Question

1. In the API 20E test each section represents which of the following? A.a test for a different enzyme within the same biochemical pathway B.a different biochemical test C.a selective media D.All of the above

2. The tests within the API 20E tubes may be performed under? A.aerobic conditions B.anaerobic conditions C.either aerobic or anaerobic conditions

3. How are the results for the API 20E interpreted? a)A reference identification code is produced from the number of positive reactions b)The positive tests are used in a dichotomous key to identify the organism

4. What are used in the Kirby-Bauer test to measure microbial antibiotic sensitivity? A.broths containing antibiotics B.discs impregnated with antibiotics C.McFarlands antibiotic D.all of the above

5. Antibiotic susceptibility is measured in terms of? A.the diameter of a zone of inhibition B.the circumference of a zone of inhibition C.the area of a zone of inhibition D.the inverse of the square of the zone of inhibition

Answer 1

1. b) a different biochemical test.

API 20E ( Analytical Profile Index ) is a biochemical panel for identification and differentiation of members of family Enterobactericeae. In API 20E, the plastic strip hold twenty mini test chambers containing dehydrated media having chemically defined compositions for each test. The kit include strips that contain 20 miniature biochemical tests. All are quick, safe and easy to perform. The test usually detect enzymatic activity, mostly related to Fermentation of carbohydrate or catabolism of proteins or amino acid by inoculated organism.

A bacterial suspension is used to rehydrate each of the well and the strips are incubated. During incubation metabolism produces color change that are either spontaneous or by adding reagents.

The API 20E test includes:

1. ONPG : test for beta galactosidase enzyme by hydrolysis of the substrate o-nitrophenyl-b-D- galactopyranoside.

2. ADH : decarboxylation of the amino acid arginine by arginine dihyrolase

3. LDC : decarboxylation of lysine by lysine decarboxylase.

4. ODC : decarboxylation of aminoacid ornithine by ornithine decarboxylase.

5. CIT : utilisation of citrate as only carbon source.

6. H2S : production of hydrogen sulphide.

7. URE : test for enzyme urease

8. TDA : detection of the enzyme tryptophan deaminase.

9. IND : indole test production from tryptophan by enzyme tryptophanase

10. VP : To detect acetoin

11.GEL : test for production of gelatinase which liquefies gelain

12.GLU : fermentation of glucose

13. MAN : fermentation of mannose

14. INO : fermentation of inositol

15. SOR : fermentation of sorbitol

16. RHA : fermentation of rhamnose

17. SAC : fermentation of sucrose

18. MEL : fermentation of melibiose

19. AMY : fermentation of amygdalin

20. ARA : fermentation of arabinose

2. A Aerobic condition.

The API 20E test is performed under aerobic condition. The LDC, ODC, ADH, H2S and URE are filled with sterile mineral oil following inoculation of bacterial suspension.

LDC, ODC, ADH are decarboxylation test, that requires anaerobic conditions for fermentation. So mineral oil is added to seal the medium from external oxygen.

URE is test for Urease detection and H2S for Hydrogen sulphide production, both require anaerobic condition for bacteria.

3. a) A reference identification code is produced from the number of positive reactions.

In API 20E test, in some chambers the colour change can be read straightly after incubation but for some reagents must be added to them before interpretation. After addition to reagents to chambers that require reagent to see the results, get the API reading Scale ( colour chart) by marking each test as postive or negative on the lid of the tray.

The wells are marked off into triplets by black triangles, for which score are given. Add up the scores for positive wells only in each triplet. Three test reactions are added together at time to give a7 digit number, which can then be looked up in the codebook. The highest score possible for a triplet is 7 and lowest is 0. Identify the organism using API catalog or apiweb.

4. B. Disc impregnated with antibiotics.

Kirby- Bauer test ( also called disk diffusion or agar diffusion test) is antibiotic sensitivity test for bacteria. It uses antibiotic disc to test whether particular bacteria are susceptible to specific antibodies.

In this test, the bacteria are grown overnight on agar plate in presence of a thin wafer ( disc) that contains a known amount of a relevant antibiotic. If the antibiotics inhibit the growth of bacteria, there will be an area around the disc where the bacterial growth is not visible. This area is called zone of inhibition. Larger zone of inhibition indicates that bacteria are more sensitive to antibiotics in disc.

Clinicians use Kirby-Bauer test to choose appropriate antibiotic for a particular infection in patients.

5. A. The diameter of a zone of inhibition.

In antibiotic susceptibility testing, the diameter of zone of inhibition is measured and compared with database of zone of standards to determine if the bacteria tested is susceptible, intermediate or resistant to the antibiotics.

​​​​​If the observed zone of inhibition is greater than or equal to the size of the standard zone, the microorganisms is considered to be sensitive. If the observed zone of inhibition is smaller than the standard size, the microorganisms is considered to be resistant. The size of zone of inhibition is inversely related to the minimum inhibitory concentration (MIC).