Unit 5 SUMMER: Differential Staining Technique: Gram Stain and Streak Isolation

Unit 5

Unit 5 SUMMER: Differential Staining Technique: Gram Stain and Streak Isolation.

By Karen Bentz, Patricia Wilber, Heather Fitzgerald, Deborah Muldavin and Andrea Peterson, 2018

Creative Commons Attribution-NonCommercial 4.0 International License.

I.Introduction

Simple Stains:

Bacterial cells are usually clear in color. To increase the chance of seeing the microbial cells under a microscope, cells are stained with dyes that are attracted to different parts of the cells. Once cells are stained, features such as overall cell size and shape, and cellular arrangements, can be determined. For a review of cell shapes and arrangements please refer to Unit 2 (Microscopes) of this laboratory manual.

Stains are pigment molecules dissolved in a solvent. The simple stains Methylene Blue, Gram’s Crystal Violet, and Safraninare all positively charged. Bacterial cell wall components (certain polysaccharides and proteins) are negatively charged. What do you know about opposites? They attract! Thus, the positively charged stains are attracted to the negatively charged bacterial cells you will stain.

Figure 5-1. Idealized view of staining of bacterial cells with Methylene Blue.Unstained, negatively charged, clear bacterial cells plus positively charged Methylene Blue stain yields blue stained cells.

Figure created by Heather Fitzgerald and Patricia G. Wilber

Figure 5-2. Actual image of bacillus-shaped bacterial cells (species Clostridium septicum) stained with Methylene Blue.

Accessed 7-9-2015. This image is in the public domain. Centers for Disease Control and Prevention's

Public Health Image Library(PHIL), with identification number#14347.

Gram Stain

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 purpleand Gram(-) cells that are pink.

Figure 5-3: Comparison of Gram(+) and Gram(-) cell walls

Figure created by Patricia G. Wilber

How the Gram stain works:

Step 1: When the primary stain, crystal violent, 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 decolorizerwill remove the stain fromGram(-) cells by removing the outer membrane and leaching stain from the thin 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 Results

II.Day 1

• Preparing a Slide with Two Organisms
• Gram Stain

Videos 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
• Gram(-) and Gram(+) growing on your cultures from last time.
• Bacterial Culture: 0.05ml Proteus vulgaris (Pv) mixed with 5ml of Streptococcus oralis (Sto) in one T-soy broth tube.
• TSA Blood plate

Activities

1. Do a new Streak Isolation

• Use the blood plate and the Bacterial Culture: 0.05ml Proteus vulgaris (Pv) mixed with 5ml of Streptococcus oralis (Sto) in one T-soy broth tube.

1. Refer to Unit 3 to review the streak isolation procedure from a broth. A broth tends to be easier to streak from than a plate.
2. STIR your mixed broth thoroughly with your sterilized loop before streaking!
3. Tap your loop to reduce bacteria before performing the streak isolation.
4. Perform your streak isolation on your blood plate.
5. Be sure to label your plate correctly.
6. Incubate this plate in a candle jar.

Figure 5-4. General procedure for the Streak Isolation Technique.

Figure by Patricia G. Wilber

1. Observe Results of Your Old Streak Isolation

Your old streak isolation plate should contain one colony type, either Pseudomonas aeruginosa(if you used the broth) or Staphylococcus aureus (if you used the plate)

1. Observe the Results of your Environmental Sample

Your environmental sample should have many different types of colonies.

1. Gram Stain: Form a hypothesis

For the Gram stain, please use the following specimens.

Gram stain sample 1:Escherichia coli growing on your slant

Gram stain sample 2:Staphylococcus aureus from your petri dish.

Sometimes theGgram(-) colonies appear slimier than the Gram(+) colonies. Based on appearance alone, hypothesis which of the above species is Gram(+) and which is Gram(-) by completing the following sentences.

I hypothesis that Escherichia coliis Gram ______because

I hypothesize that Staphylococcus aureusis Gram ______because

1. Test your Hypothesis: Gram Stainand View Cells with a Microscope

To test your hypothesis you will need to Gram stain the E.coli and the S. aureuscells An example is shown.

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

1. Touch the flat end of the dowel to one type of colony.
2. Tap (tap tap) (do not rub) the dowel on the middle-right of the slide to transfer the bacteriato 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
4. Tap that dowel end on the middle-left side of the slide, so that you get an overlap of the slimy and the less slimy colonies 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 seconds
8. Rinse your slide with DI water.
9. Place a few drops of Gram’s Iodine on the bacteria. Let sit for 30 seconds.
10. Rinse slide with DI water.
11. Drip the alcohol acetone decolorizer on the bacteriaand rinse it off right away with DI water.
12. Place a few drops of Gram’s Safranin on the bacteriaand 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 of 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.

Results

Table 5-2:Results of your Gram stain.

Conclusion

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

III.Day 2

Observe your streak isolation plate.

Do you have two types of colonies?

If yes, great!

If no, find a lab mate that does and complete the following. Use the following information to hypothesize about which of the colonies are Gram(+) and which are Gram (-). This is, in part, what you will do during your Final Project, and it is what is done in diagnostic labs as well.

Table 5.3. Characteristics of Proteus vulgaris and Streptococcus oralis

Insert a photograph of a streak isolation with two types of colonies and label the colonies you think arethe Gram(-) Proteus vulgaris and whichyou think arethe Gram(+) Streptococcus oralis.

Post-Lab QuestionsName ______

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

Time on Slide

COLOR if Gram(+)

COLOR if Gram( -)

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

Gram(+)

Gram(-)

1. What is the function of a mordant?

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

1. How could you test your hypothesis regarding which colonies are Sto and which are Pv for your mixed species streak isolation?

Unit 5 Page 1