Micro Chapter 3
Steps in order for grams staining:
(1) Primary Stain: Crystal Violet (2) Mordant: Iodine (3) Decolorizing Agent: Alcohol-Acetone (4) Counterstain: Safranin
what is the wavelegnth of the fluorescence microscope?
.1 micrometer
What is the resolution of the bright field microscope?
0.2 micro meters
What is the resolution (resolving power) of a compound light microscope
0.4 nm
Simple Staining
1 basic dye
1,000 nanometers (nm) =
1 micrometer
What are the steps to Endospore Staining?
1) Primary Stain: Malachite green, usually with heat 2) Decolorize cells: Water (steam, which allows the color to go through) 3) Counterstain: Safranin
What are the steps to making a slide?
1) make a smear 2) air dry 3) heat fix
1 Micrometer =
10⁻⁶ of a meter; i millionth of a meter
1 Nanometer =
10⁻⁹ of a meter; 1 billionth of a meter
Scanning Electron Microscopy (SEM)
20 nm
time to make cards from
: 39:01
MESOSOMES
ARE NOT TRUE STRUCTURES BUT ARE CREATED AS ARTIFACTS OF ELECTRON MICROSCOPE
Gram Negative cell wall consists of:
Abundance of Lipid Very little Murein/peptidoglycan
Gram positive cell walls consist of :
Abundance of peptidoglycan (murein) Very little Lipid
Phase-Contrast Microscopy
Accentuates diffraction of the light that passes through a specimen Live cells No staining
Differential Interference Contrast Microscopy: DIC
Accentuates diffraction of the light that passes through a specimen; Uses two beams of light Has two prisms that add color to live cells Slightly better resolution because of extra light
Which stain would be used to identify microbes in the genera Mycobacterium and Nocardia?
Acid
What is the Ziel-Neilson method used for?
Acid Fast staining
Which staining is the decolorizing step not as sensitive?
Acid-Fast
What are the 3 types of dye used for stains?
Acidic Basic Neutral
What stains would you use to look at capsules?
Acidic Dyes: India ink; nigrosin; congo red Then basic dye to see the cell
What is the 2nd step in Grams Staining?
Add Mordant: Iodine
How does fluorescent microscopy work?
Antibodies attack bacteria and fluorescent dye attaches; you must buy antibody to attack bacteria, its flurecent die will attack to the sample, if it is possitive, it will attach and you can identify it
Steps to flagella staining:
Apply mordant: Tanic Acid Stain: Carbolfuchsin
Microbacterium TTB is identified by _________
Auramine fluorescent stain
If you apply an acidic dye, what will be stained?
Background (the slide)
What happens when you heat fix a microorganism?
Bacteria Die Bacteria coagulate Bacteria shrink
In a ______ dye, the chromophore is a cation
Basic
Since bacteria have an overall negative charge, _______ dye staining is usually used
Basic dye
why does an acidic dye stain the background and not the cell?
Because the colored part has a negetive charge so it is repeled by bacteria cell wall
which is the most common of the microscopes?
Bright field microscopes
________ illumination: dark objects are visible against a bright background; light is reflected off the specimen and does not enter the objective lense
Brightfield illumination
What are the two types of Special Stains?
Capsule stain Endospore stain Flagella stain
Facts about confocal Microscopy
Cells are stained with fluorochrome dyes Short-wavelength (blue) light is used to excite the dyes The light illuminates each plane in a specimen to produce a three-dimensional image Up to 100 µm deep
How can you improve the resolution of a microscope?
Change the wavelength by using a shorter wavelength ;
Types of light Microscopy
Compound light microscopy Darkfield microscopy Phase-contrast microscopy Differential interference contrast microscopy Flourescence microscopy Confocal microscopy
______ is like a CT scan for bacteria and gives a brightly colored 3d image of the bacteria. Best resolution of all light microscopes.
Confocal Microscope
Best resolution of all microscopes
Confocal Microscope (light microscopes?)
Iris Diaphragm
Controls the amount of light entering the condenser
What is step 3 of Acid-Fast staining?
Counterstain: Methylene Blue
What is the last step in Grams staining?
Counterstain: Safranin
5 Basic Dyes:
Crystal Violet Methylene Blue Malachite Green Carbol fuschen safranin
Brightfield illumination
Dark objects are visible against a bright background Light reflected off the specimen does not enter the objective lens
Treponi syphallis / Treponema pallidum is viewed with this type of illumination
Darkfield
in _______ illumination, light reflected off the specimen enters the objective lens
Darkfield
In __________ illumination, you can see living cells because you do not need to stain. Field of view is dark.
Darkfield illumination
What is step 2 of Acid-Fast staining?
Decolorizing Agent: ACID-ALCOHOL
What is the 3rd step in Grams Staining?
Decolorizing Agent: Alcohol-Acetone
What does alcohol do to the gram positive cell wall?
Dehydrates the murein and makes the cell wall pores close and locks in the dye
What does alcohol do to a Gram Positive cell wall with a lot of Murein?
Dehydrates the peptidoglycan and makes the opening in the cell walls close and the CV complex remains inside
Acid fast falls under which category?
Differential Staining
Grams Staining falls under which category?
Differential Staining
What stains are used to distinguish between bacteria?
Differential Stains
Which microscope accentuates diffraction of the light that passes through a specimen and uses two beans of light (prisms) and can add color;
Differential interference contrast microscopy ; D.I.C
Which technique is used to differentiate between different microbes?
Differential staining
What does alcohol do to the gram negative cell wall?
Dissolves lipid, makes opening bigger; the color is lost
What is the schaeffer-Fulten method used for?
Endospore staining
______ is a highly resistant structure that forms in response to adverse enviornments
Endospore; very hard to stain
T/F: Gram positive cells change color after primary staining is done?
False, Gram pos stays purple during the whole process; holds on to the die the whole time
Which microscope would you use to study the reaction between an antigen and antibody?
Fluorescence Microscope
What are the two differential staining techniques?
Grams Acid Fast
Two types of differential Stains
Grams stain Acid fast
How do stained endospores appear?
Green
Fluorescence Microscopy is used in this technique:
Immunofluorescence / Flourescent antibody technique
Types of acidic dyes
India Ink Nigrosin Congo Red
What does alcohol do to a Gram Negative cell with a lot of lipid and little Murein?
It dissolves the lipid and makes the opening bigger and the CV complex is lost
why do you have to use heat to push the malachite green into an endospore?
It has a lot of layers, peptidoglycan, protein coat ; the dye cannot penetrate without the heat
Illuminator
Light Source
Darkfield Illumination
Light objects are visible against a dark background Light reflected off the specimen enters the objective lens Field of view Dark Living Cells Used to view Treponima Syphallis
Gram negative cells has a lot of _____ with little _____ wich is the reason it will not stain
Lipid, Peptidoglycan
Characteristics of Gram positive cell wall:
Little lipid A lot of peptidoglycan (murein)
Why stain?
Live or unstained cells have little contrast with the surrounding medium. Researchers do make discoveries about cell behavior by observing live specimens.
What does making a smear mean?
Making a thin film of solution of microbes on a slide
In Grams staining, what attaches to crystal violet to form the crystal violet complex?
Mordant: Iodine
Compound microscope has?
More than one lens
What are you suspecting a patient has if you do an acid-fast stain?
Mycobacterium Tuberculosis Nocardia Mycobacterium Lepra
Acid-Fast postitive cells contain ________ in their cell walls
Mycolic Acid (fat)
Fat in the cell wall is called:
Mycolic acid
TB and Leprosy are a __________
Myobacterium
Do you heat fix the organism in negative staining?
NO
What are the two units to measure microbes?
Nanometers Micrometer (more common)
Does the condenser magnify?
No, it gathers the light and focuses it
Simple microscope has?
Only one lens
What is the 1st step in Grams Staining?
Place Primary Stain: Crystal Violet
basic dye has a ____ charge
Positive
Steps to acid fast stain:
Primary Stain: Carbolfuchsin Decolorizing Agent: Acid-alcohol Counterstain: Methylene Blue
Objective lenses
Primary lenses that magnify the specimen
In acid fast cells, what dissolves in the mycolic acid?
Primary stain Carbolfuchsin
Cells with a capsule function as :
Protection Helps resist antibody
What does heat fixing do?
Proteins coagulate and stick; it kills the organism
____ is the color of a gram positive cell.
Purple
____ is the color of a gram negative cell
Red (pink to me)
Endospore Staining is also called this method:
Schaeffer-Fulten method
What are the 3 staining techniques?
Simple Differential Special
what procedure do you use to look for capsules?
Special Staining: negative staining + positive staining to see halo
If you apply a basic dye to a cell, what does it stain? and why?
Stains the cell; because the dye is Positive and the cell is Negative
What is step 1 of Acid-Fast staining?
Steam, Flood then add: Primary Stain: Carbolfuchsin
Neutral Dye
Sudan Black- dissolves fat
What organism are you looking for if you do an acid fast stain?
TB- microbacterium tuberculosis Mycobacterium lepra (lepracy) Nocardia
______ is the mordant used to thicken a flagella.
Tanic Acid
if you are acid-staining a cell and it does not take, what can you assume?
There is no mycolic acid
Gram negative lose their color in step _____; they then gain color in step _______ by the addition of _____ dye.
Three; Four; Counterstain Safranin
Body tube
Transmits the image from the objective lens to the ocular lens
which bacteria causes syphilis
Treponema pallidum
Fluorescence Microscopy uses ______ light
UV light
Transmission Electron Microscopy (TEM)
Ultrathin sections of specimens Light passes through specimen, then an electromagnetic lens, to a screen or film Specimens may be stained with heavy-metal salts
Special stain: Capsule
Use acidic for the background of cell (Negative) Use basic for the actual cell (positive) Shows true size and shape of cell
Electron Microscopy
Uses electrons instead of light The shorter wavelength of electrons gives greater resolution
Why are electron microscopes better than the rest?
Uses electrons that have a shorter wavelegnth than light
Endospores are almost always in the ____________ state.
Vegitative
Do you steam acid fast?
YES
Acid-Fast Stain is also known as the _______________ method
Ziehl-Nelson
What is a capsule?
a gell layer outside the cell wall composed of sugars
Nocardia causes:
a type of pnemonia
in _______ dye, the slide is stained instead of the organism
acidic
in an _____ dye, the chromophore is an anion
acidic dye
you see a clear halo in your slide, what is it?
capsule
______ disolves in the mycolic acid
carbolfuchsin
How do unstained endospores appear?
clear
auxochrome
clear
Staining
coloring microbe with a dye that emphasizes certain structures
Which microscope can you connect to a computer?
confocal
which is the critical step in grams staining?
decolorizing with alcohol-acetone
which category category would grams and acid fast staining fall under?
differential staining
In confocal microscopy, the cells are stained with __________ dyes
fluorochrome
Most microbes are not fluorescent and must use _________
fluorochromes
________ is antigenic and stimulates antibody
flurescence microscopy
________ keeps the refractive index low.
immersion oil
Genus: Bacillus clostridium
important tetnus etc
What is the mordant used in gram staining?
iodine
Refractive index
is the measure of the light-bending ability of a medium
Condenser Focuses
light through specimen
Mordant
may be used to hold the stain or coat the specimen to enlarge it
Gram _______ bacteria are more resistant to antibiotics
negative
bacteria have an overall ______ charge
negative
_______ ________ is the process of staining the background instead of the the cell
negative staining
staining of the background is called ?
negative staining
_____ dyes have no charge
neutral dyes
A bacteria is OVER 25 hours, is this appropriate to use for gram staining?
no, must be Less than 24 hours
Total magnification
objective lens x ocular lens
Gram positive cells has a lot of ________ in the cell wall
peptidoglycan / murien with very little lipid
With this microscope you can see internal details of the cell (light and dark)
phase-contrast microscope
_________________ accentuates diffraction of the light that passes through a specimen.
phase-contrast microscopy
Gram _______ bacteria tend to be killed by penicillin and detergents
positive
_____ staining is when you stain the cell and not the background
positive
Acid-Fast Staining is __________ to color loss
resistent
Transmission Electron Microscopy (TEM) Resolution
resolution 2.5 nm
what is the counterstain in gramstaining?
safranin
What microscope would you use to see a 3D image of the surface of the cells
scanning electron microscope (SEM)
________ provides greater resolution
shorter wavelengths
3 staining techniques
simple differential special
micrograph
slide of bacteria
_____ pushes dye inside the cell
steam
Resolution
the ability of the lenses to distinguish two points
compound microscope
the image from the objective lens is magnified again by the ocular lens
Flagella staining is a sensitive process because the flagella are:
thin and fragile
Which electron microscope would you use to see internal details of cells
transmission electron microscope
T/F acid fast stain, decolorizing step is not important
true
Simple Stain
use of a single basic dye
Why would you want to do negative staining?
used to look for capsules
