Experiments 5,7,11

Question 1

Identify these bacterial shapes and arrangements:

Answer 1

Question 2

Identify these bacterial shapes and arrangements:

Answer 2

Question 3

What bacterial shape is this?

Answer 3

Vibrios (bent rods)

Question 4

What bacterial shape is this?

Answer 4

Spiral (Spirilla)

Question 5

What bacterium is a rod?

Answer 5

Bacillus subtilis

Question 6

Which bacterium is larger, Bacillus subtilis or Staphylococcus epidermidis

Answer 6

Bacillus subtilis

Question 7

Of what value is a simple stain?

Answer 7

Determining cell morphology, size, and arrangement.

Question 8

What is the purpose of heat fixing the smear?

Answer 8

It helps the cells adhere to the slide so that they can be stained; it also prevents lysis.

Question 9

Another method of fixing smears is to use methanol instead of heat. How does alcohol chemically fix the bacteria?

Answer 9

Alcohol acts by removing water from the cell. As the bacterial cells are denatured, they retain their structures and adhere to the surface that they are found in.

Question 10

In heat fixing, what would happen if too much heat were applied?

Answer 10

Too much heat would cause the cell’s shape to distort.
Denatures bacterial enzymes that cause the cells to break.

Question 11

Methylene blue can be prepared as a basic stain or an acidic stain. How would the pH of the stain affect the staining of bacteria?

Answer 11

Bacteria is negatively charged, so it can only absorb stain that has an opposite positive charge (aka Basic). Acidic stains carry a negative charge and therefore the bacteria will repel the dye.

Question 12

Can dyes other than methylene blue be used for direct staining

Answer 12

Yes. Crystal violet, basic fuchsin, and safranin are all dyes that can be used in direct staining because they are cationic which means they are positively charged.

Question 13

Bacteria can be seen without staining. Why then was Koch’s recommendation for fixing and staining important in microbiology?

Answer 13

Allows bacteria to be saved and reexamined or shared.

Question 14

Infection quality control staff in a sterilization unit of a hospital used a simple stain to determine whether bacteria were present in sterilized materials. A simple stain of sterile saline used for respiratory therapy revealed the presence of bacteria. Is the saline contaminated?

Answer 14

Yes. It should be free of bacteria, however.

Question 15

What color will a gram-negative cell stain?

Answer 15

Red/pink

Question 16

What color will a gram-positive cell stain?

Answer 16

Purple

Question 17

1. Staphylococcus epidermidis
2. Bacillus subtilis
3. Escherichia coli

Of these three bacteria, which organism is the largest?

Answer 17

Bacillus subtilis

Question 18

1. Staphylococcus epidermidis
2. Bacillus subtilis
3. Escherichia coli

Which bacterial cultures were gram-positive?

Answer 18

Staphylococcus epidermidis & Bacillus subtilis

Question 19

Name this bacteria

Answer 19

Staphylococcus epidermidis

Question 20

Name this bacteria

Answer 20

Bacillus subtilis

Question 21

Name this bacteria

Answer 21

Escherichia coli

Question 22

Why will gram-positive cells more than 24 hours old stain gram-negative?

Answer 22

When bacteria die their cell walls degrade and may not retain the primary stain.

Question 23

Can iodine be added before the primary stain in a Gram stain?

Answer 23

No

Question 24

List the steps of the Gram-staining process, and identify the color of G+ and G- cells in each step.

Answer 24

Question 25

Which step can you omit without affecting determination of the Gram reaction?

Answer 25

Safranin

Question 26

Suppose you performed a Gram stain on a sample from a pure culture of bacteria and observed a fiels of red and purple cocci. Adjacent cells were not always the same color. What do you conclude?

Answer 26

Bacteria is G+, but because it could have possibly been an old sample, the cells could not retain the dye.

Question 27

Suppose you are viewing a Gram-stained field of red rods and purple cocci though the microscope. What do you conclude?

Answer 27

It is a mixed culture

Question 28

Human cells can be stained with crystal ciolet and safranin, so why can't human cells be Gram stained?

Answer 28

Human cells do not have cell walls so the primary stain will be removed easily.

Question 29

Considering you can't identify from a Gram stain, why might a physician perform a Gram stain on a sample before prescribing an antibiotic?

Answer 29

G+ and G- cells are different in the compositions of their cell walls. By determining its' properties the physician can pick the correct antibiotic that goes with that wall type.

Question 30

What will a streak plate with two species of bacteria look like?

Answer 30

You can identify two distinguishable species because they will appear different on the streak plate.

Question 31

Where will the colonies be located in the pour plate? Where will they be the largest?

Answer 31

Colonies will be embedded in the agar. Pour plates are prefferable for anaerobic bacteria so they will be largest deeper within the medium.

Question 32

How can you tell whether a mixed culture has different bacterias?

Answer 32

The appearance of microbes will allow you to distinguish whether or not a mixed culture has multiple bacterias.

Question 33

How do the colonies on the surface of the pour plate differ from those suspended in the agar?

Answer 33

Anaerobes proliferate suspended in pour plate agars, colonies on the surface tolerate oxygen

Question 34

How could your streak plate technique be improved?

Answer 34

Keep in mind a small amount of concentrated bacteria will go a long way, therefore by streaking the plate at a better angle you can isolate better

Question 35

How do you know if you got a pure culture in your subculture?

Answer 35

If the colonies appear the same.

Question 36

What is a contaminant?

Answer 36

Presence of unwanted organisms

Question 37

How would you determine whether a colony was a contaminant on a streak plate? On a pour plate?

Answer 37

Streak plate: The contaminant will be located in an area where you did not inoculate (outside of the streak line). And will also appear different Pour plate: The appearance of the contaminant will be different from the rest.

Question 38

What would happen if the plates were incubated a week longer? A month?

Answer 38

A week longer the organisms on the plate will grow more. The plate will eventually become too full, and you will be unable to isolate. A month longer the bacteria will have used up all the nutrients in the agar and die.

Question 39

Could some bacteria grow on the streak plate and not be seen if the pour plate technique is used?

Answer 39

Yes, some aerobic organims will not grow on the pour plate but will grow on the streak plate

Question 40

What is a disadvantage of the streak plate technique? Of the pour plate technique?

Answer 40

Streak Plate: Colony isolation problems Pour plate: Preparation is time consuming, reduced growth rate of aerobes, and the danger of killing heat-sensitive organisms.

Question 41

Will the isolated colonies always be in the fourth sector on the streak plate?

Answer 41

No. Depending on how well you streak you may be able to find colonies within the 3rd sector.

Question 42

Colony-forming units per ml =

Answer 42

of Colonies ————————————— Dilution x Amount Plated