Ecuaciones diferenciales como modelos matemáticos

Most bacteria appear on culture media with distinct characteristics, which allows easy identification. The features to be noted while studying the colonies on solid media include the following: shape, size, elevation, margins, surface, edges, color, structure and consistency.

There various culture media are used to identify the bacteria include the following:

Nutrient agar

Nutrient agar is the simplest medium used in most laboratories. It is made by adding 2 percent agar to nutrient broth (Fig. 3.14).

Nutrient agar contains beef extract (which supplies carbohydrates, organic nitrogen, salts, vitamins), peptone (enzymatic digest of certain proteins; provides readily available nitrogen), agar (extract of marine algae which is used as a solidifying agent) and water. It has a pH of 6.8.

Blood agar

The agar is derived from seaweed with the addition of 5 to 10 percent red blood cells, which provide nutrients,

Figure 3.14: Nutrient agar TABLE 3.6

Diagnostic stains used for preparation of smear Type of stain Organism Color

Gram stain Bacteria Gram-positive-purple Gram-negative-pink Acridine orange Bacteria Yellow-orange

Fungi Yellow-orange

Acanthamoeba

Calcofluor white Fungi Bright green Acanthamoeba Bright green cysts

Acanthamoeba Reddish orange trophozoites

Mycobacteria Acid-fast Mycobacteria Figure 3.13: Grocott-Gomori Methenamine silver nitrate stain showing

Aspergillus (Courtesy: Dr Seema Sen)

23 as well as an index of hemolysis (Fig. 3.15). This is a

standard medium for isolation of aerobic bacteria at 35°C. It also supports the growth of saprophytic fungi and Nocardia at room temperature.

Chocolate agar

Chocolate agar is incubated with 10 percent carbon dioxide to isolate facultative organisms. It is prepared by heat denaturation of blood to 56^oC to provide human and diphosphopyridine nucleotide for the growth of organisms such as Haemophilus, Neisseria and Moraxella.

Thioglycolate broth

This is a liquid media, which is incubated at 35°C for and is used for the isolation of aerobic and anaerobic bacteria. It contains a sulfhydryl compound which acts as an oxygen-reducing agent to facilitate recovery of anaerobic bacteria. Thiol is a variation of thioglycollate broth and contains special complexes and 0.1 percent semisolid agar to prevent convection currents and promote the growth of aerobic bacteria as well as obligate and facultatively anaerobic organisms.

Sabouraud agar

This consists of Sabouraud glucose and peptone agar and is a non selective media for opportunistic fungi.

Yeast extract is used to improve the nutrition and an antibiotic such as gentamicin is added to inhibit the bacterial contamination (Fig. 3.16). The medium should not contain cycloheximide as this inhibits the growth of the saprophytic fungi.

Sabouraud agar is incubated at room temperature and is used for isolation of fungi and Nocardia.

Löwenstein-Jensen Medium

Mycobacteria are specifically isolated on Löwenstein-Jensen media. Glycerol and egg mixture which are added to this media provide fatty acids and protein required for the metabolism of mycobacteria. The coagulated egg albumin provides a solid medium for inoculation purposes. It also contains malachite green as an inhibitor to microorganisms other than acid-fast bacilli. Cultures should be read within 5-7 days after inoculation and once a week thereafter for up to 8 weeks.

Thayer-Martin Medium

This is a special, selective, chemically enriched chocolate agar, which is used to isolate Neisseria gonorrhoeae by suppressing the growth of other inhibitory fungi and bacteria.

Brain-heart Infusion (BHI) broth

Brain-heart infusion broth with neopeptone is incubated at room temperature and is used to enhance the isolation of filamentous fungi and yeasts and less frequently Bacillus species.

Robertson Cooked Meat Medium

Robertson cooked meat medium provides a favorable environment for the growth of anaerobes. The muscle

Figure 3.15: Blood agar

Figure 3.16: Aspergillus growth on Sabouraud’s dextrose agar

tissue is a source of amino acids and reducing substances, particularly glutathione, which permits the growth of strict anaerobes. Glucose, Hemin and Vitamin K1 is supplemented with yeast extract, to enhance the growth of anaerobic microorganisms ( Fig. 3.17). Growth is indicated by turbidity and, with some organisms, by the presence of gas bubbles in the medium. Disinte-gration and blackening of the meat particles indicates proteolysis.

References

1. Bacon AS, Frazer DG, Dart JK, Matheson M, Ficker LA, Wright P. A review of 72 consecutive cases of Acantham-oeba keratitis, 1984-1992. Eye 1993;7:719-25.

2. McCulley JP, Alizadeh H, Niederkorn JY. The diagnosis and management of Acanthamoeba keratitis. CLAO J.

2000;26:47-51.

3. Rauz S, Tuft S, Dart JK, Bonshek R, Luthert P, Curry A.

Ultrastructural examination of two cases of stromal microsporidial keratitis. J Med Microbiol 2004;53:775-4. Joseph J, Murthy S, Garg P, Sharma S. Use of different81.

stains for microscopic evaluation of corneal scrapings for diagnosis of microsporidial keratitis. J Clin Microbiol 2006;44:583-5.

5. Forster RK, Wirta MG, Solis M, Rebell G. Methenamine-silver-stained corneal scrapings in keratomycosis. Am J Ophthalmol 1976;82:261-5.

6. Kozlowski M, Stepien PP. Restriction enzyme analysis of mitochondrial DNA of members of the genus aspergillus as an aid in taxonomy. J Gen Microbiol 1982;128:471-6.

7. Nelson PD, Toussoun TA, Marasas WF. Fusarium species: An illustrated manual for identification, University Park: The Pennsylvannia State University Press, 1983;3-48.

8. Sharma S, Silverberg M, Mehta P, Gopinathan U, Agrawal V, Naduvilath TJ. Early diagnosis of mycotic keratitis: Predictive value of potassium hydroxide preparation. Indian J Ophthalmol 1998;46:31-5.

9. Vajpayee RB, Angra SK, Sandramouli S, Honavar SG, Chhabra VK. Laboratory diagnosis of keratomycosis:

Comparative evaluation of direct microscopy and culture results. Ann Ophthalmol 1993;25:68-71.

10. Jones DB. Initial therapy of suspected microbial corneal ulcers. II. Specific antibiotic therapy based on corneal smears.Surv Ophthalmol 1979;24:97, 105-16.

11. Marines HM, Osato MS, Font RL. The value of calcofluor white in the diagnosis of mycotic and Acanthamoeba infections of the eye and ocular adnexa. Ophthalmology 1987;94:23-6.

12. Gomez JT, Robinson NM, Osato MS, Wilhelmus KR.

Comparison of acridine orange and Gram’s stains in bacterial keratitis. Am J Ophthalmol 1988;106:735-7.

13. Groden LR, Rodnite J, Brinser JH, Genvert GI. Acridine orange and Gram’s stains in infectious keratitis. Cornea 1990;9:122-4.

Figure 3.17: Robertson cooked meat medium

25

Pharmacology

4

Medical therapy forms the first line of treatment of infectious keratitis. The drugs used to treat microbial keratitis include antimicrobial agents, cycloplegics, anti-glaucoma medications and adjuvants.