Rainbow™ - Biolog | E. coli O157 Detection

For laboratory use as a selective, chromogenic culture medium to aid in the detection, isolation and presumptive identification of verotoxin-producing strains of Escherichia coli, particularly serotype O157:H7. Not for human in vitro diagnostic use.

Summary of Test

This bacterium is often endemic to cattle and spread through contaminated meat¹, and the Food Safety Inspection Service (USDA-FSIS) has declared it to be an “official adulterant” and requires meat producers to exclude it from their products².

Rainbow Agar O157 has both selective and chromogenic properties that make it particularly useful for isolating pathogenic E. coli strains. The medium contains chromogenic substrates that are specific for two E. coli-associated enzymes: β-galactosidase (a blue-black chromogenic substrate) and β-glucuronidase (a red chromogenic substrate).

-	Strain O157:H7 is typically glucuronidase negative so it forms unique and distinctive black or gray colonies.
-	Many other non-O157 toxigenic strains overproduce β-galactosidase relative to β-glucuronidase on this medium and consequently they are typically colored purple, violet or blue.
-	Most non-pathogenic E. coli strains are glucuronidase positive, and range from pink to magenta with occasional purple or blue strains.
-	Nearly all other bacterial species are either inhibited on this medium or they grow as white or cream-colored colonies.

Contents

This sealed package contains dry powder to make Rainbow Agar O157. 30 grams of powder will make 500 mL or approximately 25 plates.

Preparation

To prepare 500 mL of Rainbow Agar O157, mix 1 30 gram package of powder into 500 mL of purified water, boil gently to dissolve components and autoclave for 10 minutes at 15 lbs. pressure and 121°C. DO NOT exceed 10 minutes at this heat and pressure. For volumes less than 500 ml, autoclave for 5 minutes. Cool agar to 45-50°C before pouring or adding selective agents. If desired, add the appropriate amount of a filter sterilized stock solution of selective agent and mix well. Dispense approximately 20 ml of media into each petri plate. The medium is ready to use as soon as it has cooled, gelled and the surface has dried. The final medium should be clear and virtually colorless. No pH adjustment is needed. The final pH should be pH 7.9-8.3.

Storage

Rainbow Agar O157 powdered medium is extremely hygroscopic and should be stored at 2°C to 25°C in a dry environment in its unopened package until use. Rainbow Agar O157 plates are stable for 2 weeks when stored in the dark, under refrigeration and packaged in cellophane to inhibit moisture loss.

User Quality Control

The following organisms are recommended if quality control is desired or required. Inoculate Rainbow Agar O157 plates with the following strains by streaking for isolation, and then incubate for 24 hours at 35°C without elevated CO2. Colony color should be read from isolated colonies.

	ATCC®	Color on Rainbow
Organism	number	Agar O157

Escherichia coli O157:H7	43894	Black or gray
Escherichia coli	11775	Pink or magenta
Enterococcus faecalis	19433	White

Procedure - Low Background Flora

Inoculate the medium by streaking or spreading a sample suspected of containing E. coli on the surface of the medium. Incubate the plates for 20 to 24 hours, or longer, at 35°C without elevated CO2 and observe for the presence of colored colonies. The distinctive black or gray coloration of E. coli O157:H7 colonies is easily viewed by laying the petri plate against a white background. When O157 is surrounded by pink or magenta non-toxigenic colonies, it may have a bluish hue. Upon sub-culturing, isolated E. coli O157:H7 colonies will have their typical black or gray coloration. Because of the selective nature of the medium, the size of the colonies formed is smaller than would be seen on other media commonly used to cultivate E. coli.

Procedure - High Background Flora

To increase the selectivity of Rainbow Agar O157 use 0.8 mg/L potassium tellurite + 10 mg/L novobiocin. Tellurite is highly selective for E. coli O157:H7 and will reduce background flora considerably. Novobiocin inhibits Proteus swarming and the growth of telluritereducing bacteria. Caution must be used because rare strains of O157:H7 are tellurite sensitive3. E. coli O157:H7 colony coloration will be slightly bluer with these selective agents added.

Expected Results^a

Organism	Colony coloration^b
E. coli O157:H7	Black or gray

E. coli O157:H7 glucuronidase positive	Purple-blue
E. coli O26:H11	Purple-magenta
E. coli O48:H21	Purple
E. coli O111:H- or O111:H8	Violet or gray
Non-toxigenic E. coli	Pink or magenta^c

^aColony coloration is indicative of isolated colonies.

^bColor can vary depending upon the E. coli strain tested.

^cCan be purple or blue.

Limitations

This medium should not be used as the sole basis for identification of microorganisms. Any colony suspected of being E. coli O157 or another toxin producing serotype should be tested further to verify its identity using an approved confirmatory protocol.

Klebsiella pneumoniae and rare Hafnia alvei or Citrobacter strains may resemble E. coli O157. Klebsiella forms distinctive orangegray colonies. Citrobacter forms blue-gray colonies and rare Hafnia strains may form light blue-gray to blue-black colonies. These species can be differentiated using the indole or PYR spot tests (E. coli: indole positive, PYR negative; K. pneumoniae, H. alvei: indole negative, Citrobacter: PYR positive).

References

1.	Doyle, M.P., and J.L. Schoeni. 1987. Applied Environmental Microbiology 53:2394-2396.
2.	Okrend, A.J.G., B.E. Rose, and B. Bennett. 1990. Journal of Food Protection 53:249-255.
3.	MacRae, M., T. Rebate, and I.D. Ogden. 1997. Letters in Applied Microbiology 25:135-137.

Biolog Ordering Information

Rainbow Agar O157

Catalog Number

Description

80102

80102 Dehydrated — ~30 g

| Rainbow™ Agar 0157 | Rainbow™ Agar Salmonella | Rainbow™ Agar Shingella / Aeromonas |

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