SOP for Serial Dilution Technique in Micro. Analysis

1.0      OBJECTIVE:

To lay down a standard procedure for serial dilution technique for reliable microbial colony count.

2.0       SCOPE:

This Standard Operating Procedure is applicable to the technical staff working in the Microbiology Laboratory competent in performing the test.


            Microbiologist shall be responsible to follow the procedure mention in this SOP.


Department Head & QA Head shall be accountable for effective implementation of this SOP.

5.0       ATTACHMENTS:


6.0       PROCEDURE:

  • Prepare and sterilize the media for analysis of water as per SOP for media preparation and sterilization
  • Enter in the MLT room as per SOP for entry and exit in MLT room.
  • Operate the LAF as per SOP operation and cleaning of LAF

6.1           Large Volume Serial dilution:

6.1.1        Label 3 bottles 1:100, 1:10,000, 1:1,000,000 i.e. 10-2, 10-4, 10-6.

6.1.2        Using a sterile 1 ml pipette, transfer 1ml of broth culture into 10-2 bottle. Each bottle containing 99ml

of sterile water.

6.1.3        Tightly cap the bottle, grab it in your hand, rest our elbow on the LAF table and rapidly move round

the arm with the bottle in an arc, 25 times.

6.1.4        This should adequately disperse the bacteria evenly throughout the bottle and break up bacterial


6.1.5        The first bottle now has a 1:100 dilution of original broth culture. There are still too many bacteria in

  1. If you were to plate them, so further dilution is necessary.

6.1.6        Using new sterile 1ml pipette, transfer 1ml out of the first bottle 10-2 and add this to your bottle

labeled 10-4. Repeat the bottle shaking procedure.

6.1.7        There are probable still too many bacteria in this dilution and the dilution process needs to be

repeated once more.

6.1.8        Using a new sterile 1ml pipette, transfer 1ml from the 1:10,000 (10-6) bottle. Repeat the shaking


6.1.9        Plating:     Label two plates 0.1ml of 10-4 and 1ml of 10-4     Label the other two plates 0.1ml of 10-6 and 1ml of 10-6     Quickly shake the dilution bottle and aliquot the indicated amount from the appropriate tube onto the

centre of the plate.     Use either 1ml pipette or 100µl micropipette, depending upon the volume plating.     Disperse it in 2-3 drops around the centre of the plate.     Use a sterile blue L-shaped spreader to spread the inoculums evenly around the plate.     Do not invert the plates until all the liquid has absorbed into the surface of the agar.     Incubate the plates for appropriate time and temperature.

6.2           Small Volume Serial Dilution:

6.2.1        Label 3 screw-capped test tubes 1:100, 1:10,000, 1:1,000,000 or 10-2, 10-4, 10-6.

6.2.2        Using Sterile 10ml pipette aliquot 9.9ml of sterile water into each tube.

6.2.3        Using a 100µl micropipette and sterile tip, transfer 0.1 ml of broth culture into 10-2 tube and cap the


6.2.4        Either mix it for a few seconds on a vortex mixer or vigorously flick the tube to adequately disperse

the bacterial clumps. Do not shake the test tubes.

6.2.5        Using a sterile tip, transfer 0.1 ml from the first tube (1:100 or 10-2) and add it to your tube labeled

1:10,000 or 10-4.

6.2.6        Repeat the tube mixing procedure.

6.2.7        Using a new sterile tip, transfer 0.1ml from the tube 10-4 and add it to tube labeled 10-6.

6.2.8        Repeat the tube mixing procedure.

6.2.9        Plating:                          Label two plates 0.1 ml of 10-4 and 1ml of 10-6.     Follow plating procedure 6.1.9.

6.3           Counting of Colony forming unit:

6.3.1        Pick the plate which has between 30-300 CFU count every colony on the plate.

6.3.2        Flip the plate over, bottom side up and put pen dot on the back of each colony. This will avoid from

counting more than once or recounting.

6.3.3        Alternatively place the plate on gridded, lightened surface of Quebec colony counter and use the grid

to  keep track of which areas have counted.

 6.3.4        Calculations:

                CUFs                 X   Dilution factor  = CFU/sample weight

Sample amount plated

e.g.    150        X  100,000 = 15000,000 or 1.5X107


7.0           REFERENCES:



SOP: Standard operating procedure

CFU : Colony forming unit

LAF : Laminar air flow

QA  : Quality Assurance

QC  : Quality Control


Quality Assurance Department

Quality Control Department


Version Number Effective Date Reason for Revision



Microbiology Labs Documents