C.L.E.D. Agar w/ Bromo Thymol Blue
Intended use
Recommended for isolation, enumeration and identification of urinary pathogens on the basis of lactose fermentation.
Composition**
Ingredients Gms / Litre
Peptone 4.000
Tryptone 4.000
HM Peptone B# 3.000
Lactose 10.000
L-Cystine 0.128
Bromothymol blue 0.020
Agar 15.000
Final pH ( at 25°C) 7.3±0.2
**Formula adjusted, standardized to suit performance parameters
Directions
Suspend 36.15 grams in 1000 ml 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
On a solid medium, Sandys reported that swarming of Proteus species can be controlled by restricting the electrolytes
(1). Formerly swarming of Proteus was controlled by adding alcohol, surface-active agent, sodium azide, boric acid etc. to
the medium (1). Later on Sandy's medium was modified by Mackey and Sandys (2), by replacing mannitol by lactose
and sucrose and elevating concentration of agar and bromothymol blue. This formulation was further modified by the
same authors, called C.L.E.D. (Cystine-Lactose-Electrolyte-Deficient) by deleting the sucrose and by including Lcystine for promoting the growth of cystine dependent dwarf colony coliforms (3). 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).
Peptone, Tryptone and HM Peptone B provides nitrogenous and carbonaceous compounds, long chain amino acids,
vitamins and other essential growth nutrients. Lactose is the fermentable sugar. L-cystine supports the growth of dwarf
coliform colony. Bromo thymol blue is the pH indicator which turns yellow at acidic pH.
Bacteriuria may be quantitated by inoculating the surface of an agar medium by proper dilution. Inoculate the medium
immediately after urine collection. It can also be inoculated by calibrated loop or duplicate dilution pour plate methods (5,6).
Type of specimen
Clinical samples - urine
Specimen Collection and Handling:
For clinical samples follow appropriate techniques for handling specimens as per established guidelines (3,4).
After use, contaminated materials must be sterilized by autoclaving before discarding.
Warning and Precautions :
In Vitro diagnostic Use only. For professional 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.
R
Technical Data
Limitations :
1. Initiation of antibiotic therapy, before collection of sample, low urine pH (less than 5) etc. may result in low bacterial count
from infected patients.
2. Shigella species may not grow on this medium
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
Cream to yellow homogeneous free flowing powder
Gelling
Firm, comparable with 1.5% Agar gel
Colour and Clarity of prepared medium
Green coloured, clear to slightly opalescent gel forms in Petri plates.
Reaction
Reaction of 3.61% w/v aqueous solution at 25°C. pH : 7.3±0.2
pH
7.10-7.50
Cultural Response
Cultural characteristics observed after an incubation at 35-37°C for 18-24 hours.
Organism Inoculum
(CFU)
Growth Recovery Colour of
Colony
Enterococcus faecalis ATCC
29212 (00087*)
50-100 good-luxuriant =70% slight yellowish
or greenish
Escherichia coli ATCC
25922 (00013*)
50-100 good-luxuriant =70% yellow, opaque,
centre slightly
deeper yellow
Klebsiella pneumoniae
ATCC 13883 (00097*)
50-100 good-luxuriant =70% yellow to
whitish blue
Proteus vulgaris ATCC
13315
50-100 good-luxuriant =70% blue
Salmonella Typhi ATCC
6539
50-100 good-luxuriant =70% bluish
Staphylococcus aureus
subsp. aureus ATCC
50-100 good-luxuriant =70% deep yellow
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. 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 (3,4).
