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Lab Title:Identification of Bacteria

Name : Rachel-Ann Suraj

ID#: 811004634

Date of Report: 2-10-2012, Tuesday

Bench no. 2

Demonstrator:Aliya

Title : Identification of Bacteria

Objectives:

To identify bacteria using biochemical / physiological methods. To carry out investigations on an unknown sample to identify the microorganism present. To discuss the differences in microorganisms with relation to their morphology. To better understand the biochemical basis of various tests used to identify bacteria. To investigate the adaptations present in microorganisms to -ferment lactose -respire anearobically, facultatively, aerobically and microaerophilically -be motile -utilize citrate

Theory: Bacteria are members of the group called the prokaryotes. Bacteria were in existence on Earth since about 3.5 billion years ago. This is a relatively long period, giving rise to the myriad of different species of bacteria. It is a very diverse group of organisms and they thrive in a number of varying conditions. Their success is due to the high ability of adaptation illustrated by these organisms. Despite being single celled organisms; they are involved in many complex reactions, they play important roles to the environment and they are also utilized in research and development of microbiological technology (Dorrestyn 1998). For instance the bacteria found in the root nodes of legumes are responsible for the conversion of inert nitrogen in the atmosphere to ammonium compounds that can be utilized by the legumes. These bacteria are nitrogen fixing bacteria and they serve as an example to show how important bacteria are to the environment. There also exist bacteria that are harmful such as certain strains of E. coli that can cause diseases in humans. Biological identification of microorganisms is based on the differences in the physiological features of the microorganism. These differences can be investigated using various biochemical reactions that can be analysed by the use of biochemical reactions in broth or agar media. Microorganisms utilize various compounds for metabolism and they may have special adaptations that enable them to function at certain conditions(Claus 1989). In this experiment five tests were underwent to determine the identity of the microorganism present in an unknown sample, U2. The microorganism was tested to determine whether it utilized lactose in respiration. The second test was used to determine if the microorganism was facultative. Facultative microorganisms can respire under both aerobic and anaerobic conditions depending on the levels of oxygen present in its environment. In addition the sample was tested for motility. This was done in a semi-solid agar slant. The utilization of citrate was another test underwent were the colour change from green to blue indicated that citrate was metabolized. Methyl red test was used to identify microorganisms that were capable of the production of stable acids by mixed acid fermentation of glucose. Figures 1, 2 and 3 illustrate how the various tests can be utilized in determining the microorganisms identity.

Figure1 : Biological Tests for Gram-Positive Bacteria

Figure 2: Biological Tests for Gram-Negative Bacteria

Figure 3: Classification of motile bacteria

The observations recorded from the tests would give the basis to classify the microorganisms. The use of the Bergeys manual is employed to determine the genus and species of the microorganism based on its morphological, cultural and physiological differences. The result for the identity of the microorganism can be verified through the use of serological methods. Serology analyses the content of a fluid sample with the use of antibody-antigen reactions. The genus and the species of bacteria can all be determined from the use of the Bergeys manual but different strains of bacteria may display variations in their morphology, physiology and culture. This is why the use of the Burgeys manual may not be sufficient in the identification of the microorganism because certain novel microorganisms may not adhere to the classification scheme presented in the Burgeys Manual.

Procedure: A tube of lactose broth that contained phenol red indicator and Durham tubes was inoculated with the unknown microorganism, U2. The tube was incubated at 35C and this was observed after a period of 24-48hours. A positive and negative control was inoculated and incubated for observation. The microorganism was tested to determine whether it was a facultative anaerobe by the following method. The unknown microorganism, U2 was heavily stab inoculated unto a tube of thioglycolate medium. The tubes was capped tightly and incubated at room temperature in the dark for 48 hours. The growth characteristic was observed and recorded. Controls containing the obligataerobe (Pseudomonas aeroginosa) and the facultative anaerobe (E.coli) were similarly made for comparison. The unknown sample was tested for motility. A tube with semi-solid motility medium was stab inoculated with an inoculating needle containing U2. This was incubated for 24-48 hours and was observed for growth pattern. A motile (E. coli) and non-motile (Enterococcus sp.) control was made and these were placed under the same conditions. The results were observed. The microorganisms ability to utilize citrate was then tested. The inoculating loop was used to streak the U2 sample on the surface of a Simmons citrate agar slant. The slant was incubated and observed after 24-48 hours. The positive control of Enterobacteraerogenes and the negative control of E.coli were placed to incubate for the 24-48 hour period and observed. Comparisons of the experiments were done. MR-VP broth was inoculated with a loopful of the unknown organism. This was incubated for 3-4 days. One half the content of the tube was transferred to a new test tube. The methyl red test was performed by the addition of 3-4 drops of methyl orange to one of the tubes. Voges-Proskauer test was performed by the addition of 0.5mL of 5% -naphthol reagent to the other tube. This was observed and periodically shaked for up to 15 minutes. Positive and negative controls were set by the lab personnel for comparisons.

Results: The unknown used was U2 Experiment Temperature Time (C) (hours) 35 24 Observation

U2

Yellow colour, 1cm3 of gas produced in Durham tube Red colour, no formation of gas in Durham tube Yellow colour, gas produced in Durham tube

Control Negative 35 (Salmonella sp) Positive 35 (E.coli)

24 24

Table 1: Observations of Tubes with Lactose Broth

Experiment U2 Control Obligate aerobe (Pseudomonas aeroginosa) Facultative anaerobe ( E.coli)

Time (hours) 24 24 24

Observation Growth throughout tube Growth of bacteria at surface of broth only Growth of bacteria at the bottom of tube

Table 2: Growth Pattern of Bacteria in Thioglycolate Medium

Experiment U2 Control Motile (E.coli) Non-motile (Enterococcus sp)

Time (hours) 24

Observation Cloudy white growth about stab inoculated region Cloudy white growth No change

24 24

Table 3: Observing Motility in Semi-Solid Medium

Experiment

Temperature Time (C) (hours) 35 24

Observation

U2

Development of a blue colour change 2.5 cm in depth from the initial green colour which remained below No colour change of green agar Colour change of agar from green to blue

Control Negative 35 (E.coli) Positive 35 (Enterobactora erogenes)

24 24

Table 4: Observation of the reaction of the Simmons citrate

Experiment U2

Test Methyl red(MR) VogesProskauer test (PV)

Observation Red colour developed Pink colour developed

MR positive

MR negative

VP VP positive negative

Control E.coli

Methyl red(MR) VogesProskauer test (PV) Methyl red(MR)

Red colour developed No colour change Red colour developed Pink colour developed No colour change No colour change

Enterobacter

VogesProskauer test (PV) Pseudomonas Methyl red(MR) VogesProskauer test (PV)

Table 5: Methyl Red Test Results

Key: Characteristic displayed by bacteria

Figure 4: Determination of microorganism in U2

Result Test Malonate Methyl red Voges-Proskauer Simmons citrate Lactose Motility V + + (V) +

(V) = more than 50% positive within 48 hours, and more than 90% positive in 3 to 7 days.

Table 6: Summary of test results for U2

U2 was determined to be Enterobacter by the miniature multi-test described in Figure 4.

Discussion: There exists a large degree of variations in the characteristics of microorganisms that are classified in the same species and genus. Bacteria are considered very diverse. They can thrive in many different conditions due to their ability to adapt to a certain extent in their environment. This has encouraged their success. The identification of microorganisms helps determine the effects and the adaptations they possess to enable them to thrive in certain environments (Claus 1989). Many bacteria strains are harmful and there are those that are useful. A comprehensive knowledge of the characteristics of different bacteria could aid in exploiting bacteria to perform useful tasks such as in biotechnology or to prevent harmful bacteria from affecting their environment. This is the reason for identification of microorganisms. It distinguishes the species and genus of bacteria present. Biochemical analysis of bacteria depends on the chemical reactions that the bacteria cell is undergoing. Bacteria, like any other cell, require energy to carry out its cell cycle and reproduce. The use of certain substrates in the presence of oxygen or without the presence of oxygen is hence a very good determination of the type of microbe present in a sample. From Table 1 it was shown that U2 utilized lactose to ferment to provide energy for its cell. Figure 5 shows the breakdown of the lactose in bacteria.

Figure 5: Metabolism of Lactose in Bacteria

http://www.google.tt/imgres?hl=e24&ved=1t:429,r:2,s:20,i:136

Carbon dioxide gas is released in the metabolism of the lactose and is collected in the Durham tube. The controls were used to show that the metabolism of the lactose was dependant on the microorganism present in the tube. The positive control and U2 both underwent a colour change from the red phenol red indicator to a yellow colour which showed the presence of lactic acid. The tube containing the stab inoculation of U2 in Thioglycolate Medium showed growth throughout the solution. This indicated that U2 was a facultative anaerobe. It was able respire in the presence and without the presence of oxygen. The Thioglycolate was placed in the dark to prevent it from reacting in the presence of light radiation to become oxidized. Pseudomonas aeroginosa was an obligate aerobe and it grew at the surface of the broth and E.coli grew at the base of the broth due to it being able to respire in the absence of oxygen. Motility was another characteristic that was tested in this experiment. U2 displayed a cloudy white colony formation around the stab inoculated region of the semi-solid medium. E.coli also showed the cloudy growth around the inoculation but the negative control, Enterococcus did not have any cloudy growth around the stab inoculation. Motility is an important adaptation to microorganisms as it allows them to move from less favourable conditions to more favourable environments (Jawetz, et al. 1989). Stimuli that encourage motility in microorganisms are light, chemicals and oxygen. The movement of the microorganisms was around the inoculation as the microorganisms moved into the agar solution for nutrients for growth. This is a characteristic of Enterobacter that move via flagella. Citrate utilization was another biochemical test that can be used in the identification of microbes. Figure 6 illustrates the reaction that occurs when bacteria utilize citrate to metabolise for the production of energy. Citrate is passed through the cell membrane of the bacteria by a membrane transporter. Inside the cell is where it is converted into oxaloacetate which is converted to pyruvate. Pyruvate could be further metabolised into formate acetate, lactate and diacetyl acetoin. The utilization of the citrate was indicated by the agars colour change from green to blue. The positive control and U2 gave positive results for

this test as described in Table 4. U2 used the Simmons agar as a source of carbon, the presence of the indicator, bromothymol blue reacted to form a blue colour when the pH of rose above 7.6, indicating that the microorganism used citrate for metabolic processes.

Figure 6: Metabolism of Citrate in Bacteria

http://www.cib.csic.es/repositorio_bd/publicacion/1773/urls_documento/chapter_3_publicado.pdf

The last test underwent was the methyl red test and the Voges-Prokeur test. Refer to Table 5 which showed that U2 was MR negative and VP positive. Methyl red test is based on the ability of the microorganism to undergo mixed acid fermentation. It is usually carried out by the group of bacteria called Enterobacteriaceae. The Voges-Proskauer test yields a pink colour when positive as was undergone by U2. This indicated the oxidation of acetylmethylcarbinol. It was determined that the microorganism in U2 utilized the butanediol for fermentation. The unknown microorganism was determined to be Enterobacter as examined in Figure 4. It is in the same family of E.coli and has many similar characteristics. The flow chart was used to pin point the organisms identity. It was motile so this was considered; the microbe was motile so this branch was descended where the option of the microorganism using citrate and lactose were considered. The microorganism was MR- so it was identified as Enterobacter. To determine the genus the Bergeys Manual was used, it was determined by careful analysis that the microorganism was of the genus Enterobacter aerogenes. It is a gramnegative, rod shaped, facultative anaerobe bacteria.

This experiment was conducted in a sterile environment to prevent microorganism contamination. Another precaution was to sterilize the equipment and apparatus before usage. The tubes were carefully labelled and placed under the respective conditions. The use of Bergeys Manual had certain limitations. The classification of microorganisms is not a tidy system. It is not possible to use it to classify the higher microorganisms. It is also limited in distinguishing the variations that exist in the micro-bacteria species. Microorganisms are capable of a large degree of diversity even within the same species so the Bergeys Manual may not be able to differentiate between these differences and the wrong classification may be done. The test may also give a false negative result which will lead to the wrong microorganism identification. The experiment results could be improved by the use of other techniques to verify the results. Serological methods, the miniature multitest systems and the use of computers with a data base programme can be used to identify the unknown microorganism present in a sample.

References:

1. Beishir L. 1991. Microbiology in Practice: A Self-Instructional Laboratory Course. 5th edition. New York. Harper Collins.

2. Christine, Dorrestyn. 1998. Clinical immunology and serology. 3rd edition. New York. R. Steins Publications.

3. Claus, G.W. 1989. Understanding Microbes. A laboratory textbook for Microbiology. New York. W.H. Freeman and Co.

4. Ergeton, Hazel. 1998. Nature Encyclopedia. Singapore. A Dorling Kindersley Book.

5. Jawetz, E., et al. 1989. Medical Microbiology. 18th edition. San Mateo. Appleton and Lange.

6. Shewood, Linda M., Woolverton and Willey. 2011. Prescotts Microbiology. 8th Edition. New York. McGraw Hill Publishers.