brightfield microscopy/ LAB exam 1

Base

Sits on table and stabilizes the microscope

Arm

Raises and holds up most parts of a microscope

Rotatable head

Swivels and holds prisms

What is the power of the ocular lens?

10x Magnification

Mechanical stage

Flat platform that slide rests on

Illuminator

Light source

Condenser *

Directs light on to the sample, has a blue filter to increase resolution (the large barrel)

Diaphragm

Regulates amount of light passing to sample, located within the condenser (inside condenser)

Rotable nosepiece

Turns to change objective sense power

Objective lenses

Lenses closer to the slide (our microscope has four power options- 4X, 10X, 40X, 100X)

Ocular lens

Lens in the eyepiece (our microscope is 10X)

Coarse adjustment knob

Raises and lowers slide by large amounts to bring objects into focus (the large one)

Fine adjustment knob

Raises and lowers slide by small amounts to bring objects into focus (small one within large one)

Stage adjustment

Metal clamp that holds slide in place

Stage adjustment knobs

Controls movement of stage adjustment, top is back for back and forth, along Y axis and bottom is for one for left and right, along X axis

On/off switch

Controls power to illuminator

Light intensity control

Regulates amount of light produced by illuminator (from dim to bright)

What are the four objective lenses on our microscope?

• Scanning: 4×

• Low power: 10×

• High power: 40×

• Oil immersion: 100×

What is the equation for total magnification, and how do you calculate it?

• 4× objective → 40× total

• 10× objective → 100× total

• 40× objective → 400× total

• 100× objective → 1000× total

What is the purpose of immersion oil?

Reduces light refraction (bending), which improves the clarity and resolution of the bacterial image

Which objective lens requires immersion oil?

100× objective lens (oil-immersion lens)

Basic laboratory safety rules

No food, no gum, no drink, lab coat required, no open toed shoes, etc.

What are some of the objects that are regularly flamed to avoid contamination?

The loop and tube (Aseptic technique)

Steps of making a smear from a liquid sample (3 major steps)

1. Place two loopfuls of bacteria within target circle

2. Let the sample air dry for ten minutes

3. Heat fix: pass the sample through the flame six times

Why do we fuse the bacteria to the slide? What is this called?

Heat fix: fuses the bacteria to the slide and kills bacteria

What do we flame during the preparation of a smear?

The loop and tube

Why is loop and tube flamed ?

Because it it the aseptic technique

What is a wet mount

A microscope slide preparation where you place a living specimen in a drop of liquid and cover it with a cover slip to view under a microscope

What is the advantage of using an advantage of using a wet mount instead of a smear?

It can be used to view live organisms and you are able to see movement, live color, and morphology (size and shape)

What is a disadvantage of using a wet mount instead of a smear?

The organisms move, making them harder to observe

What microbes did you see using the wet mount that were autotrophs?

Filamentous algae and Diatoms

What microbes did you see when using the wet mount that were heterotrophs ?

Earthworms, Rotifers, Daphnia/copepods, and Ostracod

Why do we stain microorganisms?

To view morphology

How is a simple stain different from a differential stain?

Simple stain stains anything and everything one color

Differential stain uses multiple stains to differentiate between bacterial types; staining select

microbes one color while staining others a different color

What are the names and colors of the two simple stains that we use in our lab?

Safranin- pink

Methylene blue- blue

what are the four basic steps and purpose to a differential staining procedure?

1. Primary stain: colors target cell

2. Mordant: helps primary stain stick in target cell

3. Decolorizer: removing primary stain from non-target cells

4. Counterstain: add color to non-target cells

When is the simple stain used as part of the differential staining protocol?

As the counter stain to stain the non-target cell as the last step

Schaeffer-Fulton Method

the method used for endospore stains

What is the primary stain in Endospore staining?

malachite green- colors endospores

What is the mordant in Endospore staining?

steam

What is the decolorizer in Endospore staining?

water

What is the counterstain in Endospore staining?

Safranin- vegetative cells

What are two genera of common endospore formers?

• Bacillus

• Clostridium

What color are endospores at the end of this stain? (Schaeffer-Fulton Method)

green

What appears to be pink/red at the end of this stain? (Schaeffer-Fulton Method)

Vegetative cells

Non-target cells

What is the primary stain in Gram Staining?

Crystal violet (1 min)

What is the mordant in gram staining?

Grams iodine (1 min)

What is the decolorizer in Gram Staining?

alcohol/ acetone (1 min)

What is the counterstain in Gram Staining?

Safranin (1 min)

in gram staining What color is a Gram positive cell at the end of the stain?

Staphylococcus aureus- purple

in gram staining What color is a Gram negative cell at the end of the stain?

Escheria coli - pink/red

what are the shapes and colors of the two genera we used for Gram staining in our lab?

Coccus (spheres) - purple
Bacillus (rhods) - pink/red

You should know how to spell correctly the two genera we used for this stain.

- Staphylococcus Aureus - Escherichia Coli