metode Manual Bergeys manual of bacteriology sni Direct

metode • Manual (Bergeys manual of bacteriology) • sni

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

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

Direct Measurements of Microbial Growth: Viable Counts Pour plate Spread plates: 30 -100 colonies Pour plates: 30 -300 colonies Surface drop (Miles-Misra): 10 -30 colonies

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

Direct Method Filtration

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.

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

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

There Are More Accurate Methods to Determine Turbidity Levels

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 Limitation: • 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