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Exercise 4: Staining Techniques in Microbiology

1. INTRODUCTION

Studying bacteria and other microorganisms in their natural state can be difficult. Aside from being
extremely small, bacteria and microorganisms are also colorless and transparent when examined under
the microscope. To visualize them, stains or dyes are used to impart color and provide contrast to their
surroundings.

Stains used in microbiology are either basic, acidic, or neutral. They are salts composed of a positive and
a negative ion. The chromatophore is the colored ion that imparts color. In general, dyes are classified as
acidic if the chromatophore has a negative charge. Acidic dyes react with the positively charged
components of the cell like the cytoplasm. Because of this, acidic dyes are referred to as cytoplasmic
dyes. Examples of acidic dyes are nigrosin and India ink.

On the one hand, basic dyes, also called nuclear stains, have positively charged chromatophores that
stain those cell parts that are negatively charged. Basic dyes include crystal violet, methylene blue,
safranin, and malachite green. Neutral stains, on the other hand, consist of a mixture of acidic and basic
dyes.

There are three staining techniques commonly used in microbiology. These are the simple, differential,
and special or structural staining techniques. In simple staining, a single basic dye is used to color and
highlight the entire organism. Differential stains distinguish bacteria according to their reaction to
particular stains. An example of a very useful differential staining technique is the Gram stain procedure
that classifies bacteria as either Gram-positive or Gram-negative. Special or structural stains are used to
color a specific part or structure of a microorganism, like the endospore, flagellum, or capsule.

Prior to the application of a stain, a thin film of the organisms is prepared, dried, and fixed onto a clean
slide. This is called a smear.

II. OBJECTIVES

At the end of the exercise, the students should be able to:

1. know how to prepare bacterial smears,

2. differentiate staining techniques:
3. describe bacterial morphology, arrangement, and staining affinity: and
4. classify bacteria according to reaction to the Gram stain method.
II. MATERIALS AND PROCEDURES (refer to lab modules videos)

Materials

Alcohol lamp
Compound microscopes
Droppers
Glass slides
Inoculating loops

Stains and reagents:

Crystal violet
Malachite green
Methylene blue
Safranin
Acid alcohol
Gram's iodine

Bacterial cultures:

Bacillus subtilis
Enterococcus
Escherichia coli
Staphylococcus aureus
Streptococcus

Procedures

Preparation of bacterial smear

1. Place a drop of distilled water at the center of a clean slide.
2. Touch a bacterial colony lightly using a sterilized and cooled inoculating loop, Pick up a small amount
of bacteria.
3. Mix and emulsify the colony to make a thin smear about 5 inch in diameter.
4. Air dry and fix by passing the slide, smear side up, over the flame of an alcohol lamp.

Simple Staining
1. Prepare bacterial smears of the different organisms.
2. Cover the smears with the stains. Use a different stain for each smear. After one minute, gently wash
off the excess stain with water.
3. Dry the smears by gently blotting with soft tissue paper.
4. Examine the smears under the LPO, HPO, and oil immersion objectives.
Gram Staining
1. Prepare bacterial smears of different organisms.
2. Apply crystal violet on the smears. After 60 seconds, rinse with tap water. Do not allow the stain to
dry on the slide.
3. Apply Gram's iodine for one minute, then rinse with tap water.
4. Decolorize by applying acid alcohol, one drop at a time, while holding the slide at an angle to drain.
Stop applying acid alcohol when there is no more color or stain seen in the rinse. This usually takes
about 5-10 seconds only.
5. Wash immediately with tap water and counterstain with safranin seconds.
6. Wash, blot-dry, and examine the slide under the HPO and O10.

Endospore Staining

1. Prepare a bacterial smear of Bacillus subtilis.

2. Cover the smear with malachite green and steam it over a flame for five minutes. Do not boil. Make
sure that the stain does not dry out by adding more stain if needed.
3. Drain excess stain and rinse slide with water.
4. Apply safranin to the smear and leave for one minute.
5. Wash slide with water to remove excess stain and blot-dry.
6. Examine the slide under the HPO and OIO. Endospores will appear green while the other parts of the
cell will be red.

IV. ILLUSTRATIONS

Draw an example image of bacteria under the three staining method.

Simple Staining Gram Staining Endospore Staining

V. RESULTS AND DISCUSSIONS

1. In the preparation of a bacterial smear, why is there a need to fix the bacteria to the slide? Aside
from passing the slide over a flame, what are the other ways of fixing the bacteria to the slide?
- Bacteria needs to be fixed on the slide to prevent the sample from being lost during a staining
procedure. Aside from passing the slide over a flame, chemical fixation is another way of fixing the
 bacteria to the slide. It Is a technique to fix a specimen with chemicals to prevent autolysis by the action
of enzymes and deformation of morphologies during specimen preparation. Biological tissues start
autolysis caused by their enzymes immediately after stopping the activities of them.
2. What information about bacteria can be observed using only a simple stain?
- Simple staining can be used for all types of bacterial cells to give contrast to the other- wise colorless
cell in order to determine cell morphology, size, and cell grouping. This technique
is simple because only one dye is used and direct because the actual cell is stained.
3. List in the correct order the reagents used for Gram staining and the purpose of each.
- Crystal Violet- stain all cells in purple.
- Gram’s Iodine- this reagent serves as killing agent and mordant, a substance that increases the cell’s
affinity for a stain.
- Ethanol or Acetone- this reagent serves a dual function as a protein-dehydrating agent and as a lipid
solvent.
- Safranin- used to stain pink those cells that have been previously decolorized.

VI. QUESTIONS FOR RESEARCH

1. What are the advantages of using stains in microbiology?
 The most basic reason that cells are stained is to enhance visualization of the cell or
certain cellular components under a microscope. Cells may also be stained to highlight
metabolic processes or to differentiate between live and dead cells in a sample. 
2. What is responsible for the differences in the Gram stain reactions of the above
microorganisms?
  Due to differences in the thickness of a peptidoglycan layer in the cell membrane
between Gram positive and Gram negative bacteria, Gram positive bacteria (with a
thicker peptidoglycan layer) retain crystal violet stain during the decolorization process,
while Gram negative bacteria lose the crystal violet
3. What is the most important reagent in the Gram stain method? The least important?
4. What may be the effects of the following on the results of the Gram staining method?
Over-decolorization- lead to an erroneous result where gram-positive cells may stain pink
to red indicating a gram-negative result,
Under-decolorization- under-decolorizing will lead to an erroneous result where gram-
negative cells may appear blue to purple indicating a gram-positive result.
Old bacterial cultures- .Cells from old cultures may stain Gram negative even if
the bacteria are Gram positive.
5. Name another differential stain used in microbiology, and identify its purpose, the reagents
utilized, and their uses.
DIFFERENTIAL PURPOSE REAGENTS UTILIZED USES
STAIN
ENDOSPORE A differential stain used to  Malachite green  water soluble and has a low affinity
STAIN visualize for cellular material, so vegetative
bacterial endospores cells may be decolourized
with water.
 to was away from the malachite dye
 Decolorization(wate from vegetative forms.
r)  to stain the vegetative cells that lost
the primary stain.
  Safranin
ACID FAST STAIN A differential stain used to  Carbol fuchsin  used as the primary stain dye to
identify acid-fast organisms detect acid-fast bacteria because it
such as members of the is more soluble in the cells wall
genus Mycobacterium . lipids than in the acid alcohol.
 Acid alcohol   The ability of the bacteria to resist
decolorization
with acid (1%) alcohol confers acid 
-fastness to the bacterium.
 Methylene blue  The main aim of this staining is to
differentiate bacteria into acid
fast group and non-acid
fast groups.
METACHROMATI Is the one that produces in ALBERT’S A
C certain elements a color SOLUTION
STAIN different from that of  Toluidine blue  Use of toluidine blue in tissue
the stain itself. sections is done with the aim to
highlight components, such as mast
cells granules, mucins, and
 Malachite cartilage.
green  malachite green stains the
cytoplasm blue-green
 Glacial acetic
acid
 Ethyl alcohol
ALBERT’S B
SOLUTION
 Iodine
 Potassium
iodide
CAPSULE STAIN  To distinguish capsular  Crystal Violet  To act as a fixative
material from the  Increase penetration power
bacterial cell.   Stain the cells (being a basic
 The capsule
dye)
stain employs an
acidic stain and a  Decrease pH of smear
basic stain to
detect capsule productio
n.
 Nigrosin  A negative staining technique
that is essentially used to
identify the presence of
capsules. Because of its acidic
nature, India ink (or Congo
red, nigrosin) stains the
background dark.