 One way to classify bacteria is by examining its morphology. Bacteria
appear in many different shapes. We will examine the three basic
shapes but note that others exist.
 3 basic shapes:
 Bacilli (singular is bacillus), are rod-shaped.
 Cocci (singular is coccus), are spherical-shaped.
 Spirilla ( singular is spirillum), are spiral-shaped.
 Bacterial cells can appear as single cells or can be found in
arrangements. Some of these are:
 Chains (prefix strepto-)
 Clusters (prefix staphylo-)
 Pairs (prefix diplo-)
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 In 1884 Hans Christian Gram developed a staining procedure known as
Gram Staining. The composition of the cell wall of bacteria vary among
species. Due to this difference, bacteria can be divided into two groups;
Gram positive and Gram negative.
 Gram positive bacteria have thick peptidoglycan cell walls and retain a
purple color when stained with crystal violet. Gram negative bacteria
have a thin peptidoglycan inner wall and outer wall made of
carbohydrates, proteins and lipids. These cells retain the red color
when stained with safranin.
 Before anything, you need to make the heat-fix sample.
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 Obtain a slide and add one drop of distilled
water (dH2O) and place in a staining tray
Staining tray
drop of H2O
slide
 Sterilize a loop
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 With the loop transfer bacteria to your
slide and mix it with the drop of dH₂O.
Sterilize the loop once done.
loop
Bacteria colony
slide
 Heat-fix the slide by passing the slide over
an open flame several times. This
evaporates the water leaving the bacteria
sample dry and fixed on the slide.
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 Put your slide in the staining rack.
Flood the sample with crystal violet
(CV) for 1 minute.
 Wash away the excess CV using dH₂O.
 Flood the sample with iodine for 1
minute.
 Rinse off the iodine with dH₂O.
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 Decolorize with ethanol.
 Rinse slide with dH₂O.
 Ethanol must be added drop by drop to a slanted slide. Stop when first
clear drop is seen (about 4 drops). The ethanol washes out the thin layer of
protein/lipids if it is gram negative, causing it to lose the CV. If it is a gram
+, the peptoglycan layer is so thick that it will most likely retain most of it,
retaining the CV impregnated in it.
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 Flood smear with safranin (counterstain) for 1
minute. Only decolorized cells will stain red.
 Gram negative will stain red, gram
positive remain purple.
 Rinse slide off with dH₂O and blot
slide dry between sheets of bibulous
paper. Slide can be examined using the
microscope.
 Here is a 3 minute video of the staining process.
http://www.youtube.com/watch?v=YvZHHNZ8cdo
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Do you recognize this bacteria?
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Each round dot is an individual
bacteria, notice it shows in pairs. Due to
the color it is a Gram negative.
Bacteria type: Coccus
Diplococcus sp.
1000x
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Do you recognize this bacteria?
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Notice each individual bacteria is
elongated and the colony shows a
clumped form. Due to the color this is a
gram positive bacteria
Bacteria type: Bacillus
Staphylobacillus sp.
1000x
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Do you recognize this bacteria?
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Bacteria type: Spirilla
Spirillum sp.
Notice the individual bacteria has a long,
curved shape. Due to its color it is a Gram
negative
1000x
The End
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