Objectives: Carbohydrate Fermentation Exercises 5-3, 5-4, 5-21

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1

Oxidation of carbohydrates

the metabolic process by which an organic molecule acts as an electron donor (becoming oxidized in the process) and one or more of its organic products act as the final electron acceptor in respiration

2

List the primary types of products which bacteria may produce from fermentation.

Lactic acid
Ethanol
Butyric acid
Propionic acid
2,3-Butanediol
Mixed acids

3

Understand why it is important to have nutrients other than the fermentable carbohydrate in a Phenol Red Carbohydrate Broth

Because not all bacteria can utilize the fermentable carbohydrates. The ability or inability of a particular species to ferment a particular carbohydrate depends on the presence of enzymes needed for a particular fermentation pathway.

4

Understand the purpose of a durham tube in Phenol Red Broths

Gas production from fermentation is indicated by a bubble or a pocket in the durham tube where the broth has been displaced.

5

Understand why some microbiologists advocate using 1% carbohydrate rather than 0.5% carbohydrate in phenol red broths.

Some microbiologists prefer to use 1% rather than 0.5% to ensure against reversion of the reaction due to depletion of the carbohydrate (the organisms break down available amino acids after carbs are used, raises pH).

6

Understand why a Phenol Red Broth base medium would be inoculated in addition to the PRB tubes containing carbohydrates when examining the fermentative abilities of an organism

To make sure that the environment remains anaerobic.

7

Understand how Kligler Iron Agar can be used to differentiate groups of species

It is a differential medium for Gram-negative enteric organisms. It differentiates organisms based on their ability to ferment the lactose present in the media. (Note: all Gram-negative enteric organisms will ferment the glucose.)

8

Describe the difference between Kligler Iron Agar and Triple Sugar Iron Agar

TSI contains three carbohydrates: glucose (0.1%), sucrose (1%), and lactose (1%). TSI is similar to Kligler's iron agar, except that Kligler's iron agar contains only two carbohydrates: glucose (0.1%) and lactose (1%)

9

Understand why the amount of glucose is limited as compared to lactose (and sucrose) in Kligler Iron agar and TSI agar

The lower concentration of glucose allows for the detection of utilization of this substrate alone. Since glucose is a monosaccharide, it will be used first.

10

What two metabolic pathways lead to the production of hydrogen sulfide?

Hydrogen sulfide may be produced by the reduction of thiosulfate (acidic conditions must exist) in the medium or by the break down of cysteine in the peptone. Ferrous sulfate reacts with the H2S to form a black precipitate, usually seen in the butt (indication of sulfur reduction and fermentation).

11

coliform

bacteria that are a commonly used bacterial indicator of sanitary quality of foods and water. They are defined as rod-shaped Gram-negative non-spore forming bacteria which can ferment lactose with the production of acid and gas when incubated at 35–37°C.[1] Coliforms can be found in the aquatic environment, in soil and on vegetation; they are universally present in large numbers in the feces of warm-blooded animals. E. coli is one of these.

12

Enterobacteriaceae

are a large family of Gram-negative bacteria that includes, along with many harmless symbionts, many of the more familiar pathogens, such as Salmonella, Escherichia coli, Yersinia pestis, Klebsiella and Shigella. Many live in the gut. They are facultative anaerobes, fermenting sugars to produce lactic acid and various other end products. Most also reduce nitrate to nitrite. Lack cytochrome c oxidase.

13

enterics

Gut flora. They are rod-shaped gram-negative bacteria of the family Enterobacteriaceae; most occur normally or pathogenically in intestines of humans and other animals

14

Identify the group of organisms which can be differentiated by the IMViC tests

identify an organism in the coliform group

15

Understand the difference between mixed acid fermentation and butanediol fermentation. Know which portion of the MRVP reaction tests for each type of fermentation

Mixed acid fermentation is an anaerobic fermentation where the products are a complex mixture of acids, particularly lactate, acetate, succinate and formate as well as ethanol and equal amounts of H2 and CO2. It is characteristic for members of the Enterobacteriaceae family. Methyl red indicator dye will change color based on pH to test for fermentation.

Butanediol fermentation is anaerobic fermentation of glucose with 2,3-butanediol as one of the end products. It's typical for Klebsiella and Enterobacter. Acetoin reacts with reagents to turn red.

2,3-butanediol fermentation produces smaller amounts of acid than mixed acid fermentation, and butanediol, ethanol, CO2 and H2 are the end products. While equal amounts of CO2 and H2 are created during mixed acid fermentation, butanediol fermentation produces more than twice the amount of CO2 because the gases are not produced only by formate hydrogen lyase like they are in the mixed acid fermentation

16

Understand why both forms of fermentation may be tested using the same inoculated and incubated broth tube

Because both are fermenting the nutrient medium which contains 0.5% glucose. The way they ferment the medium determines the difference in the tests MR and VP.

If the microbe performs mixed acids fermentation, the medium will acquire an acidic pH. Addition of a pH indicator which changes color below pH 4 indicates presence of the extreme acidity associated with mixed acids fermentation. The reagent added to determine pH is methyl red, and so the test is called the methyl red (MR) test.

2,3-butanediol is the end product of a long fermentation pathway, but it is not easily detected. So, the test detects one of the pathway intermediates instead: acetylmethylcarbinol (acetoin). The production of 2,3-butanediol is thus indirectly detected when the pathway intermediate acetoin reacts with reagents to turn red. The test is named for its discoverers, as the Voges-Proskauer test.

17

Understand why it is necessary to repeatedly vortex the VP test over 30 minutes

to oxygenate it

18

Understand what the reversion of a reaction is and how it might affect the results of both the Phenol Red Broths and KIA (TSI) slants

TSIA is inoculated with a glucose only fermenter. As the glucose diminishes, the organisms located in the aerobic region (slant) will break down amino acids, producing NH3 and raising the pH. This process takes 18-24 hours and only occurs in the slant because of the anaerobic conditions in the butt. This is reversion. A high pH will change the color of the broth and slant to make it appear that it did not ferment. Fermentation brings down the pH and changes the color. It can give a false reading.

19

Understand the purpose of an uninoculated control when running metabolic studies

It shows the medium in an aerobic environment when uninoculated. It allows you to clearly see the fermentation in an anaerobic environment in contrast. Helps to ensure accuracy of interpretation.

20

Differentiate between the lack of sensitivity and the lack of specificity and how each might affect the interpretation of results of metabolic tests

An inability to detect small amounts of the chemical or organism in question would yield a false negative result and would be a result of inadequate sensitivity of the test.

An inability to discriminate between the chemical or organism in question and similar chemicals or organisms would yield a false positive result and would be the result of inadequate specificity of the test.

Sensitivity = True Positives / (True Positives + False Negatives)

Specificity = True Negatives / (True Negatives + False Positives)

The closer the sensitivity and specificity is to one, the more useful the test is.

21

Fermentation

a metabolic process that converts sugar to acids, gases and/or alcohol. It occurs in yeast and bacteria, but also in oxygen-starved muscle cells, as in the case of lactic acid fermentation. Fermentation is also used more broadly to refer to the bulk growth of microorganisms on a growth medium.

22

Anaerobic respiration

a form of respiration using electron acceptors other than oxygen. Although oxygen is not used as the final electron acceptor, the process still uses a respiratory electron transport chain; it is respiration without oxygen

23

aerobic respiration

a form of respiration that requires oxygen as an electron acceptor in order to generate ATP.