Q1 What other elution buffer can I use?
This is the filter elution buffer that we recommend instead of the Laureth 12.
1. 2000 mL Phosphate buffered saline (PBS)
2. 300 μL Tween 80
3. 300uL Antifoam B
1. Add 100 mls of PBS Concentrate ( X20 ) pH 7.4 @ 25 oC to 1900 mls of distilled water
2. Add the Antifoam B and Tween 80 (Use 1 ml disposable syringe for accurate measurement)
3. Mix well
4. Label Envirocheck Buffer and date
Q2 The morphology or the DAPI fluorescence of the Cryptosporidium and Giardia in samples that have been seeded with ColorSeed are not as good as the positive control in the EasyStain kit. Why is this?
The Cryptosporidium and Giardia can become damaged during the sample processing, particularly the acid dissociation in combination with drying the sample onto the slide. The positive control from the EasyStain™ kit does not go through the IMS and acid dissociation, therefore it is likely to appear less damaged. Tips on how to minimise the effects of the slide drying and acid dissociation on Cryptosporidium and Giardia staining can be found in the EasyStain™ FAQ.
Q3 Can ColorSeed be used as source of DNA for PCR assays?
The dose of gamma radiation that is used to inactivate the cysts and oocysts in ColorSeed™ is small and is unlikely to damage the DNA sufficiently to interfere with PCR analysis.The effect if any would also depend on the target of interest. The radiation is unlikely to affect results for a multicopy target. But If the PCR is detecting a single copy target or a labile target such as rRNA or mRNA then it is probably likely that ColorSeed™ may behave differently to fresh cysts and oocysts. We have no data on how stable the DNA is within the oocysts and cysts once they have been irradiated.
We do not have enough information or experience with the actual use of these products in PCR assays to provide more detailed information.