Chapter 10
Cultural Methods
Ian L. Pepper and Charles P. Gerba
1.
Student A performs a soil extraction with peptone as the extracting solution. A dilution and plating
technique is performed using the spread plate method. The following results are obtained:
a. 150 colonies are counted on an R2A plate at the 10 -5 dilution
b. 30 colonies are counted on a nutrient agar plate at 10 -4 dilution
Calculate the number of viable bacteria per gram of soil for R2A and nutrient agar. Why
are the counts for nutrient agar lower than for the R2A media?
Number of viable bacteria per gram of soil from R2A:.
Assuming 0.1 ml of soil extract is spread plated.
N = 150 × 1 / 10-6 = 1.5 × 108 CFU/g soil
Number of viable bacteria per gram of soil from nutrient agar.
N = 30 × 1 / 10-5 = 3 × 106 CFU/g soil.
Counts from nutrient agar are lower since it is a nutrient rich medium which does not allow for the
growth of oligotrophic bacteria found in soil. R2A is a nutrient poor medium that does allow for
oligotrophic growth.
2.
Student B performs a soil extraction with peptone as the extracting solution. A dilution and plating
technique is performed using the pour plate method. The number of bacteria per gram of
soil is calculated to be 8 × 108. What dilution plate contained 80 colonies?
Since the pour plate method is used, assume 1 ml of soil extract is plated.
Number of CFU / g soil 
# colonies 1
8 108
dilution
∴ for 80 colonies
80 
1
 8 10 8
dilution
Dilution 
3.
80
8 10
8
 10 7
Student C used direct microscopic methods to count bacteria in the same soil as student A
(see problem 1). How would student C’s numbers compare to student A’s
numbers and why?
The number for student C (direct counts) would exceed the number for student A
(culturable count) since the direct count also measures viable but nonculturable, viable
but difficult to culture, and also non viable bacteria.
4.
Student C also extracted the same soil in peptone but let the extraction sit overnight at
room temperature before diluting and plating. How would these numbers
compare to student A’s numbers and why?
The number for student C would most likely exceed student A’s numbers due to potential
growth of bacteria within the peptone extractant.
5.
Identify the major errors associated with dilution and plating techniques.
There are many major errors, including:
a. Only 1–10% of soil bacteria are culturable.
b. No anaerobes are cultured.
c. The assumption of 1 colony arising from 1 cell is usually invalid due to cell
clumping, resulting in an underestimation of CFUs.
d. Sedimentation of soil during preparation of dilutions can lead to an
underestimation of CFUs.
6.
What is the difference between a primary cell line and a continuous cell line? Which cell
line do you think is better for isolating viruses from the environment? Why?
In primary cell culture the cells come directly from the animal and only can be passed a
limited number of times. In contrast, continuous cells lines can be passed indefinably.