2. metode
Manual (Bergeys manual of bacteriology)
sni

11. Direct Counting Methods
Normally Viable Counts
Remember that a Colony Starts Out as 1 Bacteria that Reproduces
Colonies May Not All Be The Same Size

13. Direct Measurements of Microbial Growth: Viable Counts
Surface drop (Miles-Misra): 20 μl
( 1 ml)
VIABLE CELL:
Able to divide & form colony on suitable agar plate medium
Each viable cell => one colony => CFU

18. Direct Measurements of Microbial Growth: Viable Counts
Pour plate
Spread plates:
colonies
Pour plates:
colonies
Surface drop
(Miles-Misra):
10-30 colonies

24. Direct Measurements of Microbial Growth: Viable Counts
SOURCES OF ERRORS
IN PLATE COUNT
- The number of colonies depends on:
Inoculum size
Inoculum conditions
Selecting Culture medium
Length of incubation Temperature of incubation
Clumps of cell => 1 colony => CFU
(colony-forming-units)
ADVANTAGES
- High sensitivity
- Could be made selective

33. Direct Method
Filtration

47. Most Probable Number
Statistical Procedure used to estimate the number of bacteria that will grow in liquid media.
Gives a 95% probability that the bacterial numbers will fall within a certain range.

50. Indirect Measurements of Microbial Growth: Turbidity
PHOTOMETER: Klett units
- Simple filter generate light, relatively a narrow wavelength
SPECTROPHOTOMETER:
Optical density (540, 600 or 660 nm)
- Prisma of diffraction generate a very narrow band of wavelength
Both measure unscattered light
# Cells proportional Klett unit or
OD except at high cell density
Standard curve needed relating direct to indirect measurements
- OD vs. # Cells (e.g. viable count)
- OD vs. dry weight
ADVANTAGES:
- Quick & easy
- Do not disturb culture

51. Indirect Measurements
Turbidity
a. No turbidity = < 107 cells/ml
b. Slight = 107 – 108 cells/ml
c. High = 108 – 109 cells/ml
d. Very High = > 109 cells/ml
Metabolic Activity
Dry Weight

52. There Are More Accurate Methods to Determine Turbidity Levels

55. Direct Measurements of Microbial Growth: Total Counts direct microscopic counting
using Petroff-Hausser counting chamber
quick way of estimating cell number
known volume of sample dried on slide & in counting chamber
Living & dead cells counted; Small cells difficult to see
Precision difficult to achieve
Phase contrast microscope required w/ no staining sample
Not suitable for cell-suspension at low cell density (sample concentration)
Motile cells has to be immobilized
Limitation: