lab 11

method most probable number (MPN)

use -enumeration fo bacteria from water, milk, and other bacteria limitations -time consuming, requires proper medium for growth, provides indirect estimate of numbers

method membrane filtration

use -enumeration of bacteria from water, milk, and other products,, especially when numbers are low limitations -time consuming, requires proper medium for growth

method viable count

use -enumeration of viable cells in water, milk, and other production limitations -time consuming, requires proper medium for growth

method turbidimetric measurement

use -rapid estimation of cell density of a suspension limitations -does not differentiate between viable and nonviable cells, provides only estimate of cell density, requires > 10^6 cells/mL

method direct microscopic count

use -rapid laboratory enumeration of cell suspension limitations -requires large number (>10^6 cells/ml) of cells for accuracy

method cell activity measurement

use -research applications to follow cell metabolism limitation -involves extensive preparatory time

is Viable Cell Number an indirect or direct counting method?

viable cell number is a direct counting method

definition of growth vs reproduction

while growth is the increase in the mass of protoplasm of an organism or population, reproduction refers to an increase in the number of individuals

bacterial division figure 11.1

-bacterial cell grows to double its original volume and length -new cell wall builds along the center to separate the two parts of the cell -cell walls completely separate the two identical daughter cells

to use the micropipetter (steps)

a. dial the desired volume by turning the volume adjustment knob b. aseptically insert the end of the micropipetter into the sterile disposable tips, press firmly with a light twist and remove the tip from the box a. never touch the tips with your fingers. Use the correct tips for the micropipetter c. Using your mouth, slowly push down until you feel light resistance d. immerse the disposable tip into the sample liquid to a depth of 1-2 mm a. avoid jerky movements e. slowly allow the push button to return to its position f. wait few seconds to make sure that all the volume was suck into the tip, withdraw the tip from your sample g. dispense the liquid by gently pressing down on the button until your thumb will not go any farther. This will release the full volume of liquid from the pipette tip h. discard the used tip in a biohazard container

name one advantage and one disadvantage of the viable count method

advantage -it allows enumeration of viable cells in water, milk, and other products disadvantage -is time consuming and requires proper medium for growth

it is important to note that

cells may grow without reproduction or may reproduce without growth

How do you determine which plate will give you the most accurate count?

plates containing 30 and 300 colonies give the most accurate count

are rates of growth and reproduction equal?

rates of growth and reproduction may be unequal in a population, resulting in a variation of cell sizes

the spectrophotometer measures the _ of a sample to determine the amount of bacteria present

turbidity

method dry weight determination

use -determination of cell mass for industrial of laboratory applications limitations -time consuming, does not differentiate between viable and nonviable cells

how to use the micropipetteters -their benefits, use and job (summary -valuable tool - make microbiologist's job easier to use -volumes moved a lot faster -less material to discard -may also be more accurate than glass pipettes) -can measure small volume -lots of times volume measured can be adjusted (changed)

-the micropipetters are a valuable tool in microbiology laboratories, they make the job of the microbiologist a lot easier since the volumes can be moved a lot faster, and also there is a lot less materials to be discarded since only small plastic tips are used. Micropipetters might also be more accurate than regular glass pipettes -micropipetters are used to measure small volume. They are automatic, and a lot of times the volume measured can be adjusted (changed) The most used sizes are: -"P-1000"- for volumes between 0.2 ml and 10 ml in 2 ul increments. The first number is in red and is always 0. This should be multiplied by 10 to obtain the actual volume dispensed. -for example if the window shoes 067, the volume to be dispense will by 670 ul -"P-200"- for volumes between 0.02 ml (20 ul) and 0.2 ml (200 ul) in 1 ul increments -"P-20"- for volumes less than 0.2 ml (<20 ul) in 1 ul increments

measurement of growth figure 11.2 growth curve 1. Turbidity 2. Determination of dry weight 3. Determination of some constituents of the cytoplasm 4. Viable count 5. Direct microscopic count

1. Turbidity -as a beam of light passes through a suspension of bacterial cells, the light is scattered because of the difference in refractive index across the interface separating the cell from the medium. As a result, less light is transmitted by a tube containing a bacterial suspension than by the same tube filled with medium alone. The main factors which determine the amount of light scattered by the suspension and (1) the difference in refractive index between the cell and the medium, which depends on the concentration of solids, (2) size and shape of the cells, (3) the wavelength of light, and (4) the path length through the suspension. One may measure the light scattered rather than the light transmitted. Measurement of scattered light is more sensitive than is measurement of turbidity. Instruments that measure scattered or reflected light are called nephelometers 2. Determination of dry weight -An aliquot of the culture is centrifuged and washed free of the soluble constituents of the medium by further centrifugation from water. The washed cells are then dried to constant weight either at 105 C at ambient pressure or at lower temperature (e.g., 60 C) at reduced pressure 3. Determination of some constituents of the cytoplasm -the washed cells can be analyzed for total N, organic P, or nucleic acid. If all constituents remain in constant proportions, the measurement of any constituent should suffice as a measure of growth -a variation of this technique is to supply an isotopic compound to the medium and determine the radioactivity in the cells after separation of the cells from the medium by filtration of a sample of the culture through a membrane filter 4. Viable count -is an estimate of the number of individual units which are capable of developing into a colony if placed in a suitable medium. A clump, a chain, or a single cell score equally. The method of viable counting depends on the physiology of the organism. Obligate or facultative aerobes are counted by spreading an aliquot of a suitable dilution on the surface of an agar medium 5. Direct microscopic count -direct counts can be made by placing a suitable dilution of the culture in a hemocytometer or Petroff-Hauser chamber. The cells are allowed to settle to the bottom of the chamber and are counted using a 4-mm (dry) phase contrast objective. Since the etched lines on the bottom of the chamber define known areas and since the depth of the chamber is known, the number of cells per unit volume can be computed -Automatic particle counters obviously facilitate the direct counts of bacteria in suspension. Currently, the most popular is the Coulter counter which is based on a measurement of the electrical conductivity of liquid filling a small pore or aperture. The suspension is diluted such that the aperture is likely to contain at the most one bacterial cell, and a measured volume of the suspension is pumped through the aperture. if a cell enters the aperture, the conductivity is reduced. After the cell passes the aperture, the conductivity abruptly rises. The resulting electrical pulse is counted electronically

the _ chamber is used for direct microscopic coutns

Petroff-Hauser

bacterial count practice assume all plating use 0.1 mL (100 ul)- for this example or all? TMTC= TFTC= choose a plate and calculate VCN for the sample (give proper units) -results --- Viable cell number = VCN (calculate using the given) -# of colonies = 259 -dilution factor = 10^-5

TMTC= too many to count TFTC= too few to count VCN = # of colonies x 1/dilution factor VCN = 259 x 1/10^-5 = 2.59 x 10^7 so there are 2.59 x 10^7 CFU/ml in the original sample