Staining bacteria

Staining Bacteria
After completing this exercise you should be able to:
1. Understand the purposes of staining microorganisms.

Bacterial morphology can be examined in two ways: by observing living, unstained organisms and
by observing dead cells that are stained with dyes.

Living bacteria are almost colourless and lack sufficient contrast with the water in which they are
suspended to be clearly visible. Staining the organisms increases their contrast with their
surroundings so that they are more visible. Certain stains can help identify internal structures of
cells that would otherwise be unseen. Further, use of the oil-immersion objective of the
microscope to obtain the greatest magnification is more convenient with stained preparations than
with wet mounts.

Although bacteria do not look greatly different from their surroundings, they differ chemically.
Stain or dye reacts chemically with the bacterial cell but not with the background, enabling us to
distinguish the bacteria. Thus, the main advantages of staining are that it provides contrast
between microorganisms and their backgrounds, enables differentiation among various
morphological types, and enables study of internal structures of the bacterial cell such as the cell
wall, vacuoles, and spores.

To differentiate between different forms of microorganisms

Reasons for making and using stained preparations of microorganisms.

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 Staining bacteria

A wide range of dyes is available to the bacteriologist today and is used in various modifications of
basic staining techniques:

Simple Stains Differential Stains Structural Stains

Basic Dyes Gram Stain Endospore Stain

Acidic Dyes Acid-fast stain Capsule Stain

Indifferent Dyes Flagella Stain

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 Direct staining with basic dyes

Direct Staining with Basic Dyes

After completing this lab you should be able to:
1. Prepare and stain slides using the procedure for basic dye staining to enhance
bacterial structures for microscopic study.

To understand how a dye stains a bacterial cell, place a small drop of water on the slide and
you must first know what a dye is. Most dyes thoroughly mix with it a small bit of the
are salts, of which one of the ions is coloured. culture.
A salt is a compound composed of a positively 2. Spread the drop on the slide to form a thin film.
charged ion and a negatively charged ion. The Most students make the smear too heavy.
simple dye methylene blue is actually the salt
3. Allow the slides to dry in the air or hold them
methylene blue chloride, which dissociates as
high above a bunsen flame.
follows
4. When the film is dry, pass the slide, film side
MBC → methylene blue+ + chloride- up, three times through the bunsen flame.
Caution: Too much heat distorts the shapes
and structures of the microorganisms. The slide
The colour of the stain is in the positively charged should feel warm but not hot against the back
methylene blue ion. of your

Bacterial cells have a slight negative charge when

the pH of their surroundings is near neutrality, The purpose of fixation is to kill the
which it generally is. The negatively charged microorganisms, coagulate the protoplasm of the
bacterial cell combines with the positively charged cells, and cause them to adhere to the slide. The
methylene blue ion, with the result that the cell is ideal fixing agent preserves the structures of the
stained. The difference in charge produces an cells in their respective forms and positions
affinity between the dye and the bacterial cell. without causing the appearance of artefacts
(structures not present in the living cell).
Dyes can be divided into two groups: basic and Although gentle heating is the most commonly
acidic. If the color is in the positive ion of the dye, used method of fixation, agents such as alcohol
we call it a basic dye or stain. Thus, methylene
blue is a basic dye. If the color is in the negatively and various chemicals can also be used. Diagram
charged ion, we call it an acidic dye or stain. below illustrates how to prepare a smear for
staining.
In this lab session you will make stained
preparations of saliva. Staining with Basic Dyes
For staining you can use the basic dyes safrinin,
PROCEDURE methylene blue, crystal violet, and carbol fuchsin.
Preparing and Fixing Bacteria for Staining These dyes differ in their rate and degree of
Prior to staining, you must fix the material to be staining. Methylene blue reacts with the negatively
observed, that is, make it stick to the glass slide on charged cell at the slowest rate, taking 30-60
which it is to be stained. If a preparation is not seconds to stain a microbial preparation properly.
fixed, the film of cells washes off during the Crystal violet is more reactive and usually requires
staining procedure. The method illustrated in this only about 10 seconds. Carbol fuchsin is an even
experiment is preliminary to most staining faster dye usually requiring only 5 seconds.
methods used for bacteria. In this exercise you
*It is imperative that a slide be clean. One suggested cleaning
will use heat to kill the cells and cause them to method is to (1) place a drop of ethyl alcohol (or dilute tissue
adhere to the slide. culture glassware detergent) on the surface of a slide; (2) clean
with tissue; and (3) pass the clean slide through a bunsen
flame and cool.
1. With a wire loop place a small drop of each of
the broth cultures provided separate clean
slides.* If a culture is taken from solid media,

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 Direct staining with basic dyes

Its reactivity is so great that you may have difficulties from overstaining, especially in preparations
that contain large amounts of organic material and debris. Carbol fuchsin is a mixture of the basic dye
fuchsin and phenol.

The predominant charge on a bacterial cell (or a protein) is a function of the acidity of its environment.
Decreasing the acidity (raising pH) increases the net negative charge on the cell, providing a stronger
attraction to basic dyes. The reverse holds true for acidic dyes. Therefore, basic dyes stain poorly at a
low pH, and acidic dyes stain poorly at high pH.

Preparing a smear for staining.

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 Microbial flora of the oral cavity

Microbial Flora of the Oral Cavity

After completing this exercise you should be able to:
1. Prepare, stain and analyse a temporary slide of human saliva.
2. Prepare and label a scientific drawing of human cheek cell.

As many as 300 species of bacteria may have PROCEDURE

been isolated from the human oral cavity (as
shown in figure below). Two protozoa, 1. Collect some saliva in a sterile test tube. With a
Entamoeba gingivalis and Trichomonas tenax, sterile inoculating loop transfer a loopful of it
and a yeast, Candida albicans, are also common. to a clean slide and spread to form a thin film.
Streptococci, spheres in short chains, have been 2. Allow the slides to dry in the air or hold them
isolated from all sites in the mouth and comprise high above a bunsen flame.
a large proportion of the normal oral flora. The 3. When the film is dry, pass the slide, film side
species S. mitior is most common in dental up, three times through the bunsen flame.
plaque, in which rods and filaments are also Caution: Too much heat distorts the shapes
common. Spirochetes (spiral-shaped bacteria) and structures of the microorganisms. The
are common in scrapings from the gingival slide should feel warm but not hot against the
crevice and in dental plaque. The flora in your back of your hand
mouth is interesting and personal subjects with 4. Flood the fixed smear with approximately 5
which to practice microscopy and are drops of carbol fuchsin, leave the stain on
particularly good subjects to compare the image for 5 seconds and then wash. Dry the slide
qualities of transmitted light and phase-contrast between blotting paper.
microscopy.
5. Place the slide on the stage of your
microscope and adjust it for transmitted
light microscopy, following the
appropriate directions for your microscope.
6. Examine the specimen carefully and see if
you can identify three or more
morphological types of bacteria. A variety
of cells will be found in the saliva stain:
several forms of bacteria and fungi,
epithelial cells, salivary corpuscles, and
leucocytes. On the report sheet, illustrate
and label what you have seen.

QUESTIONS (23 marks): To be handed

in with your drawings

1. Why do you heat fix the slides? (5

marks)
2. Why are microorganisms stained by the
Some common morphological types of cells
basic dye? (5 marks)
found in the human oral cavity: epithelial
cells (A); red blood cells (B); spirochete (C);
3. Why is the mouth such a good habitat for
Bacillus (D); Streptococcus (E); and microorganisms? What are the sources of
Trichomonas tenax, a protozoan (F). nutrition for the organisms found there? (8
marks)
4. Would you expect to find the same
microbes in saliva and in plaque? (5marks)

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