Microbiology Lab Midterm

Front 1

What type of microscope do we use in class and why is it called this?

Back 1

A bright-field, compound, and binocular microscope.
Bright-field: A dark image on a field of bright light
Compound: a two-lens system
Binocular: two ocular lenses

Front 2

What is a compound microscope?

Back 2

A two-lens system:
1. The lens you look through: the ocular lens
2. The lens closest to the specimen: the objective lens

Front 3

How do you calculate the total magnification?

Back 3

ocular X objective lens magnification

Front 4

What is the purpose of a simple stain?

Back 4

To identify bacterial size, shape, and arrangement.

Front 5

What is the purpose of a gram stain?

Back 5

To differentiate between cells containing a thick (Gram +, purple) or thin (Gran -, pink) peptidoglycan layer.
Also to identify the bacterial size, shape, and arrangement.

Front 6

What is the purpose of a flagella stain?

Back 6

To identify presence and arrangement of flagella

Front 7

What is the purpose of an acid-fast stain?

Back 7

To differentiate between acid-fast and non-acid-fast bacteria

Front 8

What is the purpose of a capsule stain?

Back 8

To identify whether or not a bacterium produces a capsule

Front 9

What is the purpose of an endospore stain?

Back 9

To identify whether or not a bacterium produces and endospore.

Front 10

What is the procedure for a simple stain?

Back 10

1. Prepare heat-fixed bacterial smear
2. Add crystal violet, do not allow to dry on slide
3. Gently wash off with water
4. Blot dry

Front 11

What is the procedure for a Gram stain?

Back 11

1. Heat fix bacterial smears
2. Apply crystal violate, do not allow to dry on slide
3. Gently wash with water
4. Apply Gram's iodine, do not allow to dry on slide
(The mordant)
5. Gently rinse slide
6. Add alcohol to the slide and rock to spread
(To decolorize gram negative cells)
7. Rinse slide
8. Apply safranin (the counterstain)
9. Rinse and blot dry

Front 12

What is the procedure for a flagella stain?

Back 12

1. Mark a circle in the center of the slide using a marker
2. Put 2 drops of distilled water in circle
3. Transfer the colony to the water by lightly touching the surface of the water, and not touching the slide
4. Air dry- do not heat fix!!
5. Flood with Flagella stain and let sit
6. Rinse by running water over the slide in a dish
7. Air dry. Do not blot dry

Front 13

What is the procedure for an acid-fast stain?

Back 13

1. Mark a circle on the slide
2. Transfer bacteria to slide
3. Add water to slide
4. Heat fix
5. Add carbolfuchsin KF stain, rinse
6. Add acid alcohol to decolorize, rinse immediately
7. Counterstain with methylene blue, rinse, blot dry

Front 14

What is the procedure for a capsule stain?

Back 14

1. Place Nigrosin stain on slide
2. Apply 1-3 loopfulls of culture next to the drop
3. Mix the two, cover with a coverslip
4. Do not use heat!

Front 15

What is the procedure for an endospore stain?

Back 15

1. Make a heat-fixed bacteria slide
2. Cover with paper towel, place slide in petri dish on heater
3. Add malachite green and steam (this cooks the dye into the endospore wall because it is very resistant to staining)
4. Rinse
5. Counterstain with safranin, rinse (the running water washes the green stain out of the vegetative cells and cause them to be colorless. The counterstain dyes them red)

Front 16

What are
monotrichous
amphitrichous
lophotrichous and
peritrichous
flagella arrangements?

Back 16

Monotrichous- single flagellum
Amphitrichous- a flagellum on each end
Lophotrichous- many flagellum located at the same spot
Peritrichous- flagella projecting in all directions

Front 17

What are two types of acid-fast bacteria?

Back 17

Mycobacterium (M. tuberculosis and M. leprae) and Nocardia

Front 18

How is an acid-fast stain different from a gram stain?

Back 18

Acid-fast bacteria have a waxy property (mycolic acid, or complex lipids) which make them resistant to the crystal violet dye used in a Gram stain. Heat or detergent softens these lipids and allows the stain to penetrate. Once stained the cells resist decolorization with acid alcohol.

Front 19

What is the purpose of a capsule?

Back 19

They exclude toxic materials, some hold water to prevent dessication, and most importantly they provide protection from things like being engulfed by eukaryotic cells such as macrophages.

Front 20

How does an endospore stain differ from a Gram stain?

Back 20

Spores have a tough covering made of keratin and are highly resistant to heat and staining, so malachite green is forced into the spores by heating the cells.

Front 21

What are two medically important bacteria which produce endospores?

Back 21

Bacillus and Clostridium: they can contaminate medical materials.

Front 22

If a microbiology lab technician left the safranin out of the Gram stain procedure, what would be the result?

Back 22

Gram-positive cells would be purple, and Gram-negative cells would be colorless

Front 23

What would you anticipate seeing if you accidentally switched crystal violet and safranin while performing a Gram stain?

Back 23

All bacteria would appear purple

Front 24

If you switched the mordant and the decolorizer in a Gram stain, what would the results be?

Back 24

All bacteria would appear pink

Front 25

What is the definition of these morphologies:
coccus
bacillus
spirillum

Back 25

Coccus: spherical shape
Bacillus: rod-shaped
Spirillum: spiral shape

Front 26

What is the definition of these arrangements:
Strepto-
Staphylo-
Random
Diplo-
Pallisading
Tetrad
Sarcina

Back 26

Strepto: two or more bacteria in a straight line
Staphylo: two or more bacteria bunched like grapes
Random: random
Diplo: two bacteria together
Pallisading: rod shapes in a line next to each other/or a t
Tetrad: four bacteria in a square shape
Sarcina: bacteria in a cuboidal shape

Front 27

What is the purpose of T-streaking?

Back 27

To streak a concentrated culture to obtain individual colonies/an isolation

Front 28

How is cfu/mL or bacteria/mL calculated?

Back 28

(# of colonies on plate X dilution factor)/mL of dilution plated

Front 29

What are the two reasons aseptic technique are used? What is involved in aseptic technique?

Back 29

1. protects our cultures from being contaminated with unwanted organisms

2. protects you and the environment around you from being contaminated with the microbes you are working with

Aseptic technique involves flaming loops and test tube lids before and after use, and never putting loops or test tube lids down on the table (hold lids with your pinky)

Front 30

What is the purpose of the carbohydrate fermentation tests? (glucose and lactose fermentation)

What is in the test tube?

What does a bubble in the tube mean?
No bubble in the tube?
A yellow result?
A red result?

Back 30

Tests for the ability to ferment sugar

There is phenol red pH indicator and sugar in the tube

A bubble means gas production
No bubble means no gas production
A yellow result means an acidic pH and a positive result
A red result means alkaline pH and a negative result

Front 31

What is the purpose of a motility test, and how do you read the results?

Back 31

A motility test is looking for the ability of an organism to move through the medium. Growth throughout the medium beyond the prick indicates motility (left test tube in the picture)

Front 32

What is the purpose of a catalase test? How is the result read?

Back 32

A catalase test shows the breakdown of peroxide (H2O2) into H2O + O2-. A positive result is the presence of bubbles after adding peroxide to the culture.

Front 33

What is the purpose of a cytochrome oxidase test? How do you read the results?

Back 33

This test looks for cytochrome oxidase as the terminal step in the electron transport chain. A positive result is a purple/black color change. The disc contains the electron donor p-aminodimethylaniline monohydrochloride which when oxidized will appear purple.
(test may be performed slightly different than the picture shown, depending on your lab)

Front 34

What is the purpose of an indole test? How are the results read?

Back 34

The indole test looks for the ability of an organism to produce tryptophanase. The reduction of tryptophan results in the formation of indole, pyruvic acid and ammonia. Kovac's reagent is added and red shows a positive result and yellow shows a negative result.

Front 35

What is the purpose of the methyl red test? How are the results read?

Back 35

The methyl red test is looking for the ability of an organism to use mixed acid fermentation which results in the formation of strong, stable acids. After the addition of methyl red pH indicator, red is a positive result, yellow is a negative result.

Front 36

What is the purpose of the Voges-Proskauer test? How are the results read?

Back 36

This test is looking for the ability of an organism to ferment pyruvic acid by butandiol pathway which results in formation of the neutral products acetoin and 2,3-butandiol. Barritt's A and B (alpha-napthol and 40% potassium hydroxide) are added, and a red ring is positive and a yellow ring is negative.

Front 37

What is the purpose of a citrate test? How are the results read?

Back 37

The citrate test is looking for the ability of organisms to use citrate as a sole carbon source. An increase of pH gives a positive result and a blue color. A negative result remains green.

Front 38

What is a thioglycolate test and how are the results read?

How do they grow in a gas pack, candle jar, and air?

Back 38

Thioglycolate is an oxygen scavenger that depletes oxygen in a medium. The medium is stabbed and the results can be:
Growth throughout the medium (facultative anaerobe)
Only at the top of the medium (obligate aerobe)
Only at the bottom of the tube (anaerobe)

Gas pack:
facultative: growth
anaerobe: growth
aerobe: no growth

Candle Jar:
facultative: growth
anaerobe: no growth
aerobe: no growth

Air:
facultative: growth
anaerobe: no growth
aerobe: growth

Front 39

What is the difference between EMB (eosin methylene blue) selective and differential medium?

Back 39

Selective: contains aniline dyes that inhibit Gram+ organisms and selects for Gram- organisms (enteric organisms)

Differential: contains lactose and differentiates between lactose fermentors (purple or green metallic colonies) and non-fermentors (colorless)

Front 40

What is mycology?

Back 40

Mycology is the study of fungi

Front 41

How to fungi reproduce?

Back 41

It can vary from simple asexual fission (yeasts) to complex reproductive styles. They reproduce by producing various types of spores (not to be confused with the endospores of bacteria)

Front 42

How are fungi classified?

Back 42

They are heterotrophic eukaryotes

Front 43

Are the following unicellular or multicellular?
Yeast
Mold
Mushrooms

Back 43

Yeast: unicellular
Mold: macroscopic and multicellular
Mushrooms: multicellular

Front 44

How do filaentous fungi propagate?

Back 44

They can propagate by either outward growth or fragmentation of existing hyphae

Front 45

What is a selective medium for fungi and why is it useful?

Back 45

Sabouraud dextrose agar is a selective medium for fungi. It contains acid which inhibits the growth of most bacteria and contains glucose to promote the growth of fungi.

Front 46

Define the following:
Psychrophile
Mesophile
Thermophile

Back 46

Psychrophile: can grow in extreme cold
Mesophile: can grow in middle temperatures
Thermophile: can grow in extreme heat

Front 47

Define the following:
Acidophile
Neutrophile
Alkalophile

Back 47

Acidophile: can grow in acidic environments
Neutrophile: can grow in neutral environments
Alkalophile: can grow in alkaline environments

Front 48

Define the following:
Halophile
Halotolerant

Back 48

Halophile: organisms that live in high salt concentrations
Halotolerant: the ability to survive in a salty concentration

Front 49

Define the following conditions in a cell:
Hypotonic
Isotonic
Hypertonic
What do each of these do to a cell?

Back 49

Hypotonic: the molar concentration outside the cell is less than that inside the cell (cell expands)

Isotonic: the molar concentration outside the cell is the same of that inside the cell (normal cell)

Hypertonic: the molar concentration inside the cell is greater than that outside the cell (cell shrinks)

Front 50

What is the effect of UV radiation on bacteria?

Back 50

Slows or stops growth in most bacteria

Front 51

What is the Luisetti's test and how is it read?

Back 51

It is a differential medium which detects the presence of fluorescein-producing Pseudomonas species. A positive result will glow yellow-green when placed under a UV light.