Stain Process:
1. Match Tag with info on data sheet- write on tag- slides prepared by and date
2. Clean 10 Slides, Mark 1-10 with wax pen
-the date examined is the date used in data entry
-make note on data sheet of yellowing or brown tissue
3. Cut absorbent pads for all ten slides, place absorbent pads onto cleaned slides.
Slide 1
Absorbent pad
4. Sterilize needle
–light flame, sterilize between each tissue handling
5. Drop tissue onto napkin to sterilize medium
6. Sterilize
7. Repeat step 5, ten times, make sure all tubes stay in order
8. Get ALL tissue out of tubes
–clear= antibiotics have done their job
–Oysters that have been out of water longer will have more bacterial growth and the medium
will be clouded –too much medium discolors iodine, that’s the reason we dry with absorbent
towel
TIPS: Make note of tissue that is not completely immersed in medium, always test best available
tissue,
9. Label Petri Dishes, change data cards as needed. (abbreviation, and slide numbers ex: AB-1
16,17,18)
10. Moisten the moist chamber within Petri dish with water, repeat twice, and make sure Petri
dishes stay in order.
Moist chamber, square of folded
paper towel
11.
12.
13.
14.
Clean Teasing Needles
Decided which stain to use, NEVER USE MORE THAN TWO DROPS
Tease sample across tissue to dry
Place tissue in the middle of slide after removing absorbent pad
15. Stain
-50% lugol solution
–oysters are thin in watery in fall and the bugs are harder to see, so more stain may be needed.
16. “Tease” stain with iodine to get iodine to soak in
–Keep in small area –break up (makes easier for iodine to penetrate)
–tissue must have light brown straw color when done with teasing
17. Wipe Down Teasing Needles and repeat for each slide
–Those tissue samples that are heavily infected with bugs will have a blue black tissue
spots(mostly in summer)
–some tissue may dry on towel during this process, before you can remove, so use drop of deionized water to remove
18. Check Lightness- may need to stain more, move to fridge
19. Each Petri dish will contain 3 slides, last will contain 4
20. Move to fridge at least overnight
21. Open Petri dishes, and let dry, if needed restain
22. Read
Read publication: Technique reference: R ay,S.M. 1966. A review of the culture methods for detecting
Dermocystidium marinum, with suggested modifications and precautions. Proc. Natl. Shellfish. Assoc.
54:55-59.