Name ___________________________________________________ 8 __________ Date _________
Bacterial cells are hard to observe even at high magnification because there is insufficient contrast between the cell and the background. Therefore it is common to stain bacterial cells to improve contrast and allow their size, shape, arrangement and structures to be seen.
Differential stains use two different colored dyes to distinguish between different populations of cells or different cellular structures. Differential stains begin with the primary dye, which generally stains all cells, followed by a wash step that removes the dye from some of the cells and a secondary dye.
Heat fixing is an important part of staining. Not only does it ensure that the cells stick to the surface of the slide when they are covered in dyes and rinsed off, it also prevents cells from shrinking during staining.
The Gram stain is the most widely performed and well known staining technique in microbiology. It is a differential stain that separates bacteria into two major groups: Gram positive and Gram negative. The primary dye is retained in the thick layer of peptidoglycan in Gram positive cells. Gram negative bacteria, which have a much thinner layer of peptidoglycan covered by an outer membrane, do not retain the primary dye after the wash step, and must be stained by a dye of another color.
The steps of the Gram stain are as follows:
1.
Primary dye: All cells are stained purple by the primary stain, crystal violet.
2.
Mordant: Gram’s iodine is added to the smear, which forms an insoluble complex in the Gram positive cell’s thick peptidoglycan layer. All cells are still purple at this point.
3.
Wash: The dye-mordant complex is removed from Gram negative cells by either alcohol or acetone.
Gram positive cells remain purple, but Gram negative cells become transparent.
4.
Counterstain: Safranin is used to colorize the colorless Gram negative cells. The Gram positive cells remain unchanged because the crystal violet stain is more intense than safranin.
Materials
Streptococcus
Escherichia coli
3 glass slides
Alcohol burner
Gram’s iodine
Inoculating needle
Clothespin
Test tube
Gram’s iodine
Crystal violet
95% alcohol
Safranin
Wash bottle with water
Goggles
Groups
You will work in groups of three, preparing three slides per group. Switch off every task. One person will perform the task. Another person will read the instructions. The third group member will write down observations and notes on technique, including any sources of error. Each group will turn in one page of notes.
Each group member will turn in sketches of the stain and answers to the concluding questions.
Procedure
Part 1: Preparation of the slides
1.
Put on goggles.
2.
Clean and dry a glass slide.
3.
Rinse out your empty test tube and fill with water.

4.
Using aseptic technique as outlined below, remove two loopfuls of water and place on your slide.
5.
Using aseptic technique as outlined below, remove a tiny amount of each bacteria from the agar’s surface. If you can see the bacteria on your loop, it is too much. Remember, you will not be able to see your cells clearly if your smear is too thick. a.
Aseptic technique: Flame your loop. b.
In your spare hand, hold the tube at a 35-40°. With the hand that is still holding your inoculating loop, unscrew the lid and grasp it between your last two fingers. c.
Flame the mouth of the tube. d.
Remove a loopful of water/tiny amount of bacteria. e.
Flame the mouth of your tube and return the cap. f.
Place your water/bacteria on the slide. Using your inoculating loop, disperse and mix the organisms within a small circle.
6.
Allow the smear to dry at room temperature. You may speed dry if necessary by holding the slide high above the flame of the alcohol burner. If it is too hot for your fingers, it is too hot for the bacteria.
7.
Heat-fix the slide by passing it through the hottest part of the flame six times using a clothespin to hold the slide.
Part 2: Gram Stain
1.
Place the slide on the staining rack by the sink and cover the smear with crystal violet for 20 seconds.
2.
Gently wash with water for 2 seconds. Drain off excess water.
3.
Add Gram’s iodine to the smear for 1 minute.
4.
Hold the slide at a 45° angle and decolorize with 95% alcohol for 10-20 seconds. Thicker smears may take longer. If the solvent runs colorlessly, stop!
5.
Wash for 2 seconds.
6.
Add safranin to the smear for 1 minute.
7.
Wash for 2 seconds.
8.
Gently blot dry with paper towels and air dry.
Part 3: Observation
1.
Set up a microscope.
2.
Examine your slide under the microscope. Remember to focus the lowest magnification first and work up to a higher magnification without adjusting the course focus.
3.
When finished, wash, rinse, and dry the slide carefully. Put all of your equipment on the counter where you found it. Wipe your desk with ethyl alcohol. Wash your hands.
Lab write-up: Answer in thoughtful, complete sentences on another piece of paper.
1.
Draw your cells, using colored pencils. Label the magnification.
2.
Which of the cells are Gram-positive? Which are Gram-negative? How do you know?
3.
Was your smear successful? Explain.
4.
Identify and explain any sources of error in your technique.
5.
Give at least three reasons why the Gram-stain is so widely used.
6.
What would your stain look like if you had omitted the iodine step? Consider both Gram-negative and positive cells.
7.
What would your stain look like if you had omitted the safranin step?
8.
Suppose you introduced penicillin, an antibiotic that prevents synthesis of the peptide cross-links in pepdidoglycan, to living cultures of Streptococcus and Escherichia.
Which genus would you expect to be most harmed?
For any credit, explain your answer.