LAB 3

Bacterial Staining Techniques II

I. Differential Stains: Gram Stain and Acid-fast Stain
II. Morphological Unknown

I. DIFFERENTIAL STAINS
A. Gram Stain
B. Acid-fast Stain

A. Gram Stain
The previous lab introduced simple staining techniques that enable microbiologists to observe the
morphological characteristics of bacteria. Although simple stains are useful, they do not reveal details
about the bacteria other than morphology and arrangement. The Gram stain is a differential stain
commonly used in the microbiology laboratory that differentiates bacteria on the basis of their cell wall
structure. Most bacteria can be divided into two groups based on the composition of their cell wall:

1) Gram-positive cell walls have a thick peptidoglycan layer beyond the plasma membrane.
Characteristic polymers called teichoic and lipoteichoic acids stick out above the peptidoglycan
and it is because of their negative charge that the cell wall is overall negative. These acids are
also very important in the body’s ability to recognize foreign bacteria. Gram-positive cell walls
stain blue/purple with the Gram stain.
2) Gram-negative cell walls are more complex. They have a thin peptidoglycan layer and an outer
membrane beyond the plasma membrane. The space between the plasma membrane and the outer
membrane is called the periplasmic space. The outer leaflet of the outer membrane is composed
largely of a molecule called lipopolysaccharide (LPS). LPS is an endotoxin that is important in
triggering the body’s immune response and contributing to the overall negative charge of the cell.
Spanning the outer membrane are porin proteins that enable the passage of small molecules.
Lipoproteins join the outer membrane and the thin peptidoglycan layer. Gram-negative cells will
stain pink with the Gram stain.

This is The Most Important staining technique in Bacteriology.

Cell wall structure of Gram+ and Gram-

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GRAM STAIN
Cell Color
Procedure Reagent Gram Positive Gram Negative
Fixed cells on slide COLORLESS COLORLESS
Primary stain Crystal Violet PURPLE PURPLE
Mordant Iodine PURPLE PURPLE
Decolorizer Alcohol PURPLE COLORLESS
Counterstain Safranin PURPLE RED

An easy way to remember the steps of the Gram stain is...

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PROCEDURE: (EACH STUDENT)
1. Using a sterile inoculating loop, add 1 drop of sterile water to the slide. Prepare a mixed smear of
Escherichia coli (G- rod) and Staphylococcus epidermidis (G+ coccus).
2. Air dry and Heat fix.
3. Cover the smear with Crystal Violet (primary stain) for 1 min.
4. Gently wash off the slide with water.
5. Add Gram’s Iodine (mordant) for 1 min.
6. Wash with water.
7. Decolorize with 95% ethanol. This is the "tricky" step. Stop decolorizing with alcohol as soon as the
purple color has stopped leaching off the slide (time will vary depending on thickness of smear).
Immediately wash with water. Be sure to dispose of all ethanol waste in the appropriately labeled waste
container.
8. Cover the smear with Safranin for 30 seconds.
9. Wash both the top & the bottom of the slide with water.
10. Blot the slide with bibulous paper.
11. Using the 10X objective lens, focus first on the line and then on the smear. Follow the focusing
procedure in Lab #1. Use the focusing procedure in Lab #2 to view the smear using the 100X (oil
immersion lens).
Focus line
E. coli

S. epidermidis

Note: Escherichia coli is a tiny pink (Gram-) rod. Staphylococcus epidermidis is a purple (Gram+) sphere
or coccus.

Draw a picture of a typical microscopic field and identify both Escherichia coli and Staphylococcus
epidermidis. Record this in the results section for this lab. Colored pencils are available throughout the room
on the chalkboard trays.

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B. Acid-fast Stain
Mycobacterium and many Nocardia species are called acid-fast because during an acid-fast staining
procedure they retain the primary dye carbol fuchsin despite decolorization with the powerful solvent
acid-alcohol (95% ethanol with 3% HCl). Nearly all other genera of bacteria are nonacid-fast. The acid-
fast genera have the waxy hydroxy-lipid called mycolic acid in their cell walls. It is assumed that
mycolic acid prevents acid-alcohol from decolorizing protoplasm. The acid-fast stain is a differential
stain.

Ziehl Neelsen Acid-fast stain

ACID-FAST STAIN
Cell Color
Procedure Reagent Acid-fast Bacteria Nonacid-fast Bacteria
Primary dye Carbolfuchsin RED RED
Decolorizer Acid-alcohol RED COLORLESS
Counterstain Methylene blue RED BLUE

PROCEDURE - (EACH STUDENT)

1. Add one loopful of sterile water to a microscope slide.
2. Make a heavy smear of Mycobacterium smegmatis. Mix thoroughly with your loop. Then transfer a
small amount of Staphylococcus epidermidis to the same drop of water.
You will now have a mixture of M. smegmatis and S. epidermidis.
3. Air dry and heat fix well.
4. Cover the smear with carbolfuchsin dye. Place a piece of paper towel on top of the dye. Be sure the
paper towel is saturated with the dye. Carbolfuchsin is a potential carcinogen. Please wear gloves when
working with this dye.
5. Place the slide on the rack over dry heat for 2 minutes.
6. Cool and rinse with water.
7. Decolorize by placing a drop of acid alcohol on the slide and allowing it to sit for 15 seconds.
8. Wash the top and bottom of slide with water and clean the slide bottom well.
9. Counterstain with Methylene Blue for 30 seconds to 1 minute.
10. Wash and blot the slide with bibulous paper.
11. Focus 10X - then use oil immersion.
M. smegmatis (heavy)

Focus line
S . epidermidis
Draw a typical microscopic field and record in Results Lab 3.
Note: The acid-fast Mycobacterium retains carbolfuchsin and stains hot pink. The Staphylococcus
epidermidis is decolorized and the counterstain colors them blue. See table for acid-fast stain.

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II. Morphological Unknown: continue your investigation of your morphological unknown.
1. Collect your unknown from the side bench. Verify that the # matches that used for your direct stain.
2. Perform a Gram stain and an Acid-fast stain as described in the above sections.
3. Record your observations in the Results section of Lab #4.

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NOTES

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 LAB 3 RESULTS
I. DIFFERENTIAL STAINS
A. Gram Stain Draw and label examples of Escherichia coli and Staphylococcus epidermidis.

B. Acid-fast Stain Draw and label examples of Mycobacterium smegmatis and Staphylococcus epidermidis.
Mycobacterium & Staphylococcus
smegmatis epidermidis

QUESTIONS:
1. What is the difference between a simple and a differential stain?__________________________
_______________________________________________________________________________

_______________________________________________________________________________
2. Describe the function of each of the following in the Gram stain
Mordant: __________________________________________________________________
Primary stain:_______________________________________________________________
Decolorizer: ________________________________________________________________
Counterstain: _______________________________________________________________
3. Which step in the Gram stain is most likely to cause poor results if done incorrectly? _________
_______________________________________________________________________________

4. Why must fresh bacterial cultures be used in a Gram stain? ______________________________

_______________________________________________________________________________

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5. Briefly describe the mechanism of Gram staining. _____________________________________

_______________________________________________________________________________

_______________________________________________________________________________

6. What is the primary stain used in the acid-fast staining procedure? ________________________

_______________________________________________________________________________
7. What is the purpose of the heat/steam during the acid-fast staining procedure? _______________

_______________________________________________________________________________

_______________________________________________________________________________
8. In a clinical microbiology laboratory, the acid-fast stain would be used for diagnosis of what diseases?

_______________________________________________________________________________
9. What makes a microorganism nonacid-fast? __________________________________________

_______________________________________________________________________________

Organisms introduced in this lab:

Mycobacterium smegmatis
Mycobacterium tuberculosis
Mycobacterium leprae
Staphylococcus epidermidis

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 GENERAL MICROBIOLOGY 2210: GRAM STAIN REPORT (15 PTS)
ASSIGNMENT
Each student will have the opportunity to submit one example of a Gram stain with a brief written report
describing the staining procedure and the stain itself. A good report will likely necessitate a minimum of 2
pages double-spaced and is limited to a 3-page maximum.
OVERALL FORMAT
Please include the following in your report:
A. Title Page with the following information: (1 point) __________
--Title: A title should be brief, creative, but descriptive of the work that was done.
--Your Name
--Course and Lab Section
--Date
B. Introduction: (4 points) __________
An introduction should include pertinent background information. In this case, the history of the Gram
stain and its significance in microbiology are relevant (1 point). A discussion of the mechanism of Gram
staining and how it differentiates bacteria on the basis of their cell wall structure should also be included
(1 point). The purpose and objectives of the experiment should be stated and a hypothesis should be
made (2 points). Remember that a hypothesis need not be correct, but it must be tested by the procedure.
Good hypotheses are also grounded in background knowledge and clearly state the predicted results.
Hypotheses can be of several types. 1 We will discuss two types here:
1. Manipulated hypotheses
This type of hypothesis is used when one variable (the independent variable) is manipulated by the
experimenter and the effects on a second variable (the dependent variable) are observed. This type of
hypothesis is generally written as an if, then statement (e.g. If the smear of the unknown bacterium is
decolorized as according to the above procedure (neither over- nor under-decolorized), then all the
cells will stain a single color.) In this example, decolorization is the independent variable (the
variable controlled by the experimenter) and the color of the cells is the dependent variable (the
variable affected by the changes in the independent variable).
2. Observational hypotheses
States something about the nature of an organism (e.g. The unknown bacterium will stain Gram-
positive.)
C. Materials and Methods: (4 points) __________
The Materials and Methods section should include a description of the procedure/steps in the Gram stain,
including the preparation of a bacterial smear and heat fixation (3 points). This section must be written
in complete sentences and in paragraph form (1 point). Illustrations may assist in clarifying points.
D. Results: (1 point) __________
In the results section, describe your Gram stain/s. Include at least one illustration of your own
creation.
E. Discussion: (3 points) __________
What features of your Gram stain exemplify the Gram stain theory (1 point)? If there are features of the
Gram stain that are not as expected, what possible sources or error may be responsible (e.g. poor quality
of the smear preparation)? (1 point) Discuss whether the hypothesis made in the introduction was tested
and if so was it supported? (1 point)

1
For a discussion of hypotheses formation please visit
http://www.utas.edu.au/sciencelinks/exdesign/HF2C.HTM
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OVERALL QUALITY
Students have the opportunity to earn points for reports that are well written, organized and edited.
(2 points) __________

DUE DATE: TAs will inform you of the due date.

*Please tear this grading key out of your lab book and attach it to your lab report at the time of
submission. Please also write your name and lab section on the frosted portion of your Gram stain
slide and hand it in with your written report.

TA comments: __________________________________________________________________________
_______________________________________________________________________________________
_______________________________________________________________________________________

TA’s suggestions for improving Gram stain: ___________________________________________________

_______________________________________________________________________________________
_______________________________________________________________________________________

Points earned out of 15 possible: ____________

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