Pure culture techniques laboratory
(for bacterial cultures)
OR
Common sense laboratory!
Jeet Kalia (jkalia@iiserpune.ac.in)
The aim
To grow microorganism of choice in the
laboratory
Why grow organisms in the lab when they are
present in nature?
• Large quantity needed for detailed studies
• To generate strains of interest
• Recombinant protein expression
• Industrial applications
Common sense begins!
• To culture microorganism in lab…give them food!
• Nutrients:
1) Macronutrients (C, H, O, N, P, S, K+, Ca2+)
2) Micronutrients (Zn2+, Mn2+)
Nutrients must be supplied in the correct form
• Some molecules are unable to enter cells
• Some molecules can enter the cells but the enzymes for
metabolizing them might not be present in the
microorganism
• Not all microorganisms can use the same molecule as
nutrition. These differences are used for identification of
the microorganism.
5
Some bacteria are better synthetic chemists
than others!
• Some bacteria can make all the organic compounds needed
from a single carbon source such as glucose.
• Others need to be fed vitamins, amino acids, blood, etc.
(needs to be added to the culture medium).
In addition to food, bacteria need other things
to grow
• Temperature
• pH
• Osmolarity
Before starting on trying to grow your microorganism:
Know what nutrition and conditions it needs (read, read, read!)
General protocol for growing
(or “culturing”) bacteria
• Pouring media “plates”
• “Streaking” bacteria on media plates
• “Picking” a “colony” from the plate
• “Inoculating” a liquid culture
Everything needs to be done under sterile
conditions!!
Sterile conditions – Why?
• To prevent contamination of your bacterial sample
from environmental microorganisms
• Absolutely critical when working with pathogenic
microorganisms
Sterile conditions – How?
1) Autoclave the media (heat to 121 °C and 20 psi)
Sterile conditions – How?
2) Use sterile petri dishes, pipette tips, plastic tubes in a
laminar hood sprayed with ethanol
Sterile conditions – How?
3) Use common sense!
• Use gloves at all times
• Don’t eat/touch other things/attend to your cell
phones while handling bacterial cultures
General protocol for growing
(or “culturing”) bacteria
• Pouring media “plates”
• “Streaking” bacteria on media plates
• “Picking” a “colony” from the plate
• “Inoculating” a liquid culture
Pouring plates
• Autoclaved media (nutrients + agar) is poured
on petri dishes. Agar is a matrix that solidifies
on cooling.
Pouring plates
Freshly poured
After cooling
Examples of types of media
LB (Lysogeny Broth/Luria Bertani) Agar
1) Agar
2) Tryptone
3) Yeast extract
4) NaCl
MacConkey Agar (selective media for growing gram negative bacteria)
1) Agar
2) Enzymatic digest of gelatin, casein and animal tissue
3) NaCl
4) Bile salts and crystal violet: to inhibit Gram positive organisms.
5) Neutral Red: pH indicator (red in color at pH below 6.8)
6) Lactose
Lactose utilizing bacteria: Pink colonies
Bacteria that do not utilize lactose: White colonies
General protocol for growing
(or “culturing”) bacteria
• Pouring media “plates”
• “Streaking” bacteria on media plates
• “Picking” a “colony” from the plate
• “Inoculating” a liquid culture
“Streaking” bacteria on media plates
Streaking loop
Colonies (various sizes, shapes, colours)
General protocol for growing
(or “culturing”) bacteria
• Pouring media “plates”
• “Streaking” bacteria on media plates
• “Picking” a “colony” from the plate
• “Inoculating” a liquid culture
Picking a colony and inoculating liquid culture
Put the picked colony
in liquid media to grow
bacteria
Picking a colony
General protocol for growing
(or “culturing”) bacteria
• Pouring media “plates”
• “Streaking” bacteria on media plates
• “Picking” a “colony” from the plate
• “Inoculating” a liquid culture
Today’s agenda
• Demonstration: Streaking plates
• Experiment: Streaking plates
• Observation: Observe different types of colonies and take notes
(shape, size, colour)
• Midsem Viva: Good luck!