Eprac 3A: Enumeration Of Micro-organisms Flashcards
Measuring size of microbial pop provides info can be used to..
Determine how microbes grow
Assess microbial quality of food and beverages
Monitor industrial processes and efficacy of treatments to improve outcomes for patients
Estimate no. of spoilage or disease causing microorganisms in food and beverages
Procedures to measure no. of individuals in pop
Direct microscopic count: enumeration of total number of microbial cells in sample; both living and dead with aid of microscope
Turbidimetric method: An indirect procedure to determine pop, both living and dead using spectrophotometer
Viable count: counting no. of living cells -> colonies on agar plates or solid media
Direct microscopic counts using Helber chamber
Rapid method for enumerating micro-organisms
- Use ethanol to wash clean
- Drop 10ul of culture into engraved circle in slide
- Wet finger with water and moisten each corner with cover slip -> cover slip over circle -> culture spread around central circle
- > coloured rings
Examine slide with 10X and 40X. Count cells according to lab manual
Grid sizes for counting chambers
Helber chambers are used for bacterial counts as grid size is appropriate for very small bacterial cells
Helber chambers have Thoma grid
When counting larger cells such as wbc, Improved Neubauer is used
Different grids have different chamber volumes and thus require different calcs
Helber chamber for bacterial cell count
By counting the no. of particles in a grid of known volume, no. of microorganisms per cm^3 can be calculated
Counting chamber is marked off by double ruled lines into 9 large squares, in 3 by 3 pattern
Each large square subdivided by single lines into 16 small squares in 4 by 4 pattern making total of 144 small squares
Turbidimetric method
Is based on the principle that:
Greater the number of cells in pop, greater amt of light scattered by sample
By measuring amt of light transmitted through pop of micro-organisms, you can estimate no. in pop
Measure turbidity
Most common unit used: optical density (OD)
OD= log100 -log%T
%T=percentage of transmittance
Turbidimetric method: absorbance and optical turbidity
When measuring the turbidity of a microbial pop, absorbance os often equated with optical density
Absorbance: absorption of light by solution
OD: scattered light
In bacterial suspension, live cells, dead cells, and debris all contribute to OD reading
Spectrophotometer
Designed to operate in visible spectrum
Measure transmittance or absorption of visible light through solution -> spectrophotometers sometimes referred as colorimeters
To determine approx no. of organisms/ml, absorbances are compared to standard curve already determined for organism at various known conc.
Viable counts
Turbidimetric method and counting chamber: examples of total bacterial counts (no. of viable cells cannot be determined)
Methods to allow for viable bacterial cell counts. Methods rely on even distribution lf bacteria, for example, spread playing, layering and membrane filters. Require one agar plate per dilution
Spread plate method
-> bacteria easy to count and isolate
After colonies grown, they are counted and no. of bacteria in original sample calculated
No. of colony forming units per mL (CFU/mL)
Successful spread plate: countable no. of isolated bacterial colonies evenly distributed on plate
Small volume of suspension spread evenly over surface
Each plate is spread with single inoculum of suspension
Agar plates
Select and prepare agar medium based upon type of bacteria to be enumerated
Plates should be dried before use
Inoculations
A convenient inoculum (in terms of spreading, absorption and calculations)= 0.1ml (100ul)
Should be spread immediately after applied (some bacteria rapidly attach to agar surface)
Calculating CFU/
CFU/ml = no. of colonies x dilution factor x volume factor
Dilution factor: adjusts for dilution performed on initial
Volume factor: no. adjusts for volume inoculated onto plate
