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Technical FAQ's
Answer:
We would not recommend the use of a microwave for sterilising or re-melting culture media containing agar. The is due to the following:
We would not recommend the use of a microwave for sterilising or re-melting culture media containing agar. The is due to the following:
- Heating may be uneven across the total volume of the medium.
- Hot spots may occur in the vessel containing the medium leading to superheating and potential explosion of the container.
Answer:
We do not recommend the storage of reconstituted supplements as the quality of the supplement will begin to deteriorate from the time of preparation.
We do not recommend the storage of reconstituted supplements as the quality of the supplement will begin to deteriorate from the time of preparation.
Answer:
You should test the pH of the prepared medium when the medium is in its final form and this should be conducted at ambient temperature (25°C).
The pH meter should be sensitive to 1 decimal place. The electrode should be a gel filled, unbreakable, combination electrode with a detachable shield for cleaning. Flat electrodes may be used, but some problems have been reported by users.
The pH value should fall within the range given on the product label. When the medium is supplemented with a selective or additional reagent, the pH limits apply to the complete medium.
An acceptable pH variation of ± 0.2 is usual. This assumes a 1 litre volume produced in strict accordance with the manufacturer's instructions.
Adjustment of the pH of the medium should not be necessary if all systems are correct and the preparation process is in strict accordance with the manufacturer's instructions.
You should test the pH of the prepared medium when the medium is in its final form and this should be conducted at ambient temperature (25°C).
The pH meter should be sensitive to 1 decimal place. The electrode should be a gel filled, unbreakable, combination electrode with a detachable shield for cleaning. Flat electrodes may be used, but some problems have been reported by users.
The pH value should fall within the range given on the product label. When the medium is supplemented with a selective or additional reagent, the pH limits apply to the complete medium.
An acceptable pH variation of ± 0.2 is usual. This assumes a 1 litre volume produced in strict accordance with the manufacturer's instructions.
Adjustment of the pH of the medium should not be necessary if all systems are correct and the preparation process is in strict accordance with the manufacturer's instructions.
Answer:
The quality of water used to reconstitute dehydrated culture media can affect the performance of the medium being prepared.
Fresh, high quality water prepared by distillation, de-ionisation, or reverse osmosis is recommended. Tap water should not be used as it will contain impurities such as calcium and magnesium and their metal ion traces. In addition chlorine and fluorine used to treat potable water may alter the characteristics of selective media.
The quality of water used to reconstitute dehydrated culture media can affect the performance of the medium being prepared.
Fresh, high quality water prepared by distillation, de-ionisation, or reverse osmosis is recommended. Tap water should not be used as it will contain impurities such as calcium and magnesium and their metal ion traces. In addition chlorine and fluorine used to treat potable water may alter the characteristics of selective media.
Answer:
Plates: most plates, stored medium side up at 4°C in the dark will have a minimum life of 7 days. This can be extended up to 3-4 weeks for simple nutrient media by using some form of airtight packing. Plates containing antibiotics have a shelf life governed by the stability of the antibiotics. Generally speaking it is unwise to extend the shelf life of an antibiotic-containing medium beyond 7 days. As a medium loses moisture the ingredients of the medium will be concentrated making selective media progressively more selective. A plate with an original medium depth of 5mm will have its ingredients concentrated 20% by the time its gel has shrunk to a depth of 4 mm. Plates showing visible signs of shrinkage (drying) should not be used. Plates should be brought up to room temperature before use to avoid any ‘thermal’ shock to the bacteria. Any plates left on the bench for more than 8 hours should be discarded as unsuitable for use.
Bottled media: any medium in an airtight capped container will have a longer shelf life than in a plate. Many simple nutrient media can be stored at 15-20°C for 3 months in the dark. Indeed, many can be stored for longer but we advise repeat of a simple QC procedure after 3 months. If a medium is stored as a gel and then is re-heated before use, repeat of a simple QC procedure is recommended. Bottled media containing antibiotics will have shelf lives governed by the activity of the antibiotic.
Plates: most plates, stored medium side up at 4°C in the dark will have a minimum life of 7 days. This can be extended up to 3-4 weeks for simple nutrient media by using some form of airtight packing. Plates containing antibiotics have a shelf life governed by the stability of the antibiotics. Generally speaking it is unwise to extend the shelf life of an antibiotic-containing medium beyond 7 days. As a medium loses moisture the ingredients of the medium will be concentrated making selective media progressively more selective. A plate with an original medium depth of 5mm will have its ingredients concentrated 20% by the time its gel has shrunk to a depth of 4 mm. Plates showing visible signs of shrinkage (drying) should not be used. Plates should be brought up to room temperature before use to avoid any ‘thermal’ shock to the bacteria. Any plates left on the bench for more than 8 hours should be discarded as unsuitable for use.
Bottled media: any medium in an airtight capped container will have a longer shelf life than in a plate. Many simple nutrient media can be stored at 15-20°C for 3 months in the dark. Indeed, many can be stored for longer but we advise repeat of a simple QC procedure after 3 months. If a medium is stored as a gel and then is re-heated before use, repeat of a simple QC procedure is recommended. Bottled media containing antibiotics will have shelf lives governed by the activity of the antibiotic.
Answer:
The laboratory that routinely quality controls the media it produces will occasionally find that it has produced a batch that is not up to standard. The following is a list of potential problems and their possible causes:
The laboratory that routinely quality controls the media it produces will occasionally find that it has produced a batch that is not up to standard. The following is a list of potential problems and their possible causes:
| Soft gel | Excess heat, pH too low causing acid hydrolysis, inaccurate weighing, inadequate mixing, agar not dissolved. |
| pH incorrect | Contaminated glassware, impure water, overheating, chemical contamination, pH taken at wrong temperature, pH equipment faulty or poorly standardised, deterioration of dehydrated medium. |
| Abnormal colour | Impure water, dirty glassware, deterioration of dehydrated medium, excess heat, pH wrong. |
| Darkening | Excess heat, deterioration of dehydrated medium. |
| Precipitation | Excess heat, deterioration of dehydrated medium, impure water or glassware. |
| Toxicity | Excess heat (scorching or burning), deterioration of dehydrated medium. |
| Poor growth | Contaminated water or glassware, deterioration of dehydrated medium, incorrect weighing and mixing, excess heat. |
| Poor selective or differential properties | Contaminated water or glassware, incorrect weighing and mixing, deterioration of dehydrated medium, excess heat. |
