BI103 General Biology

Practical 2: Identifying the Bacteria by Simple and Differential Staining

Methods

Learning Objectives
The learning objectives of this practical is:
 To identify the morphology and arrangement of the bacterial cells.
 Identification and characterization of bacteria by using differential (Gram’s) staining.

I. Simple- Staining
Introduction:
Bacteria are microscopic organisms that cannot be seen with unaided eye. They can be seen even
in unstained preparations such as a wet mount or hanging drop preparation but the morphology is
not clear. Bacteria are colorless and when suspended in saline they don’t offer any contrast.
Besides, bacterial motility makes it difficult to observe the morphology clearly. Hence, bacteria
have to be stained to observe them. The dyes often used are toxic chemicals that kill the bacteria.
The process of smearing, fixing and drying often kill the bacteria. This process fixes the bacteria
to the slide and their position on slide remains unaltered. Staining can be performed with basic
dyes such as crystal violet or methylene blue, positively charged dyes that are attracted to the
negatively charged materials of the microbial cytoplasm. Such a procedure is the simple stain
procedure. An alternative is to use a dye such as nigrosin or Congo red, acidic, negatively charged
dyes. They are repelled by the negatively charged cytoplasm and gather around the cells, leaving
the cells clear and unstained. This technique is called the negative stain technique.
The simple stain can be used as a quick and easy way to determine cell shape, size and
arrangements of bacteria. True to its name, the simple stain is a very simple staining procedure
involving single solution of stain. Any basic dye such as methylene blue, safranin, or crystal violet
can be used to color the bacterial cells.

Method and Materials:

Following are the steps undertaken in laboratory while preparing microscope slide for simple
staining to be examined under the microscope. The pictures of the following steps are provided in
practical 2 worksheet, use these pictures to familiarize yourself with simple staining procedures.
Preparation of a smear and heat fixing:
1. Using a sterilized inoculating loop, transfer loopful of liquid suspension containing bacteria
to a slide (clean grease free microscopic slide) or transfer an isolated colony from a culture
plate to a slide with a water drop.
2. Disperse the bacteria on the loop in the drop of water on the slide and spread the drop over
an area the size of a dime. It should be a thin, even smear.
3. Allow the smear to dry thoroughly.
4. Heat-fix the smear cautiously by passing the underside of the slide through the burner flame
two or three times. It fixes the cell in the slide. Do not overheat the slide as it will distort
the bacterial cells.
Staining:
1. Cover the smear with methylene blue and allow the dye to remain in the smear for
approximately one minute (Staining time is not critical here; somewhere between 30
seconds to 2 minutes should give you an acceptable stain, the longer you leave the dye in
it, the darker will be the stain).
2. Using distilled water wash bottle, gently wash off the excess methylene blue from the
slide by directing a gentle stream of water over the surface of the slide.
3. Wash off any stain that got on the bottom of the slide as well.
4. Wipe the back of the slide and blot the stained surface with bibulous paper or with a paper
towel.
5. Place the stained smear on the microscope stage smear side up and focus the smear using
the 10X objective.
6. Choose an area of the smear in which the cells are well spread in a monolayer. Center the
area to be studied, apply immersion oil directly to the smear, and focus the smear under
oil with the 100X objective.

Note: The bacterial cells usually stain uniformly and the color of the cell depends on the type of
dye used. If methylene blue is used, some granules in the interior of the cells of some bacteria may
appear more deeply stained than the rest of the cell, which is due to presence of different chemical
substances.

Left: Cocci in Cluster; Right: Bacilli

(Image source: microrao.com)
Results:
Complete the table in the worksheet on steps for simple staining and describe Bacillus subtilis.

II. Differential-Staining (Gram- Staining)

Principle:
Simple stains like methylene blue 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:

I. 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.
II. 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. Gram-staining begins by getting
cells to stick on a clean microscope slide. Such a prep is called a bacterial smear. To a bacterial
smear the following chemicals are applied to make a Gram-stain:
1. Gram’s Crystal Violet Stain

The smear is flooded with Crystal Violet for 60 seconds. Crystal violet is a purple chemical that
sticks to the peptidoglycan layer of the bacterial cell wall. After 60 seconds, crystal violet is rinsed
off using distilled water.

2. Iodine

The smear is next flooded with Iodine for 60 seconds. The iodine is called a mordant- it causes
crystal violet to stick to peptidoglycan like mortar causes bricks to stick together. After 60 seconds,
iodine is rinsed off using distilled water.

3. Decolourizer (Ethanol Wash)

The smear is next flooded with alcohol for JUST 5 SECONDS. The alcohol washes crystal violet
out of the Gram-negative cell wall. The Gram-positive cell wall retains crystal violet as long as the
alcohol wash lasts not more than a few seconds. After 5 SECONDS, the alcohol is rinsed off using
distilled water. The alcohol wash is the differential step in the Gram-stain process. That is, it is the
alcohol that creates the observable difference: Gram-positive cells look purple after this step;
Gram-negative cells look clear.

4. Safranin Stain

The smear is flooded with Safranin for 90 seconds to two minutes. Safranin is a pink stain that
sticks to cytoplasmic components of the cell. All cells become stained with Safranin. Gram
negative cells, which were cleared in the previous step, end up looking pink. After 90 seconds to
two minutes (the longer the better), Safranin is rinsed off with distilled water.

5. Drying the slide

The completed Gram-stained slide is stuck into a book of bibulous paper and allowed to dry for a
minute or two. Once dry, the slide is ready for observation.

Method and Materials:

The steps of Gram staining is clearly explained in this video so watch it to understand better
https://www.youtube.com/watch?v=sxa46xKfIOY. Following are the steps undertaken in
laboratory while preparing microscopic slide for Gram staining to be examined under the
microscope.

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 aureus (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 colour
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 labelled waste container.
8. Cover the smear with Safranin for 1-2 minutes.
9. Wash both the top & the bottom of the slide with water.
10. Blot the slide with bibulous paper.
11. Use different objective lens including the oil immersion lens for better observation of the
bacterial cells on the smear.

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

Result:

Complete the task mentioned in the worksheet. Identify a typical microscopic view of both
Escherichia coli and Staphylococcus aureus. Describe cell size, shape, arrangement, and Gram-
reaction for the two microscopic images given.

Discussion Questions:

1. What colour are Gram-positive cells supposed to be after a Gram-stain? What about Gram
negative cells?
2. What characteristics can be determined in a Gram-stain?
3. List all of the things that can go happen during the Gram-staining process that could lead to an
incorrect or poor result.

Conclusion:

Write a simple conclusion.

Reference
USP library provides guide on how to reference using APA style. Use this link below to learn
how to do in-text citation and list references. https://usp.ac.fj.libguides.com/apa