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Prepared by Agrani Paudel
BSc Microbiology first year
History
• Gram stain was developed by the
Danish physician, Hans Christian Gram,
while working in Berlin in 1883. He
later published his procedure in 1884.
At the time, Dr Gram was studying lung
tissue sections from patients who had
died of pneumonia.
Cont.
• Carl Weigert (1845-1904)
• Gram did not use a
counterstain in his
procedure
• German pathologist Carl
Weigert added final step of
staining with safranin
Gram Staining
• It is one of the most important
staining method in bacteriology
• It differentiates bacterial species into
two major groups
i. Gram positive bacteria
ii. Gram negative bacteria
• Here with the help of gram staining
bacteria differs due to difference in
cell wall composition
Gram positive bacteria
• Have thick wall of peptidoglycan
• Peptidoglycan is polymer of sugar
amino acids
• In between cell wall and cell
membrane there is membrane
teichoic acid
• Very thin layer of
lipopolysaccharide is present
Gram negative bacteria
• Outer membrane of
phospholipids
• Lipopolysaccharides outside of
thin peptidoglycan layer
• Space between outer membrane
and the peptidoglycan layer is
called the periplasmic space
Principle of Gram staining
Stain is fixed due to
formation of CVI complex
All bacteria stained
purple
Bacteria
Some cells will be decolorized
while some will retain stain
Cells that retain primary stain i.e. Crystal violet
are Gram positive
And those that retains the colour
of counter stain i.e. safranin are Gram negative
Stained with
crystal violet
Addition of
Gram’s iodine
Addition of
decolorizer
alcohol or
acetone
Staining with safranin
Gram staining Gram positive and gram negative bacteria can be distinguished by Gram Staining
Difference in action in cell wall
• For Gram’s Iodine
• Forms (crystal violet iodine) CV-I complex by
binding to primary stain
• Only in Gram positive cells, CV-I complex binds to
the magnesium –ribonucleic acid component of
the cell wall forming magnesium- ribonucleic acid-
crystal violet iodine (Mg-RNA-CV-I) complex which
is more difficult to remove than smaller CV-I
complex.
• For decolorizing agent
• Functions as lipid solvent and protein dehydrating agent
• In Gram Positive cells, small amount of lipid content is readily
dissolved by the action of alcohol, forming minute cell wall pores
which later is closed by alcohol dehydrating effect. So, the larger Mg-
RNA-CV-I complex is difficult to remove and hence remains purple.
• In case of Gram negative cells, outer thick lipid layer is dissolved by
alcohol, creating large pores in cell wall that do not close appreciably
on dehydration of cell wall proteins. As a result, release of unbound
CV-I complex is facilitated leaving these cell unstained
Gram staining Gram positive and gram negative bacteria can be distinguished by Gram Staining
Basic steps of gram staining
• Applying primary stain(crystal violet) or methyl violet to a heat fixed
of bacterial culture
• Addition of mordant(Gram’s iodine)
• Rapid decolorization with alcohol or acetone
• Counterstaining with safranin or basic fuchsin
Materials and chemicals required
• Inoculating loop
• Clean grease free slide
• Bunsen burner
• Primary stain- crystal violet
• Mordant –Gram iodine
• Decolorizer- alcohol or acetone
• Counter stain- safranin
Procedure
• Lab table is disinfected applying a
disinfectant such as Lysol
• All essentials materials are
arranged in aseptic environment
• chemical resistant gloves and
chemical splash goggles is
recommended to be used if the
microorganism is highly
pathogenic
The slide( clean, dry, grease free) is marked where smear is to be prepared
step 1: smear making
For liquid culture, two loops of liquid bacterial growth is taken in the center of clean slide
One drop of distilled water is added at center of slide for solid culture being used
Step 1 smear preparation
Inoculating loop is sterilized and let it to cool, if it is too hot it may distort microorganisms
colony is added in distilled water with inoculating loop
loop is sterilized
Now smear is spread in a circular motion to about
1cm in diameter
It is left for air dry
step 2: Heat fixing
Step 2 heat fixing
slide is warmed by back and froth movement for attachment of
microorganisms to the slide
step 3 : smear is stained with crystal violet for 60 sec
Step 3 smear stained with crystal violet for about 60 s
Gram crystal violet stain is applied from an eye-dropper or pipet onto and slightly
beyond the smear area on the slide and is allowed to sit for one minute.
step 4: rinsed with distilled water
Step 4 : rinsed with distilled water Step 4 : rinsed with distilled water
slide is tilted on an angle and the slide is gently rinsed with distilled water from a dropper bottle by dropping
water above the smear and allowing it to rinse the stain off the smear. It is rinsed until the solution dripping
from the slide is clear.
step 5 : applying mordant Gram iodine
Step 5 Gram’s iodine is added for 60s
Gram iodine is applied from an eye-dropper or pipet onto and
slightly beyond the smear area on the slide and was allowed to sit
for one minute
step 6 : Rinse with distilled water
Step 6 rinsed with distilled water Step 6 rinsed with distilled water
slide is tilted on an angle and the slide is gently rinsed with distilled water from a dropper bottle by dropping
water above the smear and allowing it to rinse the stain off the smear. It is rinsed until the solution dripping
from the slide is clear.
step 7 : decolorized with acetone or ethanol
Step 7 : decolorized with alcohol or acetone
microbial smears is decolorized with 95% ethyl alcohol by applying
drop-wise for approximately 30 seconds or to a tipped slide until no
more color runs off.
step 8 : rinse with distilled water
Step 8 : rinsed with distilled water
Decolorization time is determined primarily by the thickness
of the smear. It is very important not to over-decolorize. Then
it is rinsed with water
step 9 : counter stain with safranin
Step 9 : counter stain is added for 45 s
Gram safranin counter stain is applied for 30–45 seconds. Safranin
serves to stain bacterium which are gram-negative that do not hold
crystal violet after decolorization
step 10 : rinse with distilled water
Step 10 : rinsed with distilled water
slide is tilted on an angle and the slide is gently rinsed with distilled water from a
dropper bottle by dropping water above the smear and allowing it to rinse the stain off
the smear. It is rinsed until the solution dripping from the slide is clear.
step 11 : slide is gently dried
Step 11 : gently dried Step 11 : gently dried
step 12 : oil emersion is added above the smear
Step 12 oil emersion is added
step 13 : observation is done under microscope
Step 13 : observed under microscope
Escherichia coli
Gram negative bacilli
Salmonella typhus
Gram negative bacilli
Shigella dysenteriae
Gram negative bacilli
Neisseria meningitidis
Gram negative cocci
Neisseria gonorrhoeae
Gram negative cocci
Streptococcus pneumoniae in sputum
Gram positive cocci
Bacillus anthracis
Gram positive bacilli
Actinomycetes spp
Gram positive filamentous
Streptococcus mutans
Gram positive cocci
Gram staining Gram positive and gram negative bacteria can be distinguished by Gram Staining
Precautions
• Culture selection
• Fresh and young culture should be used to avoid misleading results
because old cultures of gram positive tend to decolorize more rapidly
and show Gram negative ness ;lose the ability to retain crystal violet
iodine complex
• It is possible to report Gram negative bacteria if Gram positive
bacteria is old , dead ,damaged and the cell wall is not intact
Precautions
• Smear preparation
• Smear should be thin and uniform to avoid over populated bacteria
but sufficiently dense for easy visualization
• Even smear should be made other wise alcohol will continue to wash
violet colour from thick parts of the smear while thin parts being over
decolorized
Precautions
• Heat fixing
• If slide is not completely dried when
you pass it through flame organisms
will be boiled and destroyed
• If too much heat fixing is done ,
organisms may be incinerated and you
will see distorted cells and cellular
remains
• If you heat fix too much little , organism
may not stick and will wash off the slide
in subsequent steps
Precautions
• During decolorization
• Over decolorization should be avoided, as over treatment washes
crystal violet complex from gram positive and they appear to be gram
negative
• Acetone decolorizes faster than alcohol
• False Gram positive report, if decolorization step is omitted
• After rinsing counter stain
• care must be taken not to rub the slide with blotting paper as this
could remove the adhering bacteria
Be careful
• Genera Actinomycetes, Arthrobacter, Corynebacterium,
Mycobacterium, and Propionibacterium have cell walls particularly
sensitive to breakage during cell division resulting in Gram
negative staining of these Gram positive cells. Staining of these
organisms results in an uneven or granular appearance
• Mycobacteria contain mycolic acids and have a high GC content in
their DNA. A Gram stain cannot penetrate the waxy cell wall. The
hydrophobic lipids cause the Gram stain to give no staining or a
variable result.(Acid fast staining)

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Gram staining Gram positive and gram negative bacteria can be distinguished by Gram Staining

  • 1. Prepared by Agrani Paudel BSc Microbiology first year
  • 2. History • Gram stain was developed by the Danish physician, Hans Christian Gram, while working in Berlin in 1883. He later published his procedure in 1884. At the time, Dr Gram was studying lung tissue sections from patients who had died of pneumonia.
  • 3. Cont. • Carl Weigert (1845-1904) • Gram did not use a counterstain in his procedure • German pathologist Carl Weigert added final step of staining with safranin
  • 4. Gram Staining • It is one of the most important staining method in bacteriology • It differentiates bacterial species into two major groups i. Gram positive bacteria ii. Gram negative bacteria • Here with the help of gram staining bacteria differs due to difference in cell wall composition
  • 5. Gram positive bacteria • Have thick wall of peptidoglycan • Peptidoglycan is polymer of sugar amino acids • In between cell wall and cell membrane there is membrane teichoic acid • Very thin layer of lipopolysaccharide is present
  • 6. Gram negative bacteria • Outer membrane of phospholipids • Lipopolysaccharides outside of thin peptidoglycan layer • Space between outer membrane and the peptidoglycan layer is called the periplasmic space
  • 7. Principle of Gram staining Stain is fixed due to formation of CVI complex All bacteria stained purple Bacteria Some cells will be decolorized while some will retain stain Cells that retain primary stain i.e. Crystal violet are Gram positive And those that retains the colour of counter stain i.e. safranin are Gram negative Stained with crystal violet Addition of Gram’s iodine Addition of decolorizer alcohol or acetone Staining with safranin
  • 9. Difference in action in cell wall • For Gram’s Iodine • Forms (crystal violet iodine) CV-I complex by binding to primary stain • Only in Gram positive cells, CV-I complex binds to the magnesium –ribonucleic acid component of the cell wall forming magnesium- ribonucleic acid- crystal violet iodine (Mg-RNA-CV-I) complex which is more difficult to remove than smaller CV-I complex.
  • 10. • For decolorizing agent • Functions as lipid solvent and protein dehydrating agent • In Gram Positive cells, small amount of lipid content is readily dissolved by the action of alcohol, forming minute cell wall pores which later is closed by alcohol dehydrating effect. So, the larger Mg- RNA-CV-I complex is difficult to remove and hence remains purple. • In case of Gram negative cells, outer thick lipid layer is dissolved by alcohol, creating large pores in cell wall that do not close appreciably on dehydration of cell wall proteins. As a result, release of unbound CV-I complex is facilitated leaving these cell unstained
  • 12. Basic steps of gram staining • Applying primary stain(crystal violet) or methyl violet to a heat fixed of bacterial culture • Addition of mordant(Gram’s iodine) • Rapid decolorization with alcohol or acetone • Counterstaining with safranin or basic fuchsin
  • 13. Materials and chemicals required • Inoculating loop • Clean grease free slide • Bunsen burner • Primary stain- crystal violet • Mordant –Gram iodine • Decolorizer- alcohol or acetone • Counter stain- safranin
  • 14. Procedure • Lab table is disinfected applying a disinfectant such as Lysol • All essentials materials are arranged in aseptic environment • chemical resistant gloves and chemical splash goggles is recommended to be used if the microorganism is highly pathogenic
  • 15. The slide( clean, dry, grease free) is marked where smear is to be prepared step 1: smear making
  • 16. For liquid culture, two loops of liquid bacterial growth is taken in the center of clean slide One drop of distilled water is added at center of slide for solid culture being used Step 1 smear preparation
  • 17. Inoculating loop is sterilized and let it to cool, if it is too hot it may distort microorganisms
  • 18. colony is added in distilled water with inoculating loop
  • 20. Now smear is spread in a circular motion to about 1cm in diameter It is left for air dry
  • 21. step 2: Heat fixing Step 2 heat fixing slide is warmed by back and froth movement for attachment of microorganisms to the slide
  • 22. step 3 : smear is stained with crystal violet for 60 sec Step 3 smear stained with crystal violet for about 60 s Gram crystal violet stain is applied from an eye-dropper or pipet onto and slightly beyond the smear area on the slide and is allowed to sit for one minute.
  • 23. step 4: rinsed with distilled water Step 4 : rinsed with distilled water Step 4 : rinsed with distilled water slide is tilted on an angle and the slide is gently rinsed with distilled water from a dropper bottle by dropping water above the smear and allowing it to rinse the stain off the smear. It is rinsed until the solution dripping from the slide is clear.
  • 24. step 5 : applying mordant Gram iodine Step 5 Gram’s iodine is added for 60s Gram iodine is applied from an eye-dropper or pipet onto and slightly beyond the smear area on the slide and was allowed to sit for one minute
  • 25. step 6 : Rinse with distilled water Step 6 rinsed with distilled water Step 6 rinsed with distilled water slide is tilted on an angle and the slide is gently rinsed with distilled water from a dropper bottle by dropping water above the smear and allowing it to rinse the stain off the smear. It is rinsed until the solution dripping from the slide is clear.
  • 26. step 7 : decolorized with acetone or ethanol Step 7 : decolorized with alcohol or acetone microbial smears is decolorized with 95% ethyl alcohol by applying drop-wise for approximately 30 seconds or to a tipped slide until no more color runs off.
  • 27. step 8 : rinse with distilled water Step 8 : rinsed with distilled water Decolorization time is determined primarily by the thickness of the smear. It is very important not to over-decolorize. Then it is rinsed with water
  • 28. step 9 : counter stain with safranin Step 9 : counter stain is added for 45 s Gram safranin counter stain is applied for 30–45 seconds. Safranin serves to stain bacterium which are gram-negative that do not hold crystal violet after decolorization
  • 29. step 10 : rinse with distilled water Step 10 : rinsed with distilled water slide is tilted on an angle and the slide is gently rinsed with distilled water from a dropper bottle by dropping water above the smear and allowing it to rinse the stain off the smear. It is rinsed until the solution dripping from the slide is clear.
  • 30. step 11 : slide is gently dried Step 11 : gently dried Step 11 : gently dried
  • 31. step 12 : oil emersion is added above the smear Step 12 oil emersion is added
  • 32. step 13 : observation is done under microscope Step 13 : observed under microscope
  • 38. Streptococcus pneumoniae in sputum Gram positive cocci
  • 43. Precautions • Culture selection • Fresh and young culture should be used to avoid misleading results because old cultures of gram positive tend to decolorize more rapidly and show Gram negative ness ;lose the ability to retain crystal violet iodine complex • It is possible to report Gram negative bacteria if Gram positive bacteria is old , dead ,damaged and the cell wall is not intact
  • 44. Precautions • Smear preparation • Smear should be thin and uniform to avoid over populated bacteria but sufficiently dense for easy visualization • Even smear should be made other wise alcohol will continue to wash violet colour from thick parts of the smear while thin parts being over decolorized
  • 45. Precautions • Heat fixing • If slide is not completely dried when you pass it through flame organisms will be boiled and destroyed • If too much heat fixing is done , organisms may be incinerated and you will see distorted cells and cellular remains • If you heat fix too much little , organism may not stick and will wash off the slide in subsequent steps
  • 46. Precautions • During decolorization • Over decolorization should be avoided, as over treatment washes crystal violet complex from gram positive and they appear to be gram negative • Acetone decolorizes faster than alcohol • False Gram positive report, if decolorization step is omitted • After rinsing counter stain • care must be taken not to rub the slide with blotting paper as this could remove the adhering bacteria
  • 47. Be careful • Genera Actinomycetes, Arthrobacter, Corynebacterium, Mycobacterium, and Propionibacterium have cell walls particularly sensitive to breakage during cell division resulting in Gram negative staining of these Gram positive cells. Staining of these organisms results in an uneven or granular appearance • Mycobacteria contain mycolic acids and have a high GC content in their DNA. A Gram stain cannot penetrate the waxy cell wall. The hydrophobic lipids cause the Gram stain to give no staining or a variable result.(Acid fast staining)