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BIOLOGY 173 Lecture Notes - Lecture 2: Serial Dilution, Titer, Antimicrobial Resistance

186 views3 pages
ochrerat803
4 Apr 2016
School
U of M
Department
Biology
Course
BIOLOGY 173
Professor
Marcus Ammerlaan

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Document Summary

A key concept in ecology is that environmental context shapes biological communities. How are bacteria in the human body a good example of this? (three points) b. A key concept in evolution is that past environmental context shapes populations. Explain this using the example of antibiotic resistance in bacteria. (three points: a dilution scheme is drawn below. Answer the questions that follow. (2, 2, 1, 1, 1 points) [start with flask, approximate titer = 2 x 10^6] [transfer 0. 1 ml from flask into tube a with 9. 9 ml sterile water] [transfer 0. 1 ml from tube a into tube b with 0. 9 ml sterile water] [transfer 0. 1 ml from tube b into tube c with 0. 9 ml sterile water] [transfer 0. 1 ml from tube c into tube d with 0. 9 ml sterile water] A research paper (weiner, ecology 67(5) 1986, pp. Mean dry weight p-value for dry weight (compared to no competition)

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Related Questions

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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.

FOR #4 A & B I don't know if I did it correctly.....Please check & the other questions I'm having a heard time on. Thanks!

A. You have been given a tube of E. coli. You are asked to make 1 mL total volume of 10-1 dilution of the bacterial culture. Explain how you would do this. Show all necessary calculations.

____ ml cells + _____ ml water = 1 ml (total volume)

V1 D1 X V2 D2
1 mL X 1 = V2 X 10^-1
V2 = 1 mL/10^-1 so V2 = 10 mL.
But because V1 is part of V2. The answer is: 10 mL-1 mL= 9 mL of diluent must be added.

V1 D1 X V2 D2
1 mL X 1 = V2 X 10^-2
V2 = 1 mL/10^-2 so V2 = 100 mL.
But because V1 is part of V2. The answer is: 100 mL-1 mL= 99 mL of diluent must be added.

You have bacteria at a concentration of 5 x 108 CFU/mL. You spread 1 mL of this sample on an agar plate to obtain isolated colonies. How many colonies do you expect to find the next day after incubation at 370C? Can you count these colonies? Can you use this plate for determining concentration?

A. You have bacteria at a concentration of 1 x 103 CFU/mL (in real life – you don’t know this, but we are just working on math skills here). You transfer 1 mL of this sample into 9 mL of water and then spread 1 mL on a plate of agar. How many colonies do you expect to find the next day after incubation at 370C? Can you count these colonies? Can you use this plate for determining concentration?

You take 0.05 mL of a culture of bacteria at a concentration of 4 x 107 CFU/mL, and add 4.95 mL of water to it. What is the dilution that you have performed? What is the concentration of bacteria (CFU/mL) in the diluted culture?

A. You have diluted a sample by 1000 fold (1/1000) and plated 1 mL on an agar plate. You observe 55 colonies. What was the concentration of the original sample in CFU/mL?

A. A bacterial sample has a concentration of 3 x107CFU/mL. You make serial dilutions of 10-3 followed by 10-2 and 10-1 dilutions. You finally plate 1 mL of the last dilution on an agar plate and incubate it at 370C. What is the total dilution? How many colonies do you expect to see on the plate?

Total dilution:

# of colonies expected on plate:

C. This time, you see 10 times fewer colonies than you had expected to see.What could have gone wrong? How will you fix this problem?

A bacterial sample has a concentration of 2 x106 CFU/mL. Show a scheme of dilutions to obtain 30-300 colonies on a plate. Your scheme should contain the volume of diluent (sterile water), volume of sample transferred each time, the concentration of bacteria (CFU/mL) in each dilution. You can assume you are plating 1 mL of the dilution on plates of nutrient agar. See Figure 1 in protocol.

These are the results of the experiment described in the protocol. From the data shown in the results section of Standard Plate Count protocol, calculate the concentration of bacteria (CFU/mL) in the original sample of E. coli. Show your calculations. Hint: remember that we don’t use plates that have less than 30 or more than 300 colonies to do these calculations.