Lecture #13: Growth (Last) to Preferences Flashcards
Serial Dilutions & Counts
To get CFU’s (Colony Forming Units). Each colony comes from a cell, and each CFU is likely to represent a single cell.
To get an accurate count, want 30-300 colonies on a plate. If < 30, not dense enough to be reliable (too much variation with small numbers) and if >300, TNTC (too numerous to count). Also, likely that cells on top of cells so count is not reliable.
Have a known volume from your broth culture, and use the CFU’s to calculate the original cell density.
Spectrophotometry
A rapid and sensitive method for measuring cell mass. Depends on fact that microbial cells scatter light that strikes them. Because microbial cells in a population are of roughly constant size, the amount of scattering is directly proportional to the biomass of cells present and indirectly related to cell number.
When the concentration of bacteria reaches about a million (10^6) cells per milliliter, the medium appears slightly cloudy or turbid. Further increases in concentration result in greater turbidity, and less light is transmitted through the medium.
The extent of light scattering (i.e., decrease in transmitted light) can be measured by a spectrophotometer and is called the absorbance (optical density) of the medium.
Absorbance is almost linearly related to cell concentration at absorbance levels less than about 0.5. If the sample exceeds this value, it must first be diluted and then absorbance measured. Thus population growth can be easily measured as long as the population is high enough to give detectable turbidity.
It is quick and is reliable at low cell densities; from about 102 to 107 cells/ml. Once the suspension is cloudy, more error. If cells are too sparse, unreliable as sensitivity falls off rapidly.
Couple it with pour or spread plates to get CFU’s.
Why Would You Want To Render a Curve Linear?
Easier and more accurate when interpolating [insert (something) between fixed points].
Use a log-linear growth curve for Spec readings or cell densities to determine the doubling time of a culture.
Use a set of Spec readings from a growth lab of the past to see how this is done.
Find g, Doubling Time
= log N2 – log N1/0.301 (t) t in hours
Or from Absorbance Values as:
= A2 – A1/0.301 (t)
Or g can be found directly from the growth curve.
Cells must be in the log or exponential phase.
Direct Counts
Determining microbial numbers using a counting chamber. Gives info about the size and morphology or microbes. Petroff-Hausser counting chambers can be used for counting bacterial and archaeal cells; hemocytometers can be used for all cell types. Counting chambers consist of specially designed slides and coverslips; the space between the slide and coverslip creates a chamber of known depth. On the bottom of the chamber is an etched grid that facilitates counting the cells. The number of microbes in a sample can be calculated by taking into account the chamber’s volume and nay dilutions made of the sample before counting. One disadvantage of using counting chambers is that to determine the population size accurately, the microbial population must be relatively large and evenly dispersed because only a small volume of the population is sampled.
Viable Counting Methods or Plate Counts
Methods used to determine number of viable microbes in a sample. They only count those cells that are able to reproduce when cultured.
Two commonly used procedures are the spread-plate and the pour-plate techniques. In both of these methods, a diluted sample of microbes is dispersed over or within agar. If each cell is far enough away from other cells, then each cell will reproduce, generating a distinct colony. The samples should yield between 30-300 colonies for most accurate counting, and the count is made more accurate by use of a colony country.
Once the number of colonies is known, the original number of viable microbes in the sample can be calculated from that number and the sample dilution. For example, if 1.0 mL of a solution diluted by a factor of 1 x 10^6 yielded 150 colonies, then the original sample contained around 1.5 x 10^8 cells per mL. However, because it’s not possible to be certain that each colony arose from an individual cell, the results are often expressed in terms of colony forming units (CFU), rather than # of microbes.
To get cell densities, need volumes & dilution factors.
20 colonies (actually CFU’s) on a plate, used 0.1 mL of the culture, and it was the 10-5 dilution.
20 colonies/0.1 ml or 200 cells/1 mL*.
So, 2.0 x 102 cells/mL (at the plate).
But the 1 mL was at a 10-5 dilution, so 105 to get back to the original cell density. 2.0 x 107.
OR from *
1 mL/.00001 dilution * 200 colonies/1 mL =
2.0 x 107 cells/mL.
Plate Sample of Known Volume & at Known Dilution onto Media
Only viable cells yield colonies. Get count of living cells.
Reliable if 30-300 colonies on the plate, so have to plate out a number of dilutions. If >300, glumps. Even so, CFU’s not cells.
Can do this with filtered water samples, culture filter. If that filter is Nitrocellulose and placed on EMB Agar or an agar differential for lactose fermenters then…
Interested in Population Growth; Measure Cell Density
Let individual cells produce colonies and count the colonies. Known volume.
Use Absorbance to measure cloudiness or turbidity. Relative measure.
Centrifuge the cells into a pellet and weigh it. Mass is a relative measure.
Count the cells directly. Known volume. Counting chamber such as the C-Chip.
Continuous Culture (Open) Systems
It is possible to grow microbes in a system with constant environmental conditions maintained through continual provision of nutrients and removal of wastes through this system.
These systems can maintain a microbial population in exponential growth, growing at a known rate and at a constant biomass concentration for extended periods. Continuous culture systems make possible the study of microbial growth at very low nutrient levels, concentrations, close to those present in natural environments.
These systems are essential for research in many areas, including ecology. For example, interactions between microbial species in environmental conditions resembling those in freshwater lake or pond can be modeled. Continuous culture systems also are used in food and industrial microbiology.
Continuous culture. Input of fresh media, outflow to remove waste. Maintains population in exponential growth.
No Stationary or Death Phases. So long as the media and the temperature remain the same, no lag phases either.
Chemostat
Open system. Constructed so that the rate at which sterile medium is fed into the culture vessel is the same as the rate at which the media containing microbes is removed.
The culture medium for a chemostat possesses an essential nutrient (e.g., a vitamin) in limiting quantities. Because one nutrient is limiting, growth rate is determined by the rate at which new medium is fed into the growth chamber; the final cell density depends on the concentration of the limiting nutrient.
The rate of nutrient exchange is expressed as the dilution rate, the rate at which medium flows through the culture vessel relative to the volume.
Media dripped in. Media inflow limits growth. Cells maintained in constant growth.
Turbidostats
Open system. Has a photocell that measures the turbidity (the amount of light scattered) of the culture in the growth vessel.
The flow rate of media through the vessel is automatically regulated to maintain a predetermined turbidity. Because turbidity is related to cell density, the turbidostat maintains a desired cell density. The turbidostat differs from the chemostat in several ways.
The dilution rate in a turbidostat varies, rather than remaining constant, and a turbidostat’s culture medium contains all nutrients in excess. That is, none of the nutrients is limiting.
The turbidostat operates best at high dilution rates; the chemostat is most stable and effective at lower dilution rates.
Turbidity readings track cell density, flow of media is adjusted to keep cells growing. For a fixed cell density, input varies as needed.
Cells Natural Phase
Nutrients may never be limiting, so cells are kept naturally in log phase.
