**7.2 Smear staining and culture**

Corneal scrapings are obtained in the office under the slit lamp. A topical anesthetic agent is instilled, ideally proparacaine hydrochloride 0.5% or a preservative-free anesthetic [81]. The corneal material is obtained with a sterile platinum spatula, blade, forceps, or a calcium alginate swab moistened in thioglycolate broth. The smear stains helpful in identifying the causative organism are Gram stain, Giemsa stain, and Acridine orange are the most frequently used for detecting bacteria. The Gram stain permits identification of gram-positive and -negative coccus and rods, which is essential to choose the initial antibiotic type before the antibiogram and sensitivity profile of the microorganism in question is available. For example, cephalosporins are more appropriate for gram-positive and aminoglycosides for gram-negative bacteria [82].

In case of presumptive fungal infection, special stains like potassium hydroxide (KOH) and calcofluor white (CFW) are more reliable to initiate antifungal therapy than Gram staining is for bacterial infection (**Figure 4A** and **B**) [82, 83].

#### **Figure 4.**

*A. Potassium hydroxide (KOH) preparation of a corneal smear from a fungal CLAIK patient, showing septate, branched, hyaline hyphae characteristic of filamentous fungus. B. Sabouraud dextrose agar (SDA) plate showing white, cottony colonies consistent with Fusarium solani.*


#### **Table 3.**

*Most used microorganism identification staining techniques for the diagnostic confirmation of contact lens-associated infectious keratitis.*

Mycobacterial or *Nocardia* infection will require the acid-fast or modified Ziehl-Neelsen (1% H2SO4, cold) staining (**Table 3**).

According to the American Academy of Ophthalmology Bacterial Keratitis Preferred Practice Pattern, cultures and smears should be obtained in cases of suspected microbial keratitis in the following conditions:


Corneal scrapings should be directly inoculated into the culture media at room temperature and immediately taken to the laboratory for further processing. If culture media are not readily available, scrapings should be inoculated into transport media, including brain-heart infusion media and amies medium with charcoal. Both transport media may be used for aerobic and facultative anaerobic bacteria and, the latter, also for fungi [82]. Standard culture media include blood agar, chocolate agar, Sabouraud dextrose agar, thioglycolate broth, and mannitol salt agar. If *Acanthamoeba* is the suspected pathogen, a non-nutrient agar with *Escherichia coli* overlay must be used (**Table 4**) [82, 85]. In addition to culturing corneal scrapings, cultures of the contact lens and case can also yield positive results. Corneal scrapings *Contact Lens-Associated Infectious Keratitis: Update on Diagnosis and Therapy DOI: http://dx.doi.org/10.5772/intechopen.100261*


## **Table 4.**

*Respective culture media type used for microorganism isolation in contact lens-associated infectious keratitis.*

culture provides positive results in 34–44% cases [67, 86–88], while cultures of contact lenses are positive in 67–92%, and 80–85% for contact lens cases [66]. Studies have found an association between cultures of corneal scrapings and of contact lenses, with a concordance of up to 84% [67, 89]. Therefore, contact lens culture may guide in the identification of the causative organism in cases in which the corneal scraping culture is negative; however, contact lens cultures do not replace corneal cultures as the gold standard for the etiologic diagnosis of microbial keratitis [67].

#### **7.3 Tissue biopsy**

A corneal biopsy may be performed if there is an inadequate response to treatment or if cultures are repeatedly negative, particularly for suspicious *Acanthamoeba* keratitis (**Figure 5A**–**C**). It can be performed at the slit-lamp or in the operating room using topical anesthesia and a small 2 or 3-mm dermatologic trephine punch; the tissue obtained is then bisected and sent for culture and histopathologic analysis. A section of the corneal specimen is homogenized with trypticase soy broth and cultured on conventional blood and chocolate agar, anaerobic media, Sabouraud agar, and thioglycolate broth; in specific cases, the corneal specimen may also be plated on a non-nutrient agar with *E. coli* or Lowenstein Jensen media. The specimen section that is sent for histopathologic analysis may be processed with standard stains for bacteria, fungi, acid-fast-bacilli, and *Acanthamoeba* such as Gram and Giemsa stain, potassium hydroxide, calcofluor white and, Ziehl-Neelsen [90]. Several considerations should be taken into account to maximize the diagnostic yield of a corneal biopsy [90–92]:

• To obtain the tissue specimen, topical antibiotics must be suspended at least 24–48 hours before the procedure [90]. Also, appropriate planning and consultation with the microbiologist and pathologist is recommended (i.e., need for special stains for fastidious organisms, appropriate fixatives if electron microscopy is required) [91].

