Laboratory Safety Guidelines

Lab Safety Rules

• Maintain a clean work area and safely store personal items off the lab bench.
• Do not eat, drink, or store food in the laboratory.
• Wash hands thoroughly before handling face/hair.
• Lab coats are required and must be buttoned/zipped.
• Safety glasses are required when handling cultures/caustic chemicals/UV light.
• Long hair must be pulled back.
• Lab tables will be disinfected before each class.
• Report spills/breakage immediately to the instructor.
• Surgical scrub of hands/skin required if contact with spilled liquid occurs.
• Report injuries immediately.
• Clean up all glassware, cultures, and supplies used.
• Disinfect workstation at the end of laboratory.
• Wash hands thoroughly before leaving the lab room.
• Discuss health concerns with a physician.
• Cultures are not to be thrown in wastebaskets or poured down the sink.
• Discard cultures in test tubes by removing tape/ink, and placing tubes in discard bin baskets.
• Tape Petri dishes shut and place them in a lined biohazard container.
• Only water and rinsed-off stains can go in the sinks.
• Malachite green and crystal violet stains are to be collected separately.
• Biohazard containers are for used pipettes, slides, cotton swabs, and agglutination cards.
• Paper wrappers, towels, and gloves are to be disposed of in a trash can.

Microscopy

• Microorganisms need to be magnified hundreds of times and dyed with stains to be seen.
• Effective microscope use requires familiarity with its parts, functions, and focusing procedures.
• Key microscope parts: eyepiece, eye tube, arm, objective lens, mechanical stage knob, coarse/fine focus.

Magnification

• Eyepieces typically magnify 10X.
• Adjust eyetubes to eliminate double vision.
• The mechanical stage supports the slide and has clips to hold it in place.
• Use mechanical stage control knobs to move the slide across the X-Y axis.
• Light intensity is adjusted using the illuminator.
• The condenser/diaphragm controls the amount of light reaching the specimen.
• There are usually four objective lenses: 4X, 10X, 40X, and 100X.
• Rotate the revolving nosepiece to change objectives.
• Coarse adjustment knob brings the specimen into approximate focus quickly.
• Fine adjustment knob fine-tunes the focus, mainly with 40X and 100X lenses.
• Total magnification = (Ocular lens) x (Objective lens magnification).

Working Distance and Refraction

• Diaphragm aperture controls resolution and contrast.
• Working distance decreases with magnification.
• Light direction changes when passing through a substance.
• Different transparent materials transmit light at different speeds.
• Change in speed causes refraction.

Microscope Usage

• Carry the microscope carefully with two hands.
• Clean lenses with lens paper or microfiber cloth, not paper towels.
• Start focusing with the 10X objective.
• Adjust the condenser and illumination intensity for comfortable viewing.
• Use the fine focus knob when switching to the 40X objective.
• Place one drop of immersion oil on the slide before using the 100X objective.
• Clean oil immersion lens after use, and remove any oil from the stage.
• When finished, set the 10x lens and turn the ocular lenses inward.

Metric Units

• The metric system is based on powers of 10.
• Metric units for length used in microscopy are nanometers, micrometers, and millimeters.
• Ocular micrometers are used to measure microorganism size.
• One meter is about 39.3701 inches.

Bacterial Shapes

• Coccus: spherical.
• Bacillus: cylindrical.
• Spirillum: spiral.
• Bacterial arrangements include: single, diplo (pairs), strepto (chains), staphylo (clusters).

Ocular Micrometer Calibration

• An ocular micrometer determines organism size using an ocular and stage micrometer.
• A stage micrometer is a slide with a "ruler" etched on it, with lines precisely 0.01mm (10µm) apart.
• Calibrate the ocular micrometer for each objective by superimposing the two scales.

Ocular Micrometer Calibration Steps

• Place stage micrometer on stage
• Focus with 10x in place
• Rotate 40x into place
• Rotate eyepiece for superimposition of black and white lines. Add oil, fine adjustment knob only.
• Align lines on stage with the ocular micrometer, determine distances, and divide to get the calibration factor.
• Replace stage micrometer with slide, line up edge of cell with ocular micrometer, and count spaces the organism occupies to determine size

High Power vs Oil Immersion

• High power: 400x = If 3 stage divisions line up with 12 ocular units, therefore @ 400X, 3 ÷ (12 ×10 micrometers) = 2.5 micrometers
• Oil immersion: 1,000x = If 1 small stage division lines up with 10 ocular units, therefore @1,000X, 1 ÷ (10 × 10 micrometers) = 1.0 micrometers
• Multiply number of ocular divisions by calibration factor (2.5µm or 1.0 µm) to determine organism size.

Ubiquity of Microorganisms

• Microorganisms are everywhere
• Aseptic technique is very important
• Tryptic Soy Agar (TSA) plate is used to demonstrate.
• Swabbing and scraping are used to transfer microbes.

Broth and Agar Preparation

• Survival of microorganisms depends on nutrients and environment
• Liquid medium lacking a solidifying agent is called a broth medium.
• Agar is an extract of seaweed with a solidifying agent produces a solid or semisolid medium

Materials Used

• Trypticase Soy Broth: 30 gm dry media per 1,000 ml distilled water
• Trypticase Soy Agar: 40 gm dry media per 1,000 ml distilled water
• Be careful when using hot plate and working with the tubes.
• Clean weigh boats.

Instructions

• Turn on the heat and strir the materials together
• Heat to rolling boil for a full minute to completely dissolve

Sterilization

• Fahrenheit and Celsius are not proportional
• Conversion formulas:
• (°F — 32) ÷ 1.8 = °C
• (1.8 × °C) + 32 = °F
• Sterilization kills all; disinfection kills vegetative cells only.

Sterilization Methods

• Autoclave - moist heat, 121°C (250°F) for 15 minutes at 15 pounds per square inch (p.s.i).
• Dry Heat - oven at 170°C (338°F) for 2 hours.
• Incineration - flaming the inoculating loop.
• Chemicals - dialdehydes for a minimum of 6 hours.
• Filtration: membrane filter with control pore sizes.
• Radiation

Smear and Staining

• Smear: thin film preparation of a bacterial suspension or solid from an agar medium on a clean glass slide
• Staining makes visible transparent microorganisms for morphological studies
• Bacterial arrangements includes: chain, clusters.
• Smears are prepared differently for liquid and solid cultures.
• Gram and Acid-fast stains separate into groups
• Used for visualization of structures: Capsule, Endospore, Flagella and Nuclear stains

Staining Details

• In simple staining, a single stain or dye is used to emphasize particular structures in the specimen.
• Basic dyes have a chromogen that is positively charged
• Simple staining makes cells appear the same color, even if more organisms are present
• Basic dyes include: basic fuchsin, crystal violet, malachite green, methylene blue, and safranin

Smear Prep Details

1. Clean 4 slides with Bonami and alcohol.
2. Label the slide with your initials and the initials of the organism using a small piece of masking tape at one end.
3. Add one or two loopfuls of deionized water to the slide
4. Reflame the loop.
5. Uncap the culture tube and keep the cap in your hand. Flame the rim of the culture test tube. Add a scanty amount of culture to the water on the slide.
6. Flame your loop and then mix the culture in the water, spreading the water to the size of a nickel.
7. HEAT FIX to adhere cells to slide.
8. Stain each smear and view under oil immersion.
9. Discarded the test tubes in the rack.