1. Pure culture techniques laboratory (for bacterial cultures) OR Common sense laboratory! Jeet Kalia (jkalia@iiserpune.ac.in)

2. The aim To grow microorganism of choice in the laboratory

3. 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

4. Common sense begins ! To culture microorganism in lab…give them food! Nutrients: 1) Macronutrients (C, H, O, N, P, S, K +, Ca 2+ ) 2) Micronutrients (Zn 2+, Mn 2+ )

5. 5 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.

6. 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).

7. 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!)

8. 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!!

9. Sterile conditions – Why? To prevent contamination of your bacterial sample from environmental microorganisms Absolutely critical when working with pathogenic microorganisms

10. Sterile conditions – How? 1) Autoclave the media (heat to 121 °C and 20 psi)

11. Sterile conditions – How? 2) Use sterile petri dishes, pipette tips, plastic tubes in a laminar hood sprayed with ethanol

12. 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

13. 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

14. Pouring plates Autoclaved media (nutrients + agar) is poured on petri dishes. Agar is a matrix that solidifies on cooling.

15. Pouring plates Freshly pouredAfter cooling

16. Examples of types of media 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 LB (Lysogeny Broth/Luria Bertani) Agar 1) Agar 2) Tryptone 3) Yeast extract 4) NaCl

17. 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

18. “Streaking” bacteria on media plates Streaking loop Colonies (various sizes, shapes, colours)

19. 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

20. Picking a colony and inoculating liquid culture Picking a colony Put the picked colony in liquid media to grow bacteria

21. 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

22. 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!