Dilution Theory / Supplementary Pages:
General Problem Set
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1. One ml of a bacterial culture was pipetted into a 9 ml dilution blank. One-tenth ml of this dilution was pipetted into a 9.9 ml dilution blank. From this dilution, one-tenth ml was plated with 25 ml of culture medium. 220 colonies arose after incubation. How many colony-forming units were present per ml of the original culture?
2. Ten ml of spring water were added to a petri dish to which 40 ml of melted Plate Count Agar were added. After incubation, 35 colonies arose on the plate. What was the count of CFUs per ml of the spring water?
3. You are given eight petri dishes of Nutrient Agar and an abundance of pipettes and 9.0 ml dilution blanks, and you need to plate out a sample of milk such that plated dilutions of 10–1, 10–2, 10–3 and 10–4 are achieved in duplicate.
a. Clearly diagram how this may be done with the materials at hand. (As it may take quite awhile for large inocula to soak into plates, do not plate any amount larger than 0.2 ml.)
b. On the plates containing the 10–4 plated dilution, you count 48 colonies on one plate and 54 colonies on the other after incubation. Calculate the number of colony-forming units (CFUs) per one ml of the original milk sample.
4. Consider the following dilution scheme:
a. Report the total number of CFUs in the entire 100 ml amount of the original lake water sample. (TNTC=too numerous to count.)
b. Would you expect any change in the answer of the above problem if the first dilution was made by adding one ml of sample to 9 ml of diluent? Why or why not?
c. Would you expect the number of viable (living) cells in the original sample to be greater than, less than or the same as the number of CFUs in the sample?
Note: Be sure you know the definitions of cell, CFU and colony as given in the introduction to Experiment 1! Realize that CFU is not strictly a quantitative term. (The cells don't know if their colonies are going to be counted or not!)
d. What dilution is achieved by adding 2 ml of inoculum to 19 ml of diluent? (No, it wasn't a misprint in the diagram.)
5. You have obtained a pond water sample and wish to determine the concentration of bacteria which are gram-negative and lactose-fermenting. After making two 1/100 dilutions, you plate 0.1 ml of the second dilution onto each of two plates of MacConkey Agar. After appropriate incubation, you find that one plate contains 155 red colonies and 45 white colonies, and the other plate contains 160 red colonies and 35 white colonies. What was the concentration of gram-negative, lactose-fermenting CFUs per ml of the pond water sample?
6. One should expect the same number of CFUs in one ml of an undiluted sample as in ml of a 1/10 dilution of the same sample.
7. The same dilution can be obtained in each of the following situations:
a. The addition of 1 ml of a sample to 9 ml of sterile diluent.
b. The addition of ml of the same sample to 27 ml of diluent.
c. The addition of 11 ml of the same sample to ml of diluent.
8. You are given a flask containing a 1/10 dilution of sauerkraut juice. Without making any further dilutions, how much of this dilution of juice can be plated in order to achieve each of the follow-ing plated dilutions? (As these will be pour-plates – as opposed to the surface-inoculated plates in problem 1 – you can inoculate more than 0.1 ml to your plates.)
9. One gram of yogurt was added to 99 ml of sterile diluent. Decimal (1/10) dilutions were then made, and one-tenth ml was plated in duplicate on Plate Count Agar. After incubation, the following colony counts were made:
Using the above data, calculate the number of CFUs per gram of the yogurt.
10. One gram of hamburger was added to a 99 ml dilution blank. Two 1/10 dilutions were then made. From the last dilution made, 0.2 ml was plated onto an all-purpose medium. After incubation, 60 colonies were counted.
Each of the 60 colonies was inoculated onto a slant of Heart Infusion Agar (to grow cultures for gram staining) and into a tube of Glucose Fermentation Broth. Exactly one half of the cultures were determined to be gram-negative. Of these gram-negative cultures, twelve produced acid and gas in the broth and the rest produced only acid.
Determine the number of gram-negative, glucose-fermenting CFUs per gram of the meat.
This page has Dilution Plating Problems from the Bacteriology 102 Lab Manual formerly used in the course.