The standard plate count method Spectrophotometer turbid metric analysis The standard plate count method is an indirect measurement of cell density live bacteria The spectrophotometer analysis is based on turbidity and indirectly measures all bacteria cell biomass dead and alive ID: 265297
Download Presentation The PPT/PDF document "Many studies require the quantitative de..." is the property of its rightful owner. Permission is granted to download and print the materials on this web site for personal, non-commercial use only, and to display it on your personal computer provided you do not modify the materials and that you retain all copyright notices contained in the materials. By downloading content from our website, you accept the terms of this agreement.
Slide1
Slide2
Many studies require the quantitative determination of bacterial populations. The two most widely used methods for determining bacterial numbers are:
The standard plate count method.
Spectrophotometer (turbid metric) analysis.
The standard plate count method is an indirect measurement of cell density ( live bacteria).
The spectrophotometer analysis is based on turbidity and indirectly measures all bacteria (cell biomass), dead and alive.
IntroductionSlide3
The Plate Count (Viable Count)
However
, if the sample is serially diluted and then plated out on an agar surface in such a manner that
single isolated bacteria form visible isolated colonies
, the number of colonies can be used as a measure of the number of viable (living) cells in that known dilution.
The number of bacteria in a given sample is usually too great to be counted directly. Slide4
Keep in mind that if the organism normally forms multiple cell arrangements, such as chains, the
colony-forming unit may consist of a chain of bacteria rather than a single bacterium.
In addition, some of the bacteria may be clumped together. Therefore, when doing the plate count technique, we generally say we are determining the number of
Colony-Forming Units (CFUs)
in that known dilution.
By extrapolation, this number can in turn be used to calculate the number of CFUs in the original sample
.
bacterial counts by these
methods
are usually expressed
as colony
forming units per milliliter
(CFU/mL). Slide5
Normally, the bacterial sample is diluted by factors of 10 and plated on agar.
After incubation, the number of colonies on a dilution plate showing
between 30 and 300 colonies is determined.
A plate having 30-300 colonies is chosen because this range is considered statistically significant. Slide6
If there are less than 30 colonies on the plate, small errors in dilution technique or the presence of a few contaminants will have a drastic effect on the final count. (too few to count (TFTC
).
Likewise, if there are more than 300 colonies on the plate, there will be poor isolation and colonies will have grown together. (too numerous to count (TNTC).Slide7
Procedure
Using sterile technique, transfer 1 mL sample to the
first dilution blank. Mix the bottle by inverting
it 20 times. Label the bottle
"10-1." Slide8
Using a fresh pipette, transfer 1 mL from the first blank to the second blank. Mix as
before. Label the second bottle "
10-2."
Using a fresh pipette, transfer 1 mL from the first blank to the second blank. Mix as before. Label the second bottle "10-2“.
Using a fresh pipette, transfer 1 mL from the first blank to the
third
blank. Mix as before. Label the second bottle "
10-3“.
Using a fresh pipette, transfer 1 mL from the first blank to the
forth
blank. Mix as before. Label the second bottle "
10-4“.
Using a fresh pipette, transfer 1 mL from the first blank to the second blank. Mix as before. Label the
fifth
bottle "
10-5“.
Label the Petri dishes:
10-2, 10-3, 10-4, 10-5,
and
10-6,
respectively.Slide9
transfer liquid from the dilution blanks to
the Petri dishes. Use a separate pipette for each blank, not for each plate (i.e. if more than
one plate uses liquid from a single blank, a single pipette may be used for that blank).
One at a time, add a tube of molten nutrient agar to each Petri dish. After adding the agar
, gently swirl the dishes in pattern for 30 seconds to mix the bacteria with the agar.
After the agar has thoroughly solidified, incubate the plates at 37°C for 24 to 48 hours.
Count the number of colonies on a plate that has between 30 and 200 colonies. Any plate
which
has more than 200 colonies is designated as "too numerous to count" (TNTC).
Plates
with fewer than 30 colonies do not have enough individuals to be statistically
acceptable
. Slide10Slide11
Slide12
Colonies Forming Units {CFU}
Calculate
the number of bacteria (CFU) per milliliter or gram of sample by dividing the number of colonies by the dilution factor multiplied by the amount of specimen added to agar plate
.
To compute the number of CFU/mL, use the formula:
c
= concentration, CFU/mL n =
number of colonies
d
= dilution
blank
s
= volume transferred to plate. Slide13Slide14
CFU Calculation Example
You count 46 colonies on your plate
You put 1 ml of bacterial culture into 99 ml of saline and plated 0.1 ml
Dilution 1/100
CFU=
46
1/100 * 0.1 = 46 * 100 * 10 =46000 C
FU/mlSlide15
END OF LECTURE