HiMedia Laboratories GmbH, Marie-Curie-Str. 3, 64683, Einhausen, Germany.
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Intended Use
Recommended for isolation and differentiation of urinary pathogens on the basis of lactose fermentation.
Composition**
| Ingredients | g/L |
|---|---|
| HiVegⓇpeptone | 4.000 |
| HiVeg® extract | 3.000 |
| Tryptone | 4.000 |
| Lactose | 10.000 |
| L-Cystine | 0.128 |
| Bromothymol blue | 0.020 |
| Andrade indicator | 0.100 |
| Agar | 15.000 |
| Final pH (at 25°C) | 7.5±0.2 |
**Formula adjusted, standardized to suit performance parameters
Directions
Suspend 36.25 grams in 1000 ml of purified/distilled water. Heat to boiling to dissolve the medium completely. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Cool to 45-50°C. Mix well and pour into sterile Petri plates.
Principle And Interpretation
Sandys reported a new technique where the swarming of Proteus on an agar medium could be prevented by restricting the electrolyte content in the culture medium (1). Sandys Medium was modified by Mackey and Sandys (2), by replacing mannitol with lactose and sucrose and elevating the concentration of agar and bromothymol blue. The same authors further modified this medium by retaining the lactose (deleting sucrose) and by including L-cystine for promoting the growth of cystine-dependent dwarf coliform colony (3). This later modified medium was designated as C.L.E.D. (Cystine- Lactose- Electrolyte-Deficient) Medium. This medium is recommended for use in urinary bacteriology, promoting the growth of all urinary pathogens. C.L.E.D. Medium is also recommended for dip stick procedures and as dip inoculum transport medium for urine specimens (2,3,4). C.L.E.D. HiVeg® Agar w/ Andrade Indicator is prepared by using vegetable peptones in place of animal based peptones which make the media free of BSE/TSE risks.
C.L.E.D. Medium was further modified by Bevis (5) by incorporation of Andrades indicator. This medium provides sharper differentiation between lactose-fermenters (LF) and lactose-non-fermenters (NLF) (5). Addition of Andrades indicator enhances the appearance of colony and aids in the identification of microorganisms.
At different pH values, the colour of the medium varies from the standard medium, which is well documented by Bevis (5).
| pH | Colour of C.L.E.D. medium |
|---|---|
| 7.4 | deep blue |
| 7.0 | bluish grey |
| 6.8 | pale grey |
| 6.6 | pinkish grey |
| 6.4 | bright red with whitish tinge |
| 6.0 | bright red |
For better results, the medium should not be incubated for more than 24 hours because if lactose fermenters predominate, the entire medium may turn pink masking the presence of non-lactose fermenters. Inoculate the medium immediately after urine collection. Shigella species may not grow on this medium. Prior initiation of antibiotic therapy, low urine pH (less than 5) etc. may result in low urine count from infected patients.
Type of specimen
Clinical sample
Specimen Collection and Handling
For clinical samples follow appropriate techniques for handling specimens as per established guidelines (6,7).
After use, contaminated materials must be sterilized by autoclaving before discarding.
Warning and Precautions
Read the label before opening the container. Wear protective gloves/protective clothing/eye protection/face protection. Follow good microbiological lab practices while handling specimens and culture. Standard precautions as per established guidelines should be followed while handling specimens. Safety guidelines may be referred in individual safety data sheets.
Limitations
- Recovery depends on the urine count.
- Inoculate the medium immediately after urine collection.
- Shigella species may not grow on this medium.
- For better results, the medium should not be incubated for more than 24 hours because if lactose fermenters predominate, the entire medium may turn pink masking the presence of non-lactose fermenters.
Performance and Evaluation
Performance of the medium is expected when used as per the direction on the label within the expiry period when stored at recommended temperature.
Quality Control
Appearance
Light yellow to greyish yellow homogeneous free flowing powder
Gelling
Firm, comparable with 1.5% Agar gel
Colour and Clarity of prepared medium
Greenish blue clear to slightly opalescent gel forms in Petri plates
Reaction
Reaction of 3.62% w/v aqueous solution at 25°C. pH: 7.5±0.2
pH
7.30-7.70
Cultural Response
Cultural characteristics observed after an incubation at 35-37°C for 18-24 hours
| Organism | Inoculum (CFU) | Growth | Recovery | Colour of colony |
|---|---|---|---|---|
| # Klebsiella aerogenes ATCC 13048 (00175*) | 50-100 | good-luxuriant | >=70% | greyish green, mucoid |
| Escherichia coli ATCC 25922 (00013*) | 50-100 | good-luxuriant | >=70% | bright pink with pink halo |
| Enterococcus faecalis ATCC 29212 (00087*) | 50-100 | good-luxuriant | >=70% | orange-yellow or greenish |
| Proteus mirabilis ATCC 25933 | 50-100 | good-luxuriant | >=70% | blue-green |
| Staphylococcus aureus subsp. aureus ATCC 25923 (00034*) | 50-100 | good-luxuriant | >=70% | golden-yellow |
| Streptococcus pyogenes ATCC 19615 | 50-100 | good-luxuriant | >=70% | greyish green |
Key: *Corresponding WDCM numbers.
#- Formerly known as Enterobacter aerogenes
Storage and Shelf Life
Store between 10-30°C in a tightly closed container and the prepared medium at 20-30°C. Use before expiry date on the label. On opening, product should be properly stored dry, after tightly capping the bottle in order to prevent lump formation due to the hygroscopic nature of the product. Improper storage of the product may lead to lump formation. Store in dry ventilated area protected from extremes of temperature and sources of ignition Seal the container tightly after use.
Disposal
User must ensure safe disposal by autoclaving and/or incineration of used or unusable preparations of this product. Follow established laboratory procedures in disposing of infectious materials and material that comes into contact with sample must be decontaminated and disposed of in accordance with current laboratory techniques (6,7).
| Product Name | C.L.E.D. HiVeg™ Agar w/ Andrade Indicator |
|---|---|
| SKU | MV352 |
| Product Type | HiVeg™ |
| Physical Form | Powder |
| Origin | Animal Free (Veg) |
| Packaging type | HDPE |
| References | 1. Sandys, 1960, J. Med. Lab. Technol., 17:224. 2.Mackey and Sandys, 1965, Br. Med. J., 2:1286. 3.MacKey and Sandys, 1966, Br. Med. J., 1:1173. 4.Dixson J. M. S. and Clark M. A., 1968, Conc. Med. Assoc. J., 99 (15) 5.Bevis T. D., 1968, J. Med. Lab. Technol., 25:38. 6.Isenberg, H.D. Clinical Microbiology Procedures Handbook. 2nd Edition. 7.Jorgensen,J.H., Pfaller , M.A., Carroll, K.C., Funke, G., Landry, M.L., Richter, S.S and Warnock., D.W. (2015) Manual ofClinical Microbiology, 11th Edition. Vol. 1. |
| Customized Product Available | No |
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