A. Use Young Cultures of Bacteria - 24 Hours Old Or Younger

University of Pittsburgh at Bradford Science In Motion Biology Lab 018

Gram Staining

Introduction:

The gram stain technique is a more complex staining technique, which is used as an aid in identifying an unknown species of bacteria. The technique uses three stains: crystal violet, Gram’s iodine, and safranin. Crystal violet stains bacterial cells purple; Gram’s iodine is a mordant which means that it helps fix the crystal violet stain and makes it difficult for the crystal violet to be removed by decolorization; safranin stain is applied last and only appears if cells lose crystal violet in the decolorization process. Some bacteria retain the purple crystal violet stain and are called Gram positive. Other bacteria lose the crystal violet when decolorized and retain the pink safranin stain and are called Gram negative. The actual reason why some bacterial species are Gram + and others are Gram - is still unknown although the chemical makeup of the cell wall is probably involved. In this investigation you will prepare Gram stains of three species of bacteria and identify them as Gram + or Gram -. In order to get accurate results make sure that you:

a. use young cultures of bacteria - 24 hours old or younger. b. prepare smears that are not thick. c. do not overheat your slide when you fix the bacteria. d. use fresh reagents. e. are consistent when following lab procedures. f. do not leave excess water on the slide between stains.

Objectives:

1. Students will learn how to handle bacteria safely using aseptic techniques. 2. Students will prepare bacterial smears of the three species using techniques provided in the aseptic technique lab. 3. Students will stain bacteria using a differential staining technique called “Gram Staining”. 4. Students will observe bacterial cells using the oil immersion lens on their microscopes. 5. Students will identify several species of bacteria as either Gram + or Gram - .

Safety Notes:

1. Review and follow all safety precautions outlined in the Aseptic Techniques Lab. 2. Students must wear safety glasses since safranin may cause damage to the eye.

Materials:

Juniata College 1 disinfectant bacterial cultures(may vary) microscope slides Bacillus megaterium alcohol lamp Micrococcus luteus wire loop Escherichia coli wire needle Spirillum volutans china marking pencil red and purple colored pencils lens paper crystal violet test tube rack Gram’s iodine distilled water bottle safranin wooden sticks 95% ethanol hand soap paper towels immersion oil clothes pins

Procedure:

1. Prepare a fixed smear of each culture. Label each slide with the culture name a. Using your inoculating loop, place a loopful of water on the slide. b. Using the loop, choose a colony of bacteria and mix it in the water and spread it out. c. Allow the smear to dry. d. Heat-fix the smear by passing the slide rapidly through the Bunsen flame three times. e. Place slides on staining rack.

2. Cover the slide with crystal violet. Let stand for 20 seconds.

3. Briefly wash off the stain, using a wash bottle of distilled water. Drain off excess water.

4. Cover the slide with Gram’s iodine solution and let stand for one minute.

5. Pour off the Gram’s iodine and flood the slide with 95% ethyl alcohol for 10 to 20 seconds. Thick smears will require more time than thin ones. Decolorization has occurred when the solvent flows colorlessly from the slide.

6. Stop the action of the alcohol by rinsing the slide with water for a few seconds.

7. Cover the slide with safranin for 20 seconds.

8. Wash gently with water for a few seconds, blot with bibulous paper. Let dry at room temperature.

9. The slide may be examined under oil immersion immediately.

Name______

Juniata College 2 Gram Staining Student Evaluation

Observation results:

Use colored pencils to draw the three species of bacteria you stained in this lab. Label the bacteria with their scientific name and as being Gram + or Gram -.

Analysis/Conclusions:

1. Why do Gram + bacteria retain the purple crystal violet stain?

2. Why does the crystal violet stain “wash out” of Gram - bacteria when they are decolorized in ethanol?

3. What is the function of a mordant?

4. Why is timing so critical during the decolorizing step of the Gram staining procedure?

5. If the counter stain safranin was not applied to the bacteria, could you distinguish between Gram + and Gram - bacteria?

6. Could methylene blue stain be substituted for safranin as the counter stain in this procedure?

Juniata College 3