real life applications lecture 01

Question 1

Methods to detect microbial growth

Answer 1

1. spectrophotometer
2. Most Probably Number (MPN)
3. Viable Count or Colony Count
4. Direct microscopic count

Question 2

How does the spectrophotometer work?

Answer 2

The spectrophotometer will pass light through a liquid sample containing microbes. The percentage of light that passes through is inversely proportional to the optical density.
The top scale is the percentage of light that passes through.
The bottom scale is the optical density (absorbance)

Question 3

turbidity

Answer 3

cloudiness of a liquid
proportional to the concentration of cells

Question 4

Most Probably Number

Answer 4

Used for water and food samples
Evaluates changes in color or turbidity due to metabolic activity.
Uses a 3 set dilution series.
After incubation period the results are recorded and compared to table to give statistical determination.

Question 5

Most Probable Number Index

Answer 5

Given as a number per 100 ml

Question 6

Procedure of the MPN

Answer 6

1. Take your liquid sample and inoculate 3 sets of 5 tubes with a different concentration of sample. (10 dilution)
   Tube should be able to change colors based on a metabolic property of the microbe that may be present in the water.
2. Incubate for 24 hours at whatever temp needed
3. Look for any tubes that changed color. Evidence that the microbe of interest is present in those tubes.
4. Collect the number of tubes that are positive. Should be a set of three numbers.
5. Use the chart and three numbers to estimate the number of bacteria /100 ml that are in the sample.

Question 7

Direct Cell Count

Answer 7

Counting the number of cells on a liquid culture or colonies on a plate. A direct way of estimating the number of organisms present in a sample.
1. Direct microscopic cell count
2. Cell-counting instruments (coulter counter, flow cytometer)

Question 8

Coulter Counter

Answer 8

Does not differentiate between live and dead cells
A glass tube with an opening is placed into the container which holds the sample. An electrode is placed in the glass tube and in the container. The movement of microbes is measured and counter by the electronic device. It’s rapid. But if the solutions is too concentrated then the reading might not be accurate.

Question 9

Importance of knowing cell count

Answer 9

It’s important to know the number of microbes or colonies especially live or dead. We want to know live or dead microbes because it helps us with understanding the effectiveness of antibiotics/medicine, infection, and contamination of food and water.

Question 10

Importance of staining live and dead cells

Answer 10

Primary stain: stains cells live or dead fluorescent green attracted to nucleic acids
Secondary stain: stains cells with damaged cell membranes red = dead
The stain can be used in microscopic techniques

Question 11

Why do we perform serial dilutions before counting our number of microbes on the plates?

Answer 11

Serial dilutions ensure that we achieve a colony between 30-300 on our plates. If the sample is too diluted with microbes it’s difficult to count the number of colonies.