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(English captions by Andrea Matsumoto, University of Michigan.) [music]

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Step 1. Prepare the smear

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Prepare the smear by flaming the loop taking[br]your inoculum from a fresh culture.

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After you have put on a drop of sealant on[br]a slide.

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Take a culture, dip part of the inoculum [br]in the culture.

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Take the culture and moistify it on the slide[br]to join with the rest of the sealant to make

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a smear of about two to three centimeters.

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Step 2. Gently heat fix the slide (after it[br]has air-dried.)

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So, after air-drying, heat fix the smear by[br]passing it through the flame about three or

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four times.

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This makes the organism to die or kill the[br]organism.

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Allows you to stick onto the slide and then[br]prevents autolysis and makes the organism

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permeable to the stain.

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Step 3. Flood the smear with crystal violet

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Flood the smear with crystal violet for one[br]minute.

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Allow it to stain for 60 seconds.

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Tip off the excess crystal violet stain and[br]wash under gentle tap water.

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Step 4. Flood the slide with Lugol's iodine[br]solution

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Cover the smear with Lugol's iodine for[br]one minute.

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Allow it to stain for 60 seconds.

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Wash off the excess Lugol's iodine stain[br]with gentle tap water.

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Step 5. Decolorize briefly with acetone.

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Decolorize the smear with acetone for ten[br]seconds.

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Tip off the excess alcohol and wash under[br]tap water, gentle tap water.

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Step 6. Counterstain with neutral red (or[br]safranin).

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Use neutral red to counter-stain for one minute.

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Cover the smear with the neutral red.

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Allow it to stain for 60 seconds.

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Wash off the excess neutral red under tap[br]water.

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And blot it dry with a filter paper or a blotting[br]paper.

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Don't wipe.

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The slide is now ready to be examined microscopically[br]at 100X (oil immersion).

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Put oil immersion on the slide where the smear[br]is.

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And put the slide under the microscope.

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We are using the five hundred objective (500X[br]zoom). Examine the slide.