Corneal Cultures & Smears St 황호식/ R2 김근영

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

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

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

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

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

Slide glasses KOH Gram stain

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

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

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

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

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

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)

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

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

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

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