Cell Biology Laboratory Manual Table of Contents

  Techniques Manual Table of Contents

  Lab Safety Sheet


  Related Topics

Aseptic Technique

Sampling and Inoculation

Benedict's Test

Petri Dishes

Streaking for Isolation

Spread plates


Staining Bacteria

  Return to START!

Inoculating Broth and Semi-solid Media

Many types of experiments are done in broth cultures. We must have pure cultures for doing work in broth in order to know which bacteria are responsible for the effects we observe. To inoculate a broth culture, hold a labeled broth tube in your left hand and your loop in your right hand using the thumb and first two fingers. (This is essentially the same technique used for swabs illustrated on the sampling and inoculation page) Flame sterilize the loop. Remove the cap from the broth tube with the little finger of the right hand curling against your palm. Flame the opening of the broth tube. Touch the loop to the desired colony on the plate and then stir the loop around in the broth. Reflame the opening of the tube, return the cap, and place the tube in a test tube rack. Flame sterilize the loop.

The phenol red broth

We can quickly differentiate between aerobic and facultative anaerobes based on whether they ferment sugars. The phenol red broth test is very useful for making this determination. Phenol red is a pH indicator which turns from red to yellow as the pH goes from alkaline to acidic. Any sugar (most often glucose since it is the most readily fermented sugar) can be added to provide a target for respiration. Fermentation of sugars other than glucose (lactose, sucrose, maltose, etc) can be used to help differentiate between various fermentative bacteria.

The phenol red broth also is a rich source of amino acids, needed for the growth of bacteria which do not ferment the sugars. Amino acid metabolism does not interfere with our results, however, because they are usually broken down in Krebs cycle rather than glycolysis and so do not yield acidic waste products. In fact, amino acid utilization will usually make the media even brighter red due to the release of excess amine groups raising the pH of the media.

To inoculate phenol red broth, use the steps described in inoculating broth cultures. Color changes should be apparent within 24 hours. If the media changes to a bright yellow color, the test is positive for fermentation. If the color is unchanged or red, the test is negative for fermentation and the bacteria are probably growing on amino acids, especially if the media becomes deep red. A pale orange color indicates that there may be some slight fermentation going on, but not enough to call the test positive. You can not judge one way or the other if there is no growth in the tube. Any bacteria that grows on TSA should also grow in a phenol red broth and therefore you probably did not get a good inoculation.

Inoculating semi-solid media.

We occasionally work with semi-solid media, which is much like any other agar media except that a smaller percentage of agar is present making the agar much softer. The agar is sufficiently soft that motile bacteria can swim through the media. We can use these properties to check for motility if we are careful in our inoculation technique. We use a needle (straight wire) rather than a loop, using one stab straight down through the media. Non-motile bacteria will only grow along the stab line but motile bacteria will swim through the soft agar making the whole tube turbid.