Introduction - Central New Mexico Community College | CNM



Unit 7: Supportive, Selective and Differential Media and Streak Isolation PracticeBy Karen Bentz, Patricia G. Wilber, Heather Fitzgerald, and Andrea Peterson, 2018Creative Commons Attribution-NonCommercial 4.0 International License.Introduction Like all living organisms, bacteria require nutrients in order to grow. Basic media contain ingredients such as partially digested milk, soy, yeast extract, or beef broth, which provide nutrients for the growth of many bacteria. T-soy, which you used in solid, liquid, and plate form for your initial inoculations, is an example of a basic medium. Supportive media contain additional ingredients, such as red blood cells, which support the growth of more fastidious (picky) bacteria. In Chocolate agar, a type of supportive medium containing sheep blood cells, the RBCs have been lysed (broken open) to make their contents more readily available to bacteria for growth. Red blood cells are an excellent source of iron, amino acids, and the required bacterial growth factors NAD (factor V) and hemin (factor X).Differential media differentiate between species that grow on the media according to specific metabolic processes the bacteria may have. Differential media have a differential ingredient that allows us to determine the results of the metabolic processes. TSA-blood is an example of a differential medium. Its differential agent (=ingredient) is defibrinated red blood cells. The TSA-blood allows the growth of most bacteria and can distinguish between species based on their ability to produce the enzyme hemolysin which breaks down red blood cells. Bacteria that can hemolyze blood utilize the nutrients and iron in the RBCs for growth. Hemolysin production is associated with pathogenicity. Most of the media we will use in subsequent labs are differential.Selective media select for growth of particular bacteria and inhibit (or select against) growth of other types of bacteria. We will use two types of selective media: MacConkey’s, which selects for Gram(-), non-fastidious species, and against everything else,CNA-blood, which selects for Gram(+) coccus-shaped species, and selects against everything else.In addition to being selective, Both the MacConkey’s and CNA-blood have a differential component, making them doubly interesting.MacConkey’s agar is selective. It contains the selective agents crystal violet and bile salts. These selective ingredients inhibit (select against) the growth of Gram(+) bacteria but allow (select for) the growth of Gram(-) bacteria. The MacConkey’s medium is also differential. If a species is Gram(-) it should be selected for, and thus grow on, the MacConkey’s. If it is selected for and grows, the plate can allow us to distinguish between these species based on their ability or lack of ability to product the enzyme lactase and catabolize the sugar lactose, the differential ingredient in the medium!If an organism is selected against and does not grow, we CANNOT assess lactose utilization.The disaccharide lactose and the pH indicator neutral red are the differential ingredients in the MacConkey’s. Those bacteria that grow and produce the enzyme lactase are able to ferment the lactose sugar in the medium. This will cause a drop in the pH (to less than 7) of the medium, which causes the neutral red to become a bright fuchsia color. Both the colonies and the media will exhibit this color. Organisms that grow, but do not produce the enzyme lactase, do not ferment the ingredient lactose and growth is light purple (due to the crystal violet) or clearish.In this lab, if you have to ask yourself, “Is that fuchsia?”, the answer is, “No!”, because all the species we test that grow and ferment lactose are very good at it and the media changes color dramatically.The Gram(-) bacteria that do produce the enzyme lactase and can ferment the sugar lactose generally live in the intestines and are not pathogenic. These lactose-fermenting normal gut organisms are also called coliforms. The Gram(-) bacteria that do not produce lactase and thus do not ferment lactose, may also be able to live in the gut, but are more likely to be pathogenic.High coliform counts in drinking water and swimming areas is bad because this indicates a large number of gut bacteria in these areas. These are normal, non-pathogenic bacteria, but a high coliform count means there is contamination with sewage. We do not want to drink or swim in poo!CNA-blood agar contains the selective agents colisten and nalidixic acid. Colisten negatively affects the cell membrane of many Gram(-) species, and nalidixic acid inhibits replication of DNA in susceptible species. The result is that CNA-blood selects for Gram(+) coccus-shaped species, and against everything else. The narrow range of species that grow on the CNA-blood and the fact that some beta-hemolytic streptococci strains will develop green (alpha-looking) hemolytic zones on CNA agar is why TSA-blood plates (that are non-selective,) and not CNA-blood plates, are routinely used in diagnostic labs.The CNA-blood agar, like the TSA-blood agar is differential. The differential material in both is defibrinated red blood cells. Bacteria that can metabolize whole RBC’s do so by producing the enzyme hemolysin. In order to assess hemolysis production, (or any differential aspect), the bacterial must first grow on the medium in question. Both the TSA-blood and the CNA-blood can be used to assess hemolysis production, but many species won’t grow on the CNA-blood.If no growth has occurred, red blood cell catabolism CANNOT be assessed.DAY 1: InoculationsChocolate Agar with Blood ’s? by Corrie AndriesMaterialsmetal Inoculating loopmicroincineratorSharpieappropriate personal protective gear (lab coats, gloves, face shield, hair ties)Media 1 MacConkey, Chocolate, TSA-blood, CNA-blood per pair of students1 T-soy agar petri plate per personBacterial cultures, from which to inoculate new media Escherichia coli (Ec), Gram(-)Haemophilus haemolyticus (Hh) Gram(-)Proteus vulgaris, (Pv) Gram(-)Streptococcus oralis (So) Gram(+) Streptococcus pyogenes, (Spy) Gram(+)Staphylococcus saprophyticus, (Ss) Gram(+)ProceduresInoculating Chocolate, TSA-Blood, CNA-blood, and MacConkey AgarWork with a partner for these inoculationsUse a Sharpie to divide the bottom of your plates into thirds. Using the marker, write your initials and the initials of the bacteria on the bottom of your plates. Also include the date and the type of medium you are working with.Inoculate your labeled plate with bacteria, following the pattern for bacteria shown in the diagrams below.Use a sterile loop and for Hh, put on a whole lot of bacteria. For the other species, use a small amount of bacteria. Flame and cool your loop before and after transferring each type of bacteria.When finished inoculating all of your plate media, place them upside down in the appropriate rack. TSA-blood and CNA-blood plates should be placed upside down in a candle jar for incubation. The candle jar provides a low oxygen environment that is required for proper function of the bacterial blood hemolysins. Figure 7.1 Inoculation patterns and species for MacConkey’s, TSA-Blood, Chocolate and CNA- Blood plates.Figures by Karen BentzStreak Isolation PracticeEach student should practice the streak isolation technique on a T-soy plate.Use very little bacteria!!! Overlap less. Don’t forget to flame between each streak!Choose either E. coli or Proteus vulgaris for your streak isolation. Refer back to Unit 3 for a refresher on the streak isolation procedure.Figure 7-2: Pattern for Streak Isolation Procedure Image created by Patricia G. Wilber, 2015Day 2: Results and InterpretationChocolate Agar (Supportive)This medium is supportive because the RBCs in the medium have been partially lysed. Fastidious (picky) bacteria that will not grow on other media may grow on Chocolate agar. Figure 7-3: Sputum Sample Streak-isolated on Chocolate Agar. Accessed 8/31/2015 from but licensed for use by the American Society for Microbiology, Creative Commons Attribution – Noncommercial – No Derivatives 4.0 International license.Insert Photo of Your Chocolate Agar Plate Here:Table 7.1 Chocolate Agar Results:Name of BacteriaSupportive Feature:Did it Grow on Chocolate Agar?Describe Colony GrowthTSA-blood (Differential)This media contains the differential ingredient sheep red blood cells, which will differentiate between bacterial species depending on their ability to lyse the blood in the media. This ability is classified as Alpha (α), Gamma (γ) or Beta (β) hemolysisBeta hemolysis: Complete lysis of the red blood cells resulting in a clear halo where the red blood cells have been removed from the previously red medium underneath the bacterial growth. The red color is GONE. The bacteria produce a high level of hemolysins. The species is likely a pathogen.Alpha hemolysis: Partial digestion of the hemoglobin in the red blood cells. The red hemoglobin in the RBCs is reduced to green methemoglobin, which results in olive green colonies, and sometimes an olive green halo around and under the colonies. The bacteria produce some hemolysins. The species is likely a pathogen.Gamma hemolysis: Growth of the bacteria, but no lysis or digestion of the red blood cells underneath the bacterial growth. The bacterial growth is often a whitish color on the surface of the red medium. The bacteria grow on the medium, but do not produce hemolysins. This is not pathogenic.Figure 7-4: Bacteria showing Alpha (α), Gamma (γ), and Beta (β) hemolysis on blood agarInsert Your TSA-blood Photo Here:Table 7.2 TSA-blood Results:Name of Bacteria Differential Feature:Did it Grow on the TSA-blood?If the Bacteria Grew, What Does the Medium Under the Bacterial Growth Look Like? (clear= beta, olive-green=alpha, red=gamma)MacConkey (Selective and Differential)This medium selects for Gram(-) bacteria, and if the species grows, differentiates for ability to ferment lactose.Type of Cell Wall (selective feature): Gram(-) cells will grow, other types of bacteria will not.Ability to Ferment Lactose: (differential feature): fuchsia bacterial growth and fuschia in the media indicates lactose fermentation by the Gram(-) species. Growth of clear to purplish colonies indicates no lactose fermentation by the Gram(-) species.Figure 7-5: Growth of Bacteria on MacConkey Media. Insert Photo of Your MacConkey Plate Here:Table 7-3. MacConkey Results:Name of BacteriaSelective Feature: Did it grow on MacConkey? (write yes or no)Differential Feature:If bacteria grew, did it turn a fuchsia color? (write yes or no)If it did not grow write “cannot tell”CNA-blood (Selective and Differential)CNA-blood agar selects for Gram(+) cocci, and if the species grows, differentiates for blood hemolysis. See Figure 7.4Insert photo of your CNA-blood plate here.Table 7-4: Results for CNA-bloodName of BacteriaSelective Feature: Did it grow on CNA-blood? (write yes or no)Differential Feature:If bacteria grew, did it hemolyze the blood? (write yes or no)If it did not grow write “cannot tell”Streak Isolation ResultsIn the space below insert a photograph of the results of your streak isolation. The goal is to have eight or more isolated colonies on your plate. How many species do you expect to see on your plate?How many species do you actually see on your plate? If you have more than one species, how did this occur?InterpretationBased on all of your results, explain what you know about the metabolism, cell wall structure, and enzymes of each bacterial species that you tested. Example for a species you did not test: Table 7-5: Interpretation for Pseudomonas aeruginosa:Based on Your Test Results, What Do You Know About This Bacteria’s Cell Wall, Metabolism, and Enzymes?What is the Evidence For or Against this Organism Being a Likely Pathogen?Chocolate agar: 1. The bacteria grew; chocolate agar is a supportive medium and many species of bacteria will grow on it. The colonies were whitish. These test results gave no information about pathogenicity.MacConkey’s agar: 1. The bacteria grew, so it is a Gram(-) organism.2. The bacterial growth was clear to slightly pink/purple. It was not bright fuchsia. This means bacteria cannot ferment lactose. 3. This species lacks the enzyme lactase.Based on this test, Pseudomonas aeruginosa is a possible pathogen because it does not ferment lactose as shown by the clear to slightly pink/purple color of the colonies on the MacConkey. Lack of lactose fermentation indicates a possible pathogen. TSA-blood: 1. The bacteria grew.2. The bacteria showed Beta hemolysis, which means the blood cells were completely lysed.3. The bacteria produced the enzyme hemolysin.The complete lysis of the blood (Beta hemolysis) indicates A-blood:The bacteria did not grow.The bacteria is not a Gram(+) cocci.Table 7-6: Interpretation for Escherichia coli:Based on Your Test Results, What Do You Know About This Bacteria’s Cell Wall, Metabolism, and Enzymes?What is the Evidence For or Against this Organism Being a Likely Pathogen?MacTSATable 7-7: Interpretation for Proteus vulgaris:Based on Your Test Results, What Do You Know About This Bacteria’s Cell Wall, Metabolism, and Enzymes?What is the Evidence For or Against this Organism Being a Likely Pathogen?MacCNATable 7-8: Interpretation for Haemophilis haemolyticus:Based on Your Test Results, What Do You Know About This Bacteria’s Cell Wall, Metabolism, and Enzymes?What is the Evidence For or Against this Organism Being a Likely Pathogen?ChocolateTSATable 7-9: Interpretation for Staphylococcus saprophyticusBased on Your Test Results, What Do You Know About This Bacteria’s Cell Wall, Metabolism, and Enzymes?What is the Evidence For or Against this Organism Being a Likely Pathogen?ChocolateTSATable 7-10: Interpretation for Streptococcus pyogenes:Based on Your Test Results, What Do You Know About This Bacteria’s Cell Wall, Metabolism, and Enzymes?What is the Evidence For or Against this Organism Being a Likely Pathogen?MacCNATable 7-11: Interpretation for Streptococcus oralis:Based on Your Test Results, What Do You Know About This Bacteria’s Cell Wall, Metabolism, and Enzymes?What is the Evidence For or Against this Organism Being a Likely Pathogen?ChocolateCNAPost Lab QuestionsName: _____________________Fill in the blanks in the table below.Table 7-12 Media ingredientsMediumSelective Ingredient(s)Differential IngredientpH IndicatorCNA-bloodMacConkeyTSA-bloodnonenoneChocolate AgarnonenonenoneUsing your results and interpretation information from this lab, give the name of one bacterial species for each of the following descriptions: Be sure to write the names of your bacteria using proper scientific nomenclature. All of the following are acceptable:Staphylococcus aureus (underline genus and species names) STAPHYLOCOCCUS AUREUS (all capital letters) Staphylococcus aureus (italicized genus and species)Gram(-), lactose fermenter:What is your evidence for this choice?Requires lysed RBCs to grow:What is your evidence for this choice?Produces no hemolysin.What is your evidence for this choice?Gram(-), does not ferment lactose:What is your evidence for this choice?Gram(+), coccus, produces a little hemolysin:What is your evidence for this choice?Gram(+), coccus, produces a lot of hemolysin:What is your evidence for this choice?Create a set of study cards for the each of the four types of media that you used in this lab. Figure 7-6. An example of a study card for the MacConkey medium.Figure created by Karen BentzTable 7-13: Characteristics of Chocolate, TSA-blood MacConkey and CNA-blood agarsMediumSupportive IngredientMechanism of SupportChocolate Agarlysed sheep red blood cellsThe lysed blood provides the bacterial growth factors NAD (factor V) and hemin (factor X), which were inside red blood cells and are released by the lysing. The agar is named for the color and contains no actual chocolate.MediumDifferential IngredientMechanism of DifferentiationTSA-bloodwhole sheep red blood cellsBacteria can be differentiated according to their ability to produce enzymes called hemolysins that digest the sheep blood in the medium.Bacterial Enzyme: HemolysinBeta hemolysis: complete digestion of the blood, the medium under the bacteria is light to clear. Bacteria produce a high level of hemolysins. The bacteria is a likely pathogen.Alpha hemolysis: partial digestion of the blood hemoglobin, the medium has an olive-green color. Bacteria produce some hemolysins. The bacteria is a possible pathogen.Gamma hemolysis: no hemolysis. Bacteria grow on top of the medium, but the blood in the medium underneath the cells remains a red color. The bacteria do not produce hemolysins, and are probably not pathogenic. MediumSelective IngredientsMechanism of SelectionDifferential IngredientMechanism of DifferentiationpH indicatorOf Special NoteCNA-bloodcolistin and nalidixic acid (CNA)Growth indicates a Gram(+) coccal organism. Colistin negatively affects the cell membrane of many Gram(-) species, and nalidixic acid inhibits replication of DNA in susceptible species Defibrinated sheep red blood cellsIf the bacteria grow and produce the enzyme hemolysin, they can digest the red blood cells in the media.Bacterial Enzyme: hemolysinnoneBeta hemolysis: bacterial growth, complete digestion of the blood. The medium under and around the bacteria is white to clear. Bacteria produce high levels of hemolysin.Alpha hemolysis: bacterial growth, partial digestion of the hemoglobin, the medium and the colonies have an olive color. Bacteria produce some hemolysin.Gamma hemolysis: no hemolysis. Bacteria grow on top of the medium, but the blood in the medium underneath the cells retains red color. No production of hemolysin.MediumSelective IngredientsMechanism of SelectionDifferential IngredientsMechanism of DifferentiationpH indicatorOf Special NoteMacConkeycrystal violetandbile saltsGrowth indicates a Gram(-) organism. Gram (+) organisms will not grow because the crystal violet and bile salts interfere with the function of the peptidoglycan layer. Lactose and neutral redIf the bacteria grow and produce lactase, they can ferment lactose. The acid waste they produce will cause the pH indicator to turn a fuchsia color. Bacterial Enzyme: lactase neutral red Neutral red is a red color at neutral pH, and turns fuchsia if the pH is acidic. (<7)The bacterial cells that produce lactase and ferment lactose become permeable to the pH indicator and absorb it, turning the cells as well as the medium fuchsia. Pink (lactose fermentation) = coliform = generally non pathogen; Not pink (no lactose fermentation) = non-coliform = possible pathogen ................
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