DILUTIONS and MICROPIPETTES (2024)

FAQs

Why must pipette tips be changed between dilutions? ›

If you keep using the same tip, you might contaminate other samples, as well as your reagents. In many laboratory workflows, you will need to change tips so often, that a micropipette has a specific ejector button for this.

It is recommended to change the pipette tip after every sample to avoid the risk of contamination with another sample.

Dilution set 1: Transfer 0.5ml of blue water into the 4.5ml of water, then 1ml of tube 1 into the next tube of 9ml water. Dilution set 2: Transfer 1ml of blue water into the 4ml of water, then 0.5ml of tube 1 into the next tube of 9.5ml.

This method is known as serial dilution and plating. Before making serial dilutions, the bacteria suspension must be thoroughly mixed. This is important because unlike solutions, which remain uniformly mixed indefinitely, suspended materials eventually settle down to the bottom of their containers.

Temperature. Temperature has many effects on pipetting accuracy. The factor that has the greatest effect is the temperature difference between the delivery device and the liquid. The air gap (dead air volume) between the liquid surface and the piston experiences thermal expansion effects unique to the case.

Be sure you use the proper size tip for each pipette. Always use a new tip for each different liquid. Use the correct pipette for the volume that is to be dispensed. Never use the 200-1000 µl pipette to dispense volumes below 200 µl.

Immersing too deep may increase the aspiration volume, whereas positioning the tip too near to the liquid surface can lead to the aspiration of air into the pipette resulting in inaccurate volume [4-8].

5. In most serial dilutions, you do not need to change the pipette tip between dilutions. However, you do want to ensure that you mix your solutions. With the tip submerged in your D1 tube, press the plunger up and down a few times to thoroughly mix the solution.

Be sure to use a new pipette for each sample to prevent contamination between samples.

What is a 10x dilution? ›

A 10x dilution is obtained by mixing 1 part of a sample with 9 parts of a diluent so that the new solution is 10 times (10x) less concentrated than the original solution. The 10x dilution can then be diluted by a factor of 10 again by mixing it with 9 more parts of the diluent.

Because stock solutions are frequently acquired and stored in highly concentrated quantities, diluting solutions is a required operation in the laboratory. The solutions must be accurately diluted to a known, lower concentration before being used in the lab.

The Dilution method is used to determine the minimal inhibitory concentration of an antimicrobial to inhibit or kill the bacteria/fungi and is the reference for antimicrobial susceptibility testing.

The first step is to rinse the pipette; this ensures that any water or solution from previous use is completely removed.

Forcing the solution out of the pipet causes too much to be delivered. Using a dirty pipet causes too little or contaminated solution to be delivered. Leaving little droplets behind on the walls (except for the small amount in the tip) causes too little solution to be delivered.

Another common pipetting error is a failure to prewet pipette tips. Prewetting pipette tips allows the air cushion to equilibrate to the liquid volume to be aspirated. To avoid this pipetting error we recommend inserting the tip not too deeply into the liquid and holding it there for ≈3 seconds before aspirating.

The most cited sources for dilution and preparation errors stem from improper use and contamination. Improper use means that the volumetric is not used correctly either through mishandling or misunderstanding of the volumetric labware.

How do you know if pipetting is accurate? ›

The most common way to check your pipette accuracy is by weighing water. The density of water is 1 g/mL. This means that every microliter (µL) should weigh exactly 0.001 g using a high-precision balance.

Avoid splashes of solution on the walls of conical flask. Do not blow out the remaining drop of solution from the tip of pipette. Read the burette and pipette at eye level / avoid parallax error.

There are two fundamental principles of micropipette operation: air displacement pipettes that have an air- cushion that moves between the piston and the sample liquid, which aspirates and dispenses the sample and the positive-displacement systems that have an integrate piston in the micropipette tip coming into direct ...

Micropipettes are now a staple in laboratories and offer accuracy of within a few percent points, usually <3% of the desired measurement.

A poor pipetting technique can lead to volume inconsistencies and sample contamination, resulting in poor data and reproducibility concerns.

Micropipettes are designed to operate with accuracies within a few percent (generally <3%) of the intended value. The accuracy of a micropipette decreases somewhat when micropipettes are set to deliver volumes close to the lowest values in their range.

When doing very high dilutions (like 1/10,000 or 1/1,000,000), it is more accurate to do the dilution in a series of smaller dilutions rather than in one giant dilution. This is called a dilution series or a serial dilution.

The dilution formula is calculated by using the volume sample divided by the total volume of the sample and the diluted blank: 1/(1+9) with a dilution factor (1+9)/1 = 10. The final dilution is calculated by multiplying the dilution of the previous tube to the subsequent tube 1/10 x 1/10.

Mixing is essential for an accurate serial dilution. A mixing step using half the total volume of the intermediate step introduced after every transferring step ensures an accurate and consistent serial dilution.

If the last step of your experiment is to run a gel, you can generally use the same tip to load multiple samples on the gel. Simply rinse your tip by pipetting up and down in the running buffer between samples.

How often do you calibrate a micropipette? ›

According to the Clinical & Laboratory Standards Institute (CLSI), you should calibrate your pipettes every 3-6 months or before they're put into service. If you fail to do so, then pipette readings may become inaccurate and unreliable.

Micropipette aspiration is easy to set up with a relatively low cost, is able to apply a wide range of forces down to pN scales, and is able to probe a wide range of cells. The main disadvantage is that because it uses optical microscopy to transduce, the spatial resolution is limited to a few microns.

For example, to make a 1:10 dilution of a 1M NaCl solution, you would mix one "part" of the 1M solution with nine "parts" of solvent (probably water), for a total of ten "parts." Therefore, 1:10 dilution means 1 part + 9 parts of water (or other diluent).

Another way to look at this is to calculate the dilution factor, that is, the ratio between the initial and final concentrations. Diluting a 5M solution to a 5mM solution is a 1000-fold dilution: Therefore, you need 1 part stock solution to make 1000 parts of your final solution.

Using this terminology, a “10X” stock might be diluted by adding 100 mL of the stock to 900 mL to produce a “1X” working solution.

Two pipetting techniques are used in the lab: forward mode (also known as standard mode) and reverse mode.

“Mouth pipetting is dangerous because it can lead to accidental poisoning with chem- icals or radioactive materials or illness from infectious organisms,” reminded Helgersen.

Wrong wiping of the pipette tip causes the pipetting to be incomplete, as liquid remains in the pipette tip. Wrong aspiration angle causes the water column in the nozzle to be lower and consequently the volume larger than the set one.

Safe Work Practices for Pipetting

Drain a pipette with tip against the inner wall of the receiving vessel. Never forcibly expel any hazardous material from a pipette. Carefully eject the disposable pipette tips to minimize aerosol formation.

The immersion angle of your pipette tip should be as near to 90 degrees as possible and not deviate more than 20 degrees from vertical. If the angle is more than this, the liquid level will be lower than when the pipette was calibrated.

Why is it important to change the micropipette tip for each transfer of the soil dilutions to a petri dish? ›

To avoid carry-over contamination: Use filter tips or positive displacement tips to prevent aerosol transfer from the sample into the pipette body, and again to the next sample. Alternatively, filters can be used on pipette tip cones. Always change the pipette tip after each sample.

- Always change the pipette tip after each sample. - Regularly autoclave, or disinfect, the pipette or the components that may come into contact with the sample. This type of contamination takes place when the pipetted liquid or aerosol particles from it enter the pipette body.

To prevent carry-over contamination: Always change the pipette tip after each sample. If pipette contamination is suspected, autoclave or disinfect the pipette in line with the manufacturer's instructions. Use filter tips to stop any aerosol transfer from the sample into the pipette body and again to the next sample.

If necessary, further decimal dilutions are prepared in order to reduce the number of microorganisms per unit volume to allow, after incubation, the observation of their growth (in tubes or bottles) or the counting of colonies (on plates) in samples that contain high numbers of organisms.

You must change pipette tips between each serial dilution because trace amounts of solutions will stick to the side of the pipette tip, which can affect concentrations of dilutions.

Because PCR amplifies DNA, even the most minute amounts of DNA contamination can turn into big problems. In short, extremely small amounts of contaminating DNA can be disastrous. If a tip comes in contact with more than one reagent, throw it out.

They used to put them in soap for a couple of hours, then wash them abundantly with tap and distilled water, boil them for 2 minutes and dry them at 37C. For basic rules of safety and possible cross contamination pipette tips should not be re-used. Re-using of pipette tips is not good way.

On average, a single tip can be washed and reused between 10-25 times. Considering roughly 30 percent of a consumables budget is allocated to pipette tips alone, re-use allows for up to a 96 percent reduction in annual spend.

The second stop ensures that you've released the “last drop.” Use the tip ejector to discard the tip. WARNING: The most common - and serious - operator error is depressing the plunger to the second stop before filling the micropipette tip.