Staining
Microbiology
Professor Sidelsky
2007
Part Two - Staining
How to Make a Smear
 Stain with Simple Stain
 Stain with Gram Stain
 Acid Fast
 Specialized Staining( Endospore and
Capsule)
 Negative Staining
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How to Make a Smear
1. Label the frosted side of your slide
with your initials, the name of the
organism, and the date.
 Turn the slide over and draw an oval on
the reverse side with a Sharpie
 2. You are going to make the smear on
the frosted side
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Broth Directions for making a
smear
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*****If you are using broth follow these directions
Flame your inoculating loop.
Use aseptic technique and remove the top of the culture tube,
flame the mouth or the culture tube, and dip the loop into the
broth. Make sure that the loop is filled.
Transfer the loopful of broth and bacteria to the slide. Using
a circular motion, spread the broth on the slide.
This is now a " smear"
Allow the smear to dry
When the smear has been allowed to " air dry" , pass the
smear through the flame to " heat fix" - Heat fixation causes
the proteins and cell parts to coagulate and stick to the slide.
Let the slide cool.
How to Make a Smear- Plate or
Slant
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*****If you are using colonies from a plate( Petri
Dish)
Place a drop of water on the slide.
Using aseptic technique, " pick" a colony from the
Plate in order to study the cells that make up a
colony
If you are using a slant
Using a circular motion spread move the inoculating
loop in a circular motion in the drop of water. Start
in the center of the drop and move in a circular
motion to the outside of the drop. The objective is
to have fewer cells on the outside of the circle.
Follow the directions from above
Don't forget to " air dry" and to " heat fix" before
staining
Simple Stain
Simple Stains- Crystal violet and
methylene blue
 1. Place a drop(s) of stain over the
smear. Make sure it covers the entire
area of the smear. Leave the stain on
the smear for one minute. Rinse with
water from the bottle.
 2. Refer to page 64 for cellular
morphology.
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The Gram Stain
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Gram Stain- See Gram Stain Directions on separate page. Please
refer to pages 71-73 in your laboratory manual.
All staining work is to be done at the sink
Care should be taken to work directly over the sink
 Place drop(s) of crystal violet stain on the smear ( 1 minute)
 Rock or roll the slide to cover the area
 Use the water bottle to drip water down the slide
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Gram Stain ( II)
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Place drop(s) of iodine on the slide ( 1
minute)
Place drops of alcohol on the slide 10
seconds ( KEY – do not leave on longer than
10 seconds or it will decolorize)
Place drop(s) of saffranin on the slide for 1
minute
Rinse with water from the bottle
Let the slide air dry
The Microbe Library – A great
Resource
Animation on Gram Stains
http://www.microbelibrary.org/ASMOnl
y/details.asp?id=2020&Lang=
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Gram Stain( step by step)
http://medic.med.uth.tmc.edu/path/gr
ampro.htm
 http://www.lumen.luc.edu/lumen/Dept
Webs/microbio/med/gram/tech.htm
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Typical Gram Stain
http://www.uphs.upenn.edu/bugdru
g/antibiotic_manual/Gram3.htm
 http://www.uphs.upenn.edu/bugdru
g/antibiotic_manual/Gramstains/s
mall/tocframeset1.htm
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Staphylococcus
Gram Negative bacteria
Gram Stain animations and
information
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http://www.microbiologybytes.com/vid
eo/Gram.html
Bacterial Cell Morphology
Acid Fast Stains( Mycobacterium)
Acid- Fast Stain
 Carbol Fuschin Stain is a red stain that is soluble in a lipid
environment. The outer cell wall of the mycobacterium is
mycolic acid
 The stain can be absorbed by placing the stain over a beaker
boiling water. Heat assists the stain inits penetration of the
waxy cell wall
 Cool and rinse with cool water
 Add acid- Alcohol drop by drop until the alcohol runs alost
clear.
 Rinse with water
 Counterstain by applying methylene blue for at least two
minutes
Acid Fast Staining
A shows cells that
are stained with
methylene blue –
These re non acid
fast
 B shows cells that
are stained with
carbol fuschin
which is retained in
the lipid cell walls
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Acid Fast
Differential Staining( Spores)
Add Malachite Green to slide and steam over a
beaker of boiling water ( beaker may be placed on a
hot plate) If stain starts to evaporate, add
additional stain. Stain should remain on for 2-3
minutes
 Remove slides from hot plate and cool Rinse with
water
 Counter stain with Saffranin for a minute. Rinse
with water
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Endospore structure
Endospore
Capsule stain
Crystal violet is applied to the bacterial
smears
 The decolorizing agent is Copper sulfate(
20%) which washes the primary stain out of
the capsule without removing the stain from
the cell wall
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Capsule stain( Klebsiella pneumoniae and Streptococcus
pneumoniae)
Negative staining
Place a drop of nigrosin on the end of a
slide- Use a plain slide
 Use the inoculating loop to mix some of the
bacterial culture with the drop of stain
 Use a clean slide. Hold at a 45o angle
against the drop. The drop will spread along
this line
 Push the slide away from the previously
spread drop
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Negative staining
Negative staining