Corneal Culture Tutorial

Chris Watts, MD


Approx 7 minutes.

In this updated video Chris Watts of the University of Iowa reviews how to perform a culture of the cornea. Dr. Watts did this video while on the cornea service with the mentorship of Dr Michael Wagoner, Dr Ken Goins, and Dr Anna Kitzmann. (From the EyeRounds.org Facebook Page)

Supplies:

For Bacterial and Fungal Culture:

Calcium Alginate Swabs Sterile

For Viral Culture:

Sterile Polyester Tipped Swabs Sterile

For Viral Culture/PCR:

Pink Viral Media
Sterile Tear Film strips
Sterile Jeweler s Forceps
Sterile eppendorf tube

For Fungal Culture:

Potato dextrose slant tube

For Bacterial Culture:

Tryptan Soy Broth (aka TSB, for aerobic bacteria)
Thioglycolate Broth (for anaerobic bacteria)
Red blood agar (for aerobic bacteria)
Chocolate agar (for Haemophilus and N. Gonorrhea)

For Mycobacterial Culture:

Middlebrook 7H11 Slant Tube
or
Lowenstein-Jensen Slant Tube

Glass slides x 2 (for Gram and fungal stain)
Slide Holder
Sharpie for labeling glass slides and area of smear (with sharpie, label the side opposite of the side with the smear)

Topical Anesthetic
Patient labels for each of the media sent
Specimen bags

Procedure:

  1. Gather all of the supplies listed above
  2. Pre-label all of the different media, slides, and tubes prior to inoculation
  3. Apply topical proparacaine to eye with corneal ulcer
  4. First, smear the two slides for Gram and Fungal stain
    • 1st: Use the calcium alginate swabs dipped in TSB
  5. Collect samples for HSV/viral media
    • 1st: Hold sterile tear film strip with jeweler s forceps and obtain a sample of the patient s tear film. Try to avoid the lashes, conjunctiva, and cornea as much as possible. After a sample has been obtained, place the strip into the small Eppendorf tube.
    • 2nd: Use sterile polyester tipped swab to sample the ulcer and stir into the Pink Viral Media
      • **For all subsequent steps, dip Calcium Alginate Swab in TSB prior to swabbing ulcer**
  6. Inoculate the blood and chocolate agar plates
    • 1st: Dip the sterile calcium alginate swab into TSB prior to sampling the ulcer
    • 2nd: Sample the ulcer paying careful attention to avoid the lashes or lids
    • 3rd: Streak the surface of the agar plates making C s and avoid penetrating the agar itself
  7. Inoculate the two slant tubes (Middlebrooke 7H11 and Potato Dextrose media)
    • 1st: Dip the sterile calcium alginate swab into TSB prior to sampling the ulcer
    • 2nd: Sample the ulcer paying careful attention to avoid the lashes or lids
    • 3rd: Streak the surface of the slant tubes
  8. Inoculate the two broth cultures (Thioglycolate first, and then TSB)
    • 1st: Dip the sterile calcium alginate swab into TSB prior to sampling the ulcer
    • 2nd: Sample the ulcer paying careful attention to avoid the lashes or lids
    • 3rd: Swirl the calcium alginate swab into the thioglycolate broth
    • 4th: Dip the sterile calcium alginate swab into TSB prior to sampling the ulcer
    • 5th: Lastly, contaminate and inoculate the TSB broth itself
      • **Critical, as if TSB is contaminated prior to completion of other media inoculation, this could lead to decreased sensitivity and specificity of corneal culture
  9. With all media, broth, and agar plates and slides appropriately labeled and placed in respective specimen bags, send them to your local microbiology lab for analysis.