Microbiology: Understanding Different Types of Culture Media and Their Uses, Study notes of Microbiology

Download Microbiology: Understanding Different Types of Culture Media and Their Uses and more Study notes Microbiology in PDF only on Docsity! Exercise 14 USE OF SELECTIVE & DIFFERENTIAL MEDIA & REPLICA PLATING TECHNIQUES Introduction Heterotrophic organisms require preformed organic compounds for their sources of carbon, while autotrophic forms can synthesize all or nearly all of their essential organic compounds from inorganic materials. Organisms that are obligate intracellular parasites are referred to as hypotrophs. Most of the microorganisms worked with in this microbiology class are heterotrophic and can be cultured on various mixtures of nutritive materials. Such mixtures are called media (singular medium) and have long been an integral part of microbiology. Some important ingredients of media include, carbon and energy sources, nitrogen sources, vitamins, growth factors, and essential minerals. Culture media must be adjusted to a suitable pH, and have osmotic pressures compatible with bacterial cells (usually isotonic). Various media can be categorized as follows: Complex media = Media containing the nutrients required for microbial growth, but in crude form, i.e., the various components and their exact quantities are not known (e.g., Nutrient agar, Brain Heart Infusion, Tryptic Soy agar, etc.). Defined media = Media providing required nutrients in relatively pure chemical form and in specified concentrations (e.g., Pseudomonas and Azotobacter enrichment media). Enriched media = Media containing cells, tissue or tissue fractions (e.g., Blood agar and Chocolate agar). Differential media = Media supporting the growth of various types of microorganisms while providing an environment that makes it easy to distinguish various different forms (e.g., EMB, MAC, T-7, MSA, etc.). Selective media = Media providing a means of isolating a particular species or type or microorganisms, while inhibiting the growth of others (e.g., EMB, MAC, T-7, MSA, etc.). Some Examples of Selective and Differential Media: Mannitol Salt Agar (MSA) Mannitol Salt Agar (MSA) is a selective medium for the isolation of pathogenic and non-pathogenic Staphylococci. Growth of most other organisms is inhibited by a high salt content (7.5% NaCl). MSA also acts as a differential medium for the differentiation of organisms that can ferment mannitol from those that cannot. Staphylococcus aureus colonies grow readily and are surrounded by yellow zones (can ferment mannitol), while non-pathogenic staphylococci such as Staphylococcus epidermidis produce small colonies surrounded by red or pink zones (cannot ferment mannitol). The color differences visible in MSA are due to the presence of phenol red, a pH indicator. Acid produced by mannitol fermenting bacteria will cause the color of the phenol red indicator to change from red (neutral pH) to yellow (acidic = low pH). MacConkey’s Agar (MAC) MacConkey’s agar (MAC) is a medium that tends to select for the growth of Gram-negative organisms while inhibiting the growth of Gram-positives. At the same time, it is a differential medium that allows for the differentiation of organisms that can ferment lactose from those that cannot. MacConkey’s Agar is recommended for use in the detection and isolation of all types of dysentery, typhoid, and paratyphoid bacteria (Salmonella). It promotes the development of these organisms and at the same time differentiates them from lactose-fermenting Gram-negative bacilli such as Escherichia coli. Isolated colonies of lactose fermenting bacteria (coliforms) are opaque pink in color, while Salmonella colonies are uncolored and translucent. Differentiation is most clear with well-isolated colonies. The pH indicator present is neutral red, and accumulates within and sometimes around colonies able to ferment lactose. Note - MAC sorbitol agar (MacConkey’s agar made with sorbitol instead of lactose) may be used to differentiate E. coli strain O157: H7 (Enterohemorrhagic E. coli) from other strains because they cannot ferment sorbitol. Eosin Methylene Blue Agar (EMB) Eosin Methylene Blue (EMB) is a medium that tends to select for the growth of Gram-negative rods (bacilli). At the same time it provides a distinct differentiation between lactose fermenting colonies, and those that cannot ferment lactose. Lactose fermenting colonies appear dark (sometimes having a metallic green sheen) or have dark centers with light peripheries, while non- fermenting forms remain uncolored and nearly transparent. Fermentative colonies lower the pH, absorb the dyes present, and then refract light causing iridescent color (the metallic green sheen). Tergitol 7 Agar (T-7) Tergitol 7 agar is a selective medium for Escherichia coli and other members of the coliform group. This medium contains the pH indicator Bromothymol blue, and permits the unrestricted growth of all coliform bacteria while inhibiting Gram-negative spore-formers and Gram-positive organisms. E. coli grown on Tergitol 7 produces yellow colonies surrounded by yellow zones, Enterobacter produce large mucoid colonies usually surrounded by yellow zones, and Salmonella, Proteus, and other non-lactose fermenters produce clear (transparent) colonies usually surrounded by blue zones. Bromothymol blue is green when pH is neutral, turns yellow in acid and blue in alkaline environments. Hektoen Enteric Agar (HEK) Hektoen enteric agar is a selective and differential medium used for the isolation and differentiation of Salmonella and Shigella from other Gram-negative enterics. This medium contains the pH indicators Bromthymol blue and acid fuchsin. The presence of bile salts and indicators inhibits the growth of most Gram-positive organisms, while thiosulfate and ferric ammonium citrate cause H2S producing colonies to form black precipitate. Coliforms such as E. coli, that can rapidly ferment the carbohydrates present (lactose, saccharose and salicin), will form salmon pink to orange, opaque colonies surrounded by bile precipitate. Shigella species form semi-transparent green colonies and Salmonella (as well as some Proteus) species form green colonies with black centers. Questions: 1. What types of bacteria can grow on MacConkey’s agar, on EMB, on T-7? Why? 2. What morphological differences can you detect between the colonies growing on MacConkey’s agar and EMB? What do these morphological differences indicate? 3. Which media would you use to isolate the following: Staphylococcus, Streptococcus, Shigella and Neisseria. Why did you choose the media you did? 4. What aspect of blood agar makes it a differential medium? NOTES, OBSERVATIONS & ADDITIONAL INFORMATION Name________________________________ Lab Section________________ WORKSHEET Exercise 14 Replica Plating Techniques Goals: __________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ Materials & Methods: Replica Plating Technique Master plate: Draw a copy of the bacterial colony arrangement present on your master plate in the space provided below, and label the individual colonies as specifically as possible. Media used (list IN ORDER of use): __________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ Incubation temperature:____________ Duration of incubation:______________________________ Additional Notes: _________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ Data & Results: Complete the following table. Include whether or not the organisms grew on the medium as well as observations of colony and medium color. Type of Medium Organisms Conclusions: Was the technique successful?________ How do you know? _______________________________ ________________________________________________________________________________ Based upon your data, what can you conclude about the following organisms? Staphylococcus aureus: _____________________________________________________________ ________________________________________________________________________________ Staphylococcus epidermidis: _________________________________________________________ ________________________________________________________________________________ Bacillus: _________________________________________________________________________ ________________________________________________________________________________ Escherichia coli: __________________________________________________________________ ________________________________________________________________________________ Salmonella: _______________________________________________________________________ ________________________________________________________________________________