Culturing Known Strains of Bacteria

Culturing is used to grow your very own microbes, but one media does not fit all! Some microbes grow well on nutrient agar, while others won't grow at all. Here is a brief introduction to culturing. Interesting selective and differential medias used in the Li lab are described below.

Materials for Creating Media

     Agar powder or LB broth powder 


Materials for Culturing


Creating Broth Media

  1. 1. Determine the volume of broth you would like to create and determine how much broth powder is required.  

  1. 2. Place a piece of autoclave tape labelled with the broth type and date onto the jar you will be using. 

  1. 3. Measure the appropriate amount of powder using a weigh paper and scoopula. 

  1. 4. Place this powder in the jar. Place a stir bar in the same jar. Add the appropriate amount of DI water. 

  1. 5. Mix the powder and water using the stir plate until all powder is dissolved. 

  1. 6. Slightly unscrew the lid and run the jar on the liquid autoclave setting. 

  1. 7. After autoclaving, place the jar in storage at room temperature. 


Creating Agar Media

  1. 1. Determine the volume of agar you would like to create and determine how much agar powder is required.  

  1. 2. Place a piece of autoclave tape labelled with “Agar, date” onto the jar you will be using. 

  1. 3. Measure the appropriate amount of powder using a weigh paper and scoopula. 

  1. 4. Place this powder in the jar. Place a stir bar in the same jar. Add the appropriate amount of DI water. 

  1. 5. Mix the powder and water using the stir plate until all powder is dissolved. 

  1. 6. Slightly unscrew the lid and run the jar on the liquid autoclave setting. 

  1. 7. After autoclaving, begin pouring plates. Add enough agar to entirely cover the bottom of the plate. 

  1. 8. Leave the plates at room temperature overnight to allow them to set.  

  1. 9. Store the plates with the lid down in at 4°C until use. 


Inoculating a Liquid Culture (Aerobes)

  1. 1. Gather materials. Into a test tube, transfer 10mL of LB broth using the automatic pipette. Label this test tube with the intended bacteria contents, your name, and the date of inoculation. 

  1. 2. If you are using a liquid parent culture: transfer 200uL of the parent culture using a pipette into the new culture. Vortex sufficiently. 

  1. 3. If you are using a solid parent culture: light the Bunsen burner. Flame the inoculation loop until it is red. From the parent culture, scrape off some growth. Place the loop inside the test tube and shake the loop to release cells. 

  1. 4. Place test tubes in the shaker in the incubator at 200rpm. Grow overnight at 37°C.  

  1. 5. After growth, store test tubes in an appropriate rack at 4°C. 

 

Inoculating a Solid Culture (Aerobes)

  1. 1. Gather materials. Label the bottom of the petri dish (the side that contains the agar) with the intended bacteria contents, your name, and the date of inoculation. 

  1. 2. If you are using a liquid parent culture: transfer 200uL of the parent culture using a pipette onto the new culture plate. Proceed to step 9. 

  1. 3. If you are using a solid parent culture: light the Bunsen burner. Flame the inoculation loop until it is red. From the parent culture, scrape off some growth. Proceed to step 9. 

  1. 4. Spread the fluid with the inoculation loop (liquid) or spread the inoculation loop (solid). Spread sufficiently around the full plate. 

  1. 5. Plate the plates in the incubator at 37°C with the lid down overnight.  

  1. 6. After growth, store petri dishes at 4°C. 

The Wonders of Culturing

As noted above, not all bacteria will grow on the same media type. For example, E. coli grows will on nutrient agar, but E. faecalis does not. Interestingly, both grow well on tryptic soy agar. You may have to do some research to find out what your bacteria likes to eat. Here are some medias we have available, and bacteria types that generally grow on them.

Nutrient agar is a general purpose medium that supports the growth of a wide variety of non-fastidious organisms. It typically contains tryptone, yeast extract, agar, and sodium chloride
Tryptic soy agar (TSA) is a general purpose medium used for a wide range of applications. It typically contains tryptone, "soytone", sodium chloride, and agar.
Blood agar is a differential media. Typically, blood agar plates are supplemented TSA plates, with 5-10% mammalian blood enrichments. These plates are able to detect hemolytic activityand are used to isolate fastidious organisms.
Eosin methylene blue (EMB) is a selective stain medim for Gram-negative bacteria. The dyes in this medium are toxic to Gram-positive bacteria. This media can differentiate based on lactose fermentation: bacteria that ferment lactose will appear with a dark center.
MacConkey agar is another selective and differential media for Gram-negative and enteric bacteria. This medium also differentiates based on lactose fermentation, where lactose fermenteres appear pink or red.
Mannitol salt agar (MSA) is used to select for salt-tolerant bacteria. Because it contains a high concentration of NaCl, it is selective against most Gram-negatives and selective for some Gram-positives (Staphylococcus, Enterococcus, Micrococcaceae, etc). This medium can also differentiate based on mannitol fermentation as it contains a pH indicator for mannitol fermentation (the agar will turn yellow).