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Module: BIOM - 1009

Lecturer: Dr Ioannou

Date: 04/10/16

Bacterial Structure and Staining Methods
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Bacterial organisms can be identified by certain morphological features such as:
 Shape
 Spherical – Cocci
 Such as: Streptococcus pneumoniae
 Rod Shaped – Bacilli
 Such as: Escherichia coli
 Spiral Shaped – Spirochaetes
 Such as: Treponema pallidum
 Size
 Motility
 Structure
 Spore/capsule formation
 Toxin production

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Bacteria can be stained using the ‘Gram Stain’
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The Gram stain
technique is as follows:
 Prepare the slide
 Make sure it is free from oil or grease, and sterilise it with alcohol
 Label the slide
 Mark the area that the bacterial smear will be placed
 Prepare the smear
 Transfer a drop of the suspended culture on to a slide with an inoculation loop
 Smear it over the slide in a very thin layer
 Heat fix the slide
 Allow the smear to air dry
 Pass the hole slide through a Bunsen burner
 Heat fixing kills the bacteria and firmly adheres the smear to the slide
 Stain the slide
 Gently flood smear with crystal violet
 Wash off remaining stain
 Gently flood smear with Gram’s iodine
 Wash off remaining stain
 Decolourise using 95% ethyl alcohol or acetone
 Immediately rinse
 Gently flood with safranin
 Wash off remaining stain
 Blot dry
 View the slide under a light microscope
 Gram positive cells are purple and gram negative cells will be
pink/red
 See a gram stain to the right:
 Below is a table of medically important bacteria sorted to either
gram positive or negative:
Medically Important Bacteria
Gram Positive

Gram Negative

Staphylococcus aureus
Bacillus spp
Clostridium spp

Neisseria gonorrhoeae
Escherichia coli
Salmonella spp

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The Gram stain works the way it does due to the differences in the structure of the bacterial cell wall
 This cell wall is composed of sugars and amino acids – called peptidoglycan
 Its role is to protect the cell from lysis and so give the cell shape
 Peptidoglycan is a polymer of N-acetyl
glucosamine and N-acetyl muramic with side
chains of alternating D and L amino acids
 These D amino acids are useful targets
for antibiotics
 Peptidoglycan is highly crosslinked meaning it
will make the cell rigid
 Gram positive cells contain large amount of another
polymer named teichoic acid
 The right image is of a Gram positive cell wall
 Gram negative cell walls contain a thin layer of
peptidoglycan; this is acting as a permeability barrier
 The wall is also not highly cross-linked
 Once the bacteria are stained with crystal violet
followed by iodine, an iodine-crystal violet complex is
formed – it is thought that the addition of alcohol to
decolourise results in pores of peptidoglycan to
shrink and therefore remain a purple colour
 Gram negative cells also have lipopolysaccharides
on the outer membrane of the cell
 The right image is of a gram negative cell
wall/membrane:

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The Ziehl–Neelsen stain, also known as the acid-fast stain is another staining technique used to identify
acid-fast organisms such as Mycobacterium tuberculosis
 These acid fast organisms have a waxy lipid in their cell walls,
therefore they do not stain readily with the Gram stain
 The right image is an example of the Ziehl-Neelsen stain at
1000x magnification

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The Auramine-Phenol stain is another stain used to identify acid-fast bacteria
 Although it is not as specific as the Ziehl-Neelsen, is more affordable and so is used more often
as a screening tool
 The microbes fluoresce under a light microscope with this stain
 The image below shows the Auramine-Phenol stain at 40x magnification:



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