Visualization of microorganisms in the living state is very difficult, not just
because they are minute, but because they are transparent and almost colorless
when suspended in an aqueous medium.
To study their properties and divide microorganisms into specific groups
for diagnostic purposes, biological stains and staining procedures, in conjunction
with light microscopy, have become major tools in microbiology.
Chemically, a stain may be defined as an organic compound containing a
benzene ring plus a chromophore and an auxochrome.
Stains are of 2 types
Types of staining techniques:
- Acidic stains e.g., picric acid
- Basic stains e.g., methylene blue.
- Simple staining (use of a single stain):
This type of staining is used for visualization of morphological shape
(cocci, bacilli, and spirilli) and arrangement (chains, clusters, pairs, and
- Differential staining: (use of 2 contrasting stains)
It is divided into two groups:
- Separation into groups, Gram stain and acid-fast stain.
- Visualization of structures, Flagella stain, apsule stain, spore stain,
- Microbes are invisible to the naked eye and are difficult to see and identify,
even when using a microscope. Staining microbes makes them easier to
observe and reveals the presence of microscopic structures, because charged
portions of the stain bind to specific macromolecules within the structures.
A simple stain displays the microorganisms, and a differential stain
displays the chemical differences in cellular structures, including the cell
wall and cell membrane, because the macromolecules within the structure
bind to different components of the stain.
- An example of this differential staining is seen in staining used for blood
- Staining white blood cells with a differential stain displays the difference
between the 5 white blood cell types: basophils, eosinophils, neutrophils,
monocytes, and lymphocytes. The intracellular granules of basophils stain
dark blue because of their affinity to basic portion of the stain. Basophil
means basic loving. On the other hand, the eosinophil (acid loving) stains
red as a result of the intracellular granule’s affinity for the acidic portion
of the stain. Treatment of microbial diseases depends upon the correct
identification of microorganisms and relies upon the ability to identify
specific internal structures. Bacterial cells are commonly stained with a
differential stain called the Gram stain and protozoal cells with the
trichrome stain, in order to reveal the internal structural differences and
to identify other organisms. Properly preparing slides for staining is
important to ensure good results. Remember, you cannot see the material
you are working with, so you must develop good technique based upon
- Always start with clean slides, using lens paper to clean them. Slides can
be made from direct clinical material (a wound, sputum, knee fluid, the
throat, etc.), broth cultures, and solid media cultures. The first principle is
that some fluid is needed to emulsify the material if it is dry; however, too
much fluid may make the microbes hard to find. Slides from clinical
cultures are usually placed directly on the slide without the addition of
water, as are slides from broth cultures. Slides from solid media require
water to emulsify and separate the individual bacterial cells for better
observation, but a single drop of water is usually adequate. Next, the material must be attached to the slide so it doesn’t wash off with the
staining process. The second principle involves fixing the slide using
either a chemical fixative or heat. In this lab, a heat-fixing tray will be
- This lab will use the principles and techniques above to make and stain
bacterial slides, using a differential staining technique called the Gram
stain. Initially, 3 stock cultures (known types) of bacteria will be stained,
and then the 3 isolated unknown microbes from the environmental cultures
will be stained and examined. The environmental culture will contain a
variety of bacteria and possibly some fungi. Bacterial cells can be observed
for shape (rod, coccus, or spirilla) and arrangement (in chains, clusters,
etc.). Arrangements of cells are best observed from clinical and broth cultures
because the emulsification process disrupts the natural arrangement from
colonies “picked” from solid media.
- 3 isolation plates
- Original environmental broth culture
- Original TSA plate
- S. epidermidis,
- E. coli.,
- Bacillus sp.
- Slides, transfer loops
Preparing the Smears
- Gram stain reagents
- Crystal violet
Staining the Smears
- Collect broth and pure subcultures and observe the colony morphology: Ask the instructor to critique your isolation technique. This will be very
important in later labs. Practice the isolation technique on a new plate if
you need some more experience.
- Preparing a smear: There are 3 steps to prepare a smear for staining.
Remember to use aseptic technique and flame the loop before and after
- Preparation of the slide—Clean and dry the slide thoroughly to remove
- Preparation of the smear—From the broth culture, use the loop to spread
1 or 2 drops of specimen in the center of the slide, spreading it until
it is approximately the size of a nickel. When making a smear from
solid media cultures, start by putting a very small drop of water in the
center of the slide and then mix a loopful of bacteria from the surface
of solid media in the water, spreading it out to the size of a nickel.
- Fixation—The point of fixation is to attach the organisms and cells to
the slide without disrupting them. In this class, we will use an electric
fixing tray that will dry and fix the smears in one step. Slides must be
completely dry and fixed before staining, or they will wash off.
- In the beginning, it is wise to make a single broth slide and a single solid
medium slide, and then stain and observe these before making the other
slides. This allows you to alter your technique if the results are not optimal.
- Begin with the known culture smears (S. epidermidis, E. coli., or Bacillus sp.).
Place the smear on the staining rack over the sink.
- Cover the smear area with the crystal violet stain, leave it for 15 seconds,
and then rinse the slide with a gentle stream of water.
- Apply Gram’s iodine, covering the smear completely for 15 seconds, and
- Using Gram’s decolorizer, apply it a drop at a time to the smear area until
no more color leaves the area. Quickly rinse with water to stop the
- Apply safranin to the smear for 15 seconds, then rinse with water.
- Allow the smear to air dry or place it on the drying and fixation tray.
Perform the simple staining procedure to compare morphological shapes and
arrangements of bacterial cells.
In simple staining, the bacterial smear is stained with a single reagent. Basic
stains with a positively charged chromogen are preferred, because bacterial
nucleic acids and certain cell wall components carry a negative charge that
strongly attracts and binds to the cationic chromogen. The purpose of simple
staining is to elucidate the morphology and arrangement of bacterial cells.
The most commonly used basic stains are methylene blue, crystal violet,
and carbol fuchsin.
- Cultures: 24-hours nutrient agar slant culture of E.coli and Bacillus cereus,
and a 24-hour nutrient broth culture of S. aureus.
- Reagents: Methylene blue, crystal violet, and carbol fuchsin.
- Prepare separate bacterial smears of the organisms. All smears must be
heat fixed prior to staining.
- Place a slide on the staining tray and flood the smear with one of the
indicated stains, using the appropriate exposure time for each carbol fuchsin, 15 to 30 seconds, crystal violet, 20 to 60 seconds, and methylene
blue, 1 to 2 minutes.
- Wash the smear with tap water to remove excess stain. During this step,
hold the slide parallel to the stream of water. This way, you can reduce
the loss of organisms from the preparation.
- Using bibulous paper, blot dry, but do not wipe the slide.
- Repeat this procedure with the remaining 2 organisms, using a different
stain for each.
- Examine all stained slides under oil immersion.
Negative staining requires the use of an acidic stain such as nigrosin. The
acidic stain, with its negatively charged chromogen, will not penetrate the cells
because of the negative charge on the surface of bacteria. Therefore, the unstained
cells are easily discernible against the colored background.
The practical application of the negative staining is 2-fold: first, since heat
fixation is not required and the cells are not subjected to the distorting effects
of the chemicals and geat, their natural size and shape can be seen.
Second, it is possible to observe bacteria that are difficult to stain, such as
: 24-hour agar slant cultures of Micrococcus luteus, Bacillus cereus
- Place a small drop of nigrosin close to one end of a clean slide.
- Using sterile technique, place a loopful of inoculum from the Micrococcus
luteus culture in the drops of nigrosin and mix.
- With the edge of a second slide, held at a 30° angle and placed in front
of the bacterial suspension. Push the mixture to form a thin smear.
- Air dry. Do not heat fix the slide.
- Repeat steps 1 to 4 for slide preparations of Bacillus cerus and Aquaspirillum
- Examine the slide under oil immersion.