Bio 220: Lecture 09

Front 1

What are the two methods of cell mass?

Back 1

indirect method and direct method

Front 2

What is the direct method of cell mass?

Back 2

measuring both live (viable) and dead cells, a total measurement
• take dry weight of all cells. You must wash the cells well to eliminate media

Front 3

What is the indirect method of cell mass?

Back 3

also a total measurement, for both viable and dead cells are considered

Front 4

What are two factors that effect the indirect method of cell mass?

Back 4

nitrogen content and turbidometric assays

Front 5

What is nitrogen content?

Back 5

use of biochemical assays to measure total amounts of nitrogen in the cell culture. Knowing the percentage of the cell mass that is generally nitrogen, one can now calculate to the total cell mass

Front 6

What is turbidometric assays

Back 6

bacterial cells will absorb and/or scatter light
• The amount of light that is absorbed or scattered is proportional to the mass of cells in the culture

Front 7

How do you measure the turbity of a culture?

Back 7

a colorimeter or spectrophotometer

Front 8

What is a colorimeter?

Back 8

monochromatic or uses just one wavelength

Front 9

What is a spectrophotometer?

Back 9

can vary the wavelength
• a more versatile instrument

Front 10

What are the direct methods of measuring cell numbers?

Back 10

measuring both viable and dead cells, a total measurement

Front 11

define: microscopy

Back 11

(direct method for measuring cell numbers)
using a special slide known as Petroff-Hausser counting chamber one can count the cells of a given volume that are placed on ruled grid. Knowing the number of cells in this given volume, one can then calculate the umber of cells in the original culture tube. This can also be useful for visualizing cell arrangements, such as bacteria in chains, as the cells are not treated with stains of heat fixed.
• Drawbacks include visualization of unstained cells, and counting cells that are motile and can literally swim on and off of the counting grid

Front 12

define: particle or coulter (inventor) counter

Back 12

uses a high tech instrument that counts bacteria that are suspended in an electrolyte. A probe is place into this suspension, and a known volume of this bacterial suspension is drawn into the probe. An electrical circuit is interrupted each time an organism enters the probe. A digital counter records the numbers. The probes can be altered or changed to vary the hole size. With appropriate probes, viruses and larger cells can be counted. Also, the length of time ti requires for each interruption can be used to distinguish large and small cells for purposes of differential counts

Front 13

T/F: The indirect method for measuring cell number only measures live cells only.

Back 13

true

Front 14

define: plate counts

Back 14

only viable cells will grow into colonies. The number of colonies is proportional to the number of cells. Plates should have between 30 and 300 colonies to be statistically significant. Dense populations should be diluted using serial dilution techniques

Front 15

What are some examples of plate counts?

Back 15

pour plate technique and spread plate technique

Front 16

What are some examples of indirect methods for measuring cell number?

Back 16

1. plate counts
2. membrane filter technique
3. tube counts using MPN (most probable number)

Front 17

What is membrane filter technique?

Back 17

samples (good for air samples) can be drawn through a millipore filter (grid size can be 0.2 micrometers) using a vacuum. Bacteria are collected on the filter and are then transferred (by aseptically pressing and the removing the filter) onto a plate for growth. These plates are incubated and the colonies are counted

Front 18

What is the tube counts using MPN?

Back 18

(most probable number) samples are inoculated into a number of culture tubes using a precise protocol. The tubes are incubated and grown. The number of positive tubes is recorded. Using previously collected data and statistical probabilities, the most probable number of organisms in the original sample is determined

Front 19

What is cell activity?

Back 19

the activities of living cells are measured. All measurements are indirect, and all measurements are based upon the activities of live (viable) cells only.

Front 20

What is a chemical method or assays in measuring cell activity?

Back 20

any chemical changes in the media will be proportional to the activity of the cells. One can monitor the rate at which glucose amounts in the media decline. The faster glucose amounts decline, the more active the organisms. The same can be said of oxygen usage and decline. Carbon dioxide (waste product of metabolism) will increase at a greater rate if the organisms are active. Rates of increases in acidity (shown by a decline in pH) are also used to determine cell activity

Front 21

What are examples of physical method or assay of measuring cell activity?

Back 21

microcalormetry and radioactive isotopes

Front 22

What is microcalorimetry?

Back 22

minute temperature changes over time can be monitored to assess cell activity using sensitive thermometers called thermistors. Antiobiotic sensitivity can be tested using microcalorimetry

Front 23

What is radioactive isotopes?

Back 23

radioacively labeled carbon released from glucose during metabolic activities in the form of carbon dioxide, can be detected and counted using a scintillation counter. The greater the count, the more active the culture. Antibiotic sensitivity can also be measured using radioactive isotopes.