Name: __________________

**Quantification: Standard Plate Count Procedure and Worksheet**

**Background:**

** **We often need to count things and this is even true for microorganisms. Quantification means to determine the quantity of, basically to count the number but includes methods that are not exactly counting. In this lab we will be using a method called the standard plate count to quantify a suspension of yeast cells. Dilutions are complicated only in that they involve both very small and very large numbers which are hard to deal with. Various disciplines have come up with different ways to try to simplify the naming (nomenclature) of dilutions. If you dilute a 1 mL concentrate into 9 mL of dilutant (saline or pure water are common) this is a tenfold dilution. The diluted solution could be expressed as 0.1 or 10%. One way math abbreviates numbers like this is with the expression 1 x 10-1. In science we often just abbreviate it 10-1. In other words the dilution could be expressed as any of these and 0.1= 10% = 1 x 10-1 = 10-1. So this is a rather silly example as it is still a rather large number (at least in terms of understanding). A much more accurate example of how this might be used is with a number like this: 0.00000000000000356. Do you have issues seeing how many zeros are there? Well I do and lots of other people do. This can cost lives when it comes down to needing that dilution and not 0.000000000000356. Wait aren’t they the same? They have all the same numbers….wrong….one is ten times larger (or more concentrated). To help eliminate these types of errors we use our scientific nomenclature for dilutions, 3.56 x 10-15 which is more obviously different from 3.56 x 10-14. At least it has a different number in it. This not only works for really small numbers but also really large numbers. The current population of the world is about 7790000000 people. We often use commas to help with large numbers like this (7,790,000,000) but we could also use scientific nomenclature 7.79 x 109. When doing quantification in microbiology we often run into both very small numbers (in dilutions) and very large numbers (in numbers of colony forming units (CFUs) present). Once you get familiar with this number nomenclature it is very handy and makes it so you make less errors.

**Procedure:**

**Graphic error! Please put 2.5 mL per tube NOT 4.5 mL per tube.**

1) Gather supplies. You will need the following:

a. Gloves

b. Disinfectant

c. 3 mL sterile pipettes

d. 1 mL sterile pipettes

e. Sterile plastic tubes

f. Sterile Petri Plates with SDA

g. Sterile saline

h. Sterile swabs

i. A Petri plate or slant with *S. cerevisiae *growth

j. Parafilm for sealing plates

k. Scissors

l. Sharpie or other pen that will write on plates and tubes

2) Label sterile plastic tubes using the number scheme in the figure above. I also added a bit extra information to mine to help me remember what I was doing so I labeled them like this (S- 3 mL, 1- 5 mL, 2- 5 mL, 3- 5mL, 4- 5 mL). Note: The mL listed is the total volume that will be in that tube at the end.

3) Open the sterile saline and add the specified amounts of saline as shown to the right of each tube in the figure above to each sterile tube using a 3 mL pipette (S=3 mL, 1=2.5mL, 2=2.5mL, 3=2.5mL, 4=2.5mL).

4) Using a sterile swab gather a visible glob of yeast from a plate or a slant and add it to the S tube. Spin the swab thoroughly to get the yeast off. Put the cap back on and mix well. Dispose of swab.

5) Use a new sterile pipette to transfer 0.5 mL of S into tube #1. Draw up and down on the pipette in tube #1 to help mix it. Close cap and mix well. Dispose of pipette.

6) Use a new sterile pipette to transfer 0.5 mL of #1 into tube #2. Draw up and down on the pipette in tube #2 to help mix it. Close cap and mix well. Dispose of pipette.

7) Use a new sterile pipette to transfer 0.5 mL of #2 into tube #3. Draw up and down on the pipette in tube #3 to help mix it. Close cap and mix well. Dispose of pipette.

8) Use a new sterile pipette to transfer 0.5 mL of #3 into tube #4. Draw up and down on the pipette in tube #4 to help mix it. Close cap and mix well. Dispose of pipette.

9) Label four Petri plates with SD agar in them on the base using both the dilution of the plate (shown on plate in figure above), the number tube it came from, your initials and date.

10) Using a new pipette remove 0.1 mL of the liquid in tube #1 and pipette it onto the plate with the corresponding number.

11) Repeat with tubes #2, #3 and #4 using new pipettes each time and pipetting onto the correctly numbered plate each time.

12) Using a sterile swab spread the liquid evenly across plate #1 using tight zigzag patterns while rolling the swab. Rotate the plate 1/3 and repeat swabbing. Rotate 1/3 and repeat swabbing. Finish swabbing by using the tip of the swab to go around the edge of the plate.

13) Repeat step 12 for each of the final three plates using a new swab on each plate.

14) Incubate for approximately 5 days at 75 F.

**Results and Final Calculation:**

1) If done correctly you should have individual colonies growing on the plates. The colonies should still be very small. If you grow them for too long the colonies will begin to grow together. If you have not grown them long enough they will be invisible to the naked eye.

2) Look for a plate that has a countable number (between 30 and 300) of colonies that are not growing together.

3) Record the plate dilution in the formula below.

4) Count the number of colonies and record this number in the formula below. I find it helpful to use a sharpie to mark each colony as I count it so I don’t count them more than once. If it seems you have up near 300 colonies it might be useful to divide the plate into sections and count the sections one at a time and add them up at the end.

**Questions and Calculation:**

Place a picture of the plate you counted here:

** Use the formula below to calculate your concentration of CFUs in the S tube.**

CFUs/mL= # of colonies x 1/plate dilution

CFUs/mL= __________ x 1/_____

CFUs/mL =_______

Make sure to simplify your answer so there is only 1 number to the left of the decimal place. For example: 13 x 105 CFUs/mL should be 1.3 x 106 CFUs/mL, 245 x 104 CFUs/mL should be 2.45 x 106 CFUs/mL.

Do a sanity check. Does your number make sense?

Are we expecting a really large number here or a really small number?

Make sure to answer the questions and submit to the D2L assignment box.