4.4: Negative Stains Procedure
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Negative Stains Procedure
Name: _____________________________________________________
Course Section: ______________________________________________
The following activity will teach students how to complete a Negative 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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2 Microscope Slides
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2 Microscope Slip Covers
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2 Stock Plates
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Stock plates will contain 1 of the following bacterial species:
Bacillus cereus
or
Micrococcus luteus
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DI Water
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Nigrosin Stain
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Staining Trays and Slide Stands
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Inoculation Loop
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Incinerators
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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 1 person's initials and the bacterial species. You may abbreviate the name of the bacteria so it fits on the slide. Try to write small.
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On the bottom of the slide, you may draw a circle in the middle of the slide with 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 coin.
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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: Negative Staining
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DO NOT Heat Fix.
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Once the bacterial smear is completely dry, add 1 or 2 drops of Nigrosin Stain to the end of the microscope slide.
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DO NOT add the Nigrosin Stain to the middle of your slide or on the smear.
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Preferably, add the stain directly below the area you labeled with a sharpie.
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Keep your slide on the stands in the staining tray.
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Place a clean cover slip into the stain at a 45° angle and drag the stain across the smear to the other end of the slide.
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Now, the entire slide should be black and left to air dry.
DO NOT Heat Fix.
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When the slide is completely dry, observe your specimen using oil immersion.
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Do Not use a wet nigrosin slide on the microscope. The nigrosin dye stains glass, which means it can stain the lens of the microscope objective (damaging it).
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Wait until the stain looks like dried paint or loses its "shiny" appearance.
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Repeat Steps 1 and 2 for the remaining bacterial species.
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When all 2 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. What are the advantages of using a Negative Stain instead of a Simple Stain?
2. Why do bacterial cells appear white after application of Nigrosin?
3. If you used heat application during a Negative Stain, what would you expect to happen to the bacterial cell body.
4. Why is it important to let the Nigrosin Stain dry completely before observing the slide under a microscope?
5. You prepare a negative stain of
Staphylococcus aureus.
What would you expect cells to look like under oil immersion?
6. When performing a negative staining technique on the fungi
C. neoformans
, why does it appear to have a white halo around it?
7. What is the name of the negative staining technique used to identify
Cryptococcus?
Attributions
"Microbiology Laboratory Manual: Labs, 1.13 Capsule Stain"
by
Dr. Rosanna Hartline
,
LibreTexts: Biology
, West Hills College Lemoore 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