DILUTIONS

Performing dilutions is a common process in microbiology that is performed with the objective of reducing the concentration of microorganisms in a solution. Dilutions have two main applications in Antimicrobial Textile Testing:

  1. Reaching a specific bacterial concentration: after estimating the concentration of the Single Colony Growth, performing dilutions is required to reach a desired concentration of microorganisms.
  2. Obtaining incubated culture plates with a countable number of colonies: plating techniques are used to determine the number of CFU´s present in a sample, and by diluting the sample in a controlled stepwise way (serial dilutions) it is possible to culture a series of plates that will have a countable number of colonies (between 30 and 300). This is specially useful when trying to determine the exact concentration of the Single Colony Growth, and also when plating the bacteria recovered from the incubated samples.

PERFORMING SERIAL DILUTIONS

A serial dilution is the process of taking a sample from a certain substance and then performing stepwise controlled dilutions using a series of specified volumes of sterile diluents. Serial dilutions are usually performed as a way to determine the concentration of a bacterial solution, becoming a useful tool to use during the Single Colony Growth concentration estimation and also during the bacteria recovery process.
The most common sterile diluents used are distilled water, saline solution, and TSB.
A serial dilution can be done using a dilution factor for the entire series, or it can be done determining specific dilution factors for individual test tubes.

This is the required procedure for making ten-fold dilutions:

INSTRUCTIONS

  1. Place the 10 ml tubes on a tube rack and properly label them as follows: -1, -2, -3, -4, -5, -6, -7, -8, -9, and -10.
  2. Fill each tube with 9 ml of sterile deionized water using a 10 ml serological pipette.
  3. Vortex the Bacterial solution, grab 1 ml of that solution using a sterile pipette tip, place that volume into the -1 tube, and discard the pipette tip.
  4. Vortex the -1 tube, grab 1 ml of that solution using a new sterile pipette tip, and place that volume into the -2 tube.
  5. Repeat that same procedure for the rest of the tubes.

MATERIALS

  • Bacterial Solution
  • 90 ml of sterile deionized water
  • 10 ml tubes (10)
  • 10 ml serological pipette
  • Pipette controller
  • 1 ml Pipette tips (10)
  • Pipette
  • Vortexer

SPECIAL CONSIDERATIONS

  • The ten-fold dilution example provided above can be adapted depending on the experiment, since it is not always necessary to perform the whole range of ten dilutions in order to get countable bacteria.
  • With the use of the Hemacytometer or Spectrophotometer methods already described, it is possible to estimate the initial bacterial concentration and, therefore, determine what would be the most effective dilutions to make in order to have between 30 and 300 CFU´s on the petri dish.
  • To improve accuracy of results, it is important to vortex between each step, and also to plate the dilutions performed in a timely manner.
  • To select the diluent used it is important to take into consideration the range of time between performing the dilutions and plating them. TSB is one of the alternatives to use as diluent. However, when it is anticipated that there will be a considerable interval between the dilution and plating processes, the nutrient components of TSB that promote bacterial growth may not be the best option since they can produce continued growth during that period of time. In those cases, sometimes it is more beneficial to use sterile deionized water.

SERIAL DILUTION CALCULATOR

Oftentimes, during Antimicrobial Textile Testing experiments it is required to dilute bacterial solutions to reach a specific concentration like it happens with the Single Colony Growth. Other times, it is required to perform serial dilutions but in a smaller range that gives a more manageable number of plates when working with high numbers of samples.
For those scenarios, the ten-fold dilution example provided above might not be useful and targeted calculations need to be performed in order to find the required steps needed to reach the desired concentration, and the most effective way to perform them.
The Dilution Calculator Spreadsheet will become useful to determine the most appropriate dilution factor required to reach the desired concentration, the number of steps required, and the volume of sterile diluent required.

Open the Dilution Calculator Spreadsheet on Google Sheets LINK