agar sterilization time & temperature and expiry dates for ... · autoclave at 121°c for...
TRANSCRIPT
Agar sterilization time, temperature and expiry dates for prepared mediaJanine 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 controls2. pH3. Sterilities• to try and establish a suitable time media can be stored.
LAB M Harlequin e.coli/coliform Medium1. Soak for 10min, swirl to mix.2. Autoclave at 121°c for 15min.3. pH7.2 ±0.2
Biolab Plate Count Agar for HPCs1. 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
1. Soak for 10min, swirl to mix.2. Autoclave at 121°C for 15min.3. pH7.2 ±0.2
Date Prep Date RunSteriTemp
Av time at Max temp
Steri Time 121 ±5°c Agar/Media pH
pH range Positive Negative Blank
Logger position
2016/09/14 2016/09/14123°c12 min at 123°C 26 min Harlequin 7.257.0 ± 0.2
Blue colonies Pink colonies
No Growth External
2016/09/14 2016/09/20123°c12 min at 123°C 26 min Harlequin 7.257.0 ± 0.2
Blue colonies Pink colonies
No Growth External
2016/09/14 2016/09/27123°c12 min at 123°C 26 min Harlequin 7.217.0 ± 0.2
Blue colonies Pink colonies
No Growth External
2016/09/14 2016/10/03123°c12 min at 123°C 26 min Harlequin 7.237.0 ± 0.2
Blue colonies Pink colonies
No Growth External
2016/09/14 2016/10/11123°c12 min at 123°C 26 min Harlequin 7.167.0 ± 0.2
Blue colonies Pink colonies
No Growth External
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 RunSteriTemp
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°C11 min at 123°C 18 min PCA 6.96 7.0 ± 0.2 Pass
No Growth External
2016/06/02 2016/06/08 123°C11 min at 123°C 18 min PCA 6.93 7.0 ± 0.2 Pass
No Growth External
2016/06/02 2016/06/15 123°C11 min at 123°C 18 min PCA 6.96 7.0 ± 0.2 Pass
No Growth External
2016/06/02 2016/06/22 123°C11 min at 123°C 18 min PCA 6.96 7.0 ± 0.2 Pass
No Growth External
2016/06/02 2016/06/27 123°C11 min at 123°C 18 min PCA 7.00 7.0 ± 0.2 Pass
No Growth External
• 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 Date Dupl 1 Dupl 2QC
IndicatorAnalyst/comments
UWL UAL
08-May-17 68 81 ACCEPT 8 12 ACCEPT 49.35 75.0 100.0
09-May-17 128 147 ACCEPT 3 2 ACCEPT 25.00 75.0 100.0
10-May-17 204 228 ACCEPT 200 200 ACCEPT 0.00 75.0 100.0
11-May-17 88 94 ACCEPT 200 200 ACCEPT 0.00 75.0 100.0
12-May-17 68 54 ACCEPT 79 91 ACCEPT 37.84 75.0 100.0
13-May-17 ACCEPT ACCEPT - 75.0 100.0
14-May-17 ACCEPT ACCEPT - 75.0 100.0
15-May-17 62 78 ACCEPT 78 98 ACCEPT 61.24 75.0 100.0
16-May-17 78 84 ACCEPT 65 65 ACCEPT 0.00 75.0 100.0
17-May-17 200 200 ACCEPT 200 200 ACCEPT 0.00 75.0 100.0
18-May-17 120 111 ACCEPT 20 25 ACCEPT 35.90 75.0 100.0
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 >200 #VALUE!
Date Dupl 1 Dupl 2
20-Sep-16 >200 >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-161 2
ACCEPT
Date Dupl 1 Dupl 2
27-Sep-16121 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-16236 203
ACCEPT
Date Dupl 1 Dupl 2
03-Oct-169 8
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 >200
ACCEPT
Date Dupl 1 Dupl 2
11-Oct-16>200 >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-161 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 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 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 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 IndexObserved Lower Upper
Count (CFU) Acceptable count Acceptable count
50 32 72
51 33 73
52 33 75
53 34 76
54 35 77
55 36 78
56 37 79
57 38 80
58 38 82
59 39 83
60 40 84
61 41 85
62 42 86
63 42 88
64 43 89
65 44 90
66 45 91
67 46 92
68 47 93
69 47 95
70 48 96
71 49 97
72 50 98
73 51 99
74 52 100
Question 2. • What time is acceptable at 121°C, and what temperature deviation is
acceptable?
Date Prep Date Run Steri TempAv time at Max temp
Steri Time 121 ±5°C Agar pH pH range Positive Negative Blank Logger position
2016/06/14 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 External2016/06/14 2016/06/21 122°c 12 min at 122°c 15 min Harlequin 7.15 7.0 ± 0.2 Blue colonies Pink colonies No Growth External
2016/06/14 2016/06/27 122°c 12 min at 122°c 15 min Harlequin 7.18 7.0 ± 0.2 Blue colonies Pink colonies No Growth External2016/06/14 2016/07/04 122°c 12 min at 122°c 15 min Harlequin 7.24 7.0 ± 0.2 Blue colonies Pink colonies No Growth External
2016/06/14 2016/07/11 122°c 12 min at 122°c 15 min Harlequin 7.20 7.0 ± 0.2 Blue colonies Pink colonies No Growth External
2016/08/19 2016/08/19 123°c 18 min at 123°c 26 min Harlequin 7.12 7.0 ± 0.2 Blue colonies Pink colonies No Growth External2016/08/19 2016/08/29 123°c 18 min at 123°c 26 min Harlequin 7.15 7.0 ± 0.2 Blue colonies Pink colonies No Growth External
2016/08/19 2016/09/05 123°c 18 min at 123°c 26 min Harlequin 7.21 7.0 ± 0.2 Blue colonies Pink colonies No Growth External
2016/08/19 2016/09/12 123°c 18 min at 123°c 26 min Harlequin 7.18 7.0 ± 0.2 Blue colonies Pink colonies No Growth External2016/08/19 2016/09/20 123°c 18 min at 123°c 26 min Harlequin 7.26 7.0 ± 0.2 Blue colonies Pink colonies No Growth External
2016/09/14 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
2016/09/14 2016/09/20 123°c 12 min at 123°c 26 min Harlequin 7.25 7.0 ± 0.2 Blue colonies Pink colonies No Growth External
2016/09/14 2016/09/27 123°c 12 min at 123°c 26 min Harlequin 7.21 7.0 ± 0.2 Blue colonies Pink colonies No Growth External2016/09/14 2016/10/03 123°c 12 min at 123°c 26 min Harlequin 7.23 7.0 ± 0.2 Blue colonies Pink colonies No Growth External
2016/09/14 2016/10/11 123°c 12 min at 123°c 26 min Harlequin 7.16 7.0 ± 0.2 Blue colonies Pink colonies No Growth External
2016/12/01 2016/12/01 124.51°c 16 min at 124°c 49 min Harlequin 7.34 7.0 ± 0.2 Blue colonies Pink colonies No Growth Submerged
2016/12/01 2016/12/01 124.51°c 16 min at 124°c 49 min Harlequin 7.36 7.0 ± 0.2 Blue colonies Pink colonies No Growth Submerged
2016/12/07 2016/12/07 122° 9 min at 122°c 23 min Harlequin 7.39 7.0 ± 0.2 Blue colonies Pink colonies No Growth Submerged
2016/12/07 2016/12/07 122° 9 min at 122°c 23 min Harlequin 7.36 7.0 ± 0.2 Blue colonies Pink colonies No Growth Submerged
2017/01/19 2017/01/19 125.5°c 25 min at 125°c 34 min Harlequin 7.23 7.0 ± 0.2 Blue colonies Pink colonies No Growth Submerged
2017/01/19 2017/01/19 125.5°c 25 min at 125°c 34 min Harlequin 7.22 7.0 ± 0.2 Blue colonies Pink colonies No Growth Submerged
2017/01/20 2017/01/20 125.75°c 21 min at 125°c 34 min Harlequin 7.21 7.0 ± 0.2 Blue colonies Pink colonies No Growth Submerged
2017/01/20 2017/01/20 125.75°c 21 min at 125°c 34 min Harlequin 7.21 7.0 ± 0.2 Blue colonies Pink colonies No Growth Submerged
2017/03/07 2017/03/07 123.08°c 7 min at 123°c 16 min Harlequin 7.18 7.0 ± 0.2 Blue colonies Pink colonies No Growth Submerged
2017/03/07 2017/03/07 123.08°c 7 min at 123°c 16 min Harlequin 7.17 7.0 ± 0.2 Blue colonies Pink colonies No Growth Submerged
Date Prep Date RunSteri
TempAv time at Max
tempSteri Time 121
±5°c Agar/Media ph ph range Positive Negative BlankLogger
position
2016/06/02 2016/06/02 123°c 11 min at 123° 18 min PCA 6.96 7.0 ± 0.2 Pass N/ANo
Growth External
2016/06/02 2016/06/08 123°c 11 min at 123° 18 min PCA 6.93 7.0 ± 0.2 Pass N/ANo
Growth External
2016/06/02 2016/06/15 123°c 11 min at 123° 18 min PCA 6.96 7.0 ± 0.2 Pass N/ANo
Growth External
2016/06/02 2016/06/22 123°c 11 min at 123° 18 min PCA 6.96 7.0 ± 0.2 Pass N/ANo
Growth External
2016/06/02 2016/06/29 123°c 11 min at 123° 18 min PCA 7.00 7.0 ± 0.2 Pass N/ANo
Growth External
2016/12/01 2016/12/01 124.51°c 16 min at 124°c 49 min PCA 7.12 7.0 ± 0.2 Pass N/ANo
Growth Submerged
2016/12/01 2016/12/7 124.51°c 16 min at 124°c 49 min PCA 7.06 7.0 ± 0.2 Pass N/ANo
Growth Submerged
2016/12/01 2016/12/14 124.51°c 16 min at 124°c 49 min PCA 7.00 7.0 ± 0.2 Pass N/ANo
Growth Submerged
2016/12/06 2016/12/06 124.56°c 17 min at 124°c 41 min PCA 7.09 7.0 ± 0.2 Pass N/ANo
Growth Submerged
2016/12/06 2016/12/06 124.56°c 17 min at 124°c 41 min PCA 7.08 7.0 ± 0.2 Pass N/ANo
Growth Submerged
2016/12/06 2016/12/06 124.56°c 17 min at 124°c 41 min PCA 7.09 7.0 ± 0.2 Pass N/ANo
Growth Submerged
2017/01/19 2017/01/19 125.5°c 25 min at 125°c 34 min PCA 7.03 7.0 ± 0.2 Pass N/ANo
Growth Submerged
2017/01/19 2017/01/19 125.5°c 25 min at 125°c 34 min PCA 6.93 7.0 ± 0.2 Pass N/ANo
Growth Submerged
2017/01/19 2017/01/19 125.5°c 25 min at 125°c 34 min PCA 7.02 7.0 ± 0.2 Pass N/ANo
Growth Submerged
2017/01/20 2017/01/20 125.75°c 21 min at 125°c 34 min PCA 6.92 7.0 ± 0.2 Pass N/ANo
Growth Submerged
2017/01/20 2017/01/20 125.75°c 21 min at 125°c 34 min PCA 6.93 7.0 ± 0.2 Pass N/ANo
Growth Submerged
2017/03/06 2017/03/03 123.79°c 11 min at 123°c 19 min PCA 6.97 7.0 ± 0.2 Pass N/ANo
Growth Submerged
2017/03/06 2017/03/03 123.79°c 11 min at 123°c 19 min PCA 6.97 7.0 ± 0.2 Pass N/ANo
Growth Submerged
Date Prep Date RunSteriTemp
Av time at Max temp
Steri Time 121 ±5°c Agar/Media ph ph range Positive Negative Blank
Logger position
2016/06/30 2016/06/30 123.3°c 11 min at 123°c 22 min PCA 6.89 7.0 ± 0.2 Pass N/ANo
Growth Submerged
2016/06/30 2017/07/03 123.3°c 11 min at 123°c 22 min PCA 6.91 7.0 ± 0.2 Pass N/ANo
Growth Submerged
2017/07/03 2017/07/03 123.79°c 11 min at 123°c 20 min PCA 6.97 7.0 ± 0.2 Pass N/ANo
Growth Submerged
2017/07/03 2017/07/10 123.79°c 11 min at 123°c 20 min PCA 6.90 7.0 ± 0.2 Pass N/ANo
Growth Submerged
2017/07/13 2017/07/13 123.06°c 4 min at 123°c 18 min PCA 6.96 7.0 ± 0.2 Pass N/ANo
Growth Submerged
2017/07/13 2017/07/18 123.06°c 4 min at 123°c 18 min PCA 6.94 7.0 ± 0.2 Pass N/ANo
Growth Submerged
2017/07/20 2017/07/20 123.3°c 8 min at 123°c 20 min PCA 6.89 7.0 ± 0.2 Pass N/ANo
Growth Submerged
2017/07/20 2017/07/25 123.3°c 8 min at 123°c 20min PCA 7.00 7.0 ± 0.2 Pass N/ANo
Growth Submerged
2017/06/30 2017/06/30 123.3°c 11 min at 123°c 22 minHarlequin coliform
agar 7.14 7.0 ± 0.2Blue
coloniesPink
coloniesNo
Growth External
2017/06/30 2017/07/03 123.3°c 11 min at 123°c 22 minHarlequin coliform
agar 7.12 7.0 ± 0.2Blue
coloniesPink
coloniesNo
Growth External
2017/07/03 2017/07/03 123.79°c 11 min at 123°c 22 minHarlequin coliform
agar 7.12 7.0 ± 0.2Blue
coloniesPink
coloniesNo
Growth External
2017/07/03 2017/07/10 123.79°c 11 min at 123°c 22 minHarlequin coliform
agar 7.21 7.0 ± 0.2Blue
coloniesPink
coloniesNo
Growth External
2017/07/13 2017/07/13 123.06°c 4 min at 123°c 18 minHarlequin coliform
agar 7.15 7.0 ± 0.2Blue
coloniesPink
coloniesNo
Growth External
2017/07/13 2017/07/18 123.06°c 4 min at 123°c 18 minHarlequin coliform
agar 7.11 7.0 ± 0.2Blue
coloniesPink
coloniesNo
Growth External
2017/07/20 2017/07/20 123.3°c 8 min at 123°c 20 minHarlequin coliform
agar 7.16 7.0 ± 0.2Blue
coloniesPink
coloniesNo
Growth External
2017/07/20 2017/07/25 123.3°c 8 min at 123°c 20 minHarlequin coliform
agar 7.15 7.0 ± 0.2Blue
coloniesPink
coloniesNo
Growth External
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)