A dilution series takes a stock solution down to one or more working concentrations. Bacteriostatic water is a suitable diluent when the stock solution was itself prepared in bacteriostatic water, or when the dilutions will be held as preserved solutions and drawn from more than once. Where the assay or the material calls for a preservative-free diluent, plain sterile water or a buffer is used instead. See when the preservative interferes.
The formula
C1V1 = C2V2, where C1 and V1 are the concentration and volume of stock taken, and C2 and V2 are the concentration and volume of the dilution wanted. To prepare 5 mL at 1 mg/mL from a 10 mg/mL stock: V1 = (1 × 5) ÷ 10 = 0.5 mL of stock, made up with 4.5 mL of bacteriostatic water.
Serial vs. independent dilutions
In a serial dilution each tube is made from the previous one, for example 1 part of the previous tube plus 9 parts of diluent for a 1:10 series. It is economical with stock but any pipetting error is carried into every later tube. In independent dilutions each tube is made directly from the stock, which uses more stock but keeps each tube’s error separate. For a calibration series, independent dilutions are usually preferred; for a wide-range screening series, serial dilution is usual.
| Tube | Made from | Stock or previous tube | Bacteriostatic water | Concentration |
|---|---|---|---|---|
| 1 | stock | 0.5 mL | 4.5 mL | 1 mg/mL (from 10 mg/mL) |
| 2 | tube 1 | 0.5 mL | 4.5 mL | 0.1 mg/mL |
| 3 | tube 2 | 0.5 mL | 4.5 mL | 0.01 mg/mL |
| 4 | tube 3 | 0.5 mL | 4.5 mL | 0.001 mg/mL |
This series uses 18 mL of diluent, so a 30 mL vial covers it with margin, or a 10 mL vial if each tube is made at 1 mL instead of 5 mL. The calculators page works out vial sizes for a given total volume.
How the preservative behaves across the series
If the stock was prepared in bacteriostatic water and the diluent is bacteriostatic water, every tube contains 0.9% benzyl alcohol regardless of the dilution factor, because both components carry the same concentration. If the stock was prepared in plain water or a buffer and the diluent is bacteriostatic water, the benzyl alcohol content rises toward 0.9% as the dilution factor increases (a 1:10 dilution contains 0.81%). For any measurement in which benzyl alcohol could contribute a signal, such as absorbance near 257 nm, the blank is prepared from the same diluent so that its contribution cancels.
Technique
- Use calibrated pipettes and, for small volumes, add the smaller volume into the larger to reduce loss on the walls of the tube.
- Mix each tube by gentle inversion before drawing from it for the next tube.
- Label every tube with its concentration, dilution factor, date and initials as it is made, not afterwards. See labeling and record-keeping for prepared solutions.
- Record the lot number of the bacteriostatic water vial and the date it was first opened; the vial itself is discarded 28 days after opening. See shelf life after opening.
Related
Dissolving lyophilized research materials · Laboratory uses of bacteriostatic water
All BAC Water Supply products are supplied for laboratory research use only and are not for human or veterinary use.