Microbiology Lab Exam 1

Front 1

What is a stain?

Back 1

A stain is simply a solution to which a colored molecule called a chromogen has been added.

Front 2

A stain is simply a solution to which a colored molecule called a _______ has been added.

Back 2

chromogen

Front 3

What are the two components that make up a chromogen?

Back 3

chromophore: gives the stain its color

auxophore: possesses a charge

The two components are linked by a covalent bond.

Front 4

What determines whether or not a stain will bind to the negatively charged surface of a bacterial cell?

Back 4

the auxochrome; in a positive stain the auxochrome is positively charged. This creates an attraction between the stain and the sfc of the bacterial cell. Negative stains are repelled. Thus, bacteria will take on the color of the stain in a positive stain, but will remain colorless against a colored background in a negative stain.

Front 5

What is the proper way to dispose of a slide used in a negative stain?

Back 5

Must be disposed of in a disinfectant container. Since the slide is not heat-fixed, it is possible for the organisms to still be alive.

Front 6

Microscope:

The condenser does what?

Back 6

it is the lens that focuses the light onto the specimen

Front 7

Microscope:

What is the iris diaphragm?

Back 7

the rotating disk, which is used to vary the intensity and path of the light that is projected upwards through the specimen.

Front 8

Microscope:

What is the nosepiece?

Back 8

The revolving part that holds all the objecive lenses.

Front 9

Microscope:

What is the additional magnifying power of the ocular lens?

Back 9

10x

thus, if you are using the 40x objective lens, the total magnification is 400x.

Front 10

What is purpose of using immersion oil?

Back 10

It prevents the light coming through the specimen from being refracted. This ensures that the light path moves straight up through the objective lens.

Front 11

The most effective way to sterilize media is to....

Back 11

autoclave it

Front 12

The 3 parameters critical in obtaining sterility:

Back 12

Temperature, time, pressure.

why are all 3 parameters needed?--it is one of the only effective ways to kill endospores.

Front 13

Autoclave:

Which type of exhaust is used for dry items?

liquids?

Back 13

fast exhaust for dry items; slow exhaust for liquids

Front 14

Three Basic Categories of Microorganisms:

Back 14

Pathogenic, opportunistic pathogens, free-living microorganisms.

Front 15

5 morphological descriptors for elevation of bacterial colony:

Back 15

Raised, Convex, Flat, Umbonate, Crateriform.

Front 16

5 morphological descriptors for form of bacterial colony:

Back 16

Circular, Irregular, Filamentous, Rhizoid

Front 17

5 morphological descriptors for margin of bacterial colony:

Back 17

Entire, Undulate, Filiform, Curled, Lobate.

Front 18

Examples of positive stains:

Back 18

Crystal Violet, Methylene Blue, Safranin

Front 19

Examples of Negative Stains:

Back 19

India Ink, Nigrosin

Front 20

Pros/ Cons of negative and positives stains:

Back 20

Pros: requires only one dye; uncomplicated procedure

Cons: cannot differentiate unique cell structures or features between bacteria

Front 21

What are the 5 steps for the Gram stain?

Back 21

1. Fixation (heat fix)
2. Primary Stain (crystal violet)
3. Mordant (iodine)
4. Decolorizer (ethanol)
5. Counterstain (safranin)

Front 22

What is the purpose of the Gram stain?

Back 22

To differentiate between bacterial cell envelope types.

-Negative = thin peptidoglycan layer and outer membrane

-Positive = thick peptidoglycan layer, no outer membrane

Front 23

What is the purpose of the mordant in the gram stain?

Back 23

Adding the mordant iodine after the crystal violet primary stain produces crystal violet-iodine complexes. Fortifies the purple pigment with the peptidoglycan.

Front 24

What is the purpose of the decolorizer in the Gram stain?

Back 24

Ethanol dissolves the outer membrane in the G-negative bacteria. This combined with the thinner layer of peptidoglycan allows the color to leave the cell wall. Bacteria with thicker peptidoglycan retain the primary stain.

Front 25

What are some common errors when preparing the Gram stain?

Back 25

using older cultures. over-decolorization. under-decolorization. using thick bacterial smears.

Front 26

What is the purpose of the acid-fast stain?

Back 26

Gram stain do not work on mycobacteria due to their waxey lipid layer of mycolic acid. (mycobacterial cell wall)... causes Gram stain to be inconsistent and inaccurate.

Front 27

What type of stain is used in an acid-fast test and why?

Back 27

A carbolfuchsin stain. It is a phenolic-based primary stain that is LIPID soluable. This allows it to move more freely through the waxey layer of mycolic acid.

Front 28

What are two ways by which the ability of carbolfuchsin to penetrate the waxey cell wall of mycobacterium is enhanced?

Back 28

1. Apply Heat in the form of steam to drive the stain in

2. Increase the concentration of the phenol.

Front 29

During an acid-fast staining procedure, if heat is used, it is known as:

Back 29

the Ziehl-Neelsen Method

Front 30

During an acid-fast staining procedure, if heat is NOT used, it is known as:

Back 30

the Kinyoun Method

Front 31

Steps of the Acid-Fast Ziehl-Neelsen Method:

Back 31

1. Primary stain with carbolfuchsin; made to penetrate the cell with the use of heat for 5 minutes.

2. Decolorization with Acid-Alcohol. This is a strong decolorizer and is effective at releasing carbolfuchsin from all bacteria except those with a waxey cell wall.

3. Counterstain using methylene blue or brilliant green. Dye is taken up by non acid-fast cells, but not by waxey cells.

Front 32

Steps of the Acid-Fast Kinyoun Method:

Back 32

1. Stained with Carbolfuchsin, but this type has a higher concentration of phenol.

2. Decolorizer: Acid-Alcohol

3. Counterstained with methylene blue and brilliant green

Front 33

What is the appearance of cells that are acid-fast positive?

Back 33

pinkish/ red

Front 34

What is a capsule?

Back 34

A complex layer of sugars and proteins that tightly surrounds a bacterium. Not all bacteria have capsules. Thus, it can be used to differentiate b/t species.

Front 35

What is the role of a capsule?

Back 35

1. Protecting the bacterium against harsh environmental conditions and dehydration

2. helps the bacterium adhere to the sfc of an object

3. Helps prevent phagocytosis of bacteria by macrophages

In pathogenic bacteria, capsule is considered a virulence factor

Front 36

Name a pathogenic bacterium that has a capsule? mentioned in class.

Back 36

Neisseria meningitidis... causes meningococcal meningitis

Front 37

Can a capsule be stained?

Back 37

No... a capsule is water soluable so stains will NOT adhere to it.

The capsule stain is a differential stain that combines negative and simple stains.

Front 38

Capsule staining steps:

Back 38

1. Negative staining with congo red. An acidic dye that carries a negative charge. Like charges repel. Only the background is stained.

2. Air Drying the slide. Do not heat fix. Heating can cause the cell to shrink, resulting in halo around cell (false positive). Heating can destroy the capsule so a halo would not appear (false negative).

3. Simple staining w/ Maneval's stain. A basic dye that carries a positive charge. Sticks to bacterium.

After stain application... no halo = no capsule.

Front 39

Is the capsule stain heat fixed?

Back 39

No.

Front 40

What is the purpose of the PHB Stain?

Back 40

to determine whether or not a bacterial cell type has inclusion bodies--storage units used to store nutrients for later use. Not all bacteria have these; thus, the stain is used as another differentiation method.

Front 41

Are carboxysomes considered inclusion bodies?

Back 41

Yes. They are storage devices used to hold the enzyme ribulose 1,5-diphosphate carboxylase. Used in carbon dioxide fixation.

Front 42

inclusions that contain volutin (inorganic phosphates), which can be used for the synthesis of ATP

Back 42

Metachromatic Granules

Front 43

inclusions that contain iron oxide, which can act like tiny magnets

Back 43

magnetosomes

Front 44

inclusions that contain sulfur or sulfur-containing compounds

Back 44

Sulfur Granules. Thiobacillus use sulfur granules as source of energy.

Front 45

inclusions that typically contain glycogen and/or starch as a source of energy

Back 45

Polysaccharide Granules

Front 46

inclusions that commonly contain the lipid polymer poly β-hydroxybutyrate (PHB), which is used as both carbon and energy sources

Back 46

Lipid Inclusions

Front 47

PHB consists of

Back 47

-multiple β-hydroxybutyrate molecules linked by an ester bond between the carboxyl group of one molecule and the hydroxyl group of the next

-hydrolysis of the ester bond results in carbon-containing molecules and sufficient free energy to be harnessed ultimately into ATP

Front 48

PHB inclusions can range in diameter from

Back 48

- 0.2 to 0.7 μm

Front 49

How the PHB stain works:

Back 49

1. a smear is flooded with sudan black B stain for 5 minutes.

--sudan black is uncharged and considerd neutral. It can dissolve through the lipid components of the inclusion, but it does NOT associate with any cellular components.

3. The excess stain is removed by tilting the slide over the sink and the slide is blotted dry with B paper. It is not rinsed.

4. The sample is decolorized for 15-20 seconds using Citri-Solv. Removed by tilting slide over sink... not rinsed. Blotted dry.

--Only the inclusions will be black. the rest will be transparent.

5. Counterstain using safranin for 1 minute. rinse with water.

--cells will appear red and, if present, inclusions will appear black.

Front 50

What are the three C's of an endospore?

Back 50

the Coat, a Cortex made up of peptidoglycan, and an inner Core that caontains the bacterium's DNA.

Front 51

The process of forming an endospore is called _____.

Back 51

sporulation.

Front 52

The process of an endospore returning to a vegetative state is called ________.

Back 52

germination

Front 53

Name two species of bacteria that can form endospores.

Back 53

Bacillus (mostly non-pathogenic, exception is anthrax) and Clostridium (many non-pathogenic yet several pathogenic species)

Front 54

What information can an endospore stain provide?

Back 54

1. Whether the bacterium can OR cannot produce endospores.

2. the size, shape, and location of the endospores w/in a bacterium can help in the identification of the organism.

Front 55

Endospore stain steps:

Back 55

1. Steam is used to drive/ force the primary stain Malachite green into all bacteria as well as endospores.

2. Decolorization with water removes the Malachite green from the bacteria but NOT from the endospores.

3. Sample is counterstained with safranin, which is taken up by all bacteria, but w/o steam cannot be taken up by the endospores.

Endospores appear green w/in red bacterial cells.

Front 56

Two ways of determining whether a bacterium is motile:

Back 56

1. Hanging drop slide

2. Semi-solid motility agar

Front 57

Name and describe the 4 arrangement types of flagella:

Back 57

Monotrichous: 1 flagella at 1pole

Lophotrichous: several flagella at 1 pole

Amphitrichous: 1 flagella at each pole

Peritrichous: flagella all over

Front 58

What is used to stain for flagella?

Back 58

a mordant. It is non-specific. It will bind to anything.

Front 59

To make a motility test easy to read ___________ can be added to the media

Back 59

TETRAZOLIUM SALT (TTC); TTC is an electron acceptor that is used to dectect cellular respiration; when it is reduced by metabolically active bacteria, it forms a red insoluable compound.

Front 60

What two things does a plaque assay show?

Back 60

1. which species of bacteria the bacteriophage can infect

2. the number of bacteriophage particles in a sample

Front 61

Plaque Assay Steps:

Back 61

1. A dilution series of the bacteriophage sample is prepared

2. Portion of each diluted phage sample added to a constant volume of the host bacterium to be infected

3. Bacteria and bacteriophage mixed together and the bacteriophage allowed to attach to the bacteria (pre-adsorption)

4. : Each bacteria + bacteriophage mixture added to a tube of soft agar that has been autoclaved and cooled to 45 degrees

5. Each soft agar + bacteria + bacteriophage tube is mixed and poured onto a regular agar plate so that the soft agar spreads evenly across the plate (agar overlay).

Front 62

The concentration of a bacteriophage sample is given in

Back 62

PFU/ ml (plaque forming units per ml)