LANAGRAM™

Cat. no. Z49 LanaGram™ 15 tests/kit

INTENDED USE

Hardy Diagnostics LanaGram™ is used in confirming the gram stain reaction of gram-negative and gram-positive aerobic or facultatively anaerobic rods or coccobacilli. This test is not recommended for use with gram-positive cocci, microaerophilic or obligate anaerobic organisms (see "Limitations" below).

SUMMARY

Many organisms including Bacillus , Erysipelothrix , Lactobacillus and Listeria are classified in part on the basis of their positive reaction in gram stain. Occasionally, however, isolates are encountered in the clinical laboratory that erroneously appear to be gram-variable or gram-negative. (2) Some of these organisms will demonstrate a gram-positive stain with young cultures or exhibit some other distinguishing trait, such as spore formation which is compatible with Bacillus spp. With others, the erroneous staining reaction may lead to misidentification or the inability to generate a compatible biochemical profile.

Alternately, some organisms that are gram-negative may, at times, appear to be gram-positive, since they have been known to resist the alcohol-acetone decolorization step in the gram stain. Among these problem organisms are members of the Neisseriaceae family, which include Moraxella ( Branhamella ) catarrhalis , Neisseria spp., Kingella and Acinetobacter spp. Erroneous gram stain reactions represent one of the most frequent causes of misidentification, which can result in a delay of appropriate therapy. (6)

Additionally, older, less viable cultures of gram-positive organisms that are unable to repair their cell walls, or whose cell wall formation is compromised by beta-lactam antibiotics, may stain gram negative. Slow growing gram-positive organisms may also stain gram-negative when the cell wall becomes thinner and more permeable. (7)

Cerny described a method for distinguishing gram-negative bacteria from gram-positive. (5) Using L-alanine-p-nitroanilide (LANA) reagent, he found complete correlation between the presence of aminopeptidase and gram-negativity in aerobic and facultative organisms. The presence of cell wall aminopeptidase is detected by the hydrolysis of L-alanine-p-nitroanilide indicating a gram-negative reaction. In his comparison study of LANA and the 3% KOH method, Carlone, et al. yielded more correct gram stain determinations than with the 3% KOH method when tested against aerobic and facultative organisms. (4)

REAGENT FORMULA

LanaGram™ disks are impregnated with L-alanine-p-nitroanilide in TRIS buffer.

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, 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

For best results use fresh cultures less than 48 hours old.

Disks should appear white to light yellow prior to the addition of water.

1. Place 2-3 drops of distilled or deionized water (pH 6.8+/- 0.2) in the small test tube containing the LanaGram™ disk.

2. Add sufficient organism from 2-3 well isolated identical colonies to produce a milky suspension.

3. Incubate at 35 +/- 1.0ºC. for 5-20 minutes.

INTERPRETATION OF RESULTS

The development of a pale yellow to bright yellow color is a positive test for aminopeptidase and evidence of a gram-negative organism. No color change indicates a negative aminopeptidase reaction and that the organism is gram-positive. Negative test reactions (no color change) should be held for the full 20 minutes. See "Limitations" section below.

LIMITATIONS

LanaGram™ will not give predictable reactions for some gram-positive cocci, microaerophilic or obligate anaerobic organisms. Such genera as Campylobacter , Bacteroides and Streptococcus are likely to produce false results.

Do not perform LanaGram™ test on colonies grown on media containing large amounts of dye such as EMB or MacConkey Agar. Dye carry-over may mask the yellow color change, resulting in false-negative reactions.

Tests performed on yellow pigmented colonies may appear positive immediately, but this is due to the bacterial pigmentation and should not be recorded as positive. In this case, a saline control (with no disk) should be inoculated and the test should be observed for a deepening of the yellow color to indicate a positive reaction.

Gardnerella vaginalis may require up to 60 minutes to yield a positive aminopeptidase reaction. An extended incubation of the test may be required for bacteria isolated from vaginal specimens.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, culture media, sterile swabs, incinerators, incubators, and distilled water with a neutral pH, etc., are not provided.

QUALITY CONTROL

Test Organisms Reaction
Escherichia coli
ATCC ® 25922
Positive aminopeptidase reaction, yellow color change observed; gram-negative organism.
Bacillus subtilis
ATCC ® 6633
Negative aminopeptidase reaction, no color change to yellow observed; gram-positive organism.

User Quality Control

PHYSICAL APPEARANCE

LanaGram™ disks should appear white to light yellow in color.

Positive Reaction

Showing positive aminopeptidase reaction.
Escherichia coli (ATCC ® 25922) was suspended in 3 drops of deionized water with a LanaGram™ Disk (Cat. no. Z49) and incubated aerobically for 5 minutes at 35ºC. The yellow color change was indicative of a gram-negative organism.

Negative Reaction

Showing negative aminopeptidase reaction.
Bacillus subtilis (ATCC ® 6633) was suspended in 3 drops of deionized water with a LanaGram™ Disk (Cat. no. Z49) and incubated aerobically for 5 minutes at 35ºC. No color change was indicative of a gram-positive organism.

REFERENCES

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. Versalovic, J., 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. Carlone, G.M., M.J. Valdez and M.J. Pickett. 1983. Method for distinguishing gram-positive from gram-negative bacteria. J. Clin. Microbiol.; 16:1157-1159.

5. Cerny, G. 1974. Method for the distinction of gram-negative from gram-positive bacteria. Eur. J. Appl. Microbiol.; 3:223-225.

6. Cowan, S.T. and J. Liston. 1974. The mechanism of identification. In Bergey's Manual of Determinative Bacteriology, 8th ed. Williams & Wilkins, Baltimore, MD; p. 10-13.

7. Donnelly, J.P. 1996. The Secrets of Gram Stain, Infect. Dis. Alert.; p. 109-112.


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