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Published 12 February 2015

Bacterial identification procedure

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Here, you have six files for bacterial identification procedure in word, in pdf, and the ppt for the presentation of this experiment.

Bacterial identification procedure - Part 1 : Preparation of API miniaturized system - Protocol in word
Bacterial identification procedure - Part 1 : Preparation of API miniaturized system - Protocol in pdf
Bacterial identification procedure - Part 1 : Preparation of API miniaturized system - Practical training in ppt
Bacterial identification procedure - Part 2 : Reading of API system - Protocol in word
Bacterial identification procedure - Part 2 : Reading of API system - Protocol in pdf
Bacterial identification procedure - Part 2 : Reading of API system - Practical training in ppt

Bacterial identification procedure - Part 1 : Preparation of API miniaturized system

1. Bacterial suspension preparation

1.1 With a sterile inoculation loop, take a bacterial colony on agar Petri dish.
Warning ! The sample is realized in asepsis around Bunsen burner

1.2 Make a suspension with this bacterial colony in a steril tube of physiological water (5 mL). In the water, rub the inoculation loop on the tube side to disperse all the bacteria.
1.3 Homogenize your tube by manual rotations and write « S » on this tube.

2. Preparation of the API system

2.1 Fill up the bottom of the box with distilled water to cover all the cavities.

2.2 Make a rotational movement with the box to distribute the water in all the cavities. After that, aspire the water excess with a unsteril pipette.

2.3 With a pincer, put the API system on the the box.
2.4 Cover with lid and write your « table number » on the plastic label of the box.

3. Filling the API system

3.1 Open the box and use the lid such as a support to place the box in an inclined position.

3.2 Take 2 mL of the suspension « S » with a sterile pipette.
Warning ! The sample is realized in asepsis around Bunsen burner

3.3 Fill up all the tests with this suspension respecting this next rules :
- For an underline test or not (example : ONPG or ADH), fill up only the microtube,
- For an framed test (example CIT), fill up the microtube and the cupule.

3.4 With a sterile pipette, add oil in the 5 cupules on the underlines tests (ADH, LDC, ODC, H2S, URE).
3.5 Close the box and incubate at 37°C during 16 hours.

4. Analyse your results

The second day, after incubation, identify your bacteria (part 2).

Bacterial identification procedure - Part 2 : Reading of API system

1. Incubation validation

First, read the GLU test. A yellow color show a good incubation.

2. Add reagents

4 complementary tests must be realized:
- TDA test : add 1 drop of FeCl3 reagent.
- IND test : add 1 drop of Kovacs reagent.
- VP test : add 1 drop of alpha-naphtol reagent and one drop of KOH reagent.
- NR test : in the GLU test cupule, add 1 drop of -naphtylamin reagent and one drop of sulfanilic acid reagent.

3. Results

- For each test, observe the color.
- With the API datasheet, deduce if the test is positive or negative.
- Complete test after test, the API results card.

4. Results interpretation


- On the computer, open the bacterial identification software.
- Report your results + and – on the software cells. Press « enter » on the keyboard to move cell from cell.
- At the end of the procedure, write the name of the bacterial strain proposed by the software.

5. Final expression of your results

What is the name of your bacteria ?