**9. Laboratory methods: The support of the diagnosis in bacterial and fungal keratitis**

In all inflammatory corneal infection it is recommended in the first consultation, for diagnos‐ tic purposes, to take a small sample of corneal secretion of the surrounding or central zone of the ulcer, taking care of avoiding being so invasive. In imminent perforation risk keratitis, the best site is in the edges of the ulcer, and it is the best to take the sample with cotton swab, algi‐ nate [11] or Kimura spatula for seeding the sample in the cultures mediums.

Before taking the corneal sample, 2 drops of topical anesthetic (tetracaine 5 mgs/ml) are ap‐ plied before obtaining smears from the corneal ulcer with heat sterilized and cold Kimura spatula, or with the sterile cotton or alginate swab as mentioned before, in order to be seed‐ ed in Petri dishes with culture medium in C streaks, in a wide variety of medium; blood agar (5% sheep blood in brain heart agar base), chocolate agar (1% enrichment supplement‐ ed) incubated in 4-5% CO2 ambient at 37º centigrade for bacterial growth. For fungus cul‐ tures, Biggy agar slant for *Candida;*Sabouraud dextrose 2%, or Sabouraud Emmons both with 0.01% cloramphenicol and without cycloheximide inhibitor agar slant, with incubation at 27º centigrade and daily observation for a minimum of 3 weeks [12].

In the same way, three samples it have to be taken for making three smears in the center of each previously cleaned slide marked with a circle made with glass pencil for staining and microscopic observation.

Microscopic examination to search bacteria and fungi was made in each case by periodicacid Schiff (PAS), Giemsa and Gram or in some cases by Zihel-Neelsen for acid-fast bacilli according to Prophet [13], and in few cases with calcofluor-Evans Blue(Cellfluor) and epi‐ fluorescent light microscopy. (Figure 2)

In keratitis cases diagnosed with concomitant anterior or posterior endophtalmitis, the sam‐ ples of aqueous and vitreous humors, can be taken in the surgical room by an ophthalmolo‐ gist and sent to the laboratory in the same syringe, in which the samples were collected. These samples will be seeded in the cultures mediums as described before and in conven‐ tional mediums for anaerobic incubation. The isolated bacteria can be identified by conven‐ tional, automated or semi automated test.

The fungal yeast isolated from corneal scrap samples may be identified by AUXACOLOR ® 2 (Biorad® France) absorption sugars kit for *Candida,* cell germination forming pseudomyi‐ celium (Figure 3) and microcultures in corn meal agar with cover glass over the seed, by the characteristic organization of hyphae, and chlamydospores in each Candida specie.

**Figure 2.** Calcofluor-Evans Blue stain and florescence microscopic view of a corneal smear in a keratomycose in a case patient

**Figure 3.** *Candida albicans* pseudomicelium test stained with PAS 1000 X

#### **Recommendations**

1. The sample must be taken before the start of treatment

**8. Application area**

66 Common Eye Infections

**fungal keratitis**

microscopic observation.

fluorescent light microscopy. (Figure 2)

tional, automated or semi automated test.

The area of application of this descriptive study, is in the diagnosis of corneal infections, and spe‐ cifically related to the support of the laboratory in the diagnosis of bacterial and fungal keratitis.

In all inflammatory corneal infection it is recommended in the first consultation, for diagnos‐ tic purposes, to take a small sample of corneal secretion of the surrounding or central zone of the ulcer, taking care of avoiding being so invasive. In imminent perforation risk keratitis, the best site is in the edges of the ulcer, and it is the best to take the sample with cotton swab, algi‐

Before taking the corneal sample, 2 drops of topical anesthetic (tetracaine 5 mgs/ml) are ap‐ plied before obtaining smears from the corneal ulcer with heat sterilized and cold Kimura spatula, or with the sterile cotton or alginate swab as mentioned before, in order to be seed‐ ed in Petri dishes with culture medium in C streaks, in a wide variety of medium; blood agar (5% sheep blood in brain heart agar base), chocolate agar (1% enrichment supplement‐ ed) incubated in 4-5% CO2 ambient at 37º centigrade for bacterial growth. For fungus cul‐ tures, Biggy agar slant for *Candida;*Sabouraud dextrose 2%, or Sabouraud Emmons both with 0.01% cloramphenicol and without cycloheximide inhibitor agar slant, with incubation

In the same way, three samples it have to be taken for making three smears in the center of each previously cleaned slide marked with a circle made with glass pencil for staining and

Microscopic examination to search bacteria and fungi was made in each case by periodicacid Schiff (PAS), Giemsa and Gram or in some cases by Zihel-Neelsen for acid-fast bacilli according to Prophet [13], and in few cases with calcofluor-Evans Blue(Cellfluor) and epi‐

In keratitis cases diagnosed with concomitant anterior or posterior endophtalmitis, the sam‐ ples of aqueous and vitreous humors, can be taken in the surgical room by an ophthalmolo‐ gist and sent to the laboratory in the same syringe, in which the samples were collected. These samples will be seeded in the cultures mediums as described before and in conven‐ tional mediums for anaerobic incubation. The isolated bacteria can be identified by conven‐

The fungal yeast isolated from corneal scrap samples may be identified by AUXACOLOR ® 2 (Biorad® France) absorption sugars kit for *Candida,* cell germination forming pseudomyi‐ celium (Figure 3) and microcultures in corn meal agar with cover glass over the seed, by the

characteristic organization of hyphae, and chlamydospores in each Candida specie.

**9. Laboratory methods: The support of the diagnosis in bacterial and**

nate [11] or Kimura spatula for seeding the sample in the cultures mediums.

at 27º centigrade and daily observation for a minimum of 3 weeks [12].

2. It is nedded the use of 2 drops of topical anesthetic (tetracaine5 mgs/ml) before scraping any corneal lesion.

#### **Giemsa Cytology**

Polymorphonuclear leukocytes +++ Bacterial or fungal keratitis Polymorphonuclear leukocytes +++, Eosinophils ++ Allergic keratoconjunctivitis Lymphocytes and macrophages +++ Viral or toxic keratitis.


**Table 1.** Clinical and laboratory features in bacterial and fungal keratitis

For white filamentous and melanized fungus cultures, it may be observed for its morpholo‐ gy and pigmentation, on surface and reverse of the colony and for its final identification in microcultures for his characteristics conidial forms, prepared with lactophenol blue and di‐ rect optical microscopy observation according to the Manual of Clinical Microbiology [14] and Larone [15].
