Fundamentals of Microbiology - Term Paper Example

Fundamentals of Microbiology Answer The method to carry out Gram stain and the purpose of each of the components used in the Gram stain procedure can be described as per the following. In Gram staining, a dye is used to bind the negatively charged-cells leaving behind gram positive ones undisturbed. A mordant such as iodine is used to treat the preparation. This forms an insoluble dye-iodine matter and then, the slide is washed using alcohol. This will separate the dye-mordant from Gram negative cells keeping Gram positive ones intact. This is followed by the most critical step called differential extraction of the dye-mordant using decolorizing agent. Finally, safranin is used as a counter stain. All decolorized cells will be treated with the second dye leaving aside those that have been stained with the first dye (Caprette, 2011). Gram positive bacteria, due to ageing, become Gram negative hence it is necessary that overnight cultures are used while carrying out Gram staining. The following components are used while carrying out Gram staining procedure. Iodine: 300 ml distilled water, 1 gram iodine, 2 gram potassium iodide Iodine solution acts as a mordant in gram staining procedure. Crystal violet: 80 ml distilled water, 1% aqueous crystal violet dye; 2 gram crystal violet, 0.8 gram ammonium oxalate, 20 ml ethyl alcohol Crystal violet is a positively charged dye to bind negatively charged bacteria. Safranin: 800 ml distilled water, 4 gram safranin powder, 200 ml anhydrous ethanol Safranin acts as a counterstaining agent in the process of gram staining. Decolorizer: Isopropyl alcohol (75%) and acetone (25%) These are the solvents act as decolorizing agents. Answer 2. The biochemical test called catalase test is used to distinguish between Staphylococcus spp. and Streptococcus spp. (Catalase Test. 2010). Theory The catalase test is a biochemical test that can be used to identify between Staphylococcus spp. and Streptococcus spp. The enzyme catalase contains a heme group and detoxifies hydrogen peroxide. The basic theory used in their identification is that one is catalase positive (Staphylococcus spp.) while other one (Streptococcus spp.) is catalase negative. Methodology of Carrying out Gram Stain a. After spreading the bacteria on an agar plate the incubation process is followed for about 18-24 hours. b. Using sterile inoculating loop bacteria are collected and applied to a microscopic slide. c. One drop of hydrogen peroxide (3%) is added to the bacteria and then kept for observation. If gas formation (O2) is observed in the form of bubbles, then it ensures that the sample of bacteria has a catalase; it is an indication of presence of Staphylococcus spp. Otherwise, on no gas formation, the sample contains Streptococcus spp. Answer 3. A step-by-step protocol to aseptically inoculate a container of liquid media can be described as per the following (Inoculation of Culture Media 2012). 1. Opening the caps of the specimen gradually without any turbulent shake so as to avoid any aerosol production. 2. Container lids or caps are kept open for as short a period as possible. 3. Caps or lids should not be kept at the place of work. 4. To ensure that inoculating loops are put through the flame before putting into the specimen container. 5. Container should not come close to the face any time. 6. To ensure that loops do not contain any large particles or fluid that may cause splattering while placed in the flame. 7. Keeping container of liquid media in racks to avoid the chances of spillage. 8. Keeping the workbench clean by employing proper disinfectant at the end of work. 9. Wash hands with detergent or soap after touching infectious container (Inoculation of Culture Media 2012). Answer 4. Preparing a Streak Plate of Bacteria to Obtain Single Colonies A streak plate of bacteria can be prepared to get individual colonies. Bacteria can be streaked around the petri plate. The process is completed in four steps. Fig.1 Fig.2 Source: http://delliss.people.cofc.edu/virtuallabbook/StreakPlates/GoodStreak.html Step 1 Obtaining bacteria from a liquid culture, a small sized circle is made on the plate (fig 1). Step2 In a second loop, four lines are made moving out from the circle. These lines are denoted in red color (fig 1). Step 3 In a third loop, four more lines are made overlapping the red ones. These lines are denoted in blue color (fig 1). Step 4 In this step, previous loop (step 3) is taken to make four more lines that overlap the blue lines (fig1). In figure 2, one can see many isolated colonies of bacteria on the streaked plate (Dellis, 2011 p.9) Answer 5. Using a combination of non-selective and selective media to isolate bacteria from a mixed sample Non-selective medium is employed when organisms are present in low numbers. This also provides an indication about the type of organism present in the sample. The use of selective medium is to isolate the desired organism present in the mixed sample Nutrient agar is ideal as a non-selective culture medium to grow a large variety of microorganisms. In order to isolate microorganism from the agar plate, the part of colony is taken to a new agar plate. The streak-plate method can be employed as it reduces density of the microorganisms on the surface of the agar. This facilitates to obtain the distinct colonies of the microorganisms of our interest by using selective media (Selective and Differential Media 2012) Selective media inhibit the growth of some organism while promote growth of others. Selective inhibition is done by adding salts, dyes or specific inhibitors affecting the metabolism of the organisms. Crystal violet or penicillin will inhibit the growth of Gram-positive bacteria. Based on this philosophy, Tellurite agar is employed as media to identify for Gram-positive organisms. When gram negative organisms are to be selected or identified, the agar with penicillin (5-50- units/ml) is used (Selective and Differential Media 2012). Selective but differential media are employed to identify closely related organisms. Presence of specific dyes in the media will cause certain changes in growth patterns and that will help into identification of the bacteria. For example, staphylococci. is identified using Mannitol salt agar as a selective medium. The high salt concentration in the media does not allow the growth of most bacteria except staphylococci. Similarly, Eosin Methylene blue agar helps identify Gram-negative pathogens. Here, Methylene blue works as an inhibitor for Gram-positive organisms (Selective and Differential Media 2012). Answer 6. Outlines of an experiment to find the minimal inhibitory concentration (MIC) and minimal bactericidal concentration of an antibiotic can be listed as per the following (Microbiology Practical Techniques, 2012). The experiment begins with making a series of dilutions of the antibiotic into broth culture with each subsequent dilution is increased by 100 percent. The dilution would be made in distilled water before transferring into the broth. Then, the antibiotic stock solution is diluted further that will form a Universal Bottle. This will be used to make four more dilutions. This will provide the series of dilutions of the drug to be added to the nutrient froth. The most concentrated of these series having 256 milligrams per liter will be produced by broth inoculation using the original stock solution of drug. A control tube without any antibiotic will indicate the viability of the bacterium under test. Antibiotic-free broth is used to inoculate a colony of the bacterium. This suspension is used for the purpose of inoculating the test broths. Beginning with the most dilute, each tube is added with one drop of the bacterial suspension with the last one without any antibiotic. The tubes are left for incubation at 37° Celsius. Turbidity in the broth will indicate the growth in the inoculums; cloudiness will be visible in the antibiotic-free control indicating that bacteria are viable. Bacteria will also grow at low concentration of the antibiotic. The minimum inhibitory concentration is the one that prevents any bacterial growth indicating a clear broth. To go further to find Minimum Bactericidal Concentration, all tubes that did not stop bacterial growth are promptly discarded. Each of the tubes without any bacterial growth is taken for further sampling. Each sample is plated out onto medium that are without antibiotic. The plates are then put for incubation for 18-24 hours to produce colonies and again checked for their growth. If the drug has propensity to kill the bacteria, three will be no growth. At this instance, Minimum Inhibitory Concentration and Minimum Bactericidal Concentration will be equal. If drug only prevents bacterial growth then removing the cells to fresh medium (free of antibiotic) will again resume the growth. On incubation, colonies of bacteria will be found on the plate and that will indicate that the antibiotic is only inhibitive to growth (Microbiology Practical Techniques, 2012). Answer 7. Determining whether a given bacterium is motile or non-motile: Bacterial motility can be determined by any of the following methods. Motility Test Medium Motility test medium (semi-solid) may be used to detect bacterial motility. The agar with 0.3% concentration does not hinder motility and is a proper medium to form a soft gel. The test is based on the premise that growth of a non-motile organism takes place along the line of inoculation, when it is stabbed into Motility Test medium and also, growth is sharp along the stab line. In contrast, motile organisms have a tendency to swim away from the stab line and growth happens all along rather than along the line of inoculation. Direct Observation of Motility An actively growing broth culture may be used for observation of motility. Motility is visible when bacteria move in separate directions. With old cultures, with a large proportion of inactive bacteria, it is difficult to observe the motility. Same is true with very active cultures because microscopic view covers a very small area and it is quite likely that microbes with high motility may have left the area under focus (Alonzo, 2012). Bibliography Alonzo, C. (2012). Motility Testing [online], https://www.softchalkcloud.com/lesson/files/MoN7ruh9Qe5CkH/Lab_7_Motility_print.html [Accessed: 5.08.12] Caprette, D. R (2011). The Gram Stain. Rice University [Online], http://www.ruf.rice.edu/~bioslabs/bios318/staining.htm#gram [Accessed: 5.08.12] Catalase Test (2010). Michigan State University [Online], http://learn.chm.msu.edu/vibl/content/catalase.html [Accessed: 5.08.12] Dellis, S (2011). Streaking a plate for single colonies [Online], http://delliss.people.cofc.edu/virtuallabbook/StreakPlates/GoodStreak.html [Accessed: 5.08.12] Inoculation of Culture Media (2012). World Health Organization. [Online], http://www.searo.who.int/en/Section10/Section17/Section53/Section482_1785.htm [Accessed: 5.08.12] Microbiology Practical Techniques, (2012). The University of Leeds. [Online], http://www.fbs.leeds.ac.uk/institutes/ilse/movies/microbiology/transcript/MICology.html [Accessed: 5.08.12] Selective and Differential Media (2012). Western Washington University. [Online], http://fire.biol.wwu.edu/brodham/biol346_S07/labman_week6.pdf [Accessed: 5.08.12] Read More