Tergitol 7 WITH TTC

Cat. no. G58 Tergitol 7 with TTC, 15x100mm Plate, 20ml 10 plates/bag

INTENDED USE

Hardy Diagnostics' Tergitol 7 with TTC agar is recommended as a selective and differential medium for enumeration of coliforms in food and water samples. (1-4,7) The medium conforms to the recommendations of the APHA. (5,12)

This product is not intended to be used for the diagnosis of human disease.

SUMMARY

Previous research by Pollard shows the bactericidal action of Tergitol 7 against gram-positive bacteria. (11) Chapman later developed the medium for the selective isolation of Escherichia coli and other members of the coliform group. According to Chapman's published formula, a medium containing Tergitol 7, proteose peptone no. 3, yeast extract, lactose, and bromothymol blue permitted the unrestricted development of coliform bacteria, while inhibiting the growth of gram-negative spore-formers, gram-positive microorganisms and the swarming of Proteus spp. (1,2) He found the difference between E. coli and Enterobacter aerogenes and other coliforms to be distinct on this medium: E. coli produces yellow colonies with yellow zones and occasionally rust colored centers; E. aerogenes produces greenish-yellow colonies, while non-lactose fermenting microorganisms produce dark red colonies with bluish zones. In addition, Chapman proposed that counts of coliform organisms on Tergitol 7 Agar were found to be as much as 30% higher than on other types of selective media. (6)

Chapman later modified his original formula by adding 40mg of triphenyltetrazolium chloride (TTC) per liter, and the modified formulation was found to be helpful in rapidly differentiating E. coli and E. aerogenes . On medium containing TTC, confirmation of the presence of E. coli and E. aerogenes was possible in as few as six to ten hours. (2) Chapman also reported that Tergitol 7 Agar with added TTC was suitable for the selective isolation of Candida spp. and other contaminating fungi. (2)

Hardy Diagnostics' Tergitol 7 with TTC agar is a modification of Chapman's original formulation and contains 25mg of 2,3,5 Triphenyltetrazolium Chloride (TTC) for the rapid detection of E. coli and E. aerogenes from food and water samples. The medium is recommended by the APHA for the recovery of injured or stressed total coliform bacteria from chemically treated or contaminated waters when parellel methods indicate suboptimal recovery. (5,12,13)

FORMULA

Ingredients per liter of deionized water:*

Lactose 10.0gm
Proteose Peptone No. 3 5.0gm
Yeast Extract 3.0gm
Tergitol 7 0.1gm
Bromothymol Blue 0.025gm
2,3,5 Triphenyltetrazolium Chloride (TTC) 0.025gm
Agar 15.0gm

Final pH 6.9 +/- 0.2 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8ºC. away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

PRECAUTIONS

PROCEDURE

Specimen Collection: Consult listed references for information on specimen collection. (1,2,8-10,12)

Method of Use: Allow the medium to warm to room temperature prior to inoculation. Inoculate agar and streak for isolation using the four quadrant method. Incubate inoculated media in an aerobic atmosphere at 35ºC. for 18-48 hours. Observe plates for growth.

Spread Plate Method :

1. Prepare decimal dilutions in sterile diluent to obtain 30-300 CFU per plate.

2. Aseptically inoculate agar surface with 0.1ml of well mixed diluted sample.

3. Using a sterile spreader device, distribute the inoculum evenly over the agar surface.

4. Invert and incubate plates aerobically for 18 to 48 +/- 2.0 hours at 35ºC. and observe for typical colonies.

Note: Pour plates do not give satisfactory results. (6)

Membrane Filter Method:

1. Shake the sample bottle vigorously at least 25 times to uniformly distribute the bacteria.

2. Aseptically filter a predetermined volume of the sample through a 47mm, 0.45 +/- 0.02um pore size membrane filter and rinse the sides of the funnel at least twice with 20 to 30ml of sterile buffered rinse water.

3. Use sterile forceps to aseptically remove the membrane filter from the filter base and roll it onto the surface of the agar, filtered side up, to avoid the formation of air bubbles between the membrane and agar surfaces. Reseat the membrane if bubbles occur.

4. Incubate inverted plates aerobically at 35 +/- 0.5ºC. for 18 to 48 hours and observe for typical colonies.

INTERPRETATION OF RESULTS

Colonies of E. coli should appear yellow with yellow zones and occasionally rust colored centers. Colonies of E. aerogenes should appear greenish-yellow. In general, lactose fermenting microorganisms should appear as yellow colored colonies.

Non-lactose fermenting microorganisms, such as Salmonella enterica , should appear as red colored colonies with a blue periphery. The growth of Proteus spp. should be partially to completely inhibited and exhibit red colonies with a blue periphery and no swarming.

Gram-positive microorganisms and gram-negative spore-forming microorganisms should be inhibited on this medium.

LIMITATIONS

Incubation at 44ºC. has been recommended for improved recovery of coliform bacteria. (3,7)

Subculture typical colonies onto a non-selective medium, such as TSA (Cat. no. G60), and/or into Tryptone Broth (Cat. no. R40) or Peptone Broth (Cat. no. K151). From the solid medium, an oxidase test (Cat. no. Z119 or Z93) or spot indole test (Cat. no. Z65) can be performed. From the broth medium, an indole Kovac's test (Cat. no. Z67) can be performed.

Consider all characteristic oxidase negative colonies as coliforms. Colonies that are oxidase negative, but indole positive, should be considered positive for Escherichia coli .

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, swabs, applicator sticks, other culture media, incinerators, and incubators, etc., as well as serological and biochemical reagents are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Escherichia coli
ATCC ® 25922**
A 18-48hr 35°C Aerobic Growth; yellow colonies
Salmonella enterica
ATCC ® 14028
A 18-48hr 35°C Aerobic Growth; red colonies with blue periphery
Proteus mirabilis
ATCC ® 12453
B 18-48hr 35°C Aerobic Partial to complete inhibition; red colonies with blue periphery, no swarming
Enterococcus faecalis
ATCC ® 29212**
B 18-48hr 35°C Aerobic Inhibited

** Recommended QC strains for User Quality Control according to the CLSI document M22 when applicable.

USER QUALITY CONTROL

REFERENCES

1 Chapman, G.H. 1947. A Superior Culture Medium for the Enumeration and Differentiation of Coliforms. J. Bacteriol. ; 53:504.

2. Chapman, G.H. 1951. A Culture Medium for Detecting and Confirming Escherichia coli in Ten Hours. Am. J. Public Health ; 41:1381.

3. Mossel, D.A.A. 1962. An Ecological Investigation on the Usefulness of Two Specific Modifications of Eijkman's Test as an Element of the Methods for the Detecting of Faecal Contamination of Foods. J. Appl. Bacteriol. ; 25:20.

4. E.U. 1998. 98/83/EC of Council of 3rd November 1998 on The Quality of Water Intended for Human Consumption. Off. J. Eur. Commun. L3330:32-54.

5. Speck, Marvin L. 1992. Compendium of Methods for the Microbiological Examination of Foods , 3rd ed. American Public Health Association, Washington, D.C.

6. MacFaddin, J.F. 1985. Media for the Isolation , Cultivation , Identification , Maintenance of Medical Bacteria , Vol. I. Williams & Wilkins, Baltimore, MD.

7. Kulp, W., et al. 1953. Use of Tergitol 7 Triphenyl Tetrazolium Chloride Agar as the Coliform Confirmatory Medium in Routine Sanitary Water Analysis. Am. J. Public Health ; 43:1111.

8. Jorgensen., et al. Manual of Clinical Microbiology , American Society for Microbiology, Washington, D.C.

9. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology , C.V. Mosby Company, St. Louis, MO.

10. Isenberg, H.D. Clinical Microbiology Procedures Handbook , Vol. I & II. American Society for Microbiology, Washington, D.C.

11. Pollard, A.L. 1946. A useful selective bactericidal property of Tergitol 7. Science .; 103:758-759.

12. American Public Health Association. Standard Methods for the Examination of Water and Wastewater, APHA, Washington, D.C.

13. ISO 9308-1:1990. International Organisation for Standardisation, Geneva. ISO (1994a). Water Quality - Detection and Enumeration of Coliform Organisms, Thermotolerant Coliform Organisms and Presumptive Escherichia coli - Part 1: Membrane filtration method.


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