1. Corneal Cultures & Smears
St 황호식/ R2 김근영

2. Introduction Microbiological culture Smear
A means of identifying causative microorganism
Determining sensitivity to antibiotics
Smear
Early detection and differentiating causal organisms

3. Indication Corneal infiltrate
(large & extends to middle ~ deep stroma)
Chronic
Unresponsive to broad-spectrum antibiotic therapy
Clinical features of suggestive of fungal, amoebic, or mycobacterial keratitis

4. Culture set? (1) Topical anesthetic drops
(2) Sterile swabs or Blade (No.15)
(3) Slide glasses
(4) Culture plates (must be
fresh from laboratory)
(5) Marking pen

5. Topical anesthetic drops
Proparacaine hydrochloride 0.5%(Alcaine)
Preferred d/t minimal inhibitory effects
on organism recovery

6. Scraping instrument Heat sterilized platinum(Kimura) spatula blade
No. 15 Bard-Parker blade
Wet Dacron/calcium alginate or sterile cotton swab
Large gauge disposable needle
Mini-tip Culturette
Small trephine for corneal Bx specimens

7. Slide glasses
KOH
Gram stain

8. Culture media Blood agar
Standard medium for isolation of aerobic bacteria at 35℃
Supports the growth of saprophytic fungi and Nocardia at room temperature
Seaweed + 5~10% red blood cells to provide nutrients as well as index of hemolysis

9. Culture media Chocolate agar
Incubated at 35℃ with 10% carbon dioxide to isolate facultative organisms
Heat denaturation of blood to provide hemin and diphosphopyridine nucleotide for growth of Haemophilus, Neisseria, and Moraxella

10. Culture media MacConkey agar
selective for Gram- bacteria and can differentiate those bacteria that are able to ferment lactose

11. Culture media Sabouraud agar
Incubated at room temperature for fungi and Nocardia

12. Culture media Thioglycollate broth
Liquid medium and incubated at 35℃ for aerobic and anaerobic bacteria
Differentiation of aerobic, anaerobic, microaerophilic, and facultatively anaerobic organisms based upon growth at
various levels in the media
Fluid Thioglycollate broth is a reducing medium. It contains sodium thioglycollate, which reacts with molecular oxygen keeping free oxygen levels low. The sodium thioglycollate in the broth creates a redox potential in the tube, with higher levels of oxygen at the top of the tube, and a complete absence of oxygen at the bottom of the tube. Fluid thioglycollate broth also typically contains a redox potential indicator such resazurin, which produces a pink color in an oxidized environment. As with the BHI media, organisms will only be able to grow where their oxygen requirements are met, and will localize to the area(s) of their oxygen requirements in the fluid thioglycollate broth

13. Tube 1  Obligate Anaerobe : absence of growth in the top portion of the broth
where oxygen is present
Tube 2 Obligate Aerobe : the growth is only in the top portion of the tube
Tube 3  Aerotolerant : uniform growth from top to bottom
Tube 4  Facultative : uneven distribution of growth from top to bottom
(more growth at the top)

14. Culture media Standard media Common isolates Blood agar
Aerobic and faculatative, anaerobic bacteria, including P. aeruginosa, S. aureus, S. epidermidis, S. pneumoniae
Chocolate agar
Aerobic and facultative, anaerobic bacteria, including H. influenzae, N. gonorrhoeae, and Bartonella species
Thioglycollate broth
Aerobic and facultative, anaerobic bacteria

15. Culture media Fungi and acanthamoeba can be recovered on blood agar
Supplemental media
Anaerobic blood agar
(CDC, Schaediler, Brucella)
P. acnes, Peptostreptococcus
Lowenstein-Jensen agar slant
Mycobacteria species, Nocardia species
Middlebrook agar
Mycobacteria species
Thayer-Martin agar
Pathogenic Neisseria species
Fungi and acanthamoeba can be recovered on blood agar
For fungi : Sabouraud’s dextrose agar, brain heart infusion
For acanthamoeba : buffered charcoal yeast extract, blood agar
Propionibacterium acnes

16. Culture Procedure Topical anesthesis – proparacaine Under slit lamp
With Spatula, blade or swab
Ulcer base(unless significant thinning has occurred)
Leading edge of the infiltrate
Slide first! Then culture media
Inoculated in C streaks

18. Stain