35: Carbohydrate Use

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Page ID: 3667

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Learning Objectives

Learn different test procedures for determining carbohydrate use
Identify different end products of sugar use

SUGARS

Sugars are very important tests for a lot of microbes. Unfortunately, there are a few problems related to sugars. Whereas most biochemical media is stable, allowing you to inoculate one day and read a couple of days later, sugar is NOT. The problem is that there are other nutrients in the media that can be used by the bacteria, like proteins. Although the sugar is the primary nutrient used (if the organism uses that particular sugar), when the microbe runs out of it, protein or other nutrients will be attacked. This can cause changes in the color of the medium because there is a pH indicator added to detect acid production. When proteins are used, alkaline by-products are produced and the medium can change colors. If you let these sugar tests go for more than a day, you risk the possibility that the color will have changed and you may call the test result negative rather than positive. Therefore, it is a good idea to run into the lab and read these sugar tests somewhere between 8-12 hours, if at all possible. There are no reagents that have to be added since the pH indicator is added to the medium already.

You are going to see 2 different ways to run sugar tests: phenol red sugar broths and the sugar disc methods. Some of the sugars come in phenol red broth, already with sugar in it lactose, mannitol, glucose (dextrose), and sucrose.

For many sugars (see the media list of sugars), we can add the sugar to the phenol broth base as asked for. We have dozens of sugars available. Please just ask if you need either of these sugars.

MATERIALS NEEDED

phenol red sugar broths (lactose, sucrose, glucose/ dextrose, mannitol)
TSB
small sterile tubes
1 ml pipettes + pi-pump
sugar solutions + tubes of phenol red broth (sugars other than lactose, sucrose, glucose, mannitol)
ethanol
forceps

THE PROCEDURES

PHENOL RED BROTH

The broth can have various sugars added to it. It has a small upside down tube called a Durham tube that collects CO₂ gas.

Inoculate the phenol red glucose broth with your unknown bacteria.
Inoculate the organism into the tube with the disc and incubate at 25º C or 37º C.

SUGAR SolutionS

There are at least a couple of dozen sugars that we have in liquid form. 1 ml of the liquid sugar is added to a 2 ml sterile phenol red solution ((having no sugar). All you have to do then is inoculate with your organism and incubate.

IT IS BEST TO READ SUGAR REACTIONS WITHIN 24 HOURS BECAUSE THE COLOR CAN CHANGE AFTER THAT, giving you a false - reaction.

After incubation, look for the typical yellow color of acid production, a + test result.

Note

PLEASE ASK if you want to run a different sugar: your instructor will prepare it.

INTERPRETATION

PHENOL RED

You are looking for a change in the phenol red indicator to yellow, for acid (A) production. In addition, if there is a durham tube in the sugar broth, there may be CO₂ gas. You may notice an alkaline change to a very red color, indicating basic end products, but we don't care about basic reactions.

SUGAR DISCS

As above, you are looking for the yellow acid color. There is no way to determine gas production with this method.

QUESTIONS

The pH indicator used in most sugars is:
Why should sugars always be read within 12 hours, IF AT ALL POSSIBLE?
What is the purpose of the durham tube?