Lab Three Flashcards
What is a viable count?
counting of growth of individual cells present in the initial sample
what is the countable range of a viable plate count?
25-250 colonies
what is the major advantage to a viable count?
it only counts living cells
what is a 10-fold dilution?
dilute 1 in 10
is the most common dilution series
what is the spread plate procedure?
take a sample of known dilution and spread it onto the agar plate, using a spreader. Individual colonies can be counted after incubation.
what could cause an error in the viable count on a pour plate?
- not sterilizing the equipment
- not working near a bunsen
- not having the agar too hot
what could cause an error in the viable count on a spread plate?
- not cooling the spreader after sterilization
- not having the agar plate at room temperature
- having pooling of the inoculum on the plate surface
what is the advantage to pour plates?
they can incorporate volumes greater than 100 microlitres into a place if needed and give a similar result to the spread plate procedure
What is the pour plate procedure?
- add the bacterial culture into molten agar (not too hot)
- allow the agar to set and then invert
- Prepare positive (undiluted) and negative (saline) controls.
what are the two kinds of direct count?
turbidity measurements and the direct microscopic cell count
what is a turbidity measurement?
the greater the number of cells, the greater the turbidity, the greater the amount of light scattered and absorbed by the solution.
what is the measurement unit for turbidity?
Optical density (OD)
what is a direct microscopic count?
Involves the estimation of the total number of microbial cells in a sample using a counting chamber and a microscope
what are the advantages of direct count?
- an easy method that requires minimal equipment
- you can observe cell morphology when counting
- you can count any solution as long as it is diluted correctly
What is a 2-fold serial dilution?
The volume of culture is the same as the volume of the diluent. The number of cells is halved in each dilution
How is optical density measured?
measured using a spectrophotometer
what is the relationship between optical density and the number of cells?
the greater the optical density, the more cells present in the culture
How do you calibrate a spectrophotometer?
- load a cuvette with 1ml of diluent (blank)
2. Press CAL button and wait until the display is zero
How do you measure the absorbance on the spectrophotometer?
- load 1ml of a sample into the same cuvette in the zeroed machine
- read the absorbance
How do you plot absorbance results?
Plot the dilutions (x axis) against optical density (y axis)
what is that direct microscopic cell count?
commonly used to estimate the total number of cells
what is the structure of a counting chamber?
The counting chamber slide has two chambers, each has a microscopic grid etched on the glass surface. A glass slip is put on the top, exactly 0.1mm above the chamber floor.
The volume in each chamber is known
how do you calculate the number of cells from a direct microscopic cell count?
The volume in each chamber is known.
Count the number of colonies in 5 cells and add
Then multiply by the number of squares in the section
Multiply the total cells in the section by the inverse of the dilution
Multiply by the chamber volume
when is a direct count more useful than a viable count?
It is quicker
What does a viable count, count?
Estimates the number of living or viable cells
What does a direct count, count?
Estimates the total number of cells, both living and dead
