Legionella Agar Base

Intended Use

These media are used in qualitative procedures for isolation of Legionella species from clinical specimens and nonclinical (environmental) samples.

Summary and Explanation

Legionella Agar is a modification of the BCYE Agar formula of Edelstein. In the formula, the concentration of ACES buffer was reduced from 10.0 g/L to 6.0 g/L.
BCYE Differential Agar is used for the presumptive identification and differentiation of Legionella spp. based on colony morphology and color.5 This medium is based on the formulation of Vickers et al.,6 and consists of the dyes bromcresol purple and bromthymol blue added to BCYE Agar.
BCYE Selective Agar w/CCVC is a highly selective medium consisting of BYCE Agar supplemented with cephalothin, colistin, vancomycin and cycloheximide. This medium is based on the formulation of Bopp et al.7 They obtained improved recovery of L. pneumophila by using the selective medium in conjunction with an acid wash treatment to reduce the contaminating microbial flora present in environmental water samples.
BCYE Selective Agar with PAC was developed by Edelstein for isolation of Legionella spp. from specimens containing mixed flora.4 He found that BYCE Agar supplemented with polymyxin B, cefamandole and anisomycin enhanced the recovery of L. pneumophila from contaminated clinical specimens. In conjunction with an acid wash treatment to reduce microbial flora, it also facilitated the recovery of the bacterium from potable water.
BCYE Selective Agar with PAV is similar to the Edelstein formula, above, except that the concentration of polymyxin B is reduced by half, and vancomycin is substituted for cefamandole.

Principles of the Procedure

These media consist of a base medium (BCYE) supplemented with antibiotics or dyes. Antibiotics improve the recovery of Legionella spp. by inhibiting the growth of contaminating organisms. Dyes facilitate differentiation and identification of Legionella spp.
The base media (BCYE Agar Base and Legionella Agar Base) contain yeast extract to supply the nutrients necessary to support bacterial growth. L-cysteine HCl, ferric pyrophosphate and alpha-ketoglutarate are incorporated to satisfy the specific nutritional requirements of Legionella species. The activated charcoal decomposes hydrogen peroxide, a toxic metabolic product, and may also collect carbon dioxide and modify surface tension. The addition of the buffer helps maintain the proper pH for optimal growth of Legionella species.
Antibiotics incorporated in the various BCYE formulations have different spectra of activity. Vancomycin inhibits gram-positive bacteria; colistin and polymyxin B inhibit gram-negative bacteria, except for Proteus spp.; and cephalothin and cefamandole inhibit both gram-positive and gram-negative bacteria. Anisomycin and cycloheximide are antifungal agents.
BCYE Differential Agar contains the dyes bromcresol purple and bromthymol blue to aid in the differentiation and identification of Legionella species.

User Quality Control

NOTE: Differences in the Identity Specifications and Cultural Response testing for media offered as both Difco™ and BBL™ brands may reflect differences in the development and testing of media for industrial and clinical applications, per the referenced publications.

Identity Specifications

Difco™ Legionella Agar Base
Dehydrated Appearance: Dark gray, free flowing, homogeneous.
Solution:                       3.7% solution, insoluble in purified water. Suspension
                                    is black, opalescent.
Prepared Appearance:    Black, opaque with precipitate.
Reaction of 3.7%
Solution at 25°C:           pH 6.85-7.0 (adjusted)
Difco™ Legionella Agar Enrichment
Lyophilized Appearance: White to off-white powder, with or without traces
                                   of brown or green powder.
Solution:                      After rehydration, white to off-white, opaque,
                                  milky suspension. Some evidence of browning
                                  may be present due to ferric pyrophosphate.
Cultural Response
Difco™ Legionella Agar Base
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C under a humidified atmosphere containing approximately 2.5% CO2 for 46-72 hours.

Identity Specifications
BBL™ BCYE Agar Base
Dehydrated Appearance: Fine to coarse, homogeneous, free of extraneous material.
Solution:                       3.83% solution, soluble in purified water upon
                                    boiling. Solution is dark, black-green to grayblack,
                                    moderately hazy to opaque.
Prepared Appearance:     Dark, black-green to gray-black, moderately hazy
                                    to opaque.
Reaction of 3.83%
Solution at 25°C:            pH 6.85 ± 0.1 (adjusted)
Cultural Response
BBL™ BCYE Agar Base
Prepare the medium per label directions. For E. coli, inoculate and incubate at 35 ± 2°C for 66-72 hours. For Legionella spp., inoculate test and control lots of medium with serial 10-fold dilutions of the test organisms and incubate at 35 ± 2°C for 66-72 hours. Growth on the test lot should be within 1 log of the control lot.

Formula

Difco™ Legionella Agar Base
Approximate Formula* Per Liter
Yeast Extract.............................................................. 11.5 g
ACES Buffer................................................................. 6.0 g
Charcoal, Activated....................................................... 1.5 g
Alpha-Ketoglutarate, Monopotassium............................. 1.0 g
Agar......................................................................... 17.0 g
Difco™ Legionella Agar Enrichment
Approximate Formula* Per Vial
L-Cysteine HCl............................................................. 0.35 g
Ferric Pyrophosphate.................................................... 0.14 g
BBL™ BCYE Agar Base
Approximate Formula* Per Liter
Yeast Extract.............................................................. 10.0 g
Ferric Pyrophosphate................................................... 0.25 g
ACES Buffer................................................................ 10.0 g
Charcoal, Activated....................................................... 2.0 g
Alpha-Ketoglutarate...................................................... 1.0 g
Agar.......................................................................... 15.0 g
*Adjusted and/or supplemented as required to meet performance criteria.

Directions for Preparation from Dehydrated Product

Difco™ Legionella Agar Base
1. Dissolve 18.5 g of the powder in 500 mL of purified water.
2. Adjust to pH 7.1-7.2 with 1 N KOH. Do not heat prior to autoclaving.
3. Autoclave at 121°C for 15 minutes.
4. Cool to 45-50°C and aseptically add 5 mL rehydrated Difco™ Legionella Agar Enrichment. Mix thoroughly.
5. Check pH. If necessary, aseptically adjust to pH 6.85-7.0 with 1 N HCl or 1 N KOH.
6. Dispense into Petri dishes. Agitate while dispensing to keep charcoal in suspension.
7. Test samples of the finished product for performance using stable, typical control cultures.
BBL™ BCYE Agar Base
1. To 500 mL of purified water, add 2.4 g KOH pellets and mix to dissolve.
2. Add 38.3 g of the powder and 500 mL of purified water. Mix thoroughly.
3. Heat with frequent agitation and boil for 1 minute to completely dissolve the powder.
4. Autoclave at 121°C for 15 minutes. 5. Cool to 45-50°C and add 4 mL of a 10% filter-sterilized solution of L-cysteine HCl.
6. Mix thoroughly. Check pH; if not 6.85 ± 0.1, adjust using 1 N HCl or KOH.
7. Dispense into Petri dishes. Agitate while dispensing to keep charcoal in suspension.
8. Test samples of the finished product for performance using stable, typical control cultures.

Procedure

Use standard procedures to obtain isolated colonies from specimens and samples.
Incubate the plates in an inverted position (agar side up) at 35 ± 2°C for a minimum of 3 days. Growth is usually visible within 3-4 days, but may take up to 2 weeks to appear.

Expected Results

On BCYE Agar, Legionella Agar and the selective media, Legionella pneumophila produces small to large, smooth, colorless to pale, blue-gray, slightly mucoid colonies that fluoresce yellow-green under long-wave UV light. Consult references for morphology and color of fluorescence of other species.^8,9
On BCYE Differential Agar, L. pneumophila produces light blue colonies with a pale green tint. L. micdadei produces blue-gray to dark blue colonies.
A Gram stain, biochemical tests and serological procedures should be performed to confirm findings.

*Store at 2-8°C.

Free Shipping within the Continental USA