C.L.E.D.

Medium (with Andrade Indicator) M352

Intended Use:
C.L.E.D. Medium w/Andrade Indicator is recommended for isolation and differentiation of urinary pathogens on the basis of
lactose fermentation.
Composition**
Ingredients Gms / Litre
Peptone 4.000
HM peptone B # 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
# - Equivalent to Beef extract

Directions
Suspend 36.25 grams in 1000 ml of 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. 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

Please refer disclaimer Overleaf.

HiMedia Laboratories Technical Data

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:Urine sample

Specimen Collection and Handling

For clinical samples follow appropriate techniques for handling specimens as per established guidelines (6,7).

Warning and Precautions

In Vitro diagnostic use only. 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 clinical specimens. Safety guidelines may be
referred in individual safety data sheets.

Limitations
1. This medium is recommended for urine infection. Low urine count may be a result of antibiotic therapy, low pH of urine.
2.Recovery depends on the urine count.
3.Inoculate the medium immediately after urine collection.
4.Shigella species may not grow on this medium.
5. For better results, the medium should not be incubated for more that 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
Cultural Response
Organism Growth Inoculum Recovery Colour of
(CFU) colony
Cultural Response
# Klebsiella aerogenes good-luxuriant 50-100 >=70% greyish green,
ATCC 13048 (00175*) mucoid
Escherichia coli ATCC good-luxuriant 50-100 >=70% bright pink
25922 (00013*) with pink halo
Enterococcus faecalis ATCC good-luxuriant 50-100 >=70% orange-yellow
29212 (00087*) or greenish
Proteus mirabilis ATCC good-luxuriant 50-100 >=70% blue-green
25933
Staphylococcus aureus good-luxuriant 50-100 >=70% golden-yellow
subsp. aureus ATCC
25923 (00034*)

Please refer disclaimer Overleaf.

 HiMedia Laboratories Technical Data

Streptococcus pyogenes good-luxuriant 50-100 >=70% greyish green

ATCC 19615
Key : *Corresponding WDCM numbers.
#- Formerly known as Enterobacter aerogenes
Storage and Shelf Life
Store below 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 inorder 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. Use before expiry date on
the label. Product performance is best if used within stated expiry period.

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 clinical
sample must be decontaminated and disposed of in accordance with current laboratory techniques (6,7).

Reference
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 of
Clinical Microbiology, 11th Edition. Vol. 1.

Revision : 04/ 2018

In vitro diagnostic medical

IVD device

CE Marking

30°C Storage temperature

10°C

Do not use if package is

damaged

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Disclaimer :
User must ensure suitability of the product(s) in their application prior to use. Products conform solely to the information contained in
this and other related HiMedia™ publications. The information contained in this publication is based on our research and development
work and is to the best of our knowledge true and accurate. HiMedia™ Laboratories Pvt Ltd reserves the right to make changes to
specifications and information related to the products at any time. Products are not intended for human or animal or therapeutic use but
for laboratory,diagnostic, research or further manufacturing use only, unless otherwise specified. Statements contained herein should not
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