When you're thinking about dilution, it helps to simplify your actions into dilution factors. When we said the diluted coffee was

• 1:4 Serial Dilution Table
• Streak Plate Method
• Serial Dilution Steps

DILUTION CHART Some numbers are rounded up or down to make measuring easier PIC’s dilution ratios are written as parts. Therefore, when PIC’s label suggest a dilution ratio of 1-to-4 (1:4) that means 1 part product and 4 parts water. Some people calculate dilution by dividing by 4 (in this example), which is an incorrect answer. Note that 'serial dilution' is a special case of 'series of dilutions'. Example: A serum sample is diluted twice with buffer. A series of five dilutions is made of this first dilution by diluting it 1/10, rediluting 1/10, and then three times more, each resulting solution then. In a serial dilution, aliquots of some solution are diluted stepwise such that the first dilution serves as the source from which an aliquot is taken for the second dilution, etc. In each of the dilutions you will be performing in this lab, 0.1 mL of yeast solution will be added to 9.9 mL of sterile dH 2 O.

'1/10th as strong as the original'

that was a dilution factor. We could also have said

'the dilution factor was 1/10', or

'the dilution factor was 0.1'.

Here are a few more for you to try:

1. 1 mL coffee + 4 mL water =
2. 1 mL coffee + 9mL water =
3. 1 mL + 99mL water =

As you've probably guessed, this works exactly the same whether you're talking about caffeine or meningicocci. Here's what a dilution factor of 0.01 looks like on the lab bench:

Notice that it really doesn't matter how much of the original stock you started with, as long as you had enough to put 1 mL into the new container. What matters is how much you transfer and how much water you add.

It's possible to write an algebraic expression for the dilution factor, but it's almost more trouble than it's worth, because it sounds so complex. But for what it's worth, the dilution factor for 1 mL stock + 99 mL water is:

amount transferred / total amount =

amount transferred / (amount transferred + amount water added) =

1 / (1+99) = 1/100 = 0.01.

Below is an applet to practice finding dilution factors, and also to determine how much water to add to achieve a given dilution. You should practice this until it is second nature!!

Can you do it in your sleep? OK, then go on to the next page...

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In some experiments it is pertinent to what the biologist is studying to ensure not only a proper colony count but that there is just one type of organism being studied. In previous blogs it is seen that these minuscule microorganisms stake their claim on whatever they see fit (so long as the right environmental factors comply) so how is it that biologists are able to separate them so that they may grow an individual microorganism and study it or even count it? That is easy (ok it’s rather time-consuming but worth all the effort) by serial dilution of course! Serial dilution is a series of dilutions, usually twofold or tenfold, used to determine the titer or concentration of a substance in a solution. Once an organism has been diluted out and allowed proper incubation time, this is when one can be counted. The way in which this experiment will ask you to count colonies will be by way of the naked eye. Plates that are suitable for counting should contain no less than 30 or no more than 300 colonies. In the below experiment you will be taken through just how to go about serial dilution of the specified microorganism and from there after proper incubation time be given the ratio on just how to count the number of colonies present.

What you will need:

*24-48 hour nutrient broth culture of Escherichia coli, six 20 mL nutrient agar deep tubes and seven sterile 9 mL water blanks.

*Hot plate, water bath, thermometer, test tube rack, Bunsen burner, sterile 1 mL serological pipette, mechanical pipetting device, sterile Petri dishes.

*Disinfectant solution in a 500 mL beaker, glassware marking pencil, bent glass rod, and beaker with 95% alcohol.

Procedure:

1. Liquify six agar deep tubes by boiling in a water bath. Cool the molten agar tubes and maintain in the water bath at 45 degrees Celsius.
2. Label the E. coli culture tube with the number 1 and the seven 9 mL water blanks as numbers 2 through 8. Place the labeled tubes in a test tube rack. Label the Petri dishes 1A, 1B, 2A, 2B, 3A, and 3B.
3. Mix the E. coli culture (Tube 1) by rolling the tube between the palms of your hands to ensure even dispersal of cells in the culture.
4. With a sterile pipette, aseptically transfer 1 mL from the bacterial suspension, Tube 1 to water blank Tube 2. Discard the pipette in the beaker of disinfectant. The culture has been diluted 10 times (1-10 dilution).
5. Mix Tube 2 and with a fresh pipette, transfer 1 mL to Tube 3. Discard the pipette. The culture has been diluted 100 times (1-100 dilution).
6. Mix Tube 3 and, with a fresh pipette, transfer 1 mL to Tube 4. Discard the pipette. The culture has been diluted 1000 times (1-100 dilution).
7. Mix Tube 4 and, with a fresh pipette, transfer 1 mL to Tube 5. Discard the pipette. The culture has been diluted 10,000 times (1-10,000 dilution).
8. Mix Tube 5 and, with a fresh pipette, transfer 0.1 mL of this suspension to Plate 1A. Return the pipette to Tube 5 and transfer 1 mL to tube 6. Discard the pipette. The culture has been diluted 100,000 times (1-100,000 dilution).
9. Mix Tube 6 and, with a fresh pipette, transfer 1 mL of this suspension to Plate 1B. Return the pipette to Tube 6 and transfer 0.1 mL to Plate 2A. Return the pipette to Tube 6 and transfer 1 mL to Tube 7. Discard the pipette. The culture has been diluted 1,000,000 times (1-1,000,000 dilution).
10. Mix Tube 7 and, with a fresh pipette, transfer 1 mL of this suspension to Plate 2B. Return the pipette to Tube 7 and transfer 0.1mL to Plate 3A. Return the pipette to Tube 7 and transfer 1 mL to Tube 8. Discard the pipette. The culture has been diluted 10,000,000 times (1-10,000,000).
11. Mix Tube 8 and, with a fresh pipette, transfer 1 mL of this suspension to Plate 3B. Discard the pipette. The dilution procedure is now complete.
12. Check the temperature of the molten agar medium to be sure the temperature is 45 degrees Celsius. Remove a tube from the water bath and wipe the outside surface dry with a paper towel. Using the pour-plate technique, pour the agar into Plate 1A rotating the plate gently to ensure uniform distribution of the cells in the medium.
13. Repeat Step 12 for the addition of molten nutrient agar to Plates 1B, 2A, 2B, 3A and 3B.
14. Once the agar has solidified, incubate the plates in an inverted position for 48 hours at 37 degrees Celsius.

1:4 Serial Dilution Table

After proper incubation time, proceed with the following:

1. Observe all colonies on plates and count each colony. Statistically valid plate counts are only obtained from bacterial cell dilutions that yield between 30 and 300 colonies. Plates with more that 300 colonies cannot be counted and are designated as “too numerous to count-TNTC”; plates with fewer than 30 colonies are designated as “too few to count-TFTC.” Count only plates containing between 30 and 300 colonies. Remember to count all subsurface as well as surface colonies.
2. The number of organisms per mL of original culture is calculated by multiplying the number of colonies counted by the dilution factor:

Number of cells per mL=number of colonies X dilution factor

Results:

The results of the serial dilution – agar plate analysis are given in the table below:

¹ Bacterial count per ml of sample (CFU/mL)

² Average count per mL of sample (CFU/mL)

Colony numbers “too numerous to count” are designated TNTC

There is some agreement between these results and the outcomes expected with a successful serial dilution regimen. With each dilution, a reduced number of colonies was observed (175, 50, and 1 for plates 1A, 2A, and 3A, respectively). The reduced counts between plate’s 1A and 2A were expected, and consistent with the regimen. Plate 3A produced only 1 colony, which actual renders it “to few to count”. The numbers of colonies on plates 1B, 2B, and 3B were “too many to count” despite the dilution.

The numbers of colony forming units was determined using the following equation:

Number of cells/ml = number of colonies x dilution factor

Those counts are given in the table above.

Streak Plate Method

Since the duplications plated are replicates of each other, it was determined that the averages of the duplicate bacterial counts per ml of sample. These results are listed in Table 1 above. Since several plates produced colonies “too many to count”, the resulting averages are probably meaningless.

Conclusion:

The ability to determine with any reliability the numbers of viable organisms growing on the plates was not achieved in this experiment. Serial dilution is a method intended to reduce the numbers of colonies to a range between 30 and 300; two (2) of the six (6) plates we produced exhibited counts within the targeted range. (It should be noted that some level of dilution was achieved as demonstrated with colony counts of 175 and 50 on plate’s 1A and 2A, respectively.)

The failure to achieve dilution is probably results from incorrect technique (measurements or calculations). Inoculation and incubation were achieved as evidenced by colonies “too numerous to count.”

Serial Dilution Steps

March 30, 2014 @ 7:15 p.m.