BIOL2368_Microbiology_2023_Practical_Logbook_26012023-1

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AD012 ASSOCIATE DEGREE IN APPLIED SCIENCE: MICROBIOLOGY BIOL2368 2023 PRACTICAL LOGBOOK
PRACTICAL SCHEDULE Date Week Pracs & Assessments 6/2 1. Online Lab introduction 13/2 2. Prac 1: Introduction to Microbiology techniques There is no escape – they’re everywhere! 20/2 3. Prac 2: The effects of physical factors on microbial growth 27/2 4. Prac 3a: Basic Identification of Gram Positive Cocci 6/3 5. Prac 3b: Basic Identification of Gram Positive Cocci 13/3 6. Prac 4: Basic Identification of Gram Positive bacilli 20/3 7. Prac 5a: Basic Identification Tests of Gram Negative Bacilli 27/3 8. Assessment week (no prac) 3/4 9. Prac 5b: Basic Identification Tests of Gram Negative Bacilli with rapID One ™ 10/4 10. Easter Break No prac 17/4 11. Prac 6a&6b: Disinfectant & antibiotic Sensitivity Testing 24/4 12. Prac 7 Online prac An Examination of Fungi 1/5 13. Prac 8: A light loving microorganism Prac 9: Set up agar art 8/5 14. Collecting data Prac Pracs 6a,6b, 8 and 9 15/5 15. Recapping 22/5 16. Assessment week (no lecture) AD012 BIOL2368 Microbiology Practical Logbook 2023 Page 2 of 52
Contents Safety Guidelines for Students ............................................................................................ 4 Maintaining Your Logbook ................................................................................................... 5 Writing a Practical Report .................................................................................................... 6 Setting up the Microscope ................................................................................................... 8 Practical Exercise No. 1 There is no escape – they’re everywhere! .................................. 10 Practical Exercise No. 2 Everything old is new again! Build on your former skills to discover something new .................................................................................................... 17 Practical Exercise No. 3 The Effects of Physical Factors on Microbial Growth .................. 22 Practical Exercise No. 4 Basic Identification Tests of Gram positive Cocci ........................ 29 Practical Exercise No. 5 Basic Identification Tests of Gram positive bacilli ....................... 38 Practical Exercise No. 6a Basic Identification Tests of Gram Negative Bacilli ................... 44 Practical Exercise No. 6b Basic Identification Tests of Gram Negative Bacilli using Microbact ™ ....................................................................................................................... 50 Practical Exercise No. 7 Anaerobic Bacilli .......................................................................... 52 Practical Exercise No. 8a Disinfectant Sensitivity Testing ................................................. 54 Practical Exercise No. 8b Antibiotic Sensitivity Testing ..................................................... 57 Practical Exercise No. 9 A Light Loving Microorganism ..................................................... 62 Practical Exercise No. 10 Fabulous Fungi .......................................................................... 65 Appendix 1: Descriptions of colonies and cells ................................................................. 70 Appendix 2: Descriptions of broths, smears and stains .................................................... 73 AD012 BIOL2368 Microbiology Practical Logbook 2023 Page 3 of 52
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Appendix 3: Selected Biochemical Tests ........................................................................... 76 Practical Exercise No. 1 Introduction of microbiology techniques Flowcharts AD012 BIOL2368 Microbiology Practical Logbook 2023 Page 4 of 52
B C D AD012 BIOL2368 Microbiology Practical Logbook 2023 Page 5 of 52
Flowcharts E F AD012 BIOL2368 Microbiology Practical Logbook 2023 Page 6 of 52
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Part A: Acquaint yourself with the laboratory Method and Analysis – Part A Gram staining tray Ethanol Gram staining reagents Deionised water in small volumes Incubator Deionised water in large volumes Lab coat A loop Safety shower bacticinerator Large Biohazard bins Loop holder Small biohazard tubs gloves Saline Safety spectacles Table 1: Location of laboratory equipment AD012 BIOL2368 Microbiology Practical Logbook 2023 Page 7 of 52
Part B: Aseptic Transfer Results – Part B: Week 2 Table 2: Nutrient broth following incubation with disc at 37°C / 24h / O 2 Analysis – Part B: Week 2 1. Comment on the success of your aseptic technique Part C: To grow individual colonies from a swab using streaking technique Results -Part C General observations about colonies and medium Streak pattern Table 3: Growth of Staphylococcus epidermidis on HBA plate AD012 BIOL2368 Microbiology Practical Logbook 2023 Page 8 of 52
Analysis -Part C Week 2 1. Swap plates with a colleague, rate each other’s and check for consistent judging 5 star rating of my own plate 5 star rating of my colleague’s plate Table 4: Analysis of streaking effectiveness 3. How might you improve your streaking technique in the future? Part D: To grow bacteria using a lawn culture Analysis -Part D Week 2 1. Observe your agar plate and comment on whether it shows a ‘lawn’ of bacterial growth (confluent growth). AD012 BIOL2368 Microbiology Practical Logbook 2023 Page 9 of 52
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2. Does the lawn culture provide one type of bacteria that could be evaluated further using identification tests? Explain. Part E: Observing microorganisms in the environment Results – Part E Swab result (Site/ Object): Site swabbed Incubation Time/ Temperature Number of bacterial colonies on plate Number of fungal colonies on plate Gram stain Table 5: Analysis of environmental microorganisms sampled using a swab technique Settle plates (Air samples): Site sampled Incubation Time/ Temperature Number of bacterial colonies on plate Number of fungal colonies on plate Gram stain Table 6: Analysis of environmental microorganisms sampled using a settle plate AD012 BIOL2368 Microbiology Practical Logbook 2023 Page 10 of 52
Contact plate: Site contacted Incubation Time/ Temperature Number of bacterial colonies on plate Number of fungal colonies on plate Gram stain Table 7: Analysis of environmental microorganisms sampled using a contact plate Analysis - Part E Week 2 1. Comment on the significance of any organisms in relation to where they were found and the differences in sampling techniques Part F: Gram Staining Results – Part F Magnification __________ Table 8: AD012 BIOL2368 Microbiology Practical Logbook 2023 Page 11 of 52 Be Sure to include a line about Gram Stain, shape and arrangement You name the table
Analysis - Part F 1. Are your results as expected? Discuss 2. What might cause Gram positive organisms to appear as if they are Gram negative? 3. What might cause Gram negative organisms to appear as if they are Gram positive? AD012 BIOL2368 Microbiology Practical Logbook 2023 Page 12 of 52
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Part G: Ennumertaing bacteria Even though this belongs to experiment 1, due to time constraints, this will be performed in the second lab session Results – Part D 1. Count the number of colonies on your plate. Ensure that you count every colony, regardless of how small or insignificant. Dilution Factor 10 -4 10 -5 10 -6 10 -7 Colony Number 2. Calculate the number of organisms per mL of culture by multiplying the number of colonies by the dilution factor. For example if you counted 220 colonies on the 10 -6 dilution plate, 220 x 10 6 = 2.2 x 10 8 orgs/mL or CFU/mL 4. For the calculation what two factors must you consider? Perform the calculation. AD012 BIOL2368 Microbiology Practical Logbook 2023 Page 13 of 52
Analysis Part D 1. Compare your results to the rest of the class. Do you think there is a statistically significant difference between them? What are some possible reasons for any discrepancies in the results? AD012 BIOL2368 Microbiology Practical Logbook 2023 Page 14 of 52
Practical Exercise No. 2 The Effects of Physical Factors on Microbial Growth Flowcharts Part A Part B Part C Part D Part E AD012 BIOL2368 Microbiology Practical Logbook 2023 Page 15 of 52
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Part A: The effect of autoclaving on thermophiles with endospores Method - Part A – Week 2 Observe and record any appearance of transparency or turbidity. Results - Part A Autoclaved broth Broth not autoclaved DiaGram 1. A comparison of the growth of Geobacillus stearothermophilus in autoclaved and not autoclaved TSA broths Analysis - Part A 1. Why was Geobacillus stearothermophilus specifically chosen for the demonstration of the effect of autoclaving? AD012 BIOL2368 Microbiology Practical Logbook 2023 Page 16 of 52
Method - Part B – Week 2 Rank species in order of successful growth at the temperature your plate was incubated at. You may do this with + signs (eg, -, +. ++, +++) Compare these results with growth at all other temperatures. Results - Part B – growth after 1 week Species 4 0 C 25 0 C 37 0 C 55 0 C DiaGram 2. A comparison of the growth of 4 bacteria species at different temperatures (from pooled class data) Analysis - Part B 1. From the results, categorise each bacterium according to its favoured temperature to grow at. Use words like, psychrophile, mesophile, thermophile 2. Geobacillus stearothermophilus Escherichia coli Micrococcus luteus Serratia marcescens Table 1. Categorisation of bacteria according to their favoured growth temperature 3. Were there any differences in the appearance of Serratia at different temperatures? If yes, what was it and why do you think it occurred? AD012 BIOL2368 Microbiology Practical Logbook 2023 Page 17 of 52
Part C: The effect of moisture on bacterial growth 1. Incubate culture tubes at 35 0 C for 24hr Results - Part C E.coli B. subtilis DiaGram 4. Observation of the growth E.coli and G. stearothermophilus after an initial dehydration phase (week 2) E.coli B. subtilis DiaGram 5. Observation of the growth E. coli and B. G. stearothermophilus after the addition of nutrient broth (week 3) Analysis - Part C 1. Was the presence or absence of growth of E.coli and Geobacillus stearothermophilus after an initial dehydration phase as you expected? How did the addition of nutrient broth demonstrate if the E.coli or B. subtilis had survived the dehydration? AD012 BIOL2368 Microbiology Practical Logbook 2023 Page 18 of 52
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Part D: The effect of heat exposure over time on the death of range of bacteria Materials and methods - Part D This has been set up for you Materials and methods - Part E Summarise the effect of prolonged exposure to 60 0 C and 90 0 C heat on E.coli . AD012 BIOL2368 Microbiology Practical Logbook 2023 Page 19 of 52
Results and analysis - Part D Record the growth patterns of bacteria subjected to different temperatures over different lengths of time. Escherichia coli 0 mins at high temperature 10 20 40 30 0 C 65 0 C DiaGram 5. Observation of E.coli after prolong exposure to heat Record the growth patterns of bacteria subjected to different temperatures over different lengths of time. Record growth as +, ++ or +++. Highlight the boxes that show TDT Temp 60 0 C 80 0 C Heat exposure (time) 0 15 20 30 0 15 20 30 E. coli Table 2. Observation of the growth rate of E.coli after prolong exposure to heat
Part E: The effect of UV on bacterial growth Results - Part E Safety Note: UV light is a mutagen. Pay attention to the demonstration and have your questions prior to use to ensure safety. Controls (10mins) Tests Visible Light Plastic and UV 5secs 10secs 60secs 10mins Growth Table 3. Observation of the growth rate of S. marcescens after prolonged exposure to UV Analysis - Part E 1. Was there any observable difference in the halves of the plates that were covered by the lid? What are the implications of this? 2. Did visible light affect growth in any way?
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3. Why do you think S. marcescens was chosen for this practical? 4. Why were plates incubated with foil over them? AD012 BIOL2368 Microbiology Practical Logbook 2023 Page 22 of 52
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Practical Exercise No. 3 Initial identification tests for Gram positive bacteria Flowcharts Part A Part B Part C AD012 BIOL2368 Microbiology Practical Logbook 2023 Page 23 of 52
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Results and Analysis – Part A Week 1 1. Comment on the quality of the plates you have prepared. Suitability for use? Week 1 Species 1 Species 2 Species 3 Species 4 Species 5 Species 6 Gram stain, cell shape, cell arrangement Table 1: Gram stain, cell shape and arrangement AD012 BIOL2368 Microbiology Practical Logbook 2023 Page 24 of 52
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Week 1 & 2 NA (wk1) MAC (wk2) HBA (wk2) Species 1 Species 2 Species 3 Species 4 Species 5 Week 2 AD012 BIOL2368 Microbiology Practical Logbook 2023 Page 25 of 52 Table 2 continued: Colony and media observations
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2. What conclusions can you draw about the purpose of each medium? Part B: To consider how oxygen might affect the growth of Gram-positive bacteria Part B Results and Analysis Species Gram/Stain cell arrangement Species Gram/Stain cell arrangement 1 4 2 5 3 AD012 BIOL2368 Microbiology Practical Logbook 2023 Page 26 of 52 Table 2: Gram stains and cell arrangements for 5 unknown species
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Results: Parts B, and C Species 1 Species 2 Species 3 Species 4 Species 5 Gram stain Growth in O 2 Growth in AnO 2 MSA OF Analysis: Part A 1. Is there any evidence that any of the media are enriched, selective or differential? Explain. (Remember that some media may perform more than one of those functions). AD012 BIOL2368 Microbiology Practical Logbook 2023 Page 27 of 52 Table 2 Differences in the ways Gram positive cocci respond to oxygen and sugars and the various enzymes they might have Table 4: Oxygen and sugar usage in 4 unknown bacterial species
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2. Is there any medium that seemed not very valuable for growing Gram positive bacteria? Justify your answer. 3. How does HBA differ from NA and how has the addition of a particular ingredient been useful for differentiating between different Gram positive bacteria? Analysis: Part B 4. Is there any way to differentiate Species 2, Species 4 and Species 5, based upon their colony morphology on NA or their Gram stains? 5. Differentiate between the terms: obligate anaerobe, facultative anaerobe, obligate aerobe, and microaerophilic. Describe the species used in this experiment by one of those terms Species 2 AD012 BIOL2368 Microbiology Practical Logbook 2023 Page 28 of 52
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Species 3 Species 4 Species 5 Analysis: Part C 6. What two key ingredients does MSA contain that allows it to differentiate between different species of Gram positive cocci? 7. Explain the principle behind OF testing ensuring that you discuss colour change and the purpose of the oil AD012 BIOL2368 Microbiology Practical Logbook 2023 Page 29 of 52
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Practical Exercise No. 3b Initial identification tests for Gram positive bacteria Flowcharts Part A catalase Part A coagulase replacement/ agglutination Part Bi Part Bii AD012 BIOL2368 Microbiology Practical Logbook 2023 Page 30 of 52
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Results: Part A and B Species 2 Species 3 Species 4 Species 5 Catalase Coagulase/ agglutination DNase Urease Table 1 Differences in the surface antigens and enzymes and Gram positive bacteria Analysis: Part A and B 1. What is the purpose of catalase and how does it help in the identification of medically significant Gram Positive cocci? AD012 BIOL2368 Microbiology Practical Logbook 2023 Page 31 of 52
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2. What is the purpose of coagulase and how does it help in the identification of medically significant Gram positive cocci? 3. What causes the urea agar slant to change colour? What benefit does urease offer microorganisms? 4. What causes the zones of clearing on a DNase plate? What benefit does DNase offer microorganisms? AD012 BIOL2368 Microbiology Practical Logbook 2023 Page 32 of 52
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Practical Exercise No. 4 Basic Identification Tests of Gram positive bacilli Results Part A Bacillus subtilis Clostridium perfringens Corynebacterium diphtheriae Media and growth observations Microscope observations Table 1. A comparison of the colony morphology, appearance of media, Gram stain and cell arrangement for Bacillus subtilis, Clostridium perfringens & Corynebacterium diphtheriae Results Part C Bacillus subtilis Clostridium perfringens Corynebacterium diphtheriae Catalase result (presence of O 2 bubbles in water) Provided as a demo Table 2. A comparison of catalase activity for Bacillus subtilis, Clostridium perfringens & Corynebacterium diphtheriae
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Part D: Differentiation of genera of Gram positive bacteria by presence of spores Results Part D Note that vegetative cells are expected to appear pink. Endospores will be green. Bacillus subtilis (prepared by student) Bacillus sp (prepared commercially) Clostridium sp (prepared commercially) Microscope analysis Table 3 Spore analysis in Bacillus sp and Clostridium sp Part B: Variation of oxygen in the atmosphere Results Part B Bacillus subtilis Clostridium perfringens Corynebacterium diphtheriae Media and growth observations under O 2 Provided as a demo Media and growth observations under AnO 2 Provided as a demo Microscope observations to corroborate suspected identity Provided as a demo Table 4. A comparison of growth of Bacillus subtilis, Clostridium perfringens & Corynebacterium diphtheriae under O 2 and AnO 2 conditions AD012 BIOL2368 Microbiology Practical Logbook 2023 Page 34 of 52
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Practical Exercise No. 5a Basic Identification Tests of Gram Negative Bacilli Flowcharts Sugar testing Oxidase test Indole test ONPG AD012 BIOL2368 Microbiology Practical Logbook 2023 Page 35 of 52
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MR-VP Citrate Nitrate reductio n motility AD012 BIOL2368 Microbiology Practical Logbook 2023 Page 36 of 52
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Results Table 1 The results of biochemical tests done on Gram Negative Bacilli E.coli Enterobacter aerogenes Klebsiella pneumoniae Pseudomonas aeruginosa Proteaus mirabilis Glucose (dt) Sucrose (dt) Lactose (dt) Catalase Oxidase Indole ONPG MR VP Citrate Urease Nitrate reduction Motility
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Analysis for all parts of the experiment 1. In the sugar fermentation tests, what causes a colour change? 2. Assuming the MR-VP medium contains glucose, what are the bacteria producing from it if the MR test is positive? 3. If the MR test is negative, why can the VP test be positive? 4. Why does a positive catalase test result in the production of bubbles? What was the point of having a catalase test in this experiment? (note that this answer is different to the answer you gave when looking at Gram positive cocci) 5. What important genera of pathogenic bacteria apart from Pseudomonas are oxidase Positive? 6. What caused one species to be motile? 7. What results were unexpected?
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Practical Exercise No. 5b Basic Identification Tests of Gram Negative Bacilli using Microbact ™ No flow chart necessary Results – Week 1 Species number/colour on MAC Gram stain Catalase oxidase Table 1. Basic properties of species to be identified through rapID One System Draw/stick in your remel RapID ONE System score sheet for Klebsiella pneumoniae AD012 BIOL2368 Microbiology Practical Logbook 2023 Page 39 of 52
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Results – Week 2 Draw/stick in your remel RapID ONE System score sheet for unknown species Identity of unknown microorganism: Analysis 1. How did your results compare to the results of other students? Compare with at least two other groups. What do these collective results tell you about your technique? 2. How might your technique be improved? AD012 BIOL2368 Microbiology Practical Logbook 2023 Page 40 of 52
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Practical Exercise No. 6a Disinfectant Sensitivity Testing Method: Flow chart AD012 BIOL2368 Microbiology Practical Logbook 2023 Page 41 of 52
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Method: Week 2 Observe growth around disks and record Results E.coli S.epidermidis Disinfectant trade name Concentr ation Active ingredient Effect on E.coli growth Effect on S.epidermidis growth 1. Sterile water/control n/a n/a 2. iodine 3. Dettol 4. bleach 5. mouth wash 6. 70% alcohol Table 1. A comparison of the effect of different disinfectants on Gram positive and negative bacteria. Analysis 1. Which disinfectant was most effective on the Gram negative bacteria? Was this the same for the Gram positive bacteria? AD012 BIOL2368 Microbiology Practical Logbook 2023 Page 42 of 52
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2. Which disinfectant was least effective on the Gram negative bacteria? Was this the same for the Gram positive bacteria? 3. Of the disinfectants tested, which are you more likely to use on a wound? Explain. 4. Of the disinfectants tested, which are you more likely to use on a toilet seat? Explain. AD012 BIOL2368 Microbiology Practical Logbook 2023 Page 43 of 52
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Practical Exercise No. 6b Antibiotic Sensitivity Testing Method: Flow chart AD012 BIOL2368 Microbiology Practical Logbook 2023 Page 44 of 52
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Part A: Making the inoculum Results Antibiotic Zone of Inhibition (mm) for ATCC strain E.coli Zone of Inhibition (mm) for ATCC strain E.coli Table 1. Zones of inhibition for Gram negative bacteria Analysis i. Use the information is Table 2 to assist you in completing Table 3 Antibiotic Resistant Intermediate Susceptible Amikacin (30µg) ≤ 14 15-16 ≥ 17 Ampicillin (10µg) ≤ 13 14-16 ≥ 17 Cefazolin (30µg) ≤ 14 15-17 ≥ 18 Gentamicin (10µg) ≤ 12 13-14 ≥ 15 Tetracycline (30µg) ≤ 14 15-18 ≥ 19 Ticarcilin (75µg) ≤ 14 15-19 ≥ 20 Trimethoprim (5µg) ≤ 10 11-15 ≥ 16 Tobramycine (10µg) ≤ 12 13-14 ≥ 15 Table 2. Interpretative standards for E.coli and other enteric Gram negative bacilli. AD012 BIOL2368 Microbiology Practical Logbook 2023 Page 45 of 52
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ii. Complete the table using the words: resistant, intermediate or susceptible Antibiotic Zone of Inhibition (mm) for ATCC strain E.coli Zone of Inhibition (mm) for ATCC strain E.coli Table 3. Susceptibility and resistance to antibiotics in Gram negative bacteria Analysis Continued (Questions) 1. Why is Mueller-Hinton agar used for antibiotic susceptibility testing? Provide at least two reasons. 2. Why must the inoculum be used within 15 minutes (hint: think about a bacterial growth graph) 3. What difficulty might an observe face when measuring zones of inhibition for Proetus mirabilis ? AD012 BIOL2368 Microbiology Practical Logbook 2023 Page 46 of 52
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Practical Exercise No. 9 Fabulous Fungi Flowchart Part A Part B Part C Part D AD012 BIOL2368 Microbiology Practical Logbook 2023 Page 47 of 52
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Results – Part A Saccharomyces cerevisiae Candida Albicans Malassezia furfur Colony appearance Microscopic appearance Table 1. the macroscopic and microscopic appearances of some common yeasts AD012 BIOL2368 Microbiology Practical Logbook 2023 Page 48 of 52
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Results – Part B, C & D Pin mould Conidial moulds Dermatophytes Rhizopus oryzae Aspergillus fumigatus Aspergillus falvus Penicillium roquefortii Microsporum canis Trichophyton rubrum Colony appearance Microscopic appearance Table 2. the macroscopic and microscopic appearances of some common moulds AD012 BIOL2368 Microbiology Practical Logbook 2023 Page 49 of 52
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Anaylsis 1. How does the macroscopic and microscopic appearance of the fungi studied differ from bacteria you have studied? 2. What are the benefits of the three different techniques that were demonstrated? AD012 BIOL2368 Microbiology Practical Logbook 2023 Page 50 of 52
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Practical Exercise No. 10 A Light Loving Microorganism Record Flowchart in logbook AD012 BIOL2368 Microbiology Practical Logbook 2023 Page 51 of 52
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Results - Week 1 Diagram 1 Microscopic representation of Euglena. Results - Week 2 Diagram 2 Growth of Euglena under different light conditions. Analysis 1. What enables the movement of Euglena? 2. In what ways is Euglena different from most protozoa? 3. What coloured window was Euglena most attracted to? What do you think the reason for this is? AD012 BIOL2368 Microbiology Practical Logbook 2023 Page 52 of 52
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