Lab Final Review - Week #4 Flashcards
What types of analyses require enumeration of microorganisms?
Food, water, milk, and in some cases air
What are the 5 methods commonly used for enumeration of microorganisms?
- direct microscopic counts
- electronic cell counter (coulter counter)
- chemical methods
- spectrophotometric analysis
- serial dilution - agar plate method
What is the the specialized slide called that is used direct microscopic counts?
Petroff-Hausser counting chamber
How does a direct microscopic count work?
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
What is the major advantage to a direct microscopic count and what is the major disadvantage?
Advantage: it is rapid
Disadvantage: it counts both living and dead cells. Also it is not sensitive to populations fewer than 1 million cells.
What are breed smears?
They are a technique used to quantitate bacterial cells in milk. Stained cells in a confined 1 in2 area, pop is determined mathematically
How does an electronic cell counter work (coulter counter)?
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
What is the major advantage of using an electronic cell counter? What are the disadvantages?
Advantage: It is rapid
Disadvantages: counts both living and dead cells it is also unable to differentiate inert matter from cells
How do chemical methods for enumerating microorganisms?
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.
How does spectrophotometric analysis work?
Using turbidimetric instruments, the amount of light transmitted is converted to electrical energy and indicated on a galvanomenter.
What is a major disadvantage of the spectrophotometric analysis method?
Sensitivity is restricted to microbial suspensions of 10 million cells or more.
How are serial dilution-agar plate analysis techniques done?
- serial dilution of a bacterial suspension in sterile water (diluent of known volume)
- pour-plate technique
- colonies are counted on a quebec colony counter (usually by hand)
- # colonies per plate X dilution factor (reciprocal of dilution) = total # of cells
Which plates are suitable for counting when using a serial dilution-agar plate analysis?
Plates that have between 30-300 colonies
List the 2 advantages of doing a serial dilution-agar plate analysis?
- only viable cells are counted (only of the 5)
2. allows for the isolation of discrete colonies which can be easily studied and identified.
List the 3 disadvantages of doing a serial dilution-agar plate analysis?
- overnight incubation is required
- more glassware is used
- the need for greater manipulation -> increases chances of error and erroneous counting.
What is the usefulness of microbial cell counts in medical laboratories?
They can be used to determine antimicrobial sensitivity as well as the course of infection
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
10 X 1,000,000 X 56 = 560,000,000
How is a bacterial growth curve charted?
By plotting the increase in cell numbers versus time of incubation
What can a population growth curve be used for?
- To delineate stages of the growth cycle
2. facilitates measurement of cell numbers and growth rate of an organism
Define Generation Time
The time required for a microbial population to double
What occurs during the lag phase of bacterial growth?
Cell are adjusting to their environment, cells are increasing in size but no division is occurring yet
What occurs during the logarithmic (log) phase of bacterial growth?
The now physiologically robust cells reproduce at a rapid rate by binary fission, therefore there is a rapid exponential increase in the population size
How long is the log phase generally? What factors contributing to this length of time?
6 to 12 hours. The organism and the composition of the medium (amount of available nutrients)
What occurs during the stationary phase of bacterial growth?
The number of cells undergoing division is equal to the number of cells dying. The population here is at its maximum.
What are the factors which contribute to the occurrences of the stationary phase?
- depletion of essential metabolites
2. accumulation of toxic acidic or alkaline end products
What occurs during the death phase of bacterial growth?
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
How much time is needed for a complete bacterial growth curve to be constructed?
24 hours
How is the direct method of for plotting a bacterial growth curve performed?
By enumerating the viable cells in a serially diluted samples of the test culture taken at 30 minute intervals
How is the indirect method for plotting a bacterial growth curve performed?
By using spectrophotometric measurements of the developing turbidity at the same 30 minute intervals
Indirect determination of generation time is figured out how?
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
How is the generation time calculated directly?
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.
What is the clinical application of determining bacterial growth curves?
It allows for the determination of antimicrobial susceptibility and resistance.
What are some factors that influence generation time?
pH, temp, gaseous condition, type of microorganism, etc.
What is a negative control and why is it necessary?
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.
What is the best way to graph data if N=3
Do not average the data points, instead graph each individual data point.
