Culturing Bacteria in the

Laboratory and Estimation of

Bacterial Numbers
Krishanpal Karmodiya
Aurnab Ghosh

Slides- Dr. Matang

Bacteria do fantastic
things!

Disease

Food technology

Drug industry

Bioremediation

Not all Bacteria are bad!!

Culturing bacteria in the laboratory

Why culture bacteria in the laboratory?
Large quantity needed for detailed studies
To generate strains of interest
Recombinant protein expression
Industrial applications
What do we need to successfully culture bacteria in the
laboratory?
Medium
Water
Salts-osmolarity
Nutrients
Source of carbon-sugars, alcohols
Source of nitrogen-amino acids, ammonium salts
Source of phosphorus-phosphates, nucleotides
pH
Temperature
Aeration

Not all bacteria can be easily cultured in the

uring bacteria in the laboratory

Todays menu
Luria-Bertani medium (LB) /Nutrient broth
Per litre
10 g tryptone (Trypsin hydrolysate of casein protein)
5 g yeast/meat extract (Cell-free extract of yeast/meat)
10 g NaCl

Should we use solid or liquid

medium?
Ans: Depends on the purpose. If
you want:
High biomass use liquid
Isolation/selection of specific
bacteria use solid

Liquid and solid LB medium (PC: Wikiped

Making LB-agar
Mix appropriate amounts of desiccated LB medium power (or
constituents) in water
Heat to dissolve LB and then add 1.5-2.0 % agar powder
Autoclave the suspension at 121 C for 15-20 mins at 15 psi
This sterilises the medium and melts the agar

Pour molten agar into petri plates under sterile

conditions and allow it to set

Isolating bacteria
Why do we need to isolate
bacteria?
How do we isolate bacteria?

acteria live in communities

How do we isolate bacteria?

On solid medium bacteria form discrete colonies
Basic principle of all isolation methods-dilution such that
each colony comes from a single parent bacterium
Dilution can be achieved by mechanical separation on
the surface of the agar plate or by diluting the
inoculum in a large volume
Bacteria that form a single colony are all clones of one
another!

solating bacteria

Colony characteristics help distinguish between organisms

Estimating bacterial numbers

Why estimate bacterial

numbers?
To know how much bacteria are there in any sample
Required for food products, dairy products, etc. to report
on numbers
Water sample
Industrial processes that use bacteria need to know how
many are there at any given time
Clinical laboratories need to measure bacterial growth
rates for testing antibiotic sensitivity

Methods to estimate bacterial numbers

Direct counts
Indirect counts
Viable plate counts
Turbidity

Direct microscopic counts

Limitations
Cells clumped
Together maybe
counted as one
Dead or alive?

Indirect measurement plate count

Limitations
Time consuming
Will know only
tomorrow how
many
cells are there!

Indirect measurement turbidity

Quick and
easy
Limitations
Dead or alive?
Need to have
Already
calibrated
the method

What are we going to do

today?
Groups of 8
Measure OD for different dilutions (10-1 to 10-8) Each
student in
the group measure OD for one dilution
While measuring OD, instrument should be auto-zeroed
with the blank (plain media without any bacteria)
600nm normally used

Use spread plate to plate out 0.1 ml of one dilution

each student do
same dilution that you measured OD for
Come back tomorrow afternoon and count the
number of CFUs in
your plate
Then estimate the number of CFUs in the original
sample