MICROSCOPY AND DIFFERENTIAL STAINING

OF BACTERIA 1

INTRODUCTION
Staining is an important technique used in microbiology to enhance the clarity
of a cell, since most bacterial species are colourless. The most widely
practiced type of staining is the Gram stain, which was discovered by the
Danish scientist and physician Hans Christian Joachim Gram in 1884. Hanss
intention was to formulate a way of making bacteria more visible under a
microscope. Later on, the stain played a role in the characterisation and

classification of bacteria. Gram staining is a differential staining technique that

classifies bacteria into two groups, namely, Gram positive and Gram negative.
Gram positive bacteria retain the crystal violet stain. Gram negative bacteria
retain the safranin counterstain.
Some bacterial species do not stain easily using the Gram stain method.
Therefore, the Ziehl-Neelsen stain, also known as the acid-fast stain, is used.
The acid-fast stain is a type of differential staining discovered by the
bacteriologist Franz Ziehl and the pathologist Friedrich Neelsen. The acid-fast
stain measures the resistance of a stained cell to decolourise by acid-alcohol,
after staining has been completed. The result can either show acid-fast
positive or acid-fast negative bacteria. Acid-fast positive bacteria retains the
Methylene blue stain. Acid-fast negative bacteria retains the Ziehls carbol
fushcin stain.
The aim of this experiment is to learn to carry out the techniques of gram
staining and acid-fast staining accurately and to be able to differentiate and
gain knowledge about gram (positive and negative) and acid-fast (positive and
negative) staining properties. Differential staining helps differentiate between
bacteria.

METHODS AND MATERIALS

All methods and materials were carried out as per Bacteriology (Micr213)
Practical manual. Practical 3 (Page 28 36)
Exercise 1 was omitted.

RESULTS

Figure 1: Drawing showing Gram stain of Staphylococcus aureus (X1000)

S. aureus bacteria were small, cocci-shaped and appeared in clusters with a
violet colour showing a Gram positive reaction.

Figure 2: Drawing showing Gram stain of Bacillus subtilis (X1000)

B. subtilis bacteria were small, rod-shaped and appeared in small groups with
a violet colour showing a Gram positive reaction.

Figure 3: Drawing showing Gram stain of Escherichia coli (X1000)

E.coli bacteria were very small, rod-shaped and appeared in small groups with
a pink colour showing a Gram negative reaction.

Figure 4: Drawing showing acid-fast stain of Mycobacterium smegtis

(X1000)
M.smegtis bacteria were extremely small, rod-shaped appearing in small
groups and singly with a pink colour showing an acid-fast positive reaction.

Figure 5: Drawing showing acid-fast stain of E.coli (X1000)

E. coli bacteria were extremely small, rod-shaped and appeared in small
groups with a blue colour showing an acid-fast negative reaction.

DISCUSSION
The result of the Gram stain showed the difference between Gram positive
and Gram negative bacteria clearly. When viewed under the microscope S.
aureus and B. subtilis, shown in Figure 1 and 2 respectively, retained the
Crystal violet stain colour, showing that these bacterial species are Gram
positive. Gram positive bacteria consist of a primarily single thick homogenous
layer of peptidoglycan outside the plasma membrane. Teichoic acids and

lipoids are present which improve the rigidity of the cell wall. A smaller
volume of periplasm than gram negative bacteria.
When viewed under the microscope, E.coli did not retain the crystal violet
colour . Instead, E.coli became colourless after being decolourised and retained
a pink colour from the counterstain, Safranin, as shown in Figure 3
respectively. Gram negative bacteria have two distinct layers: A thin
peptidoglycan layer (Gram positive is thicker) covered by an outer membrane.
No teichoic acids and lipoids present. A larger volume of periplasm than gram
positive bacteria.
Decolourisation is the most important step in the Gram staining technique.
95% ethyl alcohol dissolves the outer membrane of gram negative bacteria
but not gram positive bacteria. When done correctly, gram positive bacteria
remains violet at this stage and gram negative bacteria become colourless.
Too much decolourisation will cause all bacteria to appear gram negative. Too
little decolourisation will cause bacteria to appear gram positive.
The result of the acid-fast stain of M. smegmatis showed an acid-fast positive
reaction, as shown in Figure 4 respectively. M. smegmatis is not easily
decolourised by acid alcohol. Acid-fast positive bacteria are hard to stain
because they have a cell wall containing lipids constructed from mycolic
acids, a group of hydroxyl fatty acids giving the cell wall a high lipid content.
The result of the acid-fast stain of E.coli showed an acid-fast negative
reaction, as shown in Figure 5 respectively. E.coli retained the methylene blue
counterstain. Acid-fast negative bacteria do not contain lipids constructed from
mycolic acid in their cell wall. Acid-fast bacteria are decolourised by acidalcohol.

CONCLUSION
The aim of this experiment to learn to carry out the techniques of Gram
staining and Acid-fast staining accurately, has been achieved. Differential
staining is a procedure that helps microbiologists to distinguish between
organisms based on their staining properties, on which knowledge has been
gained by completing this experiment. The hypothesis has been supported,
differential staining differentiate between bacteria.

REFERENCES
Willy, Sherwood and Woolverton. Prescotts Microbiology. 9th edition.
Bacteriology(Micr213) Practical Manual 2015
http://www.madsci.org/~lynn/micro/staining/GramStain/
http://en.wikipedia.org/wiki/Gram-positive_bacteria
http://en.wikipedia.org/wiki/Gram-negative_bacteria
http://en.wikipedia.org/wiki/Acid-fast

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