How to Prepare a Gram Stain

This show video demonstrates how to prepare a gram stain. This resource was developed by Charles Adjei Osei of the Kwame Nkrumah University of Science and Technology and Cary Engleberg of the University of Michigan. It is part of a larger learning module about laboratory methods for clinical microbiology. The full learning module, editable animation, and video transcript are available at http://open.umich.edu/education/med/oernetwork/med/microbiology/clinical-microbio-lab/2009. Copyright 2009-2010, Kwame Nkrumah University of Science and Technology and Cary Engleberg. This is licensed under a Creative Commons Attribution Noncommercial 3.0 License http://creativecommons.org/licenses/by-nc/3.0/. Video transcribed by Andrea Matsumoto (University of Michigan).

(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.