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, 2018Creative Commons Attribution-NonCommercial 4.0 International License.

I. IntroductionSimple 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 Safranin are 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

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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 purple and Gram(-) cells that are pink.

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

Figure created by Patricia G. Wilber

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How the Gram stain works:Step 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 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. Day 1

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

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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 cultures from last time.o Bacterial Culture: 0.05ml Proteus vulgaris (Pv) mixed with 5ml of

Streptococcus oralis (Sto) in one T-soy broth tube. TSA Blood plate

ActivitiesA. 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

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B. Observe Results of Your Old Streak IsolationYour 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)

C. Observe the Results of your Environmental SampleYour environmental sample should have many different types of colonies.

D. Gram Stain: Form a hypothesisFor 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 coli is Gram __________ because

I hypothesize that Staphylococcus aureus is Gram _____________ because

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Species you used: ______________

Dow many colony types do you have?

Describe your colonies.

What did you swab for your environmental sample? __________________

Dow many colony types do you have?

Describe the differences between two of the colonies types.

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E. Test your Hypothesis: Gram Stain and View Cells with a MicroscopeTo test your hypothesis you will need to Gram stain the E.coli and the S. aureus cells 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

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 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 seconds8. 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 bacteria and rinse it off right away with

DI water.12. Place a few drops of Gram’s Safranin on 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 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.

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Results

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Table 5-2: Results of your Gram stain.Write the name of the species you hypothesized was Gram(-) in D 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:__________________

Write the name of the species you hypothesized was Gram(+)in D 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:__________________

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

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III. Day 2Observe 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 oralisSpecies Proteus vulgaris Streptococcus oralis

Gram Negative Positive

Cell shape Bacillus Coccus

Appearance on TSA blood plate

Grey, slimy colonies. Tiny, green colonies.

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

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I hypothesized that Escherichia coli was Gram __________. The results of my gram stain showed….

Therefore, my hypothesis for Ec was

I hypothesized that Staphylococcus aureus is Gram _____________ . The results of my gram stain showed….

Therefore, my hypothesis for Sa was

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

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