CHROMOBRIT IU

A chromogenic culture medium, used for the isolation, enumeration, presumptive identification and simultaneous detection of different bacteria from urine samples.

Purpose

Urinary tract infections are one of the most frequent causes of medical consult. Due to the increase of antimicrobial resistance that actually show urinary pathogenic bacteria, we consider the necessity to perform urine culture tests in a simple and systematic way, to isolate the ethyologic agent and to perform the appropriate antibiogram of it.
Nowadays, urine samples constitute the principal samples (in quantities) received and processed in microbiology laboratories.
Urine cultures, are recommended to be inoculated in blood agar and Mac Conkey Agar, and in Europe and Argentina, it is widely used CLED Medium (Cystine Lactose Electrolyte Deficient Medium) Blood Agar, is considered an optimum medium for the isolation and enumeration of bacteria from urine samples, but allows a low differentiation among bacteria isolated and it is difficult the interpretation of results when swarming from Proteus species is observed.
Differential culture media (such as CLED and Mac Conkey), avoid swarming phenomenon of Proteus species, and allow the differentiation between lactose and non lactose fermenter organisms, but it is necessary to perform further biochemical tests for bacterial identification.
Few years ago, synthetic chromogenic substrates have been started to use, in the identification of organisms capable to detect through the formation of coloured compounds, the presence of specific enzymes of bacteria species.
As a rule, many groups of chromogenic compounds have been used in biochemical reactions to study kinetic properties of specific enzymes, according to the principle that a substrate is hydrolyzed by the specific enzyme, releasing a chromophore compound.
Many of these substrates, can be incorporated into culture media to obtain faster and more accurate bacterial identification.
Various investigators add these substrates into selective and differential culture media.
The interaction among organisms and chemical substances differ, and depend of the structure of chromogenic substrates used and the enzymes detected, and the compound produced by the enzymatic reaction, precipitates on microbial cells, producing specific chromogenic characteristics to the colonies.
Laboratorios Britania, has developed a new product: Chromobrit IU, which consists in a basal nutritive culture medium containing a chromogenic mix that allows to detect simultaneously different enzymatic activities in the same medium and allows to make a direct presumptive identification of the main bacteria that cause urinary tract infections, just performing a few additional biochemical tests.
As the main organisms that cause urinary tract infections are Escherichia coli, other Enterobacteriaceae members and Enterococcus spp, the new culture media must allow the direct identification of these organisms and the growth of other urinary pathogenic bacteria that are not directly identificated as typical colonies (such as Staphyloccoccus spp. and Pseudomonas spp.) so to be subcultured for bacterial identification. Chromobrit IU, allows the detection ofβ glucuronidase enzyme and β glucosidase enzyme, tryptophan deaminase activity (TDA) and indole production (by the spot technique). Detection of enzymatic activity, morphology characteristics and colour of the colonies, allow the presumptive identification of:

E. coli: β glucuronidase enzyme positive, indole positive

KES Group (Klebsiella, Enterobacter, Serratia): β glucosidase enzyme positive.

Enterococcus spp.: β glucosidase enzyme positive.

Proteus-Morganella-Providencia: triptophan deaminase enzyme positive and Proteus vulgaris is indole positive.

In this medium, yeast extract, peptone and trypteine provide nutrients for bacterial growth. Also, trypteine and tryptophan are the source for tryptophan deaminase enzyme. Starch is the source of glucose, sodium pyruvate allows to recover injured cells, and ammonium ferric citrate allows the detection of Tryptophan deaminase enzyme present in Proteae members. The chromogenic mix allows the simultaneously detection of Escherichia coli, KES group and Enterococcus spp. Escherichia coli contains the β glucuronidase enzyme and cleavages the chromogenic substrate with the consequent production of a green compound. Enterococcus spp. and KES group bacteria contains the β glucosidase enzyme wich cleavages the chromogenic subtrate with the consequent production of a pink compound.

Formula (in grams per liter)		Instructions
Starch	1.0	Melt the culture medium in boiling water. Pour approximately 15 ml in Petri dishes. This culture medium is slightly opalescent. Keep plates at 2-8 ºC, away from light.
Yeast extract	6.0
Peptone	6.0
Trypteine	14.0
L-tryptophan	1.0
Sodium pyruvate	1.0
Ammonium ferric citrate	0.02
Chromogenic mix	0.2
Agar	15.0
Final pH: 6.9 ± 0.2

Inoculation
Directly, streak on the surface of the agar. Use Britania´s calibrated loops (code B1655330).

Incubation
Aerobically, at 35-37 ºC, for 18-24 hours

Results:
Enumerate colonies and consider the volume of the urine sample previously inoculated. Report results as CFU/ml .
Detection of enzymatic activity, morphology characteristics and colour of the colonies, allow the presumptive identification of:

E. coli: Green colonies.
β glucuronidase enzyme: positive.
β glucosidase enzyme: negative.
Note: approximately 95-98 % of strains of Escherichia coli posses β glucuronidase enzyme.

KES Group (Klebsiella, Enterobacter, Serratia):pink colonies.
β glucuronidase enzyme: negative.
β glucosidase enzyme: positive.

Enterococcus spp.: pinpoint pink colonies.
β glucuronidase enzyme: negative.
β glucosidase enzyme: positive.

Proteus-Morganella-Providencia: brownish colonies.
Tryptophan deaminase enzyme: positive.
β glucuronidase enzyme: negative.
β glucosidase enzyme: negative.
Indole Test positive: Proteus vulgaris, Morganella morganii, Providencia spp.
Indole Test negative: Proteus mirabilis.

Pseudomonas aeruginosa: white or pigmented colonies.

Staphylococcus aureus: white yellowish colonies.

Streptococcus agalactiae: pinpoint colonies, generally green coloured.

Candida spp: white colonies.

Note: except E. coli β glucuronidase enzyme positive, it is necessary to make further biochemical tests for definitive bacterial identification of the organism isolated.

Quality Control

Organisms	Colonial Characteristics
E. coli ATCC 25922	Green colonies
E. coli ATCC 35218	Green colonies
E. coli O157 H7 ATCC 700728	White colonies
K. pneumoniae ATCC 700603	Pink colonies
E. cloacae ATCC 13047	Pink colonies
S. marcescens ATCC 13880	Pink and pigmented colonies
E. faecalis ATCC 29212	Pinpoint pink colonies
S. aureus ATCC 25923	White-pinkishcolonies
S. agalactiae ATCC 13813	Pinpoint green colonies
P. mirabilis ATCC 43071	Brownish colonies
P. aeruginosa ATCC 27853	White colonies
C. albicans ATCC 10231	White colonies

Limitation

-According to bibliography, 95-98 % of strains of  E. coli, possess the ß-glucuronidase enzyme.
-Some strains of Salmonella sp. Shigella spp., Citrobacter freundii and Yersinia sp., possess the ß-glucuronidase enzyme
- If desire to perform Indole Test, use p-dimethylamino cinnamaldehyde reagent.
- If desire to perform Tryptophane deaminase Test, use Phenylalanine Reagent (code: B1550461).

 

Appearance

Prepared medium: amber coloured gel.

Storage:

Prepared medium: at 10-35 ºC

Packaging
6 flasks x  50 ml	B04-215-84
12 flasks x 100 ml	B04-215-92