CLED Agar: Introduction, Composition, Principle, Preparation, Testing Procedure, Colony Characteristics, Modifications, Limitations, and Keynotes

Introduction of CLED Agar

Table of Contents

CLED stands for cysteine, lactose, and electrolyte-deficient. CLED agar or medium is a differential culture medium used for the isolation and enumeration of bacteria in urine specimens from suspected cases of urinary tract infection (UTI). It favors the growth of all potential urinary pathogens as well as a number of contaminants such as diphtheroid, lactobacilli, and micrococci. Candida also enjoys this medium. This is a sole medium alternate for routine urine culture and inoculation of specimens in a combination of blood agar (BAP) and MacConkey agar (MAC) without compromising the quality and reducing the cost.

Staphylococcus aureus (11 o'clock) Klebsiella pneumoniae (5 o'clock) and Pseudomonas aeruginosa (8 o'clock) growth on CLED agar — Fig. Staphylococcus aureus (11 o’clock) *Klebsiella pneumoniae* (5 o’clock) and *Pseudomonas aeruginosa* (8 o’clock) growth on CLED agar-Front View

Staphylococcus aureus (11 o'clock) Klebsiella pneumoniae (5 o'clock) and Pseudomonas aeruginosa (8 o'clock) growth on CLED agar-Back View — Fig. Staphylococcus aureus, *Klebsiella pneumoniae* and *Pseudomonas aeruginosa* growth on CLED agar-Back View

Composition of CLED Medium

The composition of CLED Medium is shown below in table-

S.No.	Ingredients	Amount
1.	Peptone	2 gm
2.	“Lab Lemco” Powder (meat extract)	1.5 gm
3.	Tryptone	2 gm
4.	Lactose	5 gm
5.	L-Cysteine	0.064
6.	Bromothymol blue	0.01g
7.	Agar	7.5 gm
8.	Distilled water	500 ml
	Final pH	7.3 +/- 0.2 at 25°C

Table: Composition of CLED Agar

Preparation of CLED Agar

CLED agar is available in dehydrated powder form by various manufacturers like Mast, Oxoid, Difco, Himedia, and so on.

Follow the instructions of the manufacturer to prepare the medium i.e. 18.06gm dehydrated medium in 500mL distilled or deionized water, mix properly and finally Sterilize by autoclaving at 121°C for 15 minutes.
After cooling to 50-55°C, mix well, and dispense it aseptically in sterile Petri dishes. Date the medium and give it a batch number.
Store the plates at 2-8°C preferably in plastic bags to prevent loss of moisture.

Shelf life of CLED Medium

It can be used for several weeks but should be free from any change in the appearance of the medium showing contamination, deterioration, or alteration of pH.

Principle of CLED Agar

Reduced electrolyte prevents the swarming of Proteus whereas Cystine promotes the formation of cystine-dependent dwarf colonies. Bromothymol blue is the indicator used in the agar, it changes to yellow in case of acid production during fermentation of lactose or changes to deep blue in case of alkalization. Lactose fermenting bacteria build yellow colonies. Bacteria that decarboxylate L-Cystine cause an alkaline reaction and build deep blue colonies.

Colony Characteristics of CLED Agar

Typical Colony Morphology on CLED Agar is as follows:

S.No.	Organisms	Colony Morphology
1.	Staphylococcus aureus	Deep yellow colonies of uniform color
2.	S. saprophyticus and other Coagulase Negative Staphylococci (CoNS)	Pale yellow to white colonies, more opaque than Enterococcus faecalis
3.	Enterococci	Small yellow colonies, about 0.5mm in diameter
4.	E. coli: Introduction, Identification Features, Keynotes, and Escherichia coli Footages Escherichia coli	Opaque yellow colonies with a slightly deeper yellow center
5.	Klebsiella species	Large mucoid yellow to whitish-blue colonies
6.	Proteus spp.	Translucent blue-grey colonies
7.	Pseudomonas aeruginosa	Green colonies with typical matted surface and rough periphery

Table: Colony Characteristics of Microbes on CLED Agar

Modifications of CLED Agar

Note: CLED medium with modification i.e. CLED Medium with Andrade’s Indicator now uses most commonly.
Composition is the same with a slight increment of Andrade’s indicator i.e. Andrade indicator (0.100 gm/L)
CLED Medium is further modified by Bevis by incorporating Andrade’s indicator. This medium provides sharper differentiation between lactose-fermenters (LF) and lactose-non-fermenters (NLF). The addition of Andrade’s indicator enhances the appearance of the colony and aids in the identification of microorganisms. At different pH values, the color of the medium varies from the standard medium, which is well documented by Bevis.

Color of CLED Medium

The difference in color due to different pH effects are as follows-

pH	Color
7.4	deep blue
7.0	bluish grey
6.8	pale grey
6.6	pinkish grey
6.4	bright red with a whitish tinge
6.0	bright red

Table: Color of CLED Agar

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.

Typical colony morphology on CLED Medium with Andrade’s Indicator

Typical colony morphology on CLED Medium with Andrade’s Indicator after incubation at 35-37°C for 18-24 hours is as follows:

S.No	Microbes	Colony Characteristics
1.	Klebsiella aerogenes ATCC 13048	greyish green, mucoid
2.	Escherichia coli ATCC 25922	bright pink with a pink halo
3.	Enterococcus faecalis ATCC 29212	orange-yellow or greenish
4.	Proteus mirabilis ATCC 25933	blue-green
5.	Staphylococcus aureus ATCC 25923	golden-yellow
6.	Streptococcus pyogenes ATCC 19615	greyish green

Table: Colony morphology of microbes on CLED agar

Proteus mirabilis green blue colony on CLED medium

Staphylococcus aureus golden yellow colonies on CLED agar after 4 days of incubation

Mixed growth of microbes on CLED agar in urine culture showing Candida and bacterial growth after 24 hours of incubation

Mixed growth of micobes on CLED agar in urine culture showing Candida and bacterial growth after 24 hours of incubation — Fig. Mixed growth of microbes on CLED agar in urine culture showing Candida and bacterial growth after 24 hours of incubation

Saline microscopy of a mixed growth of microbes from above culture plate showing yeast cells and bacteria

Saline microscopy of mixed growth of microbes from above culture plate showing yeast cells and bacteria — Fig. Saline microscopy of a mixed growth of microbes from above culture plate showing yeast cells and bacteria

Candida colony morphology on CLED agar

Gram-positive yeast cells of Candida

Various microbial growth on CLED agar plates of urine culture

Advantages of CLED Agar

(CLED Agar for Urine Culture)

Good discrimination of gram-negative bacteria on the basis of lactose fermentation and colony appearance.
It inhibits the swarming of Proteus species (Proteus mirabilis and Proteus vulgaris are frequently involved in urinary tract infections) due to electrolyte deficiency.
Relatively low cost as compared to the combined use of blood agar and MacConkey agar for urine culture.

Limitations of CLED Medium

This medium is recommended for urine infections. Low urine count may be a result of antibiotic therapy, and low pH of urine.
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.

Keynotes on CLED Agar

The color of the colony is directly affected by pH variance.
The colony morphology of microbes may vary according to modifications of CLED media (e.g. use of Andrade’s indicator).
It is one of the recommended culture media for urine culture replacing the use of MacConkey and blood agar which is also beneficial for the reduction of laboratory costs and labor.
There is a need for trained staff to report CLED agar due to having growth of Gram-positive, negative, and even fungi which is a common drawback of using CLED agar.