Food Microbiology Lab and Aseptic Techniques (Experiment 1)

# Experiment No: 01: Introduction of food microbiology lab and aseptic techniques. ## Principal: Introduction to Food Microbiology Lab emphasizes the crucial role of microorganisms in food safety and quality. Safety, equipment familiarity, and mastery of microbial techniques are key. Proficiency i...

# Experiment No: 01: Introduction of food microbiology lab and aseptic techniques. ## Principal: Introduction to Food Microbiology Lab emphasizes the crucial role of microorganisms in food safety and quality. Safety, equipment familiarity, and mastery of microbial techniques are key. Proficiency in media preparation, sterilization, and aseptic sampling is crucial. Quality control measures and meticulous documentation ensure experiment reliability. Aseptic techniques involve personal hygiene, surface disinfection, and precise transfer techniques to prevent contamination. Flame sterilization, Biological Safety Cabinet use, and maintaining aseptic conditions during experiments are vital. Integrating these principles equips students with essential skills for accurate food microbiology analyses and cultivates a strong foundation for scientific inquiry. ## Introduction to Microorganism: ### Microorganisms or microbes (can't see with naked eye) | | bacteria | viruses | fungi | algae | protozoa | archaea | | :-- | :------------------------------------------------------------ | :---------------------------------------------------------------------- | :-------------------------------------------- | :------------ | :-------------------------------------------------------- | :------------------------------ | | | - good & bad types | - only BAD parasite | - mold & yeast | - tiny plants | - multi-cellular & found everywhere! | - survive in extreme environments | | | - single cell | - 100x smaller than bacteria | | | | science with Bobert | | | - eat what's around them | - vitamin C kills | | | | | | | - antibiotics are a bacteria killing bomb! | - vitamin C kills | | | | | | | - vitamin C kills | | | | | | # Laboratory Safety: ## 1. Basic Safety Rules and Guidelines: - Know Your Environment: Familiarize yourself with the lab layout, emergency exits, and safety equipment. - Follow Procedures: Adhere to established protocols and procedures for handling chemicals, equipment, and biological materials. - Labeling and Storage: Properly label and store chemicals, ensuring compatibility and preventing accidents. - Waste Disposal: Dispose of waste materials according to guidelines, minimizing environmental impact. ## 2. Personal Protective Equipment (PPE): - Eye Protection: Safety goggles or glasses shield eyes from chemical splashes and debris. - Lab Coats: Protect clothing and skin from spills and splashes. - Gloves: Prevent skin contact with hazardous substances. - Closed-toe Shoes: Ensure foot protection against spills or falling objects. # II. Laboratory Equipment: ## A. Microscopes: Microscopes are essential tools for magnifying and observing microscopic organisms. Proper use involves adjusting objectives, focusing, and using appropriate illumination techniques for accurate specimen examination. ## B. Incubators: Incubators provide controlled environments for cultivating microorganisms. Temperature, humidity, and airflow regulation create optimal conditions for microbial growth, facilitating experiments like culturing and studying bacterial colonies. ## C. Autoclaves: Autoclaves sterilize equipment and media using high-pressure steam. Ensuring complete sterilization, autoclaves eliminate microbial contamination, guaranteeing aseptic conditions crucial for reliable experimental outcomes. ## D. Biological Safety Cabinets: These cabinets maintain a sterile workspace when handling hazardous materials. Airflow and filtration systems protect both the operator and the experiment, ensuring safety in microbiological work with pathogenic or genetically modified organisms. # III. Surface Disinfection - Cleaning and disinfection of work surfaces - Use of disinfectants and their concentrations - Maintaining a clean workspace # IV. Aseptic Transfer Techniques: ## A. Aseptic Pipetting: Aseptic pipetting involves precise and sterile transfer of liquids to avoid contamination. Using sterile pipettes, practitioners ensure accurate volume measurement while preventing the introduction of unwanted microorganisms. ## B. Inoculation of Media: Inoculating media requires aseptic techniques to introduce microorganisms without introducing contaminants. Proper handling and aseptic procedures during this step are critical for the success of microbial culture experiments. ## C. Handling and Transferring Cultures: When handling cultures, aseptic practices minimize the risk of contamination. Techniques involve careful manipulation and transfer of microbial samples to maintain purity and integrity throughout experiments. # V. Introduction to media: Culture media, also known as growth media, are specific mixtures of nutrients and other substances that support the growth of microorganisms such as bacteria and fungi (yeasts and molds). ## Types of culture media: Two types- 1. **Pure culture:** It is a culture in which definite species of bacteria or organisms can grow on a laboratory media. It is a population of cells or multicellular organisms growing in the absence of other species or types. A pure culture may originate from a single cell or single organism, in which case the cells are genetic clones of one another. 2. **Mixed culture:** It is a culture in which different species of bacteria or organisms can grow on a laboratory media. Mixed-culture are those in which the inoculum always consists of two or more organisms. Mixed cultures can consist of known species to the exclusion of all others, or they may be composed of mixtures of unknown species. | | Based on physical state | Culture media Based on Ingredients/ | Based on oxygen requirement | | :-------- | :------------------------ | :------------------------------ | :---------------------------- | | | Solid | Simple | Aerobic | | | Semi-solid | Complex | Anaerobic | | | Liquid | Synthetic/Define | | | | | Special | | ## Based on Physical state: ### Solid Media: - They are useful for isolating bacteria or for determining the characteristics of colonies - The solidifying agent is usually agar which at concentrations of 1.5-2.0 percent forms firm, transparent gels that are not degraded by most bacteria - Silica gel sometimes used as an inorganic solidifying agent for autotrophic bacteria - Colony morphology, pigmentation, hemolysis can be appreciated. - Eg: Nutrient agar, Blood agar ### Semi Solid: - Prepared with agar at concentration of 0.5% or less, have a soft custard like consistency - Useful for the cultivation of microaerophilic bacteria or for determination of bacterial motility - Eg: Motility medium ### Liquid media - no agar. - For inoculum preparation, Blood culture, continuous culture. - Eg: Nutrient broth ## Based on Ingredients: 1. **Simple Media/ Basal Media:** Most common in routine diagnostic laboratories Eg: Nutrient Broth, Nutrient Agar 2. **Complex Media:** They have added complex ingredients such as yeast extract or casein hydrolysate, which consist of a mixture of many chemical species in unknown proportions. It provides special nutrients and exact Chemical compositions are unknown. Example: Blood Agar 3. **Synthetic media:** Media prepared from pure chemical substances. Its exact composition is known. It is used for special studies, eg. metabolic requirements. Eg: peptone water- (1% peptone + 0.5% NaCl in water) ## Different special media: 1. **Selective media:** Media where the inhibitory substance is added to a solid media to inhibit commensal or contaminating bacteria like antibiotics, dyes, chemicals, alteration of pH is called selective media. It helps to increase in number of colonies of desired bacterium Eg: Desoxycholate citrate medium for dysentery bacilli, Mac Conkey's medium for gram negative bacteria, TCBS – for V. cholerae, Lowenstein Jenson (LJ) medium – M. tuberculosis, EMB- E. coli 2. **Differential media:** They are designed in such a way that different bacteria can be recognized on the basis of their colony color. Dyes and metabolic substances are added to the media so that those bacteria utilize them appeared as different colony color. Eg: Mac Conkey's medium for distinguishing lactose fermenter and non-lactose fermenter bacteria. Lactose fermenter provide pink colonies and non-lactose fermenter provides colorless colonies. XLD agar media for differencing Salmonella and Shigella. Salmonella species: red colonies, some with black centers. The agar itself will turn red due to the presence of Salmonella type colonies. Shigella species: red colonies. Coliforms yellow to orange colonies. 3. **Transport media:** Media used for transporting the samples. Delicate organisms may not survive the time taken for transporting the specimen without a transport media. Eg: Stuart's medium non nutrient soft agar gel containing a reducing agent & charcoal used for Gonnococci. Buffered glycerol saline – enteric bacilli