INDOLE TEST REAGENTS

Cat. no. Z65 Indole Spot Reagent 15ml
Cat. no. Z67 Indole Kovacs Reagent 15ml

INTENDED USE

Hardy Diagnostics Indole Spot Reagent and Indole Kovacs Reagent are recommended for use in determining the indole reaction of bacteria.

SUMMARY

The indole test is a qualitative procedure for determining the ability of bacteria to produce indole by deamination of tryptophan.

Using Kovacs tube method, indole combines, in the presence of a tryptophan rich medium, with p-Dimethylaminobenzaldehyde at an acid pH in alcohol to produce a red-violet compound.

In the spot test, indole combines, in the filter paper matrix, at an acid pH with p-Dimethylaminocinnamaldehyde (DMACA) to produce a blue to blue-green compound. Indole Spot Reagent (DMACA) has been reported to be useful in detecting indole production by members of the family Enterobacteriaceae and certain anaerobic species.

REAGENT FORMULA

Ingredients per liter:*

Indole Spot Reagent:
p-Dimethylaminocinnamaldehyde (DMACA) 10.0gm
Hydrochloric Acid, 37% 100.0ml
Deionized Water 900.0ml

Indole Kovacs Reagent:
p-Dimethylaminobenzaldehyde 50.0gm
Hydrochloric Acid, 37% 250.0ml
Amyl Alcohol 750.0ml

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

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-30ºC. Products should not be used if there are any signs of deterioration or if the expiration date has passed.

PRECAUTIONS

PROCEDURE

Specimen Collection: This product is not intended for primary isolation of patient specimens. This product is used in conjunction with other biochemical tests to identify cultures of isolated organisms.

Indole Spot Reagent (DMACA): Place several drops of Indole Spot Reagent on a piece of filter paper. With an inoculating loop or wooden applicator stick, pick a portion of an 18-24 hour isolated colony from a non-selective media and rub it onto the reagent saturated area of the filter paper.

Interpretation of Results: A positive reaction is denoted by the appearance of a blue to blue-green color change on the bacterial smear, or red-violet in the case of Providencia alcalifaciens , within 10 seconds. Negative reactions remain colorless or light pink.

The filter paper may appear pink to purple after the reagent is applied, but only the color change of the bacterial smear itself should be indicative of a positive reaction.

Indole Kovacs Reagent: Lightly inoculate Tryptone Broth (Cat. no. R40) or Peptone Broth (Cat. no. K151) with the test organism. Incubate 24-48 hours at 35ºC. Add 4-5 drops of Kovacs Reagent to the tube, shake gently.

Interpretation of Results: A positive Kovacs tube test reaction is denoted by the appearance of a pink to red color in the top alcohol layer. Negative reactions remain colorless or light yellow.

If Kovacs Indole is to be used with a commercial identification test strip, such as API ® or EnteroPluri, or Microgen, consult the manufacturer's literature.

LIMITATIONS

Indole tests may be used as an aid in the identification and differentiation of gram-positive and gram-negative organisms. Additional biochemical testing using pure cultures is recommended for complete identification.

The tube test is a more sensitive method of detecting indole than the spot test.

When performing a spot test, Kovacs Indole Reagent may be used as a substitute for the spot test reagent. However, Kovacs Indole Reagent, when used as the spot test reagent, is less sensitive in detecting indole than the Indole Spot Reagent (DMACA). (6)

Kovacs Indole Reagent is not recommended for use with anaerobic bacteria. The Indole Spot Reagent (DMACA) is suitable for anaerobe use.

Since peptones have been shown to vary with regard to their suitability for use with indole testing, media selected for indole determination should be tested with known positive and negative organisms to insure suitability.

Media containing glucose should not be used for indole testing due to the formation of acid end products which have been shown to reduce indole production. Mueller Hinton Agar should also not be used for this test because tryptophan is destroyed during acid hydrolysis of casein.

Media containing dye, such as MacConkey and EMB, are unsuitable sources of inoculum due to possible carryover of dye and subsequent interference of indole color interpretation.

Indole-positive colonies have been reported to cause adjacent indole-negative colonies to appear false-positive due to diffusion of indole into the media. To avoid false-positives, select colonies of different morphologies that are separated by at least 5mm for indole testing. (6)

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, Tryptone Broth (Cat. no. R40), Peptone Broth (Cat. no. K151), needles, incubators, and incinerators, etc., as well as biochemical and serological reagents, are not provided.

QUALITY CONTROL

Test Organisms Reaction
Indole Spot
Escherichia coli
ATCC ® 25922
Positive; blue to blue-green color change
Pseudomonas aeruginosa
ATCC ® 27853
Negative; no color change or pink
Indole Kovacs
Escherichia coli
ATCC ® 25922
Positive; red color change
Pseudomonas aeruginosa
ATCC ® 27853
Negative; no color change or light yellow

User Quality Control

PHYSICAL APPEARANCE

Positive indole reaction

Showing positive indole reaction.
Filter paper was saturated with Indole Spot Reagent (Cat. no. Z65) and Escherichia coli (ATCC ® 25922) growth was applied subsequently. E. coli was incubated aerobically for 24 hours at 35ºC. on a TSA plate (Cat. no. G60).

Negative indole reaction

Showing negative indole reaction.
Filter paper was saturated with Indole Spot Reagent (Cat. no. Z65) and Pseudomonas aeruginosa (ATCC ® 27853) growth was applied subsequently. P. aeruginosa was incubated aerobically for 24 hours at 35ºC. on a TSA plate (Cat. no. G60).



Positive indole reaction

Showing positive indole reaction.
Escherichia coli (ATCC ® 25922) was incubated in Tryptone Broth under aerobic conditions for 24 hours at 35ºC. 5 drops of Indole Kovac's Reagent (Cat. no. Z67) was added directly to the broth and the tube was gently shaken. The top alcohol layer shows a positive reaction.

Negative indole reaction

Showing negative indole reaction.
Pseudomonas aeruginosa (ATCC ® 27853) was incubated in Tryptone Broth (Cat. no. R40) under aerobic conditions for 24 hours at 35ºC. 5 drops of Indole Kovac's Reagent (Cat. no. Z67) was added directly to the broth and the tube was gently shaken. The top alcohol layer shows a negative reaction.

REFERENCES

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

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

3. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

4. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology, J.B. Lippincott Company, Philadelphia, PA.

5. MacFaddin, J.F. Biochemical Tests for Identification of Medical Bacteria,, Lipincott Williams & Wilkins, Philadelphia, PA.

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

7. Centers for Medicare and Medicaid, Appendix C, Survey Procedures and Interpretive Guidelines for Laboratories and Laboratory Services. Subpart K - Quality System for Non-Waived Testing. 493;1200-1265. www.cms.hhs.gov/clia.


API is a registered trademark of bioMeriuex, France.
ATCC is a registered trademark of the American Type Culture Collection.

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