(English captions by Andrea Matsumoto, University of Michigan.) [music]
Step 1. Prepare the smear
Prepare the smear by flaming the loop taking your inoculum from a fresh culture.
After you have put on a drop of sealant on a slide.
Take a culture, dip part of the inoculum in the culture.
Take the culture and moistify it on the slide to join with the rest of the sealant to make
a smear of about two to three centimeters.
Step 2. Gently heat fix the slide (after it has air-dried.)
So, after air-drying, heat fix the smear by passing it through the flame about three or
four times.
This makes the organism to die or kill the organism.
Allows you to stick onto the slide and then prevents autolysis and makes the organism
permeable to the stain.
Step 3. Flood the smear with crystal violet
Flood the smear with crystal violet for one minute.
Allow it to stain for 60 seconds.
Tip off the excess crystal violet stain and wash under gentle tap water.
Step 4. Flood the slide with Lugol's iodine solution
Cover the smear with Lugol's iodine for one minute.
Allow it to stain for 60 seconds.
Wash off the excess Lugol's iodine stain with gentle tap water.
Step 5. Decolorize briefly with acetone.
Decolorize the smear with acetone for ten seconds.
Tip off the excess alcohol and wash under tap water, gentle tap water.
Step 6. Counterstain with neutral red (or safranin).
Use neutral red to counter-stain for one minute.
Cover the smear with the neutral red.
Allow it to stain for 60 seconds.
Wash off the excess neutral red under tap water.
And blot it dry with a filter paper or a blotting paper.
Don't wipe.
The slide is now ready to be examined microscopically at 100X (oil immersion).
Put oil immersion on the slide where the smear is.
And put the slide under the microscope.
We are using the five hundred objective (500X zoom). Examine the slide.