Donny Techiera
Sunday, April 17th 2016
Leila K.
Microbiology 2300 lab
Spring 2016
Identification of unknown Number 9
Georgia State University
Introduction
Since the beginning of time, unknown bacteria has played a key role in society. Certain bacteria undergo various biochemical tests in order to determine the metabolic differences between them. Each biochemical test targets a specific biological pathway beginning with a positive-negative reaction. For example, a catalase test determines the presence of catalase, an enzyme that catalyses release oxygen from hydrogen peroxide (H2O2). It is important to be able to identify the types of bacteria using items such as dichotomous keys. Dichotomous keys are paired statements in a flowchart order, each statement pairs together to identify several different microorganisms. A dichotomous key is constructed using characteristics of known organisms, the organisms allow one to choose which pathway to follow, and then lead to a discovery of the collected specimen.
Chemical testing also enables one to learn specific details of a bacterium that ferments glucose or not, gram stains or not, and so much more. Most of the chemical tests performed in this experiment are morphology tests, lactose fermentation tests, glucose fermentation tests, and much more. These tests aid in discovery of the unknown pathogen(s). The purpose of this lab is to be able to identify the different strains of bacteria that survive in the real world, each and every unique finding leads scientists one step closer to discovering the specific bacteria. With the use of a dichotomous key and various biochemical tests, the genus of a bacterial sample will be determined and the profiles of standardized pre-identified specimen will be matched.
MATERIALS AND METHODS
Collect a sample of an unknown microorganism, notate the number of the specimen, and continuously vortex the solution. Collect tape, and Label four test tubes. Perform serial dilutions 10-2, 10-4,10-5 and 10-6. Transfer 0.1ml of an unknown sample using a pipet into a culture tube that contains 9.9 ml of sterile water creating 10-2 dilution; vortex. After vortexing the dilution, use a pipet to transfer 0.1ml of an unknown sample from 10-2 dilution into 9.9ml of sterile water, creating 10-4 solution; vortex. Later transfer 1ml of 10-4 dilution into 9ml of sterile water creating 10-5 solution; vortex.
Use a fourth pipet, transfer 1 ml of 10-5 solution into 9ml of sterile water, creating 10-6 dilution; vortex. Serial dilutions are now complete and ready for the spread plate protocol. Prepare a couple of spread plates, dip a cell spreader in ethanol, sterilize the cell spreader in flame, and let the cell spreader cool down before transferring the 10-2-10-6 solutions onto separate Trypticose soy agar (TSA) plates. Spread each sample onto TSA plates (before inverting TSA) and rotate the plates in an up and down motion, turn 90 degrees and repeat spreading before placing the plates in an incubator for later observations. During week 2-3 perform a quadrant isolation streak protocol by seperate spread plate in four different quadrants. Streak one line of bacterium, and streaking one line into the next quadrant until streaks are complete; incubate. During week 3-4, use specific biochemical tests listed on the dichotomous key to learn more about the bacteria being experimented on. The first test is very critical in learning more about the bacteria, gram staining, and morphology. In order to gram stain, check each streak plate for visible growth of two different organisms. After gram staining, screen the unknown bacteria, and pay close attention to their reaction to gram staining. Gram staining determines morphology (determining whether a bacteria is gram negative or gram positive). After learning whether the bacteria is gram positive, streak one gram positive organism onto a plate horizontally, repeat the procedure for gram negative organism, and incubate. If a specimen’s appears to be gram positive and cocci shaped, perform a catalase test, to perform a catalase test use 3% hydroxide and drop droplets onto the stained slide, touch the center of the colonies. If the catalase test performed is tested positive there will be immediate bubbling, perform a mannitol fermentation test. The mannitol fermentation test is performed in quadrants, streaking bacteria creating colonies. If the mannitol test is positive, there will bea color change indicating the production of acid. There should then enough evidence available to to take an educated guess of the bacteria by matching the dichotomous key to results. If a specimen is tested gram negative, morphology rod, collect TSA plates containing phenol red broth and perform streaks in order to incorporate a lactose fermentation test. If the lactose fermentation test is negative, perform a glucose fermentation test. According to whether the glucose fermentation test is negative or positive, there should be enough evidence to make an educated guess.
RESULTS
Unknown #9 consisted of the following morphology on both TSA plates & tubes. After multiple tests and following the path of action, it was easier to discover the unknown contained gram negative rod shaped organisms in the colonies. Streaks and slants were performed on TSA plates. The unknown bacterium were also inoculated into lactose, and glucose tubes. Table I. lists every biochemical test, the reason why they were performed, observations, and lastly results. The results are shown in order from first to last. Unknown number 9 also contained streaks of gram positive mucus colored opaque (human eye) cocci. Table II. lists biochemical testing performed.
Test
Purpose
Reagents
Observations
Results
Gram stain
To determine how the bacteria reacts.
Crystal violet, Distilled water, Iodine, alcohol and safranine
Pink Rods
Gram Negative Rod shaped bacteria.
Lactose Fermentation
To determine if the bacteria ferments lactose carbohydrates.
No reagents
Color turns orange, no acids were produced.
Tested Negative
Glucose fermentation
To determine if the bacteria produces sugar.
No reagents.
pH indicator did not drip, phenol red stayed red.
Tested Negative
Pseudomonas Aeruginosa
Table I. Tests and Results are subsequently listed in identifying Pseudomonas Aeruginosa.
Table II. Test and results are subsequently listed in identifying Streptococci Epidermis.
Test
Purpose
Reagents
Observations
Results
Gram stain
To determine how the bacteria reacts to reagents.
Crystal Violet, Iodine, Alcohol, Safranin.
Purple
Cocci
Catalase
To differentiate staphyloccocus and streptococcus species.
3% hydrogen peroxide
Colonies immediately started bubbling.
Tested Positive
Mannitol
To determine whether the bacteria ferments salt or not.
Unkown bacteria.
No triggering of the ph balances.
Tested negative.
Streptococci Epidermis
The dichotomus key is used for one to follow directions leading to bacteria. One step completed successfully increases knowledge of chacterestics of a bacteria. Figure 1. The pathway of foll owed using the dichotomous key leading to the discovery of the unknown characteristics.
DISCUSSION
The tests results led to identification of the unknown in ways that the provided accuracy. The reason it is so accurate is because scientist perform test similar (if not resembling) to the tests completed in a laboratory setting. Both guesses of the unknown were correct because the results were performed correctly. Although there were problems of contamination and broken TSA plates, more tests were performed, which prolonged the discovery time. After multiple different tests, it was safe to conclude that the unknown #9 is Staphylococcus Epidermis, and Pseudomonas Aeroginosa respectively. After performing lactose and glucose fermentation tests, all tests worked well. Pseudomonas Aeruginosa ,is an opportunistic pathogen that reproduces in immunocompromised humans. This bacteria causes a wide range of infections due to its resistance to many cheap antibiotics that are distributed in hundreds of countries worldwide. Pseudomonas aeruginosa, is extremely difficult to eradicate. Pseudomonas aeruginosa causes UTI, gastrointestinal infections and a variety of systemic infections. It also appears on patients with cancer, burns and even AIDS and HIV patients causing a high mortality rate. (Nanologix). Staphylococcus Epidermis, an opportunistic pathogen, can survive on dry surfaces for long periods of time! This bacteria lives on skin and mucous membranes located in the human body, this bacteria is only dangerous if found inside tissues where they do not belong. This bacteria can also ferment sugars in low oxygen, and grows best in anaerobic conditions. The best way to avoid infection from bacteria is to practice aseptic technique in and out of facilities. (Otto, US)
Nanologix. "Pseudomonas Aeruginosa." Pseudomonas Aeruginosa. Nanologix Information, n.d. Web. 17 Apr. 2016. <http://nanologix.com/bacteria/pseudomonas_aeruginosa.html>.
Otto, Michael. "Staphylococcus Epidermidis – the “accidental” pathogen." Nature Reviews. Microbiology. U.S. National Library of Medicine. Web. 17 Apr. 2016.