New York City Agar- Composition, Principle, Preparation, Results, Uses

New York City Agar (NYC Agar) is a type of culture medium that is used for the isolation and cultivation of pathogenic Neisseria species, such as Neisseria gonorrhoeae and Neisseria meningitidis, from clinical specimens. It is also useful for the growth of genital mycoplasmas, such as Mycoplasma hominis and Ureaplasma urealyticum, which are associated with urogenital tract infections. NYC Agar is an enriched and selective medium that provides optimal conditions for the recovery of these fastidious organisms.

New York City Agar (NYC Agar) is a complex medium that contains various ingredients to support the growth and differentiation of pathogenic Neisseria species and genital mycoplasmas. The main components of NYC Agar are:

• Peptone: A mixture of proteoses, peptones, and free amino acids that provide nitrogenous nutrients for bacterial growth.
• Corn starch agar base: A solidifying agent that also neutralizes the toxic metabolites produced by Neisseria species.
• Phosphates: Buffering agents that maintain the pH of the medium at 7.4±0.2.
• Sodium chloride: An electrolyte that maintains the osmotic balance of the medium and the bacterial cells.
• Dextrose: A carbohydrate source that serves as an energy source for some bacteria.
• Horse plasma: A source of serum proteins, growth factors, and other nutrients that enhance the growth of Neisseria species and mycoplasmas.
• Horse hemoglobin: A source of heme and iron that are required for the growth and metabolism of Neisseria species and some mycoplasmas.
• Yeast autolysate: A supplement that provides oxaloacetic acid, which is metabolized by gonococci to produce CO2, a capnophilic requirement for their growth. It also reduces the lag phase of Neisseria growth and increases the size and number of colonies.
• Antibiotics: A selective supplement that contains vancomycin, colistin, nystatin, and trimethoprim lactate. These antibiotics inhibit the growth of gram-positive bacteria, gram-negative bacilli (including Pseudomonas species), yeasts, and Proteus species, respectively. The combination of trimethoprim and colistin acts synergistically against gram-negative bacilli.

New York City (NYC) agar is a transparent medium that is designed to selectively isolate and cultivate pathogenic Neisseria species, such as Neisseria gonorrhoeae and Neisseria meningitidis, from clinical specimens. NYC agar has several components that enhance the growth of Neisseria and inhibit the growth of other bacteria and fungi.

• Proteose peptone is a source of nitrogen, carbon, and other essential nutrients for bacterial growth. It contains proteoses, peptones, and free amino acids that are readily utilized by Neisseria.
• Phosphate buffers the medium and maintains a stable pH of 7.4±0.2, which is optimal for Neisseria growth.
• Starch acts as a detoxifying agent that neutralizes the toxic metabolites produced by Neisseria, such as hydrogen peroxide and fatty acids. Starch also helps to prevent the oxidation of hemoglobin and plasma components in the medium.
• Sodium chloride provides essential electrolytes and maintains osmotic equilibrium in the medium, thereby preserving the integrity of bacterial cells.
• Selective supplement contains four antibiotics that inhibit the growth of most other bacteria and fungi that may contaminate the specimens. The antibiotics are:
  • Vancomycin, which inhibits gram-positive bacteria by interfering with their cell wall synthesis.
  • Colistin, which inhibits gram-negative bacilli by disrupting their cell membrane permeability. Colistin also has a synergistic effect with trimethoprim against gram-negative bacilli.
  • Nystatin, which inhibits yeasts and molds by binding to their cell membrane sterols and causing leakage of intracellular contents.
  • Trimethoprim lactate, which inhibits Proteus species and other gram-negative bacilli by blocking their folate metabolism. Proteus species can cause swarming on agar plates and interfere with the identification of Neisseria colonies.
• Enrichment supplement contains three ingredients that provide additional nutrients and growth factors for Neisseria. The ingredients are:
  • Horse blood cells, which provide hemoglobin as a source of iron and heme for Neisseria. Hemoglobin also enhances the color and morphology of Neisseria colonies on NYC agar.
  • Horse plasma, which provides serum proteins, vitamins, minerals, and other cofactors for Neisseria growth. Plasma also helps to maintain the moisture and transparency of the medium.
  • Yeast autolysate, which provides amino acids, peptides, nucleotides, vitamins, and other growth factors for Neisseria. Yeast autolysate also fulfills the CO2 requirements of Neisseria by providing oxaloacetic acid, which is metabolized by gonococci to produce CO2. CO2 enhances the growth of capnophilic gonococci, which are more fastidious than meningococci. Yeast autolysate also reduces the lag phase of Neisseria growth, thus increasing both the size and number of colonies on NYC agar.

To prepare New York City Agar, you will need the following ingredients and materials:

• New York City Agar powder (25.50 grams)
• Distilled water (320 ml)
• Sedimented horse blood cells (100 ml)
• Citrated horse plasma (60 ml)
• NYC Supplement (1 vial)
• Yeast Autolysate Supplement (1 vial)
• Autoclave
• Water bath
• Sterile Petri plates

The steps for preparing New York City Agar are as follows:

1. Suspend 25.50 grams of New York City Agar powder in 320 ml of distilled water in a suitable container.
2. Heat the mixture to boiling to dissolve the medium completely. Stir occasionally to avoid clumping.
3. Sterilize the medium by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Avoid overheating as this may affect the quality of the medium.
4. Cool the medium to 45-50°C in a water bath or on a cooling rack.
5. Aseptically add 100 ml of sedimented horse blood cells and 60 ml of citrated horse plasma to the medium. Mix well to ensure uniform distribution of the components.
6. Aseptically add the rehydrated contents of 1 vial of NYC Supplement and 1 vial of Yeast Autolysate Supplement to the medium. Mix well to dissolve the supplements.
7. Pour the medium into sterile Petri plates, about 15-20 ml per plate. Allow the medium to solidify before use or storage.
8. Store the plates in a refrigerator (2-8°C) in an inverted position until use. Do not use plates that show signs of contamination, deterioration, or drying.

New York City Agar is a transparent medium that allows direct observation of the colonies of Neisseria and mycoplasmas under a microscope. The colonies can be differentiated by their size, shape, color, and morphology.

• Neisseria gonorrhoeae: small (0.5-1.0 mm), grayish-white to colorless, mucoid colonies.
• Neisseria meningitidis: large mucoid colonies, colorless to bluish-gray.
• Mycoplasma hominis: small (0.1-0.3 mm), fried-egg shaped colonies with a dense center and a thin peripheral zone.
• Ureaplasma urealyticum: very small (0.02-0.05 mm), pinpoint colonies that are difficult to see without staining or dark-field microscopy.

Other bacteria and fungi are inhibited by the antibiotics in the medium, making it highly selective for Neisseria and mycoplasmas. However, some strains of Neisseria gonorrhoeae may be inhibited by the concentration of vancomycin in the medium, so it is recommended to use a nonselective chocolate agar as a backup, especially for suspect cases that are culture negative or for sterile specimens.

The results on New York City Agar should be confirmed by biochemical, immunological, molecular, or mass spectrometry tests for complete identification of the isolates.

New York City (NYC) agar is an enriched selective medium that has several uses in microbiology and clinical diagnostics. Some of the main uses are:

• Isolation and cultivation of pathogenic Neisseria species. NYC agar is designed to selectively grow Neisseria gonorrhoeae and Neisseria meningitidis, the causative agents of gonorrhea and meningitis, respectively. These bacteria are fastidious and require special nutrients and conditions for optimal growth. NYC agar provides these requirements and inhibits the growth of other bacteria that may contaminate the specimens. NYC agar can be used to culture specimens from various sites, such as genital, pharyngeal, rectal, conjunctival, cerebrospinal fluid, and blood.
• Gonorrhea screening programs. NYC agar is useful for detecting asymptomatic or symptomatic infections of gonorrhea in high-risk populations, such as sexually active individuals, men who have sex with men, commercial sex workers, and pregnant women. Gonorrhea is a sexually transmitted infection that can cause serious complications if left untreated, such as pelvic inflammatory disease, infertility, ectopic pregnancy, neonatal conjunctivitis, and disseminated gonococcal infection. NYC agar can help identify the presence of Neisseria gonorrhoeae in genital swabs or urine samples and guide appropriate treatment and prevention strategies.
• Recognition of active or asymptomatic mycoplasma infections. NYC agar can also support the growth of genital mycoplasmas, such as Mycoplasma hominis and Ureaplasma urealyticum. These bacteria are associated with various urogenital tract infections, such as urethritis, cervicitis, pelvic inflammatory disease, infertility, and adverse pregnancy outcomes. NYC agar can help diagnose these infections by culturing specimens from the genital tract or urine. However, since mycoplasmas are not visible on Gram stain and may grow slowly on NYC agar, additional tests may be needed to confirm their identification.
• Establishing the frequency of association of mycoplasmas with urogenital tract infection. NYC agar can also be used for epidemiological studies to determine the prevalence and distribution of genital mycoplasmas in different populations and regions. This can help understand the role of these bacteria in urogenital tract infections and their potential interactions with other pathogens, such as Neisseria gonorrhoeae and Chlamydia trachomatis. NYC agar can also help monitor the antimicrobial susceptibility patterns of genital mycoplasmas and guide empirical therapy.

These are some of the main uses of NYC agar in microbiology and clinical diagnostics. NYC agar is a valuable medium that can help isolate and identify important pathogens that cause urogenital tract infections and systemic diseases.

• New York City Agar is a selective medium that inhibits the growth of many bacteria other than pathogenic Neisseria species. However, some strains of Neisseria gonorrhoeae are also inhibited by the concentration of vancomycin in the selective supplement. This may lead to false-negative results in some cases of gonorrhea infection. Therefore, it is recommended to use nonselective chocolate agar along with New York City Agar, especially for sterile specimens or suspect cases that are culture negative.
• New York City Agar is an enriched medium that provides nutrients and CO2 for the growth of pathogenic Neisseria species. However, it may also support the growth of some genital mycoplasmas (Mycoplasma hominis and Ureaplasma urealyticum), which are often associated with urogenital tract infections. These mycoplasmas may appear as small, pinpoint colonies on New York City Agar and may be confused with Neisseria colonies. Therefore, it is important to perform further tests to confirm the identity of the isolates.
• New York City Agar is a complex medium that contains many ingredients, such as horse blood, horse plasma, yeast autolysate, and antibiotics. These ingredients may vary in quality and quantity depending on the source and preparation. Therefore, it is essential to use standardized and quality-controlled media and supplements to ensure consistent and reliable results. It is also advisable to check the pH and sterility of the medium before use.
• New York City Agar is a medium that requires careful handling and storage. The medium should be stored in a refrigerator (2-8°C) and used within one month of preparation. The medium should not be frozen or exposed to excessive heat or light. The medium should be warmed to room temperature before inoculation and incubated in a humidified atmosphere with 5-10% CO2 at 35-37°C for 24-48 hours. The medium should be examined for growth and hemolysis at regular intervals.

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