Microbiology Lab Safety and Sampling

• Study guide covers microbiology lab practices

General Laboratory Safety and Behavior

• Lab is likely Biosafety Level 2 (BSL-2)
• BSL-2 is standard for handling microorganisms that cause mild diseases in humans
• PPE includes lab coats, gloves, safety goggles, and closed-toe shoes, protects against chemical spills, biological hazards, and contamination
• Contaminants are discarded in biohazard waste containers
• Sharps containers are for needles and broken glass
• Clean bench with disinfectant before and after lab work to prevent contamination
• If soaked with a chemical, remove contaminated clothing, rinse with water for 15 minutes, and use emergency shower if necessary
• If chemicals get in eyes, rinse at eyewash station for 15 minutes and seek medical attention
• Appropriate clothing includes long pants, closed-toe shoes, and a lab coat
• Avoid loose clothing, jewelry, and open-toed shoes

Sampling the Environment

• Purpose is to assess presence and types of microorganisms in various environments
• Use sterile swabs, contact plates, or air sampling devices to collect samples and inoculate onto nutrient agar or other media for incubation
• Observe colony morphology (size, shape, color, texture, margin) and count colonies to estimate microbial load

Use and Care of the Microscope

• Key parts include ocular lens, objective lenses (4X, 10X, 40X, 100X), coarse adjustment knob, fine adjustment knob, mechanical stage, iris diaphragm, and light source
• Iris diaphragm controls amount of light passing through the specimen
• Coarse adjustment knob moves the stage up and down for initial focusing
• Fine adjustment knob fine-tunes focus for high magnification
• Start focusing with the 4X objective, then move to 10X, and finally to 40X/100X.
• Use only fine focus knob for 40X/100X
• Clean lenses with lens paper
• Store the microscope with lowest objective lens in place and the stage lowered
• Observe color, Gram reaction (positive or negative), cell morphology (cocci, bacilli, spirilla), and arrangement (chains, clusters, pairs)

Aseptic Transfer

• Minimize contamination by flame sterilizing inoculating loops/needles
• Minimise contamination by working near a Bunsen burner
• Minimise contamination by avoiding touching non-sterile surfaces
• Broths are liquid media for growing large volumes of microbes
• Slants are solid media in test tubes for long-term storage
• Plates are solid media in Petri dishes for isolation and observation
• Use inoculating loops for plates/slants
• Use needles for deep agar
• Use pipettes for broths

Streak Isolation

• Purpose is to isolate individual colonies from a mixed culture
• Streak sample in quadrants on an agar plate using a sterile loop, diluting the sample with each streak
• Look for isolated colonies to identify pure cultures

Gram Staining and Viewing

Creation of a Specimen Smear

• Purpose is to prepare a thin layer of cells for staining and microscopic observation
• For liquid samples, place a drop on the slide and spread
• For solid samples, mix with water
• Heat-fix to adhere cells to the slide

Gram Staining

• Purpose is to differentiate bacteria into Gram-positive (purple) and Gram-negative (pink) based on cell wall structure
• Apply crystal violet (primary stain), iodine (mordant), alcohol (decolorizer), and safranin (counterstain)
• Gram-positive stains purple, Gram-negative stains pink
• Errors include over-decolorizing or under-decolorizing

Selective/Differential Media and Fungi/Protista

Selective/Differential Media

Mannitol Salt Agar

• Purpose is to isolate and differentiate Staphylococcus species
• Selective and differential media
• Streak sample on agar
• Selective: High salt (7.5% NaCl)
• Differential: Mannitol and phenol red (pH indicator)
• Yellow indicates Mannitol fermentation (S. aureus)
• Red indicates no fermentation

Eosin Methylene Blue (EMB)

• Purpose is to isolate Gram-negative bacteria, especially coliforms
• Selective and differential media
• Streak sample on agar
• Selective: Eosin and methylene blue
• Differential: Lactose
• Metallic green indicates E. coli
• Pink indicates lactose fermenters
• Colorless indicates non-lactose fermenters

MacConkey Agar

• Purpose is to isolate and differentiate Gram-negative bacteria
• Selective and differential media
• Streak sample on agar
• Selective: Bile salts and crystal violet
• Differential: Lactose and neutral red (pH indicator)
• Pink indicates lactose fermenters
• Colorless indicates non-lactose fermenters

DNase Agar

• Purpose is to detect DNase-producing bacteria
• Differential media
• Streak sample on agar
• Differential: DNA and methyl green (indicator)
• Clear zones indicate DNase activity

Fungi/Protista

• Yeasts are unicellular and oval-shaped
• Molds are multicellular with hyphae
• Rhizopus: Sporangia with spores
• Aspergillus: Conidiophores with chains of conidia
• Penicillium: Brush-like conidiophores
• Wet Mount: Place a drop of sample on a slide, add a coverslip, and observe. Not stained or heat-fixed.
• Protist structures include flagella, cilia, pseudopods, and chloroplasts