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Questions and Answers
What formula is used to calculate CFU/mL in bacterial enumeration?
Why is turbidity measured in indirect methods of bacterial enumeration?
What does a higher turbidity in a bacterial solution indicate?
How are McFarland turbidity standards used in bacterial enumeration?
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Which method doesn't distinguish between live and dead cells in bacterial enumeration?
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What is the purpose of adjusting bacterial suspensions using McFarland standards?
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Which factor is inversely related to CFU/mL in bacterial enumeration?
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How do McFarland standards help correlate turbidity with bacterial cell density?
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Why is it important to convert microliters (uL) to milliliters (mL) when calculating CFU/mL?
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Which method correlates turbidity with CFU counts directly?
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Study Notes
Cell Counting Techniques
- High cell density can lead to overlapping cells, complicating accurate counting.
- The Petroff-Hausser cell counter features a space of 0.02 mm (1/50 mm) between the cover slip and slide.
- Each grid consists of 25 large squares, totaling 1 mm² area and volume of 0.02 mm³.
Microscopic Observation
- Cells are counted across several large squares, composed of 16 smaller squares each.
- Example average: 12 cells counted per several large squares.
Calculating Cell Density
- Formula for cells per milliliter:
- 12 cells x 25 large squares x 50 x 10³ = 1.5 x 10⁷ cells/mL.
Colony Forming Units (CFU)
- CFU counts estimate viable bacterial count through serial dilution, plating diluted samples on agar, and counting colonies.
- Each colony corresponds to a single viable bacterial cell, facilitating the assessment of cell viability and count.
Plate Counting Methods
- Two primary methods exist:
- Spread-plate method: Distributes a known volume (≤ 0.1 mL) evenly across agar; CFU counted after 24-48 hours of incubation.
- Pour-plate method: Bacterial culture is mixed with molten agar and poured into a plate.
McFarland Units (MFU)
- Standards for cell density are expressed in McFarland Units:
- 0.5 MFU equates to 1.5 x 10⁸ cells/mL.
- 1.0 MFU is 3.0 x 10⁸ cells/mL.
- 2.0 MFU is 6.0 x 10⁸ cells/mL.
- 3.0 MFU is 9.0 x 10⁸ cells/mL.
- 4.0 MFU is 12 x 10⁸ cells/mL.
Culturing Bacteria
- Bacterial growth requires nutritional media reflecting their natural environments, including nutrients, temperature, pH, and oxygen.
- Pure cultures for diagnostics depend on isolating single colonies using specific techniques.
Isolation Techniques
- The 16-streak dilution technique promotes separation of different bacterial species based on morphology.
- Well-isolated colonies arise from single cells replicating in their specific location on agar, representing a single genetic lineage.
Determining Viable Bacteria
- Viable bacteria (CFU/mL) are assessed through serial dilution and inoculation onto nutrient agar, followed by CFU enumeration post-incubation.
- Serial ten-fold dilutions are commonly employed in viability testing methods.
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Description
Learn how to calculate cell density in a Petroff-Hausser cell counter using the dimensions of the grid and the number of cells counted in multiple large squares. This quiz covers the formula and steps to determine the number of cells per millimeter of the sample.
