Cat. no. Z7081 ALA Differentiation Disks 50 disks/cartridge


HardyDisk™ ALA (delta-aminolevulinic) Differentiation Disks rapidly detect the presence of porphyrin and cytochrome compounds and are used to differentiate Haemophilus species, including Aggregatibacter aphrophilus.


Traditionally Haemophilus species have been differentiated by their varying requirements for hemin (X-Factor), NAD (nicotinamide adenine dinucleotide, V-Factor), and a combination of hemin and NAD (XV-Factor). However, erroneous results have been demonstrated when using growth factor requirement tests. These misidentifications are largely attributed to the carryover of X-Factor in the inoculum as well as the presence of trace amounts of X-Factor in the medium. (5) The use of HardyDisk™ ALA Differentiation Disk is used as an alternative method to X-Factor requirement testing. The ALA procedure is a more rapid test method as well as a more accurate method for determining hemin requirements by eliminating the erroneous results associated with X-Factor requirement tests.(8)

HardyDisk™ ALA Differentiation Disk assesses the ability of a Haemophilus strain to synthesize hemin from the ALA substrate. The test detects the presence of porphyrin compounds, which are intermediates in the hemin biosynthetic pathway.(4,5) Porphyrins are detected by the emission of a red-orange fluorescence under UV light (366nm) and indicate that the organism is capable of synthesizing hemin and is not dependent on hemin (X-Factor) for growth. Conversely, hemin requiring strains lack the enzymes to synthesize hemin and do not produce intermediate porphyrin compounds.


Each HardyDisk™ ALA Differentiation Disk is prepared by impregnating carefully controlled concentrations of delta-aminolevulinic acid onto a high quality 6mm diameter filter paper disk.


Storage: Upon receipt store at -20 to +8ºC. away from direct light. Disks should not be used if there are any signs of discoloration or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.



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

Method of Use:

1. Perform HardyDisk™ ALA Differentiation Disks only on isolates either growing on Chocolate Agar in 18-24 hours or that satellite around Staphylococcus aureus on a blood agar plate. See "Precautions" section above.

2. Prior to use, allow the disks to equilibrate to room temperature.

3. Aseptically place the disk into a sterile petri plate. Wet the disk with a small drop (0.04ml) of sterile saline (Cat. no. R45).

4. Inoculate disk with several well isolated 18-24 hour colonies to yield a visible cell paste on the disk surface. Alternatively, the disk can be touched to a colony and then placed in a petri dish.

5. Moisten a piece of filter paper with water and place it into the lid of the petri plate to ensure that the disk is kept moist during incubation.

6. Incubate the disks aerobically at 35ºC. for up to 2 hours.

7. After 2 hours, examine the disk under ultra-violet light (366nm) in a darkened room. Observe the disk for the presence of a red fluorescence while under UV light.


A positive reaction is recorded when orange/red fluorescence is observed on the HardyDisk™ ALA Differentiation Disk. This color change is a positive result for porphyrin synthesis and indicates that the organism does not require hemin (X-Factor) for growth.

A negative result is recorded when no fluorescence is observed on the disk and indicates that porphyrin was not synthesized. Consequently, the organism requires hemin for growth.

Haemophilus species Growth Factor Requirements Porphyrin Synthesis
Haemophilus ducreyi + - -
Haemophilus haemolyticus + + -
Haemophilus influenzae + + -
Haemophilus parahaemolyticus - + +
Haemophilus parainfluenzae - + +
Aggregatibacter aphrophilus (formerly H. aphrophilus and H. paraphrophilus ) - V +


In order to avoid inaccurate results it is recommended that organisms be less than 24 hours old, a heavy inoculum is used to inoculate the disk, and that the disk is kept moist during incubation.(4)

H. duceryi may not be identified using this procedure as some strains do not grow as satellite colonies on blood agar and grow more slowly, on the magnitude of several days, on enriched media.

Because similarities exist in growth factor requirements of Haemophilus species, it is not recommended that this procedure be the sole criterion for species identification.(4,5) Consult listed references for additional information regarding the recommended tests for complete identification of Haemophilus species.(2-5)

The ALA test, even in conjunction with the satellite test, does not differentiate H. influenzae and H. haemolyticus. H. haemolyticus is not considered pathogenic and is separated from H. influenzae by a positive hemolysis reaction on rabbit or horse blood agar.

Francisella spp., including Francisella tularensis, is ALA negative and grows only on chocolate agar. It can be differentiated from H. influenzae as it grows more slowly, does not satellite on blood agar, and is not V-Factor dependent.


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


Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Aggregatibacter aphrophilus (formerly H. parainfluenzae)
ATCC® 7901
* 2hr 35°C Aerobic Positive for porphyrin synthesis; orange/red fluorescence in the presence of UV light
Haemophilus influenzae
ATCC® 10211
* 2hr 35°C Aerobic Negative for porphyrin synthesis; no red fluorescence in the presence of UV light

User Quality Control


HardyDisk™ ALA Differentiation Disks are 6mm (in diameter) filter paper disks with the letters ALA printed on both sides, and should appear white in color.

ALA Differentiation Disks

Showing positive (left disk) and negative (right disk) HardyDisk™ ALA Differentiation Disks (Cat. no. Z7081) under UV light.
Disks were moistened with a drop of sterile saline and growth from 24 hour cultures was applied to the disks with a sterile loop. The disks were incubated aerobically in a petri dish for two hours. A sterile piece of filter paper soaked with deionized water was placed in the dish to maintain proper humidity.
LEFT DISK: Haemophilus parainfluenzae (ATCC® 7901) growth applied to ALA Disk. The orange/red color under UV light was indicative as positive.
RIGHT DISK: Haemophilus influenzae (ATCC® 10211) growth applied to ALA Disk. No orange/red color under UV light was indicative as negative.


1. 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.

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

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

4. Howard, B.J., et al. 1987. Clinical and Pathogenic Microbiology , 2nd ed. C.V. Mosby Company, St. Louis, MO.

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

6. Parker, R.H. and P.D. Hoeprich. 1962. Am. J. Clin. Path.; 37:319-327.

7. Wong, J.D. 1987. Porphyrin test as an alternative to benzidine test for detecting cytochromes in catalase-negative, gram-positive cocci. J. Clin. Micriobiol.; 25:2006-2007.

8. Lund M.S. and D.J. Blazevic. 1977. Rapid speciation of Haemophilus with the porphyrin production test vs. the satellite test for X. J. Clin. Microbiol.; 5:142-144.

ATCC is a registered trademark of the American Type Culture Collection.