Exercise IX Selective, Differential, and Differentially-Selective Media Different microorganisms have varied physical growth requirements which must be met for them to be successfully cultured. In addition to their physical growth requirements, each microorganism also has specific nutritional requirements. Media can also be utilized to provide added information about a group of microorganisms. By adding ph indicators, alternative sources of carbon or nitrogen, or inhibiting chemical agents, the various nutrients an organism is capable of utilizing, the by-products of metabolism produced, or the organism s ability to withstand adverse chemical agents may be determined. Selective and differential media are used to select or identify particular bacteria and aid in the primary isolation of bacteria. Selective media allows certain types or groups of bacteria to grow and prevents or inhibits others from growing. These media are used to select (isolate) specific groups of bacteria. Chemical substances are incorporated into the media that inhibit the growth of one type of bacteria while permitting the growth of another, thus facilitating bacterial isolation. Selective inhibition of unwanted microorganisms is accomplished by providing unfavorable ph environments or the incorporation of bacteriostatic agents, dyes, antibiotics, salts, or special inhibitors that affect the metabolism or enzyme systems of the particular microorganisms. Crystal violet and bile salts are inhibiting to Gram positive bacteria, but not to Gram negative bacteria. Differential media are used to differentiate closely related bacteria or groups of bacteria. A differential media incorporates certain reagents or chemicals into the media, which results in recognizable reactions following incubation. Owing to the presence of certain dyes or chemicals in the media, the bacteria will produce characteristic changes or growth patterns that are used for identification or differentiation. Differential media incorporates ingredients that cause certain bacteria to develop a different appearance from other bacteria growing on the same medium. Other differential media include a ph indicator to demonstrate that a specific nutrient has been utilized by the bacteria present, with a resulting change of ph. Carbohydrate fermentation will occur in Triple Sugar Iron (TSI) which is a differential media containing a ph indicator phenol red. If a fermentable sugar, such as glucose, is provided in the media, as well as a ph indicator, it will produce, after incubation, a visible distinction between bacteria which are fermenters and those that are non-fermenters. Differential media are especially useful when working with closely related groups of bacteria with nearly identical morphology and other biological characteristics. They incorporate chemical components that, following incubation, produce a characteristic change in the appearance of the bacteria and/or the medium surrounding the colonies, which permitted diffusion. 60
More often, media are selective and differential rather than one or the other. Differentially-selective media combine aspects of both selective and differential media. These media provide chemicals, such as dyes, that inhibit the growth of some bacteria and also provide other chemicals, such as carbohydrates, that allow the differentiation between the selected bacteria (those that are growing). Eosin methylene blue (EMB), which is selective for Gram negative bacteria because of the bacteriostatic effect of the dye on Gram positive bacteria, also contains lactose. EMB is also a differential medium; it differentiates between lactose-fermenting and non-lactose fermenting bacteria. Gram negative bacteria capable of fermenting lactose take some dye into the cell, which causes the colonies to appear dark purple and may have pink edges. Colonies of bacteria that are unable to ferment lactose appear colorless. Differential Media Triple Sugar Iron (TSI) Triple Sugar Iron (TSI) is a differential medium used for the identification of Gram negative enteric (intestinal) bacteria. The media indicates the ability of bacteria to ferment carbohydrates (lactose, sucrose (saccharose), and glucose) with the formation of acid and gas, and the ability to produce hydrogen sulfide gas. The media contains a variety of substances that enteric bacteria can utilize, including, several carbohydrates, proteins, and sodium thiosulfate. Different enteric bacteria utilize these substances differently, so they can be distinguished based on their pattern of utilization. The fermentable carbohydrates are lactose, sucrose, and glucose and TSI is designed to differentiate bacteria by their lactose fermentation, sucrose fermentation, and glucose fermentation. When carbohydrates are fermented gas is released and appears as bubbles in the butt of the tube. The gas produced may be so strong that the butt may split (fracture) and part of the slant may rise near the top of the tube. Sodium thiosulfate is the source of sulfur and reduction produces hydrogen sulfide gas (H 2 S). It contains ferrous sulfate which reacts with H 2 S to form a black precipitate of ferrous sulfide. The ph indicator in TSI is phenol red. Phenol red is red (alkaline) at the initial ph of 7.4 and turns yellow (acid) when the ph falls below 6.8. When inoculating TSI, an inoculating needle is used to stab the butt, followed by streaking the slant as the needle is removed. The following results may occur after incubation: 1. Alkaline butt/alkaline slant (ALK/ALK) No carbohydrates fermented or hydrogen sulfide produced. No acids are produced in the slant or butt to cause a drop in ph of the medium. The butt and slant remain red or turn a dark red due to the production of alkaline products from peptone in the medium. 61
2. Acid butt/alkaline slant (AC/ALK) Glucose fermentation only. Yellow butt and red slant. Glucose fermentation results in the formation of acids that lower the ph in the butt. The butt turns yellow while the slant remains red or turns dark red. 3. Acid butt/alkaline slant, H 2 S (AC/ALK, H 2 S) Glucose fermentation only with hydrogen sulfide production. The butt turns yellow and black and the slant remains red or turns dark red 4. Acid/Acid (AC/AC) Lactose and/or sucrose fermentation results in the formation of acids that lower the ph of the butt and slant. The butt and slant turn yellow. 5. Acid/Acid, H 2 S (AC/AC, H 2 S) Lactose and/or sucrose fermentation with hydrogen sulfide production. The butt turns yellow and black and the slant turns yellow. If bubbles or splitting appears in the butt, or if the entire medium has been pushed upward from the bottom of the tube, gas has been produced and acids have been produced by the fermentation of carbohydrates. Escherichia coli and Enterobacter aerogenes produce gas, whereas Serratia marcescens does not. Differentially Selective Media Eosin Methylene Blue Agar (EMB) Eosin Methylene Blue agar (EMB) is a differentially-selective media used for the detection and isolation of Gram negative enteric (intestinal) bacteria. A combination of eosin and methylene blue is used as an indicator, which gives a distinct difference between bacteria that ferment lactose and those that do not. Sucrose is also included in the medium because certain members of the coliform group ferment sucrose more readily than lactose. EMB differentiates lactose fermenters from non-lactose fermenters. Lactose fermenters have dark purple colonies, some with pink edges, and non-lactose fermenters have colorless colonies. Methylene blue also acts as an inhibitor to Gram positive bacteria. The media selects for Gram negative bacteria, while inhibiting Gram positive bacteria. 62
Escherichia coli, Enterobacter aerogenes, and Serratia marcescens are selected for on EMB, whereas Staphylococcus epidermidis, Streptococcus mutans, and Micrococcus luteus are inhibited. Escherichia coli produce colonies with a characteristic green metallic sheen. Enterobacter aerogenes produces colonies that have a dark or purple center with pink around the edges. Serratia marcescens appears similar to E. aerogenes but tends to spread more from the streak lines. MacConkey s Agar (MAC) MacConkey s Agar (MAC) is a differentially-selective media used to isolate and differentiate enteric (intestinal) bacteria and differentiate members of the Enterobacteriaceae based on their ability to ferment lactose. MAC contains lactose, crystal violet, bile salts, and the ph indicator neutral red. The growth of Gram positive bacteria is inhibited by the crystal violet and bile salts in the media. The inclusion of bile salts creates favorable conditions for the growth of intestinal bacteria. The medium selects for Gram negative bacteria such as Escherichia coli, Enterobacter aerogenes, and Serratia marcescens, while inhibiting Gram positive bacteria such as Staphylococcus epidermidis, Streptococcus mutans, and Micrococcus luteus. MAC also differentiates between lactose fermenters and non-lactose fermenters. Neutral red dye is a ph indicator that is colorless above a ph of 6.8 and red at a ph less than 6.8. Acid accumulation from the lactose fermenters turns the dye red. Gram negative bacteria that produce acid from lactose will produce pink to red colonies, while those that do not will be colorless (transparent). If an organism produces great quantities of acid rapidly, the acid will act on the bile salts in the medium and cause absorption of the neutral red indicator in the medium and the bacteria and the media will have a deep pink-red color. Escherichia coli produces greater quantities of acid from lactose than the other species. When this occurs, the medium surrounding the growth also becomes red because of the action of the acid that precipitates the bile salts, followed by absorption of the neutral red; the colonies are red to pink. Enterobacter aerogenes produces pink and mucoid colonies while Serratia marcescens produces red colonies. Mannitol Salt Agar (MSA) Mannitol Salt Agar (MSA) is a differentially-selective media used for the isolation of pathogenic staphylococci. MSA contains 7.5% sodium chloride (NACl), mannitol, and the ph indicator phenol red. Phenol red is red at a ph of 6.8 to 8.4, pink at 8.4, and yellow below 6.8. The sodium chloride makes the media selective for Staphylococcus as other organisms grow sparsely or are inhibited by the high concentration of salt. Gram negative bacteria are inhibited and other Gram positive will grow to some extent. 63
The ph indicator phenol red differentiates pathogenic staphylococci from nonpathogenic staphylococci. A yellow zone or halo forms around colonies of pathogenic staphylococci. The bacteria ferment the mannitol, forming an acid which changes the phenol red from red to yellow. Mannitol is a substrate for the enzyme system of pathogenic Staphylococcus aureus, which metabolizes mannitol, converting it to acid waste products. The acid end products of mannitol fermentation diffuse into the medium, which changes the indicator from red to yellow producing a yellow zone or halo around the colonies due to the effects of the ph change in the phenol red. Non-pathogenic Staphylococcus epidermidis forms small white colonies, and the color of the media does not change because mannitol is not fermented. Micrococcus luteus does not ferment mannitol and produces small yellow colonies. Materials Needed culture of Escherichia coli culture of Enterobacter aerogenes culture of Serratia marcescens culture of Staphylococcus epidermidis culture of Streptococcus mutans culture of Micrococcus luteus culture of Bacillus subtilis 3 sterile Triple Sugar Iron agar slants 2 sterile Eosin Methylene Blue plates 2 sterile MacConkey s agar plates 2 sterile Mannitol Salt agar plates Bunsen burner striker inoculating loop inoculating needle wax pencil test tube rack goggles gloves spray bottle of disinfectant paper towels 64
Procedure 1: Triple Sugar Iron (TSI) 1. Obtain a culture of Escherichia coli or Enterobacter aerogenes. 2. Using a wax pencil, label a sterile TSI tube with the initials of the microorganism and the initials of someone in the group. 3. Using the inoculating needle and aseptic technique obtain a sample of the bacteria. 4. Insert the inoculating needle into the sterile TSI tube without touching the sides of tube. 5. Insert the needle half way into the butt. 6. Remove the needle from the butt and streak the slant using a zigzag pattern. 7. Repeat the procedure using Serratia marcescens and either Staphylococcus epidermidis, Streptococcus mutans, Micrococcus luteus, or Bacillus subtilis. 8. Place all three tubes in a test tube rack and place a piece of tape across the top. 9. Place the test tube rack in the incubator. Procedure 2: Eosin Methylene Blue (EMB) 1. Obtain a culture of Escherichia coli and Enterobacter aerogenes. 2. Place a sterile EMB plate upside down and using a wax pencil, divide the plate in half and label with the initials of someone in the group. 3. Using a wax pencil, label each half with the initials of the bacteria to be transferred. 4. Using an inoculating loop and aseptic technique, transfer a sample of Escherichia coli to one half of the media using the streak plate method. 5. Using an inoculating loop and aseptic technique, transfer a sample of Enterobacter aerogenes to the other half of the media using the streak plate method. 6. Repeat the procedure on another sterile EMB plate using Serratia marcescens and either Staphylococcus epidermidis, Streptococcus mutans, Micrococcus luteus, or Bacillus subtilis. 7. Turn the plates upside down, tape them together and place them in the incubator. 65
Procedure 3: MacConkey s Agar (MAC) 1. Obtain a culture of Escherichia coli and Enterobacter aerogenes. 2. Place a sterile MAC plate upside down and using a wax pencil, divide the plate in half and label with the initials of someone in the group. 3. Using a wax pencil, label each half with the initials of the bacteria to be transferred. 4. Using an inoculating loop and aseptic technique, transfer a sample of Escherichia coli to one half of the media using the streak plate method. 5. Using an inoculating loop and aseptic technique, transfer a sample of Enterobacter aerogenes to the other half of the media using the streak plate method. 6. Repeat the procedure on another sterile MAC plate using Serratia marcescens and either Staphylococcus epidermidis, Streptococcus mutans, Micrococcus luteus, or Bacillus subtilis. 7. Turn the plates upside down, tape them together and place them in the incubator. Procedure 4: Mannitol Salt Agar (MSA) 1. Obtain a culture of Staphylococcus epidermidis and Micrococcus luteus. 2. Place a sterile MSA plate upside down and using a wax pencil, divide the plate in half and label with the initials of someone in the group. 3. Using a wax pencil, label each half with the initials of the bacteria to be transferred. 4. Using an inoculating loop and aseptic technique, transfer a sample of Staphylococcus epidermidis to one half of the plate using the streak plate method. 5. Using an inoculating loop and aseptic technique, transfer a sample of Micrococcus luteus to the other half of the media using the streak plate method. 6. Repeat the procedure on another sterile MSA plate using Bacillus subtilis and either Escherichia coli, Enterobacter aerogenes, Serratia marcescens, or Streptococcus mutans. 7. Turn the plates upside down, tape them together and place them in the incubator. Clean-up Procedure 1. Return the cultures, inoculating needle and loop and other materials to the appropriate place. 2. Spray and wipe the table top with disinfectant. 66
Procedure - Second Lab Period 1. Obtain the TSI slants, EMB plates, MAC plates, and MSA plates from the incubator. 2. Note the color of the colonies, type of growth, and the color of the media for each of the bacteria on the TSI slants, EMB plates, MAC plates, and MSA plates. 3. Dispose of the slants and plates in the waste bin. 4. Spray and wipe the table top with disinfectant. 67
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