COLLECTION OF SAMPLES - Microbiological Investigations (2022)

The samples should be collected at the initial presenta-tion prior to the start of anti-microbial therapy. Thetreatment can be initiated based on the results of smearexamination and, if required modified in accordancewith the culture and sensitivity results.

Various types of samples may be collected to aid thediagnosis of corneal ulcer (Table 6.2). The most impor-tant sample for microbiological examination is thecorneal scraping1,2 (Fig. 6.2). The samples should alsobe obtained from the contact lenses, contact lens caseand contact lens solutions if the patient is a contact lenswearer. If there is any lacrimal sac discharge, it shouldalso be sent for microbiological investigations. Althoughit has also been recommended to obtain samples suchas a conjunctival swab and an eyelid swab, it has notproved to be of much help in our experience.

Corneal Scraping

Corneal scraping is the most valuable specimen in cases

of corneal ulcer and its examination is the main stay inthe diagnosis and subsequent management (Fig. 6.1).


Corneal scraping is performed under topical anesthesiapreferably after instillation of two drops of 0.5 percentproparacaine in the lower fornix of the affected eye.Topical 0.5 percent proparacaine is least bactericidal ascompared to other anesthetic agents such as tetracaineand xylocaine.1,3 Proparacaine provides adequateanesthesia within one minute and does not cause intensestinging on first installation.

General anesthesia and sedation may be required inchildren, uncooperative adults or mentally impairedpatients.3, 4


Corneal scraping is obtained using a Kimura’ s spatula(Fig. 6.3). The other instruments for corneal scraping,are 26-gauge needle, Bard Parker blade #57 (Becton


Samples for diagnosis of corneal ulcer

Eyelid swab- Not of much useConjunctival swab - Not of much useCorneal scraping- Most important

Contact lens, contact lens case and solution- Must in contactlens wear

AC paracentesis (Hypopyon)- Deep ulceration or wheninsufficient material is present

Figure 6.2: Fixation of smear by heating

Figure 6.3: Kimura’s spatulaFigure 6.1: Corneal scraping


Dickinson, Franklin Lakes, New Jersy), hypodermicneedle, surgical blade no 15 and calcium alginate swab.The platinum spatula has been traditionally used forcorneal scraping. It is rapidly sterilized with a Bunsenburner and cools rapidly between scrapings. A modifiedplatinum spatula is also available with a roundedflexible tip, which is modified with a honing stone tocreate a narrow tapered roughened edge to enhanceremoval of corneal material.3

We routinely use a 23-gauge needle to scrap the ulcer.It is easier to get good quantity of corneal scrapings dueto the sharpness of this instrument. However extremecaution is need in cases of dry and deep ulcers especiallythin corneas.


A lid speculum is applied gently to separate the lids.Care should be taken not to rub the cornea against thesuperior flange especially while applying the speculum.The scraping may be done under a slit lamp or underan operating microscope. Under direct illumination, theulcer is inspected. Any mucous or debris on and aroundthe ulcer is carefully cleaned with a sterile swab stick.Then, using a Kimura Spatula or a Bard Parker Knife ora 23-gauge needle, the leading edges and base of theulcer are scraped. Streptococci pneumoniae is more readilyfound at the edge of the ulcer whereas Moraxella is morelikely to be present at the ulcer base.1 Since the materialobtained from corneal scraping may not be adequate, itshould be directly inoculated into the culture mediarather than placing it first into the transport media.4 Careshould be taken to ensure that the instrument is movedin one direction only. Multiple scrapings must beobtained to enhance the yield of the organisms. Oneshould be careful not to touch the eyelids or the lasheswhile collecting the sample to avoid contamination.

More recently, calcium alginate swabs moistenedwith trypticase soy broth provides another method ofcollecting corneal specimens.6 Studies have demonstra-ted higher yield of bacteria7 as well as fungi8 when thismethod was used as compared to the platinum spatulas.

Difficulties in Collection of Corneal Scrapings

There may be various situations where it is difficult toobtain the samples for corneal scraping.9

In cases of small corneal ulcers, which are less severe,and in cases of non-suppurative cases of keratitis theremay be insufficient material to inoculate.

In advanced keratitis with severe keratolysis anddescemetocele, it may not be possible to obtain multiplescrapings.

In some cases the overlying epithelium may be intactand it may be required to disrupt the corneal epitheliumusing a surgical blade. In cases of deep stromal keratitis,microsurgical scissors, a no. 11 Bard Parker blade or asmall trephine may be used to obtain an adequatesample.


Smears are prepared by scraping the ulcer and gentlytransferring the material on the spatula on to the glassslide over an area of approximately 1 cm in diameter.An etched or wax pencil mark on the slide obviates theneed to search for the area smeared under the micro-scope. At least two slides are prepared, one for Gramstaining, and the second for KOH wet preparation. Anadditional smear may be prepared for special stains suchas Giemsa, periodicacid-Schiff, calcofluor or Gomorimodified methenamine silver stain for which gelatincoated slide is preferred. 9 KOH wet mount preparationis undertaken to identify the fungal organisms. Gramstaining of the corneal smears is done in all casesroutinely to identify the organisms (Table 6.3). Specialstains may be needed only in certain circumstances(Table 6.3).

In order to use the smear to guide the anti-microbialtherapy, an adequate material should be there andcareful staining techniques should be followed. Prior useof anti-microbial agents, an insufficient sample, excessiveheat fixation, mechanical damage to the cell wall, poorstaining techniques and failure to examine the wholeslide may give erroneous results.4

Routine Smears and Stains

Potassium Hydro-oxide Wet Mount Preparation

The scraped material is spread out as thinly as possiblewith the help of spatula on the slide. One drop of10 percent KOH solution is put on the scrapings and aslide cover is placed. The slide is examined under amicroscope. The KOH helps in loosening the cornealstromal lamellae and exposing more fungal filaments(Fig. 6.4A). It also stains the filaments in a very lightyellow color. 10 percent KOH mount examined byconventional microscope is a useful test in helpingidentification of fungi and Acanthamoeba. The test hashigh sensitivity (92%) and a high specificity (96%), can

Overall, Gram’s stain is accurate in 61 percent ofcases of bacterial keratitis.4 If performed correctly,Gram’s stain identifies the organism correctly in upto75 percent of the cases caused by a single organism andin 37 percent cases of polymicrobial keratitis.12

Difficulties in Gram Staining

Sometimes only small number of organisms is presentin the smear, which may be difficult to identify as theseare usually present in areas which contain necroticepithelial cells and numerous large polymorphonuclearcells.

Gram-negative organisms are more difficult toidentify than the Gram-positive organism due to their

Figure 6.4A: KOH wet mount preparation Figure 6.4B: Gram positive cocci

Figure 6.4C: Gram negative bacilli Figure 6.4D: Acid fast Mycobacteria

be performed in outpatient area and does not involvetoo much of cost.10,11

Gram’s Staining

Gram’s stain classifies the bacteria into two major groupsbased on the cell wall of the bacteria (Table 6.4). Gram-positive bacteria retain the gentian violet-iodine complexand appear blue-purple (Fig. 6.4B), whereas the Gram-negative bacteria lose their gentian violet-iodine complexwith decolorization step and appear pink whencounterstained with safranin (Fig. 6.4C). A 5-minuteGram staining procedure as well as a 5-second Gramstaining procedure is also available.12,13 We prefer to usethe 5-minute Gram staining procedure (Table 6.3).


lighter color. Gram-negative organism may appeargram-positive if decolorization is inadequate.

Caution is also mandatory against various artifacts,which may accompany Gram’s stain such as staineddeposits, carbon particles, talcum powder, sodiumchloride, crystals, melanin and granules. Precipitatedgentian violet may mimic gram-positive cocci.13 If theGram stain reagents are not used frequently, yeast maygrow in the solutions and periodic filtering helps toremove these particles.4

Special Stains

Giemsa Staining

The Giemsa stain is usually used to determine the typeof inflammatory cells present. We do not recommendits use routinely. This stain differentiates bacteria fromfungi, and also identifies chlamydia inclusion bodies andcysts and trophozoites of Acanthamoeba species. Itidentifies the normal and inflammatory cells. WithGiemsa technique the bacteria appear dark blue in color.The yeast cells and fungal hyphae absorb the stain andappear purple or blue while the cell walls and theseptations do not stain. The conventional Giemsa staintakes 60 minutes to perform, although a rapid 15-minutemodification of the stain is also available.

Ziehl-Neelsen Acid-fast Stain

Special stains include the use of carbol-fuchsin or Ziehl-Neelsen acid-fast stain for identification of suspected

Mycobacteria, Actinomyces or Nocardia. Mycobacteria are

acid fast (Fig. 6.4D), Nocardia stain variably, whereas

Actinomyces are non-acid fast.

The use of this stain is based on the resistance of themycobacterial species and certain strains of Nocardia todecolorization by strong mineral acids after stainingwith basic carbol fuchsin. This resistance is due to thepresence of intact cell walls that contain specific lipidunsaponifiable wax fraction.

Calcofluor White

Calcofluor white binds to chitin and cellulose. Becausethe cell walls of the yeast and filamentous fungi arecomposed of chitin and cellulose, these organisms stainbright green with calcofluor white under epiflorescentmicroscope (Fig. 6.5).14 Blankophone stain, which issimilar to calcofluor can also be used to identify fungalhyphae (Fig. 6.6). The cysts of Acanthamoeba likewise alsohave chitin and cellulose and also stain bright green.The trophozoites of Acanthamoeba stain reddish –orangein color.15

Acridine Orange

The acridine orange is a chemoflorescent dye, whichstains fungi and bacteria yellow-orange against a greenbackground when the pH is acidic and an epi-flourescent microscope is used to visualize theseorganisms. It identifies gram-positive and gram-negativebacteria, yeast and hyphal forms of fungi and both the


Gram Stain Procedure

Fix smear by either of the following methods:

Place in methanol for 5-10 min and allow to air dry:preferred method

Pass the slide, through flame two or three times. Allowcooling.

Flood the slide with crystal violet stain. - Allow stain toremain on for 1 minute

• Rinse gently with tap water.

Flood slide with Gram’s iodine solution. Allow solution-to remain on for 1 minute.

• Rinse gently with tap water.

Decolorize with decolorizer solution until the color stopsrunning from the smear

• Rinse gently with tap water.

Flood the slide with Safranin stain. Allow stain to remainon for 30 seconds.

• Rinse gently with tap water.• Allow to air dry.


Gram stain morphology of organisms

Type of stain Organism Color of thevisualized organism

Gram stain Bacteria Gram positive-purple

Gram negative-pinkAcridine orange Bacteria Yellow–orange

Fungi Yellow-orange


Calcofluor white Fungi Bright green

Acanthamoeba cysts Bright greenAcanthamoeba Reddish orangetrophozoites

MycobacteriaAcid fast Mycobacteria

Figure 6.5: Fungal hyphae stained with calcofluor white (Courtesy:

Dr H Sheorey, Dept. of Microbiology, St Vincent hospital, Melbourne)

Figure 6.6: Fungal hyphae stained with blankophore (Courtesy:

Dr H Sheorey, Dept. of Microbiology, St Vincent hospital, Melbourne)

trophozoite and cyst form of Acanthamoeba. The Gram’sstain can be directly done on a slide where prior stainingwith acridine-orange has been done without destaining.The acridine orange stains accurately predicts cultureresults in 71 to 84 percent of cases in comparison to 62to 79 percent for Gram’s stain.16,17

Modified Grocott-Gomori MethenamineSilver Nitrate Stain

For fungal infections, this stain is more reliable than theGram, Giemsa, or KOH stain. The specimens should bespread onto gelatin-coated slides. With the methena-mine-silver nitrate stain, fungus cell walls and septablack and can be easily seen against the background,which is a faint transparent green.

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