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LAB 9: Single Colony Isolation

Partial Materials in the Final Lab Exam (Nov. 28/29): Labs #9-23 (All labs after the first lab exam) Please also read the review sheets I handed out on Nov. 20 in lab. I will have office hour M/Th: 9:15 a.m. - 10:15 a.m. at DH 553 or DH 543 (my lab). LAB 9: Single Colony Isolation.

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LAB 9: Single Colony Isolation

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  1. Partial Materials in the Final Lab Exam (Nov. 28/29): Labs #9-23 (All labs after the first lab exam)Please also read the review sheets I handed out on Nov. 20 in lab. I will have office hour M/Th: 9:15 a.m. - 10:15 a.m. at DH 553 or DH 543 (my lab)

  2. LAB 9: Single Colony Isolation Know how to obtain single colonies through the “Streak for Isolation” on an agar plate technique.

  3. Streak Plate technique

  4. Lab #9 Micrococcus luteus Staphylococcus aureus All in NA plates Serratia marcescens Klebsiella rosea

  5. Blood agar(BA) is a differential medium. - Some bacteria produce an enzyme that is able to lyse RBCs – this process is hemolysis. - By growing bacteria on blood agar we can determine if the bacteria produce hemolysin and thus lyse the RBCs. - Blood agar is NA to which sheep RBCs have been added. - If hemolysin is produced by the bacteria it will be secreted into the medium and the RBCs will be lysed (the medium will be clear rather than red). - So presence of clearing around the bacterial growth indicates hemolysis. - Growth on BA differentiates between the hemolytic and non-hemolytic bacteria.

  6. Gamma hemolysis = No hemolysis Alpha hemolysis = Partial Beta hemolysis = Complete

  7. Lab 10: Stock and Working Slants • Why did we prepare a stock and a working stock slant for the unknown? • Why did we grow the unknown in different media and under different conditions?

  8. “Working” “Stock”

  9. Lab 11: Simple Staining & Bacterial Smear Understand simple, negative, and positive staining. Know how to prepare a bacteria smear

  10. Demos: simple stains of: Neisseria (diplococci) Pseudomonas (bacilli)

  11. Lab 12: Differential Staining (Gram Stain) Know the entire Gram Staining procedure and the function of each step. Know the Endospore procedure (in Appendix, p. 121)

  12. GRAM STAIN E coli (Gm -) Staph epidermidis (Gm+)

  13. ENDOSPORE STAIN Bacillus megaterium See Appendix IV, p. 121 Outcome for endospore + for Micr20 Bacillus anthracis Clostridium tetani

  14. Cell Arrangements:

  15. Lab 13: Selective and Differential Media • EMB: Eosin-Methylene Blue a. Differential and selective properties. b.Contains bile salts and the dyes eosin and methylene blue; all inhibitory to Gram-positive bacteria (e.g. Staphylococcus aureus). c. Selects for Gram-negative bacteria (e.g. Escherichia. coli). d.Differentiates lactose fermenting (dark color with metallic sheen) from non-lactose-fermenting (colorless) bacteria.

  16. Salmonella pullorum E. coli Staph. epidermidis Staph. aureus

  17. Lab 13: Selective and Differential Media TGA: Tellurite Glycine Agar a.Selects for coagulase-positive staphylococci. b.Differential: coagulase-positive cocci form black colonies. c.Coagulase-negative cocci are generally inhibited. The ones that grow are gray. d.Most Bacilli and Pseudomonas (Gm+) are inhibited. e.Proteus sp rarely grows and form brown colonies.

  18. Staph. aureus E. coli Staph. epidermidis Salmonella pullorum

  19. Know all the media we covered in Micro20 since lab #9: The purpose of the medium, how to read a positive and a negative result, what those results mean, and the MAJOR components of the medium.

  20. Lab 14: Antibiotic Sensitivity • Antibiotics are chemicals that are produced by other bacteria/fungi • that have the ability to prevent other organisms (bacteria) from • growing or killing them. • Sensitivity X Resistance to antibiotics. • Bacteriostasis (stopping bacterial growth) X bacteriocide (killing of bacteria). • Broad spectrumantibiotics- effective against a wide range of bacteria (G+ and G-). • Narrow spectrumantibiotics - effective against a small specific group of bacteria (either G+ or G-).

  21. Lab 14: Antibiotic Sensitivity; Disc Diffusion Method

  22. LAB 15: Catalase, Amylase, Gelatinase (Proteinase), MRVP • MRVP, see Appendix IV, p.118-119

  23. CATALASE H2O2 Negative Positive Enterococcus faecalis Streptococcus aureus

  24. Amylase: Starch Hydrolysis BEFORE AFTER Flood with Iodine solution E.coli Bacillus subtilis E coli - (neg.) Bacillus subtilis + (pos.)

  25. Gelatinase test: Plate was flooded with Frazier’s Developer Gelatinase + Gelatinase negative

  26. LAB 16: Urease, SIM agar, Citrate

  27. UREASE • UREASE TEST: Urease is an enzyme that breaks the carbon-nitrogen bond of amides (e.g. urea) to form carbon dioxide, ammonia, and water. Members of genus Proteus are known to produce urease. When urea is broken down, ammonia is released and the pH of the medium increases (becomes more basic). This pH change is detected by a pH indicator that turns pink in a basic environment. A pink medium indicates a positive test for urease.

  28. SIM agar • SIM = Sulfide, Indole, Motility. • INDOLE TEST: Indole is a component of the amino acid tryptophan. Some bacteria have the ability to break down tryptophan for nutritional needs using the enzyme tryptophanase. When tryptophan is broken down, the presence of indole can be detected through the use of Kovacs' reagent. Kovac's reagent, which is yellow, reacts with indole and produces a red color on the surface of the test tube.

  29. SIM agar

  30. MOTILITY Motile bacterium Non-motile bacterium (e.g. Staph aureus) (e.g. Pseudomonas aeruginosa)

  31. Citrate: The Simmon’s Citrate medium tests the ability of the bacteria culture to be able to use citrate as the sole C source. Bacteria that are able to produce the enzyme citrase are able to transport the citrate into the cell and use it as a source of C. Since the medium does not contain any other source for C, only those bacteria that can produce citrase are able to grow in this medium. When cultures are able to use the citrate they break it down, producing sodium bicarbonate, which changes the pH of the medium to alkaline. The pH indicator in the medium (bromothymol blue) changes to a blue color from its original green color. +

  32. LAB 17: Carbohydrate Utilization

  33. Lab 17: KIA medium • C = Uninoculated 3 = Glucose fermenter + H2S producer • 1 = Non-fermenter 4 = Glucose+Lactose fermenter, gas • 2 = Glucose fermenter 5 = Gluc + Lact ferm + H2S producer

  34. Lab 17: Fermentation of Carbohydrates F- tubes

  35. SUGAR Fermentation Detection is based on acid production due to sugar fermentation. The pH indicator is PHENOL RED. Phenol red turns yellow under acidic conditions. Hence, yellow means a positive result.

  36. The ability to ferment specific sugars is dependant on the ability of the bacterium to produce the specific enzymes required for the transport and metabolism of that particular sugar. Thus fermentation of various sugars can be used to characterize bacteria. The F-tubes use phenol red in the medium as pH indicator and the use of inverted tubes to detect production of gases. Results are recorded as Negative (no metabolism); Acid (+ reaction); Acid + Gas (+ with gas production).

  37. LAB 18: Unknown & Single Colony Isolation Know how to use the Dichotomous Key to identify a bacterium based on morphology, Gram staining, endospore production, and various metabolic reactions. See p.58-61of lab manual.

  38. LAB 19: Pour Plate • Pour Plate Technique • Serial Dilution • Colony Forming Unit (CFU) • Quantification of Bacteria in Cell/ml

  39. 1 2 3 4 5 • *Best 30-300 CFU • CFU = 100 • Dilution = 1000 • Hence 100 X 1,000 = • 100,000 = 1x105 (1:1 1:10 1:100 1:1000 1:10,000) TMTC 1000 400 100* 20 (CFU) Lab 19: Pour Plate Dilution Series: 100 10-1 10-2 10-3 10-4 (Dilution) • Bacteria Enumeration 1ml 1ml 1ml 1ml

  40. Bacteria Enumeration 1x10-5 1x10-6 Cell /ml= (CFU X dilution factor) / volume

  41. LAB 20: Most Probable Number • MPN method, MPN table • Durham tubes • Presumptive, Confirmed, and Completed tests

  42. Lab 20: Most Probable Number (MPN) Bacteria Enumeration (Presumptive) MPN method: 1st- Presumptive test: growth on lauryl tryptose broth 2nd - Confirmed test: on Eosine-Methylene Blue Agar (EMB) 3rd - Completed tests

  43. LAB 21: Phage Characterization and Quantification • Plaque, Plaque Forming Unit (PFU) • Serial Dilution, Phage quantification • T1phage

  44. BACTERIOPHAGE 1x10-4 Dilution Plaque (clear zone) 1x10-6 Dilution

  45. LAB 22: Bacterial Aggutination & Immunoprecipitation

  46. Immunoprecipitation • - is the reaction between a soluble antigen and its specific antibodies • soluble antigens are smaller and in solution; complexing with antibodies make these • bigger and they fall out of solution as a precipitate –visible to the eye. Antibody specificity known (toxin, protein, etc.) Antigen presence or identity not known (?) Precipitationreaction between antibody and soluble antigen

  47. Immunoprecipitation Antibody specificity known (toxin, protein, etc.) Antigen presence or identity not known (?) Precipitationreaction between antibody and soluble antigen

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