Bacterial Quantification by

Culture:
Count bacteria with serial dilution
Aguilar, J.M., Canicula, J.K., Menorca, G.R.,
Nacalaban, C., Pascual, P.J., Sanchez, R. III
 BACTERIAL GROWTH
In ideal conditions, a bacterial population can expand at an
exponential rate.
With an optimal generation time of roughly 20 minutes, a
single E. coli bacteria can spawn a population over 1
billion strong in a 10-hour timeframe.
Generation Time - gives a good estimation of the health of
your culture and the optimization of the growth conditions
This growth is, however, neither spontaneous nor constant
and follows distinct growth curves.
In this Labster simulation, we were able to...

 Find a novel antibiotic compound

 Learn about Inquiry-based approach to
quantify bacteria
 Learn to not waste time and agar plates
 Learn how to quantify the bacterial growth
 BACTERIAL GROWTH CURVE
The bacterial growth starts with the inoculation of fresh
growth media with a small bacterial population.

1. Lag Phase – the bacteria are expected to be adapting to

the new media and resources, with increased metabolic
activity, a potential increase in mass and volume and
changes in gene regulation.
2. Exponential Phase – or the ‘log’ phase, is where there
is an actual rapid rise of the bacterial population; where
the bacteria are the wealthiest.
3. Stationary Phase – growth is not sustained.
4. Death/Decline Phase – due to shortage of resources or
overpopulation, bacteria are badly harmed and start
dying at an exponential rate.
 COLONY FORMING UNIT
A measurement used to estimate the number of viable
bacteria or fungal cells in a sample.
Cells are considered viable if it can multiply via binary
fission under controlled conditions.
Can be calculated by spreading a specific volume of
the microbial culture onto agar plate.
0.1 mL of bacteria culture results in 25 colonies.
Cfu/mL= colonies/volume speed.
 COLONY FORMING UNIT
Also defined also as a single cell or group of
cells that upon plating in or on solid media
replicates to form a visible as well as well-
defined area of growth.

Can form starting with any number of cells and

that the resulting colony will look pretty much
the same unless it was started with an extremely
large number of cells.
 SERIAL DILUTION
A technique use to quantify the effect of a novel
antibiotic compound on bacterial growth.

How to dilute a serial

multiply the dilution factors for each step
Dilution is the process of diluting or mixing two
or more substances or even compounds.
Dilution factor = 1/ (final concentration/ initial
concentration) = 1/ (0.001M/ 1M) = 1000X
 ADVANTAGES OF SERIAL DILUTION
It helps to reduce a dense culture of cells to a more
usable concentration.
The number of colonies cultured of the sample are
counted to estimate the concentration of an unknown
sample.
CHALLENGES:
Transfer inaccuracies result in less effective and
precise distributing with each sequential serial dilution
phase.
It does not separate bacteria like a streak plate.
Overnight incubation is required.
 STERILE TECHNIQUE
Is used to ensure a “clean” lab environment.

Steps used to keep laboratory work sterile:

Laboratory doors and windows should be kept close.
Wire loop and glass spreader should be sterilized before and
after use.
Lids from bottle should be held when removed.
Neck of bottle and tubes must be heated immediately.
Bottle or tube should be opened for minimum time possible.
Media and equipment should be sterilized.
 STERILIZATION
Refers to the removal of all microorganism
in a material or on an object.

Sterilizing media
The medium and its container must be
sterilized by autoclaving before use.

Sterilizing Equipment
Lab equipment can be sterilized by
autoclaving, heating, use of disinfectant.
 ANTIBIOTIC SELECTION
Mutations
Every time the bacterium goes through this process there is a chance
that errors occur.
Are random and can be located anywhere in the DNA.
Can form due to external factors like radiation or harmful chemicals.
Can result in antibiotic resistance in bacteria.
Treating bacterial population with an antibiotic, only the resistant
bacteria will be able to multiply; the antibiotic selects for them.

Natural selection
While some mutations are harmful to the bacteria, others can provide an
advantage given the right circumstances.
 ANTIBIOTIC RESISTANCE
HOW ANTIBIOTIC RESISTANCE
Normally occurs when a microbe is exposed to sub-lethal HAPPENS
concentrations of a particular antibiotic.
The surviving microbes can evolve resistance mechanisms
through mutations in their DNA over many generations as a
result of the selective pressure.
Researchers use antibiotic resistance genes to identify
bacteria’s plasmid containing their gene of interest.
These resistance mechanisms can be sorted into four
categories: (1) modification of the primary target of the
antibiotic, (2) inactivation of the antibiotic compound itself,
(3) preventing the antibiotic from reaching the target, and (4)
using alternative metabolic pathways.
Superbugs - a bacterial species that are resistant to all but the
most extreme therapies.
 E.COLI
Escherichia coli, E. coli, in short, is a
rod-shaped bacterium that is commonly
found in the lower intestine of warm-
blooded organisms.

Most E. coli strains are harmless, but

some serotypes can cause serious food
poisoning in their hosts and are
occasionally responsible for product
recalls due to food contamination.
 E.COLI
E. coli can be grown and cultured easily and inexpensively in a laboratory setting.

Model Organisms
Prokaryotes:
E. Coli
Eukaryotes:
Yeast
C. Elegans
Fruit Fly
Arabidopsis thaliana
Chicken
Mouse