Lab Manual 2

Staining Techniques
LABSTER: The Gram Stain: Identify and differentiate bacteria
Laboratory Workflow
 Read the lab manual
 Attend the face-to-face laboratory session and write your results in your lab
notebook
 Write your laboratory report and turn it on Blackboard before the due date
Objectives Following the completion of this lab module students should…
 Understand the concepts of direct stain or indirect stain.
 Learn to differentiate between gram-positive and gram-negative bacteria under
the microscope.
 Learn necessary troubleshooting skills of gram stain.
Introduction
(1) Direct Stain and Indirect Stain
Bacteria are trans lucid microscopic organisms which are challenging to observe even
while under a microscope. Special staining techniques are required to differentiate
bacteria from the background. Furthermore, other staining techniques are used to
determine bacterial structures.

There are two types of staining techniques: direct stain and indirect stain.

The direct stain technique is used to stain the bacterial cytoplasm. This technique
requires the use of basic dyes, which are positively charged and interact with the
negative charges present in the bacterial cytoplasm. The reaction stains the bacterium,
trapping the basic dye inside the cell (Figure 1). In comparison, the background remains
translucid after the dye is washed. Some examples of basic dyes include crystal violet,
safranin, basic fuchsin, and methylene blue.

The steps of the direct stain technique are described as follows:

1. With a sterile bacterial loop, spread a drop of liquid culture on a glass slide.
2. Wait until the sample dries over the glass.
 3. Pass the glass slide over a Bunsen burner flame two or three times (Heat
fixing).
4. Add some drops of the basic dye into the slide.
5. Wait one minute, remove the excess dye with water.
6. Let the slide dry and view it under the microscope.

Heat fixing is the most crucial step during this technique. A brief heat fixing step may
cause the bacteria to be washed during the method. Slide overheating may cause the
bacteria to explode, modifying its morphology. Heat fixing a wet slide might interfere
with the bacteria morphology as well.

The indirect stain technique is used to stain the background instead. The dyes used for
this technique are called anionic dyes. Acidic dyes are negatively charged and are
repelled by the negative charges inside the cell. The cell's background is stained while
the bacterium remains translucid (Figure 2). Some examples of acidic dyes are
nigrosine, Indian ink, and Congo red.

The steps of the indirect stain technique are described as follows:

1. Add a drop of liquid culture close to one of the glass slides borders.
2. Add a drop of acidic die to the bacterial culture.
3. Take a second glass slide and place it over the sample on a 45° angle.
a. Make sure that the bacterial sample is mixed while touching the
second slide.
4. With a single movement, spread the bacteria forming a smear over the slide
surface.
5. Let the slide dry and view it under the microscope

The smear step is the most crucial step during this technique. Spreading the bacteria in
multiple movements may create artifacts in the microscope. Uneven distribution of the
sample over the slide would make the sample interpretation challenging.
Figure 1 Basic and Acidic dye interaction with the bacterial cell.

(2) The Gram Stain

The Gram stain is a differential staining technique. It divides bacteria into two types:
Gram-positive bacteria and Gram-negative bacteria. During Gram stain, bacteria are
dyed according to their cell wall structural characteristics.
The Gram stain was developed by the Danish bacteriologist Hans Christian Gram. This
technique is essential in a microbiology laboratory. Dr. Gram developed the staining
technique while working with lung tissue from patients who had died from pneumonia.
He discovered that bacteria fall into two distinct categories after being stained
sequentially with crystal violet, iodine, decolorizer (acetone-alcohol solution), and a
counterstain.
One group of bacterial cells retained the crystal violet after being washed with the
decolorizer (Gram-positive), while the other group did not (Gram-negative). Dr. Gram
covered the bacterial cells that did not retain crystal violet with a second dye
(counterstain). Safranin was used as a counterstain. The bacterial cells stained red with
counterstain dye.
As mentioned before, Gram stain differentiates bacteria into the two distinct categories
is based on structural differences in their cell wall.
Gram-positive bacteria have a thick cell wall composed of a peptidoglycan layer (Figure
2). These cells retain the crystal violet during decolorization and are dyed purple (Figure
3).

Gram-negative bacteria have a thin cell wall instead. The peptidoglycan content of the
wall is less than in Gram-positive bacteria. Furthermore, a Gram-negative bacterium
has two cytoplasmic membranes (Figure 2). These cells do not retain the crystal violet
during decolorization and are dyed pink with the counterstain dye safranin (Figure 3).

Figure 2. Gram-Negative and Gram-Positive bacterial cell wall.

Figure 3. Gram stain procedure.

(3) Troubleshooting the Gram Stain technique.
The Gram stain technique accuracy relies on the decolorization step. Excess of
decolorizer may wash the crystal violet retained inside the Gram-positive bacteria. The
decolorized Gram-positive bacteria will get dyed pink with safranin. The event would
create a false negative that would interfere with our results. However, not adding
enough decolorizer is also a problem. Gram-negative bacteria that are not appropriately
decolorized still retain the crystal violet. These cells are dyed purple and are interpreted
as a false positive.
The decolorization step should be standardized to obtain reliable results. Adding the
same amount of decolorizer and letting it act the same time for each sample should
decrease these errors.

Protocol
Each team will be provided with three liquid bacterial cultures:
1. Escherichia coli
2. Staphylococcus aureus
3. Unknown culture
Part I: Direct stain
1. Prepare a heat-fixed smear of the culture you would like to examine.
a. Sterilize the inoculating loop in the flame of the Bunsen burner until it turns
red.
b. Let the loop cool.
c. Place the loop into the bacteria liquid culture.
d. Touch microscope slide with the loop and spread the liquid across the
slide to coat an area in which you will be able to stain.
e. Let the slide air dry.
f. Once dried, pass the slide through the flame of the Bunsen burner to heat-
fix the bacteria to the slide, keep on passing the slide a couple of times
until the slide is warm to the touch.
NOTE: Do not hold the slide in the flame of the Bunsen burner.
2. Cover the smear with the safranin dye.
3. Allow the dye to remain on the smear for one minute.
4. Wash the excess dye off the slide with distilled water.
a. Pick up the slide by one end and hold it at a 45° angle over the staining
tray.
 b. Using the bottle of distilled water, gently wash off the excess dye from the
slide by directing a gentle stream of water at the top of the slide. Let the
distilled water run down the slide until it is clear.
5. Let the slide air dry. If needed, the excess water may be blotted off by gently
placing the slide in a folded KimWipe® and press down gently.
*NOTE: Do not rub the slide.
6. Examine the slide under the microscope under 10x, then 40x
7. Record the bacterium shape and arrangement.
8. Take a picture of the view under the microscope.
Part II: Indirect stain
1. Place a small drop of the nigrosin dye near the end of the slide.
2. Transfer one loop full of the liquid culture of bacteria you would like to examine
into the dye drop and mix the two together.
a. Sterilize your loop just as you did in Part I Step 1 a-f.
3. Hold a second, clean, slide at a 20° angle to the first slide.
4. Touch the edge of the clean slide to the mixture so that the mixture spreads
across the edge.
5. Push the bacteria-dye mixture across the surface of the slide by firmly pushing
the second slide across the first slide while making sure that the smear is thin.
*NOTE: Smear the bacterial solution in a single move for a better result.
6. Let the slide air dry.
7. Examine the slide under the microscope under 10x, then 40x.
8. Record the shape and arrangement.
9. Take a picture of your view
Part III Gram stain of the bacteria
1. Prepare a heat-fixed smear of the liquid culture of bacteria.
a. Sterilize your loop just as you did in Part I Step 1 a-f.
2. Place the slide on a staining rack and carefully cover the smear with crystal
violet. Let the dye remain on the slide for one minute.
3. Pick up the slide by one end and hold it at a 45° angle over the staining tray.
4. Using the bottle of distilled water, gently wash off the excess dye from the slide
bydirecting a gentle stream of water at the top of the slide. Let the distilled water
rundown the slide until it is clear. NOTE: Do not aim the water directly at the
smear as it will wash the smear off the slide.
5. Place the slide back onto the staining rack and carefully cover the smear with
Gram’s iodine. Let the dye remain on the slide for one minute.
6. Pick up the slide by one end and hold it at a 45° angle over the staining tray.
7. This time, using the decolorizer, gently wash off the excess dye from the slide by
directing a gentle stream of the acetone-alcohol solution at the top of the slide.
8. Let the decolorizer run down the slide until it is clear. NOTE: Do not aim the
decolorizer directly at the smear as it will wash the smear off the slide.
 9. Place the slide back onto the staining rack and carefully cover the smear with
safranin. Let the dye remain on the slide for one minute.
10. Using the bottle of distilled water, gently wash off the excess dye from the slide.
11. Let the slide air dry NOTE: Do not rub the slide with paper towels.
12. Examine the slide under the microscope under 10x, then 40x
13. Record the shape, arrangement, and gram of the organism.
Required Lab Report Sub-Sections & Content Expectations
Note:
Consult your AI/TA to meet the lab report expectations.