lab report

41

EXCERCISE 1.4

A Differential Stain: The Gram Stain

Getting Started Differential stains usually involve at least two dyes and are used to distinguish one group of

organisms from another. For example, the Gram stain determines whether organisms are Gram

positive or Gram negative.

The Gram stain is especially useful as one of the first procedures employed in identifying

organisms. It reveals not only the morphology and the arrangement of the cells but also

information about the cell wall.

In the late 1800s, Christian Gram devised the staining procedure when trying to stain

bacteria so that they contrasted with the tissue sections he was observing. Many years later, it

was found that purple (Gram-positive) bacteria had thick cell walls of peptidoglycan, while pink

(Gram-negative) bacteria had much thinner cell walls of peptidoglycan surrounded by an

additional lipid membrane. The thick cell wall retains the purple dye in the procedure, but the

thin wall does not (table 3.1).

In the Gram stain, a bacterial smear is dried and then heat-fixed to cause it to adhere to

the glass slide. It is then stained with crystal violet dye, the primary stain, which is rinsed off

and replaced with an iodine solution. The iodine acts as a mordant-that is, it binds the dye to

the cell. The smear is then decolorized with alcohol and counter, stained with safranin. In

Gram-positive organisms, the purple crystal violet dye, complexed with the iodine solution, is

not removed by the alcohol, and thus the organisms remain purple. On the other hand, the

purple stain is removed from Gram-negative organisms by the alcohol, and the colorless cells

take up the red color of the safranin counterstain. [1]

Table 3.1. Appearance of the Cells After Each Procedure. [1]

42

In this exercise, you will prepare smears of bacteria for staining and then stain the

smears with the gram stain procedure to infer the cell wall structures of microorganisms on

your plate.

Special Notes to Improve Your Gram Stains

1. Gram-positive organisms can lose their ability to retain the crystal violet complex when they are old. This can happen when a culture has been incubating 18 hours or more. The genus

Bacillus is especially apt to become Gram negative. Use young, overnight cultures whenever possible. It is interesting to note that Gram-positive organisms can appear Gram negative,

but Gram-negative organisms almost never appear Gram positive.

2. Another way Gram-positive organisms may appear falsely Gram negative is by over decolorizing in the Gram-stain procedure. If excessive amounts of alcohol are used, almost

any Gram-positive organism will lose the crystal violet stain and appear Gram-negative.

3. If you are staining a very thick smear, it may be difficult for the dyes to penetrate properly. This is not a problem with broth cultures, which are naturally quite dilute, but be careful not

to make the suspension from a colony in a drop of water too thick.

4. When possible, avoid making smears from inhibitory media such as eosin methylene blue (EMB) because the bacteria frequently give variable staining results and can show atypical

morphology.

5. The use of safranin in the Gram stain is not essential. It is simply used as a way of dying the colorless cells so they contrast with the purple. If you are color-blind and have difficulty

distinguishing pink from purple, try other dyes as a counterstain.

[1]

Objectives

1. Describe the Gram-stain procedure. 2. Differentiate between Gram-positive organisms and Gram-negative organisms.

43

Materials

Staining kit per team containing bottles of:

Crystal violet, 1

Gram’s iodine, 1 Alcohol 95%, 1

Safranin, 1

Per student:

Microscope, 1

Forceps, 1

Glass slide, 1

Inoculating loop, 1

LB agar plate with colonies (TA streak colonies from one of your petri-dishes in Exercise 1.3 to

a new petri dish just one day before the lab so your group will have fresh colonies for gram

staining)

Bunsen burner, 1

Striker, 1

Large clean beaker, 1

Dropper pipettes as needed

Cleansing powder such as Boraxo or Bon Ami

Waterproof marking pen

Disposable gloves

Wash bottle or glass beaker filled with tap water, 1

Ethanol-water wash bottle, 1

Bibulous paper or paper towels as needed

Lens paper as needed

Goggles or safety glasses, 1

Procedure ***Before completing this exercise, please carefully read Appendix 4: Use of a Bunsen burner and Appendix 6: Aseptic Technique.***

Please make sure each group member has a chance to perform the protocol from start. Preparation of Smears

6. Sanitize your workspace with ethanol.

7. Be sure to wear goggles throughout this exercise as the fumes from the chemicals used here have the potential to burn your eyes.

44

8. If the glass slide is covered with dust, clean a glass slide by rubbing it with slightly moistened cleansing powder such as Boraxo or Bon Ami located at sink stations. Rinse

well and dry with a paper towel. New slides should be washed because sometimes they

are covered with a protective coating.

9. Draw one circle with a waterproof pen on the underside of the slide.

10. Using a dropper pipette, add a drop of water to the slide on top of the circle. Use your loop to transfer tap water or use water from a dropper bottle. This water does not need

to be sterile. Although there are some organisms in municipal water systems, they are

non-pathogens and are too few to be seen.

11. Using instructions given in Appendix 4, light the Bunsen burner with the striker and adjust the valves so that there are two distinct blue zones, the inner of which is a bright

blue cone. Keep in mind that when sterilizing loops and other materials, aim for the hottest part of the flame located just above the tip of the inner bright blue cone.

12. Sterilize a loop by holding it at an angle in the flame of the Bunsen burner. Heat the entire wire red hot, but avoid putting your hand directly over the flame or heating the

handle itself.

13. Allow the loop to cool a few seconds, then remove a small amount of a bacterial culture from just one of your plates by lightly touching the culture with the loop and suspend it

in one of the drops of water on the slide. Continue to mix in bacteria until the drop

becomes slightly cloudy. If your preparation is too thick, it will stain unevenly, and if it is

too thin, you will have a difficult time finding organisms under the microscope. In the

beginning, it may be better to err on the side of having a slightly too cloudy preparation

– at least you will be able to see organisms and you will learn from experience how dense to make the suspension.

14. Heat the loop red hot again. It is important to burn off the remaining organisms so that you will not contaminate your benchtop. If you then rest your loop on the side of your

Bunsen burner, it can cool without burning anything on the bench.

15. Permit the slide to dry. Do not heat it in any way to hasten the process, because the cells will become distorted. Place the slide off to the side of the bench so that you can

proceed with other work.

16. When the slide is fairly dry (in about 5-10 minutes), heat-fix the organisms to the slide by using forceps to quickly pass it through a Bunsen burner flame two or three times so

that the bottom of the slide is barely warm. This step causes the cells to adhere to the

glass so they will not wash off in the staining process.

17. Extinguish the Bunsen burner flame using information given in Appendix 4 and set aside.

45

Gram Stain

1. Working over an empty beaker, hold the slide with forceps. Flood the slide with crystal violet until the slide is completely covered. Leave the stain on for 30-60 seconds. The

timing is not critical. Rinse the dye completely from the slide with water from a wash

bottle or with gently running tap water into a waste beaker. Avoid directly spraying the smear with water when rinsing as the bacteria from the smear could wash off.

2. Flood the slide with Gram’s iodine for a few seconds to a minute or longer and then wash off the iodine with tap water into a waste beaker.

3. Timing is critical in the decolorization step of the Gram Stain. Hold the slide at a 45° angle and carefully drip 99% ethanol, the decolorizer, over it until no more purple dye

runs off. Immediately wash the slide with tap water. Thicker smears may take longer

than thinner ones, but ethanol should usually be added for about 20 seconds.

4. Flood the slide with safranin and leave it on for 10-30 seconds – timing is not important. Wash with tap water into a waste bottle. Safranin is a counter-stain because it stains the

cells that have lost the purple dye.

5. Blot the slide carefully with bibulous paper or paper towel to remove the water, but do not rub from side to side. When it is completely dry, observe the slide under the

microscope. Focus the slide on low power and then again on high power. Note that no

coverslip is used when looking at stained organisms. Compare your stain with color

plate 5 in Appendix 1.

6. Describe the appearance of your stained bacteria in the Results section of the Laboratory Report.

7. Important note: Do not dump the contents of your beaker in the sink under any circumstance. The contents are considered hazardous waste and must be poured into a

bottle marked for hazardous waste. Please consult your instructor or TA for the location

of these bottles.

8. It is unnecessary to boil stained slides after use because the staining process kills the organisms. You can simply throw your slide in the specially marked glass container in the

front of the lab. [1]

46

Results

Record your observations under a microscope below (Include a sketch of the organisms you

observed and indicate whether the organisms are gram-positive or gram-negative):