micro b lab 3- identification of microorganisms

7/31/2019 micro b lab 3- identification of microorganisms

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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


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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 identifybacteria.

To investigate the adaptations present in microorganisms to-ferment lactose

-respire anearobically, facultatively, aerobically and microaerophilically

-be motile-utilize citrate


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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 canbe 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 ofE. 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 agarslant. 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.


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Figure1 : Biological Tests for Gram-Positive Bacteria

Figure 2: Biological Tests for Gram-Negative Bacteria


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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.


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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 negativecontrol 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 loopwas used to streak the U2 sample on the surface of a Simmons citrate agar slant.

The slant was incubated and observedafter 24-48 hours. The positive control of

Enterobacteraerogenes and the negative control ofE.coliwere 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.


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Results:

The unknown used was U2

Experiment Temperature

(C)

Time

(hours)

Observation

U2 35 24 Yellow colour, 1cm3

of gas produced in

Durham tube

Control

Negative

(Salmonella sp)

35 24 Red colour, no formation of gas in Durham

tube

Positive

(E.coli)

35 24 Yellow colour, gas produced in Durham

tube

Table 1: Observations of Tubes with Lactose Broth

Experiment Time

(hours)

Observation

U2 24 Growth throughout tube

Control

Obligate aerobe

(Pseudomonas aeroginosa)

24 Growth of bacteria at surface of broth only

Facultative anaerobe

( E.coli)

24 Growth of bacteria at the bottom of tube

Table 2: Growth Pattern of Bacteria in Thioglycolate Medium


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Experiment Time

(hours)

Observation

U2 24 Cloudy white growth about stab inoculatedregion

Control

Motile (E.coli) 24 Cloudy white growth

Non-motile (Enterococcus

sp)

24 No change

Table 3: Observing Motility in Semi-Solid Medium

Experiment Temperature

(C)

Time

(hours)

Observation

U2 35 24 Development of a blue colour change 2.5 cm

in depth from the initial green colour which

remained below

Control

Negative

(E.coli)

35 24 No colour change of green agar

Positive

(Enterobactora

erogenes)

35 24 Colour change of agar from green to blue

Table 4: Observation of the reaction of the Simmons citrate


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Experiment Test Observation MR

positive

MR

negative

VP

positive

VP

negative

U2 Methyl

red(MR)

Red colour

developed

Voges-

Proskauer

test (PV)

Pink colour

developed

Control

E.coli Methyl

red(MR)

Red colour

developed

Voges-

Proskauer

test (PV)

No colour

change

Enterobacter Methyl

red(MR)

Red colour

developed

Voges-

Proskauer

test (PV)

Pink colour

developed

Pseudomonas Methyl

red(MR)

No colour

change

Voges-

Proskauer

test (PV)

No colour

change

Table 5: Methyl Red Test Results


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Figure 4: Determination of microorganism in U2

Key:

Characteristic displayed by bacteria


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Test

Result

Malonate V

Methyl red -

Voges-Proskauer +

Simmons citrate +

Lactose (V)

Motility +

(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 Enterobacterby the miniature multi-test described in

Figure 4.


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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 adaptto 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

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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.coligrew 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.colialso 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 encouragemotility 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 ofEnterobacter

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


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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 Enterobacteras examined in

Figure 4. It is in the same family ofE.coliand 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. Themicroorganism 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 gram-

negative, rod shaped, facultative anaerobe bacteria.
http://www.cib.csic.es/repositorio_bd/publicacion/1773/urls_documento/chapter_3_publicado.pdfhttp://www.cib.csic.es/repositorio_bd/publicacion/1773/urls_documento/chapter_3_publicado.pdfhttp://en.wikipedia.org/wiki/Enterobacteriaceaehttp://en.wikipedia.org/wiki/Enterobacteriaceaehttp://en.wikipedia.org/wiki/Enterobacteriaceaehttp://www.cib.csic.es/repositorio_bd/publicacion/1773/urls_documento/chapter_3_publicado.pdf


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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.


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References:

1. Beishir L. 1991. Microbiology in Practice: A Self-Instructional LaboratoryCourse. 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 forMicrobiology. New York. W.H. Freeman and Co.

4. Ergeton, Hazel. 1998. Nature Encyclopedia. Singapore. A Dorling KindersleyBook.

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.8

thEdition. New York. McGraw Hill Publishers.

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micro b lab 3- identification of microorganisms