Compound light microscope two main objectives
magnification & resolution
Magnification
enlargement of an image.
Resolution
the ability to see fine detail.
Relationship of magnification & resolution
indirectly related, as magnification increases and resolution decreases.
Resolution is measured using a term called
Resolving power
Resolving power
(rp) means that two points at a specified distance appear as two points rather than one. A resolving power= 2nm, means that when two points are 2nm apart you can distinguish them as two separate points.
Ocular Lens
Eyepeices.
Standard is 10x; may be changed to 15x or 20x
Objective lens
4x scanning; 10x low power; 40x high power; 100x oil immersion
To calculate the total magnification:
multiply ocular strength times objective strength.
Highest magnification possible in a compound microscope?
2000x
At magnifications higher than 2000x the light rays bend to the point that the image become distorted and the adequate resolution is impossible.
As magnification is increased in a light microscope it becomes necessary to increase the amount of light
What is the purpose of oil immersion?
Placing a drop of oil on the slide causes a closed system to be created between the specimen and the objective. (Note that the oil oozes up around the sides of the objective). This prevents light from escaping to the environment and places more light on t
Parts of a compound microscope
from the light to the eye.
illumination source (light)
condenser
stage ( specimen on slide)
objective lens
prism
ocular lens
Brightfield microscopy
most often used with a compound microscope. The specimen is dark or stained and the field is bright and light.
The electron microscope
Emerged in the 1940's as a very powerful tool in microbiology
Uses beams of electrons instead of beams of light
Specimen appears on a screen; no eyepieces
Made it possible for very small microbes to be seen, including viruses
Two types of electron microscopes
1.)Transmission- Magnifies up to 100,000 times, capable of resolving objects only .2 nm apart.
2.)Scanning- Magnifies up to 10,000 times. Yields striking three-dimensional images
(allows us to see things that are too small, make bigger doesn't use light.)
Staining
coloring the slide in order to change the refractive index of the specimen
Refractive index
This term refers to the speed at which light passes through an object. The refractive index of a glass slide and the refractive index of an unstained specimen are the same. This means the light from the microscope illumination source passes through the sl
Staining is done by
applying dyes to the specimen
Dyes
are salts, composed of positively charged components (positive ion or cation) and a negatively charged component (negative ion or anion)
in each dye either a positive ion or the negative ion is responsible for the actually coloring process.
Basic Dyes
positive ion acts as the coloring agent
Acidic dyes
negative ion acts as the coloring agent
Bacterial cells have a slightly overall _____ surface charge.
negative
What dyes are needed to stain bacteria
Basic, bc the positively charged coloring agent in a basic dye is attracted to the negative cell surface of the bacteria. (opposites attract)
When are acidic dyes used to study bacteria?
Acidic dyes (with their negatively charged coloring agents) are used to stain the slide background. This leaves the bacteria colorless are allows them to be seen against a stained background. This is called "negative staining." A capsule stain is an examp
Making a smear
Place a drop of water on a clean slide. (very small drop)
Using a sterile loop plae a small amount of bacteria in the drop of water and make a smear approximately the size of a quarter.
Allow the smear to airdry
Fix the smear
After the smear is completely dried, pass it back and fourth through a female twice. This is called fixing the smear.
Two reasons smears must be fixed.
Kill the bacteria.
Adhere the smear to the glass.
Main staining techniques in microbiology
Simple stains.
Differential stains.
Structural or Special stains.
Simple Staining
2
Allows us to determine the presence or absence of bacteria
Allows us to determine the morphology 9shape) of bacteria
Apply a basic dye, wait 60 seconds and wash the slide
The bacteria present will be the color of the dye applied.
Differential staining
Allows us to divide a bacteria into two groups based upon a characteristic of the bacteria.
Examples of differential staining.
1. gram stain (4) *
2. Acid-fast stain
Gram Stain
Named after the man who devised the procedure Hans Christian Gram.
Divides bacteria into 2 groups based upoN the amount of PEPTIDOGLYCAN found in the bacterial cell wall.
Bacterial cells with a thick layer of peptidoglycan will be purple or violet at the
Gram Positive vs Gram Negative cell walls
Gram Positive- cell wall composed of mainly of a thick layer of peptidoglycan.
Gram Negative- cell wall composed of a thin layer of peptidoglycan. An outer membrane composed of: phospholipids, lipoproteins, lipoplysaccharides
Gram staining involves the application of 4 different solutions.
1. Crystal violet- primary stain (basic dye)
2.Gram iodine- mordant (not dye)
3.Decolorizer-alcohol/acetone; ethyl alcohol
4.Safranin- counterstain (basic dye)
What happens to gram positive bacteria?
1.Crystal violet (CV) enters the cell
2.Gram's iodine (I) enters the cell.
3.Inside the cell, crystal violet and iodine bind together, forming the CV-I complex which is a larger molecule than CV or I is alone.
4.Decolorizer is added but has very little ef
What happens to gram negative bacteria?
1.Crystal violet enters the cell.
2.Gram's iodine enters the cell.
3.Inside the cell, crystal violet and iodine bind together, forming the CV-I complex which is a larger molecule than CV or I is alone.
4.Decolorizer is added, dissolving the outer layer of
What is the purpose of the mordant?
traps the crystal violet in the cell.
Is crystal violet a basic or acidic dye?
basic
Is safranin a basic or acidic dye?
basic
Is gram's iodine a basic or acidic dye?
not a dye
In each case how do you know?
basic color bacteria. stain
In theory what color are gram positive bacteria after the addition of the:
Primary Stain:
Mordant:
Decolorizer:
Counterstrain:
purple
purple
purple
purple
In theory what color are gram negative bacteria after the addition of the:
Primary stain
mordant
decolorizer
counterstain
purple
purple
colorless
pink
If the mordant was omitted, what color would gram positive bacteria be at the end of the gram staining procedure?
Pink
If the mordant was omitted, what color would gram negative bacteria be at the end of the gram staining procedure?
Pink
If the decolorizer was omitted what color would gram positive bacteria be at the end of the gram staining procedure?
purple
If the decolorizer was omitted what color would gram negative bacteria be at the end of the gram staining procedure?
purple