Cardiac technology

1. Bio Practicals
Nagaraj Balasubramanian
Gram Staining of Bacteria
Thursday 29 August 13

2. 1884
• Statue of Liberty presented to US in Paris
• First England-Australia Test series played
• The first edition of the Oxford English Dictionary
was published
• Robert Koch and Friedrich Loeffler formulate
Koch's postulates on the causal relationship
between microbes and diseases.
• First president of India Rajendra Prasad was born
Thursday 29 August 13

3. Gram staining developed by
Hans Christian Gram, a
Danish doctor working in
Berlin
While examining lung tissue from patients who had
died of pneumonia, he discovered that certain stains
were preferentially taken up and retained by
bacterial cells.
Gram devised his technique to enable bacteria to be
seen more readily in stained sections of lung tissue.
He published his method in 1884, and included in his
short report the observation that the Typhus bacillus
did not retain the stain.
Thursday 29 August 13

4. Gram staining is a bacteriological laboratory
technique used to differentiate bacterial species into
two large groups
(Gram-positive and Gram-negative)
based on the physical properties of their cell walls.
2013
Thursday 29 August 13

5. Cell envelope seen in bacteria.
The cell envelope may be defined
as the cell membrane and cell wall
plus an outer membrane if one is
present.
The cell wall is the tough, usually
flexible but sometimes fairly rigid
layer that surrounds cells. It is
located outside the cell membrane
and provides these cells with
structural support and protection.
Thursday 29 August 13

6. Cell wall in Bacteria.
The cell wall consists of the peptidoglycan layer and attached structures.
Most bacterial cell envelopes fall into two major categories
a) Gram positive and b) Gram negative.
This is based on Gram staining characteristics that reflect major
structural differences between the two groups.
Thursday 29 August 13

7. The peptidoglycan is a single bag-shaped, highly cross-linked
macromolecule that surrounds the bacterial cell membrane and provides
rigidity.
It is huge (billions in molecular weight; compare proteins which are
thousands in molecular weight).
Peptidoglycan consists of a glycan (polysaccharide) backbone
consisting of N-acetyl muramic acid and N-acetyl glucosamine with
peptide side chains containing D- and L- amino acids and in some
instances diaminopimelic acid.
The side chains are cross-linked by peptide bridges.These peptide
bridges vary in structure among bacterial species
Peptidoglycan Cell wall in Bacteria.
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8. Peptidoglycan Cell wall in Bacteria.
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9. Gram Positive Cell wall in Bacteria.
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10. Gram Negative Cell wall in Bacteria.
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11. Gram Positive Gram Negative
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12. Gram Staining kit has...
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13. ‣Addition of iodine (mordant) forms crystal violet
iodine complex within the cell wall.
‣The complex formed is larger than crystal violet so it
cannot be easily washed out from the intact
peptidoglycan layer.
Primary stain, crystal violet stains all the cells purple.
‣Application of alcohol (decolorizer) decolorizes the stain.
‣ Since gram negative organism have thin peptidoglycan layer and
have additional lipopolysaccharide layer which gets dissolved due to
the addition of alcohol, so gram negative organism fails to retain the
complex and gets decolorized
‣To observe the decolorized cells secondary stains like Basic
fuchsin or Safranin is added which stains the gram negative
organisms pink
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14. Gram Positive vs Gram Negative Bacteria.
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15. Thursday 29 August 13

16. 100x Oil Immersion Objective
The definition of numerical aperture (N.A.)
N.A. = n sin a
where n is the index of refraction of the intervening medium between object and lens and a is the half-angle
defined by the limiting ray (a-air or a-oil).The “light-gathering” power of the microscope’s objective lens is thus
increased by increasing the refractive index of the intervening medium.
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17. ocular
micrometer
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18. ocular
micrometer
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