Unknown Bacteria Reagent

An unknown bacterium was handed out by Dr. Honer. The appropriate tests were prepared and applied. The first procedure that was done was the gram stain. Under a microscope, if the gram stain is purple, the bacterium is gram positive, if the stain is red, it is gram negative. The next test was the fermentation tests for glucose, sucrose and

The purpose of this lab was to identify an unknown bacteria culture using differential tests. The identification of the unknown culture was accomplished by identifying the bacteria based on its specific metabolic characteristics and morphology. It is suggested that culture 11 is a sample of Enterobacter aerogenes.

A mixed culture of two unknown bacteria was provided by the instructor. The methods used for

The next step of the project included preparing a Gram stain to discover the cell shape, arrangement, and if the bacteria is gram positive or

In a laboratory setting, it often becomes necessary to identify an unknown organism. In this experiment, researchers classified an unidentified bacterium based on its physical structure, colony morphology, optimal conditions and metabolic properties. A Gram stain using crystal violet, iodine, and safranin and a simple stain using methylene blue characterized the organism’s cell wall. Cultural behavior was classified by inoculating the organism onto nutrient agar and incubating it at 37° C for 48 hours, and observing its behavior, as well as using SIM medium to test for motility. Optimal growth temperature was

The purpose of this lab was to identify two unknown bacteria cultures using various differential tests. The identification of these unknown cultures was accomplished by separating and differentiating possible

There are many reasons for knowing the identity of microorganisms. The reasons range from knowing the causative agent of a disease in a patient, so as to know how it can be treated, to knowing the correct microorganism to be used for making certain foods or antibiotics. This study was done by applying all of the methods that have been learned so far in the microbiology laboratory class for the identification of unknown bacteria. The identification process can be completed with a series of deferential stains and biochemical tests. Creating a dichotomous key helps to limit the amount of biochemical tests done on an unknown organism and by observation

For the Urease test, I incoluated my Urea test tube with my unkown bacteria from a TSA plate using and inoculating loop. The Urea tube was then incubated at 37 degrees Celsius for 8 days to observe for a color change. The Urea tests for the ability of a bacteria grown in urea broth produces urease. This medium contains the pH indicator phenol red. If urease is produced the pH of the media will raise thus causing the phenol red to change from yellow to a pink color.

Two smears of the unknown bacterium #5 were inoculated while the second smear was used for a back up. The unknown bacterium dried for at least forty minutes. After the smears dried, the slides were heat fixed two times to ensure the stability of the organism. The slide was placed on top of the staining rack then over the small sink.

Microorganisms are ubiquitous and play a large role in the environment. Microorganisms help some other organism in symbiotic relationships and they can also harm other organisms by causing them disease. Tests have been developed to help determine the characteristics of many microbes so that their special capabilities can be utilized for benefit. While these tests have also helped to develop substances and strategies to reduce the populations of microbes that have detrimental effects. The experiment that was performed used some of these techniques to determine the identity of some common microbes.

Gram staining differentiates between Gram positive and Gram negative bacteria and provides cell morphology. From this, pink bacilli were observed, providing a Gram negative result. Thus the unknown was narrowed down to E. coli and P. aeruginosa. Next was the Fluid Thioglycollate Media (FTM) which consists of a sugar broth and determines the oxygen requirements for the unknown. Since the turbidity was observed at the top of the tube, thus the unknown was an aerobe, just like P. aeruginosa and not E. coli which is a facultative anaerobe. Then a Durham tube with a glucose broth was used to determine whether the bacterium produces acid and gas from carbohydrate fermentation. Since only a red broth with some turbidity at the top was observed it could not have

First, take the prepared slide of bacterial smear and examined it under the microscope in order to become familiar with the morphological types of bacteria. Once familiarized, initialed a microscope slide with a grease pencil. Then, place a drop of tap water on the center of the slide. Next, pick up a very small amount of

Gram Stain: The Gram Stain test was ran. The unknown bacteria F#1729 was stained and the result was Gram Negative (-). This result identifies the layer of peptidoglycan being thin. This thin outer membrane is composed of lipopolysaccharides that are not able to retain the crystal violet iodine used, instead they slough off when rinsed with the ethanol alcohol. This leaves the color unretained and the counterstain safranin is absorbed, staining the bacteria pink. Once the slide was ready, we were able to observe the bacterial morphology under the microscope. We identified the bacterial shape as bacillus under 100X magnification with oil immersion the morphology was verified.

Extremely similar to Eosin Methylene Blue Agar, MacConkey Agar is a selective and differential media used to isolate organisms based on their lactose fermentation ability. It contains lactose, bile salts, neutral red, and crystal violet (Leboffe, A Photographic Atlas for the Microbiology Laboratory). Just like the Eosin Y and Methylene Blue dyes in the EMB plate, the bile salts and crystal violet in the MacConkey plate encourage the growth of Gram-negative bacteria while preventing the growth of Gram-positive bacteria. The neutral red is used as a pH indicator, therefore if the bacteria is acidic and has a pH less than 6.8 the growth will turn pink; whereas, if the bacteria is neutral or basic and has a pH higher then 6.8 the growth will remain the same color, usually a cream color, or turn the same color as the media. The crystal violet is used to control Proteus or similar bacteria, so they do not obstruct with other results (Leboffe, Microbiology Laboratory Theory and Application). The major difference between the EMB and MacConkey media is that EMB uses lactose and sucrose sugars to test fermentation while, MacConkey only uses the lactose sugar when testing organism fermentation; because the EMB plate and the MacConkey plate tests are homogenous, identical results were found. All four bacteria were positive for growth; two of the four bacteria were positive for lactose fermentation. It was previously mentioned that Enterobacter aerogenes is slightly

A complex between crystal violet and iodine (CV-I) is formed within the cell. The structure that determines the Gram reaction is the cell wall structure and not that shape. Bacillus cereus and Staphylococcus aureus are stained purple in the Gram staining as they have a high amount of peptidoglycan which forms the outer layer of the cell. This thick peptidoglycan layer is able to trap the purple CV-I complex even after alcohol treatment. Escherichia coli is stained pink in the Gram staining and it is a Gram-negative bacteria. Gram-negative bacterias usually have a thin peptidoglycan layer compared to Gram-positive bacterias. The peptidoglycan layer is located between the plasma membrane and an outer membrane containing lipopolysaccharide and this outer layer is dissolved during the alcohol treatment which results in a loss of the CV-I complex, hence the pink safranin counterstain is trapped by the peptidoglycan layer (ASM, 2004).