Download Unit 5: Differential Staining Technique: Gram Stain (Summer Term)

Unit 5
Unit 5 SUMMER: Differential Staining Technique: Gram Stain
and Streak Isolation.
By Karen Bentz, Patricia Wilber, Heather Fitzgerald and Deborah Muldavin.
Copyright Central New Mexico Community College, 2016
Introduction
Most microbial cells that laboratory workers deal with are too small to be seen with the
naked eye. Additionally, even using a microscope, most microbes need to be magnified at
least 400X before individual cells can be distinctly seen. Cells viewed at this level are
generally clear in color. Therefore, to increase the chance of seeing the microbial cells using
standard microscope optics, cells are stained with dyes that are attracted to different parts
of the cells. Once cells can be seen easily, features such as overall cell size and shape, and
cellular arrangements, can be determined. For a review of shapes and arrangements please
refer to Unit 2 (Microscopes) of this laboratory manual.
Stains are pigment molecules dissolved in a solvent. Commonly used stains in this lab
include Methylene Blue, India Ink, Gram’s Crystal Violet and Safranin. These are all simple
stains, which are stains that contain only one pigment type (or dye).
Stains and cells contain charges. The simple stains Methylene Blue, Gram’s Crystal Violet,
and Safranin are all positively charged. They are classified as basic stains because they are
positively charged (+) and have a high pH. Many bacterial and fungal cell wall components
(certain polysaccharides and proteins) are negatively charged. What do you know about
opposites? They attract! Thus, the positively charged simple stains are attracted to the
negatively charged bacterial cells you will stain.
Nucleic acids also have an overall negative charge and are thus attracted to this type of stain.
You may remember from BIO 1492 that you stained your own cheek cells with Methylene
Blue to produce a blue nucleus due to the attraction of the dye for nucleic acid.
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Figure 5-1. Idealized view of basic staining of bacterial cells with Methylene blue. (A)
Unstained, negatively charged, clear (they are there—look closely) bacterial cells (cocci,
staphylo) plus (B) positively charged Methylene Blue stain. Together these will yield (C) bluestained bacterial cells due to the interaction of the charged particles.
+
(A) Unstained cells
+
+
+
+
+
(B) Methylene blue 
(C) cells stained blue
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 utilizes two simple stains and is one of the most important differential staining
techniques used in a medical lab. Knowing whether a pathogen has a Gram(+) (Gram Positive)
or Gram(-) (Gram Negative) cell wall structure will influence the choice of antibiotic used to
eliminate it. Since Gram(+) cell walls are composed of a very thick peptidoglycan layer,
antibiotics that disrupt the structural integrity of the peptidoglycan will be more effective
against these bacteria than against Gram(-) bacteria.
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Figure 5-1: Comparison of Gram(+) and Gram(-) Cell Walls
Outer Membrane of
polysaccharide, lipid A
and phospholipid
Peptidoglycan
Plasma membrane
Gram(+) Cell Wall
Gram(-) Cell Wall
This is how the Gram Stain works. When the primary stain, crystal violent, is applied to the
bacterial smear, all of the cells present will become purple. The crystal violet penetrates the
peptidoglycan of both cell types. The brief, gentle water rinse that follows the application of
the crystal violet is intended to wash off any stain that has not adhered to a cell.
When the mordant, Gram’s iodine, is applied, it floods into the peptidoglycan layers of the
Gram(+) and the Gram(-) cells and binds ionically to the crystal violet forming the crystal
violet-iodine complex. This larger molecular complex sticks inside the thick peptidoglycan
layer of the cell even better than the crystal violet alone. The second water rinse washes
excess iodine off the slide, but it does not remove crystal violet or the crystal violet-iodine
complex that is in the peptidoglycan layer. All of the cells continue to be purple.
The next step, decolorizing, impacts the Gram(+) cells differently than the Gram(-) cells. Recall
that Gram(-) cell walls have a very thin peptidoglycan layer with an outer membrane. The
decolorizing agent, alcohol acetone, is a solution that will destabilize the outer membrane
causing the thin peptidoglycan layer underneath to become vulnerable to the effect of the
decolorizer. When done correctly, the Gram(-) cells will become colorless and the Gram(+)
cells will retain enough of the Crystal Violet-Iodine Complex to stay purple. The quickly applied
water rinse in this case stops the decolorization and rinses off the alcohol acetone.
The final step is simply intended to stain the colorless cells. A counterstain, safranin is applied.
The Gram(+) cells are so dark that the safranin does not change their appearance. The Gram(-)
cells will take on a pale pink to reddish color. The last water rinse is used to get rid of the
excess safranin.
Even though there is only one technique used on both cell types, the outcomes will be
different based on whether cells have a simple thick peptidoglycan layer (Gram(+)) or a thin
one with an outer membrane(Gram(-)) . Thus, the Gram Stain is a differential stain.
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Streak Isolation: In medicine, samples from patients are rarely delivered in a pure culture,
so perfecting a streak isolation and being able to recognize different colonies is really
important! A good streak isolation with two species could hopefully look like this plate by
Leanna Gutierrez, Bio 2192, Spring 2016. It has two very distinct and isolated colony types!
I.
Gram Stain.
Video Links:


Preparing a Slide with Two Organisms https://youtu.be/S2CC-b5fQwI
Gram Stain https://youtu.be/Y4qpwTUf33Q
Videos created by Corrie Andries and Karen Bentz
Materials (per person):





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Slide
Dowel
Water bottle
Staining Solutions: Gram Crystal Violet, Gram Iodine, Gram Decolorizer, Safranin
1 TSA with Blood plate
Bacteria Cultures
o Your environmental sample from prior lab (grown on a petri dish)
o Your Staphylococcus aureus (Sa) on a slant from prior lab
o Your streak isolation of Pseudomonas aeruginosa (Pa) or Escherichia coli (Ec)
on a Chocolate from prior lab
o Mixed Ec and Sa in a Tsoy broth (for streak isolation)
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Procedure
Prepare one slide containing BOTH Escherichia coli (or you could use Pseudomonas
aeruginosa) and Staphylococcus aureus.
NOTE: Don’t put too much culture on the slide, as a little goes a long way!
E.g. Staphylococcus aureus (Left) & Escherichia coli (Right); Mix in middle.
1. Obtain the Ec plate and the Sa plate.
2. Touch the flat end of the dowel to one Gram(-) bacterial colony on your Gram(-) E.
coli plate.
3. Tap (tap tap) (do not rub) the dowel on the middle right of the slide to transfer the
Gram(-) bacteria to the slide.
4. Turn your dowel over and touch the clean end to a Gram(+) bacterial colony on the
Gram(+) S. aureus plate.
5. Tap that dowel end on the middle left side of the slide, so that you get an overlap of
Gram(-) and Gram(+) in the middle, Gram(+) alone on the left and Gram(-) alone on
the right.
6. Dispose of the dowel in the sharps container.
7. Go to the sink and place your slide on the slide holder.
8. Place a few drops of Gram’s Crystal Violet stain on your slide, covering all the
bacteria (but not flooding the world) and let it sit for about 30 seconds
9. Rinse your slide with DI water.
10. Place a few drops of Grams Iodine on the slide, covering the bacteria. Let sit for 30
seconds.
11. Rinse slide with DI water.
12. Place the alcohol acetone decolorizer on the slide and rinse it off right away with DI
water.
13. Place a few drops of Gram’s Safranin on the slide, covering the bacteria, and let it sit
on the slide for 1 minute.
14. Rinse slide with DI water. Pat dry with a kimwipe. DO NOT rub slide, or you may wipe
off your bacteria.
15. Examine the stained bacteria with your microscope at 1000X TM.
16. Make a drawing of your two bacterial types, noting shape and arrangement of the
cells, and whether they are Gram(+) or Gram(-).
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17. Estimate the size of your two bacterial types.
18. Dispose of your slide in the sharps container when you are done.
Table 5-1: Gram Stain Procedure and Expected Results
Time
Color of Gram(+)
Primary stain:
Crystal violet
Rinse with DI water
Mordant:
Gram’s Iodine
Rinse with DI water
Decolorizer:
Acetone Alcohol
Secondary Stain:
Safranin
Rinse with DI water
Reminders:
II.
Color of Gram(-)
30 secs
Purple
Purple
5 secs
30 secs
Purple
Purple
Purple
Purple
5 secs
Put on, rinse off
Purple
Purple
Purple
clearish
1 minute
Purple
Pink
5 secs
Purple
Pink
Close the lids on the stains when finished
Refill DI water from reservoir
Report any compounds that are in low supply.
Streak Isolation from a mixed broth
Materials
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
1 blood plate per student.
Bacterial Culture: 0.05ml Escherichia coli (Ec) mixed with 5ml of Streptococcus mitis
(Stmi) in one T-soy broth tube.
Procedure
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.
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General procedure for the Streak Isolation Technique.
B, 10 streaks through A
A, 1 cm
smear
C
E
D
Figure by Patricia G. Wilber
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III.
Results and Interpretation
Gram stain.
Figure 5-2: Gram(+) Cocci
Figure 5-3: Gram(+) Rods
Accessed_7/20/2015_from_http://commons.wikimedia.org/wik
i/File:Purulent_inflammation,_Gram_stain_3.jpg. The author is
listed as “Patho” and this file is licensed under the Creative
Commons Attribution-Share Alike 3.0 Unported license.
Figure 5-4: Gram(-) Rods
Accessed_6/20/2015_from_http://commons.wikimedia.org/wi
ki/File:Bacillus_species.jpg The author is Dr. Sahay and this file is
licensed under the Creative Commons Attribution-Share Alike
3.0 Unported license.
Figure 5-5. Gram(-) Spirilli
Accessed_6/20/2015_from_http://commons.wikimedia.org/wi
ki/File:E_choli_Gram.JPG The author is Bobjgalindo and this file
is licensed under the GNU Free Documentation License.
Accessed 11/19/15:
"Botony Exam 1 004" by pookypoo87 - Taken personally in
Peace College Lab. Licensed under Public Domain via Commons
https://commons.wikimedia.org/wiki/File:Botony_Exam_1_00
4.JPG#/media/File:Botony_Exam_1_004.
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Bacteria: _______________________
Bacteria: _______________________
TM used:_____________
TM used:_____________
Field diameter: __________
Field diameter: __________
Number of cells that fit across: ______
Number of cells that fit across: ______
Cell size in mm:___________
Cell size in mm:___________
Cell size in μm:___________
Cell size in μm:___________
Cell shape: ______________
Cell shape: ______________
Cell arrangement:__________________
Cell arrangement:__________________
Cell color_____________
Cell color_____________
Gram(+) or Gram(-)? _______________
Gram(+) or Gram(-)? _______________
Table 5-2: Results of Gram Stain
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Day Two
Streak isolation Results
Draw or photograph your streak isolation results here:
Do you have two different types of colonies?
If yes, briefly describe each colony type.
Hold the plate up to the light. Can you see through the plate around any of the colony
types?
Note which types have this clearing.
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Post-Lab Questions
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. What is the purpose of a streak isolation?
6. How did your streak isolation from the mixed broth compare to your first streak
isolation?
7. Did you get isolated colonies? If not, what could you do differently next time?
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The authors of this lab unit would like to thank Andrea Peterson and Deyanna Decatur for testing new media and
organisms, our associate dean Linda Martin for many kinds of aid, Michael Jillson and Alex Silage for IT support, and our
dean John Cornish.
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