Simple Stains Procedure
Name: _____________________________________________________
Course Section: ______________________________________________
The following activity will teach students how to complete a Simple Stain using bacteria that have different cell shapes and arrangements.
Read the following procedure carefully before starting the activity.
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Materials
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3 Microscope Slides
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3 Stock Plates:
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Stock plates will contain 1 of the following bacteria:
Bacillus cereus, Staphylococcus epidermidis, or
Escherichia coli
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DI Water
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Crystal Violet Stain
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Bibulous Paper
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Hot Plate
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Clothes Pins
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Staining Trays, Slide Stands
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Inoculation Loop
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Incinerators
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Timers
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Sharpies and/or Wax Pencils
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Microscopes: Lens Paper, Lens Cleaner, Immersion Oil
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Procedure
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Step 1: Prepare A Bacterial Smear
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Get into groups of 2.
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Label the edge of the microscope slide with the bacterial species. You may abbreviate the name of the bacteria so it fits on the slide.
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On the bottom of the slide, draw a circle in the middle with a sharpie or wax pencil so you know where to make your smear. This is optional.
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Sterilize your inoculation loop and use it to pick up a small amount of DI water. Place the water on top of the slide, in the middle or in the circle if you drew one.
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Sterilize your loop again and pick up a small bacterial colony from the stock plate.
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Gently mix the bacteria into the water drop on the slide. You want to spread the water and bacteria out to about the size of a quarter.
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Let the water and bacteria smear completely air dry. The slide should look dry and "crusty" before moving to Step 2.
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Step 2: Heat Fixation
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Once the bacterial smear is completely dry, use a clothes pin to clip the end of your slide.
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Place your slide directly on a hot plate for 30 seconds.
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After 30 seconds, place your slide onto the slide stand in your staining tray.
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Don't touch the slide, it will be hot. Don't place the slide on the cold table, the glass slide could crack.
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Once your slide is completely cool, move on to Step 3.
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Step 3: Simple Staining
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With your slide on the stand
and
in the staining tray, add a few drops of Crystal Violet Stain to the smear.
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You do not need to cover the entire slide with stain, focus on the area you added bacteria to.
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Let the stain sit on the slide for 1 minute.
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Lift one end of the slide up to about a 45° angle.
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Apply DI water slightly above your smear so that it flows down across your bacteria and into the staining tray.
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Wash the slide until the water run-off is clear.
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Never apply water directly to the smear, you can wash the bacteria off the slide.
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Place the clean slide between 2 sheets of bibulous paper and gently pat dry.
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Don't rip paper from the booklet, you can place your slide directly on the pages.
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Observe your specimen using oil immersion.
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Repeat Steps 1 through 3 for the remaining bacterial species.
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When all 3 slides are done, clean your microscope and place it back in the cabinet.
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Rinse your staining tray in a sink and put all materials back in their labeled locations.
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Observation Results
General Questions
1. Simple stains are used in microscopy to increase which principle of microscopy?
2. Why is it important to create a thin bacterial smear (a smear with a small amount of water and small amount of bacteria)?
3. What is the goal of heat fixation in a simple stain?
4. You prepare a simple stain of
B. subtilis.
When you observe your slide under the microscope, you don't see any cells or stain. What could have gone wrong in your simple stain prep that may have caused this result?
5. You prepare a simple stain of
S
treptococcus pneumoniae.
When you observe your slide under the microscope, the cells appear to be in a single, mono arrangement. What could have gone wrong in your simple stain prep that may have caused this result?
Health-Related Questions
1. Based on the genus name
Staphylococcus
, what can we infer about the shape and arrangement of
Staphylococcus aureus
?
2. Bacterial shape and arrangement should never be used as the ONLY evidence for a disease diagnosis. Why is this the case?
Attributions
"Microbiology Laboratory Manual: Labs, 1.9 Simple Stain"
by
Dr. Rosanna Hartline
, West Hills College Lemoore,
LibreTexts: Biology
is licensed under
CC BY-NC-SA 4.0
"General Microbiology Lab Manual: Labs 3, Simple, Negative, and Gram Stain"
by
Nazzy Pakpour and Sharon Horgan
,
LibreTexts: Biology
,
Coastal State University, Easy Bay
is licensed under
CC BY-SA
"Microbiology Textbook: Chapter 2, How We See the Invisible World"
by
Openstax
, Digital ISBN 13: 978-1-947172-23-4 is licensed under
CC BY 4.0
Image Citations
Procedure Image, Modified From: