Microb LAB 4 ACID GAS Sarah PDF

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Summary

4. After incubation, the color of the tubes were observed. Yellow indicates acid production. The Durham tubes were observed. Partial or complete filling of the Durham tube indicates positive gas production....

Description

STB 1093: MICROBIOLOGY PRACTICAL 4

ASSAYS FOR SPECIFIC METABOLIC ACTIVITIES Group 21 (Group A)

NAME

1. NURUL MAISARAH BINTI ZULKIFLI

(63724)

2. NUR SHUADA ADZILLA BINTI AFFANDI

(63645)

3. ZAKIAH YUSRA BINTI ZULKIFLI

(63875)

4. NUR IRSHA HUMAIRA BT HADI

(63617)

LECTURER

DR. MICKY AK VINCENT

DATE

24TH APRIL 2019

4A. ASSAYS FOR SPECIFIC METABOLIC ACTIVITIES INTRODUCTION Microorganisms have several central metabolic pathways involved in the flow of energy and carbon through the cell. Knowledge of the ability of bacteria to dissimilate different certain substrates and to synthesize various product are frequently used to identify bacterial spesies. Biochemical characteristics of the bacteria are ability to utilize sources as food, the waste products that result, and what enzymes are generated. Heterotrophic microorganisms exhibit two basic metabolic strategies, fermentation and respiration. Ability of certain bacteria to utilize carbohydrate for energy source either by oxidative or fermentative metabolism used to identify their spesies. Fermentation is the term applied to the anaerobic breakdown of carbohydrates or sugars. The end products of fermentation often vary from one organism to another depending on the pathways involved. However, the end products are usually acids of various type or acids and gas. There are species of bacteria that have the ability to use citrate as the only carbon source. The capability to utilize citrate is determined by inoculating the bacteria on Simmon’s Citrate agar. The bacteria will use the enzyme citrase to degrade the citrate and also convert the ammonium phosphate to ammonia and ammonium Hydroxide. Both of these molecules will cause the agar to become alkaline and produce a blue color as they raise the pH of the slanted agar to alkaline.

OBJECTIVES 1. To examine several specific metabolic activities of microorganism that are frequently used to identify bacterial species. 2. To detect metabolic pathways that differentiates microorganisms.

A.ACID AND GAS PRODUCTION FROM CARBOHYDRATE FERMENTATION Introduction

The main goal of this experiment is to identify the formation of acid and the production of gas from carbohydrates by innoculating them with microorganisms E. coli and Enterobacter. Fermentation of carbohydrate is a metabolic process where organic molecule plays role as electron donor while its products act as electron receptor. Microorganisms may produce acidic end products like lactic acid and carbon dioxide or neither acid nor gas from a particular carbohydrate substrate. The products rely on the microorganism ability to utilize the particular substrate and the fermentation pathways that it is competent to carry out. The recognition of the acid and gas production can be achieved by using a medium that contains a pH indicator. Phenol Red broth is used as a diferential test medium which is prepared as a foundation to which carbohydrate is added.

Objective

1. To identify the ability of E. coli and Enterobacter to ferment the given carbohydrate. 2. To determine the ability of E. coli and Enterobacter to produce gaseous end products in fermentation.

Materials Tubes of sterile purple broth base with inverted Durham tubes (Alternative: tubes of sterile phenol red broth with inverted Durham tubes) Filter sterilized solutions of carbohydrates (10% wt/vol): glucose. fructose, lactose, sucrose, mannitol Pipettes

Procedures 1. I mL quantities of the specific carbohydrates was added to the purple broth or phenol red broth tubes. These media were already contained a protein source to support bacterial growth and a pH indicator. Purple broth base contained bromcresol purple, which turns from purple to yellow at acid pH; phenol red turns from red to yellow at acid pH. The tubes were labeled. 2. A set of carbohydrate-supplemented tubes were innoculated with each of the test bacteria. 3. The tubes were incubated at 37°C for 24-48 hours. 4. After incubation, the color of the tubes were observed. Yellow indicates acid production. The Durham tubes were observed. Partial or complete filling of the Durham tube indicates positive gas production. 5. Results were recorded.

Results CARBOHYDRATE FERMENTATION:

Microorganism

Carbohydrate

Acid production

Gas production

No visible

Red  Yellow

There is visible gas

changes

No visible changes

Discussion

production

Red  Pink

There is no visible gas production

The term fermentation is often used to describe the breaking down or catabolism of a carbohydrate under anaerobic conditions. Therefore, bacteria capable of fermenting a carbohydrate are usually facultative anaerobes. It should also be noted that while the terms "carbohydrate" and "sugar" are often used interchangeably, the term sugar might not indicate the true chemical composition of certain substrates such as in the case of dulcitol and mannitor (Reiner, 2013). The microorganism may produce acidic end product for example lactic acid and carbone dioxide, or neither acid nor gas from particular carbohydrate substrate. The product rely on the ability of the microorganism is skilled to carry out. Identification of the production of acid and gas can be carried out by using a medium.

Questions

1. Your microorganism grows in the tube with glucose as shown by an increase in turbidity but the tube does not turn yellow. Why? It was because the microorganism did not produce acidic end products that could decrease the pH of the broth, hence the color of pH indicator does not turn yellow.

2. What gas other than carbon dioxide might be produced and collected in the Durham tube? Other than carbon dioxide, nitrous oxide, nitrogen gas, hydrogen sulfide or methane might be produced and collected in the Durham tube.

3. How could you detect acid and gas production using a solid medium? Place the pH indicator into the solid medium to detect the acid production. A crack on the medium indicate if there is gas production by the microorganism.

B. VOGES-PROSKAUER TEST

Introduction Reaction of voger paskeur was developed by Danial Wilhelm Otto Voges and Bernhard Proskauer, German bacteriologists in 1898 at the Institute for Infectious Diseases. In1898, Voges and Paskeur characterized the fermentation of sugars by various bacterial isolates (Voges and Proskauer. 1898). They showed that the gas produced during fermentation was a mix of CO 2 and H 2 and that by adding KOH to cultures that had grown in glucose peptone media for prolonged incubation in the presence of oxygen, some organisms produced a red fluorescent color. Although the nature of the coloration was not clear at the time, this method was suggested for differentiation between bacterial isolates that produced the color and those that did not. The purpose of this analysis is originally were used to distinguish between members of the family Enterobacteriaceae, but now they are used to characterize other groups of bacteria including Actinobacteria (Schumann, Behrendt, Ulrich and Suzuki. 2003). Voges-Proskauer tests are part of a battery of biochemical tests known as IMViC used in the clinical lab. Material and methods Sterile tubes of MR-VP medium Barritt's reagent A (a-naphthol) Barritt's reagent B (40% KOH) Pipettes

Procedures 1. Tubes of MR-VP broth were inoculated with each of the test cultures. Label all tubes. 2. The inoculated tubes were incubated at 37°C for 24-48 hours. 3. After incubation, aseptically pipette 1 mL of culture asepticated to a clean test tube. To the 1 mL of culture, add 0.5 mL of Barritt's reagent A and 0.5 mL of Barritt's reagent B. Shake vigorously for 30 seconds. 4. The tubes were observed for the gradual formation of a pink to red color (positive test for acetoin). Some cultures react within 1 minute, but others may take 1-hourss. Results were recorded. Results

Microorganism Microorganism a

Reaction The colour was not changing. It is negative

Microorganism b

reaction. The colour is changing fromyellow to red. It is positive reaction.

VP tube a- negative proskeur

VP tube for microorganism b- positive proskeur

Discussion

Voges–Proskauer or VP is a test used to detect acetoin in a bacterial broth culture. The VP tube A has negative proskeur while VP tube B has positive proskeur.

The test is

performed by adding alphanaphthol and potassium hydroxide to the Voges-Proskauer broth which has been inoculated with bacteria. A cherry red color indicates a positive result for test tube B , while a yellow-brown color indicates a negative result test tube A. Organisms such as members of the Enterobacter group produce acetoin as the chief end product of glucose metabolism and form smaller quantities of mixed acids. This test depends on the digestion of glucose to acetylmethylcarbinol. When glucose is being broken down, it will react with alpha-naphthol (VP reagent 1) and potassium hydroxide (VP reagent 2) to form a red color (Hemraj, Dipta & Gupta, 2013). Alphanaphthol and potassium hydroxide are chemicals that detect acetoin. The red complex form in the test tube B is because of the presence of the atmospheric pressure and 40% of potassium hydroxide, acetoin converted to diacetyl(Voges Proskauer (VP) Test: Principle, Procedure and Results). Questions

1. If the Voges –Proskeur test is run too early or too late, it will be negative even if the microorganism is carrying the butanediol fermentation pathway. Why ? The test depends on the digestion of glucose to acetylmethylcarbinol. If glucose is being broken down, it will react with alpha-naphthol (VP reagent 1) and potassium hydroxide (VP reagent 2) to form a red color. Alpha-naphthol and potassium hydroxide are chemicals that detect acetoin. The VP test detects organisms that utilize the butylene glycol pathway and produce acetoin. When the VP reagents are added to MR-VP broth that has been inoculated with an organism that uses the butylene glycol pathway, the acetoin end product is oxidized in the presence of potassium hydroxide (KOH) to diacetyl. Creatine is also present in the reagent as a catalyst. Diacetyl then reacts to produce a red color. Therefore, red is a positive result. If, after the reagents have been added, a copper color is present, the result is negative. The reagents used for the VP test are Barritt's A (alphanapthol) and Barritt's B (potassium hydroxide). When these reagents are added to a broth in which acetyl methyl carbinol is present, they turn a pink-burgundy color (a positive VP test). This color may take 20 to 30 minutes to develop. E. coli does not produce acetyl methyl carbinol, but Enterobacter and Klebsiella do. The MR and VP tests are particularly useful in the identification of the Enterobacteriaceae. As for the V.P, often that needs to go for longer than 2 days typical protocols may specify 5 days. Also, be sure to put parafilm over the tube and shake it vigorously several times over several minutes, before deciding it's negative. The reaction needs oxygen. 2. Why is the evoges proskeur is important in microbial taxanomy? Because, the test is to identify an organism. The Voges-Proskauer test determines the capability of some organisms to produce non-acidic or neutral end products, such as acetyl methyl carbinol, from the organic acids that result from glucose metabolism. The reagent used in this test is Barritt's reagent, consists of a mixture of alcoholic a-naphthol and 40% potassium hydroxide solution. Detection of acetyl methyl carbinol requires this end product to be oxidized to a diacety compound. This reaction will proceed in the presence of the anaphthol catalyst and a guanidine group that is present in the peptone of the MR-VP medium. As a result, a red complex is formed, imparting a rose color to the medium. Development of a deep rose color in the culture 1 minutes following the addition of Barritt's reagent is indicative of the presence of acetyl methyl carbinol and represents a positive result. The absence of rose color is a negative result.

C. METHYL RED TEST

Introduction Methyl red test is used to identify bacteria that carried out mixed acid fermentation of glucose such as Escherichia coli. The mixed acid fermentation pathway produces a variety of end products, including ethanol, acetate, formate, molecular hydrogen, and carbon dioxide. The proportions of the products vary depending on the bacterial species. Assuming that this is the primary fermentation pathway of a bacterial species, sufficient acidic products accumulate to reduce the pH drastically. The accumulation of lactic, acetic, succinic, and formic acids readily drops the pH below 5.0, even when the broth is slightly buffered with peptone and potassium phosphate. At pH values less than 5.0 methyl red reagent is red in color, and this forms the basis of the methyl red test.

Materials

Sterile tubes of MR-VP medium Methyl red solution

Procedures 1. To perform the methyl red test, the remaining broth from the cultures grown for the Voges-Proskauer test had been used. 2. A few drops of methyl red solution were added to the cultures. An immediate red reaction signifies a mixed acid fermentation; a yellow reaction signifies a negative test. The results were recorded.

Results

Microorganism

Reaction

Microorganisms A

The colour was changing from yellow to red. It is positive reaction. The test shown there was formation of acid fermentation..

Microorganisms B

The colour was remain in yellow colour. It is negative reaction. No formation of acid fermentation.

Questions

1.

Can a microorganism carry out both the butanediol and mixed acid fermentations? - Yes. Some microorganisms can carry out both the butanediol and mixed acid fermentations

2.

Can a microorganism be both methyl red and Voges-Proskauer positive? Explain. -Some bacteria cannot be both both methyl red and Voges-Proskauer positive. Bacteria which tested negative in methyl red test, were positive in the Voges-Proskauer reation. This observation established the general observed correlation between the production of hydrogen ions and acetoin. Organisms that ferment glucose producing high levels of hydrogen ions do not produce acetoin as a fermentation intermediate.

Discussion

Methyl red is widely used as pH indicator. As methyl red is a weak acid, a small amount is needed so that it not affect much on the original pH (Christian, 1986). In this experiment, microorganisms A (positive result), is a low-ratio microorganisms (Clark and Lubs, 1915 ). It ferment sugars by the mixed acid pathway resulting in a low ratio of carbon dioxide to hydrogen gas produced by fermentation. The end product produce by mixed acid pathway were acidic products (lactic and acetic acid), neutral fermentation product (ethanol), carbon dioxide, hydrogen of glucose fermented. The large quantity of acids produced causes a significant decrease in the pH of the culture medium. In contrast, microorganisms B (negative result), is a high-ratio microorganisms, those that produce a high ratio of carbon dioxide to hydrogen from the fermentation of glucose. It ferment sugars via the butanediol fermentation pathway, producing only 1 mol of acid per mol of glucose.

The pH indicator of methyl red has been found to be suitable to measure the concentration of hydrogen ions between pH 4.4 (red) and 6.0 (yellow). To prove a microorganisms can carried out a mixed acid fermentation, sufficient acids should be produced to reduce the pH to below 5.0 to allow methyl red test shows a positive reaction. In contrast, a microorganisms that carry put butanediol fermentation would cannot change the colour of methyl red. It will remain in yellow as no or small amount of acidic end products are produced.

In our experiment result, we determined that microorganisms A is Escherichia coli as it able to carried out mixed acid fermentation. Escherichia coli changed the colour from yellow to red. While microorganisms B is Enterobacter aerogenes as it cannot carried out mixed acid fermentation. Colour of Enterobacter aerogenes was remained in yellow.

D. CITRATE UTILIZATION Introduction Citrate utilization test is commonly employed as part of a group of tests, the IMViC (Indole, Methyl Red, VP and Citrate) tests, that distinguish between members of the Enterobacteriaceae family based on their metabolic by-products (Aryal & Jennifer, 2018). Citrate utilization can be used to distinguish between coliforms such as Enterobacter aerogenes (+ve) which occur naturally in the soil and in aquatic environments and fecal coliforms such as Escherichia coli (-ve) whose presence would be indicative of fecal contamination. The purpose of citrate utlilization test is used to determine the ability of bacteria to utilize sodium citrate as its only carbon source and inorganic (NH4H2PO4) is the sole fixed nitrogen source. When an organic acid such as citrate is used as a carbon and energy source, alkaline carbonates and bicarbonates are produced ultimately. In addition, ammonium hydroxide is produced when the ammonium salts in the medium are used as the sole nitrogen source. Utilization of exogenous citrate requires the presence of citrate transport proteins (permeases). Upon uptake by the cell, citrate is cleaved by citrate lyase to oxaloacetate and acetate. The oxaloacetate is then metabolized to pyruvate and CO 2.Citrate utilization test is often part of a battery of tests used to identify gram-negative pathogens of Enterobacteriaceae family and environmental isolates (Microbeonline, 2013). Material Slants of Simmon's citrate agar Procedures 1. Agar slants of Simmon's citrate medium was ioculated with each of the test microorganisms. 2. The tubes was incubateat 37°C for 24-48 hours. 3. The color of the tubes was observed after incubation. A royal blue color is a positive test; a green color is a negative test for citrate utilization.The results was recorded.

Results Microorganism E. coli

Reaction There is no growth visible. No colour change will occur. The medium will remain the deep forest green color of the uninoculated agar. Only bacteria that can utilize citrate as the sole carbon and energy source will be able to grow on the Simmons citrate medium, thus a citrate-negative test culture will be virtually indistinguishable from an uninoculated slant. It is negative reaction.

Enterobacter

The growth is visible on the slant surface and the medium will be an intense Prussian blue. The alkaline carbonates and bicarbonates produced as by-products of citrate catabolism raise the pH of the medium to above 7.6, causing the bromothymol blue to change from the original green color to blue. Thus, the colour is changing.It is a positive result.

Bottle A- negative reaction

Bottle B- positive reaction

Discussion Citrate utilization is a test to determine the ability of bacteria to utilize sodium citrate as its only carbon source and inorganic (NH4H2PO4) is the sole fixed nitrogen source. The citrate utilization labelled A is a negative reaction while citrate utilization labelled B has positive reaction. The butt remains green because some organisms are capable of growth on citrate and do not produce a colour change. Growth is considered a positive citrate utilization test, even in the absence of a colour change. The test is performed by inoculate Simmons citrate agar lightly on the slant by touching the tip of a needle to a colony that is 18 to 24 hours old. Then it is incubated at 35 oC to 37oC for 18 to 24 hours. The alkaline carbonates and...