Lab Final Review - Week #4

Question 1

What types of analyses require enumeration of microorganisms?

Answer 1

Food, water, milk, and in some cases air

Question 2

What are the 5 methods commonly used for enumeration of microorganisms?

Answer 2

1. direct microscopic counts
2. electronic cell counter (coulter counter)
3. chemical methods
4. spectrophotometric analysis
5. serial dilution - agar plate method

Question 3

What is the the specialized slide called that is used direct microscopic counts?

Answer 3

Petroff-Hausser counting chamber

Question 4

How does a direct microscopic count work?

Answer 4

An aliquot of a bacterial suspension is counted and the total number of cells is then determined mathematically: # of cells per mm = # of cells counted X dilution X 50,000

Question 5

What is the major advantage to a direct microscopic count and what is the major disadvantage?

Answer 5

Advantage: it is rapid
Disadvantage: it counts both living and dead cells. Also it is not sensitive to populations fewer than 1 million cells.

Question 6

What are breed smears?

Answer 6

They are a technique used to quantitate bacterial cells in milk. Stained cells in a confined 1 in2 area, pop is determined mathematically

Question 7

How does an electronic cell counter work (coulter counter)?

Answer 7

Cells in a conducting fluid are counted by passing them through an electrical current, cells are nonconducters… so the electrical resistance is used to determine the amount of cells present

Question 8

What is the major advantage of using an electronic cell counter? What are the disadvantages?

Answer 8

Advantage: It is rapid
Disadvantages: counts both living and dead cells it is also unable to differentiate inert matter from cells

Question 9

How do chemical methods for enumerating microorganisms?

Answer 9

They indirectly measure increases in both protein conc. and DNA production. Cell mass can be estimated too. Measurements of metabolic parameters (O2, CO2 conc) can be used as well.

Question 10

How does spectrophotometric analysis work?

Answer 10

Using turbidimetric instruments, the amount of light transmitted is converted to electrical energy and indicated on a galvanomenter.

Question 11

What is a major disadvantage of the spectrophotometric analysis method?

Answer 11

Sensitivity is restricted to microbial suspensions of 10 million cells or more.

Question 12

How are serial dilution-agar plate analysis techniques done?

Answer 12

1. serial dilution of a bacterial suspension in sterile water (diluent of known volume)
2. pour-plate technique
3. colonies are counted on a quebec colony counter (usually by hand)
4. # colonies per plate X dilution factor (reciprocal of dilution) = total # of cells

Question 13

Which plates are suitable for counting when using a serial dilution-agar plate analysis?

Answer 13

Plates that have between 30-300 colonies

Question 14

List the 2 advantages of doing a serial dilution-agar plate analysis?

Answer 14

1. only viable cells are counted (only of the 5)

2. allows for the isolation of discrete colonies which can be easily studied and identified.

Question 15

List the 3 disadvantages of doing a serial dilution-agar plate analysis?

Answer 15

1. overnight incubation is required
2. more glassware is used
3. the need for greater manipulation -> increases chances of error and erroneous counting.

Question 16

What is the usefulness of microbial cell counts in medical laboratories?

Answer 16

They can be used to determine antimicrobial sensitivity as well as the course of infection

Question 17

If 0.1 mL of a 1 X 10 -6 dilution plate contains 56 colonies, calculate the number of cells per mL of the original culture

Answer 17

10 X 1,000,000 X 56 = 560,000,000

Question 18

How is a bacterial growth curve charted?

Answer 18

By plotting the increase in cell numbers versus time of incubation

Question 19

What can a population growth curve be used for?

Answer 19

1. To delineate stages of the growth cycle

2. facilitates measurement of cell numbers and growth rate of an organism

Question 20

Define Generation Time

Answer 20

The time required for a microbial population to double

Question 21

What occurs during the lag phase of bacterial growth?

Answer 21

Cell are adjusting to their environment, cells are increasing in size but no division is occurring yet

Question 22

What occurs during the logarithmic (log) phase of bacterial growth?

Answer 22

The now physiologically robust cells reproduce at a rapid rate by binary fission, therefore there is a rapid exponential increase in the population size

Question 23

How long is the log phase generally? What factors contributing to this length of time?

Answer 23

6 to 12 hours. The organism and the composition of the medium (amount of available nutrients)

Question 24

What occurs during the stationary phase of bacterial growth?

Answer 24

The number of cells undergoing division is equal to the number of cells dying. The population here is at its maximum.

Question 25

What are the factors which contribute to the occurrences of the stationary phase?

Answer 25

1. depletion of essential metabolites

2. accumulation of toxic acidic or alkaline end products

Question 26

What occurs during the death phase of bacterial growth?

Answer 26

The decrease in population is due to the amount of cells dying being much greater than the cell undergoing division. At this point only the more resistant cells are able to survive

Question 27

How much time is needed for a complete bacterial growth curve to be constructed?

Answer 27

24 hours

Question 28

How is the direct method of for plotting a bacterial growth curve performed?

Answer 28

By enumerating the viable cells in a serially diluted samples of the test culture taken at 30 minute intervals

Question 29

How is the indirect method for plotting a bacterial growth curve performed?

Answer 29

By using spectrophotometric measurements of the developing turbidity at the same 30 minute intervals

Question 30

Indirect determination of generation time is figured out how?

Answer 30

by simple extrapolation form the log phase. 2 points are selected that represent the doubling of turbidity. The generation time is the amount of time between those to points

Question 31

How is the generation time calculated directly?

Answer 31

The number of generations can first be calculated by taking the log # cells at a specified time and subtracting the log # cells at zero from it. Then divide all of this by the log 2. The number of generations is then divided by the specified time and this is the generation time.

Question 32

What is the clinical application of determining bacterial growth curves?

Answer 32

It allows for the determination of antimicrobial susceptibility and resistance.

Question 33

What are some factors that influence generation time?

Answer 33

pH, temp, gaseous condition, type of microorganism, etc.

Question 34

What is a negative control and why is it necessary?

Answer 34

a negative control is used to ensure that the medium onto which bacteria is being placed is not being affected by things like temp or pH. This is important so that nay changes observed in the media can be contributed solely to the bacteria and not to environmental factors.

Question 35

What is the best way to graph data if N=3

Answer 35

Do not average the data points, instead graph each individual data point.