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Answer the following questions regarding a 10x Serial Dilution experiment using yeast cell cultures from tube A.

Tube A contains 1 ml of yeast culture. 3.6 ml of sterile water is placed into 4 tubes (B, C, D, and E). 0.4 ml of yeast culture is removed from tube A and put into tube B.. 0.4 ml is transferred from tube B to tube C, and from tube C to tube E.

1.) What is the final volume in tubes C & E after the dilution?

C ____ml.

E _____ ml

When 0.1 ml of Dilution D was plated on each of the four separate YED plates; 125, 140, 137, and 830 colonies were observed for each plate after 48 hours of incubation. When 0.1 ml of of dilution E was plated on each of the four sepearte YED plates; 12, 10, 11 and 8 colonies were observed on each plate after 48 hours of incubation.

2.) What is the cell denisity (titer) in tube D? (show your calculation and unit)

3.) What is the cell denisity (titer) in tube A? (show your calculation and unit)

4.) What is the cell denisity (titer) in tube A if the volume of cell culture plated on each plate was 50 microliters? (show your calculation and unit)

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27 Jan 2018
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Step 16.
A.

Don appropriate personal protective equipment (PPE).
B.

Aseptically transfer 1 mL from dilution tube 2 to dilution tube 3 and mix well. This is a 10-5 cumulative dilution.
C.

Aseptically transfer 1.0 mL from dilution tube 4 to dilution tube 5 and mix well. This is a 10-7 cumulative dilution.
D.

Aseptically transfer 0.1 mL from dilution tube 1 to dilution tube 2 and mix well. The cumulative dilution of the culture is 10-4.
E.

Allow the inocula to soak into the plates before proceeding.
F.

Aseptically transfer 0.1 mL from dilution tube 2 to plate A1. Using the spread plate technique, disperse the sample evenly over the entire surface of the agar.
G.

Aseptically transfer 1 mL from dilution tube 3 to dilution tube 4 and mix well. This is a 10-6 cumulative dilution.
H.

Obtain eight plates, organize them int four pairs and label them A1, A2, B1, B2, etc.
I.

Keep accurate records in your lab notebook.

Clean up the lab benches, doff PPE, and wash hands.
J.

Repeate the procedure with plate A2 and label both plates "10-5mL of original sample," or simply "10-5"

Notice that transferring 0.1 mL counts as a dilution!! The tube was a 10-4 dilution, but the plate is a 10-5 dilution!!
K.

Invert the plates so they are "agar up" to prevent condensation from ruining the plates.
L.

Repeat the aseptic transfer, spread plate technique, and label schematic for dilutions tubes 3, 4, and 5, and plates B, C, and D.
M.

Incubate the plates at 35℃ for 24 to 48 hours.
N.

Obtain five dilution tubes and label them 1–5, respectively. Make sure they remain covered until needed.
O.

Aseptically add 9.9 mL sterile water to dilution tubes 1 & 2. Cover when finished. Aseptically add 9.0 mL sterile water to dilution tubes 3, 4, and 5. Cover when finished.
P.

Mix the broth culture an aseptically transfer 0.1 mL to dilution tube 1. Mix dilution tube 1 well. This is a 10-2 dilution

You have been provided with 100 grams of a yogurt culture. You are asked to perform a plate count to determine the number of viable organisms present in the sample. You estimate that there may be as many as 10^8 cfu/g in the culture. Understanding that a countable number of cfu on a plate is 30 t0 300.....

1. Which of the following dilution series is appropriate for this essay if 1.0 mL was plated from the last dilution tube? (circle the correct answer and show your reasoning)

a) (0.1 gram of sample): 9.9 - 1 mL - 9 - 1 mL - 9 - 1 mL - 9 - 1 mL - 9

b) (10 grams of sample): 9 - 1 mL - 9 - 1 mL - 99 - 1 mL 9

c) (10 grams of sample): 90 - 10 mL - 90 - 10 mL - 90 - 10 mL - 90

d) (1 gram of sample): 9 - 1 mL - 99 - 1 mL - 99 - 1 mL - 9 - 1 mL - 9 - 1 mL - 9

2. Referring to the diargram/scheme you chose above, assume that 90 cfu/g grew on the plate from the last dilution. Calculate the total number of bacteria (cfu) in the entire yofurt culture. Show all work, including the determination of the bacterial concentration in the original sample.

3. In the above example, if 0.1 mL had been plated from the last tube of the dilution series instead, how many cfu would you anticipate on the plate? Show your work.

4. Looking at the tubes in row C, calculate the final overall dilution in the last tube of the series if each tube contained 49 mL of diluent and 1 mL was transferred between each tube. Show your work.

You are trying to determine the concentration of a bacterialculture. You set up 7 1/10 serial dilutions of you culture byadding 1 ml of culture from the more concentrate sample into 9 mlof broth. You then plate 0.1 mL of each dilution on an agar plate.The next day you observed growth on each plate. The plate that camefrom the 7th dilution tube contain 12 colonies. The plate that camefrom the 6th dilution contained 135 colonies. The plate that camefrom the 5th dilution contain 1200 colonies (that was a lot ofcounting). All other plates contain lawns of bacteria (to many tocount: TMTC). Report the concentration of the original culture.

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