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SOP for Biomerieux Kit

Standard operating procedure to calibrate the Biomerieux Kit used in microbiology laboratory.


To lay down a procedure for Operation and Calibration of Biomerieux Kit (mini API, Densimat and Electronic Pipette)


This is applicable to Microbiology lab


Microbiology personnel


Head of Department


5.1 Operation of mini API instrument:

5.1.1 Principle

The principle of mini API depends upon two types of readings.
a) Turbinephelometric reading:
Turbinephelometric reading is used for test strips containing assimilation tests. Ex: ID32GN, ID32 C
Turbidimetry: - Measurement of the intensity of transmitted light (T), which is inversely proportional to the amount of bacterial growth.
Nephelometry: - Measurement of the intensity of light scattered (S) at 30 degrees directly proportional to the amount of bacterial growth.
b) Colorimetric reading:
Colorimetric reading is used for strips containing chromogenic substrates. Ex: ID32 STAPH, RAPID ID32 E, RAPID ID32 A, RAPID ID32 STREP.
Mini API measures the transmission of light for each cupule in four regions of the visible spectrum.


5.2.1 Cleaning of mini API Instrument To remove the dust from the surface of the machine:
Wipe gently the surface of the instrument with a soft, dry cloth lint-free cloth. If this is not sufficient, use a special plastic casing cleaner on a soft cloth. To clean the disc use the 3.5" disc cleaning kit. To clean the printer use a soft -bristled brush, taking care to remove all dust or dirt.

5.3 Operation of mini API

5.3.1 Before reading the strips follow the given steps for operating the mini API

Step 1: Switch on mini API using the ON/OFF switch at the back of the Instrument
Step 2: Wait for 15 minutes before starting to read the strips (Preheating)
Preparing the strips for reading
Remove the strip lids.
Add the reagents required for the development of certain tests.
Step 3: Pull out the protection rail.
Step 4: Place the strip on the strip carriage.
Step 5: Reading the strips
The mini API software initiates strip reading. Processing of the strips is automatic.
The strip code is read and the results are interpreted. This generates the type of reading corresponding to the strip (Turbinephelometric or Colorimetric).
Note: When mini API is switched on, the internal system configuration is tested. The instrument beeps twice indicating that mini API has successfully carried out internal tests.
The mini API software presentation page is briefly displayed on the screen before the main menu appears.
For a Turbinephelometric reading, the strip carriage enters and exits once.
For a Colorimetric reading, the strip carriage enters and exits twice.
At the end of the reading cycle, the strip carriage stays out. It automatically goes in when you quit the result entry module of the mini API software.
" Troubleshooting" in the mini API procedure manual, otherwise. Refer to technical engineer.
The instrument is equipped with a fan, which allows it to remain switched on when it is not being used.

5.4 Cleaning of Densimat

5.4.1 Take a dry cotton swab and dip in filtered 70 % (v/v) Isopropyl Alcohol.
5.4.2 Swab on the photosensors situated in the right section of the reading block.
5.4.3 Densimat should be cleaned when
a) Ampoule has been broken inside the reading block.
b) The results are too high.
5.4.4 Principle: Densimat is designed to measure the bacterial density in an ampoule of liquid Medium
5.4.5 Densimat enables precise determination of bacterial density by two measurements.
a) Scattered light S (Two photosensors) b) Transmitted light (One photosensor)
5.4.6 The correspondence McFarland scale / Bacterial concentration

5.5 Operation of Densimat

5.5.1 Densimat automatically switching on when an ampoule is inserted into the optical block.
5.5.2 Densimat goes into automatic standby mode after 30 seconds if there is no change in the OD.
5.5.3 Standby mode is indicated by "McF" on the display.
5.5.4 Densimat come out of the standby mode when the ampoule is removed.
5.5.5 Wipe the ampoule with a clean and dry lint-free cloth.
5.5.6 Turn the ampoule until you obtain the minimum stable reading on the scale.

5.6 Adjustment of inoculum density

5.6.1 Add the desired isolated colony with the help of sterile loop into the specified medium.
5.6.2 Adjust the bacterial suspension to obtain the recommended standard.
5.6.3 Insert the ampoule into the specified place on Densimat & note the density in McF.
5.6.4 If the density is higher than the standard required, the bacterial suspension should be diluted by adding a certain volume of medium.
5.6.5 If the density is lower than the standard required, more bacterial culture should be added.

5.7 Operation of Electronic pipette

5.7.1 Cleaning of Electronic pipette
Clean the external surface of the Electronic pipette with a lint-free cloth soaked in filtered 70 % (v/v) Isopropyl Alcohol.
5.7.2 Control devices
The pipette's control devices include
a) ON / OFF switch b) Keyboard c) Liquid Crystal display
d) Start button e) Tip ejection lever
5.7.3 Keyboard
M Key: To select the type of strip to be inoculated (No. of cupules / Vol. per cupule).
S ► Key: To program the inoculation speed in the CONTINUOUS mode.
▲ (select up) Key: Numerical value displayed increased by 1.
▼ (select down) key: Numerical value displayed decreased by 1.
* Key: To select the dispensing mode: CONTINUOUS OR STEP
E Key: To initialize or confirm after selection of a parameter.

5.7.4 Procedure for use The START button is used to start the pipette
a) The suction phase for 55 μl volumes to be inoculated (or)
The homogenate phase for 135μl volumes to be inoculated.
b) The dispensing operation if the STEP dispensing mode has been selected. (or)
The dispensing cycle if the CONTINUOUS dispensing mode has been selected.
c) Interrupt the dispensing cycle if the CONTINUOUS dispensing mode has been selected. Stopping the Pipette: To stop the pipette, put the ON / OFF switch in the OFF position.

5.8 Performance qualification of BIOMERIEUX KIT

5.8.1 Performance qualification of mini API

The Performance qualification of mini API shall be conducted with the following microorganisms
Rapid ID 32E ═► for Enterobacteriacae (Salmonella sp.)
ID32 STAPH ═► for Gram-positive organisms (S.aureus)
ID32 E ═► for Enterobacteriace (E.coli / Salmonella sp.)
ID32 GN ═► for Gram-Negative bacteria (Pseudomonas aeruginosa)
ID32 C ═► for Yeasts (Candida albicans)
5.8.2 Operate the instrument as per operating procedure and record the result details as per Annexure-1 Performance qualification of mini API (Biomerieux) "
5.8.3 ACCEPTANCE CRITERIA: Above all known microorganisms should be identified by mini API, by using respective strips.
5.8.4 Frequency: Once in a Year and whenever there is any major maintenance activity.
3 Standard organisms - E.coli / Salmonella species / Pseudomonas aeruginosa - Bacillus subtilis / Staphylococcus aureus - Candida albicans

5.9 Calibration of Densimat accuracy

5.9.1 Select an ampoule of Suspension medium (One ampoule of 2.5 ml minimum)
5.9.2 Place it in Densimat. The values displayed shall be between 0 and 0.1 McFarland.
5.9.3 Prepare a suspension of Escherichia coli in 0.22 micron filtered water, using a spectrophotometer (550 nm), adjust the turbidity to an optical density of 0.125 = 0.5 McFarland.
5.9.4 Transfer this suspension to the previously emptied ampoule.
5.9.5 Place the ampoule in Densimat, 0.5 McF±0.1 McFarland shall be displayed.
5.9.6 Enter the details in Annexure "Densimat [Biomerieux] Calibration Record"
5.9.7 If the readings are out of range, clean the photosensors
5.9.8 ACCEPTANCE CRITERIA: McFarland scale shall be 0.5 ± 0.1
5.9.9 Frequency: Once in a year and whenever the readings are not within the range.

5.10 Calibration of Electronic pipette

5.10.1 Take dry beakers previously washed & rinsed with water for injection (WFI) and fill it with freshly generated and cooled WFI.
5.10.2 Place a dry beaker on the weighing pan of the weighing balance, which is previously calibrated, and tare the weight of dry beaker.
5.10.3 Fix the required microtip with the micropipette and pipette out the water for injection and deliver it into the beaker placed on the weighing pan.
5.10.4 Wait for the stable display on the screen and then press the printer button to get the print of the weight. Tare the weight.
5.10.5 Take the readings by following above procedure at a minimum set value (16/ 55μl microlitre) and maximum set value (32 / 135μl microlitre) and simultaneously take its printout. Enter the observation in "Electronic Pipette [Biomerieux] calibration Record "
Quantity of WFI in ml = Weight of dispensed WFI
                                      Density of Water at 25°C
5.10.6 Calculate the percentage of error and Relative Standard Deviation.
5.10.7 Acceptance Criteria: Relative Standard Deviation should not be more than 2 % and % Error should not be more than 0.98 for 135μl and 1.46 for 55 μl.
5.10.8 Frequency of Calibration: Once in a year and whenever there is any major maintenance activity.

5.11 Variability identification between different analysts

5.11.1 Coded samples (2 Standard organisms - Bacteria, Candida and one environmental isolate shall be given to the analyst for identification by the microbiology section in-charge. (Name of the organism, which shall be allotted to the analyst shall keep secret)
5.11.2 Perform the analysis as per SOP.
5.11.3 Acceptance Criteria: The % ID of the identified standard culture shall be NLT 90 %. The % ID of the identified organism “standard culture / Environmental isolate” shall be NMT 20% between different analysts.
5.11.4 Frequency of Calibration: For every new analyst

5.12 System check

5.12.1 The identification kit which is meant for gram-negative bacteria shall be inoculated with gram-positive bacteria (Known culture/ Standard culture) and vice versa. The same shall be identified.
5.12.2 Identification shall be done for 2 Standard organisms (Gram - ve Bacteria & Gram + ve Bacteria)
5.12.3 Frequency of Calibration: Once in a year and whenever there is any major maintenance activity.


5.13.1 Do not use corrosive products to clean the surface of mini API and the screen, as they could damage the components.
5.13.2 Do not use alcohol or solvents to clean the printer, as they could damage components.
5.13.3 Do not allow water to run into the printing mechanism or on the electronic components.
5.13.4 Do not use hard-bristled brushes or abrasive materials.
5.13.5 Do not spray lubricants inside the printer.
5.13.6 Do not use Compressed air to clean inside the printer
Note: In Performance qualification isolates shall be taken in addition to laboratory cultures

6.0 Abbreviations

6.1 SOP - Standard operating procedure
6.2 WFI - Water for injection
6.3 % - Percent
6.4 °C - Degree Centigrade
6.5 -ve - Negative
6.6 +ve - Positive
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