature was over 124°C no failures were noted on controls or pH. The average run time at 121°C after the last calibration sits roughly at 22 minutes, with no failures and very similar readings on controls and pH. We have also started to run a pH with the daily controls with each batch of Plate Count Agar used.

No failures were detected on any of these checks. The pH is very consistent and within range. A duplicate was run on a weekly basis to compare freshly prepared agar with the stored plates. All these duplicates passed as per the 95% Confidence range that we use to check our duplicates on.

95% Confidence Index Observed Lower Upper Count (CFU) Acceptable count 50 32 72 51 33 73 52 75 53 34 76 54 35 77 55 36 78 56 37 79 57 38 80 58 82 59 39 83 60 40 84 61 41 85 62 42 86 63 88 64 43 89 65 44 90 66 45 91 67 46 92 68 47 93 69 95 70 48 96 71 49 97 98 99 74 100

Question 2. What time is acceptable at 121°C, and what temperature deviation is acceptable? Date Prep Date Run Steri Temp Av time at Max temp Steri Time 121 ±5°C Agar pH pH range Positive Negative Blank Logger position 2016/06/14 122°c 12 min at 122°c 15 min Harlequin 7.11 7.0 ± 0.2 Blue colonies Pink colonies No Growth External 2016/06/21 7.15 2016/06/27 7.18 2016/07/04 7.24 2016/07/11 7.20   2016/08/19 123°c 18 min at 123°c 26 min 7.12 2016/08/29 2016/09/05 7.21 2016/09/12 2016/09/20 7.26 2016/09/14 12 min at 123°c 7.25 2016/09/27 2016/10/03 7.23 2016/10/11 7.16 2016/12/01 124.51°c 16 min at 124°c 49 min 7.34 Submerged 7.36 2016/12/07 122° 9 min at 122°c 23 min 7.39 2017/01/19 125.5°c 25 min at 125°c 34 min 7.22 2017/01/20 125.75°c 21 min at 125°c 2017/03/07 123.08°c 7 min at 123°c 16 min 7.17

Date Prep Date Run Steri Temp Av time at Max temp Steri Time 121 ±5°c Agar/Media ph ph range Positive Negative Blank Logger position 2016/06/02 123°c 11 min at 123° 18 min PCA 6.96 7.0 ± 0.2 Pass N/A No Growth External 2016/06/08 6.93 2016/06/15 2016/06/22 2016/06/29 7.00   2016/12/01 124.51°c 16 min at 124°c 49 min 7.12 Submerged 2016/12/7 7.06 2016/12/14 2016/12/06 124.56°c 17 min at 124°c 41 min 7.09 7.08 2017/01/19 125.5°c 25 min at 125°c 34 min 7.03 7.02 2017/01/20 125.75°c 21 min at 125°c 6.92 2017/03/06 2017/03/03 123.79°c 11 min at 123°c 19 min 6.97

Harlequin coliform agar Date Prep Date Run Steri Temp Av time at Max temp Steri Time 121 ±5°c Agar/Media ph ph range Positive Negative Blank Logger position 2016/06/30 123.3°c 11 min at 123°c 22 min PCA 6.89 7.0 ± 0.2 Pass N/A No Growth Submerged 2017/07/03 6.91   123.79°c 20 min 6.97 2017/07/10 6.90 2017/07/13 123.06°c 4 min at 123°c 18 min 6.96 2017/07/18 6.94 2017/07/20 8 min at 123°c 2017/07/25 20min 7.00 2017/06/30 Harlequin coliform agar 7.14 Blue colonies Pink colonies External 7.12 7.21 7.15 7.11 7.16

Decision: Temperature: Looking at historical sterility checks we’ve always been working on a 121°C ±3°C, and our recent data shows that we have been maintaining the required temperature. Sterilization time: The data shows that we have times ranging from 18-30 minutes at 121°C ±3°C, and that the agar is still performing very well under these conditions. No failures were detected on controls or pH’s. Sterilization time is set at 15-25 minutes.

NLA Results Looking at our NLA results, we’ve had very good results over the past couple of years. Agar used in Round 5 2016: Both Harlequin and PCA was prepared on the same day, 2/09/2016 and the NLA was run on 9/09/2016. Temperature: 123.39°C Sterilization time @ 121°C ±3°C: 26 minutes Harlequin pH: 7.11 (7.0 ± 0.2) PCA pH: 6.97 (7.2 ± 0.2)

NLA Results 2016 Round 5