Transcript MICROBIOLOGY LECTURE TITLE: Measuring Bacterial Growth

Counting Bacteria
Serial Dilutions
Because of their very small size, counting
the number of bacteria in a sample can be
difficult
Although direct counts are possible with
a microscope, they require a lot of time
and expertise
Why Count the Number of
Bacteria??
• Drug Companies:
– How effective drugs are in killing
microorganisms?
• Hospitals:
– How many MO in CSF, blood, urine
• Public Health:
- Contaminants in food, milk,
water
Total Cell Count
• The most common method of
enumerating the total microbial
cells (dead and alive)
• The direct counting of cell
suspension in a counting
chamber of known volume using
a microscope
Total Cell Count – Both
living and dead MO
• Disadvantage: Unknown
potential for continuing
infection
• Advantage: Toxins can come
from both living and dead MO
Total Cell Count – Both
living and dead MO
• Neubauer counting chamber
• Special slide & cover slip,
microscopic marks on slide,
known area
•
Counting Chamber
Counting Chamber
•Use special pipette for
dilution
•Count specific areas of
chamber, multiply by dilution
factor
Instrumentation
• * Spectrophotometer (Light Meter)
•
Each single MO stops some light
from passing through a special test
tube.
•
One side light source, then test
tube containing MO, other side light
meter.
•
Needle reading from scale, then
conversion table telling # of MO in test
tube
Coulter Counter
• Another method for counting
cells
• An electronic instrument
“electronic eye”
• Has a tiny tube (one cell
diameter) that lets through only
one cell at a time
Viable Cell Count –
Living Cells Only
Viable Count – Living
Cells only
An easier method is to spread bacteria
over a wide area (nutrient agar plate)
and count the number of colonies that
grow
If the bacteria are spread out enough,
each bacterial cell in the original sample
should produce a single colony
Serial Dilutions
Bacterial samples must be diluted
considerably to obtain reasonable
counts
To determine the number of cells in a
bacterial culture carry out serial
dilutions
Serial Dilutions
Bacterial cell numbers are usually very high in
your original sample
Plating out this sample, undiluted, would lead
to the creation of a bacterial lawn
Bacterial Lawn - many individual bacteria
colonies that are all growing next to or on top
of one another
Serial Dilutions
Bacterial cell numbers need to be reduced
This is done by repeatedly diluting the amount
of bacteria you have in your sample
A small amount of bacteria sample is mixed
with a diluent solution (sterile broth or water),
and then successive dilutions are made
Serial Dilutions
A small amount of each of the diluted
bacteria samples is then spread onto an
agar plate
The numbers of bacterial colonies that
grow on each plate are counted
Serial Dilutions
Work backwards using multiplication with
the "dilution factor”
Dilution Factor - the number of times that
you have diluted the bacteria sample with
the diluent solution
Make a determination of the numbers of
bacteria in your original sample.
Serial Dilution
Serial Dilutions
*Make serial dilutions
*Spread a small amount from
each tube onto agar plate
*Culture bacteria for a couple of
days
*Count colonies
Each living MO = 1 Colony
Membrane Filtration
• Membrane filters can also be
used to determine the bacterial
numbers.
• In this method cells are filtered
onto membrane filter which is
then placed over a nutrient agar
plate.
Membrane Filtration
• The methodology is similar to
conventional total plate counts.
• Membranes have a printed
millimeter grid and colonies can
be counted under a binocular
microscope.
Factors essential for bacterial growth
--proper nutrients
--proper environment (space,
temperature)
--No inhibiting factors
Factors that could inhibit bacterial
growth
--lack of nutrients
--lack of proper environment
--presence of an inhibiting factor