Midterm Lab Exam

Question 1

What are the steps for preparing a slide?

Answer 1

1. Spread culture in thin film over slide
2. Dry in air
3. Pass slide through flame to heat fix
4. Flood slide with stain; rinse and dry
5. Place drop of oil on slide; examine with 100x objective lens

Question 2

What is culture media?

Answer 2

A liquid or gel designed to support designed to support the growth of microorganisms

Question 3

What is the difference between making a smear with solid culture and liquid culture?

Answer 3

when using a solid culture, you must add a loopful of H2O to the slide

Question 4

Explain the importance of heat fixing and air-drying specimens on slides.

Answer 4

• heat fixing kills the bacteria in the smear and firmly adheres them to the slide
• air drying removes excess water and allows for better heat fixation

Question 5

Compare and contrast simple staining and differential staining.

Answer 5

• simple staining involves one stain

- differential staining involves more than one stain

Question 6

Recognize the different bacterial morphologies.

Answer 6

-cocci
-bacilli
-

Question 7

What is the difference between gram-positive and gram-negative cells?

Answer 7

• gram-positive cells have a thick layer of peptidoglycan
• gram-negative cells have a thin layer of peptidoglycan
• gram-positive cells stain purple
• gram-negative cells stain pink

Question 8

Understand the purpose of a streak plate and how you would do one.

Answer 8

• a streak plate is used to isolate bacterial colonies
• flame the loop in between each quadrant streak
• there should be 4 quadrant streaks

Question 9

Define pure culture and CFU.

Answer 9

• a pure culture is a population of cells arising from a single cell
• a CFU is a colony forming unit

Question 10

Know the purpose of a gelatin stab, how you conducted this test, and what a
positive reaction for gelatinase production (gelatin hydrolysis) would look like.

Answer 10

-gelatinase

Question 11

Describe endospores, their function, and know which genera produce them.

Answer 11

• endospores are structures within certain bacteria that allow them to survive harsh conditions
• the two genera that produce endospores are bacillus and clostridium

Question 12

Understand the Schaeffer-Fulton endospore staining procedure (know the primary
stain and counterstain), why heat is used as a mordant, and what endospores and
their producers look like under the microscope after being stained.

Answer 12

• primary stain is malachite green
• counterstain is safranin
• mordant is heat and is used to penetrate the primary stain into the endospores
• endospores appear green and vegetative cells appear pink

Procedure:

1. Set up hot water bath and bring the water to a boil
2. Using aseptic technique, make a heat fixed smear
3. Put a small piece of paper towel directly on top of the smear
4. Saturate the piece of paper towel with malachite green
5. Place the slide on top of the beaker of the hot water bath and steam the slide for 5 minutes.
6. Gently rinse the smear with water
7. Cover the smear with safranin for 30 seconds
8. Gently rinse the smear with water
9. Gently blot the slide with a paper towel
10. Observe the endospore stain under 1000x magnification (oil immersion)

Question 13

Know which genera of bacteria are acid-fast and know the characteristic that
causes them to be acid-fast.

Answer 13

• the genera of bacteria that are acid fast are mycobacterium
• the characteristic that causes them to be acid fast is the waxy mycolic acids in their cell wall

Question 14

Describe the acid-fast stain (primary stain, decolorization, counterstain) and know
how acid-fast cells and non-acid fast cells appear after staining.

Answer 14

• primary stain is carbolfuchsin
• decolorizer is acid alcohol
• counterstain is methylene blue
• acid fast cells appear red/pink
• non-acid fast cells appear blue

Question 15

Describe bacterial capsules and their functions.

Answer 15

• a bacterial capsule is a protective outer structure related to virulence
• bacterial capsules protect the bacteria from macrophages

Question 16

Describe the use of negative staining to visualize capsules and know how capsules
appear after using this method.

Answer 16

Procedure:

• place a small drop of Maneval’s A near one end of a well-cleaned and flamed slide
• remove a small amount of the culture from the slant with an inoculating loop and disperse it in the drop of stain
• use another clean slide to spread the drop of stain containing the organism using the following technique
• rest one end of the clean slide on the center of slide with the stain
• tilt the clean slide toward the drop

Question 17

Understand the method we used to visualize capsules, including the purpose of
Maneval’s A and Maneval’s B.

Question 18

Understand the methods we used to see motility and how motility (or lack of) was
determined.

Question 19

Of the bacteria that were tested for motility, be able to name which bacterium was
motile and which one was non-motile.

Answer 19

E. coli is motile and M. luteus is nonmotile

Question 20

What is the functional type, type of colony growth, and description of colony appearance, and mechanism of action for MAC?

Know the functional type, mechanism of action, what type of colonies will grow,
and how they can be distinguished on the following media: MacConkey Agar
(MAC), Phenylethyl Alcohol Agar (PEA), and Blood Agar.

Answer 20

• functional type: selective and differential
• type of colony growth: gram-negative
• description of colony appearance: lactose fermenters appear pink and non-lactose fermenters appear tan/cream
• mechanism of action: bile salts and crystal violet inhibit the growth of gram positive organisms; lactose provides a source of fermentable carbohydrate, allowing for differentiation

Question 21

What is the functional type, type of colony growth, and description of colony appearance, and mechanism of action for PEA?

Answer 21

• functional type: selective
• type of colony growth: gram-positive
• description of colony appearance: will all look the same
• mechanism of action:

Question 22

What is the functional type, type of colony growth, and description of colony appearance, and mechanism of action for blood agar?

Answer 22

• functional type: enriched and differential
• type of colony growth: gram-positive and gram-negative
• description of colony appearance: gamma-hemolytic: no change in color, alpha-hemolytic: green color, beta-hemolytic: yellow ring of bacteria around
• mechanism of action: allows the detection of hemolysis (destroying the RBC) by cytolytic toxins secreted by some bacteria

Question 23

Understand how we conducted and graphed a bacterial growth curve.

Answer 23

• we measured the absorbance of the broth using a spectrophotometer
• we made a graph of absorbance v. time to see the bacterial growth curve

Question 24

Know the typical phases of growth for a population of bacteria growing in a batch
culture.

Answer 24

• lag: initial growth, flat line
• exponential: doubling at a constant rate
• stationary: population stops dividing due to lack of nutrients or space
• death: exponential decrease in the number of viable cells

Question 25

For each of the following tests, know the purpose, what a positive reaction would look like, what media and/ or reagents were used, and how the media and/or reagents work to show negative and positive reactions (ex. methyl red in MRVP, turns red when the pH drops to 5 or less indicating mixed acid fermentation). Carbohydrate (Glucose) Fermentation

Answer 25

- purpose: to determine whether the bacteria can ferment carbohydrates - media: carbohydrate broth (glucose) with Durham tube - reagent: phenol red - positive test: yellow broth, gas bubble