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OCR A Level Biology A (H420) Biology B (H422) PAG 7: Microbiological techniques Practice Exam Questions and Mark Scheme

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OCR A Level Biology A (H420) Biology B (H422) PAG 7: Microbiological techniques Practice Exam Questions and Mark Scheme This resource includes exam questions from both Biology A and Biology B.... It is not designed as a topic test but is instead intended as a document to support the teaching and learning of Module 1 – Development of practical skills in biology. 1. Serial dilutions can be used to estimate the size of a bacterial population in a culture. A scientist used 20 cm3 of a bacterial culture that contained 1.0 × 106 bacterial cells. • 5% of the 20 cm3 culture was transferred to a new test tube and made up to 10 cm3 with water. • An additional ten-fold dilution was carried out, which produced a final 10 cm3 solution. • 0.1 cm3 of the final 10 cm3 solution was transferred to an agar plate. i. Each colony that developed on the agar plate was assumed to represent a single bacterial cell in the bacterial culture. Estimate the number of colonies that you would expect to develop on the agar plate. number of colonies = .......................................................... [3] ii. 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.     [1] 2(a). A student was asked to measure the population density of bacteria in a broth. The student was supplied with a broth culture of the bacterium Bacillus subtilis. The teacher suggested that the student should measure the population by transferring a sample of the broth to an agar plate then incubating the plate for 24 hours. The bacterial colonies could then be counted. The student took certain precautions to avoid contaminating the cultures. Explain how each precaution shown in the table below helped to avoid contamination. Write your answers in the spaces provided on the table. [2] (b). After incubation for 24 hours, the student studied the agar plate. The plate was completely covered by a film of bacteria and it was not possible to count colonies. Describe a modification to the procedure that would enable the student to estimate the population size. [2] 3(a). Two students investigated the growth of bacteria at different temperatures. Three flasks containing identical solutions of nutrient broth were used. • Flask 1: inoculated with 1 cm3 of broth containing the bacterium Bacillus subtilis and incubated at 20 °C. • Flask 2: inoculated with 1 cm3 of broth containing B. subtilis and incubated at 30 °C. • Flask 3: inoculated with 1 cm3 of broth containing no bacteria and incubated at 30 °C. Aseptic techniques were used throughout. At set times over the next 3 days the students removed samples from each flask and measured the number of viable bacteria present. State one further variable the students should have controlled in their investigation in order to produce valid results. [1] (b). The students used the following procedure to determine the number of viable bacteria in each flask at a given time. From each flask, 0.1 cm3 was removed and mixed with 9.9 cm3 of sterile water in a test tube. This was labelled Tube A. A serial dilution then proceeded, as shown in Table 19.1.  Tube Contents  B 1 cm3 of Tube A mixture 9 cm3 of sterile water  C 1 cm3 of Tube B mixture 9 cm3 of sterile water  D 1 cm3 of Tube C mixture 9 cm3 of sterile water  E 1 cm3 of Tube D mixture 9 cm3 of sterile water  F 1 cm3 of Tube E mixture 9 cm3 of sterile water Table 19.1 From each tube, A–F, 0.1 cm3 of mixture was cultured on nutrient agar for 24 hours at 30 °C. The results from Flask 2 after 7 hours of incubation are shown in Fig. 19. Fig. 19 The students used Tube F to calculate the number of viable bacteria present in the original sample. i. Use Tube F to calculate the number of viable bacteria present in the original 0.1 cm3 sample from Flask 2 after 7 hours of incubation. Give your answer in standard form. Answer .......................................................... [2] ii. The students disagreed about which tube’s result to use as a starting point for their calculation. Discuss whether the petri dish resulting from Tube F was the most appropriate for them to use. [3] (c). The processed results from the students’ investigation are shown in Table 19.2.  Time after  incubation  started (hours) Number of viable bacteria present in Flask 1 at 20 °C Number of viable bacteria present in Flask 2 at 30 °C 0 7.0 × 102 7.1 × 102 2 6.8 × 102 7.4 × 102 4 4.7 × 104 2.5 × 106 8 6.5 × 107 9.2 × 1010 12 2.4 × 109 1.8 × 1011 18 7.8 × 1010 1.8 × 1011 24 9.2 × 1010 5.5 × 108 36 8.6 × 1010 4.2 × 104 48 6.0 × 109 6.7 × 102 60 5.7 × 107 5.2 × 102 72 1.3 × 105 3.1 × 102 Table 19.2 i. No bacteria were detected at any time in the flask that was inoculated with nutrient broth that did not contain bacteria. Explain the purpose of this flask. [2] ii. The teacher told the students they should not investigate the growth of bacteria at 35 °C. Suggest why the teacher told them not to grow bacteria at 35 °C. [1] iii. The teacher also suggested that the students should have carried out the investigation using three flasks at each temperature. Explain how this suggestion would have improved the students’ investigation. [3] 4. Laboratory techniques are used by workers in various professions, and by scientists. A patient has been coughing blood, and it is suspected that bacteria will be found in the blood. A medical technician cultures the blood on an agar plate. What measures should the technician take to keep the agar plate culture sterile? [2] 5. i. Streptococcus pneumoniae is a Gram-positive bacterium commonly found in the throat of humans. These bacteria can spread from person to person and may cause pneumonia. What name is given to a disease that is spread between different individuals? [1] ii. A sample of S. pneumoniae was prepared for viewing under a light microscope by Gramstaining a sample taken from a colony on an agar plate. Suggest three steps that should have been taken to make sure that the procedure was carried out aseptically. 1. 2. 3. [3] 6(a). Researchers were studying the possible association between smoking and the respiratory tract infections, pneumonia and bronchitis. The study included an analysis of the infection figures for all admissions to a hospital. • Data from 5018 patients were collected. • 4022 of these patients were non-smokers. • Each patient had only one infection. Some of the data from this study are shown in Table 5.1. Number of patients admitted with a respiratory tract infection Number of patients admitted with other type of infection Pneumonia Bronchitis Smokers 335 230 431 Non-smokers 1166 526 2330 Table 5.1 A sample of bacteria from a patient with pneumonia was collected and a pure culture was prepared. A Kirby-Bauer test was carried out to allow the doctor treating the patient to decide on the correct concentration of antibiotic to use. Six different concentrations of the antibiotic were used, as shown in Table 5.2. Kirby-Bauer disc Concentration of antibiotic (a.u.) A 0.0001 B 0.0010 C 0.0100 D 0.1000 E 1.0000 F 10.0000 Table 5.2 Describe the apparatus and method necessary to produce the six different concentrations. You are supplied with an antibiotic solution of 10.0000 a.u. concentration and distilled water. [4] (b). Fig. 5.2 shows the result of the Kirby-Bauer test. Fig. 5.2 Use the information in Fig. 5.2 to advise the doctor on the most appropriate concentration of antibiotic to use. Explain your choice. [3] END OF QUESTION PAPER Mark scheme Question Answer/Indicative content Marks Guidance 1 i 50 ✓✓✓ 3(AO2.8) If the answer is incorrect, award one mark for (1,000,000 / 20 =) 50000 / 5 x 104 and one mark for (50,000 / 10 =) 5000 / 5 x 103 Examiner’s Comments This was a difficult question for many candidates, involving a multi stepped calculation, where a few candidates did not attempt. Many candidates who miscalculated either the second or third division still scored one or 2 working marks (for 50000 or 5000), and many gained full marks. OCR support The mathematical skills handbook can be used to support candidates with serial dilutions: https:/www.ocr.org.uk/Images/294471-biology-mathematical-skills-handbook.pdf ii idea of (with low colony numbers) small (random) differences (in plating) produce large errors when estimating / scaling ✓ 1 (AO3.3) DO NOT ALLOW assumes bacteria are equally distributed when removing the sample Examiner’s Comments Very few candidates recognised the implications of the low colony count for generating error when scaling up. Several candidates knew that too many dilutions had occurred, but found it difficult to express the correct idea clearly. Total 4 2 a flaming the tube causes air to expand and pushes bacteria away so less likely to settle into tube (1) kills bacteria on neck of tube (1) holding lid of petri dish over agar plate avoids infection / inoculation with bacteria in the air (1) 2 b dilute the sample taken from the colony (1) multiply result from agar plate by dilution factor (1) 2 ALLOW for serial dilution, choose the correct plate (highest number of colonies without colonies merging) Total 4 3 a 1 2 3 4 5 6 volume of broth (in flask) ✓ pH (of broth) ✓ oxygen (concentration in flask) ✓ number / concentration , of bacteria in , broth at beginning / AW ✓ volume removed (from each flask) ✓ (standard) stirring / mixing , before withdrawal of samples ✓ 1 max (AO3.4) IGNORE ‘amount’ throughout 4 ALLOW batch of broth / starting population of bacteria 4 IGNORE volume / mass Examiner’s Comments Most candidates were aware that conditions ought to be as similar as possible and around half achieved a mark by stating one of marking points 2 to 5. As usual in these questions, the vague word ‘amount’ did not achieve any credit. Irrelevant responses such as ‘size of flask’ and ‘light’ were seen but received no credit. Some candidates described a control sample and gained no credit. b i 6.0 / 6 , × 107 ✓✓ 2 (AO2.8) Max 1 if answer not given as standard form ALLOW 1 mark for 6 × 106 / 6 × 108 Examiner’s Comments Less than 20% of responses achieved both marks. Candidates fell into one of two similarly-sized groups: those who clearly knew how to calculate number of bacteria and were either correct or out by a factor of 10 or 100, and those who did not know how to approach the calculation at all. Some candidates ignored the instruction to write the answer in standard form and so were limited to 1 mark. ii 1 2 3 4 5 6 should have used E ✓ (has) most / more , (countable) colonies ✓ idea that anomalies will have smaller effect ✓ more representative / larger , sample ✓ (fewer serial dilutions) decreases chance of error ✓ F (is appropriate) because , colonies / AW , are countable ✓ 3 max (AO3.1) (AO3.4) 1 Other points can be awarded in the context of plates other than E 2-5 ora for F 4 ALLOW estimate will be more accurate 4 IGNORE valid / reliable / repeatable 6 ALLOW bacteria as AW for colonies for this marking point only Examiner’s Comments Most candidates gained at least 1 mark but it was rare for a response to achieve all 3. Many candidates thought that F was the most appropriate and this limited their score to 1 mark. Candidates who discussed reasons why F might not have been the best tube often achieved a mark for discussing diluting error or marking point 3 in some form. However, many such candidates seemed reluctant to clearly state that E would have been the best tube to use. Marking point 2 was rarely credited because few candidates used the word ‘colonies’. A significant minority of candidates seemed to think that E would be inappropriate because someone might make a mistake when counting; such responses were not credited. Candidates are advised that answers to practical questions that cite avoidable human error are not likely to attract marks. Exemplar 7 This is an example of a response that suggests that human counting error is the main concern when selecting which tube to use and hence achieves no marks. Typically, this type of response often achieved marking point 6 but this one does not explicitly state that the colonies in tube F are countable, hence the omission mark, so no marks were credited. i control ✓ idea of checking for contamination ✓ 2 (AO3.3) (AO3.4) DO NOT CREDIT control , group / variable / condition ALLOW shows growth due only to B. subtilis ALLOW e.g. to ensure conditions were aseptic / if the flask had bacterial growth the results would be invalid Examiner’s Comments Most could establish it was a control, although some confused this control with a control variable or control group. Many were able to link this to the idea of checking for unintended bacterial growth. The context should have clearly signalled the idea of contamination so, on this occasion, responses that gave generic answers about comparisons were not credited. ii idea that it could encourage the growth of human pathogens ✓ 1 (AO3.3) ALLOW harmful microbes could grow DO NOT CREDIT refs to denaturation IGNORE bacteria will grow rapidly which could be dangerous Examiner’s Comments About a third of candidates achieved this mark. Incorrect responses often referred to enzymes denaturing (which is unlikely at 35°C) or rapid uncontrollable growth. iii reduce impact of , anomalous / AW , results ✓ measure / increase / show / ensure , repeatability ✓ allow , calculation of standard deviation / (named) statistical test ✓ (calculated) mean likely to be , more accurate / closer to true value (than individual value) ✓ 3 max (AO3.3) IGNORE identify / ignore / exclude ALLOW reliability IGNORE valid / accurate ALLOW any named statistical test Examiner’s Comments Correct use of the term ‘repeatable’ and appropriate use of ‘accurate’ were able to gain credit as part of a correct explanation; in the context of this question, ‘valid’ was not relevant. Help with the correct use of the language of measurement is available in the OCR Practical Skills Handbook. Most responses achieved one mark, usually for reference to the idea of repeatability. All marking points were credited, but marking point 2 was by far the most frequently credited. It is worth reminding centres that discarding results purely on the basis of their being different from the mean is a very questionable practice. Anomalous results should only be excluded if there is a clear explanation for why they occurred. If not, while they might trigger further replicates or affect the outcome of a statistical test, they ought to be included in any calculated means. Replicates will, however, minimise the impact of any outlying data on those means. Total 12 4 two from work in an inoculating cabinet / maintain minimum plate-opening time (1) flame inoculating loop / use sterile, pipette tip / implement of transfer (1) seal the plates for incubation (1) 2 IGNORE refs to safety – question is about sterile practice. IGNORE autoclave, irradiation etc., as done before technician gets sample. Total 2 5 i communicable / transmissible ✓ 1 ACCEPT infectious / contagious Examiner's Comments Most candidates got (i) correct and many candidates answered (ii) in a way that suggested that they have carried out the practical work and discussed aseptic techniques a lot. However, three marks was rarely achieved because candidates were giving three general laboratory rules from the first mark point or candidates were writing too generally about tools or equipment being sterilised and not slides or loops. ii example of general lab hygiene ✓ idea of work close to, a Bunsen burner / UV light ✓ only lift lid slightly ✓ flame the, spreader / loop ✓ safe disposal of, slides / agar plates ✓ idea of prevention of anaerobic conditions ✓ 3 Max e.g. use of disinfectant to wash hands / bench, use sterilised slide, use of lab coats, gloves, goggles ACCEPT use lid as umbrella e.g. hypochlorite / autoclaving / bleach Total 4 6 a states apparatus, to hold solution AND measure volumes ✔ ‘F’ is, stock / initial, solution used in the (serial) dilution ✔ correct volumes ✔ correct sequence ✔ 4 Apparatus could include • suitable vessels for holding solutions, e.g. test tubes • volumetric apparatus, e.g. measuring cylinder, syringe, (graduated) pipette, burette ALLOW 10.0000AU in place of ‘F’ ALLOW description (serial) dilution of ‘F’ e.g. • measurement of one aliquot (antibiotic solution) and, adding to / mixing with, water • removal of one aliquot of new solution and, adding to / mixing with, water (repeated five times) Volumes one tenth of each solution forms the aliquot and is mixed with nine aliquots of water ALLOW ‘parts’ in place of absolute volumes IGNORE scale of volumes Sequence must result in correct dilutions being produced, i.e. start with stock solution and proceed in order Examiner’s Comments Q5(d) was well answered in the main. Some candidates however were unable to provide appropriate apparatus and/or give even the most basic of descriptions of serial dilutions. Some candidates described serial dilutions but did not give appropriate volumes to generate the concentrations of the antibiotic required e.g. gave dilutions of 50%, 25% etc. rather than by diluting by factors of 10 in each case. b E and F have same sized, zone of inhibition ✔ most appropriate (concentration) is, 1.0000 a.u. / E ✔ reason given for not choosing, 10.0000 a.u. / solution F OR reason given for choosing, 1.0000 a.u. / solution E ✔ 3 DO NOT ALLOW E is optimum concentration ALLOW • 10.0000 a.u./ solution F, is not optimal because it is wasteful / expensive • may cause (worse) side-effects • greater % of antibiotic isn’t absorbed in gut / AW • greater % is egested OWTTE • idea that it is good practice to prescribe the lowest effective dose Examiner’s Comments In Q5(e) to obtain full credit candidates needed to work through all three steps of identifying E and F as equally effective in this test, a reason for preferring E over F and a clear statement that E was the most appropriate. 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