Bacteria Counting

Bacteria counting
If we need to count penciles we

will say…..
One.. Two .. Three .. .. .. ..
But when we need to count
bacteria this will be impossible ...
so…. HOW???!!!
Importance :
*Knowing how to count organisms and
understanding their growth cycles is often
important in treating infections.
*By counting individual organisms and
experimenting, we can determine how many
it takes to cause disease.
* Counting bacteria is also important in
environmental microbiology; to control
environmental conditions or enhance growth
to obtain desired results.
**A single tiny drop
of nutrient broth
incubated overnight
may contain
5 000 000 cells – this
is
a lot to count.
**1cm3 may contain
108 cells.. so
**In order to estimate
*Direct methods :
With direct methods we count individual cells or
colonies that are assumed to have apart or arisen
through the division of a single cell.
1- Counting Chamber
The haemocytometer is a specialized
(Hemocytometer) :
microscope slide used to count cells.
The centre portion of the slide has etched grids
with precisely spaced lines.
To get an accurate count there should be
between 40 and 70 cells in a 1 mm square. If not
you dilute or concentrate the cell suspension as
Calibrated Stained Smear
Petroff- Hauser Counting Chamber
To get the final count in cells/ml, first divide
the total count by 0.1 (chamber depth) then
divide the result by the total surface area
counted , then divide the result by 5 mm-
squared, which is the total area counted (each
large square is 1 mm-squared).
There are 1000 mm-cubed per ml, so you
calculate cells/ml. Sometimes you will need to
dilute a cell suspension to get the cell density
low enough for counting.
In that case you will need to multiply your
final count by the dilution factor.
Hemocytometer Counting using a counting

chamber.
2- Coulter Counter :
electronic counting (this
machine detects the
difference in current as
individual
microorganisms pass
It is Very
through fast , orifice).
a small easy
to use but;
Very EXPENSIVE.
3- Viable count assays (Colony
Counting) :
Colony counting after plating dilutions of
the sample onto growth medium.
Standard plate counts using spread and
pour plate techniques (cfu for “colony
forming unit”) .
This is the method we will be using to quantify

our samples.
Count only those cells capable of growing
Viable counts can be accomplished by such
techniques as pour plating.
Assumption each viable cell gives rise to a
colony.
We will use two viable count assays:
1- Spread plates
A diluted sample is spread onto the
surface of an agar plate
2- Pour plates
Microorganisms are mixed with molten
agar and poured into a petri dish.
*Advantages:
- The method can be made to be very
sensitive.
- One count can be subsets of the
population.
*Disadvantages:
- Colony-forming units may underestimate cell
numbers because of clumping or chains of cells.
- Counts require at least a few hours, usually
overnight, for incubation.
4- Serial
Dilution
*Why use :Serial Dilutions?
- Bacteria undergo exponential growth , thus
many may be present in each sample.
- In order to count the number of bacteria, each
colony must be single and distinct.
- The number of countable colonies per plate is
30-300
- Serial dilutions allow one to dilute a sample of
bacteria to the point that the total number of
bacteria can be counted on an agar plate.
ow to Perform Serial Dilutions :
SERIAL DILUTION
1ml into 9ml = 1:10 dilution
conc. 10-1 10-2 10-3 10-4 10-5 10-6
*Indirect
Methods
Indirect : often rely on the results of
methods
metabolic tests or other growth characteristics.
- Measurement
And it’s to: of metabolic
activity.
-Gas or Acid
Production.
- Turbidity using a
spectrophotometer.
-spectrophotometry,
using a spectrophotometer .
These Indirect counts :
- depend on the effects of the organisms to
estimate their numbers.
- As organisms grow they make the nutrient
broth turbid.
- This turbidity can be measured with a
colorimeter
- The more organisms
the greater the
optical density of the
solution.
- The Coulter counter is a probe which measures
the change in conductivity of a solution as a
bacteria passes through a narrow gap.
- Problem with
INDIRECT COUNTS
and DIRECT COUNTS
is that they can not tell
living cells apart from
dead cells.
- Advantage of INDIRECT
COUNT is that the
Epifluorescence Microscopy
• Nucleic acid specific dye
– 3-6 tetramethyldiamino acridine
• Binds between base pairs of DNA or RNA
– Intercalation
• Fluoresces when bound
– DS-DNA -> Green
– SS-DNA -> Red Orange
– Degraded DNA -> Red Orange
Epifluorescence
• Approximate viable cells /total cells
– Viable cells green
– Dead cells orange
• Count low numbers
– cells fluorscent against a black background
Dilution Series: dilution
1ml 1ml 1ml 1ml
1ml
9ml broth
1/10 1/1,000 1/100,000

1/100
1/10,000
Dilution Series: plating
1ml 1ml 1ml 1ml 1ml
1/10 1/100 1/1,000 1/10,000 1/100,000

Dilution Series: Colony counts
1/10 1/100 1/1,000
1/10,000 1/100,000
Membrane filtration
• Membrane filter
– fixed pores 0.45 m 0.22 m
– bacteria trapped on filter
• Filter known amount of fluid
– ALL bacteria trapped on membrane
• Place filter in Petri dish with appropriate medium
• Incubate
• Count number of colonies
Membrane filtration
• Number of Viable cells
– Each colony from a single bacterium
• Sensitive
– eg filter entire bottle of pop
– <1 bacterium / bottle
• Growth media
– selective or differential
• Combine with Epifluorescence
– Black polycarbonate filters
Electronic Measurement
• Turbidity
– Broths with many bacteria become turbid
– turbidity increases light scattering
– increased absorbance in spectrophotometer
• Cell counters
– fluid pumped through a micro pipette
– pumped past an electronic sensor
– records number of cells

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Bacteria counting

If we need to count penciles we

Hemocytometer Counting using a counting

This is the method we will be using to quantify

1/10 1/1,000 1/100,000

1ml 1ml 1ml 1ml 1ml

1/10 1/100 1/1,000 1/10,000 1/100,000

1/10 1/100 1/1,000

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