SOP for Identification of Microbial Cultures using Biomeriux Identification System

1.0 PURPOSE

To lay down the procedure for identification of microbial cultures and isolates identified during various microbiological tests.

2.0 SCOPE

It is applicable to Microbiology lab.

3.0 RESPONSIBILITY

Microbiologist

4.0 ACCOUNTABILITY

Head of Department

5.0 PROCEDURE

5.1 Precaution to be taken during the identification
5.1.1 Prepare pure culture before identification procedure.
5.1.2 Kits and reagents to be used shall be within its shelf life.
5.1.3 Follow the manufacturer instruction for identification procedure.
5.1.4 The kits and reagents shall be handled carefully.
5.1.5 All staining shall be performed as per SOP for microbial staining procedures.
5.1.6 Ensure the electronic pipette is fully charged.
5.1.7 Check the cleaning status of the instrument.

5.2 Procedure for identification

5.2.1 Pick the suspected colony from any source such as environment, water and product.
5.2.2 Prepare a suspension of the suspected colony in a 10 ml of sterile normal saline.
5.2.3 Take a loop full of prepared suspension on media agar plate for streaking.
5.2.4 Streak the suspension in such a way as to get an isolated colony.
5.2.5 Prepare pure culture and observe the morphological characteristic of the colony.
5.2.6 For colony morphology refer Appendix-I
5.2.7 Pick the purified colony and perform the gram staining according to SOP.
5.2.8 For cell morphology and arrangement refer Appendix-II and for color refer Appendix-III
5.2.9 Proceed for identification as shown in the chart below.

5.3 Culture suspension preparation with the help of DENSIMAT

Note: The ratio S/T is directly proportional to the density of the bacterial suspension. The results are displayed on a liquid crystal display on densimat.
5.3.1 DENSIMAT will be in ‘OFF’ mode before use. It automatically turns ‘ON’ when inserting an ampule and automatically turns ‘OFF’ when the ampule is removed.
5.3.2 Prepare a homogeneous bacterial suspension in a minimum volume of 2.5 ml for large ampule or suitable quantity.
5.3.3 Wipe the ampule from the outer surface with tissue paper.
5.3.4 Place the ampule in the DENSIMAT slot provided it turns ‘ON’ automatically.
5.3.5 Turn the ampule until you obtained the minimum stable reading on the scale
Note: Only the minimum stable valve obtained is of significance and shall be considered.
5.3.6 Use the bacterial suspension with a density equal to a McFarland valve. If it is higher than adjust with the same diluent.

5.4 Inoculating the strip with the help of Electronic pipette.

5.4.1 Take the pipette out of its stand.
5.4.2 Place the sterile tip firmly on the nose cone and insert tip into the inoculum prepared.
5.4.3 Perform suction or homogenisation of the bacterial suspension.
5.4.4 To activate the suction or homogenisation phase, press the start button.
5.4.5 Suction: keep the tip at the bottom of the ampule.
5.4.6 Raise the pipette slightly so that the tip is pressed against the wall of the ampule above the liquid.
5.4.7 Rotate the inoculum ampule around the tip, keeping the tip against the wall of the ampule.
5.4.8 Put the tip of the pipette back into the bottom of the ampule.
5.4.9 When suction or homogenisation is completed, press the start button. Remove the tip from the ampule.
5.4.10 Perform inoculation of the strip cupules in CONTINUOUS or STEP mode.
5.4.11 When inoculation is finished, the remain inoculum in the tip shall be placed back into the ampule.
5.4.12 Press the start button: the liquid remaining in the tip is emptied into the ampule.
5.4.13 Squeeze the EJECTION LEVER the tip is ejected from the piston into the ampule
5.4.14 Perform inoculation of another strip or put the pipette back onto its stand.
5.4.15 Incubate the strip as per the manufacturer instruction.
Related: SOP for Biomerieux Kit

5.5 Reading the strip

5.5.1 Switch on mini API using the ON/OFF switch at the back of the instrument.
5.5.2 Note: Protection rail shall be pulled completely to allow the strip carriage to come out.
5.5.3 Wait 15 minutes before starting to read the strips (preheating)
5.5.4 Remove the strips lids.
5.5.5 Add the reagents required for the test (refer to the package inserts for the strips)
5.5.6 Pull out the protection rail.
5.5.7 Place the strip on the strip carriage, comes out from mini API.
5.5.8 Enter the required data.
5.5.9 The mini API software initiates strip reading.
5.5.10 Processing of strips is automatic.
5.5.11 The strip code is read and the results are interpreted. Instrument will automatically generates the type of reading corresponding to the strip: Turbinephelometric or colorimetric.

5.6 To create a record

5.6.1 When mini API is turned on after few minutes the main screen will come.
5.6.2 Choose from ENTER, DISPLAY, COMM., UTTL., api/ATB
5.6.3 Use the (¬) and (®) keys to position the cursor on (ENTRY)
5.6.4 Enter the record, sample, technician name, and date of analysis, sample origin, and sample type.
5.6.5 After reading the strip collect the print.
5.6.6 Switch off mini API after completion of analysis,
5.6.7 ‘Push in’ the protection rail in then, Press (DEL)
5.6.8 The screen display: C:\MINIAPI>
5.6.9 Write cd.. and enter
5.6.10 Switch off the mini API instrument from backside.

5.7 Acceptance criteria For % of Identification

For Isolates: NLT 80 %
For Pure Culture: NLT 95 %
5.7.1 If in case of Isolates the % of identification is Less than 80 %(Unacceptable profile) then again purify the isolate for identification and proceed as per point No.5.2 to 5.5, if again % of identification of isolates is Less than 80 % (Unacceptable profile) of test, then send the isolate to IMTECH (Institute of Microbial Technology) or equivalent Contract Laboratory having facility of identification of isolates up to DNA sequencing.
5.7.2 If in case of Pure Culture the % of identification is found less than 95 % then repeat the identification of pure culture as per point No. 5.2 to 5.5 if the result is not meeting the acceptance criteria, then discard the pure culture.

5.8 Cleaning of mini API

5.8.1 Do not use the corrosive material to clean the surface of mini API and its screen.
5.8.2 Wipe gently with a soft, dry cloth.
5.8.3 Use a soft bristled brush, to remove all the dust and dirt.
5.8.4 Do not use alcohol or the solvent to clean the printer.

5.9 Cleaning of DENSIMAT

5.9.1 Use an alcohol-impregnated swab.
5.9.2 Swab the photo sensors situated in the right section of the reading block.
5.9.3 Test the instrument by referring to “Testing DENSIMAT” if the results are incorrect, repeat the cleaning procedure.

5.10 Cleaning of ELECTRONIC PIPETTE

5.10.1 Unscrew and remove the nose cone.
5.10.2 Check that the two holes in the nose cone are unobstructed and clean them if necessary.
5.10.3 Start a dispensing cycle so that the piston comes out as far as possible from the ejection.
5.10.4 Check whether the piston is clean, clean and grease if necessary.
5.10.5 Screw the nose cone back into position.
5.10.6 Check the O-ring on the nose cone is properly lubricated and grease it if necessary.
5.10.7 Enter the usage of the instrument in SOP

5.11 Record numbering

5.11.1 Record numbering shall be done in the following manner
Each record number consist of 12 characters
QCM/YY/00001
First three characters ‘QCM’ stands for quality control microbiology
Next character is ‘/’ for separation.
Next characters are ‘YY’ stands for last two digits of the current year.
Next character is ‘/’ for separation.
Next, five characters are number starts from 00001 and so on
5.12 Numbering of isolates
5.12.1 The isolates identified shall be numbered in the following manner,
Each isolates number consists of 13 characteristics.
QCM/EXX/0001
First three characters ‘QCM’ stands for quality control microbiology
Next character is ‘/’ for separation.
Next characters are ‘E’ stands for Environments isolates.
Next characters are ‘XX’ stands for MI, CP, LO and TB.
Where:
MI is Microbiology isolates and CP is Capsule, LO is liquid oral, TB is the tablet.
Next character is ‘/’ for separation.
Next four character are number starts from 0001 and so on.
5.12.2 In case of water, numbering shall be done in the following manner,
Each isolate number consists of 12 characteristics.
QCM/WI/0001.
First three characters ‘QCM’ stands for quality control microbiology
Next character is ‘/’ for separation.
Next characters are ‘WI’ stands for Water isolates.
Next character is ‘/’ for separation.
Next four characters are number starts from 0001 and so on.
5.13 When regular isolates are identified enter the details.
5.14 When new isolates are identified enter the details of isolates.

6.0 ABBREVIATION

6.1 SOP - Standard Operating Procedure
6.2 QCM - Quality Control Micro

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