ANSWERS TO QUESTIONS

1. Define the following terms and give and example for each:
a. Counterstain
Counterstain is a stain with color contrasting to the primary stain. It is used to stain
the gram negative bacteria after decolorization. One common example is pink safranin
which is used to counterstain with the blue crystal violet. (Bruckner, 2012)
b. Decolorizer
Decolorizer is a chemical used to remove the primary stain. It dehydrates the
peptidoglycan layer, shrinking and tightening it. It is used to determine gram positive and
gram negative bacteria. The gram positive bacteria will retain the primary stain, the gram
negative will not. Alcohol is a common decolorizer used in gram staining. (Bruckner,
2012)
c. Mordant
Mordant is a chemical used to increase the affinity of the bacterial cell walls to stain,
by forming a complex with the primary stain. An example of a mordant is iodine which
forms a complex with crystal violet stain that has attached to the cell wall of bacteria.
(Bruckner, 2012)
d. Primary stain
The primary stain colors all the cells at the start of the gram staining procedure. After,
decolorization, the primary stain is used to detect the gram positive bacteria. One
common example used as a primary stain is crystal violet stain. (Bruckner, 2012)
e. Simple stain
Simple stain is a staining technique that uses only one dye on a fixed slide for a given
period of time. A simple stain only shows the arrangement and basic morphology of the
bacteria. Examples are methylene blue and malachite green, which stain blue and green
respectively. (Lindh et al., 2009)
f.
Differential stain
Diferential stain is a staining technique that uses more than one dye to stain different
types of microorganism. The most common differential stain used is gram staining. Gram
staining is used to differentiate two groups of bacteria based on their cell wall
constituents. (Lindh et al., 2009)
g.
Smear
Smear is a film containing microorganisms that are spread over the surface of
microscopic slides for microscopic examination. A bacterial smear, for example, is
commonly used in microbiology.
2. Explain the similarities and differences between Gram-positive and Gram-negative cell
walls.
Gram positive and gram negative cell walls share one thing in common that is unique to
bacteria, the peptidoglycan layer. The peptidoglycan layer contains chains of Nacetylglucosamine and N-acetylmuramic acid which are cross linked by short peptide chains.
The cell wall of gram-positive bacteria has a very thick outer peptidoglycan layer making it
rigid. Peptidoglycan accounts for about 90% of the cell wall and the rest are polysaccharides.
On the other hand, the cell wall of a gram negative bacteria is thinner but more complex than
gram positive cell wall. It accounts for only 10-20% of peptidoglycan. It is mainly composed

of lipoproteins and lipid polysaccharides making it elastic. A periplasmic space is present

between the inner surface of the cell wall and cell membrane of a gram negative bacteria.
(Sing et.al, 2008)

Figure. The differences between Gram positive and Gram negative cell walls
(image source: http://www.microbiologyinfo.com/differences-between-gram-positiveand-gram-negative-bacteria/)
3. Why is it recommended to use 18-24 h-old cultures of bacteria in Gram-staining? What is
a Gram-variable reaction? What factors contribute to Gram-variability
Physiological damage or ageing can make the cell wall of the bacteria leaky causing the
escape of the dye during gram staining. Therefore, gram staining should be performed only
in cultures up to 24 hours old (Sing et.al, 2008). Gram variable reaction can also occur due to
overageing of bacterial culture. Gram variable reaction happens when the bacteria may stain
either gram positive or gram negative (James, 2016). Other factors that contribute to gram
variability are improper heat fixing of the smear, cell density of the smear, concentration and
freshness of the gram stain reagents, and period of decolorization.
4. Explain briefly the working principle behind the Gram-staining method.
Gram staining differentiates two groups of bacteria based on their cell wall structure and
composition. In gram staining, the primary stain gives a blue or violet color to the bacteria.
The mordent, iodine forms a complex with the crystal violet inside the cell wall of the
bacteria. The cells are then washed with a decolorizer. After decolorization, the gram positive
bacteria will retain the dye complex and remain purple because the thick peptidoglycan layer
within the bacterial cell wall. On the other hand, the gram negative bacteria will be
decolorized due to its thin peptidoglycan layer of the cell wall. The gram negative will then
be counterstained with pink safranin to easily differentiate them with gram positive bacteria.
(Bisen et al., 2012)
5. What is the purpose of heat-fixing during smear preparation? What would happen if too
much heat is applied during heat-fixing?
Heat fixing is important in the denaturation of bacterial proteins or enzymes preventing
autolysis. It also increases the adherence of the bacteria to the slide so that the bacteria will
not be washed off during the staining process. (Aneja, 2005) During heat fixing, too much

heat can damage the microorganisms and alter their staining reaction. In the case of gram
staining, it may cause gram positive bacteria to stain gram negatively. (Harisha, 2006)

References:
Aneja, K.R. (2005). Experiments in Microbiology, Plant Pathology and Biotechnology. 4th ed.
New
Delhi:
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%20microbiology&f=false
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