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:
Remove 0.5 mL from original suspension (100) into a new tube containing 4.5 mL Ringer's. Mix -> This is 10-1 dilution.
Remove 0.5 mL of 10-1 suspension to further 4.5 mL -> 10-2 dilution.
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
Medium
Toothbrush
Sink
Tap-water
CBA
-3, -4
-3, -4
0
PYO
0
0
0
ME
0, -1
-1, -2
0
R2A
-2, -3
-2, -3
0
LTA37
0
0
0
LTA44
0
0
0
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/Group
Toothbrush
Sink
Tap-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.