Selective And Differential Media For Identifying Microorganisms - Term Paper Example

 1. Describe the method you would use to carry out a Gram Stain and explain the purpose of each of the components that are used in the Gram stain procedure. Staining procedure that make visible the differences between bacterial cells or parts of a bacterial cell are termed differential staining techniques. One of the most and widely used differential staining techniques in microbiology is Gram staining. This technique was introduced by Christian Gram in 1884. In this process the fixed bacterial smear is subjected to the following staining reagents in the order -crystal violet, iodine solution, alcohol (decolorizing agent) and safranin or some other suitable counter stain. Bacteria stained by the Gram method fall into two groups- Gram positive bacteria which retain the crystal violet and hence appear deep violet in color and Gram negative bacteria which lose the crystal violet, are counterstained by the safranin and hence appear red in color. The most plausible explanations for this phenomenon are associated with the structure and composition of the cell wall. Difference between the thickness of cell walls of Gram-negative and Gram-positive bacteria is imperative. The cell walls of Gram negative bacteria are generally thinner than those of Gram-positive bacteria. Gram-negative bacteria contains a higher percentage of lipid than do Gram-positive bacteria. Experimental evidence suggests that during staining of Gram-negative bacteria, the alcohol treatment extracts the lipid, which results in increased porosity or permeability of the cell wall. Thus the CV-I complex can be extracted and Gram-negative organism is decolorized. These cells subsequently take on the color of the safranin counterstain. Since walls of Gram-negative bacteria have a smaller amount of peptidoglycan, which is less extensively cross-linked than that in the wall of Gram-positive bacteria, therefore the pores of the cell wall of Gram-negative bacteria remain large even after ethanol treatment to allow alcohol to extract CV-I complex. The cell walls of Gram-positive bacteria because of their different composition (lower lipid content), become dehydrated during treatment with alcohol. The pore size decreases, permeability is reduced and the CV-I complex cannot be extracted. Therefore, these cells remain purple-violet. Alcohol causes diminution in the diameter of the pores in the cell-wall peptidoglycan (Web. Gram Staining). (http://www.austincc.edu/microbugz/gram_stain.php) 2. Describe the biochemical test you would use to distinguish between Staphylococcus spp. and Streptococcus spp. include both the methodology and the theory behind the test in your answer. Staphylococcus spp. occur most commonly as irregular clusters of spherical cells and Streptococcus spp. are cocci that divide in a single plane forming chains. Both these organisms are Gram-positive. The biochemical test that distinguish both Staphylococcus spp. and Streptococcus spp. is Catalase test. Catalase is an enzyme that converts hydrogen peroxide to water and oxygen. Staphylococcus spp. possess the enzyme (Catalase positive). The presence of catalase as evidenced by frothing on addition of hydrogen peroxide indicates bateriuria and a positive result is obtained. 2H2O2 Catalase 2H2O + O2 The Streptococcus spp. do not possess the enzyme (Catalase negative) (Web. Catalase Test). (http://iws2.collin.edu/dcain/CCCCD%20Micro/Catalase.jpg) 3. Write a step-by-step protocol that you would use to aseptically inoculate a container of liquid media. Depending upon the growth requirement of the microorganism, culture media are prepared encompassing a source of carbon, nitrogen (provided by peptones), inorganic salts, minerals and vitamins in traces. Buffers are used to stabilize the pH of the medium. Required amount of chemicals are added to the measured volume of distilled water one by one and reconstituted. Pour the measured amount of liquid media in the vials or test tubes and then they are sealed with cotton plug. Wrapped by a paper to prevent cotton from getting wet which otherwise allow the water of the autoclave to enter the test tube. Sterilization is done by heating the media in an autoclave at 121°C at the pressure of 15 lb/sq. in for the period of 15 minutes. This temperature generates steam under pressure to kill all the life forms including spore. The media is then allowed to cool. Aseptic techniques are the prerequisites of microbiological work as it reduces the probability of microbial contamination. The steps involved in aseptic liquid media inoculation are- a. Disinfection of working areas, use of laminar flow and flame to eliminate the presence of bacteria. Inoculation is done near the flame. b. The vial or broth tube is marked with the name of the organism and other required features. c. Universal screw-capped bottles are held at an angle. Remove the lid to pass the mouth of bottle through the flame. Insert a sterile loop (sterilized on flame) and take a drop of liquid. Pass the mouth of the vial through the flame and close it with the lid. d. The loop with inoculum is transferred to broth in the vial with fresh, autoclaved media by passing the vial through the flame. e. The vial is flamed and sealed. Bring the wire loop on the flame and heat till it gets red hot in order to kill the remaining bacteria. The vial is then incubated at the set temperature in incubator. The broth becomes cloudy as the growth progresses (Web. Experiments To Show The Growth of Bacteria- Basic Techniques). 4. Explain, with a diagram how you would prepare a streak plate of bacteria to obtain single colonies. The inoculum to be inoculated is taken on the sterile transfer loop and plates are streaked in four directions as shown in the diagram to get the single colonies. The plates are then incubated upside down at 37°C. To store the culture, the plates must be sealed with Parafilm and stored at 4°C. (Web. General Microbiological Techniques) 5. Describe how you would use a combination of non-selective and selective media to isolate a bacteria from a mixed sample. Many special-purpose media are needed to facilitate recognition, enumeration and isolation of certain types of bacteria. To meet these needs the microbiologists have made available numerous media which, on the basis of their application of function may be classified as- Non-selective media is used for the cultivation of microorganisms for routine purpose. Instead, certain complex raw materials such as peptones, meat extract and yeast -extract are used and the resulting media support the growth of a wide variety of heterotrophic bacteria. Agar is included as a non-nutritive solidifying agent when a solid medium is desired. E.g. Nutrient agar and nutrient broth. Selective Media provide nutrients that enhance the growth and predominance of a particular type of bacterium and do not enhance (and may even inhibit) other types of organisms that may be present. For instance, a medium in which cellulose is the only carbon source will specifically select for or enrich the growth of cellulose-utilizing organisms when it is inoculated with a soil sample containing many kinds of bacteria. In order to select a particular microorganism the sample is first inoculated in the non-selective media at a particular temperature. Microbes able to proliferate at that temperature will grow. From this mixed population a measured amount of the inoculum is taken and then the inoculum is directly added to the selective media. Since selective media is incorporated with a particular nutrition supplement, only the desired organism will grow (Selective and Differential Media for Identifying Microorganisms). 6. Outline an experiment that you could use to determine the minimum inhibitory concentration and minimum bactericidal concentration of an antibiotic. Aim- To find out the MIC and MBC of the given antibiotic (Ampicillin) for the bacterial strain (E. coli). Material Required: Ampicillin, nutrient broth tubes, test tubes for making dilutions, sterile distilled water, micropipette etc. Theory: Minimum Inhibitory Concentration (MIC) is the lowest concentration of the drug that inhibits microbial growth. The minimum bactericidal concentration (MBC) is the lowest concentration of the drug that kills the bacterium. Procedure: 1)Prepare the stock solution of the given antibiotic (for instance Ampicillin). From the stock solution prepare dilutions of the antibiotic using serial dilution technique. Make dilutions ranging from 300µg to 10µg. 2) Inoculate the bacterial strain in each of the media tube. Mark the tubes with the concentration of the antibiotic being added to the culture. 3) Add the diluted antibiotic solution to the media tube. Mix well and incubate at 37°C overnight to see the bacterial growth. 4) Bacterial growth can be checked by taking loop of the culture media and by streaking on the agar plate (solid media). Mark the plate appropriately (mention the concentration of the antibiotic solution from which the inoculum was taken). Observation: After overnight incubation, the minimum inhibitory concentration (MIC) is read by noting the lowest concentration of the drug that inhibits growth. This is read when least number of bacteria are observed in the tube. In order to find out the minimum bactericidal concentration (MBC), a slightly higher concentration of the drug is used in gradation. Result: Results must be interpreted as- S. No. Concentration of the drug No. of Colonies (Web. ATS LABS: Excellence in Antimicrobial Testing) 7. Explain how you would determine whether a given bacterium is motile or non-motile. Many bacteria possess flagella that allow them to move from one location to other. Bacteria are able to swim all the way through water-based surroundings, such motility can be observed in a wet mount or in Motility Agar Stab where soft agar is used in a test tube (not slant). Culture of the bacteria to be tested for motility is inoculated by means of stab-inoculation into the agar (not on the top). If the bacteria is motile it will grow along the stab line and reach to the surface. However, if the bacteria is non-motile then it will display growth confined only to the inoculated area (Web. Motility). References ATS LABS: Excellence in Antimicrobial Testing. [online] Available at: . [Accessed 19 July 2012]. Catalase test Picture. [online] Available at: [Accessed 19 July 2012]. Catalase Test. [online] Available at: [Accessed 19 July 2012]. Experiments To Show The Growth Of Bacteria- basic techniques. [online] Available at: [Accessed 19 July 2012]. General Microbiological Techniques. [pdf]. Available at: [Accessed 19 July 2012]. Gram Staining. [pdf]. Available at: [Accessed 19 July 2012]. Gram Staining Picture. [online] Available at: [Accessed 19 July 2012]. Motility. [online] Available at [Accessed 19 July 2012]. Selective and Differential Media for Identifying Microorganisms. [online] Available at: [Accessed 19 July 2012]. Read More