Buccal Smear

Buccal Smear Lab

The cells which line the inside of your cheeks are classified as a stratified squamous epitheliumtissue and are the surface of a mucous membrane. These flat, scale-like buccal cells (pronounced “buckle”) are shed constantly as the tissue is renewed. By gently scraping the inside of your cheek, these cells can be harvested, and when smeared and stained, may be used to illustrate a number of important biological phenomena including cell and tissue structure, oral bacterial floraand morphology, etc. This tissue is non-keratinized and therefore the surfacecells are living and still possess their nuclei, in contrast with shed epidermalcells. See DiFiore’s Atlas of Histology, 9th Ed, pp 326&327 for similar tissue found lining the vagina. (Note that the keratinized surfacecells of the epidermis have no nucleus.) Here is a labeled image of a buccal smear stained with methylene blue.

Compare the following steps with those in the Bact. Smear and Staining Protocol.

Equipment and Supplies

Soap and Water
Microscope Slides
Dropper Bottle of dH2O
Dropper Bottle of 0.3% Methylene Blue
Toothpicks (optional)
Bunsen Burner or Alcohol Lamp, Striker
Bibulous Paper or Paper Towel

Preparation of Slide and Fingernail:

Wash Slide
Wash Slide

 

Clean a microscope slide well with soap & water, dry with a non-linty paper towel.

 

 

Clean Finger Nail
Clean Finger Nail

 

Cleanse very thoroughly under the nail of your index finger.

 

 

Add Small Drop of Water
Add Small Drop of Water

 

Place a small drop of dH 20 in the center of the very clean slide.

 

 

Harvest the Cells, Prepare the Smear:

Collecting Cheek Cells with Finger
Collecting Cheek Cells

GENTLY scrap the inside ofyour cheek to pick up some of the shed stratifiedsquamous cells. Do NOT scrape chunks. A toothpick may be used if you have no fingernails. Gentle scraping is the watchword, there should be no discomfort.

 

Express the cheek cells from the fingernail
Expressing the cells

 

 

 

 

Press Cells onto Slide
Press Cells onto Slide

 

Express the material from under your nail by pressing with your thumb, and press the material into the drop of water on the slide, mix and spread the material around to the size of a dime.

 

Spread Material on Slide dime sized
Spread Material on Slide

 

 

 

 

Fix the Smear

Fix Specimen on Slide
Fix Specimen on Slide

 

Pass the slide through the flame several times to fix the smear. Do NOT heat the slide above a temp which is comfortable. You are merely “gluing” the smear to the slide.

Stain the Smear

Add Methylene Blue to Slide
Add Methylene Blue

Place a drop of 0.3% methylene blue on the specimen. Let sit for 1 minute.

 

 

Rinse off Slide
Rinse off Slide

Rinse off the excess stain with tap water. (Do not splash on your white shirt!)

 

 

Blot Dry Slide
Blot Dry Slide

Blot dry with an non-linty paper towel or bibulous paper. Do not rub.

 

 

Blot Dry Slide
Blot Dry Slide

 

 

 

Dry Slide with burner
Dry Slide

Flame again briefly to dry slide.

 

 

Examine Under Microscope

Buccal Smear
Buccal Smear

Examine first with the 4x objective, scanning the entire field to find a well-distributed region. Avoidregions where cells may be piled up to thickly . Then view with the 10x and 40x objectives, illustrating the view at both powers. Note 1) the nucleus, 2) nucleolus, 3) cell boundary and 4) the variety of bacteria colonizing the surface of the cells.
For Microbiology only: you will be instructed on oil immersion use , then illustrate bacterial morphologies with the 100x oil immersion objective.

Clean Up

Washed Slides
Washed Slides

When finished, scrub the slide well in hot soapy water, rinse well and drain dry in a plastic test tube holder.

Isolation of Buccal Cell DNA

Isolation of Buccal Cell DNA

This procedure is used to isolate individual DNA to be used in future PCR probes. Completely non-invasive and straight forward, it is a simple method to isolate small amounts of DNA from buccal cells.

EQUIPMENT:
15 mL sterile polypropylene centrifuge test tube
two sterile 1.5ml Eppendorf tube
5mL pipettor + sterile tips
1000 uL micropipettor + sterile tips
200 uL micropipettor + sterile tips
clinical centrifuge, balance
1.5 mL test tube holder (> 10 holes)
boiling water bath in 1000 mL beaker
microcentrifuge

SUPPLIES PER STUDENT:
10 mL 0.9% saline aliquoted into the 15 mL centrifuge tube
package of beverage straws
0.5 mL 10 % Chelex resin beads in sterile dH2O

LABEL TUBES:
Saline centrifuge tube
1.5 mL Eppendorf for separating Chelex
1.5 mL Eppendorf for storage of DNA
HARVEST BUCCAL CELLS:
Label a 15 mL polypropylene test tube and the top of a 1.5ml Eppendorf tube with your name and/or seat number.

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Pour the 10 mL of saline solution into your mouth and vigorously swish against your cheeks for 10 seconds.
With a beverage straw, deliver the saline wash solution back into the labeled 15 mL polypropylene test tube.

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SPIN DOWN CELLS
Place your test tube, with others, in a balanced array in the clinical centrifuge. Centrifuge at 2000 x g for 10 minutes. (Top speed, setting number 7 on the tabletop clinical centrifuge.)

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pelleted buccal cells spun down from saline

The cells form a firm pellet below the saline supernatant.

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SAVE THE PELLET, DISCARD THE SUPERNATANT by decanting into the sink with running water, taking care not to disturb pelleted cheek cells at the bottom of the tube.

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Drain all of the saline supernatant.

ADD CHELEX BEADS: Chelex is an ion exchange resin which removes polyvalent metal ions which might break down DNA during boiling or inhibit PCR reactions (next experiment).
Enlarge the aperture of the end of a 1000 uL pipetor tip (blue) by clipping off 2 mm from so that particulate matter will not stop it up. Use this prepared tip to resuspend the 10% suspension of Chelex resin beads by pipetting the beads in and out of the micropipettor. Before resin settles, pipet 500 uL of Chelex into the 15 mL tube containing your buccal cell pellet. Save pipet tip.

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RESUSPEND CHEEK CELLS WITH CHELEX Using the same prepared blue tip, resuspend the cells in the pellet by pipetting in and out several times. (If the tip stops up, snip off 2 mm of the tip.)

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Examine the suspension carefully to ensure that no visible clumps remain.

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Using the same prepared tip, transfer a 500 uL aliquot of the cells and resin suspension to a clean 1.5 mL Eppendorf tube labeled with your name.

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chelex treated cells 100 C for ten minutes

BOIL FOR 10 MINUTES: The cells are lysed and proteins denatured by exposing to 100 C for ten minutes: place your sample, along with other samples from the group, into a 1.5 mL floating test tube holder and float in a boiling water bath for 10 min.

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Time the ten minutes in the boiling water bath.

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CHILL ON ICE: After the heat treatment, transfer all samples to crushed ice.

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SPIN DOWN CHELEX: Place your chilled sample, along with others, in a balanced array in a microcentrifuge and spin for 30 to 60 seconds at top speed.

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The chelex precipitates along with the denatured protein. The DNA is in the supernatant.

OLYMPUS DIGITAL CAMERA

SAVE 200 uL CLEAR SUPERNATANT: Use a fresh pipet tip to transfer 200 uL of the clear supernatant to a clean 1.5 mL Eppendorf tube labeled with your:
Seat Number
name
date
cheek DNA
Take care not to pick up any of the cheek cell debris or resin from the bottom of the tube. Store your sample for a few minutes or hours on crushed ice or for days at -20 degrees Centigrade until you are ready to proceed to Set up and run PCR reaction