There are numerous occasions when it is necessary to either
estimate or determine the number of bacterial cells. Determination
of cell numbers can be accomplished by a number of direct or
indirect methods. The methods include: 1- The standard plate count.
2- Turbidity. 3- Direct microscopic counts.
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I-Standard Plate Count (Viable Counts) I-Standard Plate Count
(Viable Counts) The number of bacteria in a given sample is usually
too great to be counted directly. However, if the sample is
serially diluted single isolated bacteria will form visible
isolated colonies. We are determining the number of Colony-Forming
Units (CFUs) in that known dilution. The number of colonies can be
used as a measure of the number of viable (living) cells in that
known dilution.
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A viable cell: A viable cell: Is defined as a cell which is
able to divide and form a population (or colony). Indirect viable
cell counts, also called plate counts: 1- A viable cell count is
usually done by diluting the original sample. 2- Plating aliquots
of the dilutions onto an appropriate culture medium. 3- Then
incubating the plates under proper conditions so that colonies are
formed. 4- After incubation, the colonies are counted and from a
knowledge of the dilution used, the original number of viable cells
can be calculated.
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Materials 1- 6 tubes each containing 9.0 ml sterile saline. 2-
3 plates of suitable media. 3- 2 sterile 1.0 ml pipettes. 4-
Pipette filler. 5- Turntable and bent glass rod. 6- Dish of
alcohol. 1.0 Milliliter (ml) Pipette
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Procedure Procedure A- Dilution of bacterial sample: 1.Flame
the sample flask. 2.Insert the pipette to the bottom of the flask,
and withdraw 1.0 ml of the sample. 3.Flame the first dilution tube.
4.Dispense the 1.0 ml of sample into the tube. 5.Draw the liquid up
and down in the pipette several times to rinse the pipette and to
properly mix. 6.Re-flame and cap the tube.
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7.Mix the tube thoroughly by holding the tube in one hand and
vigorously tapping the bottom with the other hand.
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8.Using the same procedure, aseptically withdraw 1.0 ml of the
sample from the first dilution tube and dispense into the second
dilution tube. Continue doing this from tube to tube.
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B- Plate out on agar surface: 1.Aseptically transfer 0.1ml from
each of last 3 dilution tubes onto the surface of the corresponding
plates. 2.Note that since only 0.1 ml of the bacterial dilution is
placed on the plate, the bacterial dilution on the plate is 1/10
the dilution of the tube from which it came. 3.Place the bent
portion of the glass rod on the agar surface and start to turn the
plate at complete 360 o. 4.Repeat for each of the 3 plates.
5.Incubate the 3 agar plates.
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1- Using a Pipette to Remove Bacteria from a Tube. Bacteria
from a Tube. 2- Using a Vortex Mixer to Mix Bacteria Throughout Mix
Bacteria Throughout a Tube. a Tube.
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3- Using a Pipette to Transfer Bacteria to an Agar Plate. 4-
Using a Bent Glass Rod and a Turntable to Spread a Turntable to
Spread a Bacterial Sample. Bacterial Sample.
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C- Results after incubation (Counting): 1- Choose a plate that
appears to have between 30 and 300 colonies. Sample 1/100,000
dilution plate (Figure a). Sample 1/1,000,000 dilution plate
(Figure b). Sample 1/10,000,000 dilution plate (Figure c). 2- Count
the exact number of colonies on that plate using the colony
counter. 3- Calculate the number of CFUs per ml of original
sample.
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Results (Figure a) (Figure b)(Figure c)
Slide 18
For accurate determination of the total number of viable cells:
The total number of viable cells is usually reported as
Colony-Forming Units (CFUs) rather than cell numbers. A plate
having 30-300 colonies is chosen because this range is considered
statistically significant. 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.
Likewise, if there are more than 300 colonies on the plate: there
will be poor isolation and colonies will have grown together.
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The number of CFUs per ml of sample = The number of colonies
(30-300 plate) X The dilution factor of the plate counted To
determine the number of CFUs per milliliter (ml) of sample:
Advantage: This method of enumeration is relatively easy to perform
and is much more sensitive than turbidimetric measurement.
Disadvantages: A major disadvantage, however, is the time necessary
for dilutions, platings and incubations, as well as the time needed
for media preparation. Only living cells develop colonies that are
counted. Clumps or chains of cells develop into a single colony.
Colonies develop only from those organisms for which the cultural
conditions are suitable for growth.
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A sample of E.coli diluted according to the above diagram. The
number of colonies that grew is indicated on the petri plates. How
many CFUs are there per ml in the original sample?
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II- Turbidity When you mix the bacteria growing in a liquid
medium, the culture appears turbid. This is because a bacterial
culture acts as a colloidal suspension that blocks and reflects
light passing through the culture. The instrument used to measure
turbidity is spectrophotometer.
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The ability of the culture to block the light can be expressed
as either percent of light transmitted through the tube or the
amount of light absorbed in the tube. The percent of light
transmitted is inversely proportional to the bacterial
concentration (The greater the percent transmittance, the lower the
number of bacteria). The absorbance (or optical density) is
directly proportional to the cell concentration (The greater the
absorbance, the greater the number of bacteria.)
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III- Direct Microscopic Count Petroff-Hausser counting chambers
can be used as a direct method to determine the number of bacterial
cells in a culture or liquid medium. It has squares 1/20 of a mm by
1/20 of a mm and is 1/50 of a mm deep. The volume of one square
therefore is 1/20,000 of a cubic mm or 1/20,000,000 of a cubic
centimeter (cc). In this procedure, the number of cells in a given
volume of culture liquid is counted directly in 10-20 microscope
fields. The average number of cells per field is calculated and the
number of bacterial cells ml -1 of original sample can then be
computed.
Slide 25
Procedure: Procedure: Count the number of bacteria in five
large double-lined squares. Divide by five to get the average
number of bacteria per large square. This number is then multiplied
by 20,000,000, since the square holds a volume of 1/20,000,000 cc,
to find the total number of organisms per cc in the original
sample. If the bacteria are diluted (such as by mixing with dye)
before being placed in the counting chamber, then this dilution
must also be considered in the final calculations.
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Counting: number of bacteria per cc = the average number of
bacteria per large double-lined square X the dilution factor of the
large square (20,000,000) X the dilution factor of any dilutions
made prior to placing the sample in the counting chamber
Slide 27
Advantage of direct counts: Is the speed at which results are
obtained. Disadvantage: Since it is often not possible to
distinguish living from dead cells, the direct microscopic count
method is not very useful for determining the number of viable
cells in a culture.