Lab Safety and Procedures

Microbiology

• Microbiology studies living organisms too small to see with the naked eye, including bacteria, archaea, fungi, and viruses.

Safety in the Lab

• Sharps containers are for sharp objects only, like needles, glass slides, cover slips, broken glass, Pasteur pipettes, and syringes.
• Biohazard containers are for soft contaminated objects, such as paper towels, cotton, tongue depressors, plastic petri dishes, and all gloves.
• Regular trash is for anything not contaminated, like paper towels used for washing hands.
• Necessary personal protective equipment (PPE) includes a lab coat, safety glasses, and goggles.

Spill Cleanup

• For large spills, notify the TA.
• For spills of bacteria, blood, or fungi:
  • Cover the area with paper towels and gently flood with Amphyl.
  • After 20 minutes, clean carefully, placing broken glass in sharps containers and paper towels in the step-on can.

Cleaning the Bench

• Put supplies away with the equipment.
• Put microscopes away clean, on the lowest magnification, and with the stage back.
• Ensure burners are off.
• Disinfect the desk with Amphyl and paper towels.
• Empty gram stain waste and pipette tips waste.
• Always wash your hands.

Lab Safety Dos and Don'ts

• DO tie hair back and wear lab coats at all times.
• DO keep bags and books off the floor, use shields for procedures with fluids, and wear safety goggles and gloves when working with chemicals.
• DO know which chemicals are hazardous.
• DON'T wear dangling jewelry or sandals.
• DON'T mouth pipette or discard anything not yours.
• DON'T apply makeup in class or vape/chew tobacco.

Microscopy

• Total magnification is determined by multiplying the objective magnification by the eyepiece magnification.
• The eyepiece magnification is typically 10x.
• Resolving power is the ability to distinguish between two very small and closely spaced objects on a specimen.
  • Higher resolving power is needed when objects are closer together.
• Numerical aperture measures the objective's ability to capture light and resolve fine specimen detail at a fixed object distance.
  • Refractive index indicates how much light bends passing from one medium to another.
• A larger numerical aperture results in higher resolution and brightness.
• Oil immersion bends more light into the lens, capturing more "image information."
  • The refractive index of oil is 1.5, compared to air's 1.
  • This provides more "image information" and finer details.
  • The 100x lens is the only one that should contact with oil.

Types of Microscopes

• Brightfield Microscope
  • The object appears dark on a bright field.
  • It is good for viewing stained specimens but living objects in wet mounts may appear washed out.
• Darkfield Microscope
  • A special condenser causes light to be reflected off the specimen.
  • The object is light on a dark background.
  • It is very good for observing unstained live specimens.
• Phase Contrast Microscope
  • Uses refracted and un-refracted light waves to enhance the contrast of the specimen without staining.
  • Shows different refractive indices, making internal structures visible.
• Fluorescence Microscope
  • Detects microbes in tissues and other samples.
  • Samples must be specially stained to fluoresce and are irradiated with light of a specific wavelength.
  • Exhibits a high degree of specificity.
• Stereomicroscope
  • Good for observing large objects like colonies of bacteria & fungi.
  • Used to study the surfaces of solid specimens and carry out close work.
  • Uses two eyepieces and two objectives with different viewing angles, producing a 3D visualization of the sample.
• Electron Microscopy
  • Uses electrons instead of light to visualize an image and is performed in a vacuum, which provides higher resolving power.
  • Electromagnetic lenses control the electron beam to form an image.
  • There are different types of EM.
    • Transmission electron microscopy (TEM) passes electrons through a very thin section of material.
      • The specimen is partly transparent to electrons and partly scatters them.
      • Scattered electrons are collected in the magnetic lens, and a computer creates an image from the electron "pattern".
    • Scanning electron microscopy (SEM) produces images by probing the specimen with a focused electron beam.
      • The electron beam interacts with the specimen, losing energy.
      • It is scanned across a rectangular area, creating a 3D image.

Protozoa

• Protozoa belong to the protist kingdom.
• They are single-celled eukaryotes, mostly lacking cell walls.
• Obtain organic carbon for growth by consuming other organisms.
• Some are human parasites.
• Classified based on their means of locomotion: flagellates, ciliates, and ameboids.

Algae

• Algae are eukaryotes that can be unicellular or multicellular.
• They photosynthesize with chloroplasts (autotrophic).
• Examples include diatoms and dinoflagellates.

Eubacteria

• Eubacteria are prokaryotic microorganisms and are mostly unicellular.
• This is a large and extremely diverse group.
• They lack a nucleus, and their DNA is a single circular chromosome.

Cyanobacteria

• Cyanobacteria are prokaryotes that can produce oxygen.
• Known as blue-green algae, obtaining energy through photosynthesis.
• Important role in nitrogen fixation, incorporating atmospheric N into organic compounds.
• Often exist as colonies, either filaments or hollow balls.

Hay Infusion

• A hay infusion is made by soaking dried plant material (hay) in water to rehydrate cysts/endospores of dormant microbes.
• Bacteria appear first and are most abundant.
• Protozoa consume the bacteria later.
• The appearance of organisms depends on pH, light, temperature, etc.

Fungi and Yeast

• Fungi are classified into two separate classes: unicellular and multicellular.
• Multicellular fungi have four subclasses based on hyphae & spores.
  • Zygomycota: Includes Rhizopus, bread molds, and mucor.
  • Ascomycota: Includes Neurospora, yeast, and sac fungi.
  • Basidiomycota: Includes mushrooms, rusts, and smuts.
  • Deuteromycota: An additional sub class of fungi
• Hyphae are extensions that fungi grow to absorb nutrients, reproduce sexually and/or asexually
  • Aerial hyphae (on surface) often produce conidia, sometimes enclosed in a sac (sporangium) called endospores.

Reproduction of Fungi

• Sexual reproduction: Involves two contributing genetic materials.
• Asexual reproduction: Offspring are created by a single parent.
  • Yeast is classified as unicellular fungi.
  • Yeast reproduces asexually by budding or fission, resulting in two new cells.
• Key characteristics include the ability to form pseudohyphae.

Opportunistic Pathogens

• Pathogens: Microbes that can cause disease in healthy people.
• Opportunistic pathogens: Cause disease when the host is compromised (e.g., suppressed or compromised immune system).

Bacteria Morphology

• Bacteria are identified by shape:
  • Cocci (spherical or round).
  • Bacilli (rectangular or rod-shaped).
  • Spirilla (curved or helical).
  • Vibrio (curved rod or comma-shaped).
  • Spirochete (corkscrew shape).

Aseptic Technique

• Aseptic technique is the process of growing bacteria in pure cultures to avoid contamination and maintain safety.

Tools for Aseptic Technique

• Inoculation loop: Used to transfer liquid cultures, pick colonies from a plate, or streak bacteria on agar surfaces.
• Bunsen burner: Used to sterilize materials, create a sterile field; the tip of the inner cone is the hottest part.

Culture Media

• Pure media: Contain a single species of microbe.
• Contaminated media: Have unwanted microbes introduced.
• Inoculated media: Have a microbe introduced into/on the media.
• Media contains water, carbon, nitrogen, minerals, and growth factors, with specific pH levels.

Types of Media

• Agar: Galactose sugar polymer ideal for solid media, incubated at 37 degrees C
• General purpose: Nutrient agar grows a variety of microorganisms.
• Selective media: Only allows certain bacterial growth, e.g., columbia media.
• Differential media: Contains substances that cause bacteria to take on a different appearance, e.g., EMB, Blood agar plates (BAP).

Smear Preparation

• A smear is a thin layer of specimen on a glass slide, used to transfer bacteria for microscope slides, which helps with staining.

Smear Prep Methods

• Broth: Use an inoculation loop for easier smear preparation but it is difficult to control microbe quantity.
• Agar: Apply a drop of DI water to the slide and use an inoculation needle for easier quantity control, but it is easier to add too much microbe. When preparing a smear, broth or agar can be used.

Heat Fixing

• Allows bacteria on slide to dry completely.
• Pass the slide over a Bunsen burner quickly in a ½ times L shape to kill bacteria and ensure they adhere.

Simple Staining

• Bacteria are very small and transparent, the dye used to analyze specific characteristics.
• Staining improves the contrast between specimen and background, which allows observation of morphology and arrangement.
• Most bacteria have anionic (negative charge) cytoplasmic membranes, meaning cationic (positively charged) dyes are effective.
• The dye molecule will ionically bind to the membrane, effectively coating it with color.

This quiz covers essential lab safety protocols, including spill cleanup, waste disposal, microscope handling, and PPE use. It emphasizes the importance of following established procedures to ensure a safe and effective laboratory environment.