Identifying an unknown organism from a cell phone by using a variety of different lab methods throughout the semester.
Bacteria are singular bacterium that consist of large domains of prokaryotic organisms. Their cell structure is simple due to the fact that they have no nucleus or membrane bound organelles and their genetic information is located in a single strands of DNA. Bacteria range in a variety of shapes such as spherical (cocci), rods (bacilli), and spirals (spirilla), comma (vibrios) or corkscrew (spirochetes).
Microbial cultures are a method used to grow and multiply organisms in a culture under a controlled laboratory setting. These cultures are used to determine the type of organism, its morphology, arrangement, whether it is gram negative or gram positive, its ability to ferment, its abundance and is also used to determine the cause of infectious diseases by allowing the bacteria to multiply in the medium. A medium is used to cultivate bacteria in the lab. Mediums can be strictly chemical or organic. Microorganisms growing on the medium form a culture. A pure culture occurs when only one type of organism is present and a mixed culture occurs when multiple organisms are present.
Methods & Results
1. Isolation of Microbes: The Streak Plate Method
The streak plate method was used to obtain a pure culture. A portion of the agar was streaked with the inoculum and then streaked onto a quadrant of the plate in attempt to dilute the inoculum across the plate until each colony is separated out. The streak plate process requires you to use the aseptic technique so introducing any new contaminated cells can be avoided. To avoid contamination, the inoculated loop must be flamed between every new streak to kill leftover cells. Streaking into numerous quadrants allows the bacteria to be more diluted after each streak, eventually leaving you with single cells in the final quadrant.
1. Using Bunsen burner, sterilize whole loop until cherry red. Keep loop underneath flame so airborne contaminants are less likely to contaminate loop while it cools.
2. Remove lid from culture tube and pass lip of tube through flame to sterilize
3. Dip cooled, flame-sterilized loop into broth culture to retrieve some cells
4. Flame lip of tube after inoculating loop from tube then replace the cap
5. Inoculate a small region of plate by spreading cells across surface of agar in a zig-zag fashion with loop. Keep loop at 45-degree angle to prevent gouging into agar
6. Flame loop to kill leftover cells from first streak, allow time for loop to cool
7. Return to original zig-zag inoculum on plate and drag loop through it, creating a second zig-zag streak
8. Flame loop again, the streak through the second streak dragging bacteria into third quadrant using same zig-zag pattern
9. Flame loop again and then streak from the spread out inoculum previously created across the rest of the surface of agar plate throughout final fourth quadrant
My results included a plate with isolated colonies ranging from sizes of about 1mm to 5mm and had white and yellowish color to them. Since all the colonies had the same appearance, it is safe to say there was no contaminant species. If contaminant species were present it would mean that the streak plate method was not done accurately whether it be not sterilizing the loop properly causing it to obtain contaminated species or another reason. Since there is only one bacteria present on the plate the culture is a pure culture, “a single population of identical cells derived from a single common ancestor”, as opposed to a mixed culture, a culture obtained from the natural environment.
2. Motility Agar
Motile bacteria are bacteria that have the ability to move around in response to the environment. In a motility-agar medium, tetrazolium salt is included and as the organism grows the dye grows with it. When the organism grows, the dye combines with the bacterial cells and creates an insoluble red pigment. This red color forms only in the area where bacteria are growing. Motile bacteria migrate away from the line of inoculation leaving red pigment visible in the area where the cells grew.
1. Following aseptic technique, stab inoculate each tube
a.) Sterilize inoculating needle and let it cool
b.) Remove lid from culture tube and using Bunsen Burner, flame lip of glass tube
c.) Retrieve culture on needle, re-flame lip of tube prior to replacing cap, then stab the agar
The inoculate tube showed my cells “swimming” off the stab line throughout the agar making it appear cloudy. Since the dye turned red, we can verify that bacteria was present and motile. The zig zag line in the center of the tube was also a huge indicator of motility.
3. Cell Morphology & Arrangement
To be able to view a microbe under a microscope it must be stained to be able to view the cell morphology. There are two different staining methods, simple and differential. Simple staining consists of a single dye and provides information such as cell shape and size and arrangement of bacterial cells. Differential stains consist of two or more dyes and differentiate between an organism’s cellular components.
1. Use an inoculating loop and aseptic technique to prepare smears
2. Place loopful of water in center of slide ans then remove a small amount of bacterial growth from the slant and mix with the water and spread to cover ½ in area on slide
3. Allow slides to completely air dry
4. Pass the smear through Bunsen Burner flame 2-3 times to heat-fix bacteria to slide
5. Stain with Methylene blue for 60 secs
7. Wash stains off by rinsing with water until no more stain washes off slide and then blot slides with bibulous paper
8. Examine slide under oil immersion lens (100x)
After following the lab procedure and viewing my bacteria under the microscope I was able to come to the conclusion that the unknown appeared to be small cocci and categorized by staphylococci based on arrangement. The organism also appeared to be a dark pink/purple color.
4. Gram Stain (-/+)
Bacteria can be divided into two groups, gram positive and gram negative. This is determined by how bacteria react when the gram stain is used and the differences in cell wall chemistry between the two. There are four steps hat take place during gram staining.
1. Heat fixed smear is flooded with primary stain, crystal violet
2. Gram’s iodine is used to form a water-insoluble complex with the crystal violet
3. Cells are washed with decolorizer, 95% ethanol and the decolorizer dissolves the outer membrane in gram negative organisms enhancing the removal of the CV-I complex through the cell wall, gram negative organisms then become colorless and since gram positive organisms contain a thick layer of peptidoglycan and no outer membrane the 95% ethanol dehydrates these cells and prevents the removal of the CV-I complex through the cell wall allowing it to remain its purple color
4. Bacteria is counterstained with safranin which stains the colorless cells (gram negative) and gives them their pink color, but does not change the gram positive organisms
My bacteria stained purple allowing me to conclude that it was a gram positive organism and has a cell wall composed of 60%-90% peptidoglycan causing it to have a thick cell wall.
5. Carbohydrate Fermentation
Fermentation is a process where bacteria can continue glycolysis even when oxygen isn’t fully present in the environment. We are able to determine an organism’s ability to ferment sugars by using the phenol red experiment which measures the production of acid and CO2 gas. If the pH of the phenol red is below 6.8, it will turn yellow and under alkaline conditions, when the pH is higher than 7.4, it will turn magenta. If the organism you have is a fermenter, then the color will change from red to yellow meaning acid was produced and fermentation occurred. The phenol red test also contains a Durham tube which is able to capture CO2 gas release during fermentation.
1. Using a loop and following aseptic technique, inoculate glucose broth with unknown
2. Using a loop and following aseptic technique, inoculate lactose broth with unknown
(Read results within 24 hours so sugars don’t completely catabolize causing reversion)
My results indicated that my organism was unable to ferment both glucose and lactose. The color of the mediums remained red meaning it could not ferment lactose or glucose and had a pH greater than 6.8. The Durham Tube showed that gas was produced in both the glucose and lactose indicating that CO2 was being produced.
Sugar Growth? Color of Medium Acids? Gas?
Glucose yes red no yes (a little)
Lactose yes red no yes (a little)
In the Mannitol Salt Agar, a selective and differential medium, staph species are selected and growth of all other species are inhibited. The MSA contains carbohydrate mannitol and phenol red as an indicator dye. If a bacterium can ferment mannitol, it will produce acids causing the pH of the medium to drop enabling the phenol red to change to yellow. Since my unknown changed from the phenol red to yellow we can conclude that my unknown was a mannitol fermenter.
The MacConkey Agar is a selective and differential medium and is selective due to crystal violet and bile salts that inhibit the growth of gram positive and some gram negative bacteria. The fermentation of lactose reduces the pH of the medium which contains the pH indicator, red. My unknown did not show on this medium indicating that it is indeed a gram positive organism.
The Eosin Methylene Blue is a differential and partially selective medium that inhibits the growth of gram-positive organisms. Eosin and Methylene Blue are dyes that form precipitates at a low pH. This means that if a microbe is capable of fermenting lactose within the medium, the pH will decrease forming a dark purple color where E. coli will form a metallic green due to even more vigorous fermentation. Since my organism was gram positive, no growth was shown on the medium for Eosin Methylene Blue.
6. Drug Susceptibility
The Kirby-Bauer Test is the most popular test used to determine an organism’s susceptibility to an antibiotic. In this test, am agar plate is swabbed with the unknown organism and paper discs containing the antibiotics are placed on the agar. During incubation, the organisms grow within the agar and we can determine if the antibiotic is effective based on the zone of inhibition that is made. If the organism is effective, a noticeable zone of inhibition will appear. The zone of inhibition is then measured and compared to standard tables which indicate organism’s susceptibilities to a variety of antibiotics.
Amikacin Amoxicillin Bacitracin Doxycycline Polmyxin B Sulfisoxazole Vancomycin
25mm 24mm 0mm 26mm 10mm 29mm 14mm
S S R S R S I
The diameters of each of the antibiotics effect on the unknown allowed me to determine whether my unknown was susceptible or resistant to that bacteria. Not all zones of inhibition were provided in the lab book so some needed to be looked up on the internet but the zones of inhibitions for each organism varied based on numerous answers on the web but the results on susceptibility and resistance for each antibiotic is shown in the table above (S= susceptible, R=resistant, I=intermediate).