Micro Chapter 3

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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


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