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LANCASTER UNIVERSITY

DEPARTMENT OF BIOMEDICAL AND LIFE SCIENCES
BIOL274: MICROBIOLOGY TECHNIQUES
LENT TERM 2026 PRACTICAL GUIDE
Module Organiser: Prof Jackie Parry

INTRODUCTION TO MICROBIOLOGY TECHNIQUES

  • Overview:
    • Introduction to microbiological techniques involving handling material that can cause diseases in humans. Importance of personal responsibility in ensuring safety in the laboratory.

SAFETY INSTRUCTIONS

  • Personal and Environmental Hygiene:
    • Essential to prevent infection and contamination of cultures.
    • Rules to Observe:
      • Always wear a lab coat and gloves.
      • No eating, drinking, or use of mobile phones in the laboratory.
      • Long hair must be tied back.
      • Keep broken skin covered.
      • Wear goggles during practicals 2 and 3.
      • Always wash hands before leaving the lab.
      • Keep bench space clean and tidy.
      • If a spill occurs, ask a demonstrator for cleanup instructions.

CONTROL OF SUBSTANCES HAZARDOUS TO HEALTH (COSHH)

REGULATIONS 1988
  • Assessment of Work:
    • Department: Biomedical and Life Sciences
    • Personnel involved: Undergraduates
    • Laboratory: Biology Teaching Lab B
    • Experimental Procedure: Isolation and identification of bacteria using various microbiological techniques.

DETAILS OF MICRO-ORGANISMS

  • Micro-organisms Likely to be Present:
    • ACDP Hazard Group
      • Group 1: Acinetobacter baumannii, Aeromonas hydrophila, Chromobacterium violaceum, Citrobacter braakii, C. freunii, Cronobacter sp., Escherichia coli, Pantoea sp., Raoultella ornithinolytica, Pseudomonas luteola, Serratia idorifera, S. liquefaciens, S. marcescens, S. rubidaea.
      • Group 2: Bacillus cereus, Enterobacter cloacae, E. gergoviae, Klebsiella oxytoca, K. pneumoniae, Pasteurella pneumotropica, Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus.

SPECIFIC CONTROL MEASURES

  • Aseptic Technique: Key to minimizing contamination during microbiological work.
  • Methods of Disinfection:
    • Surfaces (e.g. benches): Trigene diluted 1:10.
    • Equipment: Chloros.
    • Hands: Disinfect with soap and hot water.
    • Disposal Methods:
      • Solid waste: Autoclaving before disposal in clinical waste bins.
      • Liquid waste: Soak in sink with disinfectant then dispose down the drain.

PRACTICAL 1: INTRODUCTION

  • Objectives:
    • Assess competency in:
      • Autonomous sampling.
      • Following detailed written instructions.
      • Performing 10-fold dilutions.
      • Preparing spread plates.

SAMPLING

  • A) Collecting Samples:
    • Homes: Separate sheet provided on Moodle.
  • B) Preparing Suspensions:
    • Toothbrush Sample:
      • Detach toothbrush from base.
      • Place into a 50 mL Falcon tube with 10 mL Ringer's solution.
      • Sonicate for 10 minutes, remove toothbrush head using forceps.
    • Sink Sample:
      • Label bijou bottle with initials, vortex for 3 minutes, remove swab using forceps and squeeze out suspension.
    • Tip Water Sample:
      • Label tube with initials.
      • Dispose of toothbrush handle in normal waste; place head and swab in autoclave bag.

PROCESSING SAMPLES

  • C) Processing of Samples: Start with Sink Sample:
    • Ca) Diluting the Sample:
      • Perform 10-fold dilutions.
      • Designate original suspension as 100 (undiluted).
      • Procedure for dilution:
      1. Remove 0.5 mL from original suspension (100) into a new tube containing 4.5 mL Ringer's. Mix -> This is 10-1 dilution.
      2. Remove 0.5 mL of 10-1 suspension to further 4.5 mL -> 10-2 dilution.
      3. Continue until 10-4 dilution is achieved.
    • Cb) Collecting Agar Plates and Labelling:
      • Collect specified plates, noted below.
      • Plates:
      • 2 x PYO (Pseudomonas agar) - Incubate at 37°C for 24 hours.
      • 4 x CBA (Columbia Blood Agar) - Incubate at 37°C for 24 hours.
      • 4 x R2A (Reasoner's 2A Agar) - Incubate at 25°C for 6 days.
      • 4 x ME (Malt Extract Agar) - Incubate at 25°C for 6 days.
      • 4 x LTA (Sodium Lauryl Tryptose Agar) - Incubate at both 37°C (coliforms) and 44°C (faecal coliforms) for 24 hours.
      • Cc) Labelling Plates with Sample Under Investigation:
      • Label each plate (underside) with 'S' for sink sample and initials.
      • Cd) Labelling with Relevant Dilutions:
      • Follow Table 1 for which plates to inoculate from each dilution.
Table 1: Dilutions of Suspension from which Two Spread Plates Should be Prepared
MediumToothbrushSinkTap-water
CBA-3, -4-3, -40
PYO000
ME0, -1-1, -20
R2A-2, -3-2, -30
LTA37000
LTA44000
  • Ce) Inoculating Plates:
    • Inoculate each plate with 0.2 mL of corresponding dilution.
    • Spread suspension evenly across agar surface.
    • Allow plates to dry.
    • Tidy workspace: disinfect test tubes, dispose of used spreaders.
  • Cf) Incubating Plates:
    • Once dry, replace lids and incubate as follows:
      • LTA44: 44°C incubator.
      • PYO, LTA37, CBA: 37°C incubator.
      • R2A and ME: 25°C incubator.

PRACTICAL 2: INTRODUCTION

  • Objectives:
    • Determine the colony-forming units (CFU) of different microbial groups.
    • Identify coliform isolates using the API 20E system.
    • Prepare a streak plate for practical 3.
    • Aseptic techniques will be mandatory due to handling cultured organisms.
DATA COLLECTION
  • Process:
    • Retrieve inoculated plates from last week.
    • Sort plates into respective groups (toothbrush, sink, tap water) without opening Petri dishes.
    • Select the optimal dilution for counting (30-50 colonies preferred).
    • Count colonies on replicate plates, calculate average.
    • On ME plates, separate counts for filamentous fungi (hairy) and yeasts (matt-like).
    • Calculate CFU mL-1 for each microbial group in original samples, complete Table 2.
    • Calculation: Average colonies x 5 (for 0.2 mL plated) ÷ dilution (e.g. for 10-2 dilution, divide by 100).
Table 2: Colony Forming Units (CFU) mL-1 in the 100 Suspensions
Medium/GroupToothbrushSinkTap-water
CBA
R2A
PYO
LTA37
LTA44
ME Yeast
ME Filamentous
Further Calculations for CFU
  • Toothbrush: CFU mL-1 = CFU head-1 after deriving from original suspension.
  • Sink Surface: CFU mL-1 directly converts to CFU cm-2.

PRACTICAL 3: BACTERIAL IDENTIFICATION

  • Classical Techniques:
    • Involves biochemical tests for identification which can be time-consuming.
    • The API method simplifies this by combining multiple tests on one strip.
    • API 20E System:
      • Contains 20 mini-test tubes.
      • Utilizes saline suspension for inoculation and incubation (18-24 hours at 37°C).
      • After incubation, read the color reactions and convert them to a seven-digit code entered into the manufacturer's database.
      • Highly reliable in food and clinical labs.
Bacterial Isolation Methodology
  • Selection of Colony:
    • Choose a large colony from LTA agar; if absent, select from R2A agar.
  • Oxidase Test:
    • Use an oxidase strip to identify if the bacterium is oxidase positive or negative; continue until an oxidase negative strain is found.
  • Preparation of Streak Plate:
    • Prepare on nutrient agar, label appropriately, and incubate at 37°C.
  • Bacterial Suspension for API:
    • Inoculate 6 mL of Ringer's solution, prepare API strip for bacterial testing.
    • Ensure anaerobic conditions for specific tests by adding mineral oil.
    • Incubate API strips for two days at 37°C.
    • Dispose of bacterial suspension in disinfectant sink.

CELL-CELL SIGNALING

  • Understanding Signaling in Bacteria:
    • Some Gram-negative bacteria signal using N-acyl-homoserine lactones (HSLs).
    • Detection of these molecules by mutant of Chromobacterium violaceum (CV026), which lacks HSL production ability but can respond to HSLs by producing purple pigment.
    • Selection of colonies from previous plates to test for signaling; includes positive and negative controls for experiment validation.

Experimental Process

  • Grid Pattern on LB Plate:
    • Aseptically streak CV026 growth into grid pattern for testing.
    • Label plates appropriately for identification.

PRACTICAL 4: TESTING DISINFECTION AND ANTIBIOTIC SUSCEPTIBILITY

Disinfection Testing

  • Objective:
    • Validate effectiveness of mouthwash against identified bacteria by determining Log10 reduction in CFUs.

Experimental Procedure:

  • Prepare bacterial suspension from selected coliforms or Bacillus cereus.
  • Conduct tests comparing the effect of mouthwash (test) and sterile water (control).

Antibiotic Susceptibility Testing

  • Purpose:
    • Determine streptomycin sulphate concentration using the agar-well diffusion method.
    • Compare zones of inhibition for test solution against known standards (2.5, 5.0, 10 µg mL-1).
Methodology:
  • Seed agar with bacterial suspension, create wells for solution placements, ensure proper diffusion conditions before incubation.

COMMUNICATING SCIENCE THROUGH BIOART

  • Definition and Relevance:
    • Effective communication of scientific concepts can use visual arts; bioart uses live microorganisms on agar plates.
    • Colonies display diverse biochemical interactions, exceeding mere aesthetic appeal.

Practical Application of Bioart

  • Microorganisms Selected:
    • Example microorganisms from practical investigations carry attributes relevant to demonstrating key scientific concepts like quorum sensing.
  • Experimental Design:
    • Inoculate agar plates creatively to depict specific scientific principles.
    • Showcase interactions such as antibiosis or signaling patterns among bacteria.

PRACTICAL 5: SUBMISSION OF RESULTS

  • Prepare all data, including CFU calculations, antibiotic susceptibility results, and bioart display, for review and submission to Prof. J Parry.
SCHEDULE FOR TASK COMPLETION
  • Ensure all samples and experimental results are comprehensively documented before leaving the lab.
  • Lab Procedures:
    • Tidy workstations, disinfect, and place all spent plates in autoclave bags for proper disposal.