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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