BIO 120 LAB EXAM 1 PREP

Covers Lab Manual Guides for labs we did Weeks 1-5

Microscope

Instrument for magnifying and looking at small/microscopic things.

Binocular Compound Light Microscope

• Description

• Function

Description

• “Binocular” = microscope has 2 eyepieces/oculars

• Compound microscopes have at least 2 lenses or mirrors for producing an image

• Visible light is the source of illumination

Function

• Enlarges the resolved image to dimensions that can be perceived by the human eye

In a light microscope, the source of illumination is.

Visible/natural light

Specimens viewed with a compound light microscope must be _____________.

Explain what this means.

Specimens viewed with a compound light microscope must be transparent.

Meaning light is able to pass through them. 

Slide

Very thin glass used for observing specimens

The slide rests on the microscope’s _________ and is held in place by the __________________-.

The slide rests on the microscope’s stage and is held in place by the mechanical stage clips.

The image seen with a microscope is produced when…

light from the illuminator below the stage passes through the specimen —> through the body tube —> through the ocular lens to your eye 

Objective lenses attach to the ______________________ .

Objective lenses attach to the revolving nosepiece.

Name the objective lenses from smallest to largest.

(1) 4x — Scanning Objective

(2) 10x — Medium Power Objective

(3) 40x — High Power Dry Objective

(4) 100x — Oil Immersion Objective

Briefly describe the function of the Scanning Objective Lens.

• shortest objective lens

• used to quickly locate your specimen by viewing large area of your slide 

When using a microscope, in addition to the objective lens you use, the __________  further magnifies the image by _______.

When using a microscope, in addition to the objective lens you use, the ocular lens further magnifies the image by 10x (ten-fold).

Ocular Lens/Eyepiece

• magnifies image seen w/ microscope by 10-fold

• adjustable to accommodate your interpupillary distance (distance b/w the pupils of your eyes)

• correctly positioned when you see a single circle of light

• if you see 2 circles of light, continue moving oculars until the circles merge together

• for binocular microscope —> keep both eyes open when viewing your slides

T or F: It’s best to keep both eyes open when using a binocular microscope.

True

If you see two circles of light when looking through a binocular microscope — your _______________ need to be adjusted.

If you see two circles of light when looking through a binocular microscope — your ocular lenses need to be adjusted.

What role does the microscope’s body play in the virtual image you see?

• Contains mirrors that bend light into the ocular lenses

• Orientation of the microscope’s optical elements causees the image to be reversed (right to left) & inverted (top to bottom) 

How do you switch objective lens you are using correctly?

• Rotate the revolving nosepiece to switch the objective lens that is above the stage

• You should heard clicking sound when the objectives are correctly aligned.

• Never grab the objectives itself

What is the purpose of the objective lenses?

Contain lenses that magnify the image produced by the microscope.

Purpose — Arm

• Supports the nosepiece

• Sometimes contains the on/off light switch 

• Contains the focus adjustment knobs

Mechanical Stage

• Description

• Purpose

Description: 

• flat platform on which slide rests

• silver/black apparatus

Purpose

• used to move the slide to position (over the ___-) on the stage

Stage Adjustment Knobs

• Description 

• Purpose

• Description:

  • Located below the stage

  • Contains 2 knobs

• Purpose:

  • One knob moves mechanical stage from right to left

  • Other knob moves mechanical stage up and down

Light Intensity Control

• Description 

• Purpose

Description:

Purpose:

Substage Condense

• Description

• Purpose

Description:

Purpose:

Aperture Diaphragm Control

• Description 

• Purpose

Description:

Purpose:

Field Diaphragm Lever

• Description 

• Purpose

Description:

Purpose:

Light Sources

• Description 

• Purpose

Description:

Purpose:

Base

• Description 

• Purpose

Description:

Purpose:

Fine Focus Adjustment Knob

• Description 

• Purpose

Description:

Purpose:

Coarse Focus Adjustment Knob

• Description 

• Purpose

Description:

Purpose:

Microscope’s 3 Distinct Properties

A microscope has 3 distinct properties related to its function:

1. Magnification — number of times image is enlarged

2. Contrast — striking difference b/w similar objects

3. Resolution — ability of a lens to show small, close-together objects that are no smaller than 0.1 mm (aka 100 micrometers)

The unaided human eye cannot distinguish objects that are smaller than __ mm which is _______________.

The unaided human eye cannot distinguish objects that are smaller than 0.1 mm which is 100 micrometers.

Magnification is limited by _________.

Magnification is limited by resolution.

The limit of useful magnification by a light microscope is about _____.

The limit of useful magnification by a light microscope is about 1500x.

Magnification in a light microscope beyond ______ produces blurry and useless images.

Magnification in a light microscope beyond 1500x produces blurry and useless images.

Resolution…

• Define

• Is limited by what?

• Is dependent on what?

Definition: Resolution refers to the ability of a microscope (or any optical system) to distinguish two closely spaced objects as separate. The higher the resolution, the clearer and more detailed the image.

Resolution is limited by —> Wavelength of the illumination source

Resolution is dependent on —> physical characteristics (numerical aperture) of the lenses

• Numerical aperture = measure of a lens’s ability to gather light and resolve fine specimen detail at a fixed distance.

T or F: Most bacteria in their natural state are uncoloured. 

True

When using light to illuminate your specimen, objects smaller than half the wavelength of light are not visible to the observer.

When using light to illuminate your specimen, objects smaller than half the wavelength of light are not visible to the observer.

Why are most bacteria uncoloured? What is the impact in microbiology lab?

Because they…

• have clear & watery cytoplasm 

• are contained w/in uncoloured cytoplasmic membrane & cell wall

Impact in microbiology lab: 

• Makes them challenging to view w/ microscope —> can be remedied by increasing the contrast b/w cells and their background on slide

Increasing the contrast between cells and the background on a slide makes it easier to see the cells.

Increasing the contrast between cells and the background on a slide makes it easier to see the cells.

Contrast

• Definition 

• What 2 methods are used to create contrast when using light microscope?

Definition

• Measure of difference in appearance b/w 2 objects

What 2 methods are used to create contrast?

1. Staining —> stains cells w/ colour, increasing contrast b/w cells and the bright/light background used in light microscopy

2. Adjusting light passing through the specimen

• Lower magnification = Less light needed for good contrast

• Higher magnification = Higher level of light needed for good contrast

In a light microscope:

The higher the magnification the higher the level of light needed for good contrast. The lower the magnification the lower the level of light needed for good contrast.

In a light microscope:

The higher the magnification the higher the level of light needed for good contrast. The lower the magnification the lower the level of light needed for good contrast.

Magnification 

• Definition

Definition: Magnification refers to how many times larger the image produced by microscope appears compared to actual size of the specimen. 

Total Magnification of an Image

(magnification of ocular lens) x (magnification of objective lens)

Resolution

Magnification

Parfocal Lens

Depth of a field

Field of view

Working Distance

Distance b/w tip of objective lens & the stage.

Why are agar plates incubated upside down?

To prevent condensation from dripping onto the agar surface, which could cause contamination or colony dispersion.

Where should agar plates be labeled, and why?

On the bottom (agar-containing side), so the sample remains identified even if the lid is separated.

What technique can be used to lift the lid of a plate while streaking?

The "clamshell" method (opening lid at 45 degree angle without completing taking it off to prevent contamination

At what temperature are most bacteria incubated in the lab?

37°C (human body temperature).

What is the purpose of a quadrant streak plate?

To isolate pure colonies of bacteria.

What is important to remember when transferring bacteria between multiple plates or tubes?

Stay focused, be systematic, and label correctly to prevent mix-ups.

(a) What is a smear? 

(b) What’s its purpose? 

(a) Smear: Thin layer of microbial sample spread on slide for microscopic observation.

(b) Purpose: Bacteria from a liquid broth culture or bacteria from agar media.

What are the basic steps of a smear preparation?

Grow culture → collect sample aseptically → apply to slide → heat fix → evaluate with stain.

Why is heat fixation important in smear prep?

It adheres the bacteria to the slide and kills any potentially infectious organisms.

What simple stain is often used to evaluate smear quality?

Methylene blue.

Why is staining necessary in microscopy of bacteria?

To add contrast so bacterial cells and structures can be visualized more clearly.

What is an agar slant?

Solidified agar in a test tube set at an angle, providing a large surface for growth.

Colony

Visible cluster of microbes that arose from single cell.

What are CFUs?

• Colony Forming Units

• Measure of viable bacterial or fungal cells capable of forming colonies

What does “pure culture” mean?

A culture containing only one species of microbes.

What is meant by “isolation” in microbiology?

Separating individual microorganisms to obtain pure cultures.

What is the clamshell method?

Gently heating a smear to adhere cells to the slide and kill them.

What is agar media?

A nutrient-rich gel made from agar, used to grow microorganisms.

What is an agar plate?

A petri dish filled with solid agar media for culturing bacteria.

What is an agar slant?

Agar solidified in a test tube at an angle to provide a larger surface for bacterial growth.

Sterile

Completely free of living microorganisms.

What is streaking?

The process of spreading microorganisms across agar to isolate colonies.

What is a vegetative cell?

A: A metabolically active bacterial cell (not in spore form).

Q: What is bacterial cellular arrangement?

A: The physical grouping of bacterial cells (e.g., chains, clusters, pairs).

Q: What is bacterial cellular morphology?

A: The shape and structure of bacterial cells (e.g., cocci, bacilli, spirilla).

Q: What is methylene blue?

A: A simple stain used to visualize bacterial cells under a microscope.

Q: What is the purpose of an inoculating loop?

To transfer microbes between media without contamination.

2 Purposes of Bunsen Burner

1. Sterilize tools

2. Create updraft that reduces airborne contamination

Purpose of Pipettes

To measure & transfer precise volumes of liquid culture

Purpose of Forceps

To transfer sterile objects without contaminating them

A gram stain is a __________.

A gram stain is a differential stain.

Gram Stain

• differential stain 

• used to differentiate b/w bacteria based on their cell wall structure

  • gram-positive —> stains purple (thick peptidoglycan layer that retains crystal violet stain)

  • gram-negative —> stains pink (thin peptidoglycan layer & outer membrane that retain safranin stain)

Describe each step of gram staining in detai.

(1) Apply crystal violet 

• primary stain

• 60 secs

• stains all cells purple initially

(2) Apply Gram’s iodine

• mordant stain

• 60 secs

• forms a complex w/ crystal violet that locks in the stain into Gram-positive bacteria’s cell walls

(3) Apply alcohol or acetone 

• decolourizer

• 24 secs

• removes stain from gram-negative cells

(4) Safranin 

• counter stain 

• 60 secs

• stains gram-negative cells pink

Which objective lens is best for viewing bacteria.

100x — Immersion oil objective

Purpose of Oil Immersion

Reduces light refraction —> Improving resolution at high magnification

INCOMPLETE—> ADD DETAILS FROM LAB MANUAL