In this video, we will demonstrate how to perform a gram stain.
It is a technique that helps you to identify and differentiate bacteria based on cell wall structure.
Here is what we will need.
Before you begin, always disinfect the lab bench using 70% ethanol
Make sure the air port is closed on the Bunsen burner.
Turn on the gas supply, use the sparker to light the Bunsen burner.
You will see a wild yellow flame.
To control the flame, turn the needle valve at the base, clockwise to increase gas flow
and counterclockwise to decrease gas flow.
You should aim to produce a flame that is approximately 6 inches high.
Adjust the air port until you observe a blue flame of approximately 2 inches high.
Remember to never control the gas flow directly using the gas supply valve.
Prepare the microscope slide near the Bunsen burner to maintain a sterile environment.
Clean the microscope slide using 70% ethanol and wipe dry with kimwipes.
Be careful to hold the slide by the edge.
Next, using the wax pencil,
draw two circles with approximately 1 cm diameter on the microscope slide
and this will be the bottom of the slide.
The circles act to guide you while you observe under the microscope.
Next, sterilize the inoculation loop by flaming it in the blue zone of the flame,
until a red-orange color is observed on the loop and a segment of the wire.
Hold the loop to cool for about a minute.
To obtain a sample, carefully unscrew the cap from the screw cap tube,
flame the top of the tube, insert the loop to obtain a sample,
flame the top of the tube and put the cap back on.
Apply the sample on the microscope slide in a thin smear to allow the slide to dry quickly.
If your sample solution does not spread easily,
you may need to add another drop of water to mix with the sample.
Leave the slide air dry.
Sterilize the inoculation loop by flaming it before putting back on the test tube rack.
When the microscope slide is dry, use the wooden clip to hold the slide,
sample side up, and gently heat fix over the flame three times.
Do not overheat as you may rupture the bacteria.
Turn off the Bunsen burner by turning off the main gas valve.
The gram stain station is set up by the sink area.
Put your heat fixed slide on the gram stain tray slide holder.
Add crystal violet solution to the slide and wait for a minute.
Rinse the slide gently with ddwater.
Please note that you do not want to wash off your samples.
Next, add gram iodine solution to the slide and wait for a minute.
Then, rinse the slide gently with ddwater.
Add the decolorizer dropwise for 10 seconds or until the runoff is clear.
Rinse the slide gently with ddwater, add safranin as a counterstain to the slide, and wait for 30 seconds.
Finally, rinse the slide gently with ddwater and let air dry on a paper towel.
When the slide is dry, you may set up the slide to observe under the microscope.