Introduction:

 

    In unit two "from Proks to Plants" we investigated Bacteria, Aseptic technique as well as Gram Stain technique. Prokaryotes were Earth’s sole inhabitants about 3.5 to 2 billion years ago. A prokaryotic is a single-celled organism that lacks membrane-bound organelles and complex compartmentalization. There are two domains of prokaryotes; Archaea and Bacteria, and Eukaryote; which all vary in shapes and sizes. The three most common shapes of bacteria which are spheres (cocci), rods (bacilli), and spirals. Most bacteria are harmless to humans however pathogenic prokaryotes are not as they releasing exotoxins or endotoxins, which causes about half of all human diseases.

 

     One of the most important features of nearly all prokaryotes is their cell wall, which maintains cell shape, provides physical protection, and prevents the cell from bursting in a hypotonic environment. Bacteria can be categorized by their ability to accept a gram stain while exposed to several staining agents; Crystal Violet, Gram’s Iodine, and Safranin. Through a process called Gram stain discovered by Danish bacteriologist, Hans Christian Gram in 1882. This techniques states that if the bacteria possesses a cell capsule, a pink color will be visible resulting in it being a “Gram Negative” bacteria. But if the bacteria lacks a cell capsule, a purple color will appear. This is referred to as a “Gram Positive” bacteria.

 

    Most prokaryotes are microscopic, however what they lack in size they make up for in numbers. Prokaryotes can reproduce every one–three hours through asexual reproduction, usually by binary fission. Asexual reproduction is defined as the formation of offspring that are genetically identical from the cell of a single parent. Transduction, conjugation, and transformation are not replicative processes however these processes are responsible for Genetic exchange and transference of DNA from one cell to another known as rapid reproduction and horizontal gene transfer and recombination.

 

    Prokaryotes thrive almost everywhere unlike many other organisms; places too acidic, too salty, too cold, or too hot.  Which is why aseptic techniques are fairly important when culturing. Aseptic technique refers to the procedures that are performed under sterile conditions. The goal of aseptic techniques is to prevent contamination of bacterial culture by other unwanted microorganisms in its environment or its surrounding. By using the Aseptic technique to transfer microbiological cultures from one medium to another, we hypothesized that two or more colonies would be formed, due to the surface we swabbed was high traffic areas.

 

Materials

 

     These materials where collected in order to successfully carry out the Gram Staining Technique and Aseptic Technique investigation.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Methods 

 

    Our Gram-Staining technique that we mention below is not our main focus, but we briefly describe the steps needed in order to carry out this experiment. Our main focus was on the ‘Swabbing Experiment’ (Aseptic Technique) that is also mentioned below.

 

    Firstly, before starting the Gram Staining technique, three slides containing bacteria such as Escherichia Coli and Bacillus was placed into a staining tray. In order to start the procedure three solution was gather also three 600ml beaker was collected containing deionized water. Firstly, we covered all the slide containing the bacteria entirely with crystal violet stain and allowed it to sit for 60 seconds. After that with a clothes peg, we gently waved each slide into the first beaker until it reached the third beaker when it was fully cleaned. Secondly, we then covered the same crystal violet stained slides with Grams iodine solution and also letting it sit for 60 seconds. Before we repeat the rinse procedure, we refilled the beaker with clean deionized water, so the purple dye from the crystal violet would not affect the slides. In case you didn’t know, the iodine solution was used to fix the crystal violet inside the cell walls of the bacteria. Than we done a skill called discoloration, were we took a bottle of ethanol solution and squirted it completely over each slide until the ethanol solution that was running off of the slides was colorless. The ethanol rinses away the outer lipopolysaccharide membrane of the gram negative bacteria along with the crystal violet and iodine complex from the now exposed peptidoglycan layer beneath. Next, we counterstain the slides by covering it with safranin-o solution and allowed it to sit for 30 seconds. Finally we repeated the rinse procedure with clean deionized water again and dried each slide using bibulous paper. We than examined our slides under a microscope.

 

 Gram Staining: http://virtuallab.nmsu.edu/stain.php

           

    Secondly, by wiping down our work space area with disinfectant solution, we was preparing for the focus of our Swabbing Experiment (Aseptic technique).With a marking pen, label each petri dish on the bottom with the sample organism's name and the date of inoculation. Remove the swab from packaging and swab sample organism. Partially lift the lid of the petri dish just enough to insert the swab onto the media. The media is to aid in the recovery, growth, isolation and identification of the microorganisms under investigation.  Do not completely open the lid and expose the surface to the air. Seal the petri dish around the edges with tap. Repeat steps for each sample. Once all 6 samples are completely prepared place them in the incubator to culture 48 hours. Once all 6 samples were completely prepared, we placed them into the incubator at 25̊c in order to culture for 48 hours.

 

Aseptic Technique link: http://biolabs.tmcc.edu/Micro%20Web/AsepticTechnique.pdf

 

Results

 

    In a two part lab we performed two different experiments on bacteria. The first procedure was Gram-Staining technique. These are the results we gathered after performed our experiment in lab. Our first slide, labeled ‘A’ had gram positive and gram negative in the shape of cocci staph. Slide two labeled ‘B’ had majority gram negative and our last slide labeled ‘C’ was all gram positive with a shape of bacilli. Our results showed us that our slide ‘A’ had a combination of both types of bacteria. On the other hand, slides ‘B’ and ‘C’ only had gram negative or gram positive.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

The second procedure was the Aseptic Technique. This process is used frequently to study the growth of organism. The chart below, displays our results after two days of bacterial growth.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Discussion

 

        In this lab we are investigating Prokaryotes (bacteria) using two techniques, Aseptic and Gram Staining. Firstly let’s begin to discuss the Gram Staining before getting to the Aseptic Technique. Gram staining is the most important staining technique in microbiology. It is a common staining technique used to differentiate two groups of bacteria based on their cell walls. In this lab we were able to determine the different shapes of bacteria using either rods, bacilli, cocci (staph and strep), or spirillum, and also to determine if the bacteria were either gram positive or gram negative. Gram positive bacteria have a thick cell wall which is made up of peptidoglycan, which stains with the crystal violet stain, causing the gram positive bacteria to look purple under the microscope. On the other hand gram negative bacteria have a thin layer of peptidoglycan. Therefore when rinse with alcohol its stain appears reddish/pink under the microscope. From our results slide ‘A’ contained gram positive and gram negative while on the other hand, slides ‘B’ and ‘C’ either had gram negative or gram positive.

 

   Secondly, Aseptic technique is a procedure to prevent sterile cultures from becoming contaminated by other microorganisms. During, the second part of the Aseptic Technique lab. We came up with five different surfaces to swab around the college.  The results for the experiment were quite diverse with different colonies of bacteria and fungi (which are a visible mass of microorganisms growing on an agar surface). The first surface we swab was a bathroom toilet handle which surprising had no growth on the petri dish. There can be thousands of bacteria in a bathroom, but specifically the toilet handle belongs to four phyla: Actinobacteria, Bacteriodetes, Firmicutes and Proteobacteria. Moreover, human skin bacteria is commonly associated with this surface and other bacteria including mouth, gut, and urine are common on a bathroom toilet handle.  Also there can be bacteria on the toilet handle that can be found on people’s shoes which can be many colonies of different kinds.

 

  During one of our swabbing we swabbed the surface of an air-conditioning vent however, we had to swab this surface twice due to a crack in one of the petri dish. We kept both petri dishes one with the crack and one without the crack to compare the results. The results in petri dish without the crack had a small circular yellowish colonies, a large beige circular colony, and also irregular beige colony and a pale yellow cluster, resulting in fungi. The petri dish with the crack however, had a yellowish cloudy coverage and a yellowish grey irregular shape blob. We concluded that both petri dishes had fungi but the petri dish with the crack had other unwanted microorganism that caused contamination. We also notice a variety of colonies on the chief’s hand, we determined that it had resulted from high traffic areas that he had touched. Some microorganisms live on normal skin and cause no harm, such as Staphylococcus (S), Micrococcus, Corynebacterium, Brevibacterium, Dermabacter, Malasezzia these can be found in the upper parts of the epidermis and hair follicles. The chef could also have bacteria colonies on his hand from high traffic kitchen areas. The skin provides ideal examples for of various microenvironments.

 

Conclusion

 

In conclusion, our hypothesis was completely wrong that two or more colonies would be formed on the bathroom toilet handle since this surface is a high traffic area. However, our results proved the hypothesis to be untrue. The highest traffic surface area was the computer lab keyboard.   Gram staining and aseptic are both great techniques that go hand in hand. They are both used to look at bacteria. Gram staining is a common staining technique used to differentiate two groups of bacteria based on their cell wall, Gram-negative and Gram-positive. Did you know some Gram-negative bacteria can cause many types of infections? According to the National Institution of Allergy and Infectious Diseases, “Gram-negative bacteria can also cause respiratory infections, such as certain types of pneumonia, and sexually transmitted diseases, including gonorrhea. Yersinia pestis, the Gram-negative bacterium responsible for plague, is transmitted to people through the bite of an infected insect or handling an infected animal”. Whereas the aseptic technique is a procedure is to prevent sterile cultures from becoming contaminated by other microorganisms.