CLED agar, also known as Cysteine-Lactose-Electrolyte-Deficient agar, is a type of culture medium that is commonly used in microbiology for the isolation and identification of bacteria from urine specimens. It contains a combination of nutrients that support the growth of most urinary tract pathogens while inhibiting the growth of commensal bacteria.
Uses of CLED Agar:
In pathology laboratories, CLED agar is primarily used for the isolation and identification of urinary tract pathogens from clinical specimens. Urinary tract infections (UTIs) are a common medical condition, and laboratory diagnosis of UTIs is essential for appropriate management and treatment. CLED agar is a selective and differential medium that can support the growth of a variety of urinary tract pathogens, such as Escherichia coli, Klebsiella species, Proteus species, Enterococcus faecalis, and others.
Some of the specific uses of CLED agar in pathology laboratories include:
- Urine culture: CLED agar is a commonly used culture medium for urine culture, which is a standard laboratory test for the detection and identification of UTIs. A urine sample is inoculated onto the surface of CLED agar and incubated for a specified period of time. The presence of bacterial growth on the medium is then examined and identified by various biochemical tests.
- Antibiotic susceptibility testing: CLED agar can be used for antibiotic susceptibility testing to determine the most effective antibiotic for the treatment of UTIs. This involves testing the bacterial isolates from urine cultures for their susceptibility to different antibiotics using various methods, such as disk diffusion or broth microdilution.
- Quality control: CLED agar is used as a quality control medium in pathology laboratories to ensure that the media and laboratory procedures are functioning properly and to detect any potential contamination.
In summary, CLED agar is a valuable tool in pathology laboratories for the diagnosis and management of UTIs. Its selective and differential properties make it a suitable medium for the isolation and identification of urinary tract pathogens, while its ability to support antibiotic susceptibility testing can aid in the selection of appropriate treatment.
Composition of CLED Agar:
The composition of CLED agar includes:
- Peptone: 04.0 grams/Liter:
a source of amino acids and nitrogen for bacterial growth - Lactose: 10.0 grams/Liter:
a carbohydrate that allows for the differentiation of lactose-fermenting and non-lactose fermenting bacteria - Agar: 15.0 grams/Liter:
a solidifying agent that provides a solid surface for bacterial growth - Bromothymol blue: 0.02 grams/Liter:
a pH indicator that changes color from green to yellow in response to acid production by lactose-fermenting bacteria - Cysteine hydrochloride: 0.128 grams/Liter:
a source of sulfur that is essential for the growth of some bacteria - Beef Extract: 03 grams/Liter:
Beef extract which provide the nitrogen, vitamins, minerals, and amino acids essential for growth.
The exact concentrations of these components may vary depending on the manufacturer, but the overall composition of CLED agar is designed to create an environment that promotes the growth of urinary tract pathogens while inhibiting the growth of commensal bacteria.
Principle of CLED Agar:
The principle of CLED agar is to provide a selective and differential medium for the isolation and identification of urinary tract pathogens from clinical specimens, such as urine samples.
The cysteine in the medium provides a source of sulfur that is essential for the growth of some bacteria, while lactose is used as a carbohydrate source to differentiate between lactose-fermenting and non-lactose fermenting bacteria. Electrolytes such as sodium chloride and potassium chloride are included to maintain a proper pH balance and prevent osmotic stress on the bacterial cells.
The medium is designed to support the growth of urinary tract pathogens while inhibiting the growth of commensal bacteria, which can be present in urine samples and interfere with the isolation and identification of the target pathogens. The medium is selective against gram-positive bacteria, which are inhibited by the low pH of the medium, as well as non-urinary tract pathogens that do not grow well in this environment.
Differentiation of lactose fermenters and non-lactose fermenters is based on the ability of lactose-fermenting bacteria to produce acid, which lowers the pH of the medium and changes the color of the colonies from green to yellow. Non-lactose fermenters produce alkaline reactions and remain green in color.
Overall, the principle of CLED agar is to provide a simple and cost-effective culture medium that can support the growth of urinary tract pathogens while inhibiting the growth of commensal bacteria and allowing for differentiation between lactose-fermenting and non-lactose fermenting bacteria.
Preparation of CLED Agar
The preparation of CLED agar involves the following steps:
- Weigh out the above amount of CLED agar powder OR according to the manufacturer’s instructions and add it to a suitable container, such as a flask or bottle.
- Add the One Liter OR according to the manufacturer’s instructions of distilled water to the container, and stir well to dissolve the agar powder completely.
- Heat the mixture on a hot plate or in a microwave until the agar is completely dissolved and the solution is clear.
- Adjust the pH of the medium to the appropriate level (usually around pH 7.2 to 7.5) using a suitable pH indicator and a pH meter or pH paper.
- Sterilize the medium by autoclaving at 121°C for 15 minutes or by using a suitable filtration system.
- Allow the medium to cool to around 45-50°C before pouring it into sterile Petri dishes or tubes.
- Allow the medium to solidify before use.
- Note that the exact preparation method may vary depending on the manufacturer’s instructions and the specific requirements of the laboratory. It is important to follow the manufacturer’s instructions carefully and to use sterile techniques throughout the preparation process to avoid contamination of the medium.
Colony Characteristics on CLED Agar:
On CLED agar, the colony characteristics of urinary tract pathogens can vary depending on the species of bacteria and their ability to ferment lactose.
Lactose-fermenting bacteria, such as Escherichia coli and Klebsiella pneumoniae, will produce yellow colonies due to the production of acid from lactose fermentation, resulting in a drop in pH and a change in color of the pH indicator. The colonies of these lactose-fermenting bacteria are typically small to medium-sized, smooth, and convex, with entire margins.
Non-lactose fermenting bacteria, such as Pseudomonas aeruginosa and Proteus mirabilis, will produce green colonies due to the lack of acid production, and the pH of the medium remains alkaline. The colonies of these non-lactose fermenting bacteria are typically larger in size, rough or irregular in texture, and may have a distinct odor.
Other bacterial species may produce a variety of colony morphologies on CLED agar, making it important to confirm the identification of any isolates using additional tests, such as Gram staining, biochemical tests, or molecular methods.
It is also worth noting that CLED agar is not selective for all urinary tract pathogens and may not be effective in detecting some fastidious organisms, such as Mycoplasma and Ureaplasma species. As such, other culture media and diagnostic tests may be required to accurately identify and characterize bacterial isolates from urine samples.
| Organism | Colony Morphology |
| Escherichia coli | Large, elevated, yellow, opaque colonies with a center more intense yellow; yellowish medium |
| Klebsiella species | extremely mucoid colonies varying in color from yellow to whitish-blue. yellowish medium. |
| Proteus species | translucent blue colonies are usually smaller than Escherichia coli. blue-green to blue medium. |
| Salmonella species | flat blue colonies |
| Pseudomonas aeruginosa | green colonies with a typical matte surface and rough periphery. “Sweet” odor. Blue-green agar |
| Enterococcus faecalis | yellow colonies about 0.5 mm in diameter. yellow medium |
| Staphylococcus aureus | deep yellow colonies about 0.75 mm diameter, uniform in color. yellow medium |
| Corynebacteria | very small grey colonies |
| Lactobacilli | similar to corynebacteria but with a rougher surface |
Escherichia Coli Characteristics on CLED Agar:
Escherichia coli is a lactose-fermenting bacteria commonly associated with urinary tract infections, and it is known to produce characteristic colony morphology on CLED agar.
On CLED agar, E. coli forms small to medium-sized colonies that are yellow in color due to the production of acid from lactose fermentation. The colonies are typically smooth and convex with entire margins, and may sometimes exhibit a slightly mucoid appearance.
E. coli is also capable of producing beta-glucuronidase, which can be detected using chromogenic substrates that result in the production of blue-green colonies. This characteristic can be helpful in differentiating E. coli from other lactose-fermenting bacteria, such as Enterobacter and Citrobacter species, which do not produce beta-glucuronidase.
Overall, the combination of yellow colony color and beta-glucuronidase positivity is highly suggestive of E. coli on CLED agar, but additional tests, such as biochemical tests or molecular methods, may be required for definitive identification.
Klebsiella species Characteristics on CLED Agar:
Klebsiella species, including Klebsiella pneumoniae, are commonly found in urinary tract infections and can be identified on CLED agar based on their characteristic colony morphology.
On CLED agar, Klebsiella species produce small to medium-sized colonies that are yellow in color due to the production of acid from lactose fermentation. The colonies are typically smooth, shiny, and convex with entire margins.
In addition to lactose fermentation, Klebsiella species are also known to produce a thick polysaccharide capsule that gives the colonies a mucoid or slimy appearance. This characteristic can be observed on CLED agar as a mucoid appearance of the colony.
Klebsiella species may also exhibit an unusual appearance on CLED agar when exposed to high levels of salt or other inhibitory factors. In some cases, they may produce small colonies with a darker center or exhibit swarming motility, a rapid form of bacterial movement across the agar surface.
Overall, the combination of yellow colony color, mucoid appearance, and other morphological characteristics can be helpful in identifying Klebsiella species on CLED agar, but additional tests, such as biochemical tests or molecular methods, may be required for definitive identification.
Proteus species Characteristics on CLED Agar:
Proteus species, including Proteus mirabilis, are commonly found in urinary tract infections and can be identified on CLED agar based on their characteristic colony morphology.
On CLED agar, Proteus species produce medium to large-sized colonies that are green in color due to the inability to ferment lactose, resulting in an alkaline environment that maintains the pH indicator at its original green color. The colonies are typically irregular and have a rough texture, with irregular or wavy margins.
Proteus species are also known for their swarming motility, a rapid form of bacterial movement across the agar surface that results in a distinct pattern of concentric rings or finger-like projections around the colony. Swarming is particularly evident on CLED agar, where the high nutrient content and alkaline pH promote this type of motility.
In addition to swarming, Proteus species may also exhibit a characteristic odor resembling that of ammonia or old sneakers due to the production of urease, an enzyme that breaks down urea to ammonia and carbon dioxide.
Overall, the combination of green colony color, irregular colony morphology, swarming motility, and characteristic odor can be helpful in identifying Proteus species on CLED agar, but additional tests, such as biochemical tests or molecular methods, may be required for definitive identification.
Salmonella species Characteristics on CLED Agar:
Salmonella species are gram-negative bacteria that are commonly associated with foodborne illnesses and can sometimes cause urinary tract infections. These bacteria have characteristic colony morphology on CLED agar.
On CLED agar, Salmonella species produce medium-sized colonies that are typically colorless or white with a slightly opaque or milky appearance. The colonies may appear slightly raised and have a smooth or slightly rough texture.
Unlike most other Enterobacteriaceae, Salmonella species do not ferment lactose and are unable to produce acid, which results in the pH indicator of the CLED agar remaining green. However, Salmonella species are capable of producing hydrogen sulfide (H2S) which can be observed as black precipitation in the center of the colony or as a black halo around the colony.
In addition to colony morphology, Salmonella species can be identified using biochemical tests and molecular methods. Some of the commonly used biochemical tests include the triple sugar iron (TSI) agar test, lysine iron agar (LIA) test, and urease test, among others.
Overall, the combination of non-lactose fermenting colonies, milky white appearance, and the production of hydrogen sulfide can be helpful in identifying Salmonella species on CLED agar, but additional tests are necessary for definitive identification.
Pseudomonas aeruginosa Characteristics on CLED Agar:
Pseudomonas aeruginosa is a gram-negative bacteria commonly found in urinary tract infections and can be identified on CLED agar based on its characteristic colony morphology.
On CLED agar, Pseudomonas aeruginosa produces medium to large-sized colonies that are typically greenish-blue or bluish-green in color. The colonies may appear flat or slightly raised and have a smooth, shiny, and sometimes mucoid appearance.
Pseudomonas aeruginosa is also known for its characteristic fruity or grape-like odor, which is due to the production of volatile organic compounds such as pyocyanin and pyoverdine.
In addition to colony morphology and odor, Pseudomonas aeruginosa is known for its ability to produce a variety of pigments, including pyocyanin, pyoverdine, and pyorubrin, which can give the colonies a variety of colors ranging from green to yellow to red.
Overall, the combination of bluish-green colony color, fruity odor, and pigment production can be helpful in identifying Pseudomonas aeruginosa on CLED agar, but additional tests, such as biochemical tests or molecular methods, may be required for definitive identification.
Enterococcus faecalis Characteristics on CLED Agar:
Enterococcus faecalis is a gram-positive bacterium commonly found in the gastrointestinal tract and can sometimes cause urinary tract infections. It can be identified on CLED agar based on its characteristic colony morphology.
On CLED agar, Enterococcus faecalis produces small to medium-sized colonies that are typically cream to yellow in color. The colonies may appear circular, convex, and slightly raised with smooth, entire margins.
Enterococcus faecalis is also known for its ability to grow in the presence of high levels of salt, bile, and other inhibitory substances, making it a common cause of urinary tract infections in patients with compromised immune systems or underlying conditions.
In addition to colony morphology, Enterococcus faecalis can be identified using biochemical tests, such as the bile-esculin test, which is positive for the hydrolysis of esculin in the presence of bile, and the PYR (pyrrolidonyl arylamidase) test, which is positive for the production of the enzyme PYR.
Overall, the combination of cream to yellow colony color, circular colony morphology, and the ability to grow in the presence of inhibitory substances can be helpful in identifying Enterococcus faecalis on CLED agar, but additional tests, such as biochemical tests or molecular methods, may be required for definitive identification.
Staphylococcus aureus Characteristics on CLED Agar:
Staphylococcus aureus is a gram-positive bacterium commonly found on the skin and in the nose of humans, and can sometimes cause urinary tract infections. It can be identified on CLED agar based on its characteristic colony morphology.
On CLED agar, Staphylococcus aureus produces small to medium-sized colonies that are typically yellow in color. The colonies may appear circular, convex, and slightly raised with smooth, entire margins.
Staphylococcus aureus is also known for its ability to produce the enzyme coagulase, which can clot blood plasma and is used as a key diagnostic feature for this organism.
In addition to colony morphology and coagulase production, Staphylococcus aureus can be identified using other biochemical tests, such as the catalase test, which is positive for the production of the enzyme catalase, and the DNase test, which is positive for the production of the enzyme DNase.
Overall, the combination of yellow colony color, circular colony morphology, and coagulase production can be helpful in identifying Staphylococcus aureus on CLED agar, but additional tests, such as biochemical tests or molecular methods, may be required for definitive identification.
Corynebacteria Characteristics on CLED Agar:
Corynebacteria are gram-positive bacteria commonly found on the skin and mucous membranes and can be identified on CLED agar based on their characteristic colony morphology.
On CLED agar, Corynebacteria produce small to medium-sized colonies that are typically gray or beige in color. The colonies may appear circular, convex, and slightly raised with smooth, entire margins.
Corynebacteria are also known for their unique cell morphology, which can be observed using Gram staining. Corynebacteria have a characteristic “club-shaped” appearance due to the presence of irregularly shaped cells called palisades.
In addition to colony morphology and cell morphology, Corynebacteria can be identified using other biochemical tests, such as the catalase test, which is positive for the production of the enzyme catalase, and the nitrate reduction test, which is positive for the reduction of nitrate to nitrite.
Overall, the combination of gray or beige colony color, circular colony morphology, and the presence of palisades on Gram staining can be helpful in identifying Corynebacteria on CLED agar, but additional tests, such as biochemical tests or molecular methods, may be required for definitive identification.
Lactobacilli Characteristics on CLED Agar:
Lactobacilli are gram-positive bacteria commonly found in the human gastrointestinal tract, mouth, and female genital tract. They are also used commercially in the production of fermented foods and beverages. Lactobacilli can be identified on CLED agar based on their characteristic colony morphology.
On CLED agar, lactobacilli produce small to medium-sized colonies that are typically white to cream in color. The colonies may appear circular, convex, and slightly raised with smooth, entire margins.
Lactobacilli are also known for their ability to produce lactic acid through the fermentation of carbohydrates, which contributes to their sour taste and is used in the production of fermented foods and beverages.
In addition to colony morphology and lactic acid production, lactobacilli can be identified using other biochemical tests, such as the catalase test, which is negative for the production of the enzyme catalase, and the oxidase test, which is negative for the production of the enzyme oxidase.
Overall, the combination of white to cream colony color, circular colony morphology, and lactic acid production can be helpful in identifying lactobacilli on CLED agar, but additional tests, such as biochemical tests or molecular methods, may be required for definitive identification.
Limitations:
While CLED agar is a useful culture medium for the isolation and identification of urinary tract pathogens, there are some limitations to its use. Some of the limitations include:
- False negatives: CLED agar may not be able to detect all types of urinary tract pathogens, leading to false negative results. For example, some bacteria may not grow well on CLED agar or may require additional nutrients for growth.
- Overgrowth of non-pathogenic bacteria: CLED agar is not selective for urinary tract pathogens and may allow the growth of non-pathogenic bacteria. This can make it difficult to identify the target bacteria and can lead to false positive results.
- Interference from urine contaminants: Urine specimens may contain various contaminants, such as skin flora, which can interfere with the growth of urinary tract pathogens on CLED agar. This can result in misleading or inconclusive results.
- Limited information on antibiotic resistance: While CLED agar can be used for antibiotic susceptibility testing, it does not provide comprehensive information on antibiotic resistance patterns. Additional tests, such as minimum inhibitory concentration (MIC) testing, may be required for more detailed information on antibiotic resistance.
- Limited shelf life: CLED agar has a limited shelf life and must be stored properly to ensure optimal performance. Improper storage can lead to changes in the composition of the medium and can affect its ability to support bacterial growth.
Overall, while CLED agar is a valuable tool for the isolation and identification of urinary tract pathogens, it should be used in conjunction with other diagnostic tests and should be interpreted with caution. Proper quality control and adherence to laboratory protocols are essential for accurate and reliable results.
FAQs:
What is CLED agar?
Answer: CLED agar is a culture medium that is commonly used for the isolation and identification of urinary tract pathogens.
What does CLED agar stand for?
Answer: CLED agar stands for cystine lactose electrolyte-deficient agar.
How is CLED agar prepared?
Answer: CLED agar can be prepared by dissolving the dehydrated powder in distilled water, sterilizing the medium, and then pouring it into sterile petri dishes.
What are the colony characteristics of Escherichia coli on CLED agar?
Answer: Escherichia coli typically produces small, round, and yellow colonies on CLED agar.
What are the limitations of CLED agar?
Answer: The limitations of CLED agar include false negatives, overgrowth of non-pathogenic bacteria, interference from urine contaminants, limited information on antibiotic resistance, and limited shelf life.
What are the uses of CLED agar in the microbiology laboratory?
Answer: The uses of CLED agar in the microbiology laboratory include the isolation and identification of urinary tract pathogens, antibiotic susceptibility testing, and quality control.
What is the pH of CLED agar?
Answer: The pH of CLED agar is around 7.3 to 7.5.
How long does it take to incubate CLED agar plates?
Answer: CLED agar plates are typically incubated for 24 to 48 hours at 35-37°C.
What are the colony characteristics of Staphylococcus aureus on CLED agar?
Answer: Staphylococcus aureus typically produces small, round, and white to cream-colored colonies on CLED agar.
What are the colony characteristics of Klebsiella species on CLED agar?
Answer: Klebsiella species typically produce large, mucoid, and pink to red colonies on CLED agar.
How is CLED agar used for urine culture?
Answer: CLED agar can be used for urine culture by inoculating a urine sample onto the surface of the medium and incubating it for a specified period of time.
What are the colony characteristics of Pseudomonas aeruginosa on CLED agar?
Answer: Pseudomonas aeruginosa typically produces small, flat, and greenish-blue colonies on CLED agar.
What are the colony characteristics of Enterococcus faecalis on CLED agar?
Answer: Enterococcus faecalis typically produces small, round, and white to cream-colored colonies on CLED agar.
How is CLED agar used for antibiotic susceptibility testing?
Answer: CLED agar can be used for antibiotic susceptibility testing by testing the bacterial isolates from urine cultures for their susceptibility to different antibiotics using various methods, such as disk diffusion or broth microdilution.
What is the composition of CLED agar?
Answer: The composition of CLED agar includes pancreatic digest of casein, beef extract, cystine, lactose, and agar.
What are the colony characteristics of Salmonella species on CLED agar?
Answer: Salmonella species typically produce small, round, and colorless to pink colonies on CLED agar.
What are the colony characteristics of Corynebacteria on CLED agar?
Answer: Corynebacteria typically produce small, round, and gray to white colonies on CLED agar.
How is CLED agar used for quality control?
Answer: CLED agar can be used for quality control by ensuring that the media and laboratory procedures are functioning properly and detecting any potential contamination.
What are the advantages of CLED agar?
Answer: The advantages of CLED agar include its ability to selectively support the growth of urinary
Conclusion:
In conclusion, CLED agar is a commonly used culture medium in the microbiology laboratory for the isolation and identification of urinary tract pathogens. Its unique composition, which is deficient in electrolytes, and the inclusion of lactose, allow for the selective growth of urinary tract pathogens while inhibiting the growth of other bacteria. CLED agar has various uses in the laboratory, including antibiotic susceptibility testing, quality control, and the isolation of different bacterial species. However, it also has some limitations, such as false negatives and overgrowth of non-pathogenic bacteria, which must be taken into account when interpreting results. Overall, CLED agar is a valuable tool in the diagnosis and treatment of urinary tract infections.
References:
- “CLED Agar | Culture Media | Microbe Notes.” Microbe Notes, 10 Nov. 2019, https://microbenotes.com/cled-agar/.
- “Cystine Lactose-Electrolyte Deficient Agar (CLED Agar) for Urine Culture.” Thermo Fisher Scientific, 2022, https://www.thermofisher.com/order/catalog/product/OXOIDCM0509.
- “CLED Agar – Principle, Composition, Uses and Preparation.” LaboratoryInfo.com, 8 Sept. 2020, https://labinfo.in/cled-agar-principle-composition-uses-and-preparation/.
- “CLED Agar | Thermo Fisher Scientific – UK.” Thermo Fisher Scientific, 2022, https://www.thermofisher.com/uk/en/home/clinical/microbiology/microbiology-learning-center/microbiology-resource-library/protocols/cled-agar.html.
- “CLED Agar.” HiMedia Laboratories, https://himedialabs.com/TD/M070.pdf.
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