Made By: Duaa Mohammed EL- Boh.
ID: 22042048.
Supervised By:
DR. Abdelraouf Elmanama.
Contents and Overview :
we will see methods of counting:
*Direct methods:
- Counting chamber.
- Coulter counter.
- Colony counting (Viable Count):
(How ,Advantages, Disadvantages)
- Serial Dilution
*Indirect methods.
If we need to count penciles we will
say…..
One.. Two .. Three .. .. .. ..
But when we need to count bacteria
this will be impossible ...
so…. What???!!!
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
numbers it is necessary to
dilute the sample.
*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 (Hemocytometer) :
The haemocytometer is a specialized 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 necessary .
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 through
a small orifice).
It is Very fast , 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.
How 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 methods often rely on the results of metabolic
tests or other growth characteristics. And it’s to:
- Measurement 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 process
can be automated.
*References :
*http://sciencelinks.jp/jeast/article/200517/000020051705A0706126.php.
*http://whyfiles.org/shorties/count_bact.html.
*http://msucares.com/livestock/dairy/bacteria.html.
*http://www.freesciencefairproject.com/biology/bacteri
a_counting.html.
*http://www.disknet.com/indiana_biolab/b038.htm.