API 20 kit | Microorganism | Microbiology

January 20, 2016 | Author: Anonymous | Category: Documents
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Analytical Profile Index. The API tests (kit) can identify a wide range of microorganisms. The API's comprise plastic st...

Description

Analytical Profile Index The API tests (kit) can identify a wide range of microorganisms. The API's comprise plastic strips that generally contain 20 miniature tubes. Almost all bacterial groups and over 550 different species can be identify used these API's tests. The identification is quite easy way and the API's tests give accurate identification results. The identification system have extensive databases of characteristic biochemical reactions of micro-organisms and are standardized.

(photo: www.biomerieux-usa.com) There are commercial products for bacteria identification: Product

Characterisation of test

API 20E

Species and subspecies identification of Enterobacteriacae and groups and species identification of non-fermenting

API 20NE

Identification of gram-negative non-Enterobacteriaceae

API Rapid 20E

Identification of Enterobacteriaceae

API NH

Identification of Branhamella catarrhalis and Neisseria haemophillus

RAPIDEC Staph

Identification of the commonly occurring staphylococci

API 20 Strep

Identification of streptococci and enterococcs

API Staph

Identification of clinical staphylococci and micrococci

API Coryne

Identification of Corynebacteria and coryne-like organisms

API 20A

Identification of anaerobes

and others

As per manufacturer's directions, the plastic sterile strip (API test) is inoculated with isolated a pure culture of microorganism suspension. This process also rehydrates the dessicated medium

in the miniature tubes. And some probes (tubes) are overlaid with sterile mineral oil (anaerobic reactions - ADH, LDC, ODC, H2S, URE (see Table B) The results are read after incubation (24 hours or less - depending which API test is used) in a humidity chamber, the color reactions are read (some of the tubes will have color changes due to pH differences and some with the aid of added reagents to detect end metabolic products), and the reactions (plus the oxidase reaction done separately) are converted to a seven-digit code (see Table B). The code is into the manufacturer's database (codebook), which gives the identification, usually as genus and species of microorganisms. There are possibilities to get the information about the identification of microorganism from API test producer via touch-tone telephone system.

Learn how to perform and interpret the miniaturized, multi-test technique for bacterial identification (by: Jackie Reynolds, Richland College, some documentations from BioMerieux and some modification by autors of webside)

MATERIALS NEEDED: - agar plates of bacterial species - 0.85% NaCl solutions (5ml) - sterile Pasteur pipettes + bulbs - sterile mineral oil - API 20E test strip (for oxidase - gram negative rods) - API test strip incubation chamber AFTER INCUBATION: 10% FeCl3, Barrett's reagents A and B, Kovac's reagent PROCEDURE: Step 1. Prepare a suspension of the bacteria in the saline tube 1. Inoculate a large colony (2-3mm diameter) of the bacterium (pure culture) into the 0.85% NaCl solution, making sure that the suspension is homogenous and without clumps of floating bacteria. 2. Use a McFarland barium sulfate standard #3 to quantitate the suspension. Step 2. Inoculate the API strip 1. Holding the strip at a slight angle up from the table top, you will now inoculate the bacterial suspension into each well with the sterile pipette (see Fig. A). 2. Touch the end of the pipette to the side of the cupule, allowing capillary action to draw the fluid into the well as you slowly squeeze the bulb. This should eliminate any bubbles

forming in the wells. Each well should be filled up to the neck (see Fig. B). 3. CIT, VP, and GEL have boxes around their names. These test wells will be filled all the way up to the top of the well. 4. LDC, ODC, ADH, H2S, and URE are filled as described in point 2 (Step 2), but they will then be filled up to the top with sterile mineral oil.

Figure A

Figure B

Step 3. Incubate the strip in its chamber 1. The bottom of the incubation chamber has small indented wells in the bottom: fill it with water just enough to fill these indentations. 2. Place the strip into this bottom. There should not be so much water that it slops onto the API strip. 3. Place the top of the incubation chamber over the bottom, and label it. 4. Place the strip at 37° C for 18-24 hours. INTERPRETATION: 1. Add the proper reagents to the compartments: o 1 drop of Kovac's to the IND (read within a couple of minutes) o 1 drop of Barritt's A and B to VP (a + reaction may take up to 10 minutes) o 1 drop of FeCl3 to TDA 2. Read all other tests as described without reagents (see point 3). 3. Record results on the diagram handed out to you in lab (see Fig. C and Photo A, Table A.) 1, 2, or 4 points for + *positive* reaction for first, second and third tube in the each triad, 0 points for - *negative* reaction for first, second and third tube in the each triad). !The oxidase test reaction should be negative, and is added as the last test result! 4. Three test reactions are added together at a time to give a 7-digit number, which can then be looked up in the codebook.

Figure C

Photo 1 Table A (for sample, not according to real Photo A) Triad

I

Tube

II

III

IV

V

VI

VII

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 oxidase

Reaction

+ + + - - - + + - +

-

-

-

+ +

-

-

+ +

-

+

Point

1 2 4 0 0 0 1 2 0 1

0

0

0

2

0

0

4

0

4

Add

7

0

3

1

7-digital Code

4

6

4

1

5

7031645

READING THE API 20 Table B. TESTS SUBSTRATE REACTION TESTED

- RESULTS *negative*

+ RESULTS *positive*

ONPG

ONPG

beta-galactosidase

colorless

yellow

ADH

arginine

arginine dihydrolase

yellow

red/orange

LDC

lysine

lysine decarboxylase

yellow

red/orange

ODC

ornithine

ornithine decarboxylase

yellow

red/orange

CIT

citrate

citrate utilization

pale green/yellow

blue-green/blue

H2S

Na thiosulfate

H2S production

colorless/gray

black deposit

URE

urea

urea hydrolysis

yellow

red/orange

TDA

tryptophan

deaminase

yellow

brown-red

IND

tryptophan

indole production

yellow

red (2 min.)

VP

Na pyruvate

acetoin production

colorless

pink/red (10 min.)

GEL

charcoal gelatin

gelatinase

no diffusion of black

black diffuse

GLU

glucose

fermentation/oxidation

blue/blue-green

yellow

MAN

mannitol

fermentation/oxidation

blue/blue-green

yellow

INO

inositol

fermentation/oxidation

blue/blue-green

yellow

SOR

sorbitol

fermentation/oxidation

blue/blue-green

yellow

RHA

rhamnose

fermentation/oxidation

blue/blue-green

yellow

SAC

sucrose

fermentation/oxidation

blue/blue-green

yellow

MEL

melibiose

fermentation/oxidation

blue/blue-green

yellow

AMY

amygdalin

fermentation/oxidation

blue/blue-green

yellow

ARA

arabinose

fermentation/oxidation

blue/blue-green

yellow

OX

oxidase

oxidase

colorless/yellow

violet

Characteristic reactions of some common bacterial species used API E20 test TESTS

Escherichia coli

Klebsiella pneumoniae

Proteus vulgaris

Salmonella sp.

ONPG

+

+

-

-

ADH

-

-

-

-

LDC

+

+

-

+

ODC

+

-

-

+

CIT

-

+

-

-

H2S

-

-

+

+

URE

-

+

+

-

TDA

-

-

+

-

IND

+

-

+

-

VP

-

+

-

-

GEL

-

-

+

-

GLU

+

+

+

+

MAN

+

+

-

+

INO

-

+

-

-

SOR

+

+

-

+

RHA

+

+

-

+

SAC

+

+

+

-

MEL

+

+

-

+

AMY

-

+

+

-

ARA

+

+

-

+

Links: More information about API's tests and micro-organisms identification: - www.biomerieux-usa.com - www.jlindquist.net - www.web.indstate.edu - www.rlc.dcccd.edu - www.som.soton.ac.uk References 1. Alderson, G., Amadi, E.N., Pulverer, G. and Zai, S. (1991) Recent advances in the classification and identification of the genus Micrococcus. In: Jeljaszewicz/Ciborowski, (Ed.) The Staphylococci, Zentralblatt für Bakteriologie Supplement 21, pp. 103-109. Stuggart: Gustav Fisher Verlag. 2. Alexander, B. and Priest, F.G. (1990) Numerical classification and identification of Bacillus sphaericus including some strains pathogenic for mosquito larvae. Journal of General Microbiology 136, 367-376. 3. Carson, J., Wagner,T., Wilson,T. and Donachie, L. (2001) Miniaturized tests for computer-assisted identification of motile Aeromonas species with an improved probability matrix. Journal of Applied Microbiology 90, 190-200. 4. Clayton, P., Feltham, R.K.A., Mitchell, C.J. and Sneath, P.H.A. (1986) Constructing a database for low cost identification Gram negative rods in clinical laboratories. Journal of Clinical Pathology 39, 798-802. 5. Cox, R.P. and Thomsen, J.K. (1990) Computer-aided identification of lactic acid bacteria using the API 50 CHL system. Letters in Applied Microbiology 10, 257-259. 6. Feltham, R.K.A., Wood, P.A. and Sneath, P.H.A. (1984) A general-purpose system for

6. 7. 8. 9.

characterizing medically important bacteria to genus level. Journal of Applied Bacteriology 57, 279-290. Geary, C., Stevens, M., Sneath, P.H.A. and Mitchell, C.J. (1989) Construction of a database to identify Staphylococcus species. Journal of Clinical Pathology 42, 289-294. Kämpfer, P. and Kroppenstedt, R.M. (1991) Probabilistic identification of streptomyces using miniaturized physiological tests. Journal of General Microbiology 137, 1893-1902. Langham, C.D., Williams, S.T., Sneath, P.H.A. and Mortimer, A.M. (1989) New probability matrices for identification of Streptomyces. Journal of General Microbiology 135, 121-133. Williams, S.T., Locci, R., Vickers, J.C., Schofield, G.M., Sneath, P.H.A. and Mortimer, A.M. (1985) Probabilistic identification of Streptoverticillium species. Journal of General Microbiology 131, 1681-1689.

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