MICRO 351 FALL 2016

Discovering the Identity of Unknown #13

Jordan Holst

Lab Section 1003

Alex Karbachev

Unknown #13
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Abstract

This course was designed to prepare and educate the student with valuable skills

regarding microbiology in the laboratory. The instruction in the lab prepared the student to

identify unknown bacteria. Methodology of the environment needed for a bacterium to grow,

how to transfer a bacterium without contamination, isolating colonies, and further tests to

discriminate a bacterium were understood. The student was required to utilize the methods

learned to show competency in identifying an unknown bacteria given to the student early in the

semester. This student was given a broth containing two unknown bacteria labeled ‘number

thirteen’. It is this student’s goal to test, document, and identify one of these organisms via the

methods learned from this class. Hopefully, the secret list of microbes given to students, hidden

under lock and key, will confirm the suspicions of this student.
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Section I – Introduction and Definition of a Bacterial Species

During the early weeks of instruction, the student was educated on the techniques used in

the lab for proper research to be conducted. Among these were the following; aseptic technique,

microscopic examination, types of growth media, the need for positive and negative controls,

understanding the bacterial species and biological species concept, DNA-DNA hybridization,

horizontal gene transfer and finally 16S rRNA sequence data. Using these techniques the student

will then identify one of two unknown bacteria that will be given to the student in a liquid broth

culture. This report details these techniques and how they were used in identifying an unknown

bacterium.

The first topic discussed in the lab was in regard to the aseptic technique. This method

utilizes great care in controlling contamination. The technique is very useful when transferring

microbes from one container to another and ensuring that the possibility of contamination is

reduced, if not eliminated. The method utilizes extreme heat from a microinsinerator to sterilize a

tool that is used for transfer of microbes. The use of gloves and careful movements ensure

nothing is touched that is not meant to be. Gentle flaming, or passing in front of the incinerator,

of each container after the cap or lid is removed and replaced also helps in preventing

contamination. The aseptic technique is used in all aspects of the lab environment. It is an

important part of streaking a plate with bacteria to grow isolated colonies.

The streak plate method of isolation relies heavily on the aseptic technique. When an

unknown bacterium is to be identified, first one must isolate the bacterium. One way of doing

this is by streaking a sample of a culture on a streak plate that contains a mixture of nutrients.

The streak plate is divided into four quadrants. The method of streaking across the plate is done
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in such a way to create a dilution gradient. This is accomplished by dragging the first streak

across the plate in the first quadrant, followed by flaming the tool and then streaking again from

the first quadrant into the second. The tool is flamed in the microinsinerator before each streak is

made from one quadrant to the next. If the aseptic technique was used and care was taken to

streak the plate, then after an incubation period, isolated growths of bacteria will be present.

Isolating the bacteria is essential when attempting to identify an unknown culture. The

growths that were isolated on the streak plate are called colonies. A colony is a group of identical

bacteria that have replicated over and over again until they are visible to the eye. Each family of

bacteria colonizes in a different way. This is referred to as its morphology. The appearance of a

colony can be used in rough predictions as to what type of bacteria is being observed. A colony

could be described by its;

- Shape – round, irregular, or punctiform (tiny, pinpoint)

- Margin – entire (smooth), undulate (wavy), lobate (lobed), filamentous, or rhizoid

(branched or root like)

- Elevation – flat, raised, convex, pulvinate (very convex), umbonate (raised in the

center), plateau

- Texture – moist, mucoid, or dry

- Pigment production – opaque, translucent, shiny, or dull

- Color

- Once a colony has been isolated it can be observed microscopically.

The next topic mastered by the student was microscopic examination. In the lab the

student became familiar with the ‘light’ or ‘bright field microscope’. It is so named because the
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light source comes from beneath the specimen being observed. The total magnification of this

microscope is one thousand times that of the original image. This magnification is compounded

by the 10x magnification of the ocular and the 100x of the objective used. The limitation of this

microscope is in the resolving power of the lens and light being used; the best image resolved

being that of 0.2μm (micrometers). For the student to view the bacteria that was isolated, it must

be transferred to a glass slide that will then be placed on a mechanical stage on the microscope.

The stage is above a light that shines through the specimen making it visible through the ocular.

Being able to isolate a colony is necessary for more than just microscopic examination. It

is also necessary for cultivating a pure sample from which other studies can be performed. A

pure culture contains only one type of microbe. To further study an unknown bacterium, one

could see if it will grow in different environments. This can be done with different growth media.

A growth medium contains nutrients for a bacterium to use in its growth and agar; a semi-solid

gelatin substance. Selective and differential media can be used to study a bacterium.

Selective media is used to identify whether or not the bacterium in question is capable of

surviving under certain conditions. Using growth media that have specific nutrient or salt

contents is a useful way of identifying a bacterium. If the bacterium grows in one medium and

not the other, information about the bacterium is known. This information is useful in identifying

it but more study is still needed. Differential media is another aid in the identification process.

A differential medium contains chemicals or dyes to see how an organism reacts during

growth. The characteristics of growth can be used in identification or differentiation of the

bacterium. The medium is normally used to differentiate closely related bacteria. Types of

differential media discussed in this lab are Mac Conkey agar and Mannitol salt agar.
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Identifying an unknown bacterium is the goal of this lab. However, in order to do that

several different exercises need to be performed. To determine if the unknown is growing under

the right conditions each time, a positive and negative control must be used in tandem for

relevant data to be collected. The use of a positive and negative control confirms that unknown

bacterium is growing or not growing on the intended media. This is an essential part of

performing a well thought out experiment. Without the use of controls, the outcome of the

experiment could not be explained with confidence. A positive and negative control yield

expected results. Much information has been gathered at this point regarding the unknown

bacteria but stating the identity of this bacterium still cannot be done. This is because of several

factors.

One such factor is the classification of species. This is an ongoing argument in the

scientific community as new technology is introduced and more information is gathered.

Methods of classification range from biological to bacterial, phenotype to genotype, horizontal

gene transfer, to 16S rRNA and DNA-DNA hybridization.

The ability to breed within a group, but not outside of it, falls under the definition of the

biological species concept. This is what Ernst Mayr was referring to when he said “the words

‘reproductively isolated’ are the key words of the biological species definition” (1). However,

this is a limited definition in classifying species as it does not account for other methods of

reproduction or the uptake of genetic information that could better identify a species. The

biological species concept differs from that of the bacterial species concept.

The bacterial species concept relies on three methods of identification; genotype,

phenotype, and phylogeny. Genotype refers to the bacterial genome. Phenotype is more complex
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in that it takes into account the morphology and physiology as well as the chemical makeup of

the cell. Phylogeny is where genotype and phenotype find middle ground and complement each

other (2). This complement accounts for DNA-DNA hybridization, horizontal gene transfer and

significant commonality amongst a 16S rRNA sequence (2). It is because of these specific

methods of identification that the biological species concept is not sufficient to classify bacteria.

To be more specific and concise, the advancement of technology has allowed for the

scientific community to better classify species. The discovery of deoxyribonucleic acid (DNA),

and its subsequent and ongoing study, has led scientists to an understanding of how traits are

passed down from one species to another. DNA contains information that is responsible for

characteristic traits in a species. In order to use DNA to compare and identify a species, a

technique is used to measure the commonality among strains. This technique is referred to as

DNA-DNA hybridization.

The methodology of this technique labels the DNA of one species and assays it against

that of another. This is done by heating the DNA so that it splits in two, then a cooling process

allows the DNA to come together again. The sequences that come together are called hybrids

because they are no longer the same. Through the use of labeling, one could see the amount of

hybridization and measure it. Similarity between the two organisms is the key to defining the

species. “A 70% or less genomic hybridization … between the two organisms is … evidence that

the two are a distinct species” (2). This method alone though is still not enough for classification

of species by today’s standards. Horizontal gene transfer (HGT) is a relatively new explanation

of how genes can move from one species to another without traditional reproduction among

bacteria (3).
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Through this unconventional transfer, bacteria from other species can exchange

information by means of; transformation, transduction, and conjugation. Transformation is the

uptake of free DNA from one bacterium to another (2). Transduction is a method of DNA being

transferred by a courier virus between bacteria (2). Conjugation requires contact between the two

bacteria and a conjugate plasmid in the donor cell (2). Through these mechanisms bacteria are

able to acquire new genes that give them new abilities and can muddy the water in identifying a

species.

The significant piece of the puzzle in identifying a species lies in the 16S rRNA sequence

of the bacteria. 16S rRNA (r referring to ribosomal) is a piece of the 30S subunit in prokaryotes.

It is a sequence that is slow to change and thus helps in identifying a single recent common

ancestor (2). This sequence is compared to other bacteria and if it is 97% identical to another,

then they are considered to be of the same species (2).

In searching for the identity of the unknown assigned, research was done on the twenty

eight possible genera the unknown could be. Several databases were exasperated to know

beforehand what positive and negative results were expected from each test performed. After

acquiring all the relevant information on each genus, a plan of attack was made to identify the

unknown. In order to identify the unknown, the following tests were performed; streak plate

method of isolation, gram stain, microscopic examination and cell morphology, an oxidase test, a

catalase test, a test for indole production, urea hydrolysis, and citrate permease. These tests gave

definitive results in identifying the unknown as Klebsiella oxytoca which was confirmed by 16S

rRNA in a BLASTn search using a database hosted by the NCBI.

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Section II- Material and Methods

- Streak Plate Method of Isolation

The test tube containing Unknown 13 was delivered in a test tube containing two separate

bacteria. In order to identify one of these, the bacteria needed to be isolated and plated to

produce a pure sample of only one bacterium. In order to isolate the colonies, the bacterial broth

had to be transferred to a complex medium (Tryptic Soy Agar) using the streak plate method

mentioned earlier and incubated for 24 hours at 30-37° C. After incubation, only one bacterial

species was growing in the medium. It is possible that the nutrients were used by the dominant

bacteria and that the other bacterium lost the battle for the available resources. It is also possible

that there was only one bacterium to begin with. The colony morphology was round, smooth,

entire, convex, mucoid, moist, shiny and white.

Early in the lab, this experiment had been performed using Escherichia coli,

Staphylococcus aureus, Bacillus subtilis, Staphylococcus epidermidis, and Enterococcus

faecalis. E. coli had morphology of irregular, raised, mucoid, and opaque. S. aureus had

morphology of round, smooth, entire, raised, convex, mucoid, shiny. B. subtilis had morphology

of irregular, filamentous, flat raised margin, mucoid, and dull. S epidermidis had morphology of

round, smooth, entire, convex, dry, and dull. E. faecalis had morphology of round, smooth,

convex, moist, shiny and opaque. Of these E. faecalis looked to be similar to the unknown that

was isolated.

- Microscopic examination

After isolating a pure sample of the unknown through the streak plate method of

isolation, examination under a microscope was necessary. Before proceeding with this the
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bacteria was transferred to a slide as an emulsion. The slide was allowed to dry and was heat

fixed. Heat fixing ensures that the bacteria are dead and aids in the staining process. The

emulsion was stained using the Grams method. Using the bright-field compound light

microscope the bacterium was observed under oil immersion at 1000x magnification. The

bacteria appeared pink and were rod shaped.

Early in the lab the student performed the gram stain on two known bacteria; S.

epidermidis and E. coli. The difference between gram positive and gram negative bacteria is the

structure of the cell wall. In gram positive bacteria a thick peptidoglycan layer traps the primary

stain with the aid of a mordant (crystal violet and iodine) preventing it from being decolorized by

alcohol. Gram negative bacteria have higher lipid content and its peptidoglycan layer is thinner

than that of gram positive. After the crystal violet-iodine complex is washed with alcohol, it is

decolorized for its counterstain; safranin. Safranin colors the bacteria pink (4).

Observation at 1000x magnification under oil immersion with a microscope showed S.

epidermidis to be purple (gram positive) and round (cocci). Under the same viewing conditions

E. coli showed pink (gram negative) rods (bacilli). By comparison of these results the unknown

bacterium was gram negative and bacillus. The unknown bacterium was very similar to that of E.

coli under the microscope.

- Oxidase test

This test reveals whether a bacterium produces the enzyme cytochrome c oxidase. It

differentiates between Enterobacteriaceae (oxidase negative) and Pseudomonadaceae (oxidase

positive) (4). This test uses a commercially available cotton swab which contains tetramethyl-p-

phenylenediamine (a chromogenic reducing agent) (4). Oxidation of the reducing agent is

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indicated by a color change from white to blue/purple. An oxidized swab is indicative of a

positive result when it turns purple, while a negative result lacks a color change (4). The controls

used in this lab were A. faecalis (positive control) and E. coli (negative control). The results

were negative; there was no color change on the oxiswab indicating the absence of cytochrome c

oxidase.

- Catalase test

In an oxygen rich environment there are toxic byproducts such as superoxide, hydrogen

peroxide and hydroxide. This test is to identify whether or not the bacterium has the ability to

combat the effects of an oxygen rich environment. The enzymes superoxide dismutase, catalase,

and peroxidase aid in a bacterium’s ability to stave off the negative effects of oxygen reduction.

A positive test results in bubbles being present after the addition of hydrogen peroxide. In the lab

the positive control was Micrococcus luteus and the negative control was Enterococcus faecalis.

The result of this test on the unknown was positive.

- Indole test

Indole production happens when the amino acid tryptophan is hydrolyzed by the enzyme

tryptophanase. Tryptophan is in the broth (TSB) that contains the unknown. A test using Kovac’s

reagent detects the presence of indole through the production of rosindole dye (a pink color). Six

drops of Kovac’s reagent is added to the test tube and the reaction is occurs quickly (4). The

controls used in the test were E. coli (positive) and Enterobacter aerogenes (negative). The result

of this test on the unknown was positive.

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- Urea hydrolysis

This test is for the presence of the enzyme urease found in some bacteria, notably Proteus

(4). The hydrolysis of urea by the enzyme urease produces carbon dioxide and ammonia; this

will increase the pH (4). Phenol red, a pH indicator, and yeast, a nutrient source, are included in

the urea broth. As the pH increases above 8.4, the urea will change color to pink. This color

change is a positive result for urease (4).

The urea broth is to be inoculated with the bacterium utilizing the aseptic technique to

prevent contamination. After inoculation of the broth, an incubation period of 24 hours at 37° C

ensues. Observation of the broth after incubation will either show a color change or not. In the

lab the control bacteria were E. coli (a negative control) and E. aerogenes (a positive control).

The unknown bacterium had a negative result for urease.

- Citrate test

The production of citrate-permease is being tested for. A defined medium, Simmons

Citrate Agar, is poured into a test tube that is placed in a slant rack for the medium to dry. The

medium contains sodium citrate as the only carbon source, ammonium phosphate and a pH

indicator, Bromthymol blue dye. A bacterium is transferred to this medium and incubated for 48

hours at 37° C. If the bacteria are able to grow in this environment a color change from green to

blue will be observed. The color change indicates citrate-permease is present. The positive

control for this experiment is E. aerogenes showing a growth in the medium and a color change

to blue. The negative control is E. coli showing no growth and no color change (4). The

unknown bacterium grew on this medium and a color change to blue was observed, this is a

positive result for citrate permease.

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- 16S rRNA Sequence Analysis

During the course of this lab a tool was revealed to the students that references a

computer database of nucleotide sequences of organisms. This tool is found on the National

Center for Biotechnology Information’s (NCBI) website and is called BLAST. BLAST stands

for the Basic Local Alignment Search Tool and it compares sequences of information between

nucleotides or proteins. This program takes the comparison and calculates an Expect value (E

value), which is the statistical biological significance.

Using this tool the student is going to run a BLASTn search, n standing for nucleotide,

and record the top three results. The 16S rRNA sequence received was from group A2 as

follows: atgtctgtgc agttaatcga ctgggatctg gccctgatcc agaaatataa ctattccggg ccgcgttata cttcgtatcc

caccgcgctg gagttttctc cagaatttgg ccccgctgaa tttgatgcgg cggttgcccg ctatccgcag cgtcccctct

cgctgtacgt gcatatcccg ttttgccata. After running the search the top three results for unknown #13

were Klebsiella oxytoca strain JKo3 (Ident: 100%, E value: 2 x 10-93), Klebsiella oxytoca

CAV1374 (Ident: 99%, E value: 1 x 10-91), and Klebsiella sp.LTGPAF-6F (Ident: 99%, E value:

5 x 10-90). The low E value and high identity value signify biological significance.

The tests performed in the lab are congruent with these findings. Cell morphology of the

demo of K. oxytoca was similar to that of the unknown bacterium. Comparing the results from all

of the tests performed on the unknown yield the same results for K. oxytoca which is gram

negative, rod shaped or bacillus in shape as viewed under the microscope, negative for oxidase,

positive for catalase, positive for indole production, negative for urease, and positive for citrate.
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Section III- Results and Discussion

Unknown 13
Gram (+) Gram (-)
Kurthia, Propionibacterium,
Staphylococcus, Bacillus,
Streptococcus, Enterecoccus,
Sporosarcina, Micrococcus, Coccus Bacillus Spirillium
Arthrobacter, Lysinibacillus, Neisseria,
Lactobacillus, Lactococcus, Veilonella
Listeria, Planococcus

Oxidase (-) Oxidase (+)

Pseudomonas,
Alcaligenes,
Flavobacterium
Catalase (-)
Catalase (+)
N/A

Indole (-) Indole (+)

Enterobacter, Serratia,
Salmonella, Shigella
Urea (-) Urea(+)
Proteus

Citrate (-)
Citrate (+) Escherichia

Klebsiella
Citrobacter Erwinia
oxytoca

16S rRNA sequence (BLAST)

As seen above in the flow chart, each positive and negative result allowed for the

elimination of all the possible bacteria that could be the unknown. These tests were chosen to

identify morphology, biochemical and physiologic properties of the unknown. Each test was

significant in eliminating a possible unknown. It can be seen that the citrate negative result left

three possible bacteria to identify. The bacteria Citrobacter and Erwinia had very little
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information to research which is why they were never eliminated along the way. These bacteria

stayed in the possible identity category until the BLASTn search was performed.

With the completion of the chosen test and the research performed, the likely candidate

for unknown #77 was Klebsiella and following the BLASTn search this was confirmed. The

species was Klebsiella oxytoca with 100% identity and an E value of 2x10-93.

Section IV – Conclusion

Unknown #13 is Klebsiella oxytoca of the genus Klebsiella a member of the family

Enterobacteriaceae and of the species oxytoca. K. oxytoca is an Indole producing (5)(6)(7)(8),

gram negative bacillus (5)(7)(9)(10), and class A extended-spectrum β-lactamase–

producing bacterium (6)(10).

Confusion has plagued the nomenclature and classification of Klebsiella for many years

but as classification practices have improved, so has the identification of this bacterium

(5)(7)(8)(11). K. oxytoca was originally “described “Bacillus oxytocus perniciosus” from old

milk” (5), by Flugge in 1886. Oxytocus comes from oxus- sour milk and –tokos – producing. It

was later named “Aerobacter oxytocum” in 1923 followed by Klebsiella oxytoca in 1956 (5).

K. oxytoca is commonly found the healthcare industry acquired from environmental

surroundings (7)(8)(9)(10)(11). There have been documented outbreaks of this pathogen in

Toronto Canada (10) and Taipei, Taiwan (6). K. oxytoca is primarily involved in nosocomial

infections (7)(8)(9)(10)(11) that are becoming more frequent (8)(9)(10). K. oxytoca can be

difficult to treat because of its ability to produce β-lactamase which prevents it from being

treated by some antibiotics (6).

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However, it is frequently found in livestock where it is associated with sepsis, urinary tract and

respiratory infections and mastitis (11). This is a financial burden for livestock farmers as some

cattle do not survive infection. (11)

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Section V – Unknown Work Sheets

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