Turbidimetry or Tube Assay

Turbidimetry or Tube Assay

Learning objectives

At the end of this lecture, student will be able to:

•       Explain tube assay principle

•       Describe the method for tube assay

•       Identify the significance of controls in microbiological tube assay

Turbidometric” or “Tube” assay

•       The turbidimetric method (Method B) depends upon the inhibition (or promotion) of growth of a microbial culture in a uniform solution of the antibiotic (or vitamin) in a fluid medium that is favourable to its rapid growth in the absence of the antibiotic.

•       In case of growth promoting substances, turbidity increases as the concentration increases

•       In case of growth inhibiting substances, turbidity decreases as the concentration increases

Tube assay – Method

For the standard curve

•       Prepare five different concentrations of the standard solution for preparing the standard curve by diluting the stock solution of the Standard Preparation of the antibiotic & increasing step wise in the ratio 4:5. 

Test sample dilution

•       Select the median concentration & dilute the solution of the   substance being examined (unknown) to obtain approximately this concentration.

•       Test concentration almost equivalent to the median concentration (S3)

•       Place 1ml of each concentration of the standard solution and of the sample solution in each of the tubes in duplicate.

·         Add 9ml of nutrient medium, previously seeded with the appropriate test organism

At the same time prepare three control tubes,

1. containing the inoculated culture medium (culture control),
2. Inoculated culture media but treated immediately with 0.5ml of dilute formaldehyde solution (blank) 
3. Uninoculated culture medium.
4. Place all the tubes, randomly distributed or in a randomized block arrangement, in an incubator or water-bath and maintain them at the specified temperature, for 3 to 4 hours.
5. After incubation add 0.5ml of dilute formaldehyde solution to each tube.
6. Measure the growth of the test organism by determining the absorbance at about 530 nm of each of the solutions in the tubes against the blank.

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