Lab Midterms Review: Safety, Techniques, and Staining

PPE

Personal protective equipment used in the lab.

Sharps waste

Placed into the designated sharp waste container.

Biological waste

Placed into designated biowaste container.

Regular non-hazardous waste

Placed into trash can.

BSL

Biosafety level classifications of microorganisms represent the potential of the organism to cause disease and the conditions under which the organism should be safely handled.

BSL-1

Not likely to pose a disease risk to healthy adults; no special precautions; basic teaching labs.

BSL-2

Poses a moderate risk to healthy adults; unlikely to spread throughout community; effective treatment readily available; need lab coat, gloves, and eye protection.

BSL-3

Can cause disease in healthy adults; may spread to community; effective treatment readily available; biosafety cabinets to prevent airborne transmission.

BSL-4

Can cause disease in healthy adults; poses lethal risk and does not respond to vaccines or antimicrobial therapy; sealed, negative pressure 'Hot zone'-- exhaust air is filtered twice through HEPA filters.

Ubiquity

The presence of microorganisms in various environments.

Bacterial colonies vs. molds

Bacterial colonies tend to be smooth, smaller, and vary in color, while molds tend to be fuzzy, larger, and white/gray/green.

Contamination measurement

The number of colonies indicates how many bacterial cells are present while the size of bacterial colonies shows the rate of growth.

Bacteria on skin

Bacteria on the skin is not a concern, depending on what kind of bacteria; they are a part of normal skin flora.

Microbial control on skin

Wash with body or hand soap.

Microbial control on surfaces

Use disinfectants.

Microbial control in air

Filtration by HEPA filtration system (purifiers).

Bacteria vs. eukaryotic microorganisms size

Bacteria are smaller in size and eukaryotes are larger in size.

Bacteria vs. eukaryotic microorganisms genetic material

Bacteria DNA is located in nucleotide and eukaryotes' DNA is located in nucleus.

Bacteria vs. eukaryotic microorganisms ribosomes

Bacteria have 70 ribosomes in total and eukaryotes have 80 ribosomes in total.

Bacteria vs. eukaryotic microorganisms cell wall

Bacteria cell wall is composed of peptidoglycan.

Bacteria vs. eukaryotic microorganisms respiration and photosynthesis

Bacteria lack membrane bounded organelles and nucleus, but can use organic/inorganic chemicals or photosynthesis for energy.

Bacteria vs. eukaryotic microorganisms motility mechanisms

Bacteria use flagella or pili, while eukaryotes use flagella, cilia, or pseudopods.

Agar plates incubation

Agar plates are incubated 'upside down' / inverted.

Colony

Visible mass of cells usually resulting from the division of a single cell.

Flat (Elevation Type)

Even and level with agar surface.

Raised (Elevation Type)

Slightly elevated but smooth.

Convex (Elevation Type)

Dome-shaped, gently rounded.

Pulvinate (Elevation Type)

Very convex, cushion-like.

Umbonate (Elevation Type)

Raised in the center, like a tiny volcano.

Entire (Margin Type)

Smooth, well-defined edges.

Undulate (Margin Type)

Wavy, gently curving edges.

Lobate (Margin Type)

Deeply indented, almost like flower petals.

Filiform (Margin Type)

Hair-like or thread-like strands.

Curled (Margin Type)

Rings or concentric patterns near the edges.

Circular (Shape Type)

Round and uniform edges.

Irregular (Shape Type)

Uneven, non-symmetrical edges.

Filamentous (Shape Type)

Thread-like extensions—almost feathery.

Rhizoid (Shape Type)

Root-like branches spreading from center.

Swarming (Shape Type)

Spreading motility pattern, often seen in Proteus species.

Ocular lens

The eye piece to look at specimen.

Nosepiece

The rotable piece to move the objective lens in order to switch magnification.

Objective lens

The piece that magnifies the image.

Condenser adjustment knob

Raises or lowers the condenser for optimal lighting.

Condenser

Located beneath the stage; focus light onto the specimen.

Diaphragm

Adjusts the amount of light that reaches the specimen; helps improve contrast.

Fine adjustment knob

Fine-tunes the focus for sharpness (especially with 40x and 100x objectives).

Coarse adjustment knob

Moves the stage up and down rapidly for general focusing (used with 4x and 10x objectives).

Illuminator

Supplies light that passes through the condenser, then the specimen, and finally through the objective and ocular lenses to form an image.

Aperture

Opening in the condenser (beneath the stage) that controls how much light passes through the specimen.

Limit of resolution (light microscope)

0.2 um.

Limit of resolution (unaided eye)

0.2 mm.

Oil immersion lens

This objective lens provides the highest magnification.

High dry lens

This objective lens provides the second-highest magnification.

Low-power lens

This objective lens provides the lowest magnification.

Working distance

The working distance of objective lens decreases as the magnification power increases.

Diopter adjustments

Diopter adjustments can be made to this lens.

Coarse focus knob

The coarse focus knob should be adjusted only when using this objective lens.

Immersion oil

The immersion oil forms a continuous lens system that limits the loss of light due to refraction.

Maximum resolution procedures

Blue filter placed over light source, condenser kept at the highest position, diagram should not be stopped down too much, use immersion oil between the slide and the 100x objective lens.

Starting with low power lens

It is advisable to start with lower power lens because it is easier to focus on the objective due to its big working distance.

Cleaning objective lens

Only lint-free, optically safe tissue should be used to wipe off microscope lenses.

Total magnification capability

The total magnification capability of a light microscope is only limited by the magnifying power of the lens system.

Parfocal microscope

A microscope that maintains focus when the objective magnification is increased.

Total magnification with 100x lens

The total magnification achieved when using a 100x oil immersion lens with 10x binocular eyepieces is 1000x.

Increasing image contrast

The most useful adjustment for increasing image contrast in low-power magnification is closing down the diaphragm.

Resolving power of a microscope

The resolving power of a microscope is a function of the magnifying power of the lenses, the numerical aperture of the lenses, and the wavelength of light.

False statement about acetone

Acetone is not the safest solvent for cleaning an objective lens.

False statement about coarse focus knob

The coarse focus knob can be used to adjust the focus when using any of the objective lenses.

True statement about focus

Once focus is achieved at one magnification, a higher-power objective lens can be rotated into position without fear of striking the slide.

Aseptic techniques

Set of practices used to prevent contamination, protect specimen, and protect you and others by unwanted microorganisms.

Streak plate method

A technique used to isolate individual colonies from a mixed culture.

Significance of a streak plate

Helps obtain pure colonies from a mixed culture, check culture purity, prepare for further testing, and study colony morphology.

Good smear

A thin, evenly spread of bacterial cells.

Chromophore

Color-bearing ionic groups.

Basic dye

Dyes that have positively charged chromophores; they stain negatively charged bacterial cells.

Acidic dye

Dyes that have negatively charged chromophores; they stain the background, leaving cells clear.

Difference between basic and acidic dye

Basic dyes stain negatively charged bacterial cells, while acidic dyes stain the background.

Reason for using basic dye on bacteria

Bacterial cells are negatively charged and basic dyes are positively charged, leading to effective staining.

Simple stain

Uses 1 dye to increase contrast of cells, determining size, shape, and arrangement of cells but cannot differentiate between types of bacteria.

Differential stain

Uses 2 or more dyes to differentiate between organisms or between cell structures.

Steps to improve contrast

Adjust iris diaphragm, lower light intensity, raise/lower condenser, use stains, use phase-contrast/darkfield microscopy.

Steps to improve resolution

Use higher NA objectives, use immersion oil (100x), clean lenses, proper focusing, quality slides/cover slips.

Steps for making a smear from liquid media

Place loopful of culture directly on slide, air dry smear, heat fix.

Steps for making a smear from solid media

Place dot of water on slide, mix dot of water with colony of bacteria, air dry, heat fix.

Purpose of a streak plate

Isolate individual colonies and check culture purity.

Observation of pure colonies

You can tell if a culture contains only one species based on uniform colony appearance.

Uses of pure colonies

Can be used for gram staining, biochemical testing, or DNA analysis.

Characteristics observed in colony morphology

Shape, color, size, and texture of different bacteria.

Gram stain purpose

To differentiate bacteria into Gram-positive and Gram-negative.

Gram negative stain result

Pink.

Gram positive stain result

Purple.

Crystal violet

Primary stain that stains all cells purple (30 secs).

Iodine

Mordant that forms CV-I complex to fix dye in thick walls (1 min).

Alcohol/ethanol

Decolorizer that removes stains from Gram-negative cells (5-15 secs).

Safranin

Counterstain that stains Gram-negative cells pink/red (1 min).

Spore stain purpose

To detect the presence of endospores.

Spore staining results

Spores - Green (retain malachite green) & Vegetative cells- Pink/red (stained with safranin).

Malachite green

Primary stain used in spore stain.

Negative stain purpose

To observe cell shape and size without distortion and to highlight capsules (if present).

Negative stain results

Background: Dark. Cells: Appear clear or light-colored against the dark background. Capsules (if present): Appear as halos around cells.