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)

NLA Results 2016 Round 5