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,
- containing the inoculated
culture medium (culture control), - Inoculated culture media but
treated immediately with 0.5ml of dilute formaldehyde solution (blank) - Uninoculated culture medium.
- 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. - After incubation add 0.5ml of
dilute formaldehyde solution to each tube. - 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.
