Poor staining of Cryptosporidium oocysts occurs when the antibody does not adequately fix to the surface of the oocysts. To optimize Cryptosporidium staining with EasyStain™, ensure that you:
- Use the Fixing Buffer that is included in the EasyStain™ kit.
- Use the Fixing Buffer directly from the refrigerator or place on ice whilst using. The Fixing Buffer must be applied to the slide cold.
- Leave the Fixing Buffer on the slide for at least 2 minutes prior to washing off.
- Only use the Mounting Medium included in the EasyStain™ kit. The Mounting Medium has been optimized to reduce fading of the EasyStain™ antibody.
Inefficient neutralization of the acid used in the IMS procedure can damage the cell surface of the Giardia cysts, causing variable staining of Giardia. Drying the samples rapidly or keeping the sample cold ensures that the surface of the Giardia cysts is not damaged. To optimize this process, ensure that:
- Once the sample has been neutralized, dry the slide rapidly at 37oC (maximum drying time of one hour) and then stain immediately.
- If there will be a delay in staining after IMS neutralization:
- Dry the slide rapidly at 37oC and then place in a refrigerator overnight. Stain the following day.
- Alternatively, dry the slide (uncovered) in a standard household refrigerator overnight at 4oC. Slides dried in a cool room may not dry as some cool rooms maintain a humid environment.
Methanol fixation assists permeabilization of Cryptosporidium and Giardia prior to DAPI staining. Some customers have reported that the methanol fixation step improves DAPI staining. Others report Cryptosporidium and Giardia appearing crumpled under the microscope and a minor detrimental effect on FITC staining.
The methanol fixation step of the EasyStain™ protocol can be omitted at the discretion of the laboratory.
FAQ list - last updated October 2008