Lab Midterms Review: Safety, Techniques, and Staining

PPE

Personal protective equipment

Lab safety

Wearing lab coats, gloves, safety glasses or goggles, and closed toe shoes

Sharps disposal

Sharps is placed into the designated sharp waste container

Biological waste disposal

Biological waste is placed into designated biowaste container

Regular waste disposal

Regular non-hazardous waste is 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

Review worksheet questions

Bacterial colonies vs molds

Texture, size, color. Molds tend to be fuzzy larger white/gray/green bacterial colonies tend to be smooth, smaller, and varies in color.

Contamination measurement

The number of colonies indicate how many bacterial cells are present while the size of bacterial colonies show 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, on environmental surfaces, and in air

On the skin, wash with body or hand soap. On surfaces in the environment, use disinfectants. In the air, filtration by HEPA filtration system (purifiers)

Size comparison

Bacteria- smaller in size and eukaryotes- larger in size

Genetic material organization

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

Ribosome comparison

Bacteria- 70 ribosomes in total and eukaryotes- 80 ribosomes in total

Cell wall composition

Bacteria cell wall composed of peptidoglycan

Respiration and photosynthesis

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

Motility mechanisms

Bacteria- flagella or pili and eukaryote- flagella, cilia, pseudopods

Agar plate incubation

The condensation on the lid of the agar plate can drip onto the agar and spread the bacterial colonies → messing up the colony shape and size + causes contamination

Colony

What is a colony?

Flat Elevation

Even and level with agar surface.

Raised Elevation

Slightly elevated but smooth.

Convex Elevation

Dome-shaped, gently rounded.

Pulvinate Elevation

Very convex, cushion-like.

Umbonate Elevation

Raised in the center, like a tiny volcano.

Entire Margin

Smooth, well-defined edges.

Undulate Margin

Wavy, gently curving edges.

Lobate Margin

Deeply indented, almost like flower petals.

Filiform Margin

Hair-like or thread-like strands.

Curled Margin

Rings or concentric patterns near the edges.

Circular Shape

Round and uniform edges.

Irregular Shape

Uneven, non-symmetrical edges.

Filamentous Shape

Thread-like extensions—almost feathery.

Rhizoid Shape

Root-like branches spreading from center.

Swarming Shape

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.

Coarse focus knob

The coarse focus knob should be adjusted only when using this objective lens: Low-power.

Ocular

This lens, also known as the eyepiece, often comes in pairs.

Diopter adjustments

Diopter adjustments can be made to this lens: Ocular.

Acetone

True or False: Acetone is the safest solvent for cleaning an objective lens. False.

Lint-free tissue

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

Total magnification capability

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

Coarse focus knob usage

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

Focus adjustment

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

Resolving power of a microscope

The resolving power of a microscope is a function of: A) The magnifying power of the lenses. B) The numerical aperture of the lenses. C) The wavelength of light. D) Both (a) and (b) are correct. E) Both (b) and (c) are correct.

Focus adjustment knobs

The coarse and fine focus knobs adjust the distance between: A) The objective and ocular lenses. B) The ocular lenses. C) The ocular lenses and your eyes. D) The stage and the condenser lens. E) The stage and the objective lens.

Parfocal microscope

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

Total magnification with oil immersion

The total magnification achieved when using a 100x oil immersion lens with 10x binocular eyepieces is: A) 10x. B) 100x. C) 200x. D) 1000x. E) 2000x.

Image contrast adjustment

The most useful adjustment for increasing image contrast in low-power magnification is: A) Closing down the diaphragm. B) Closing one eye. C) Opening up the diaphragm. D) Placing a drop of oil on the slide. E) Using a blue filter.

Oil immersion preparation

Before the oil immersion lens is rotated into place, you should: A) Center the object of interest in the preceding lens. B) Lower the stage with use of the coarse focus adjustment knob. C) Place a drop of oil on the slide. D) Both (a) and (c) are correct. E) All are correct.

Aseptic techniques

Aseptic techniques: set of practices used to prevent contamination, protect specimen, and protect you and others by unwanted microorganisms.

Streak plate method

How is a streak plate done? Label the plate on the bottom (agar side) with your name, date, and specimen. Sterilize the loop by flaming it until red-hot; let it cool.

Improve Contrast Techniques

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

Improve Resolution Techniques

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

Sterilize the loop

Flaming it until red-hot and letting it cool.

Streak plate significance

Isolate individual colonies to obtain pure colonies from a mixed culture.

Check culture purity

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

Prepare for further testing

Pure colonies can be used for gram staining, biochemical testing, or DNA analysis.

Study colony morphology

Helps observe characteristics like shape, color, size, and texture of different bacteria.

Negative stain example

Nigrosin or india ink.

Omitted step in staining for cell dimensions

Heat fixation because it can distort the bacterial cells.

External bacterial cell structures demonstrated by a negative stain

Capsules.

Color of gram positive bacteria after heat fixation

Colorless.

Color of gram positive bacteria after crystal violet staining

Purple.

Color of gram positive bacteria after applying iodine

Purple.

Color of gram positive bacteria after decolorizing with ethanol

Purple.

Color of gram positive bacteria after counterstaining with safranin

Purple.

Color of gram negative bacteria after heat fixation

Colorless.

Color of gram negative bacteria after crystal violet staining

Purple.

Color of gram negative bacteria after applying iodine

Purple.

Color of gram negative bacteria after decolorizing with ethanol

Colorless.

Color of gram negative bacteria after counterstaining with safranin

Pink.

Why is gram stain considered a differential stain?

Differential staining reactions take advantage of the fact that cells or structures within the cell display dissimilar staining reactions due to different cell wall structures.

Most error-prone step in gram stain procedure

The decolorizing step- leaving alcohol on too long or not enough.

Function of a mordant

Fix or bind the primary stain to the target structure, enhancing contrast and making the stain more permanent.

Functions of endospores in bacteria

Allow bacteria to survive extreme conditions such as heat, UV radiation, desiccation, chemical disinfectants, and nutrient depletion.

Protective barrier on endospores

Spore coat composed of proteins.

Water content in endospores compared to vegetative cells

An endospore contains about 10-30% of the water found in a vegetative cell.

Mordant in the spore stain

Heat.

Stimulus for endospore production

Harsh environments like low carbon or nitrogen, high temperature, desiccation, high cell density.

Diseases caused by endospores

Bacillus anthracis - anthrax; Clostridium tetani - tetanus; Clostridium botulinum - muscle paralysis.

Are endospore structures reproductive structures?

No, they're dormant structures.