Culturing microorganisms

Culturing microorganisms on nutrient agar requires aseptic techniques.

What is not part of a standard aseptic procedure when culturing microorganisms?

A Disinfect the work area before starting.

B Ensure equipment does not touch any unsterilised surfaces before use.

C Pass the agar plate through a Bunsen flame before use.

D Work near a Bunsen flame.

Aseptic conditions are important when culturing microorganisms.

Which option is not a correct part of the procedure for spreading bacteria on an agar-filled Petri dish?

A Remove the lid of the Petri dish and rest it upside-down on the work surface.

B Replace the lid of the Petri dish and secure it with tape.

C Sterilise the area surrounding the experiment with disinfectant.

D Work near a Bunsen flame to create an upward draft of air.

A standard growth curve for a population of bacteria of the same species in a closed system involves a death phase.

Which option explains the death phase?

A Interspecific competition for nutrients, such as glucose, is high.

B Intraspecific competition for resources, such as oxygen, begins.

C Production of toxic waste products is at its highest level.

D Reproduction stops due to exhaustion of resources.

Escherichia coli is a bacterium that is used widely in scientific experiments and in biotechnology.

This is a transmission electron micrograph of E. coli.

Name the structure labelled A.

Based on your knowledge, estimate the diameter of the E. coli cell.

Give your answer in m.

A student carries out a serial dilution of an E. coli culture.

This is the method the student uses:

• Transfer 10 cm³ of E. coli culture to a sterilised test tube from an original culture that has a volume of 50 cm³.

• Carry out four 10-fold serial dilutions. Each dilution involves transferring 1 cm³ of culture from one test tube to another test tube containing 9 cm³ of distilled water.

• Transfer 1 cm³ of the final 10 cm³ diluted culture to an agar plate. Evenly spread 1 cm³ of liquid across the plate using a sterilised spreader.

• Use a micropipette for each transfer.

• Incubate the agar plates for 24 h.

• Count the number of colonies that develop on the plate. Each colony is assumed to develop from a single bacterium.

• Estimate the E. coli population in the original 50 cm³ culture.

Describe two improvements to the student’s method, other than using different equipment, that could improve the accuracy of their population estimate.

The student counts 22 colonies on the agar plate.

Calculate the total number of E. coli cells in the original 50 cm³ culture.

Give your answer in standard form.

Total number of E. coli cells in 50 cm³ =........

E. coli can be genetically modified to produce useful proteins.

State two other reasons why E. coli is a suitable microorganism to use in biotechnology.

The graph below shows the population of viable bacteria in a flask over 48 hours.

Which time, A to D, has the highest bacterial death rate?

Some students investigated the effect of time on the growth of bacterial populations.

The students prepared a large flask of bacterial culture.

They divided this large culture into a number of smaller flasks each containing 50 cm³ of bacterial culture.

They then incubated the smaller flasks at 20 °C for up to 48 h.

Every 4 h the students removed one of the flasks and counted the bacteria.

The students recorded the total number of bacteria and the number of viable bacteria in each flask.

When growing bacteria in culture, it is important that aseptic techniques are used.

State why it is important that the technique used for culturing microorganisms be aseptic.

The students prepared the culture by adding a suspension of bacteria to a flask containing nutrient broth.

List two precautions that should be taken when preparing a bacterial culture in order to ensure that the procedure is aseptic.

When counting the number of bacteria, the students performed serial dilutions on samples removed from each small flask. In each serial dilution, the students removed 0.1 cm³ and added it to 9.9 cm³ of water.

To estimate the total number of bacteria, the students used a light microscope to count the number of bacterial cells in a 0.01 cm³ sample of the final serial dilution.

To estimate the number of viable bacteria, the students spread 0.1 cm³ of the final serial dilution on an agar plate and counted the number of colonies that had grown after 24 h.

The students shook each flask before they removed the samples for counting.

Suggest why the students shook the flasks.

It can be more difficult to count bacterial cells using a light microscope than it is to count human cells.

Suggest one reason why bacterial cells are difficult to count using a light microscope.

In one 0.01 cm³ sample the students counted 52 bacterial cells under the microscope. (iii)

Describe the calculation steps the students would then need to make to estimate the total number of bacteria in the small flask.

The graph shows the students’ results.

Explain, with reference to the graph, why the students used a logarithmic scale on the y-axis.

Calculate the percentage decrease in the viable population between 40 h and 48 h.

Give your answer to 3 significant figures.

Explain, with reference to the graph, the decrease in the population of viable bacteria between 40 h and 48 h.

Bacteria and fungi can be used to make food for human consumption. The use of microorganisms in food production creates fewer ethical issues than the use of animals.

On an industrial scale, microorganisms can be cultured using either batch fermentation or continuous fermentation.

The table below lists statements about industrial culturing of microorganisms.

Place ticks (✓) in the table to indicate whether each statement applies to batch or continuous fermentation.

A student carried out a different serial dilution to estimate the size of another bacterial population.

The serial dilution resulted in four colonies developing on an agar plate.

Explain why the student’s estimation of this bacterial population is likely to be inaccurate.

Some microorganisms can be used by humans in industry. Some microorganisms are pathogenic.

Pathogenic microorganisms are transmitted in various ways.

Complete the following passage about the transmission of pathogenic microorganisms using the most appropriate terms.

Some pathogens are carried between host organisms by animals, which are often insects.

These animals suffer no symptoms of the disease and are known as........

Other pathogens, such as P. infestans that causes potato blight, produce reproductive structures

called........, which can be carried on air currents to infect other hosts.

Bread contains a mixture of polypeptides known as gluten.

Gluten consists of two types of polypeptide: gliadins and glutenins.

The table below contains statements about the structures of gluten polypeptides.

In the boxes next to each statement, write the level of protein structure (primary, secondary, tertiary, or quaternary) to which the statement refers.

Coeliac disease is caused by an immune reaction to gliadins in a person’s digestive system. The immune system produces antibodies that bind to part of the gliadin polypeptides, which causes inflammation.

Some people who stop eating foods that contain gluten still occasionally experience the symptoms of coeliac disease.

What can you conclude about:

• the structure of the antibody that causes coeliac disease; and

• what the antibody binds to when producing the symptoms of coeliac disease?

Gluten helps to trap carbon dioxide within bread dough. This enables bread to rise when it is baked.

The carbon dioxide is produced by baker’s yeast, Saccharomyces cerevisiae. This species of yeast is able to convert ethanol to acetyl CoA at low glucose concentrations.

Fig. 2 shows the oxygen consumption and carbon dioxide production of a population of S. cerevisiae grown in batch culture. The population was provided with glucose as their only initial source of carbon.

Fig. 2

Suggest and explain what conclusions can be drawn from Fig. 2 about the factors that affected the rate and type of respiration carried out by S. cerevisiae in this batch culture.

Describe two practical considerations to ensure the S. cerevisiae population grows successfully when the initial culture is established.

A group of students were designing an experiment to investigate the effect of temperature on the respiration rate of S. cerevisiae.

Their planned method included the following:

• S. cerevisiae yeast suspension will be divided into six equal volumes to form the experimental groups.

• Six temperatures will be tested: 15 °C, 20 °C, 25 °C, 30 °C, 35 °C and 40 °C.

• Beakers of S. cerevisiae will be placed in water baths to control the temperature.

• Respiration rate will be measured by using a pH probe to monitor changes in the pH of the suspensions.

• The experiment will be repeated four times.

Evaluate whether the students’ method is likely to produce valid results.

The students used a Student’s t-test to compare the results at 30 °C and 35 °C.

They calculated a t value of 2.200.

The critical value for p = 0.05 is 2.306.

Assuming their final method was valid, what can the students conclude from the result of the t-test?