Niacin test: negative - left and positive - right.
Commercial niacin detection kit
(Microxpress Niacin Drop Test)
Niacin accumulation test
Niacin (nicotinic acid) is produced by all mycobacteria but some of them lack the
enzymes to convert it further into nicotinamide adenine dinuceotide (NAD). Thus,
niacin accumulates in the culture medium  from which it can be extracted with sterile
water or physiologic saline.
M. tuberculosis  accumulates the most, but other species
may also return positive results:
M. africanum, M. microti, M. simiae and some strains
M. chelonae, M. marinum, and M. ulcerans.

In the presence of citric acid, chloramine-T and potassium thiocyanate will react to
form cyanogen chloride. This compound will break apart the pyridine ring of niacin to
produce gamma-carboxy glutaconic aldehyde and joins an aromatic amine to form a
yellow color.

NIACIN REAGENT COMPOSITION: potassium thiocyanate 30.0 g,
chloramine-T 25.0 g, p-aminosalicylic acid 5.0 g, citric acid trihidrate 4.0 g,
ethyl alcohol 50.0 ml, demineralized water 150 ml.

  • Puncture the culture slant with a loop or with a Pasteur pipette. Niacin
    production is extracellular and accumulates in the medium so, you have to
    break the cell mass so that the medium is exposed.
  • Add 1 ml of sterile distilled water in the culture tube.
  • Place the tube horizontally so the fluid covers the entire surface of the medium.
  • Leave the tube at room temperature for 30 minutes then turn the slants upright
    for 5 minutes to let fluid to drain to the bottom.
  • Transfer 0.5 ml  of fluid in a new empty screw cap test tube then add the
    reagents or insert the impregnated strip (depending on the kit you are using).
    Some kits require centrifugation (3000 rpm for 15 min.) of the fluid and use of
    the supernatant
  • Leave at room temperature for 15-20 minutes, agitating the tube occasionally.
  • Observe the colour of the liquid in the bottom of the tube against a white
    background. Positive result: a yellow color appears. Negative result: no color.

Positive control:
Mycobacterium tuberculosis ATCC 25177,
Negative control:
Mycobacterium fortuitum ATCC 6841.

- cultures less than 3-4 weeks old or in small quantities may contain less amount of
niacin therefore may produce weak or negative reaction.
- niacin-negative M. tuberculosis strains may be found in patients who have
received long-term chemotherapy
1. Young, W.D.; Maslansky, Alvin; Lefar, Lefar; Kronish, Donald (December 1970). "Development of a paper strip test for detection of
niacin produced by mycobacteria". Applied Microbiology. 20 (6): 939–945.
2. Niacin Reagent Strip. Remel leaflet.
3. Niacin Drop Test
. Biochemical Test for Identification of Mycobacterium tuberculosis. Microxpress a division of Tulip Diagnostics (P)
LTD. product leaflet.

4. Bhalla, G. S., Sarao, M. S., Kalra, D., Bandyopadhyay, K., & John, A. R. (2018). Methods of phenotypic identification of non-tuberculous
mycobacteria. Practical Laboratory Medicine, 12, e00107.

5. Costin Stoica.
Mycobacterium tuberculosis in Regnum Prokaryotae, accessed 02 Feb 2021.
Culture media
Biochemical tests
(c) Costin Stoica
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