Gram Staining
The Most Commonly Used Differential Stain
Advantages:
• Can observe size and morphology (like other staining)
• Can find out additional information about the organism- primarily what
type of cell wall/envelope it has
• This determination leads to more information about the cell, for example,
toxin production, enzyme production, characteristic symptoms during
infections, types of antibiotics for treatment, and more
(A) Gram Negative
Cell Membrane
(B) Gram Positive
Gram Negative
Gram Positive
Step 1: Start with a smear prep:
• Think of your smear preps from last lab,
• Were the smears from broths too sparse? Add more this time – if the
organisms are too sparse, it is difficult to find them and evaluate them
• Were the smears from solids too thick? Add less this time – if it is too thick,
decolorization and interpretation is too challenging
• You should be able to adjust your smears based on your previous results
• Try to get the smear evenly distributed
• The lab book suggests using a positive control (a known Gram positive
bacterium) and a negative control (a known Gram negative bacterium) along
with what organism you are trying to determine. This increases the amount
of time it takes to make a smear prep and does complicate the process. I will
suggest some organisms to start with as an alternative.
• Remember – a “smear prep” always finishes with heat fixation
Step 2: Flood your slide with crystal violet
(primary stain)
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All the Gram stains are in the Gram staining kits at each table (there are 3 or 4 per table)
Rinse off after one minute (longer than one minute will not cause any problems)
Gently tap off the excess water
All bacteria are stained purple at this time
Step 3: Flood your slide with Gram’s iodine
(mordant)
• This forms an insoluable complex with the crystal violet and attaches to the
peptidoglycan layer
• Rinse off after one minute (longer than one minute will not cause any problems)
• Gently tap off the excess water
Next – Decolorize with Gram’s decolorizer
(alcohol/acetone)
• This is the critical step
• Hold your slide at an angle and drip decolorizer onto your slide until it runs
clear
• Immediately stop the decolorization by rinsing the slide with water
• If your smear is uneven, it causes part of the smear to be either over or
under decolorized
• You have to decolorize longer if it is thick, therefore over decolorizing the thinner
areas
• Or if you decolorize the thinner areas perfectly, you may have under decolorized the
thicker areas
• Only YOU can be the judge of YOUR slide, so pay attention to your technique
• Practice makes you better!
• Positives are purple and Negatives should be colorless at this step
Lastly – Flood the smear with safranin
(counterstain)
• Time should be around 1 -2 mins (longer will not cause a problem)
• Rinse off with water and blot in bibulous paper
• Be sure to rinse stain off the back of the slide as well
Observe UNDER 100X WITH OIL IMMERSION
This is a DIFFERENTIAL staining procedure – you get 2 results:
1) Purple (crystal violet) is POSITIVE
2) Pink (safranin) is NEGATIVE
Used slides are put in sanisol dishes, add additional sanisol if needed
Close all stain bottles and return to them to the Gram staining kits
POSITIVE GRAM STAIN
• This reaction is associated with bacteria that have a specific cell wall – a
thick peptidoglycan layer which combines with the crystal violet/iodine
complex and is not easily decolorized.
• Results are best on younger cultures such as 24 hr. cultures
• When cells die off, they don’t pick up the stain as well, such as an older
culture
• Interpret your stain results by considering all of the variables (age of
culture, thickness of smear, difficulty of decolorizing, numbers of organisms
that appear purple or pink)
• Sometimes you will be wrong – you get better with practice
• Remember this is a skill – these are living organisms – some results will not
be explained – be open minded – make your best assessment – its simple –
do you think it is positive or negative?! (Just like – Is it a rod or a cocci?) In
other words, don’t overcomplicate it!
Gram Positive Cell Wall
Examples of Gram Positives
Notice everything is not purple – but it is
still obvious that the majority is purple
Still seeing pink around the edges, that is
just safranin collecting or staining cellular
or other debris
More Examples of Gram Positives:
Example of a Gram positive with some
variable stained cells
Colorless spores inside cells – all known
spore formers are Gram positive
Negative Gram Stain
• This reaction likely occurs because the peptidoglycan layer is thin and the outer
membrane outside of the peptidoglycan layer is easily destroyed by the
decolorizer, allowing the crystal violet to be removed easier.
• The bacterial cells were decolorized (they had been stained with the crystal
violet, but it was washed away and they became colorless) and then they were
stained with the counterstain, safranin
• They are now pink
• Thicker areas may have purple still, some cells may seem purplish, you simply
have to decide, based on the thickness of the slide, the difficulty in decolorizing,
the age of the culture, etc.
• As a possible rule – it is more likely that Gram positive organisms get over
decolorized as a problem, Gram negative organisms are easily decolorized and
even if you over decolorize them, the results are still the same – they are still
pink. If you undercolorize them, they would appear Gram positive.
• Only YOU can interpret YOUR Gram stain results! You get better with practice!!
Gram Negative Cell Wall
Examples of Gram Negatives
Notice some areas are dark but it’s still
pink
Coccobacilli – small rods are common
for the Gram negative rods
More Examples of Gram Negatives
Notice some of the rods look purple
but the majority are pink
Sometimes they look this light – so be
careful not to turn your light up too bright
Debris and Stain Crystals: notice the size and
shapes of the unwanted artifacts