Basic Microbiology Techniques Lecture 15 PDF

Basic Microbiology Techniques Lecture 15 Sampling for Microbial Analysis BMT_LT15_AS/CE_ExamSeries_Sep24 1  Purpose of Microbial Sampling To detect, identify or enumerate microorganisms (bacteria, fungi, viruses) present in various materials, such as food, wa...

Basic Microbiology Techniques Lecture 15 Sampling for Microbial Analysis BMT_LT15_AS/CE_ExamSeries_Sep24 1  Purpose of Microbial Sampling To detect, identify or enumerate microorganisms (bacteria, fungi, viruses) present in various materials, such as food, water, environmental samples, and clinical specimens. 2 Challenges of Microbial Sampling Techniques Microbial sampling and analysis must be carried out systematically and accurately to ensure the following: Representativeness Samples should accurately represent the larger microbial population. Microorganisms may not be evenly distributed causing samples to be unrepresentative and not reflect the true microbial composition. Sensitivity Samples may not detect low numbers of microorganisms. Consistency Too much variability in the data may not lead to meaningful comparisons. No Contamination / Interference Presence of solid particles from food, soil, etc. may interfere with detect of microorganisms. Improper handling and storage may lead to contamination and produce false results. 3 Types of Microbial Sampling Techniques Air Sampling Solid Sampling Assesses the concentration of Used to detect the presence of viable microbes in the air, such contamination in food, soil, etc.. as in hospitals or clean rooms. Liquid Sampling Surface Sampling Used to test for the presence of Used to detect the presence of microbial contamination in contamination on surfaces, liquids, including water, including equipment, utensils, beverages, and pharmaceuticals. workspaces & counter tops. 4  Air Sampling There are two types of air sampling Active Monitoring Passive Monitoring Air sampler physically draws a Agar plates are exposed to air for known volume of air through or over specific time periods. These are known a particle collection device. as “settle plates” Microorganisms are measured Microorganisms/spores settle on the in colony forming units (CFU) per agar surface, grow and form colonies. cubic meter of air. Measurement: Assess microbial Equipment used: Surface Air System contamination by counting the (SAS) Sampler or Impaction device. colonies/colony forming units (CFU). Equipment: Sterile nutrient medium plates 5  Liquid Sampling Methods Liquid / water samples may be collected from tap water, swimming pools, reservoirs, beverages, etc. Collection equipment/vessels must be sterile and stored appropriately. The following techniques may be performed in order to enumerate the microbes present in the liquid sample Direct Plating on to agar – using sterile inoculating loop / micropipette. Dilution of liquid samples before plating on agar. Membrane filtration 6  Solid Sampling Techniques Solid samples (e.g. food) undergo homogenisation - blended with a diluent to create a homogenized / uniform mixture. Dilutions of the homogenised mixture are plated on sterile agar media. Required equipment: Blender / Stomacher 7  Surface Sampling Techniques Swab Sampling Method Swabs are used to collect samples from surfaces over at fixed area. Swabs are streaked onto agar plates or immersed in liquid media. Contact Plates Method Agar plates (contact plates) are pressed onto surfaces to collect microbial samples. Tape Lift Method: Adhesive tape is applied to surfaces and then transferred to agar plates. Colonies / CFUs are counted after incubation. 8 Proper Labelling Practices for Microbial Sampling Each sample container should be labelled with the following information: Sample ID/Code: - To act as a unique identifier. Sampling Date and Time: - To help track sample history. Sampling Location/Place of collection - Where the sample was collected. Nature/type of Sample: - Air, liquid, food or surface. Storage condition (e.g. temperature) or Medium Used: - If applicable. 9 Aseptic Sampling Designed by Freepik Image: https://www.freepik.com/free-vector/hand-drawn-germs-cartoon-illustration_49545897.htm 10  Aseptic Sampling Maintaining aseptic conditions and practising aseptic techniques* critically important in sampling for microbial analysis Use of sterile sampling equipment & tools to ensure sterility Perform any necessary sterilisation of Photo by Pexels equipment & tools (if reusable) 11  Why is aseptic handling so important in the sampling process for the purpose of microbial analysis? Compliance with Regulatory Standards Many industries, such as food production, pharmaceuticals, and healthcare, are subject to strict regulatory standards governing microbial analysis. Aseptic techniques mandated to ensure compliance with regulatory standards to uphold:  quality and safety of products (eg, food, drugs)  effectiveness of environmental monitoring (eg. testing of soil, sea water) 12  Why is aseptic handling so important in the sampling process for the purpose of microbial analysis? Preventing Contamination of Sample Aseptic techniques ensure sample collected is not contaminated by unwanted microorganisms and substances from:  environment  person collecting the sample  other samples (cross-contamination) 13  Why is aseptic handling so important in the sampling process for the purpose of microbial Preventing Contamination of Sample analysis? Microbial integrity of sample refers to profile (presence/absence, identity, characteristics) of microorganisms in sample Aseptic techniques maintain integrity of microbial population being studied by preventing introducing contaminants that can:  alter composition of microorganisms in sample (addition of other microbes)  affect viability of microorganisms in sample (eg. addition of 14  Why is aseptic handling so important in the sampling process for the purpose of microbial Preventing Contamination of Sample analysis? Contamination introduces errors in analysis results, leading to inaccurate conclusions about microbial population present in sample Contamination make it harder to obtain reproducible analysis results; comparisons of results between different samples becomes unreliable Important to obtain accurate, reliable and meaningful microbial analysis data to help make decisions in various fields, eg. healthcare, food safety, environmental monitoring, biotechnology 15  Why is aseptic handling so important in the sampling process for the purpose of microbial analysis? Protecting Personnel & Environment Protect laboratory personnel and surrounding environment from exposure to potentially harmful microorganisms in samples. Particularly important when dealing with samples that may contain pathogenic and hazardous microorganisms. 16 Pre- Analysis Sample Processing & Handling Designed by Freepik Image: https://www.freepik.com/free-vector/hand-drawn-germs-cartoon-illustration_49545897.htm 17  Pre-Analysis Sample Processing & Handling Before microbial analysis, proper sample treatment, preservation, transport and storage are essential to preserve microbial integrity of samples and ensure accurate results Adhere to standard protocols and regulatory guidelines to ensure quality and reliability of microbial analysis data 18  Sample Treatment Grinding or blending  Solid samples may need to be ground or blended to extract or release microorganism from sample Homogenization  If sample is heterogenous, thorough mixing is needed to obtain representative portion for analysis  Eg. pathogenic microbes not necessarily homogenously distributed throughout different regions (rim, core) of sausage  An example of homogenization: Stomaching (blending by movable paddles) Filtration  Some samples, such as water or air, may require filtration to concentrate 19  Sample Storage and Transport Example of a sampling process flow (clinical sample) Samples collected may need to be transported to another testing site (different from sampling site) for analysis  Samples to be preserved appropriately during transportation Samples may need to be stored after collection (pending transportation) or upon arrival at testing site (pending analysis); https://pocketdentistry.com/use-of-the-microbiology-laboratory/ samples to be persevered and20  Preservation Methods Refrigeration  For short-term storage, samples refrigerated at temperatures typically between 2-8°C  Slow down microbial growth; preserve microbial integrity of samples Freezing  For long-term storage, samples frozen at temperatures below -20°C  Effectively halting microbial growth; preserve microbial integrity of samples for extended periods Chemical Preservatives  Adding chemical preservatives (eg. sodium azide, ethanol, glycerol) can inhibit microbial growth and preserve microbial integrity of samples 21  Transport Methods Insulated containers  Use insulated containers to maintain stable temperatures during transportation, especially for samples that require refrigeration or freezing. Cold Packs / Dry Ice  Use cold packs to keep samples at refrigeration temperature during transit  Use dry ice to keep samples at freezing temperature during transit Secure Packaging  Ensure samples are securely packaged to prevent leakage or contamination during transportation.  Use leak-proof containers and seal them properly. 22  Storage Conditions Temperature Control  Store samples at recommended temperature (eg. refrigerator, freezer) based on the chosen preservation method (refrigeration or freezing) to prevent microbial growth or sample degradation. Light Protection  Protect light-sensitive samples from exposure to direct sunlight or artificial light by storing them in opaque containers or dark storage areas, or by wrapping containers with aluminium foil  Light can kill photosensitive microbes In Air-Tight Containers  Store samples in air-tight containers to prevent contamination of sample and minimize exposure23to the environment that could affect microbial integrity of sample  References Understanding and Evaluating Microbiological Sampling and Testing https://www.fsis.usda.gov/sites/default/files/media_file/2020-08/1-EIOA-Micro-Sampling-Testing.p df Microbiological analysis and testing at Campden BRI https://www.campdenbri.co.uk/services/microbiological-testing.php Food Sampling - Microbiological - Environmental Health https://www.eh.org.au/associations/best-practice-manual/119-food-sampling---microbiological Food Safety and Standards Authority of India: https://fssai.gov.in/upload/uploadfiles/files/Notice_Comments_Guidelines_Sampling_Microbiology _14_10_2021.pdf Cropnuts.com https://cropnuts.helpscoutdocs.com/article/909-sampling-method-of-water-for-microbiology-testing 24