Chapter 25: MICROSCOPIC FINDINGS

Uses

To evaluate for the presence of cells, casts, and crystals.

Materials

Freshly collected urine specimen, centrifuge, graduated centrifuge tubes, glass microscope slide, and coverslip.

Method

1. Pour 10 mL of freshly collected urine into a graduated centrifuge tube.

2. Centrifuge at ×400 to ×450 gravity for 5 minutes.

3. Decant 9 mL of supernatant, leaving 1 mL in the tube.

4. Resuspend the centrifuged pellet in the remaining 1 mL of urine by stirring with a pipet.

5. Place one drop of resuspended urine on a glass microscope slide.

6. Overlay with a coverslip.

7. Examine initially using scanning ×10 power, emphasizing the periphery of the coverslip, since urinary elements tend to gather at the edges.

8. Switch to ×40 power to focus on specific urinary elements such as cells, casts, and crystals. Use ×100 power as needed for specific identification.

Uses

To determine the presence of uric acid crystals (in patients with gout) or calcium pyrophosphate crystals (in patients with pseudogout) in joint fluid.

Materials

Freshly collected joint fluid, glass microscope slide, coverslip, and polarizer.

Method

1. To prevent interference from polarizing artifacts, clean the slide and coverslip with alcohol prior to using them.

2. Using freshly collected unspun joint fluid, place a drop of joint fluid on the glass microscope slide.

3. Overlay coverslip.

4. View the slide using the polarizer.

5. Scan at ×10 power; ×100 power is needed to see intracellular crystals.

Uses

The first step in the identification of a predominant bacterial organism in a specimen. Classifies an organism by its cell wall’s ability to retain crystal violet dye during solvent treatment. Morphology of the organism is also identified.

Materials

Freshly collected specimen to be examined, glass microscope slide, crystal violet, Gram iodine, acetone-alcohol (acetone, 30 mL, and 95% alcohol, 70 mL), safranin, and Bunsen burner.

Method

1. Put specimen on dry, clean glass microscope slide and allow to air dry.

2. Heat-fix specimen by gently passing over flame.

3. Cover specimen with crystal violet for 1 minute.

4. Rinse off completely with water; do not blot.

5. Cover specimen with Gram iodine for 1 minute.

6. Rinse off completely with water; do not blot.

7. Decolorize for 30 seconds with gentle agitation in acetone-alcohol.

8. Rinse off completely with water; do not blot.

9. Cover with safranin for 10 to 20 seconds.

10. Rinse off completely with water and let air dry.

Uses

To evaluate a lesion (chancre, mucous patche, condyloma lata, skin rash) for the presence of Treponema pallidum.

Materials

Compound microscope with dark-field condenser (dark-field microscope), glass microscope slide, coverslip, and physiologic saline.

Method for Obtaining and Viewing the Specimen

1. From chancre or condyloma lata:

   1. Gently abrade the lesion with a dry gauze.

   2. Dab away any bleeding.

   3. Touch slide to exudative fluid in base of lesion.

   4. Overlay coverslip and view immediately under dark-field microscope using ×40 and ×100 objectives.

2. From mucous patch:

   1. Touch slide to mucous patch.

   2. Overlay coverslip and view immediately under dark-field microscope using ×40 and ×100 objectives.

3. From skin lesion:

   1. Gently scrape surface of skin lesion with edge of a number 15 scalpel blade.

   2. Dab away any bleeding.

   3. Touch slide to exudative fluid rising from skin lesion.

   4. Overlay coverslip and view immediately under dark-field microscope using ×40 and ×100 objectives.

Uses

To examine for clue cells, Trichomonas, and sperm.

Materials

Aqueous sodium chloride, glass microscope slide, and coverslip.

Method

1. Place a drop of saline onto the middle of the glass slide. (Alternative method: Place several drops of saline in a small glass test tube and place the swab in the tube. The swab can then be wiped onto a slide at a later time.)

2. Mix a small amount of vaginal fluid to be examined into the saline drop.

3. Overlay a coverslip.

4. Examine directly through microscope at ×40 and ×100 (oil immersion).

Uses

To examine for yeast and fungus.

Materials

Aqueous potassium hydroxide (KOH) 10%, glass microscope slide, and coverslip.

Method

1. Place a drop of KOH onto the middle of the glass slide.

2. Suspend a small amount of vaginal fluid into the drop of KOH.

3. Overlay a coverslip.

4. Let sit at room temperature for 30 minutes; as an alternative, gently heat the slide over a Bunsen burner but do not boil.

5. Examine under microscope for hyphae and spores.

Uses

To evaluate a patient’s stool sample for the presence of fecal leukocytes.

Materials

Freshly collected liquid stool specimen, glass microscope slide, coverslip, and methylene blue.

Method

1. Place a drop of liquid stool onto the glass slide.

2. Add two drops of methylene blue to the stool specimen.

3. Mix thoroughly.

4. Overlay with a coverslip.

5. Place the edge of a piece of filter paper adjacent to the coverslip to absorb any excess methylene blue.

6. Examine using ×10 objective to scan specimen and ×40 and ×100 to identify specific leukocytes.

Uses

To determine fungal dermatoses or skin infestations.

Materials

Fresh skin scraping, glass microscope slide, coverslip, and 10% potassium hydroxide or mineral oil.

Method

1. Specimen collection:

   1. Gently scrape skin lesion with edge of a number 15 scalpel.

2. Slide preparation:

   1. Pediculosis may be seen grossly clinging to individual hair or under low power. Live nits may fluoresce with a Wood lamp.

   2. For scabies, place a drop of KOH or mineral oil onto the slide.

   3. Suspend a small amount of the scraping onto the drop.

   4. Overlay a coverslip.

   5. Let sit at room temperature for 30 minutes; as an alternative, gently heat the slide over a Bunsen burner but do not boil.

   6. Examine under microscope for hyphae, spores, or infestations.

Uses

To examine cerebrospinal fluid for organisms with capsules, particularly Cryptococcus neoformans.

Materials

India Ink, glass microscope slide, and coverslip.

Method

1. Lightly centrifuge cerebrospinal fluid to concentrate cells at bottom of tube (1-2 minutes).

2. Pour off excess fluid (retain if further testing may be necessary).

3. Take a drop from the bottom of the centrifuge tube and place it in the middle of a glass microscope slide.

4. Place a drop of India Ink into the specimen drop; gently mix.

5. Overlay a coverslip.

6. Examine at ×10 to screen specimen, use ×40 objective to confirm findings.

Uses

To evaluate for the presence of ring trophozoites.

Materials

Air-dried blood smear, Coplin jar of Wright stain, slide rack, pH 7.2 buffer, and blotting paper.

Method

1. Place a drop of blood on the middle of a slide.

2. Hold another slide evenly on top of the slide at a 45-degree angle and drag the slide over the drop of blood to the opposite edge to spread the blood evenly.

3. Allow the blood to dry for 5 to 10 minutes.

4. Stain air-dried smears in a closed Coplin jar of Wright stain for 5 minutes.

5. Place the slide on a rack.

6. Rinse and treat with pH 7.2 buffer primed with 1 mL Wright stain per 400 mL for 3 minutes.

7. Rinse in pH 7.2 buffer for 20 seconds.

8. Blot dry and mount on microscope at ×100 (oil immersion).

Uses

To distinguish between amniotic fluid due to membrane rupture and normal vaginal secretions in patients beyond the 20th week in preganacy who present with spontaneous vaginal fluid passage. Characteristic arborization, or typical ferning pattern, confirms amniotic fluid and spontaneous membrane rupture.

Materials

Sterile speculum, vaginal fluid, microscope slide, microscope, and serile swab or pipette.

Method

1. Place patient in dorsal lithotomy position.

2. Do not use lubricants or cleaning agents.

3. Place sterile speculum into vaginal vault.

4. Obtain sample of vaginal secretions from posterior vaginal pool using a pipette or steril swab.

5. Place a drop of vaginal fluid on a microscope slide.

6. Spread the specimen evenly so that a thin smear is formed.

7. Allow the fluid to air dry for 5 to 10 minutes. Do not apply heat.

8. Examine slide under low power (10×) for ferning pattern.

Uses

To detect the presence of schistocytes or blast cells in a peripheral blood smear. Schistocytes are fragmented red blood cells due to shearing forces in microarterioles lined or meshed with fibrin strands. They are found in patients with disseminated intravascular coagulation, throbotic thrombocytopenic purpura/haemolytic uremic syndrome, microangiopathic haemolytic anemia, uremia, and carcinoma. Turbulent blood flow due to congestive heart failure, artificial heart valves, or vavlular stenosis may cause schistocyte formation. Greater than 1% of forms or greater than two schistocytes per high-powered field suggest schistocytosis.

Peripheral blood smear blasts are circulating immature white blood cells. When elevated white blood cell count and circulating blasts are seen in addition to anemia and thrombocytopenia, acute leukemia is suspected. As leukemia blast cells build up in the bone marrow, they allow less room for production of healthy white blood cells, red blood cells, and platelets. Although the diagnosis of leukemia can usually be made from the peripheral smear, bone marrow examination (aspiration or needle biopsy) is routinely done for definitive diagnosis.

Materials

Two glass microscope slides, drop of blood, pipette, Wright stain, Giemsa stain, immersion oil, and microscope.

Method

Smear Preparation

1. Agitate sample well, by inversion of tube or mechanical rocker.

2. Place a 2- to 3-mm drop of whole blood ¼ in from the right edge of a 1 × 3 in slide using a wooden applicator stick.

3. Place the slide on a flat surface and hold securely.

4. Grasp a second slide (spreader slide) in the right hand between thumb and forefinger.

5. Place the spreader slide onto the lower slide in front of the blood drop, and pull the slide back until it touches the drop.

6. Allow the blood to spread by capillary action almost to the edges to fht lower slide.

7. Push the spreader slide forward at a 30-degree angle, using a rapid even motion. The weight of the spreader slide should be the only weight applied. The drop of blood must be spread within seconds or the cell distribution will be uneven.

8. Allow to air dry.

Wright Stain of Peripheral Smear

1. Completely cover peripheral smear with Wright stain using a pipette. The stain layer should be ⅛ in thick.

2. Wait for 2 minutes.

3. Cover with equal amount of Giemsa solution.

4. Blow gently on the slide to mix solutions.

5. Allow to stand for 4 minutes.

6. Wash slide for 30 seconds with distilled water.

7. Allow to air dry

8. View smear with oil emersion at high (100×) power.

Uses

To detect the presence of eggs of Enterobias vermicularis in patients who present with nocturnal perianal pruritis or concern for pinworm infestation.

Materials

Microscope slide, clear transparent tape, tongue blade, microscope, or a cellulose-tape slide preparation.

Method

1. Affix ½ in of a 4-in piece of tape to the underside of a microscope slide.

2. Hold the slide against the tongue depressor 1 in from the end and lift the long end of the tape from the slide.

3. Loop tape over end of the depressor to expose gummed surface.

4. Hold tape and slide firmly against the tongue depressor.

5. Press gummed surfaces of the tape against several areas of the perianal area.

6. Affix long portion of the tape to the slide.

7. Smooth tape with cotton gauze.

8. View specimen under low (10×) power.

Note: Specimens are best obtained several hours after going to sleep or upon waking before a bowel movement or bath.

Uses

To detect the multinucleated giant cells confirming the presence of a herpes infection.

Materials

Microscope, Bunsen burner, glass microscope slide, 5% methylene blue or Wright stain, or Geimsa stain, immersion oil, and sterile scalpel or hypdermic needle.

Method

1. Unroof a fresh, uncrusted vesicle with a sterile hypodermic needle or scalpel.

2. Scrape the floor of the vescicle with the scalpel and smear scrapings of the lesion onto a glass microscope slide.

3. Let air dry.

4. Fix specimen with absolute alcohol or gentle heat.

5. Stain with blue stain (5% methylene blue, Wright stain, Geimsa stain) for 5 seconds, rinse, and air dry.

6. View preparation through immersion oil at high power (40-50×).