Indoxyl Acetate Disk Test: Introduction, Principle, Test Requirements, Procedure, Result-Interpretation, Limitations, and Keynotes

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Introduction of Indoxyl Acetate Disk Test 

Indoxyl is a tryptophan product of putrefactive decomposition in the intestines of humans by a bacterial esterase. This Indoxyl Acetate Disk test uses to distinguish Campylobacter species. Positive tests are Campylobacter jejuni and Campylobacter coli whereas Campylobacter lari and Campylobacter fetus are negative.

Campylobacter fetus Colony Morphology on Chocolate agar
Fig. Campylobacter fetus Colony Morphology on Chocolate agar
Campylobacter fetus in Gram staining of culture showing poor stain
Fig. Campylobacter fetus in Gram staining of culture showing poor stain
Campylobacter fetus in Basic Fuchsin staining of culture
Fig. Campylobacter fetus in Basic Fuchsin staining of culture

Principle of Indoxyl Acetate Disk Assay

The presence of esterase enzyme present in organisms can be confirmed in vitro by the bacterial hydrolysis of indoxyl acetate to release indoxyl. Indoxyl then combines with oxygen to spontaneously form indigo and the Indoxyl Acetate Disk test becomes blue i.e. positive

Requirements for the Test 

  • Indoxyl acetate Disk
  • Test organisms

Oxidase-positive, motile, curved, gram-negative  bacilli or  rods suggestive of Campylobacter species and related organisms (Helicobacter and Arcobacter)

Gram-negative, oxidase-positive diplococci grow on blood agar as white colonies that remain together when lifted with an inoculating loop or needle.

  • Sterile wooden sticks or inoculating loop
  • Slide or Petri dish
  • Sterile distilled water or  normal saline
  • Control strains
  •  Positive Control (PC): C. jejuni ATCC 33560 or Moraxella catarrhalis ATCC 25240
  • Negative Control (NC) Campylobacter hyointestinalis ATCC35217or C.fetus ATCC27374

Procedure of Indoxyl Acetate Disk Assay

  1. Put the indoxyl acetate disk in a plastic petri dish or on a slide and transfer a large loopful of 24- 48 hours of growth from the plate onto the disk.
  2.  Add a drop of sterile distilled water or deionized water to the disk for Campylobacter. For Moraxella, premoisten the disk with saline prior to inoculation.
  3. Incubate the disks at room temperature for up to 30 minutes for Campylobacter and up to 3 minutes for Moraxella.

Result and Interpretation

Test positive: Development of blue color on disk in 3 minutes for Moraxella and 5 to 10 minutes for Campylobacter.

Negative test: No color change.

Weakly positive reactions:  Formation of pale blue in 10 to 30 minutes

C. jejuni ATCC 33560 or Moraxella catarrhalis ATCC 25240 : Test positive

 Campylobacter hyointestinalis ATCC35217or C.fetus ATCC27374: Test Negative

Indoxyl Acetate Disk Test-Positive and Negative Results
Fig. Indoxyl Acetate Disk Test-Positive and Negative Results

Keynotes

  1. Indoxyl acetate-positive organisms are  C. jejuni, C. coli, Campylobacter upsaliensis, Arcobacter cryaerophilus, and Helicobacter fennelliae.
  2. Indoxyl acetate-negative organisms are C. lari, Helicobacter pylori, Helicobacter cinaedi, and Helicobacter pullorum.
  3. Report as Moraxella catarrhalis if oxidase-positive, gram-negative diplococci grow on blood agar as colonies that remain together when sampled and are strongly indoxyl acetate positive in 3 minutes.

Limitations of the Indoxyl Acetate Disk test 

  • The disk test is more rapid and reliable than the tube method.
  • The test is reliable regardless of the media used for growth.

Bibliography 

  1. Cowan and Steel’s, manual for the identification of medical bacteria
  2. Lynne S. Garcia, Clinical Microbiology Procedures Handbook
  3. https://catalog.hardydiagnostics.com/cp_prod/content/hugo/IndoxylAcetateDisks.html
  4. https://www.fishersci.ca/shop/products/remel-indoxyl-acetate-disc/r21087

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