Good Morning! Please pick up the yellow Gram- Staining Lab worksheets from the overhead cart. Grab some goggles and a lab apron (in box underneath goggles). Start reading the introduction and the procedure.

Today’s Objective: We’re going to be gram-staining today! I didn’t get to do this until my college microbiology lab, so you guys are very lucky. This lab experience will be very helpful if you plan on taking AP Biology or become a science major in college.

Introduction to Gram-stains In 1884, Hans Christian Gram, a Danish doctor accidentally stumbled on a method that differentiates bacteria. While examining lung tissue from patients who had died of pneumonia, he discovered that certain stains were preferentially taken up and retained by bacterial cells. Gram developed a staining procedure which divided almost all bacteria into two large groups - the Gram stain.

Why is this important? Bacterial cells without a stain are almost transparent. Adding a stain to these cells helps us see the bacteria much better than just looking at them without a stain. Gram-staining also helps us differentiate types of bacteria. Knowing whether they are gram-positive or gram-negative gives us information about their cell walls. Gram-positive: thicker layer of peptidoglycan Gram-negative: thin layer of peptidoglycan Peptidoglycan gives the cell structural strength

Gram-Staining in the Real World Gram-staining used in hospitals and clinics when trying to diagnose infections. Some antibiotics target specific types of bacteria. For example, penicillin only affects gram positive bacteria. Knowing whether the infection is caused by a gram positive or gram negative bacteria can help the doctor determine the best antibiotic to prescribe. Method is also used in scientific research.

Gram-Staining Procedure http://www.youtube.com/watch?v=syJTy 8C_BG0

Gram Positive: purple Gram Negative: pink Your product: Gram Positive: purple Gram Negative: pink

Station 1: Making a Bacterial Smear Step 1 on the lab procedure, station is at the back of the classroom. Write down your bacteria on your paper. Get a microscope slide and place one drop of water on the slide. Choose a type of bacteria and open the vial. Take inoculating loop and scrape some of the bacteria from the vial onto the loop. Do not pierce into the agar. Spread the bacteria on your loop into the water droplet. You do not need to cover the entire slide. Heat fixing the bacteria kills the bacterial cells.

Station 2: Crystal Violet Step 2 on the lab procedure. Station is next to the pencil sharpener. Place 1 drop of crystal violet on the slide. Let it sit for one minute and then wash with water from the sink. Tilt slide away from you so you don’t get any of the stain on your hands. Crystal violet is the primary stain in this procedure. If the cell is gram positive it will take in this stain.

Station 3: Iodine Step 3 on your lab procedure. Station is located next to the sink closest to your grades. Place 1-2 drops of iodine on the smear. Wait one minute then wash the slide. Iodine is called a mordant in this procedure. This means it allows the stain to stick very well to the bacterial cells.

Station 4: Alcohol Step 4 on your lab procedure. Located near the goggles. Tilt slide at 45 degree angle away from you. Add alcohol onto the slide until the color stops flowing. (You should get runoff that is clear at the end) The alcohol is a de-colorizer. If the cell is gram positive, the stain will stick. If it is gram negative the stain will wash away.

Station 5: Safranin Step 5 of lab procedure. Located next to the notebook cabinet. Add 1 drop of safranin to the smear, wait one minute then wash. Safranin is the counterstain. It will only stain gram negative bacteria, and dyes the cell pink.

Station 6: Microscope Steps 6 of lab procedure. Located at the back of the classroom. Blot dry the slide. Then place it on the stage of the microscope. Start at the lowest magnification (4X) then move to the highest (100X).

Question to Answer Would penicillin be an effective antibiotic to prevent the spread and growth of your bacteria? When you are done with the lab, please answer the analysis questions on your worksheet. If you are waiting for your turn or are finished with the questions please work on the build-a-virus activity and the viruses and bacteria worksheet.