Microbial techniques and culturingEdexcel A-Level Biology B: Subtopic test

1. 1
   A technician transfers a sample of a harmless strain of Escherichia coli from a nutrient broth culture to a nutrient agar plate using an inoculating loop, then incubates the plate at 25 °C.

   (a)

   Why is the inoculating loop heated until it is red hot and then allowed to cool before it touches the culture?

   [1 mark]

   • ATo allow bacterial colonies to form
   • BTo kill microorganisms on the loop that could contaminate the culture
   • CTo make the agar liquid
   • DTo increase the growth rate of the bacteria

   (b)

   Why is the plate incubated at 25 °C rather than 37 °C?

   [1 mark]

   • APathogens that are harmful to humans grow best near 37 °C, so a lower temperature reduces the risk
   • BBacteria cannot grow at 37 °C
   • CAgar melts at 37 °C
   • DThe bacteria would be killed at 37 °C

   (c)

   The technician worked close to a lit Bunsen burner and lifted the lid of the Petri dish only slightly. Explain why these precautions reduce contamination.

   [2 marks]

   Total for question 1: 4 marks

2. 2
   A microbiologist wants to isolate a salt-tolerant bacterium from a seawater sample that contains many species of bacteria.

   (a)

   Which medium would be selective for salt-tolerant bacteria?

   [1 mark]

   • ANutrient broth
   • BNutrient agar with no additives
   • CNutrient agar containing a high concentration of sodium chloride
   • DDistilled water with agar

   (b)

   Which statement about agar is correct?

   [1 mark]

   • AIt is a liquid broth that becomes cloudy as bacteria grow
   • BIt is the main source of carbon digested by most bacteria
   • CIt is a protein that sets at 40 °C
   • DIt is a polysaccharide that sets as a gel, so that separate colonies form on its surface

   (c)

   Describe how streak plating could be used to obtain isolated colonies of the bacterium from a mixed culture.

   [2 marks]

   Total for question 2: 4 marks

3. 3
   A broth culture of bacteria was serially diluted by transferring 1 cm³ into 9 cm³ of sterile diluent five times, giving a dilution of 10⁻⁵ in the final tube. A 0.1 cm³ sample of this final dilution was spread on nutrient agar and incubated, and 46 colonies were counted.

   (a)

   Calculate the number of viable bacteria per cm³ in the original culture. Give your answer in standard form.

   [3 marks]

   (b)

   Suggest why a dilution plating count is likely to be lower than a total cell count made using a haemocytometer, and explain why the culture had to be diluted.

   [4 marks]

   Total for question 3: 7 marks

4. 4
   A flask of nutrient broth was inoculated with a bacterium and incubated at 37 °C, and viable counts were made for 30 hours. Between hour 2 and hour 7 the number of cells increased exponentially from 2.0 × 10⁴ to 3.2 × 10⁷ cells per cm³. After hour 7 the number continued to rise more slowly, levelled off at about hour 14 and began to fall after about hour 24.

   (a)

   Describe and explain the changes in the number of viable bacteria in the four phases of the growth curve for this culture.

   [6 marks]

   (b)

   Calculate the exponential growth rate constant (in generations per hour) for the bacterium between hour 2 and hour 7, and the mean generation time in minutes. Use log₁₀ 2 = 0.301.

   [6 marks]

   Total for question 4: 12 marks

End of questions