Transcript Cells and Gram Stain

Class Six – June 8,2006
Microbiology
2006
Part Two - Staining
How to Make a Smear
 Stain with Simple Stain
 Stain with Gram Stain
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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
smaer
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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
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
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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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
Bacterial Cell Morphology