MONIS, Clemcy Pearl A.

BSN – 1A
MICROBIOLOGY AND PARASITOLOGY

BACTERIAL SIMPLE STAIN PROCEDURE

1. What is simple stain of bacteria? (5 pts)

Preliminary Staining of Bacteria: Simple Stains

Simple staining involves directly staining the bacterial cell with a positively charged
dye in order to see bacterial detail, in contrast to negative staining where the bacteria
remain unstained against a dark background. Staining is almost always applied to color
certain features of a specimen before examining it under a light microscope. Some
staining techniques involve the application of only one dye to the sample; others require
more than one dye. In simple staining, a single dye is used to emphasize particular
structures in the specimen. A simple stain will generally make all of the organisms in a
sample appear to be the same color, even if the sample contains more than one type of
organism. The term simple staining sometimes interchangeable with the terms like direct,
positive or monochrome staining. Now let us understand why simple staining is called by
such alternative names.

• Direct staining: Because it is a direct method that directly stains the bacterial cell
with a colorless background.
• Positive staining: Because it uses positively charged basic dyes that bind with the
negatively charged bacterial cell.
• Monochrome staining: Because it adds contrast to the specimen by using a single
stain only.
ILLUSTRATION OF SIMPLE, DIFFERENTIAL, and SPECIAL STAINS

2. Enumerate the purposes of conducting simple staining of bacteria (5 pts)

Simple Staining: Purposes, Procedure and Uses

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 a single solution of stain. Any basic dye such as methylene blue,
safranin, or crystal violet can be used to color the bacterial cells.

PURPOSES:
 • To elucidate the morphology and arrangement of bacterial cells.
• To highlight the entire microorganism so that cellular shapes and basic
structures are visible.
• To allow visualization of bacteria by examination of the shape and
arrangement.
PROCEDURE:

Preparation of a smear:

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.

USES:

Diagnostic microbiology laboratory generally does not perform simple staining

methods but simple staining can be useful in the following circumstances.

1. To differentiate bacteria from yeast cells: When endocervical swab culture is done
in blood agar both Staphylococcus spp and yeast cells may give similar-looking
colonies in Blood agar (a common error for a new technologist or microbiologist
with less experience). Performing the wet mount technique or simple staining from
the isolate can be helpful.
2. To presumptively identify the bacterial isolate: Due to their ubiquitous presence,
Bacillus spp may present as a contaminant in the culture plates. In some
circumstances (e.g. growth in Blood Agar but no growth in MacConkey Agar),
identifying the shape of the bacteria (rod or cocci) may help to eliminate the isolate
as possible contaminants (e.g., Bacillus spp) or further process as a potential
pathogen (cocci).

3. Discuss the different steps in doing simple staining of bacteria. (5 pts)

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. Saturate the smear again but this time with Iodine. Iodine will set the stain
5. Wash of any excess iodine with gently running tap water. Rinse thoroughly.
 6. Wipe the back of the slide and blot the stained surface with bibulous paper or with a
paper towel.
7. Place the stained smear on the microscope stage smear side up and focus the
smear using the 10X objective.
8. 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.

Left: Cocci in Cluster; Right: Bacilli

4. The magnification of the lens in 100x. Explain its meaning (3 pts)

MAGNIFICATION OF THE LENS

The objective and ocular lenses are responsible for magnifying the image of the
specimen being viewed. So for 10X objective and 10X ocular, Total magnification = 10 X 10
= 100X (this means that the image being viewed will appear to be 100 times its actual size).
MAGNIFICATION TOTAL MAGNIFICATION

Scanning 4x 40x

Low Power 10x 100x

High Power 40x 400x

Oil Immersion 100x 1000x

5. Identify the two solutions used in simple staining of bacteria and its color (2 pts)

You may choose from methylene blue and Gram crystal violet. Basic stains, such as
methylene blue, Gram safranin, or Gram crystal violet are useful for staining most
bacteria.