Simple Stain Procedure:
1. Clean and dry microscope slides thoroughly.
2. Flame the surface in which the smear
is to be spread.
3. Flame the inoculating loop.
4. Transfer a loop full of tap water to
the flamed slide surface.
5. Reflame the loop making sure the entire
length of the wire that will enter the tube has been heated to redness
6. Remove the tube cap with the fingers
of the hand holding the loop.
7. Flame the tube mouth.
8. Touch the inoculating loop to the inside
of the tube to make sure it is not so hot that it will distort the
then pick up a pinhead size sample of the bacterial growth without digging
into the agar.
9. Reflame the tube mouth, replace the can,
and put the tube back in the holder.
10. Disperse the bacteria on the loop in the drop
of water on the slide and spread the drop over an area the size
of a dime. It should
be a thin, even smear.
11. Reflame the inoculating loop to redness including
the entire length that entered the tube.
12. Allow the smear to dry thoroughly.
13. Heat-fix the smear cautiously by passing the
underside of the slide through the burner flame two or three
times. Test the
temperature of the slide after each pass against the back of the hand.
It has been heated
sufficiently when it feels
hot but can still be held against the skin for several seconds. Overheating
distort the cells.
14. Stain the smear by flooding it with one of
the staining solutions and allowing it to remain covered with the
stain for the time designated
Methylene blue - 1 minute
Crystal violet - 30 seconds
Carbol fuchsin - 20 seconds
During the staining the
slide may be placed on the rack or held in the fingers.
15. At the end of the designated time rinse off
the excess stain with gently running tap water. Rinse thoroughly.
16. Wipe the back of the slide and blot the stained
surface with bibulous paper or with a paper towel.
17. Place the stained smear on the microscope
stage smear side up and focus the smear using the 10X objective.
18. Choose an area of the smear in which the cells
are well spread in a monolayer. Center the area to be studied,
apply oil directly to
the smear, and focus the smear under oil with the 100X objective.
19. Draw the cells observed.
Go To Results of Simple Stain