Overview:
CLED Agar (Cystine-Lactose-Electrolyte-Deficient Agar), also known as BROLACIN Agar or Bromothymol-Blue Lactose Cystine Agar, is a differential, non-selective solid medium developed by Monsur in 1961 for the isolation, enumeration, and presumptive identification of bacteria from urine specimens, particularly urinary tract pathogens. It features a low-electrolyte formulation to inhibit swarming by Proteus species, while providing cystine as a sulfur source for cysteine-dependent organisms like Salmonella and Shigella. Lactose serves as the primary carbohydrate, with bromothymol blue as a pH indicator that differentiates lactose fermenters (blue-green colonies) from non-fermenters (yellow or colorless). The medium supports the growth of a broad spectrum of Gram-positive and Gram-negative bacteria, including Escherichia coli, Enterococcus spp., Klebsiella, and Staphylococcus, making it ideal for routine urinalysis in clinical microbiology labs per CLSI and ISO 15189 standards.
Applications:
?? Isolation and enumeration of urinary tract infection (UTI) pathogens from clean-catch, catheter, or suprapubic urine samples
?? Differentiation of lactose-fermenting (e.g., E. coli, Klebsiella) vs. non-fermenting (e.g., Proteus, Pseudomonas) bacteria in diagnostic workflows
?? Screening for asymptomatic bacteriuria and quantitative colony counts (>10^5 CFU/mL) per CDC and APHA guidelines
?? Support for subculture from enrichment broths or direct plating for antimicrobial susceptibility testing
Key Features:
?? Electrolyte-deficient base prevents Proteus swarming, enabling isolated colonies without overlay techniques
?? Cystine supplementation enhances recovery of fastidious pathogens like Salmonella typhi and Shigella spp. in urine
?? Bromothymol blue indicator provides clear differentiation: blue-green for fermenters, yellow for non-fermenters after 18-24 hours at 35-37�C
?? Broad-spectrum support for >95% of urinary pathogens, validated for ATCC strains like E. coli 25922 and P. mirabilis 7002 in CLSI protocols
?? Opaque, greenish medium with smooth surface; compatible with automated colony counters for high-throughput labs
Composition (per liter):
Gelatin Peptone � 14.0 g
Proteose Peptone � 4.0 g
Lactose � 10.0 g
Sodium Chloride (NaCl) � 5.0 g
Disodium Phosphate (Na?HPO?) � 3.0 g
L-Cystine � 0.128 g
Agar � 15.0 g
Bromothymol Blue � 0.02 g
Final pH: 7.3 � 0.2 at 25�C
Preparation:
Suspend 51.148 g of the dehydrated medium in 1 liter of purified/distilled water. Heat with gentle agitation to boiling until completely dissolved (do not overheat to avoid caramelization of lactose). Dispense into tubes or flasks if needed, then autoclave at 121�C (15 psi) for 15 minutes. Cool to 45-50�C in a water bath and pour into sterile Petri plates (20-25 mL per plate). Allow to solidify at room temperature and dry the surface briefly to absorb excess moisture. The prepared medium should appear greenish-blue, opaque, and homogeneous without precipitates. For use, streak 0.001 mL urine directly or via calibrated loop for quantitative plating; incubate aerobically at 35-37�C for 18-24 hours.
Storage:
Dehydrated medium: Store in a tightly closed container at 10�30�C, protected from moisture, light, and humidity; stable until expiry date (typically 3-5 years).
Prepared plates: Store at 2�8�C in sealed plastic bags; use within 4-8 weeks. Discard if dehydrated, contaminated, discolored, or if indicator fades.
?? For laboratory and research use only. Not for diagnostic procedures without proper validation. Not for human or veterinary consumption. Handle under biosafety level 2; wear gloves, lab coat, and eye protection�use Universal Precautions for urine specimens. Autoclave all waste before disposal. Over-inoculation may cause confluent growth; confirm isolates biochemically for definitive ID. Not suitable for anaerobes or fastidious organisms requiring CO?.