Lab Exam 1

Question 1

Aspergillus Conidiophores

Answer 1

Question 2

Penicillium

Answer 2

Question 3

Colony Morphology includes:

1. ________ (name 3 subcategories)
2. ________ (name 5 subcategories)
3. ________ (name 5 subcategories)
4. ________ (name 3 sub categories)
5. ________ (name 4 subcategories)

Answer 3

1. Shape
   
   1. Circular
   2. Irregular
   3. Punctiform (tiny)
2. Margin
   
   1. Entire (smooth, mo irregularities)
   2. Undulate (wavy)
   3. Lobate (lobed)
   4. Filamentous
   5. Rhizoid (branched like roots)
3. Elevations
   
   1. Flat
   2. Raised
   3. Convex
   4. Pulvinate (very convex)
   5. Umbonate (raised in the center)
4. Texture
   
   1. Moist
   2. Mucoid
   3. Dry
5. Pigment production
   
   1. Opaque
   2. Transluscent
   3. Shiny
   4. Dull)

Question 4

With regards to bacterial growth, what does the term pure colony or colony-forming unit mean?

Answer 4

Pure colony: Microbial culture containing only one species

Colony-Forming Unit: Cell or groups of cells that produces a colony when transferred to a plated media

Question 5

What are the important precautions taken to avoid contamination?

Answer 5

• Do not perform transfers over books, study materials, etc
• Be organized with your materials and be sure they are not set up in ways that might result in contamination
• Plase all media tubes i the test tube rack when not in use whether they are sterile or not
• Take your time
• Never hold tube culture by it’s cap
• Try not to breathe over cultures
• Minimize talking with peers to avoid contamination

Question 6

In which hand should the tube cap be held while transferring cultures from one medium to another?

Answer 6

In your dominant hand, same as the loop.

Question 7

Why should Petri dishes be incubated in the upside down position?

Answer 7

Moisture can build up on the lid if the plated culture is set with the culture on the bottom part. To avoid this you place the dish upside down.

Question 8

Why is the mouth of the test-tube passed over the flame after opening and before closing the tube cap?

Answer 8

To sanitize the tube and the air around it.

Question 9

Why is the inoculating loop flamed before and after use?

Answer 9

To sanitize and avoid contamination.

Question 10

What is the pH of nutrient agar?

Answer 10

6.6-7.0 at 25º C

Question 11

What are the ingredients of a nutrient agar and a nutrient broth?

Answer 11

Nutrient agar: 3.0 g beef extract, 5.0g peptone, 15.0g agar, 1.0L distilled or deionized water

Nutrient broth: 3.0 g beef extract, 5.0g peptone, 1.0L distilled or deionized water

Question 12

What is the limit of resolution and how do you calculate it?

Answer 12

Limit of resolution: The closest two points can be together for the microscope lens to make them appear separate; two points closer than the limit of resolution will blur together

(Note: the resolution improves as the limit of resolution is made smaller)

Formula:

D (r_transmitted in the image): the minimum point at which two points can be resolved (best is usually 2 µm in electron microscope) this is the value you are normally trying to calculate

λ: (lambda) measurement of wavelengths in nm, normally provided in the problem

Na_condenser: the numerical aperture of the condenser

Na_objective: the numerical aperture of the selected objective lens (this is not the same value as the magnification, it will look like this: 10x/1.30 - use the 1.30 value)

Question 13

How do you calculate total magnification?

Answer 13

Objective lens magnification x Ocular lens magnification

Example:

Low power objective lens x Ocular lens

10 x 10 = 100x magnification

Question 14

What is the magnification of the Ocular lens?

Answer 14

10x

Question 15

What are the magnifications of the three objective lenses?

Answer 15

10x, 20x, and 40x

Question 16

Objective lens

Answer 16

The microscopic

Question 17

Examlpes of simple stains

Answer 17

Methylene blue

Safranin

Crystal Violet

Question 18

Why does a basic stain color the cells?

Answer 18

Because the positive charge of the auxochrome of a basic stain allows its attachment to the negative charge of most bacterial cell surfaces, thus the cell becomes colored.

Question 19

Steps of Gram Staining procedure.

What is the other name for the reagant?

Answer 19

1. Heat fixed emulsion
2. Cover smear with crystal violet stain for 1 min, rinse with distilled water
3. Cover smear with iodine stain for 1 min, rinse with distilled water
4. Decolorize smear with ethanol/acetone for about 10 seconds, rinse with distilled water
5. Counterstain smear with safranin for 1 min, rinse with distilled water
6. Pat dry and view under microscope

Other name: iodine?? (need to update)

Question 20

What is the function of each substance used for gram staining?

Answer 20

Crystal violet: primary stain for both gram negative and gram positive bacteria

Iodine: acts as mordant for keeping crystal violet dye in gram positive bacteria

Ethanol/Aceton: to decolorize gram negative bacteria

Safranin: to re-stain the gram negative cells for visual comparison

Question 21

Identify Gram positive vs Gram negative bacteria under a microscope

Answer 21

Question 22

Question 1 from 3.6

Predict the effect of the following mistakes made when performing Gram stain.

a. failure to add the iodine
b. failure to apply the decolorizer
c. failure to apply the safranin
d. reversal of crystal violet and safranin stains

Answer 22

a. the crystal purple die will not fix with the material of the cell and the dye could leave
b. the dye will not be removed from the gram neg cells and both neg and pos will appear purple
c. you will not be able to see the gram neg cells as they will have no dye
d. both gram pos and gram neg cells will be purple and indistinguishable

Question 23

Endospore Staining

Answer 23

A differential stain used to visualize bacterial endospores

Question 24

What are the steps and stains used in the Endospore staining process

Answer 24

1. Start with heat-fixed emulsion.
2. Cover smear with a strip of bibulous paper, aple malachite green stain and steam for 5 minutes
3. Rinse slide with water
4. Counterstain with safranin for 1 min
5. Gently blot dry

Malachite green is forced into the spre while it is being steamed, breaking down the keratin of the endospore. **Malachite green is water-soluble and has low affinity for cellular material, so vegetative cells and spore mother cells can be decolorized with water and counterstained with safranin.

The safranin counterstain is to stain bacterial cells so there is contrast between themselves and the endospores under the microscope

Question 25

What stain can stain vegetative cells?

Answer 25

Safranin

Question 26

What is the purpose of steaming the slide during the staining process?

Answer 26

To expedite the ability of the malachite green to enter the endospores.

Question 27

Identify vegetative cells and endospores on a microscopic slide.

Answer 27

Blue arrows indicate vegetative cells/spore mother cells (pink)

Black arrow indicates endospores (blue)

Question 28

Question 1 from 3-9 Lab (Endospores)

Why does this exercise call for an older (5-day) culture of Bacillus?

Answer 28

So the bacterial cells are well into the stabilization phase and have plenty of endospores created for us to stain and view.

Question 29

Be able to determine motility results by looking at a stab culture in semi-solid agar (Example of non-motile bacteria - Micrococcus luteus; Example of highly motile bacteria - Proteus mirabilis)

Answer 29

Question 30

What is the agar concentration in a typical nutrient agar medium and in a semi-solid medium?

Answer 30

Typical nutrient agar medium: 1.5%

Semi-solid medium: 0.4% (just enough to maintain fom while allowing movement)

Question 31

Acid-fast staining

Answer 31

A differential stain used to identify acid-fast organisms such as members of the genus Mycobacterium . Acid-fast organisms are characterized by wax-like, nearly impermeable cell walls; they contain mycolic acid and large amounts of fatty acids, waxes, and complex lipids.

Question 32

Steps of Acid-fast staining

Answer 32

1. Obtain 3 motility stabs. Label two tubes, each with the name of the organism, your name, adn the date. Label the third tube “control.”
2. Stab-inoculate two tubes with the test organisms. Do not inoculate the contro.
3. Incubate the tubes aerobically at 35 degrees celsius for 24-48 hours
4. Examen growth patterns

Question 33

What is the use of carbolfuschin in acid-fast staining?

Answer 33

When carbolfuschin is used as the primary stain because it is lipid-soluble and penetrates the waxy cell wall. It also stains acid-fast negative cells. Staining of acid-fast cells by carbolfushin i further enhances by steam heating the preparation to melt the wax and allw the stain to move into the cell

Question 34

Recognize acid-fast and non-acid-fast bacteria.

Which stain stains acid-fast bacteria?

Which stain stains non acid-fast bacteria?

Answer 34

Carbolfuschin stains acid-fast bacteria

Methylene blue stains non acid-fast bacteria

Question 35

Why is myobacterium acid fast?

Answer 35

Because it has wax-like, nearly impermeable cell walls; they contain mycolic acid and large amounts of fatty acids, waxes, and complex lipids.

Question 36

How does heating the bacterial smear during a ZN stain promote entry of carbolfuschim into the acid-fast cell wall?

Answer 36

The steam melts the waxy wall allowing the carolfuschin to enter the cell and stain it.

Question 37

Are acid-fast negative cells stained by carbolfuschin? If so, how can this be a differential stain?

Answer 37

Yes, it can stain acid-fast negative cells which can be used to identify a genus and then help start determining the species.

Question 38

Why do you suppose the acid-fast stain is not as wifely used as the Gram stain? When is it more useful than the gram stain?

Answer 38

Because it is not always necessary to break down a thick, waxy outer membrane. some gram neg bacteria have more porous membranes;

Question 39

Aerobic bacterium from a facultative anaerobe by looking at the thiglycollate test results.

Answer 39

Question 40

What is the purpose of adding thioglycollate to the medium?

Answer 40

It is a liquid medium designed to promote growth of a wife variety of fastidious microorganisms. It can be used to grow microves representing all levels of oxygen tolerance; however, it generall is associated with the cultivation of anaerobic and microaerophilic bacteria.

Question 41

What are the ingredients of FTM and what is the use of each?

Answer 41

Yeast extract - provides nutrients

Pancreatic digest of casein - provide nutrients

Dextrose - notmentioned

Sodium chloride - notmentioned

Sodium thioglycollate - reduce oygen to water

L-cystine - reduce oxygen to water

Agar - slow oxygen diffusion and stabilize growth

Resazurin - indicator

Distilled or deionized water - notmentioned

Question 42

What is the indicator used int hemedium?

Answer 42

Resazurin

Question 43

How are bacteria classified based on the oxygen requirements?

Answer 43

Microaerophiles: survive only in environments containing lower than atmospheric level sof oxygen

Aerotolerant: microorganisms that don’t require oxygen and are not adversely affected by it

Facultative anaerobes: grow in the presence or absence of xygen. when xygen is available, they respire aerobically.

Obligate anaerobes: organisms for which even small amounts of oxygen are lethal, and therefore will be seen only in the lower regions of the medium

Obligate anaerobes: organiss that require oxygen for respiration, grow at the top where xygen is most plentiful

Question 44

Nostoc

Answer 44

Question 45

Rhizopus Nigricans

Answer 45

Question 46

Saccharomyces