Microbiology Ch. 3 Part 1

Front 1

Bacterial flagella are too small to see with the eye. MIcrobiologists use the number and arrangement of flagella to properly diagnose.

what types of procedures do you use in testing flagella?

Back 1

-a mordant
-stain carbolfuchsin

*these two build up the the diameters of the flagella until they become visible

Front 2

Special tests:

*used to color and isolate various structures, each as capsules, endospores, and flagella, sometimes used as a diagnostic aid.

what three types of special tests are there?

Back 2

1. Negative
2. Endospore
3. Flagella

Front 3

Negative (special) testing:
used to determine the presence of....

Back 3

capsules

*because capsules do not accept stains, they appear as unstained halos around bacterial cells AGAINST a contrasting background.

Front 4

Endospore (special) testing.
used to determine the presence of...

Back 4

Endospores in bacteria.
-Malachite green is applied to a fixed heat smear
-stain penetrates bacteria and makes them GREEN.
-safranin is applied and makes the rest of the cells pink or red.

Front 5

Flagella (special) testing
used to determine the presence of...

Back 5

flagella.

-A mordant is used to build up the diameters of flagella until they become visible when stained with carbolfuchsin.

Front 6

Differential stains (X2)

-Acid fast
-Gram staining

Back 6

two tests used to destinguish different types of bacteria.

Front 7

-Acid fast (differential) staining
used to determine the presence of...

Back 7

Mycobacteriuam and Nocardia.

-once stained with carbolfuchsin and treated with ACID-ALCOHOL, remain PINK or RED because they retain the carbolfuchsin.

Front 8

Non acid fast bacteria do not remain PINK or RED because....

Back 8

-are stained with methylene blue, appear blue because they lose the carlfuchsin stain and then are able to accept the methylene blue stain.

Front 9

Gram (differential) stain:
used to determine the presence of...

Back 9

gram negative or gram positive bacteria

Front 10

Gram positive bacteria retain...

Back 10

the crystal violet stain and appear purple

Front 11

Gram negative bacteria retain...

Back 11

do not retain the purple color from the crystal violet stain

-they remain colorless until counterstained with safranin and appear PINK.

Front 12

Simple stain (methylene blue, carbolfuchsin, crystal violet, safranin)
used to determine...

Back 12

highlight microorganisms to determine cellular shapes and arrangements.
-Aqueous or alcohol solution of a single basic dye stains cells.

-Sometimes a mordant is used to intensify the stain.

Front 13

An endospore is a special resistant, dormant structure formed within a cell that protects a bacterium from adverse environmental conditions.

-although relativley uncommon in bacteria, they can be formed by a new genera of bacteria.

Back 13

-Endospores CAN NOT be stained by ordinary methods, such as simple staining or Gram staining, because the dyes do not penetrate the capsules.

-

Front 14

the most common endospore stain is the....

Back 14

Schaeffer Fulton endospore stain

-Malachite green is the primary stain and added to a heat fixed smear and heated for 5 min.
-preperation washed for 30 seconds and the green is washed away except for the endospores.
-next safranin is applied, (a counter stain), to stain the other parts of the cells that are not endospores.

Front 15

In a properly stained endospore, the cell appears

Back 15

pink with green endospore.

-Use the Schaeffer fulton endospore stain

Front 16

Demonstrating the presence of a capsule in a means of determining the organisms virulence, which means...

Back 16

the degree to which a pathogen can cause disease

Front 17

why is capsule staining more difficult?

Back 17

-because the capsules are soluble in water and maybe dislodged or removed during vigorous washing.

-use collodial suspension of colored particles (usually India ink or nigrosin) to provide contrasting background and then stain with simple stain safranin.

-capsules appear as a halos because they do not retain dyes or safranin

Front 18

Mycobacterium bovis bacteria that have been acid stained tested appear red with an acid fast stain. Non acid fast cells (staphloccus) are stained with what to appear blue?

Back 18

methylene blue counterstain

Front 19

Special stains use ______ and isolate various parts of microorganisms such as endospores and flagella and to reveal the presence of capsules.

Back 19

-color

-negative (capsule)
-endospore
-flagells

Front 20

what does acid fast staining test for?

Back 20

Mycobacterium tuberculosis
Mycobacterium leprae
Nocardia

-waxy material in cell walls

Front 21

what test is this:

1. red dye carbolfuchsin is applied to a fixed smear
2. Slide is heated for several min (intensify dye color)
3. Side cooled and washed with water
4. Smear treated with acid-alcohol (decolorizer) removing the red stain from cells that do not have lipid in walls (washing away the carbolfuchsin)
5. The acid-fast microorganisms retain the carbolfuchsin because it retains the color in the lipid layer
6. Non acid fast bacteria are colorless after the acid alcohol wash because teh carbolfuchsin is not retained. These cells will appear blue (after being colorless) because methylene blue will color them (the counterstain)

Back 21

Acid fast testing

for testing waxy lipids in cell walls such as Mycobacterium leprae Nocardia, etc.

Front 22

The two stains most used to identify bacteria are...

Back 22

-Gram stain
-Acid fast stain

Front 23

1. A heat fixed smear is covered with basic purple dye, crystal violet. Because the purple stain imparts its color to all cells, it is referred to as a primary stain.

2. After a short time, the purple dye is washed off, and the smear is covered with iodine, a MORDANT. When the iodine is washed off, both gram positive and gram negative bacteria appear dark violet or purple.

3. The slide is washed with alcohol or alcohol acetone solution. This is a decolorizing agent. Removes the purple from some cells and not from others.

4. Alcohol is rinsed off, the slide is stained safranin, a basic RED dye. The smear is washed again and blotted

Back 23

Gram stain procedure

Front 24

Because gram negative bacteria are colorless after the alcohol wash, what stain is used to color them red or pink?

Back 24

safranin (counterstain)

-because gram positive bacteria retain the original purple stain, they are not affected by the safranin counterstain.

Front 25

-a thicker peptidoglycan cell wall
-contain a layer of lipid lipopolysaccharide as a part of their cell wall
-crystal violet and iodine penetrate the cell making it retain the purple color
-alcohol does not wash out the color because the combo of crystal violet and iodine form to create CV-1

Back 25

gram positive bacteria

Front 26

-the alcohol wash disrupts the outer lipopolysaccharide layer, and the CV1 complex is washed out through the thin layer of peptidoglycan.
-as a resulte these bacteria are colorless until stained with the counter stain, safranin, which turns them PINK OR RED.

Back 26

-gram negative bacteria

Front 27

The gram reaction is most consistent when it is used on ...

Back 27

young growing bacteria

-gram pos. tend to be easily killed by penecillia
-gram neg. more resistant because antibiotics can not penetrate the lipopolysaccharide layer.

Front 28

A chemical is added to intensify the stain, this is otherwise knows as a ....

Back 28

mordant

Front 29

1. application of crystal violet
2. application of iodine
3. application of alcohol wash with decolorization
4. application of safranin

Back 29

Gram staining

Front 30

The primary purpose of this type of stain is to highlight the entire microorganism so that cellular shapes and basic structures are visible.

it is an aqeuous or alcohol solution of a single basic dye.

Back 30

Simple stain: methylene blue, carbolfuchsin, crystal violet, and safranin.

Front 31

Before microorganisms can be stained they must be __________ (attached) to the slide, this simultaneously kills the microorganisms. It also preserves the various parts of the microbes in their natural state with only minimal distortion.

Back 31

fixed

Front 32

After staining and fixing the specimen to the slide, a film is spread over the surface of the slide which contains the microorganism.

the film is otherwise called a ______, which is allowed to air dry.

Back 32

smear

Front 33

In most staining procedures, the slide is then fixed by passing it through the flame of a bunsen burner several times, smear side up, or by covering the slide with methyl alcohol for 1 min.
-stain is applied and then washed off with water, then the slide is blotted with absorbent paper.
-Without fixing the stain might wash the microbes off the slide.

Back 33

fixing procedure

Front 34

Stains are salts composed of a positive and negative ion, one of which is colored and known as the...

Back 34

chromosphore.

Front 35

The color of the basic dyes is in the

Back 35

positive ion

Front 36

The color of the acidic dyes is in the

Back 36

negative ion

Front 37

bacteria are slightly negatively charged at a pH of 7.

thus the colored positive ion in basic dye is attracted to the negatively charged bacterial cell.
-basic dyes include crystal violet, methylene blue, malachite green and safranin

Back 37

yes

Front 38

acidic dyes are not used that much because the dyes negative ions are repelled by the neg. charged bacteria surface, so the stain colors the background instead.

-examples of acidic dyes are eosin, acid fuchsin, and nigrosin.

Back 38

acidic dyes--used for background staining

Front 39

Preparing colorless bacteria against a colored background is called......

used with acidic dyes

Back 39

negative staining

Front 40

the standard unit for measurment is metic system is the meter.
-factors of 10
*1m= 10 decimeters
*1m= 10 decimeters or 100 millimeters
*micrometers or nanometers are used to measure microorganisms

Back 40

micrometers or nanometers are used to measure microorganisms

Front 41

a micrometer is eqyam to 0.000001 m (10X -6 m).

a nanometer is equal to 0.000000001 m (10-9 m).

Back 41

yes

Front 42

Van Leeuwenhoek was the best lens grinder to his day. he was the first person to see bacteria.

-Robert Hooke built compound microscopes which have multiple lenses.

-Lister made the best one and was the fonder of the modern compound microscope.

Back 42

yes

Front 43

-Light microscopy refers to the use of any kind of microscope that used visible light to observe specimens.

Back 43

light microscopy

Front 44

1. light rays pass through an illuminator (the light source)
2. then it passes through a condenser, which has the light rays pass through the specimen.
3. Light rays pass through the objective lenses, the lenses closest to the specimen.
4. the image of the specimen is magnified again by the occular lens, or eyepiece.

Back 44

Compound light microscope

Front 45

calculate the total magnification of a specimen by...

multiplying...
objective lens magnification (power) X occular lens magnification

Back 45

yes

Front 46

________ is the ability to distinguish fine detail and structure.

Back 46

Resolution--used to distinguish two points a specified distance apart.

for example, if a if a microscope has a resolving power of .4nm, it can distinguish two points if they are .4nm apart.

Front 47

the shorter the wavelength of light used in the instrument, the greater the......

Back 47

resolution

Front 48

the white light in compound microscope has a long wavelength and can not resolve structures smaller than

Back 48

.2um.

Front 49

the refractive index is the measure of the light bending ability of a medium.

-we change the refractive index of specimens by staining them, light rays increase visibility by passing through two mediums (specimen and stain), and the image is greatly magnified.

Back 49

refractive index

Front 50

to preserve direction of light rays at the highest magnification, immersion oil is placed between the glass and oil immersion objective lens. -immersion oil has the same refractive index as glass, so the oil becomes part of the optics of the glass and microscope.

Back 50

immersion oil

Front 51

The oil has the same effect as increasing the objective lens in diameter--improving the resolving power of the lens.

Back 51

immersion oil

Front 52

this microscope is used to examine the live microscopes that are invisible to the ordinary light microscope, or distorted by staining.

-uses a darkfield condenser that contains an opaque disk.

Back 52

-darkfield microscopy

Front 53

-because there is no direct background light, specimens appear light against a black background.

-useful for specimens in suspended in liquid, examination of very thin spirochetes, such as Treponeuma pallidum which causes syphilis.

Back 53

-darkfield micrscope

Front 54

-permites examination of living microorganisms
-don't need to attach or fix microorgansims
-form image of specimen on occular lens containing areas that are light an shades of gray, to black.
-internal structures are more closely defined.

-uses differences in refractive lenses

Back 54

Phase contrast microscopy

Front 55

-uses differences in refractive lenses (like phase contrast microscopy), but BUT uses TWO different beams of light instead of ONE.

-resolution is higher than standard phase contrast microscope, and the image appears 3D.

Back 55

Differential Interference Contrast (DIC)

-along with Phase contrast microscope, it uses differences in refractive lenses.

Front 56

The ability of substances to absorb short wavelengths of light (ultraviolet) and give off light at a longer wavelength (visible).

-stained with florochromes, they appear as luminescent bright objects agains a black background.

Back 56

Flourescence Microscopy

Front 57

Immunofluorescence is especially useful in diagnosing syphilis and rabies

Back 57

Fluorescence Microscopy

Front 58

Because the refractive indexes of the glass microscope slide and immersion oil are the SAME, the light rays do not refract when passing from one to the other when an oil immersion objective lens is used.

Use of immersion oil is necessary at magnification grater than _____

Back 58

900X

Front 59

type of microscopy used to reconstruct 3D images.

-specimen is stained with fluorochromes so they will emit light.
-ONE plane of a small field is illuminated with a short wavelength (blue) light.
-successive planes are illuminated until the entire specimen has been scanned.
-uses pinhole aperature, thus, eliminates BLURRING.
-improved visibility up to 40% compared to other microscopes.

Back 59

Confocal Microscopy

Front 60

-used with conjunction with computers to make 3D images.
-used to evaluate cellular physiology by monitoring the distributions and concentrations of substances such as ATP and calcium ions.

-can image cells only up to a depth of less than 100um.

Back 60

Confocal Microscopy

Front 61

Two photon microscopy and Confocal Microscopy use what?

Back 61

fluorochrome

Front 62

-uses long wavelengths (red) light and two photons instead of ONE are needed to EXCITE the flurochrome to emit light.
-longer wavelength allows for imaging of cells in tissues up to 1mm (100um) deep
-longer wavelengths less likely to generate singlet oxygen which can damage cells.

-can track activity of cells in REAL TIME.
-OBSERVE cells of immune system in REAL TIME.

Back 62

Two Photon Microscopy (TPM)

Front 63

-this microscopy consists of interpreting the action of a sound wave sent through a specimen.
-A sound wave of a specific frequency travels through the specimen, and a portion of it is reflected back every time it hits an interference within the material.

-resolution is about 1um.
-used to study living cells attached to another surface, such as CANCER cells, artery plaque, and bacterial biofilms that foul equipment.

Back 63

Scanning Acoustic Microscopy (SAM)

Front 64

-looks at viruses or structures of cells in .2um.
-beam of electrons are used, thus shorter lengths, (100,000 smaller than wavelength of light).
-images are always BLACK AND WHITE
-uses electromagnetic lenses
-transmission and scanning type

Back 64

Electron Microscope

Front 65

A type of flurochrome is combined with antibodies against a specific type of bacterium.
-Antibiodies attach to the bacterial cells and the cells fluorescent when illuminated

Back 65

principle of immunofluroescence.

Front 66

-a finely focused beam of electrons from an electron gun passes through a ultra thin section of specimen.
-beam is focused on a very specific area of specimen by electromagnetic condenser of a light microscope-directing beam of electrons in a straight line to illuminate the specimen.
-specimen placed on a copper mesh grid.
-electrons are focused by a electromagnetic lens (rather than occular lens) onto a flourescent screen or photographic pate.
-final image, the transmission electron micrograph appears as many light and dark areas depending on the number of electrons absorbed by specimen.

Back 66

Tansmission Electron Microscopy

Front 67

-contrast is weak
-can use stain that absorbs electrons
-salts of heavy metals such as led, osmium, tungsten, and uranium are commonly used as stains.
-metals can be fixed onto specimens (positive staining) or can used to increase electron opacity of the surrounding field (negative staining).

Back 67

Transmission Electron Microscopy

Front 68

Negative staining is useful for the study of very small specimens, such as virus particles, bacterial flagella, and protein molecules.

-can resolve images as close together as 10pm, and objects are magnified 10,000 to 100,000X.

Back 68

Transmission Electron Microscopy

Front 69

Viewed with this technique where a heavy metal such as platinum or gold is sprayed at an angle of about 45 degrees so that it strikes the microbe from only one side.
-the metal piles up on one side of the specimen, and the uncoated area on the opposite side of the specimen leaves as clear area behind it as a shadow.
-3D image

-disadvanages: only a very thin section can be studied (100nm)
-specimen thus has no 3D aspect
-specimens must be fixed, dehydrated, and viewed under high vacume
-kill specimen
-addtional structures may appear as a result of preparation distorting image
-artifacts--structure that appear as a result of preparation

Back 69

Transmission electron microscopy

Front 70

-this microscope overcomes the Trasmission electron microscopy problem.
-uses primary electron beam to make 3D images
-electromagnetic lenses are shot over the surface of specimen, knocking electrons out fo the surface of the specimen, and the secondary electrons thus produced are transmitted to an electron collector, amplified,d and used to produce an image on a viewing screen or photographic plate.
--studies surface areas of intact cells or viruses
-can resolve objects as close together as 10nm, and objects are generally magnified 1000 to 10,000X.

Back 70

Scanning electron microscopy

Front 71

-use probes to examine the surface of a specimen using electric current, which does NOT modify the specimen or expose it to damaging high radiation.
-map atomic and molecular shapes, to characterize magnetic and chemical properties, and determine temperature variations inside cells.

Back 71

Scanned probe microccopy

2 types:
1-sanning tunneling
2-Atomic FORCE

Front 72

-uses thin metal (tungsten) probe that scans specimen and produces an image revealing bumps and depressions of the atoms on the surface of specimen.
-resolving power of STM is much greater than that of an electron microscope; it can resolve features that are only about 1/100 the size of an atom.
-special preparation of specimen not needed
-detailed views of molecules and DNA.

Back 72

Scanning Tunneling Microscopy

Front 73

-metal and diamond probe gently forced onto a specimen
-movements are recorded as prove moves onto specimen, and 3D image is recorded.
-does not require special specemin preparation (along with STM).
-used to image biological substances (in atomic detail) and molecular processes (such as assembly of fibrin, a component of blood clot).

Back 73

Atomic FORCE Microscopy

Front 74

Which two microscopes do not require special preparation?

Back 74

Scanning Tunneling Microscopy (STM) and Atomic Force Microscopy (AFM).