Unit 5

Unit 5 FALL AND SPRING: Differential Staining Technique: Gram StainBy Karen Bentz, Patricia Wilber, Heather Fitzgerald, Deborah Muldavin and Andrea Peterson, 2018Creative Commons Attribution-NonCommercial 4.0 International License.

I. Introduction:The Gram stain is a differential stain because it will differentiate between Gram(+) (Gram positive) and Gram(-) (Gram negative) cells based on their different cell wall compositions. Gram(+) cell walls are composed of a very thick peptidoglycan layer, while Gram(-) cell walls have a thin layer of peptidoglycan with an outer lipid membrane. (See Figure 5-1 below).

The Gram stain is one of the most important differential staining techniques used in a medical lab. Knowing whether a pathogen has a Gram(+) or Gram(-) cell wall structure will influence the choice of treatment.

When completed correctly, the Gram stain will result in Gram (+) cells that are purple and Gram(-) cells that are pink.

Figure 5-1: Comparison of Gram(+) and Gram(-) cell walls

Figure created by Patty Wilber, 2015

How the Gram Stain WorksStep 1: When the primary stain, crystal violet, is applied to the bacterial smear, the crystal violet penetrates the peptidoglycan of both cell types making them both purple.

Step 2: When the mordant, Gram’s iodine, is applied, it locks the crystal violet stain into the peptidoglycan layers of both cell types. Both cell types continue to be purple.

Step 3: The next step is decolorizing with alcohol acetone. Decolorizing is the most important step of the Gram stain, and it differentiates between Gram(+) and Gram(-) cells based on the thickness of their peptidoglycan. The decolorizer will remove the stain from Gram(-) cells by removing the outer membrane and leaching stain from the thin

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peptidoglycan layer, leaving the cells colorless. The thick peptidoglycan layer of the Gram(+) holds the stain, and the Gram(+) cells will remain purple.

Step 4: Lastly, the secondary stain, safranin, is applied. The Gram(+) cells are so dark that the safranin does not change their appearance and they remain purple. The safranin will penetrate the thin peptidoglycan layer of the Gram(-) cells and they will become pink.

Table 5-1: Gram stain procedure and expected resultsTime Color of

Gram(+)Color of Gram(-)

Primary stain:Crystal violet

30 secs Purple Purple

Rinse with DI water 5 secs Purple PurpleMordant:Gram’s Iodine

30 secs Purple Purple

Rinse with DI water 5 secs Purple PurpleDecolorizer:Acetone Alcohol

Put on, rinse off Purple clearish

Secondary Stain:Safranin

1 minute Purple Pink

Rinse with DI water 5 secs Purple Pink

II. Lab Work

Preparing a Slide with Two Organisms https://youtu.be/S2CC-b5fQwI Gram Stain https://youtu.be/Y4qpwTUf33QVideos created by Corrie Andries and Karen Bentz

Materials (per person): Slide Dowel Water bottle Staining Solutions: Gram Crystal Violet, Gram Iodine, Gram Decolorizer, Safranin Bacteria Cultures

o Gram(-) and Gram(+) growing on your streak isolation plate.

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ProcedureA. Observe Results of your Streak IsolationYour streak isolation plate should contain two distinct colony types, one that is Gram(-)and one that is Gram(+). If you do not have two colony types, look at the plates of the students sitting near you at the lab table.

For the two distinct COLONY types for this lab, describe the differences between them.

B. Form a Hypothesis: Observe your colonies and use the following information to hypothesize about which of the colonies is Gram(+) and which is Gram (-). This is what you will do during your Final Project, and it is what is done in diagnostic labs as well.

Table 5.2. Characteristics of Proteus vulgaris and Streptococcus oralisSpecies Proteus vulgaris Streptococcus oralisGram Negative PositiveCell shape Bacillus CoccusAppearance on TSA blood plate

Grey, slimy colonies. Tiny, green colonies.

Which of your colonies do you think is Proteus vulgaris and which do you think is Streptococcus oralis?

C. Test your Hypothesis: Gram Stain and View Cells with a MicroscopeTo test your hypothesis you will need to Gram stain cells from each of your two different types of colonies. Be sure to label which side of the slide has which colony type. An example is shown.

Species 1 from the tiny green colonies was tapped onto the left side and Species 2 from the slimy gray colonies was tapped onto the right side. They were overlapped in the middle.

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After staining, cells from one of the colonies will be purple and cells from the other will be pink, and you will then be able to evaluate your hypothesis.

NOTE: Don’t put too much culture on the slide, as a little goes a long way!

1. If you have two distinct colony types: Touch the flat end of the dowel to one type of colony. If you do NOT have two distinct colony types, get the type you are missing from the plate of someone that does.

2. Tap (tap tap) (do not rub) the dowel on the middle-right of the slide to transfer the bacteria to the slide. MARK YOUR SLIDE so you know which colony sample is on the right side!

3. Turn your dowel over and touch the clean end to the other bacterial colony type on your streak isolation plate.

4. Tap that dowel end on the middle-left side of the slide, so that you get an overlap of the Gram(-) and Gram(+) in the middle. MARK YOUR SLIDE so you know which colony sample is on the left side!

5. Dispose of the dowel in the sharps container.6. Go to the sink and place your slide on the slide holder, or attach a clothespin to the

slide and hold the clothespin.7. Place a few drops of Gram’s Crystal Violet stain on your slide, covering all the

bacteria, and let it sit for about 30 seconds8. Rinse your slide with DI water. 9. Place a few drops of Gram’s Iodine on the slide, covering the bacteria. Let sit for 30

seconds.10. Rinse slide with DI water.11. Drip the alcohol acetone decolorizer on the slide and rinse it off right away with DI

water.12. Place a few drops of Gram’s Safranin on the slide, covering the bacteria, and let it sit

on the slide for 1 minute.13. Rinse slide with DI water. Pat dry with a Kimwipe. DO NOT rub the slide, or you may

wipe off your bacteria.14. Examine the stained bacteria with your microscope at 1000X TM. 15. Make a drawing (rather than a photograph) of your two bacterial types, noting shape

and arrangement of the cells, and whether they are Gram(+) or Gram(-).16. Estimate the size of your two bacterial types.17. Dispose of your slide in the sharps container when you are done.18. Use a Kimwipe and lens cleaner to clean all of the oil off the 40X and 100X lenses.

Follow all other steps to prepare your microscope for return to the cabinet. 19. Have your instructor check your microscope before you put it back in the cabinet.

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Results

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Table 5-3: Results of your Gram stain.Give the description of one of the original colonies from the Procedure Part A above: _____________________________

After staining record:

Cell color: _____________

Gram(+) or Gram(-)? _______________

TM used: 1000X

Field diameter: __________

Number of cells that fit across: ______

Cell size in mm:___________

Cell size in μm:___________

Cell shape: ______________

Cell arrangement:__________________

Give the description of the other original colony from the Procedure Part A above: _____________________________

After staining record:

Cell color: ______________

Gram(+) or Gram(-)? _______________

TM used: 1000X

Field diameter: __________

Number of cells that fit across: ______

Cell size in mm:___________

Cell size in μm:___________

Cell shape: ______________

Cell arrangement:__________________

D. ConclusionCompare the results of your Gram stain to your hypothesis regarding colony types.

Was your hypothesis correct or not? ___________. Explain how you know this.

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Post-Lab Questions Name ___________________

1. Fill in the following table as it relates to timing, cell appearance and chemical solutions used during the Gram staining procedure.

STEP CHEMICAL SOLUTION USED

Time on Slide

COLOR if Gram(+)

COLOR if Gram( -)

Primary Stain

Mordant

Decolorizer

Secondary Stain

2. Differentiate between Gram(+) and Gram(–) bacteria by briefly describing the molecular composition of their cell walls. (1pt)

Gram(+)

Gram(-)

3. What is the function of a mordant?

4. What step in the Gram stain is differential, and why?

5. Was your hypothesis regarding which colony was which supported or refuted? Explain fully.

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