CHAPTER 3 Observing Microorganisms Through a Microscope 69
washed off, both gram-positive and gram-negative bacteria
Simple Stains
A simple stain is an aqneous or alcohol solution of a single basic
dye. Although different dyes bind specifically to different parts of
cells, the primary purpose of a simple stain is to highlight the
entire microorganism so that cellular shapes and basic strncturcs
are visible. The stain is applied to the fixed smear for a certain
length of time ru1d then washed off, and the slide is dried and
exrunined. Occasionally, a chemical is added to the solution to
intensify the stain; such an additive is called a mordant One
func­tion of a mordant is to increase the affinity of a stain for a
biolog­ical specimen; another is to coat a structure (such as a
flagellum) to make it thicker and easier to see after it is stained
with a dye. Some of the simple stains commonly used in the
laboratory are methylene blue, carbolfuchsin, crystal violet, and
safranin.
Differential Stains
Unlike simple stains, differential stains react differently with
different kinds of bacteria and thus can be used to distinguish
them. The differential stains most frequently used for bacteria
are the Gram stain and the acid-fast stain.
Gram Stain
The Grani stain was developed in 1884 by the Danish bacteriol­
ogist Hans Christian Gram. It is one of the most useful staining
procedures because it classifies bacteria into two large groups:
gram-positive ru1d gram-negative.
In this procedure (Figure 3.12a),
0 A heat-fixed smear is covered with a basic purple dye,
usually crystal violet. Because the purple stain imparts its
color to all cells, it is referred to as a primary stain.
f) After a short time, the purple dye is washed off, and the
smear is covered with iodine, a mordant. Vvhen the iodine is
appear dark violet or purple.
8 Next, the slide is washed with alcohol or an alcohol-acetone
solution. This solution is a decolorizing agent, which
removes the purple from the cells of some species but not
from others.
0 The alcohol is rinsed off, and the slide is then stained with
safranin, a basic red dye. The smear is washed again, blotted
dry, and examined microscopically.
The purple dye and the iodine combine in the cytoplasm of
each bacterium and color it dark violet or purple. Bacteria that
retain this color after the alcohol has attempted to decolorize
them are classified as gram-positive; bacteria that lose the dark
violet or purple color after decolorization are classified as grani­
negative (Figure 3.12b). Because gram-negative bacteria are
colorless after the alcohol wash, they arc no longer visible. This is
why the basic dye safranin is applied; it turns the gram-negative
bacteria pink. Stains such as safranin that have a contrasting
color to the primary stain are called counterstains. Because
gram-positive bacteria retain the original purple stain, they are
not affected by the safranin cou□terstain.
As you will see in Chapter 4, different kinds of bacteria react
differently to the Gram stain because structural differences in
their cell walls affect the retention or escape of a combination of
crystal violet and iodine, called the crystal violet-iodine (CV-I)
complex. Among other differences, grrun-positive bacteria have a
thicker peptidoglycan (disaccharides and amino acids) cell wall
thru1 gram-negative bacteria. ln addition, gram-negative bacteria
contain a layer of lipopolysaccharide (lipids and polysaccha­
rides) as part of their cell wall (see Figure 4.13, page 86). When
applied to both gram-positive and gram-negative cells, crystal
violet and then iodine readily enter the cells. Inside the cells, the
crystal violet and iodine combine to form CV-I. This complex is
larger than the crystal violet molecule that entered the cells, and,
because of its size, it cannot be washed out of the intact peptido­
glycan layer of gram-positive cells by alcohol. Consequently,
gram-positive cells retain the color of the crystal violet dye. In
gram-negative cells, however, the alcohol wash disrupts the outer
lipopolysaccharide layer, and the CV-I complex is washed out
through the thin layer of peptidoglycan. As a result, gram­
negative ceUs are colorless until counterstained with safranin,
after wh.ich they are pink.
ln summary, gram-posit.ive cells retain the dye and remain
purple. Gram-negative cells do not retain the dye; they are color­
less until counterstained with a red dye.
The Gram method is one of the most important staining
techniques in medical microbiology. But Grrun staining results
are not universaUy applicable, because some bacterial cells stain
poorly or not at all. The Gram reaction is most consistent when
it is used on young, growing bacteria.
70 PART ONE Fundamentals of Microbiology

■■
I
t<E.Y

Crystal violet
Iodine
□
■ Alcohol
Safranin

Gram-positive
..____.---..---, Gram-negative

O Application of O Appl ication of

-
f) Application of E) Alcohol wash
crystal violet iodine (mordant) (decolorization) safranin (counterstain)
(a) (purple dye)

- �,
' Rod
/) (gram-positive)

/I
t
Coccus

• ,j - \
(gram-positive)
);(
�

,)(,
-..s;_ A '1
{ r"-
�-
'-

f. --r:;
,,., .. �� \lte�
)
) J'r\ - -,�
/. Lvibrio Figure 3.12 Gram staining. (a) Procedure. Cb) Micrograph of
(gram-negative) , gram-stained bacteria. The rods and cocci (purple) are gram-positive. and the
'f';;fJ,-'-' ' \ 4
.., -
#' sp1rilla (pink) are gram-negative.

(b) Q How can the Gram reaction be useful In prescribing antibiotic treatment?

The Gram reaction of a bacterium can provide valuable infor­
mation for the b·eatment of disease. Gram-positive bacteria tend to
be killed easily by penicillins and cephalospori1.1S. Gram-negative
bacteria are generally more resistant because the antibiotics cannot
penetrate the lipopolysaccharide layer. Some resistance to these
antibiotics among both gram-positive and gran1-negative bacteria
is due to bacterial inactivation of the antibiotics.
Acid-Fast Stain (Ziehl-Neelsen (ZN) stain)
Another important differential stain (one that differentiates bac­
teria into distinctive groups) is the acid-fast stain, which binds
strongly only to bacteria that have a waxy material in their cell
walls. Microbiologists use this stain to identify all bacteria in the
genus Mycobacterium, including the two important pathogens
Mycobacteri1-1m tuberculosis, the causative agent of tuberculosis,
and Mycobacterium leprae (lep' ri), the causative agent ofleprosy.
This stain is also used to identify the pathogenic strains of the
genus Nocardia (n6-kar' de-a). Bacteria i.n the genera
Mycobacterium and Nocardia are acid-fast.
Q.
&A
In the acid-fast staining procedure, the red dye car­
bol fuchsin is applied to a fixed smear, and the slide
is gently heated for several minutes. (Heating enhances penetra­
tion and retention of the dye.) Then the slide is cooled and
washed with water. The smear is next treated with acid-alcohol,
a decolorizer, which removes the red stain from bacteria that are
not acid-fast. The acid-fast microorganisms retain the red color
because the carbolfuchsin is more soluble in the cell wall lipids
thru1 in the acid-ale.oho] (Figure 3.13). In non-acid-fast bacteria,