MICROBIOLOGY 101 LABORATORY MANUAL

EXERCISE #5: GRAM STAIN AND STREAKING #2

NAME, ID #:_______________________________________________.

NAME of TA: ______________________________

REVISED: 08/04/99

INTRODUCTION

In 1883-4 Dr. H. C. GRAM, a physician who was working with R. Koch, discovered that bacteria fell into two distinct categories when stained sequentially with CRYSTAL VIOLET followed in sequence by a bath in an IODINE SOLUTION, a wash with a destaining agent and a COUNTER-STAIN, if the cells were bathed following an initial treatment with the crystal violet. One group of cells RESISTED the removal of the crystal violet when washed with ETHANOL or ACETONE (DECOLORIZING AGENTS), whereas the second group was readily DECOLORIZED by a brief rinse with these reagents. To visualize the decolorized cells Gram briefly exposed them to a COUNTER-STAIN, or a stain of a different color from the crystal violet. Gram settled on the red counter staining dye SAFRANIN. Thus cells which resisted decolorization remained DEEP PURPLE or BLUE and came to be referred to a GRAM POSITIVE cells, whereas cells that easily lost the crystal violet dye were red after counter staining. These red cells came to be called GRAM NEGATIVE cells.

Gram and others drew several important conclusions from these staining results. First, they realized that DIFFERENTIAL STAINING of cells and cell components was possible. Secondly, they recognized that this staining was DIAGNOSTIC and could be used to IDENTIFY CELLS and SUBSTANCES. Thirdly, they reasoned that cells and cell components DIFFERED CHEMICALLY as evidenced by their differential staining. Ultimately these realizations led to the idea of a MAGIC BULLET, which eventually led Fleming to recognize the significance of penicillin, which finally brought the world antibiotics and CHEMOTHERAPEUTIC AGENTS to treat diseases.

cellwall.gif (4556 bytes)
Figure 1. Comparison of G+ and G- cell walls. NOTE the differences in the thickness of the peptidoglycan & the absence of an OUTER MEMBRANE in the G+ cell wall. For more information on the difference between G+ and G- cells click here.

We now know that G+ BACTERIAL CELLS are very different from G- CELLS. These differences have proven useful in understanding the physiology of these two categories of bacteria. We do not understand why or when they evolved and we are unlikely to ever know this. The gram characteristic is almost as fundamental to a bacterial description as its morphology. By knowing this characteristic we automatically know a lot about a given bacterium; it is very much like knowing the "sex" of an organism because that one "fact" tells you so much.

Performing a good gram stain is easy, but it does require some experience. For example, the original "bacterial smear" must not be TOO THICK or TOO THIN or your results will be poor. Also the age of the culture is important. Very young and very old cells often produce poor results, whereas mid-log cells that are healthy and growing optimally, tend to give dependable results. The media the cells are grown in and the environmental conditions may also effect the outcome of a gram stain because these ultimately reflect on the chemical nature of the cell. As the clique states, "the devil is in the details"! That is, the timing of the various steps, the quality of the reagents and the experience of the person performing the procedure all influence the final results. While many bacteria are "cleanly" G+ or G- a large number are to some degree GRAM VARIABLE.

In this exercise you will carry out a classical gram stain in which you will determine the gram characteristic of an unknown bacterium. Before going on view this site to see how to do a gram stain.

PURPOSE OF LABORATORY:

  1. To complete the first streaking experiment and to study colonial morphology.
  2. To learn about the Gram stain.
  3. To learn how to perform & interpret a Gram stain.
  4. To further improve your streaking technique.

RELATIONSHIP TO LECTURE MATERIAL

GENERAL INSTRUCTIONS:

  1. Draw the required representations of what you've seen in the circles below.
  2. Prepare your smears and carry out the Gram stain procedure first.
  3. While the slide is drying carry out a second streak plate.
  4. Examine your streak plates from Exercise #3 if you have not done so and describe the colonies based on Fig. 2, Exercise 4. View them with the dissecting scope.

GRAM STAINING PROCEDURE

GRAM STAIN

  1. Read Section 4, pg. 27-28, especially Fig. 4.1, in A Photographic Atlas for the Microbiology Laboratory. Also click here to see how the staining process actually looks while it is being done.
  2. Divide a clean slide into 3 sections. Obtain the cultures indicated by the instructor, including a G+ and a G- control and a mixed culture.
  3. Aseptically place loops of the known G+ and G- control cells at either end of the slide.
  4. Place a loop of the mixture in the middle of the slide.
  5. Allow the 3 smears to dry and heat fix as before.
  6. Flood the 3 smears with the crystal violet solution for 1 min.
  7. Wash gently with tap water, but don't dry; shake off most of the water.
  8. Flood the smears with the iodine solution for 1 min. & rinse again with water and shake almost dry.
  9. Rinse with the decolorizing reagent as demonstrated by the instructor.
  10. Immediately, rinse the smears with water to remove the last bit of the crystal violet/decolorizing reagent. At this point the G+ cells will still retain their crystal violet and will thus be purple (like your fingers at this stage?), whereas the G- cells will be UNSTAINED. Shake off the excess water.
  11. Flood the smears for about 30 seconds with the safranin counter stain. Finally wash with water and blot dry.
  12. Examine the respective G+ and G- smears at 40X until you locate a visible cluster of bacterial cells. Then add immersion oil and switch to 100X. Focus and observe the color and form of the bacteria. If the G+ control is clearly purple with few to no red cells and the G- control is mostly red with few to no blue cells, you can go ahead and examine the unknown. If either of these criteria are not met, discard the slide and start over again.
  13. Have the instructor approve your final result. It should look like the sample in the A Photographic Atlas for the Microbiology Laboratory, pg. 28.
  14. Click here to compare your Gram-stain results with those seen in a medical school. Your gram negative sample should be red and the gram positive blue.

STREAKING #2

  1. If necessary reread pg. 9 and examine the figures of streaks on pg. 1-4 in A Photographic Atlas for the Microbiology Laboratory.
  2. Two sterile agar plates per student.
  3. Cultures of the appropriate bacterial cultures.
  4. Streak one culture on each plate as indicated by the instructor.
  5. Label the plates with the date and your name, the sample and turn them into the instructor for incubation & subsequent grading.
  6. At the next lab examine the colonies and compare them with those show in the Atlas.
  7. The instructor will also grade the plates A-F. Those who get a "C- to F" will have to streak another plate in the next lab and turn it into the instructor for assessment. If it still does not pass muster, (B or better) you will be docked 2 points and will have to continue streaking plates until your technique is approved. Each subsequent failure will cost 2 points from your total.

SAMPLE QUESTIONS: You should be able to answer these questions at the conclusion of this laboratory.

TEST OF YOUR KNOWLEDGE OF THE GRAM STAIN

Click here to take the test.

Other sites with information/pictures on the Gram stain:

Copyright © Dr. R. E. Hurlbert, 1998.
This material may be used for educational purposes only and may not be duplicated for commercial purposes.
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