Analytical Profile Index (API) & Automated Machines

Analytical Profile Index (API) & Automated Machines. principle and procedure

1. Analytical Profile Index (API) & Automated machines Zari Esa M.Saleh MSc. Medical Microbiology

2. Analytical Profile Index (API) Analytical Profile Index or API are commercial miniaturized biochemical test panels that cover a significant number of clinically-important groups of bacteria, as well as food- and water-associated microorganisms. API is a classification of bacteria based on experiments, allowing fast identification (rapid identification). The system is developed for quick identification of clinically relevant bacteria. Only known bacteria can identified.

3. Analytical Profile Index (API) API test strips consists of microtubes (cupules) containing dehydrated substrates to detect the enzymatic activity or the fermentation of sugars by the inoculated organisms. During incubation, metabolism produces colour changes that are either spontaneous or revealed by the addition of reagents When the carbohydrates are fermented, the pH within the cupule changes and is shown by an indicator.

4. Analytical Profile Index (API) API Gram negative Identification API 20E. 18-24 hour identification of Enterobacteriacae and other non-fastidious gram negative bacteria. API Rapid 20E. 4-hour identification of Enterobacteriaceae . API 20NE. 24 to 48-hour identification of Gram negative non- Enterobacteriaceae . API NH. 2-hour identification of Neisseria Haemophilus and Branhamella catarrhalis . API Campy. 24-hour identification of Campylobacter species

5. Analytical Profile Index (API) API Gram positive Identification API Staph. Overnight identification of clinical staphylococci and micrococci. RAPIDEC Staph. 2-hour identification of the commonly occurring staphylococci. API 20 Strep. 4 or 24-hour identification of streptococci and enterococci. API Coryne . 24-hour identification of Corynebacteria and coryne -like organisms. API Listeria. 24-hour identification of all Listeria species

6. Analytical Profile Index (API) Other API System include: Anaerobe Identification API 20A. 24-hour identification of anaerobes. Rapid ID 32A. 4-hour identification of anaerobes API Yeast Identification API 20C AUX. 48 to 72-hour identification of yeasts. Others API 50 CH. Performance of carbohydrate metabolism tests. API ZYM. Semiquantitation of enzymatic activities

7. The API 20E Strip, For Identification Of The Enterobacteriaceae and other Gram negative rods, it consists of 20 microtubules containing dehydrated substrates. These tests are inoculated with a bacterial suspension that reconstitute the media. During incubation, the metabolism of the organism produces color changes that are either spontaneous or revealed by the addition of reagent. Analytical Profile Index (API)

8. Analytical Profile Index (API) Material Needed. API 20 E Strips Incubation boxes Report sheets Disposable Plastic pipettes Disposable plastic inoculating loop 5 ml sterile distilled water Mineral Oil MacConkey agar plate. Reagent needed. TDA reagent James reagent VPI reagent VP2 reagent

9. Preparation of the inoculum Open an ampule of API NaCl 0.85 % Medium (5 ml) or use any tube containing 5 ml of sterile saline or sterile distilled water, without additives. Use colony from an isolation plate. It is recommended to use young cultures (18-24 hours old). Carefully emulsify to achieve a homogeneous bacterial suspension. This suspension must be used immediately after preparation.

10. Dispense about 5 ml of tap water into the tray Vortex mix the saline suspension to get uniform dispersal Inoculate all the tubes on the test strip with the pipette by depositing the suspension into the cupules as you tilt the API tray Preparation of the inoculum

11. Create anaerobiosis in the tests ADH , LDC , ODC , H2S and URE . by overlaying with mineral oil. Since the media in |CIT| , |VP| , and |GEL | compartments require oxygen, completely fill both the cupule and tube of these compartments. Fill only the tube (and not the cupule) of the other tests. Inoculate and streak MacConkey purity plate. Close the incubation box. Incubate the box along with the MacConkey agar at 37°C for 18-24 hours . Preparation of the inoculum

12. Results and interpretations Evaluation of tests, all reactions will be recorded on the laboratory report and test reagents will be added to some compartments. The seven digit profile number will be determined so the unknown organism can be looked up in the API 20E analytical profile index . If the MacConkey purity plate give mix culture, then repeat the test.

13. Results and interpretations Reveal the test which require the addition of reagent as follows: Vp test: add one drop of VPI and VP2 reagent and wait for 10 min for. color development. The pale pink color that occurs immediately has no significance . A positive reaction is dark pink or red. TDA test: add one drop of TDA reagent. A positive reaction brown-red will occur immediately. A negative reaction color is yellow. IND test: add one drop of James reagent. Look for the positive red ring reaction within 2 minutes. After several minutes the acid in the reagents reacts with the plastic cupule to produce a color change from yellow to brownish-red , which consider negative .

14. Results and interpretations Read the API strip according to the interpretation table, and record the result on the report sheet . On the report sheet , the test are separated into groups of three and number 1 , 2 or 4 is allocated for each test. By adding the numbers corresponding to the positive reaction within each group, a 7-digit profile number is obtained for 20 tests of the API 20E strip.

15. Results and interpretations The 7-digit profile is then compared with the numerical profile in the API 20 E analytical profile index book to obtain the organism identification.

16. Results and interpretations TEST REACTION NEGATIVE POSITIVE ONPG  - galactosidase Colourless Yellow (maybe pale) ADH Arginine dihydrolase Yellow Orange or red LDC Lysine decarboxylase Yellow Orange or red ODC Ornithine decarboxylase Yellow Orange or red CIT Citrate utilisation Light green Blue-green or blue H 2 S H 2 S production Colourless Black URE Urea hydrolysis Yellow Pink TDA Tryptophan deamination Yellow Dark brown IND Indole production Colourless reagent Pink VP Acetoin production Colourless Pink or red GEL Gelatin hydrolysis Colourless Black diffuse pigment GLU Glucose fermentation Blue Yellow MAN Mannitol Blue Yellow INO Inositol Blue Yellow SOR Sorbitol Blue Yellow RHA Rhamnose Blue Yellow SAC Sucrose Blue Yellow MEL Melibiose Blue Yellow AMY Amygdalin Blue Yellow ARA Arabinose Blue Yellow Oxidase Cytochrome oxidase Colourless Purple

17. VITEK 2 syste m The VITEK 2 system has everything healthcare laboratories need for fast, accurate microbial identification, and antibiotic susceptibility testing. The VITEK 2 compact system is a fully automated system that performs bacterial identification by biochemical analysis using colorimetry. It focuses on the clinical microbiology laboratory and provide increased levels of automation and capacity for higher volume laboratories.

18. VITEK 2 syste m The VITEK 2 compact system offers Advance Colorimetric technology that allows for the identification of 98% of clinical isolates.

19. VITEK 2 syste m VITEK 2 system can accurate identify of some bacterial strains in as little as two hours. In addition to being able to identify bacteria, the VITEK 2 compact system is able to identify multiple species of yeast. They also provide an option of automatic pipetting and dilution for antimicrobial susceptibility testing.

20. VITEK 2 syste m There are currently four reagent cards available for the identification of different organism classes as follows: GN. Gram-negative fermenting and non-fermenting bacilli. GP. Gram-positive cocci and non-spore-forming bacilli. YST. Yeasts and yeast-like organisms. BCL. Gram-positive spore-forming bacilli

21. VITEK 2 system The reagent cards have 64 wells that can each contain an individual test substrate . Substrates measure various metabolic activities such as acidification, alkalinization , enzyme hydrolysis, and growth in the presence of inhibitory substances .

22. VITEK 2 system The reagent cards have 64 wells that can each contain an individual test substrate . Substrates measure various metabolic activities such as acidification, alkalinization , enzyme hydrolysis, and growth in the presence of inhibitory substances .

23. Phoenix Phoenix automated microbiology system is intended for the in vitro rapid identification (ID) of Gram Positive bacteria from pure culture belonging to the genera Staphylococcus , Enterococcus, and other Gram Positive cocci and Gram Positive bacilli. The Phoenix Automated Microbiology System is also intended for the quantitative determination of antimicrobial susceptibility by minimal inhibitory concentration (MIC) of most Gram Positive bacteria from pure culture belonging to the genera Staphylococcus and Enterococcus.