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ID-
HIPPURATE
INTENDED
USE
The
Gibson ID- Hippurate kit is intended for use as a
rapid colorimetric test for the detection of hippurate hydrolysis
by microbial enzymes.
SUMMARY
AND EXPLANATION
The
differentiation of microorganisms on the basis of hippurate
hydrolysis is applicable to a variety of species. Streptococcus
agalactiae (group B) can be difterentiated from other
beta-h emolytic streptococci on the basis of this test.
Hydrolysis of hippurate is also a distinguishing characteristic
of Gardnerella vaginalis. Also, Campylobacterjejuni
can be separated from other campylobacter species by
this test.
PRINCIPLES
Glycine
and sodium benzoate are produced upon the hydrolysis of
sodium hippurate by the enzyme hippuricase. Ninhydrin reagent
is used to detect the glycine end product. The formation
of a purple color is positive for glycine.
MATERIALS
PROVIDED
Each
Gibson Rapid Hippurate kit contains the following reagents
sufficient for 10 tests:
10-Test
Vials with paper disc
Formula:
Sodium Hippurate 150 gms
Phosphate
Buffer 1000 mIs
1- 3m1
bottle Ninhydrin Reagent
Formula:
Ninhydrin 35gms
Butanol
500mls
Acetone
500mls
1-3m1
bottle Deionized Water
PRECAUTIONS:
This test is intended for use by those trained in appropriate
laboratory and bacteriological procedures. This test is
for IN VITRO DIAGNOSTIC USE only. Precautions should be
taken against the dangers of microbiological hazards. Specimens,
containers, media, and test vials should be sterilized after
use. Reagents should not come into contact with skin, eyes,
or clothing. Do not inhale or ingest reagents. In case of
an accident, seek medical attention immediately.
STORAGE
INSTRUCTIONS: The ID- Hippurate kit must be stored at
2-8C to prevent breakdown of the substrates and reagents.
Avoid exposing other cups to fluctuations in temperature.
Do not use this product if the expiration date has passed.
EVIDENCE
OF DETERIORATION: Materials provided should not be used
if the expiration date has passed. If any deficiencies are
noted with this product notify the manufacturer.
SPECIMEN
COLLECTION
Information
on specimen collection and procedures for isolation and
purification can be found in standard reference texts.
PROCEDURES
(1)
MATERIALS
REQUIRED BUT NOT PROVIDED: The standard clinical microbiological
equipment such as loop, burner, deionized water and incubator
are needed for procedures involving the use of this product.
Other materials required include the following: American
Type Culture Collection (ATCC) strains- Streptococcus
agalactiae (13813) and Streptococcus pyogenes (19615).
Use
one vial for each specimen tested.
1.
Remove cap from test vial.
2.
Add 6 drops of deionized water to each vial.
3.
Select 4-5 well isolated, morphologically similar colonies
and inoculate the test vial.
4.
Mix until a cloudy suspension of the test organism is
produced.
5.
Replace cap and incubate at 37C for 2 hours.
6.
Remove cap and add 6 drops of the ninhydrin reagent. Do
not shake vial.
7.
Replace cap and re-incubate test cup at 37C for 10 minutes.
Do not incubate longer than 1/2 hour: false positives
may occur.
INTERPRETATION
USER
QUALITY CONTROL
Daily
quality control should be performed in accordance with proper
laboratory procedures using
organisms
that will produce known positive and negative reactions.
Follow steps under
PROCEDURE
section. If quality control results are false clinical results
should not be recorded.
Notify
the manufacturer if proper results are not obtained. The
following American Type Culture
Collection
strains are recommended:
Streptococcus
agalactiae ATCC 13813 -- Purple color change (Positive)
Streptococcus
pyogenes ATCC 19615 -- No color change (Negative)
LIMITATIONS
OF PROCEDURE
Avoid
exposing unused vials to fluctuations in temperature. Occasional
strains of C. jejuni may be hippurate negative.(2-3) Nearly
all (99%) group B streptococci, whether they are beta-hemolytic
or non-hemolytic, hydrolyze hippurate and therefor react
positively.(4) Group D streptococci may also react positively
but are distinguished by the Bile Esculin test. Beta hemolytic
streptococci other than group B do not hydrolyze hippurate.
(5)
REFERENCES
1.
Facklam, R. Streptococci and Aerococci. In: Len
nette, Balows, Hausler, and Truant, Manual of Clinical
Microbiology, 3rd ed., ASM, Washington, D.C. 1980.
2.
Morris, G.K., M.R. El Sherbeeny, C.M. Patton, H. Kodaka,
G.L. Lombard, P. Edmonds, D.G. Hollis, and D.J. Brenner.
1985. Comparison of four hippurate hydrolysis methods
for identification of thermophilic Campylobacter spp.
J. Clin. Microbiol. 22:714-718.
3.
Totten, P.A., C.M. Patton, F.C. Tenover, T.J. Barrett,
W.E. Stamm, A.G. Steigerwalt, J.Y. Lin, K.K. Holmes, and
D.J. Brenner. 1987. Prevalence and characterization of
hippuratenegative Campylobacter jejuni in King County,
Washington. J. Clin. Micrbiol. 25:1747-1752.
4.
Hwang, M.N., and G.M. Ederer, 1975. J. Clin. Microbiol.
1:114-115.
5.
Lennette, Balows, Hausler, Shadomy. 1985. Manual of clinical
microbiology. ASM
Catalog
# 60040
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