Chromogenic UTI Medium: Introduction, Principle, Composition, Preparation, Procedure, Colony Morphology, Uses and Keynotes

Chromogenic UTI Medium: Introduction, Principle, Composition, Preparation, Procedure, Colony Morphology, Uses and Keynotes

Introduction of Chromogenic UTI Medium

A chromogenic UTI Medium is applicable for the presumptive identification and differentiation of all the main micro-organisms that cause urinary tract infections (UTIs).

Principle of Chromogenic UTI Medium

Chromogenic UTI Medium is a differential agar that provides presumptive identification of the principal pathogens that cause infection of the urinary tract.  Peptone is a complex mixture of partially digested proteins. It contains proteoses, amino acids, polypeptides, phosphates, minerals (K, Mg), and accessory growth factors like nicotinic acid and riboflavin. Agar acts as the solidifying agent.  Peptone used in this medium is rich in tryptophan content. Water is an essential ingredient for the growth and reproduction of organisms and also serves as a transport medium for the agar’s various substances. The composition contains two specific chromogenic substrates that are cleaved by enzymes produced by organisms like Enterococcus, E. coli, and coliforms. In addition, it contains tryptophan which indicates tryptophan deaminase activity (TDA), indicating the presence of Proteus spp.  X-glucoside (chromogen) is cleaved by ß-glucosidase and allows the specific detection of enterococci through the formation of blue colonies while red-galactoside ( another chromogen) is targeted by the enzyme ß-galactosidase which is produced by E. coli, resulting in pink colonies. Cleavage of both the chromogens (X-glucoside and red-galactoside)  by members of the coliform group results in purple colonies. The medium also contains tryptophan that acts as an indicator of tryptophan deaminase activity (TDA), causing halos around the colonies of genera like  Proteus, Morganella, and Providencia spp.

 

Composition of Chromogenic UTI Medium

Formula       gm/litre

  • Peptone : 15.0
  • Chromogenic mix:  13.0
  • Agar : 15.0

Final pH 7.0 ± 0.2

 Preparation of Chromogenic UTI Medium,

  1. Suspend 43.0 grams in 1 liter purified/distilled or deionized water.
  2. Mix the medium properly.
  3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
  4. After autoclaving,  leave for cooling to 50°C.
  5. Pour Chromogenic UTI Medium into each plate and leave plates on the sterile surface until the agar has solidified.
  6. Store the plates in a refrigerator at 2-8°C.

Storage and Shelf life of Nutrient agar

  • Store at 2-8ºC  and away from direct light.
  • Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination.
  • The product is light and temperature-sensitive; protects from light, excessive heat, moisture, and freezing.

Test Requirements

Procedure of Chromogenic UTI Medium

  1. Allow the plates to warm at 37°C or to room temperature, and the agar surface to dry before inoculating.
  2. Inoculate and streak the specimen as soon as possible after collection.
  3. If the specimen to be cultured is on a swab, roll the swab over a small area of the agar surface.
  4. Streak for isolation with a sterile loop.
  5. Incubate plates aerobically at 35-37ºC. for 18-24 hours.
  6. Examine colonial characteristics.

Colony Morphology of Chromogenic UTI Medium

Escherichia coli -1-2mm pink colored colonies.

Enterobacter aerogenes -1-2mm purple colored colonies

Proteus mirabilis-1-2mm straw/brown colonies may exhibit some
swarming

Citrobacter freundii -3mm blue/purple colonies

Enterococcus faecalis -Pinpoint-1mm blue/green colonies

Staphylococcus aureus -white  cream colonies

Control strains-

Escherichia coli ATCC 25922 and Staphylococcus aureus
 ATCC 25923- Growth with pink and cream colors respectively
Negative control: Uninoculated medium-No change

Uses of Chromogenic UTI Medium

A chromogenic UTI Medium uses for the cultivation of urinary tract infections causing etiological agents. It is highly useful for uropathogens as compared to CLED agar and using blood agar and McConkey agar combination for urine culture and easy to interpret the findings due to different color formation by different genera.

 

Keynotes on Chromogenic UTI Medium

  • The appearance of dehydrated Chromogenic UTI Medium is a free-flowing straw-colored powder while the prepared medium is a straw-colored, transparent agar.
  • This medium should only be used for in vitro diagnostic purposes and do not use beyond the stated expiry date, or if the product shows any sign of deterioration.
  • A chromogenic UTI Medium shows inherent characteristics for demonstrating polymicrobial growth and it is easy for rapid identification by distinct colony colors is unique.
  •  CLED agar provides a valuable non-inhibitory diagnostic agar for plate culture of uropathogens, whilst preventing the swarming of Proteus spp.
  • It should be noted that bacteria with atypical enzyme patterns may give anomalous reactions. e.g. in a trial, over 45% of Enterobacter cloacae were shown to lack ß-glucosidase, resulting in pink colonies which were non-differentiable from E. coli. In such conditions,  an indole test can be beneficial using DMACA indole (Kovac’s reagent is prohibited since the color of the E. coli colonies may interfere with the red color of a positive indole test). The test should be done on filter paper, ignoring directly on the plate. This test will differentiate between E. coli and Enterobacter spp, and also between Proteus mirabilis and other species.
  • HiCrome™ UTI Agar modified medium composition is slightly different from chromogenic UTI medium (Clear)-Oxoid due to extra ingredient incorporation like HM Peptone B and tryptone that made accessible for clinical samples  ( urine, feces), food samples, and also water samples.
  • The advantage of Chromogenic media is faster results,  reliable visual detection, and additional testing possible directly from the agar

Limitations of Chromogenic UTI Medium

  1. Colony characteristics only provide presumptive identification and thus biochemical, immunological, molecular, or mass spectrometry testing be performed on colonies from pure culture for final identification.
  2. As it is an enzyme-substrate-based reaction, the intensity of color may vary with isolates.
  3. Serial inoculation may be required to assure adequate isolation of mixed flora samples.

Further Readings on Chromogenic UTI Medium

  • http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp
  • https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4163227/
  • https://himedialabs.com/TD/M1418.pdf
  • https://www.sigmaaldrich.com/overview-chromogenic-media
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