MICLAB-030 Media Preparation in Microbiology Laboratory

Care must be taken at all times when using the hot plates, hot Distilled Water and dispensing of media powders.  Heatproof gloves and facemasks are available.

1. General Preparation

On receipt of dehydrated media into the Microbiology Laboratory, record the date received clearly on the outside of the container

Distilled Water must be used in the preparation of all media and must be collected on the day of use.

Upon initially opening a container of media the date must be written clearly on the container.

Preparation details are to be recorded in log book.

If the batch is prepared in different vessels, e.g. 20L in two 10L buckets, then each 10 L is considered to be a separate batch.  This is identified in the log book by the use of Lot No. in the batch record.

It is important that each batch remains separate as individual pH, positive control and negative control results will be required.  When labelling, ensure that each lot is identified, as it will be necessary to select the correct lot when entering test details in log book.

Mixing is an important requirement for all media preparation. When preparing media with Tween additional mixing is essential.  All media containing Tween will need to be heated as this will decrease the amount of time required for mixing.

All agar preparations must be soaked in cold distilled water for a minimum of 15 minutes prior to heating.  This allows the agar to swell and absorb the water.  Agars need to be brought to the boil to allow the agar to be dispersed. Burning of agar reduces the effectiveness of the agar ie SDA, clarity reduced.

The media is to be re-constituted according to the manufacturers’ directions on the container (with the exception of those below), have the pH determined and bought to within specification (as outlined in section 2) and volumes sufficient for current testing methods are to be distributed into suitable containers.  These volumes and containers are best determined by observing those specified in individual product and raw material control methods and by referring to Appendices 1 and 2.

1.1.         Peptone Water: Diluent prepared from Peptic Digest of Animal Tissue.
Prepare a 0.1% w/v solution.

Peptone Water + 5% v/v Tween 80
Prepare 0.1% w/v solution to which 5% v/v Tween 80 is added.  Certain control methods specify the addition of glass beads to particular volumes of this solution.

Peptone Water + 1% v/v Tween 80
Prepare 0.1% w/v solution to which 1% v/v Tween 80 is added.  Certain control methods specify the addition of glass beads to particular volumes of this solution.  The temperature of the water on the time of collection has an effect on color of peptone water from batches to batches.

1.2.         Trypticase Soy Broth (TSB) + 0.5% Tween 80
Prepare according to manufacturer’s directions, adding 0.5% v/v Tween 80.  Heating is necessary for dissolving powdered media.  Do not overheat or boil.

TSB + 5% v/v Lecithin
For a solution to contain a 5% v/v Lecithin Supplement, prepare the TSB according to manufacturers’ directions.  When preparing 1 liter of this media, add 50 mL of Amyl Supplement SP430 to 1L of Distilled Water used for reconstituting the media.

If Amyl Supplement is not available, prepare this medium using 0.5% w/v Lecithin (soaked and blended) and 4% w/v Tween 20 instead of Polysorbate.

1.3.         Fluid Thioglycollate Medium (FTM) + 0.5% Tween
Prepare according to manufacturer’s method adding 0.5% v/v Tween 80.  It is critical that this solution be brought to the boil with constant stirring before dispensing.

FTM +0 5% v/v Lecithin
To prepare a solution containing a 5% v/v Lecithin Supplement prepare FTM according to the manufacturer’s instructions.  When preparing 1 litre of this media, add 50 mL of Amyl Supplement SP430 to 1L of Distilled Water used for reconstituting the media and ensure that the media is brought to the boil.

FTM + 10% Tween 80
To obtain a solution of FTM containing 10% Tween 80 prepare FTM according to the manufacturers directions, add the Tween 80 and stir to mix thoroughly, bring to the boil ensuring that both the FTM and Tween are dissolved.

Prior to dispensing into containers ensure that the solutions are thoroughly mixed in order to obtain a uniform content of Tween throughout the solution.

When dispensing into required containers ensure that all bottles are above two‑thirds filled.  If, when this medium is about to be used, more than the top fifth of the content is Pink, heating on a boiling water bath may restore the medium, until this Pink colour is discharged.  This restoration process must only be performed once on each bottle and this must be noted on the bottle and in the log book.

After autoclaving, agitate the bottles whilst cooling to remix the Tween in the medium if necessary, being careful to avoid oxygenating the medium.  Unnecessary agitation of this medium is to be avoided.

1.4.         EE Broth

1.4.1.     Weigh out required mass of powdered media and add appropriate volume of water into a large plastic beaker.  Stir thoroughly to disperse media.

1.4.2.     pH sample.

1.4.3.     Pour into pre-sterilized 100ml glass bottles and fill with 90ml, (just below shoulder of bottle).  Screw caps on loosely.

1.4.4.     Place these filled bottles into approximately 5cm of boiling water in a metal beaker on a hotplate.  Boil for 30 minutes.  Take care when placing bottles into boiling water.

1.4.5.     Remove 100ml bottles from boiling water and place carefully into approximately 5cm of cold water in a plastic tray.  Screw down caps tightly.  Take care when removing bottles from hot water, use heatproof gloves.  Bottles may crack when placed into cold water – these should be discarded.

1.4.6.     pH sample after sterilizing.

1.4.7.     Once bottles are cool, label and incubate as outlined in point 5 below.

1.5.         Tryptone Soy Agar + Supplement
TSA+L+P (TSA + 0.1% w/v Lecithin + 0.7% w/v Polysorbate) or add 5 mL Amyl Supplement SP430 to 100 mL.

1.6.         Reinforced Clostridial Agar + Supplement
RCA+L+P (RCA + 0.1% w/v Lecithin + 0.7% w/v Polysorbate) or add 5 mL Amyl Supplement SP430 to 100 mL.

1.7.         LA + SP430 Supplement
50ml of SP430 Supplement per 1L of Distilled Water.  Bring to the boil.  After autoclaving, gently swirl the bottles to disperse the supplement.

2. pH

Calibrate the pH meter before proceeding any further.

Check the pH of liquid Media and Peptone waters prior to dispensing into bottles.  Use a small sample from the bulk media.  Ensure that the media is cooled to approximately 25°C before testing. Agars do not require a pH measurement.  Adjust if necessary using either 1M NaOH (­) or 1M HCl (¯) to ensure that the pH lies within + 0.2 units of the manufacturer’s stated pH.  The pH ranges for media can be found on the bottles of media.  This result is then entered in the log book.

NOTE: If batches are being put in different autoclave loads and or batches are being made in 2 lots (e.g. 20L as 2 x 10L), ensure that each load has a ph measurement and both 10L buckets has a pH measurement prior to dispensing.

A designated 100ml bottle, (use a 20ml bottle for batches being bottled in 20ml McCartney bottles), for all batches should be labelled as the “pH sample” and autoclaved with the batch.  After autoclaving, this pH sample should be allowed to cool and re-tested to check that pH results are still within specification.

Media outside the specified pH range should not be used in testing.  An OOS investigation is to be conducted under a DR if the media outside of specification needs to be used, this is with approval from the Microbiology Manager or senior Technician.  Comments are to be recorded in the ‘Comments’ section in log book.

3. Pre-Autoclaving Procedure

The 20ml and 100ml bottles are placed in wire trays.  Each tray is labelled with a piece of autoclave tape stating the type of media and expiry date.  Caps are left loose.

Bottles larger than 100ml are placed in wire trays.  Each tray is labelled with a piece of autoclave tape stating the type of media and expiry date.  Caps are left loose and a piece of autoclave tape is placed on each cap.

600ml Schott bottles for use within the sterility rooms are to be placed in wire trays.  Each tray is labelled with a piece of autoclave tape stating the type of media and expiry date.  Caps are over-wrapped with Kraft paper and a piece of autoclave tape.

4. Autoclaving

All bottles of Media and Peptone waters prepared in the Microbiology Laboratory are to be autoclaved within 4 hours of preparation at 121°C with the caps left loose.  The exception to this is EE Broth – refer to point 1.4 for details.

After autoclaving, the bottles are to be removed from the autoclave and the caps tightened.

Note: Media bottles after autoclaving are not to be put straight into the 30úC hot room.

5. Labelling

All batches of media prepared within the Laboratory are given a 3 month expiry date from the date of preparation.  Each bottle should be individually labelled with the automatic labelling gun.  If for some reason the gun is unavailable then the media is to be labelled by hand.  The label should include the Lot number if there is more than 1 lot of media made at the same time.  This is important later when details of Sterility Test Sessions are entered into log book.

6. Charts

The Chart Recording used to monitor the autoclave cycles used in the preparation of media must be checked daily, signed and filed.

7. Positive Control “Stasis” (growth promotion testing)

Each batch of media prepared and each batch of a different autoclave load, requires Stasis tests to be conducted.  It is the responsibility of the person preparing the media to place the required number of media bottles in the Stasis cupboard.  See Appendix 1 for the number of bottles to be set aside and MICLAB 090 for details of Stasis Testing.

Media that does not conform to the stasis requirements is not to be used for testing.  An OOS investigation is to be conducted under a Deviation Report if the media outside of specification needs to be used, this is with approval from the Microbiology Manager or senior Technician.  Comments are to be recorded in the ‘Comments’ section in Log Book.

8. Negative Control Incubation

8.1.         Media and Peptone Water used for Sterility Testing

8.1.1.     All media used for Sterility Testing is required to have 2 bottles from each autoclave load incubated for 14 days.  The temperatures for incubation are as follows:

TSB – 2 bottles at 25°C(22.5°C±2.5°)

FTM – 2 bottles at 30°C (32.5°C±2.5°)

Peptone Water (600ml) – 1 bottle at 25°C and 1 bottle at 30°C.

8.1.2.     The remainder of the batch is to be incubated at 30°C for 5 days.  Media may be used prior to the end of this incubation period providing a sample of the batch remains for the full 14 days and that the stasis results has been completed.

8.1.3.     These incubation completion dates are entered into log book at the time the media is removed from the Hot room.

8.1.4.     The media may be signed “ready to use” after the 14 day incubation period has been completed providing that stasis results have been entered.

8.1.5.     Note: If the Negative control media becomes contaminated, the Microbiology Manger is to be informed and the positive result investigated, under a Deviation Report.  If the negative control is positive and stasis result fails, the media should not be used.  Results should be entered into log book and comments should be made.

8.2.         All other solid and liquid media

8.2.1.     All other media used within the Microbiology Laboratory is required to have 2 bottles from each autoclave load incubated for 14 days at 30°C.  These are labelled with the media type and end of incubation date.  The 14 day completion date is to be written in the Media Preparation Diary, which is to be checked daily by a Microlab Technician.

8.2.2.     The remainder of the batch is to be incubated at 30°C for 5 days.  Media may be used prior to the end of this incubation period providing a sample of the batch remains for the full 14 days and that the stasis results has been completed.

8.2.3.     These incubation completion dates are entered into log book at the time the media is removed from the hot room.

8.2.4.     The media may be signed “ready to use” after the 14 day incubation period has been completed providing that stasis results have been entered.

8.2.5     Note: If the Negative control media becomes contaminated, the Microbiology Manger is to be informed and the positive result investigated, under a DR.  If the negative control is positive and stasis result fails, the media should not be used.  Results should be entered into log book and comments should be made.

9. Recording Batch Details for Media Prepared in the Laboratory

All batches of media that are prepared in the Microbiology Laboratory need to have their preparation details recorded in log book/work book.

Details for media prepared within the Microbiology Laboratory are entered in the appropriate work books or other recording system.

The initial incubation date should be entered at the time of preparing the media and the 5 day and 14 day completion dates at the time when the incubation period has been completed, (NOT at the time of media preparation).

10. Details required for Media purchased ‘ready-made’

10.1.      All media received into the Microbiology Laboratory needs to be recorded in the log book/work book.

10.2.      Pre-poured plates on arrival into the Microbiology Laboratory are to be stored on the shelving in the storage room or in the refrigerator.

10.3.      All ‘ready-made’ media are to be supplied with a Quality Certificate from the supplier.  This is to be stored in the “Quality Control Certificates File”.

10.4.      Pre-poured media is signed “to use” immediately upon receipt of the media as the supplier conducts the relevant growth promotion tests and the goods are supplied with a Certificate of Analysis.

11. Approval of Media/Peptone Water for use

When all relevant Quality Control checks such as pH, Negative control and Positive control details are complete for a batch of Media or Peptone Water it can be approved by a Laboratory Technician for use in conducting testing.

11.1.      NOTE: Care must be taken that the 14 day incubation period is completed and all stasis work has been conducted prior to signing media ready for use.

11.2.      The same person cannot prepare the Media or Peptone Water and then sign for use.  This information is recorded in log book.

11.3.      If a QC check does not meet the specifications, the Microbiology Manager or senior Technician is to be consulted and the batch of Media or Peptone Water is not to be used without their authorization.

12. Storage of Media

Media is stored in the either Storeroom and the temperature of the area is to be monitored daily with a designated thermometer and recorded on to Form 660.

Media is not to be used until it has completed all QC testing and meets the requirements, until then when stored on shelves in either storeroom there should be a Quarantine sign, stating in process QC testing.

13. Checklist for Media Preparation

13.1.      Each morning

Have you:

a. Checked the hot room for media that has completed the 5 day incubation period?

b. Checked the Media Preparation Diary for media that has completed the 14 day incubation period?

c. Entered these end incubation dates into log book?

d. Signed the “ready to use” field, providing that all other details including stasis results have been entered?

13.2.      Media Preparation

Have you:

a. Labelled wire trays?

b. Measured and adjusted the pH prior to filling (for liquid media)?

c. Labelled the required number of bottles for stasis?

d. Labelled a bottle for pH testing after autoclaving (for liquid media)?

e. Labelled 2 bottles from each autoclave load for a 14 day incubation and entered the projected finish date into the Media Preparation Diary?

f. If required – attached autoclave tape to lids or over-wrapped lids with Kraft paper?

g. Entered all details into log book; including the number of bottles filled?

13.3.      After Autoclaving

Have you:

a. Tightened all lids and labelled all bottles?

b. Measured the pH and entered the result into log book?

c. Incubated the batch at the required temperature?

14. Appendix 1 – Media Preparation Table: Broths

KEY:  St = for Sterility Testing; N/S = for Non-Sterile Testing.

Note: Put 2 Bottles at 30^oC for the Media that is not used in Sterility testing.

NOTE: If in the one batch size of media you fill more than one size bottle, then only one set of bottles is required for stasis.

15. Appendix 2 – Media Preparation Table: AGARS

16. Summary of Changes

Daily Store Room Temperatures form