do a 300 words minimum lab report.

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Acid and gas production during carbohydrate fermentation

Enteric species, as well as other groups of Gram negative and Gram positive bacteria, can be characterized in 3 ways that describe how they ferment a particular carbohydrate:

1. Does the organism grow? (ie. can it utilize the carbohydrate present?)

If the answer to #1 is yes……...

2. Does the organism produce an acidic by-product from fermentation of the carbohydrate?

3. Does the organism produce gas from fermentation of the carbohydrate?

Growth: Carbohydrate fermentation media is in the form of broth. If an organism is capable of utilizing the carbohydrate, the media will become turbid. If turbidity is not obvious following the incubation period, swirl the tube to make sure that cells have not grown and settled to the bottom of the tube. Cells of some bacterial species (such as some Bacillus species) will precipitate out of the media and form a pellet at the bottom. You could incorrectly assume that growth did not occur in this case.

Acid production: As mentioned in earlier labs, the process of fermentation is characterized by incomplete digestion of some catabolite, usually a carbohydrate. Instead of totally mineralizing the catabolite, as happens in the process of respiration, fermentors only partially break down the food resulting in the formation of reduced by-products such as acids, alcohols, and other solvents. Some organisms produce acidic by-products upon fermenting a particular carbohydrate, whereas others produce neutral pH or alkaline by-products. Carbohydrate fermentation media contains the pH indicator phenol red, which turns from red at neutral pH to yellow at acidic pH. The rule of thumb is this; you should score a test positive for acid production ONLY if the media turns totally yellow from top to bottom. Some organisms will cause a weak acid reaction (yellowish-red color) throughout the tube, whereas other organisms will form a yellow color only at the top or bottom of the tube. These are considered false positive reactions.

Gas production: Carbohydrate fermentation tubes also contain a small inverted vial called a Durham tube. Some fermenting organisms produce gas in addition to reduced by-products when fermenting certain carbohydrates. If the organism produces gas, bubbles will be trapped in the inverted tube for you to see. Although most manuals suggest that the presence of any amount of gas should be considered a positive reaction for gas production, a tiny bubble most likely had nothing to do with gas production by the bacteria. You will be able to tell the difference. When inoculating fermentation tubes, you must do so gently so you do not accidentally force bubbles in the Durham tube and cause a false positive.

If carbohydrate fermentation results were very dependable (which, I am afraid, they are not) we could use the method to differentiate several of our enteric organisms. See the table of expected results below which reflects that seen in many manuals. For example, using lactose alone, notice how we could differentiate: E. coli from Proteus from Klebsiella & Enterobacter from Salmonella & Shigella. Each of these 4 groups gives a different profile based upon growth / acid / gas results. Notice the lactose profile for E. coli. E. coli is theoretically the only organism on our list to give positive results for growth, acid and gas on lactose (but this does not work in practice, at least in our lab). For this reason, we say the E. coli is a fecal coliform, which is defined as a Gram negative, oxidase negative rod that produces acid and gas from lactose, and is bile resistant. E. coli is the representative of the fecal coliforms. E. coli is used as the indicator organism for fecal contamination in treated wastewater, drinking water, and the food industry.

Inoculate each organism that you choose to use into 1 fermentation tube of each carbohydrate type. Use a very small amount of cells as an inoculum. If you add too many cells, the media will be turbid from the beginning and you will not be able to determine for sure whether the organism grew in the media or not. Incubate the tubes at 37oC for 24-48 hours. Examine and score each tube for growth, acid production and gas production.

FOR UNKNOWNS the most useful results for separating enteric genera include:

1. Acid production from sucrose: Citrobacter + vs Salmonella –

2. Gas production from glucose: Shigella - vs all other enterics +

SEE IMAGES:

Carbohydrate fermentation: left to right: positive acid and gas, negative acid and gas

Carbohydrate fermentation: left to right: positive acid and negative gas, negative acid and gas

Expected results – DO NOT trust these results

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G = glucose L = lactose S = sucrose

NOTES:

*We will only use this test to differentiate Gram negative rods.

* Did you notice the results for E. coli, the “fecal coliform,” on lactose in the table above? Remember why we say that E. coli is a fecal coliform.