MBIO 251 Lab 1 Notes: Lab Safety, Aseptic Techniques, and Streak Plate

Lab Safety Overview

• Lab safety is important to protect:

  • a) you from the microorganisms that you are working with in lab

  • b) people inside the lab from the microorganisms that you are working with in lab

  • c) people outside the lab from the microorganisms that you are working with in lab

  • d) your experiment from microorganisms naturally present in and on the human body

  • e) your experiment from microorganisms naturally present in environmental locations (e.g., soil, air, and water) and on other organisms

• A full list of lab safety guidelines and procedures is covered in a separate document available on Canvas (read that document):

  • a) Store backpacks and personal items on the windowsills

  • b) Disinfect lab benches upon arrival and before leaving lab

  • c) Wash hands after disinfecting bench before and after lab

  • d) Return lab common items to the appropriate drawer

  • e) Place used cultures and materials as well as old media onto the discard cart

  • f) Place all new media into the walk-in incubator

  • g) Adhere to proper aseptic techniques and treat all bacteria as a potential pathogen

  • h) Alert lab instructor if you spill or drop a culture, break any glassware, or make a mistake

• Types of Microorganisms

  • Cellular forms of microbial life

  • Prokaryotic cells

    • Bacteria and Archaea

    • Cells do not contain a nucleus or organelles

    • Cells are $10\times$ smaller than eukaryotic cells

  • Eukaryotic cells

    • Yeasts, Molds, Algae, and Protists

    • Cells contain a nucleus and organelles

    • Cells are $10\times$ larger than prokaryotic cells

  • Acellular forms of microbial life

  • Phage and Viruses

    • Phage typically infect either Bacteria or Archaea

    • Viruses typically infect a eukaryotic organisms/cells

  • Prions are infectious proteins that only cause disease in animals

  • Viroids are infectious RNAs that only affect plants

• Ubiquity of Microorganisms

  • Microorganisms are almost everywhere in the world

  • In soil, air, and the atmosphere

  • In freshwater, seawater, and polar ice

  • Inside and on humans, other animals, and plants

  • Locations typically contain tens to hundreds of different species that may cooperate or compete

  • Most microorganisms do not cause disease; many are beneficial to human health

  • Involved in food production

  • Part of the human immune system

  • A small percentage are pathogenic (disease-causing)

  • Many microorganisms have been identified, but only a small percentage have been successfully grown or cultured in the lab

• Risk-based classification of microorganisms (Bio-Safety Levels, BSL)

  • Microbes are separated into different BSLs based on risk to human health

  • BSL-1: least danger, no disease risk for humans

  • BSL-2: minimal risk for healthy individuals; moderate risk possible if inhaled, swallowed, or skin exposure

  • BSL-4: most dangerous; significant risk of death; no available treatment, therapy, or vaccine

• Lab procedures for BSL-1 microbes

  • Standard microbiological practices (covered later with aseptic techniques)

  • Hand washing sink required

  • Work on an open lab bench

  • No PPE required beyond standard lab attire

  • Lab doors separate from rest of building

  • Microbes used in lab this semester (BSL-1):

  • Bacillus subtilis

  • Enterobacter aerogenes

  • Escherichia coli (nonpathogenic lab strain)

  • Lactococcus lactis

  • Staphylococcus epidermidis

• Lab procedures for BSL-2 microbes

  • Standard microbiological practices

  • Hand washing sink and eyewash station required

  • Work on an open lab bench

  • Restricted access to laboratory space

  • Autoclave required for disposal of contaminated materials

  • PPE (e.g., lab coats, eyewear, gloves) as needed

  • Microbes used in lab this semester (BSL-2):

  • Pseudomonas aeruginosa

  • Staphylococcus aureus

• Standard microbiological practices and aseptic techniques

  • Aseptic techniques are designed to accomplish two goals:

  • Prevent contamination of media, surfaces, people, and property

    • Contamination = presence of unwanted microorganisms

    • Do not touch media with nonsterile items

    • Do not leave media exposed to air for too long

  • Limit the spread of microorganisms to media, surfaces, people, and property

    • Avoid spilling liquid cultures

    • Keep lab bench area free of clutter

    • Clean up spills properly

• Growing or culturing microorganisms in the lab

  • Bacteria are grown using media

  • Media = nutrient-containing substance allowing growth

  • Media can be liquid or solid

    • Liquid media = broth; used in test tubes, flasks, bottles

    • Turbidity (cloudy appearance) indicates growth; increases with cell number

  • Solid media contains agar

  • Agar is derived from seaweed and used as a solidifying agent for media

  • Solid media typically in plates (and sometimes in test tubes)

  • Solid media growth forms colonies or large masses of growth

  • A colony typically arises from a single cell sufficiently separated on an agar surface

  • Colonies can also arise from a cell aggregate or an endospore

    • Cell aggregate: cells that always grow in pairs or large groups

    • Endospore: a survival structure produced by some bacteria

  • Inoculation (intentional introduction of microorganisms to media by the lab worker)

  • Inoculated cultures can become contaminated if aseptic techniques fail

• Levels of microbial control and asepsis

  • Media must be made sterile before use

  • Sterilization = highest level of control; kills/removes all living microorganisms, viruses, prions, and endospores

  • Endospores are survival structures produced by a subset of bacteria

  • Methods of sterilization (primary method):

  • Autoclaving: steam (moist heat) under pressure; primary method for sterilizing media

    • Autoclave = equipment that combines steam under pressure

    • Other items to sterilize via autoclave include control disks, cell spreaders, etc.

  • Incineration: sterilizes inoculating loop or needle using a Bunsen burner

    • Flame should be adjusted until inner blue cone and outer blue cone are visible

    • Outer blue cone height should be approximately $3\,\text{in}$

  • Radiation or certain chemicals can be used but are rarely used for sterilization in this context

  • Disinfection (second-highest level of control): kills most pathogenic bacteria and viruses

    • Methods include boiling, UV exposure, or certain chemicals

    • Bac-Down is a chemical used for disinfecting lab benches

  • Degerming: removal of microorganisms from skin using soap and water before handling tubes and plates

  • Handwashing and degerming do not kill microorganisms on the skin; they remove them

• Aseptic technique during work with cultures and sterile media

  • When working with cultures and sterile media, the Bunsen burner should be lit, and work within a $1 ext{-}2\ \text{ft}$ radius around the flame

  • The flame heats the air and hot air rises, helping prevent particles from falling onto work areas

  • When removing a cap (test tube) or lid (bottle), pass the opening through the flame several times to slightly heat the glass and the air above the opening

  • This heating helps prevent air-borne contaminants from entering as soon as the cap is removed

  • The cap or lid should never be placed on the lab bench; keep it in one hand while performing another action with the other hand

  • Re-pass the opening through the flame AA to 2–3 times before replacing the cap or lid

• Inoculating cultures

  • The loop is used to inoculate liquid media and to perform the streak plate method

  • The inoculum can come from a liquid culture or from an agar plate

  • The needle is used to inoculate agar deep tubes

• Laboratory etiquette and labeling

  • Good lab behavior complements sterilization and disinfection

  • Stay attentive to the instructor and surroundings to prevent accidents and contamination

  • Proper labeling before inoculation and incubation:

  • Test tubes labeled with tape including name/initials, date, lab section, and bacterium used

  • After incubation, remove tape from tubes

  • Agar plates labeled on the bottom with a Sharpie (labels should be on the bottom surface of plates, not on the lid)

  • Remove media and other lab materials exposed to cultures onto the discard cart when no longer needed

  • Return common materials (e.g., loops, markers, Bunsen burners) to appropriate drawers after use

• Streak plate method

  • The streak plate method is one of the most important techniques in microbiology

  • Purpose: obtain a pure culture

  • Pure culture = cells arising from a single cell; all cells in the culture are identical

  • The most common method is the quadrant streak technique

  • Performed on an agar plate

  • Draw an “X” on the bottom of the plate to divide into four equal quadrants

    • Some methods use three or two quadrants

  • Quadrants labeled $1,2,3,4$

  • The technique starts with a high number of cells in quadrant $1$ and progressively fewer in quadrants $2,3,4$, acting as a dilution method

  • Goal: visible individual colonies in quadrant $2$ or $3$; often little or no growth in quadrant $4$

• Pre-lab background questions & activities (concepts to know)

  • Definitions to know:

  • Colony —> comes from a single cell duplication, they are all the same.

  • Contaminate / contamination —>

  • Degerming

  • Disinfect / disinfection

  • Incubate / incubation

  • Inoculate / inoculation

  • Media / medium

  • Pathogen / pathogenic

  • Sterile / sterilization

  • Turbidity

  • Identify the two aspects of aseptic techniques

  • What two conditions are used during the autoclave process? (steam under pressure; duration/temperature specifics are part of procedure)

  • What does the acronym BSL stand for?

  • a) Which BSL category includes the most dangerous microbes?

  • b) Which BSL category includes the least dangerous microbes?

  • What equipment is used to sterilize the inoculating loop and needle?

  • Difference between sterilization and disinfection:

  • a) Which level of control is better?

  • b) What are the four “things” destroyed by sterilization?

  • c) What are the two “things” killed by disinfection?

• Post-lab questions & activities

  • What is the streak plate used for?

  • Diagram the standard procedure for performing a four-quadrant streak plate

  • Demonstrate the proper method for sterilizing the inoculating loop or needle

  • Demonstrate the proper method for removing culture from a test tube using the inoculating loop

• Inoculation tools

  • The loop is used for inoculating liquid media and performing the streak plate method

  • The needle is used to inoculate agar deep tubes

• Streak plate concept and practical outcomes

  • A well-isolated colony in quadrants enables isolation of a single species for further study

• Important practical notes

  • Always follow aseptic practices to prevent contamination and ensure experimental validity

  • Keep work areas organized and clean to minimize cross-contamination

  • Dispose of contaminated materials properly via the discard cart

  • Dispose of all used media and cultures in accordance with lab safety guidelines

• Chemical, physical, and procedural controls used in the lab

  • Sterilization: autoclave, incineration, radiation (rare), certain chemicals (rare)

  • Disinfection: boiling, UV exposure, chemicals (e.g., Bac-Down)

  • Degerming: removal of microorganisms from skin using soap and water before handling samples

  • Physical controls (e.g., heat, flame) help maintain a sterile field by creating a barrier of hot air and preventing air-borne contamination

• Additional notes on growth and culture terminology

  • Media can be liquid (broth) or solid (agar)

  • Turbidity is a proxy for growth, increasing as cell density increases

  • A colony may originate from a single cell, an aggregate, or an endospore

• Quick references to safety levels and organisms used in this course (as of this lab)

  • BSL-1 organisms used this semester: $\text{Bacillus subtilis}, \text{Enterobacter aerogenes}, \text{Escherichia coli (nonpathogenic)}, \text{Lactococcus lactis}, \text{Staphylococcus epidermidis}$

  • BSL-2 organisms used this semester: $\text{Pseudomonas aeruginosa}, \text{Staphylococcus aureus}$

Key terms and definitions (glossary pulled from pre-lab section)

• Colony

• Contaminate / contamination

• Degerming

• Disinfect / disinfection

• Incubate / incubation

• Inoculate / inoculation

• Media / medium

• Pathogen / pathogenic

• Sterile / sterilization

• Turbidity

Common procedural snapshots (from post-lab prompts)

• Four-quadrant streak plate procedure (diagramed steps)

• Sterilizing the inoculating loop or needle (through flame, ensuring sterile technique)

• Removing culture from a test tube using the inoculating loop (maintain aseptic technique throughout)

Practical tips for the lab session

• Always ensure labeling is clear and compliant with lab policy

• Keep the lab bench clean and uncluttered to minimize accidental contamination

• Perform aseptic technique steps in the order specified by the instructor and lab manual

• If you are unsure, ask the instructor before proceeding with a procedure