Unit 5: Cultivation of Bacteria

Importance

We frequently conduct experiments on bacteria. This is because they are relatively easy to culture and the presence of plasmid enable simple gene editing. In order for bacteria to express protein for us, we have to culture bacteria nicely so that they live healthy enough.

Solid culture medium

Part of the content from Teach.Genetics (http://teach.genetics.utah.edu/content/microbiology/plates/)

Bacteria is cultured on agar plates. Agar plates are the standard solid support material for growing microorganisms. Microbial growth media contains nutrients and an energy source to fuel the microbes as they grow, and agar to keep the media in a semi-solid, gel-like state. Chemically, agar is a polymer made up of subunits of the sugar galactose, and is a component of the cell walls of several species of red algae that are usually harvested in eastern Asia and California. Dissolved in boiling water and cooled, laboratory agar looks gelatinous. Unlike gelatin, won't be degraded (eaten) by bacteria. Also, agar is firmer and stronger than gelatin.

Preparing agar plate

Agar does not come as a semi-solid gel form. It comes as dry powder, called Luria-Bertani (LB) nutrition powder. We need to dissolve the powder by boiling it in hot water. Cooling it down to about 50℃, we then pour it into petri dishes and then cool them down. Agar is gel-textured when cooled to room temperature.

The procedure of preparing LB agar plate can be found in The Protocol.

The concentration of the agar plate, the skill of pouring the liquid LB into petri dishes affects the quality of the LB plate, which affects the growth of bacteria. Pouring plate requires a lot of practice. Below are some examples of poorly poured plates.

Liquid culture medium

Liquid LB broth contains the same nutrients as that in LB agar. The only difference is that LB is in liquid state. It is usual used in boiling tubes or flasks, where competent bacteria is prepared. We can collect cells at the bottom of tubes or flasks after growing then precipitating the mixture.

Incubation

Incubating bacteria on its culture medium let bacteria develop healthy bacterial colonies. E.coli bacteria is cultured and incubated generally at 37℃, that is the optimum temperature of E.coli growth. To further promote the growth of bacteria, oxygen is blended into the liquid LB broth by shaking the container. In general, we incubate E.coli for 24 hours to harvest healthy colonies for further experimental procedures.

Passing the colonies

Spreading

Spread plates are used to distribute bacterial cells evenly across the surface of an agar plate. Bacterial cells suspended in a small drop of liquid are distributed using a sterile spreader. If the concentration of bacteria is low, individual colonies will result from the spreading process. Colonies can be picked to carry out further experimental procedures.

Streaking

Preventing contamination

The most common type of contamination on agar plates are moulds and other bacteria. Contamination may affect the growth of targeted cell type and experimenter may pick the unwanted cell type. Most contamination happens on LB plates without antibiotics. However, if agar plates with antibiotics are contaminated, the contaminant is antibiotic resistance. The gain of antibiotic resistance in wild-type microorganism is dangerous to human health. Therefore it is crucial to prevent contamination on agar plates.

Spotting contamination

(part of the content from Career Trendhttps://careertrend.com/how-6101553-detect-contamination-agar-plates.html)

Fungal contamination

Look for signs of fungal contamination. Fungal contamination will appear as fuzzy, filamentous, or hair-like growths, and should be visible to the unaided eye. Fungal contamination often occurs right along the edge of an agar plate.

https://eurekabrewing.wordpress.com/2012/08/24/yeast-banking-2-agar-plates/

Inspect for signs of bacterial contamination. If the plate has not been inoculated, the presence of any bacterial colonies indicates contamination. On an inoculated plate, look for colonies that display morphology different than what you would expect from the type of bacteria used to inoculate the plate.

Bacterial contamination

Antiseptic procedures

Creating a sterile space

Connect Bunsen burner to town gas tap. Close the air hole.
Spray 70% alcohol on working bench.
Light the Bunsen burner. Open the air hole.
Wait for half a minute or until area around the Bunsen burner is warm. Warm area is a sterile space.
Do experimental procedures inside the sterile space.

Preparing agar plates

When opening the flask of liquid agar or LB broth, sweep the mouth of the flask over the flame before removing the aluminium foil.
When closing flasks, sweep the mouth of the flask over the flame before covering it with aluminum foil.

Spreading and streaking

Set up a sterile space.
Put streaker rack inside the sterile space.
Immerse spreaders or streakers in to 70% alcohol
Sweep the spreaders or streakers over Bunsen flame. Wait until the alcohol is completely burnt that no flame is present on the spreaders or streakers.
Assign the spreaders or streakers on the rack. Let cool the spreaders or streakers and make sure they do not melt the agar.
Spread or streak

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