HAEMOPHILUS ID QUADPLATE

Cat. no. J82 Haemophilus ID Quadplate, 15x100mm Quadplate,
5ml/quadrant
10 plates/bag

INTENDED USE

The Hardy Diagnostics Haemophilus ID Quadplate is recommended for the differentiation of Haemophilus species.

SUMMARY

Members of the genus Haemophilus are differentiated by requirements for X- and V-factors and hemolytic reactions on either rabbit blood, horse blood or RTF Medium with sheep blood. RTF Medium allows Haemophilus to grow and show appropriate hemolytic patterns with Sheep Blood. The Haemophilus ID Quadplate consists of four separate formulations, which supply the requirements as follows:

RTF Casman Medium, Modified X- and V-factors
Tryptic Soy Agar (TSA) with Hemin X-factor
Tryptic Soy Agar (TSA), Modified with NAD V-factor
Chocolate Agar X- and V-factors

FORMULA

Viewing from the top of the plate, in clockwise order beginning with the red quadrant, the media formulas per liter of deionized water are as follows:*

Quadrant I:
RTF Casman Medium, Modified 43.0gm
Sheep Blood 50.0ml
Quadrant II:
Tryptic Soy Agar (TSA) 40.0gm
Hemin (X-factor) 0.02gm
Quadrant III:
Tryptic Soy Agar (TSA), Modified 40.0gm
NAD (V-factor) 0.10gm
Quadrant IV:
GC Agar Base 36.0gm
Hemoglobin 10.0gm
Koenzyme (X- and V-factors) 10.0ml

Final pH 7.2 +/- 0.2 at 25ºC.

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

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 (shrinking, cracking, or 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

Specimen Collection: Infectious material should be submitted directly to the laboratory without delay and protected from excessive heat and cold. If there is to be a delay in processing, the specimen should be inoculated onto an appropriate transport media and refrigerated until inoculation.

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 organisms.

Method of Use: Prior to inoculation, the medium should be brought to room temperature. Select one or two well isolated colonies that resemble Haemophilus species both by gram stain and morphology and dilute in 5ml of sterile Tryptic Soy Broth (Cat. no. R30) or sterile Saline, 0.85% (Cat. no. K59). Vortex to mix. Do not inoculate directly from the colony to the quadplate. Due to the possibility of carryover of growth factors, do not cool the inoculating loop in the primary isolation medium before selecting colonies. Do flame the loop between inoculation of each quadrant. Be careful not to pick up any culture media on the loop when picking colonies for broth inoculation. Incubate the plate in 5-10% CO 2 at 35-37ºC. for 18-24 hours. Examine plates for growth, no growth and hemolytic reactions.

INTERPRETATION OF RESULTS

Quadrants are identified by starting with the red quadrant and proceeding in clockwise order.

Disregard very light growth in quadrants containing TSA with Hemin (quadrant 2) and TSA, Modified with NAD (quadrant 3) compared to the growth of the RTF Casman, Modified (quadrant 1) and Chocolate Agar (quadrant 4).

Growth in quadrants 2 and 3 must be as great as quadrant 4 in order to be called positive; i.e., slight growth is interpreted as a negative.

Organisms RTF Casman, Modified
(Quadrant 1)
X-Factor
TSA with Hemin
(Quadrant 2)

V-Factor
TSA, Modified with NAD
(Quadrant 3)

X- and V-Factors
Chocolate Agar
(Quadrant 4)

Growth Hemolysis
H. influenzae + - - - +
H. haemolyticus + + - - +
H. parainfluenzae + - - + +
H. parahaemolyticus + + - + +
H. aphrophilus * + - V - +
H. paraphrophilus* + - - + +
H. aegyptius + - - - +
H. segnis * + - - + +

KEY: +  =  Growth or hemolysis            -   =  No growth            V   =   Variable

* H. aphrophilus and H. paraphrophilus were recently reclassified as Aggregatibacter aphrophilus and H. segnis was recently reclassified as Aggregatibacter segnis .

LIMITATIONS

Clinical specimens may contain more than one species of Haemophilus ; therefore, strict attention to colony morphology and hemolytic reaction is necessary when selecting colonies from primary isolation media.

Avoid excess inoculum. Refer to " Inoculation Procedures " on the Hardy Diagnostics website for a description of the proper inoculation method. Some strains of Haemophilus influenzae may appear to have no X-factor requirement due to traces of hemin compounds carried over from a heavy inoculum. Care should be taken during inoculation of specimens onto culture media in order to prevent nutrient carryover.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, other culture media, V-Factor Disk (Cat. no. Z7041), swabs, applicator sticks, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Haemophilus influenzae
ATCC ® 10211
B 24hr 35°C CO 2 ** RTF Casman, Modified:
Growth with no hemolysis
TSA with Hemin:*** Growth only around the V-Factor Disk
TSA , Modified with NAD: No growth
Chocolate Agar: Growth





Haemophilus parahaemolyticus
ATCC ® 10014
B 24hr 35°C CO 2 ** RTF Casman, Modified:
Growth with hemolysis
TSA with Hemin: No growth
TSA , Modified with NAD: Growth
Chocolate Agar: Growth





Note: When interpreting results, growth in quadrants 2 and 3 must be as great as quadrant 4 in order to be called positive; i.e., slight growth is interpreted as negative. Always prepare a suspension of the organisms before inoculation. When inoculating, the loop must be flamed between streaking of each quadrant.

** Atmosphere of incubation is enriched with 5-10% CO 2 .

*** For QC purposes, after streaking the test organism on the plate, a V-Factor Disk (Cat. no. Z7041) should be placed on the center of the quadrant.

User Quality Control

PHYSICAL APPEARANCE

H. influenzae growing on Haemophilus ID Quad Plate

Haemophilus ID Quad Plate (Cat. no. J82).
Showing growth of Haemophilus influenzae (ATCC ® 10211) colonies on RTF Casman, Modified (no hemolysis) and Chocolate Agar sectors. No growth on TSA with Hemin or TSA, Modified with NAD sectors. This growth pattern was indicative of an organism that requires both X-factor and V-factor for growth. This was consistant with H. influenzae . Incubated in CO 2 for 48 hours at 35ºC.

H. parahaemolyticus growing on Haemophilus ID Quad Plate

Haemophilus ID Quad Plate (Cat. no. J82).
Showing growth of Haemophilus parahaemolyticus (ATCC ® 10014) colonies on RTF Casman, Modified (with hemolysis); TSA, Modified with NAD and Chocolate Agar sectors. No growth on TSA with Hemin sector. This growth pattern was indicative of an organism that does not require X-factor for growth but does require V-factor. This was consistant with H. parahaemolyticus . Incubated in CO 2 for 48 hours at 35ºC.

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

5. Quality Assurance for Commercially Prepared Microbiological Culture Media, M22. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS), Wayne, PA.


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