1.2.4 Determination of cell Numbers Flashcards
3 ways in determining cell numbers?
- Total cell count
- Viable count / Plate count
- Measuring the density or turbidity of cells
Can be used to estimate the number of bacteria present in a specimen. It does not distinguish between live and dead cells
Total cell count (Direct counting)
What microscope is used in Total cell count?
Phase-contrast micorscope
What microscope is used in Total cell count?
Phase-contrast microscope
Used to determine the number of colony-forming units per milliliter (CFU/mL of bacteria). This method provides a count of viable cells only.
Viable Count or Plate count
How Plate count is done?
Serial dilution then plate each of diluted
Common amount of colonies required in Plate count?
30-300 colonies
The density of a bacterial broth culture in log phase can be correlated to CFU/mL of the culture. This method is used to prepare a standard inoculum for antimicrobial susceptibility testing
Density Measurement
What equipment is use to determine the turbidity of a sample?
Spectrophotometer
When is density measurement is standard?
Antimicrobial susceptibility testing
Conventional method of Density measurement
Compare it to 0.5 Mcfarland solution
How to measure Exponential phase or lag phase that is to be used in Density measurement?
Optical density 600 (0.02 - 0.05)
2 ways in performing plate count
Spread-plate method
Pour-plate method
Steps in Spread-plate method?
- Sample is pipetted onto surface of agar plate (0.1 mL or less)
- Sample is spread evenly over surface of agar using sterile glass spreader
Steps in Pour-plate method
- Sample is pipetted in to sterile plate (0.1 - 1 mL)
- Sterile medium (Molten Agar 45 -50 C) is added and mixed well (Swirl) with inoculum
- Solidify 15-20 mins
- Incubate (35 +- 2) - 18 - 24 hrs
Most valid count of colonies?
30 -300 colonies
If there are too much colonies, what should be the interpretation?
TNTC (To numerous to count)
Most common dilution to dilute the bacteria in Plate count?
10-Fold dilution
How 10-fold dilution is done?
0.5 of sample with 4.5 mL of diluent or 1.0 ml of sample with 9.0 mL of diluent
How 100-fold dilution is done?
0.05 mL can be mixed with 4.95 mL of diluent or 0.1 mL with 9.9 mL of diluent
Alternatively, a 10^-2 dilution can be achieved by making two successive 10 fold dilution`
What is the diluent use for 10 fold dilution?
Phosphate buffer solution
