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Cities of Mandaluyong and Pasig
COLLEGE OF ARTS AND SCIENCES

BIO221L
MICROBIOLOGY-LABORATORY
LABORATORY EXERCISE 3
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Laboratory Exercise 3: Preparation of Bacterial Smears and Introduction to

Staining

Objectives
1. Learn the differences between simple staining and differential staining techniques.
2. Learn how to prepare a bacterial smear from cultured organisms.
3. Learn the differences between gram-positive and gram-negative bacteria.
4. Learn how to perform the gram stain procedure.
5. Use microscopy to examine gram stained cells.
6. Learn about some special staining procedures, and view examples of these under oil immersion.

Key Terms: Gram stain, bacterial smear, simple stain, differential stain, Gram positive, Gram
negative, Gram variable, capsule, spirochete, flagella, negative staining, silver stain

Introduction
Most types of cells do not have much natural pigment and are therefore difficult to see under the light microscope
unless they are stained. Several types of stains are used to make bacterial cells more visible. In addition, specific
staining techniques can be used to determine the cells’ biochemical or structural properties, such as cell wall type
and presence or absence of endospores. This type of information can help scientists identify and classify
microorganisms, and can be used by health care providers to diagnose the cause of a bacterial infection.

One type of staining procedure that can be used is the simple stain, in which only one stain is used, and all types
of bacteria appear as the color of that stain when viewed under the microscope. Some stains commonly used for
simple staining include crystal violet, safranin, and methylene blue. Simple stains can be used to determine a
bacterial species’ morphology and arrangement, but they do not give any additional information.

Scientists will often choose to perform a differential stain, as this allows them to gather additional information
about the bacteria they are working with. Differential stains use more than one stain, and cells will have a different
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appearance based on their chemical or structural properties. Some examples of differential stains are the Gram
stain, acid-fast stain, and endospore stain. In this lab you will learn how to prepare bacterial cells for staining, and
learn about the gram staining technique.

The Gram Stain

This very commonly used staining procedure was first developed by the Danish bacteriologist Hans Christian
Gram in 1882 (published in 1884) while working with tissue samples from the lungs of patients who had died
from pneumonia. Since then, the Gram stain procedure has been widely used by microbiologists everywhere to
obtain important information about the bacterial species they are working with. Knowing the Gram reaction of a
clinical isolate can help the health care professional make a diagnosis and choose the appropriate antibiotic for
treatment.

Gram stain results reflect differences in cell wall composition. Gram positive cells have thick layers of a
peptidoglycan (a carbohydrate) in their cell walls; Gram negative bacteria have very little. Gram positive
bacteria also have teichoic acids, whereas Gram negatives do not. Gram negative cells have an outer membrane
that resembles the phospholipid bilayer of the cell membrane. The outer membrane contains lipopolysaccharides
(LPS), which are released as endotoxins when Gram negative cells die. This can be of concern to a person with
an infection caused by a gram negative organism.

Figure 1. shows the major differences between the Gram-positive and Gram-negative cell walls (also refer to your
textbook for additional information). The differences in the cell wall composition are reflected in the way the
cells react with the stains used in the Gram stain procedure.

Gram stains are best performed on fresh cultures—older cells may have damaged cell walls and not give the
proper Gram reaction. Also, some species are known as Gram-variable, and so both Gram positive and Gram
negative reactions may be visible on your slide.

Although the vast majority of bacteria are either Gram positive or Gram negative, it is important to remember that
not all bacteria can be stained with this procedure (for example, Mycoplasmas, which have no cell wall, stain
poorly with the Gram stain)
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Figure 1.
Teichoic acids
Gram Positive Cell Wall

Peptidoglycan

Cell membrane

Gram Negative Cell Wall

LPS Porin

Outer membrane

Cell membrane

Special Stains

There are a variety of staining procedures used to identify specific external or internal structures that are not found
in all bacterial species (see table at the end of this exercise for a comparison of staining procedures). You will do
some of these staining procedures in the next lab (acid-fast staining and endospore staining). In today’s lab, you
will observe prepared slides of special stains: a capsule stain (Klebsiella pneumoniae), flagella stain (Proteus
vulgaris) and spirochete stain (Treponema pallidum).

Capsule Stain

Some bacteria secrete a polysaccharide-rich structure external to the cell wall called a glycocalyx. If the
glycocalyx is thin and loosely attached, it is called a slime layer; if it is thick and tightly bound to the cell, it is

called a capsule. The glycocalyx can protect the cell from desiccation and can allow the cell to stick to surfaces
like tissues in the body. They may also provide cells with protection against detection and phagocytosis by
immune cells and contribute to the formation of a biofilm: in this way a glycocalyx can act as a virulence factor;
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(contributes to the ability of an organism to cause disease).

Capsules can be detected using a negative staining procedure in which the background (the slide) and the bacteria
are stained, but the capsule is not stained. The capsule appears as a clear unstained zone around the bacterial cell.
Since capsules are destroyed by heat, the capsule staining procedure is done without heat-fixing the bacteria.

Silver Stain

Flagella (long whip-like structures used for bacterial motility) and some bacteria (e.g. spirochetes) are too thin
to be observed with regular staining procedures. In these cases, a silver stain is used. Silver nitrate is applied
to the bacteria along with a special mordant; the silver nitrate precipitates around the flagella or the thin bacteria,
thus thickening them so they can be observed under the light microscope.

Procedures

A. Preparation of a Bacterial Smear and Gram Staining

This semester you will be performing three staining procedures: Simple stain, Gram stain, and endospore stain.
All three of these staining procedures begin with the preparation of a bacterial smear.

Materials

• Clean microscope slides

• Staining trays and newspaper

• Gram stain reagents: crystal violet, Gram’s iodine, safranin, 95% ethanol

• Water bottle (for rinsing)

• Bacterial cultures: (sub cultured from exposed Petri dishes)

How to make a bacterial smear

1. Label a clean glass slide as demonstrated by your instructor.

2. Add a small drop of saline to the slide (you will usually put two bacteria on one microscope slide- Follow
your instructor’s specific instructions). This can be done by placing a drop of saline onto your inoculation
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loop and then transferring it to the slide. If you use the saline dropper directly on the slide, do not release a
full drop.

3. With an inoculation loop or needle, pick up a small amount of bacteria. Mix it well with the saline and
spread the mixture over a wider area of the slide.
• Be careful not to have the two smears run into each other.
4. Air dry the bacterial specimen on the slide (slide warmers may also be used).
5. When slides are completely air-dry, heat fix the bacterial specimen by passing the slide slowly over the
flame twice (your instructor will demonstrate this).
• Heat fixing kills cells, and adheres them to the slide.
• Cells will be rinsed off the slides if they are not heat fixed properly.
• Be careful not to overheat the slides in this procedure

After heat-fixing is complete, you are ready to gram stain your slide.

Figure 2. Heat fixation

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GRAM STAINING PROCEDURE

• For all steps in the gram staining procedure, add enough of the solution to cover the areas of the slide
that have bacteria on them. You do not need to flood the entire slide.
• All staining should be done over a staining tray. Be sure to put newspaper under the tray in case of
spillage.
• Gloves should be worn while staining and removed before working with the microscope.

STEPS EXPLANATION
1. Add a few drops of crystal violet (primary
stain) to the smear and let it sit for 60 seconds.
All cells are purple after this step. Stopping
2. Rinse the slide with water.
here would be a simple stain.
3. Add a few drops of Gram's iodine (mordant) to Gram’s iodine forms a complex with crystal
the smear and let it sit for 60 seconds. violet
All cells are purple after this step.
4. Rinse the slide with water.

5. Decolorize with 95% ethanol: let the alcohol

Gram positive cells retain crystal violet and
run over surface of slide until no more crystal
remain purple
violet color comes out of the smear (time
Gram negative cells lose crystal violet and
varies—no more than 5-10 seconds).
are now colorless

6. Rinse with water.

Water rinse stops the decolorization process
7. Add a few drops of safranin (counterstain) and
Safranin is a pink/red dye
let it sit for 60 seconds.
8. Rinse with water. Blot dry on bibulous paper. Gram positive cells remain purple; gram
Be careful not to wipe off the bacteria. negative cells are now pink/red
9. Observe your slide under the microscope. For each organism, determine morphology,
arrangement and Gram reaction
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Gram Stain Results

Draw sketches for each type of bacteria that you observe. Identify its morphology, arrangement, and Gram
reaction.

Species Species
Morphology Morphology
Arrangement Arrangement
Gram reaction Gram reaction

Species Species
Morphology Morphology
Arrangement Arrangement
Gram reaction Gram reaction
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Simple Stain Results

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Endospore Stain Results

Observe the special stains set up at the demo microscopes at the back of the lab. Make sketches below.

Species Species
Staining technique Staining technique

Comments: Comments:

Species
Staining technique

Comments:
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Summary of Common Bacterial Staining Techniques

Simple Stains • Used to provide color to otherwise transparent
bacterial cells
Crystal Violet, Methylene Blue, Safranin • Can be used to determine cell size, morphology
and arrangement

Gram Stain • Common differential stain

• Gram reaction (positive or negative) reflects cell
Primary stain – crystal violet wall properties
Mordant – iodine; decolorizer- 95% Ethanol • Also used to determine cell size, morphology and
arrangement
Counterstain – Safranin

Acid-Fast Stain • A differential stain used to detect bacteria with

Primary stain – Carbol fuchsin mycolic acid cell walls (genera Mycobacterium
and Nocardia)
Decolorizer – acid alcohol • Developed to detect the bacterial species that
causes tuberculosis
Counterstain – Methylene blue
• Acid-fast organisms resist decolorization with
acid-alcohol

Endospore Stain • Endospores resist staining with basic stains

Primary stain: Malachite green • Endospores stain with malachite green;
vegetative cells stain with safranin
Counterstain: safranin

Capsule Stain (Negative staining) • Negative stains are neither heat-fixed nor rinsed
Uses an acidic stain: (Congo red or Nigrosin) • The background of the slide is stained by acidic
stains (capsule remains unstained)
and a basic stain: (crystal violet or safranin) • The cells within the capsule are stained with
Basic stains
• Examples of encapsulated cells: Bacillus
anthracis, Streptococcus pneumoniae, and
Klebsiella pneumonia

Flagella Stain • Used to see bacterial flagella that are too slender
Silver nitrate to be seen with other staining techniques
• Silver nitrate makes flagella appear larger than
they are
• Can be used to determine arrangement of flagella
for identification.
o Ex: Proteus vulgaris has peritrichous
flagella
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Spirochete stain • Used to visualize slender spirochetes like

Silver nitrate Treponema pallidum

Review Questions
1. Explain the major differences between the gram positive and gram negative cell wall.

2. Salmonella typhi is a gram negative organism.

a. What color will it appear when simple stained with crystal violet?

b. What color would it be if it was gram stained correctly?

3. Explain how bacterial cells would look in the gram staining procedure if the following mistakes were made:
a. Decolorizer left on too long

b. Decolorizer not left on long enough

c. Slide not heat-fixed before staining

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4. What is the difference between a simple stain and a differential stain?

5. Explain why it is important to use only a small amount of bacteria when preparing a smear.

6. What are the two things that are stained in a capsule stain?

7. What is NOT stained in a capsule stain?

8. Why would a health care provider be interested in knowing the gram stain results of a bacterial sample?
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Cities of Mandaluyong and Pasig
COLLEGE OF ARTS AND SCIENCES