Bacterial Fermentation Lab
Background: (Summarize for your lab notebook)
Because of their microscopic size, bacteria are difficult to identify by direct observation. One way of identifying a bacterium is to determine the biochemical pathways it uses. The ability or inability of a bacterium to ferment specific sugars provides such information. Students will observe carbohydrate fermentation on five different bacterial organisms.
Sugar fermentation is a process by which bacterial cells produce energy that is used as the fuel source for other metabolic pathways. This process requires that sugar molecules be broken down into smaller organic end products. These end products are usually strong organic acids produced under anaerobic conditions. The types of sugars that bacteria are able to ferment as well as the end products produced are important characteristics used in the identification of bacteria.
The number and types of sugars that a given microbe can assimilate will determine to a large extent the environments in which it can survive and grow. Citrobacter freundii is a gram-negative enteric rod. Enterics are bacteria found in the intestinal tract of humans and other animals. Those enterics that can ferment sugars are known as fermenters, while enterics that cannot ferment any sugars are called nonfermenters.
Serratia liquefaciens is a gram-negative rod found in soil, water, on plant surfaces and occasionally as an opportunistic human pathogen.
Bacillus subtilis can ferment more than one type of sugar, such as sucrose and dextrose, but cannot ferment other types of sugars, such as lactose. B. subtilis is a gram-positive rod often found in soil and decomposing organic matter; it is a common laboratory contaminate. Enterococcus
(Streptococcus) faecalis is a gram-positive coccus that is found in the intestines of humans and warm-blooded animals. It is common in many food products and often unrelated to direct fecal contamination. Some bacteria, such as Micrococcus luteus, cannot ferment any type of sugar. M. luteus is a gram-positive coccus found on the skin of humans and other animals.
Each phenol red broth tube contains a nutrient medium that will support growth of most bacteria, a single sugar source (such as dextrose, lactose, or sucrose), phenol red as a pH indicator, and an inverted Durham (or fermentation) tube.
Some bacteria ferment the sugar, producing acids, and/or gaseous end products. If the pH is lowered below 6.8, that is, if the acidity of the medium increases, the phenol red will turn from red to yellow. Some acidic end products are acetic, formic, lactic, butric, succinic, and propionic acids. Some bacteria can grow in the nutrient medium but cannot ferment the sugar, while other bacteria can ferment the sugar, producing nonacidic and/or gaseous end products. In both cases, the medium will remain alkaline, or red. Some of the nonacidic end products of fermentation are acetone, butanol, and ethanol. Some of the gasous end products produced are carbon dioxide and hydrogen.
Any gases formed in the inoculated media are trapped in the Durham tube.
Fermentation media are used to determine metabolic differences among bacteria.
These data supplement other observations that collectively identify the bacterial species.

Purpose: (Write in your lab notebook)
Describe the method and products of bacterial fermentation and explain how they are used to identify bacteria.
Materials (See website)
Bacillus subtilis
Citrobacter freundii
Enterococcus
(Streptococcus) faecalis
10-Phenol Red Dextrose Broth
10-Phenol Red Lactose Broth
10-Phenol Red Sucrose Broth
Metal Inoculating Loops
Autoclavable disposable bag Micrococcus luteus
Serratia liquefaciens
Other materials needed but not included are Bunsen burners, an incubator, and a disinfectant such as Lysol or 70% ethanol.
Procedure: (See website)
1.
Wipe down the work area with a disinfectant such as Lysol or 70% ethanol and wash hands thoroughly before beginning inoculations.
2.
Each station’s teams should separately inoculate one phenol red dextrose broth, one phenol red lactose broth, and one phenol red sucrose broth with the bacterial culture placed at the station.
3.
Begin inoculation by carefully inverting each tube of medium to eliminate air from the Durham fermentation tube.
4.
For each separate inoculation, remove the caps from the bacterial culture and a tube of phenol red broth. Hold the metal inoculating loop in the flame of the
Bunsen burner until it is red-hot. Hold the mouths of the tubes in the flame of the Bunsen burner for a few seconds. Scrape in small amount of bacteria from the bacterial culture and smear along the inside of the phenol red broth tube below the level of the broth. Flame the inoculating loop and repeat this procedure for the other two phenol red tubes.
5.
Flame the mouths of the tubes and replace the caps.
6.
Label the cultures with the name of the bacterium and the group number.
7.
Dispose of the used bacterial stock cultures in the autoclavable disposal bag.
8.
Incubate the inoculated tubes at 37 degrees C for 24 hours with the caps
9.
loosened.
After incubation, the teams at each station should compare their results and record them in Table 1.
10.
Dispose of the inoculated tubes in the autoclavable disposable bag.
11.
Compare the results to the expected kit results and record in Table 1.

Data: (Record in your lab notebook)
Table 1
Student’s Results- Observed
Bacterium Dextrose Lactose Sucrose
Table 2
Expected Results
Bacterium Dextrose Lactose Sucrose
A/N-Acid, no gas accumulation
A/G-Acid, gas accumulation
N/N-No acid, no gas accumulation
N/G-No acid, gas accumulation
Observations: (Record in your lab notebook)
Analysis and Conclusion (Answer the following questions using full sentences in your lab notebook)
1.
Explain the purpose of doing this type of test.
2.
Describe the process of bacterial fermentation. Why do most bacteria ferment sugar?
3.
Why did the tubes change color? Describe how the indicator works.
4.
Describe the purpose and procedures of aseptic technique.
5.
Compare the expected and student results. Why were there differences for some of the bacteria? Describe at least two sources of error and ways to solve these in the future.