Microbiology Lab Assignment

Shakira Jarvis
Microbiology Lab Assignment

Laboratory Assignment Outline

1. Check in & The Microscope a. Review of proper lab etiquette. i. Review laboratory syllabus and b. Review of the Parts of a Microscope ii. Review of lab exercises about different types of Microscopes 2. Survey of Microorganisms c. Viewing, drawing, and describing several types of fungi, algae, and Bacteria iii. Chlamydomonas iv. Spirogyra v. Penicillum mycelium condidiophores vi. Cladophora vii. Volvox viii. Paramecium caudatum ix. Escherichia coli x. Bacillus subtilis xi. Staphylococcus aureus xii. Aspergillus condidiophores xiii. Amoeba Proteus
xiv. Budding Yeast xv. Diatoms ( Marine) xvi. Peziza Apothecium xvii. Coprinus Mushroom xviii. Tryanosoma lewisi xix. Plasmodium malariae 3. Aseptic Technique, Smear Preparation, Simple & Negative Staining d. Aseptic Technique xx. Work area disinfection xxi. Flaming loops/ needles before and after use xxii. Flaming the top of tubes xxiii. Washing hands before and after lab e. Smear Preparation xxiv. Can be prepared from either broth of agar plate 1. Broth a. No water added b. Flame loop every time it needs to be reintroduced to the culture c. Spread all over slide d. Let dry & heat fix 2. Agar e. Add drop of water to CLEAN slide f. Flame loop, let cool, get loop full of bacteria g. Spread bacteria through out drop of water h. Spread all over slide i. Let air dry then Heat fix f. Simple Stain – xxv. one dye ( Methylene blue) stains the organism xxvi. can only determine morphology and arrangement xxvii. Procedure 3. Prepare smear 4. Add Methylene blue ( 30 sec. ) 5. Wash w/ water 6. Blot dry 7. View g. Negative Stain – xxviii. One dye ( Nigrosin = [acidic dye w/ negative charge] ) does not stain the organism [also negatively charged], but rather the environment xxix. Can determine size. xxx. Procedure 8. Prepare smear j. DO NOT HEAT FIX! 9. Add Nigrosin 10. Let air dry 11. View 4. Differential Stains (Gram Stain, Acid Fast Stain, Endospore Stain) h. Differential Stains – Stains that classify the differences in two organisms i. Gram Stain – Differentiates between organisms with a thick layer of peptidoglycan in their cell wall versus organisms with a thin layer of peptidoglycan. xxxi. Procedure 12. Prepare smear 13. Stain with primary stain Crystal Violet for 30 seconds 14. Wash w/ water 15. Add mordant, Gram’s Iodine for 1 minutes 16. Wash w/ water 17. Add decolorizer, 95% ethanol for 10 to 15 seconds 18. Wash w/ water 19. Add secondary stain Safranin for 1 minute 20. Wash with water 21. Blot dry 22. View xxxii. Results 23. Gram Negative – Appears pink ( ex. Escherichia coli) 24. Gram Positive – Appears purple ( ex. Staphylococcus aureus) j. Endospore Stain – Differentiates betweens endospores and vegetative cells in bacteria that form spores ( ex. B. subtilis) xxxiii. Procedure ( Schaeffer fulton Method) 25. Prepare smear 26. Put slide of steam and add Primary stain (Malachite Green) to slide via a piece of paper towel for 5 minutes 27. After 5 minutes, add counter stain , Safranin for 30 sec. 28. Blot dry 29. View xxxiv. Results 30. Endospore- appears green 31. Vegetative cell – appears pink k. Acid Fast Stain – Differentiates between cells that contain Mycolic acid in their cell walls verses those that do not. xxxv. Procedure ( Ziehl- Neelson) Method 32. The same procedure as the Schaffer- Fulton Method but … k. The Primary stain is Carbolfushin l. The Carbolfushin is not administered through paper towel m. The Carbolfushin is decolorized with acid alcohol n. The counter stain is Methylene Blue. xxxvi. Results 33. Acid Fast Bacteria – Appear Pink ( from the Carbolfushin) ( ex: M. smegmatis) 34. Non Acid Fast Bacteria – Appear Blue (S. aureus) 5. Review of all staining methods | Simple Stain | Negative Stain | Gram Stain | Endospore Stain | Acid Fact Stain | Primary Stain | Methylene Blue | Nigrosin | Crystal Violet | Malachite Green | Carbolfushin | Mordant | - | - | Iodine | Heat | Heat | Decolorizer | - | - | 95% ethanol | - | Acid Alcohol | Counter Stain | - | - | Safranin | Safranin | Methylene Blue |

6. Motility Determination: Wet Mount & Tube Method l. Proper presentation of Genus and Species xxxvii. Micrococcus luteus 35. When typed the name of the bacteria is italicized 36. When written the bacteria name is underlined 37. The Genus (first name) is Capitalized 38. The species (second name) is NOT capitalized m. Motility Determination xxxviii. Flagella arrangement | Description | Motility Description | Example | Monotrichous | Single flagella | Motile but not highly so | V. choleraePseudomonas | Amphitrichous | One flagella on each end | Adequate motility | - | Peritrichous | Many flagella all over | Highly motile | P. vulgaris | Lophotrichous | Many flagella on one end | Highly motile | Spirillum | xxxix. Chemotaxis – the movement of bacteria toward a desirable chemical and away from and undesirable chemical xl. Brownian Motion – Caused by currents under the cover glass, appears as a “jiggling” movement, NOT TRUE MOTILITY xli. Axial Filaments – “endoflagella” extending beyond the cell wall. n. Preparing a Wet mount xlii. Drop of viable cells on a side xliii. Place a cover slip xliv. View under microscope xlv. Work fast so mount does not dry out. o. Motility Medium/Tube Method xlvi. Semi Solid Media ( SIM) 39. w/ agar concentration of 0.4% xlvii. Procedure 40. Gather bacteria on a sterile inoculating needle 41. Stab media, straight down, ¾ of the way down. 42. Exit media on the same track as going in xlviii. Reading the media 43. Motile – Haziness around and away from the inoculation stab. o. Ex: P. vulgaris 44. Non- Motile – growth only within the inoculation stab and on he top of the media. p. Ex: M. luteus 7. Pure Culture Technique – Pour Plate Method p. Goal to get isolated colonies on media q. Pour Plate Method xlix. Procedure 45. Melt agar tubes , let cool ( to 45°C ; use cheek test) 46. Take loop of bacteria from Agar plate and place in tube 1 47. Flame loop 48. Take loop full from Tube 1 Tube 2 49. Flame loop 50. Take loop full from Tube 2 Tube 3 51. Flame Loop 52. Take loop full from Tube 3 Tube 4 53. Label empty, sterile, agar plates 1-4 54. Pour tube 1 Plate 1 55. Pour tube 2 Plate 2 56. Pour tube 3 Plate 3 57. Pour tube 4 Plate 4 58. Let solidify, then incubate @ 37° 8. Streak Plate Quadrant + Cultivation of anerobes r. Streak Plate Method l. Use a sterile loop to collect a bacterial sample li. Streak one quadrant of a sterile agar plate. lii. Flame loop. liii. Using a sample from the first quadrant, streak the second quadrant liv. Flame and repeat for third and fourth quadrants lv. Incubate s. Cultivation of Anaerobes lvi. Oxygen Requirements 59. Obligate Aerobes- Require Oxygen for growth q. Ex. Bacillus, Micrococcus 60. Obligate Anaerobes – Cannot grow in the presence of oxygen r. Ex: Clostridium 61. Facultative Aerobes – grow well in the presence of Oxygen, but can also grow without Oxygen s. Ex: E. coli 62. Aerotolerant anaerobes – Do not require Oxygen but can grow in its prescens t. Enterococcus faecalis lvii. Media – 63. Fluid Thioglycollate Medium u. Grows aerobic and anaerobi bacteria v. Dye resazurin. Turns pink in the presence of oxygen 64. Brewer’s Anaerobic Agar w. Cultivates anaerobic bacteria x. Contains Thioglycollate and resazurin y. Must be cultivated in a Gas Pak lviii. Results – 65. Grew Aerobically z. E.coli {. B. Subtilis 66. Did not grow Aerobically |. C. sporogenes 9. Bacterial Population Count t. Direct Method – Serial Dilution and Standard Plate Count (SPC) lix. Serial Dilution Blanks 67. PBS – Phosphate buffer saline 68. 9 ml volume lx. Spread Plate lxi. Procedure 69. Take 1 ml of bacterial sample ( using a sterile pipette) and put it in the first blank [Dilution factor of 10-1]. 70. Throw away pipette 71. Take 1 ml of the first blank and place it in the second blank [Dilution factor 10-2] 72. Repeat until desired dilution factor 73. Plate desired dilution factors }. Use pipette to measure 1 ml of sample ~. Inject ON ( not in, do not break the agar) the agar plate. . Use sterile glass spreader to spread liquid into agar . Incubate. lxii. Determining the count 74. Count the colonies on the plate, once it has been incubating long enough 75. Statistically viable counts – Between 30-300 colonies 76. ( # of colonies ) (dilution of the sample ) =(# of bac. / ml) . Value MUST be in SCIENTIFIC NOTATION u. Indirect method lxiii. Based on turbidity lxiv. Cannot distinguish between dead and viable cells lxv. Uses a spectrophotometer 10. Identification of An Unknown Bacteria v. Several tests to determine the identity of an Unknown bacteria lxvi. Preparation of a working culture 77. Streak Plate inoculation lxvii. Preparation of a stock culture 78. Stock culture – agar slant, used to store the bacteria in the event the working culture is compromised or depleted. . Procedure for Stock culture inoculation i. Use sterile loop to obtain bacterial sample ii. Lightly streak the slant of the agar slant. lxviii.
Tests done Test | Purpose | Reagents | Result (Positive) | Result (Negative) | Gram Stain | 1. To determine the cell wall formation ( thick layer peptidoglycan or thin layer peptidoglycan) | Crystal violet, iodine, 95% Alcohol, Safranin | Purple specimen- Gram Positive, cell wall has thick peptidoglycan layer | Pink Specimen- Gram Negative, cell wall has thin peptidoglycan layer | Catalase Test | To determine the production of catalase enzyme. The enzyme that catalyzes H2O2 | Hydrogen Peroxide | Bubbles produced – enzyme present | No bubbles – enzyme not present | Oxidase Test | To determine the presence of cytochrome oxidase. Enzyme used when O2 is used as a terminal electron acceptor | Tetramethyl- pphenylenediamine | Color change from yellow to purple – cytochrome present | No change, reagent remains yellow- cytochrome not present | Glucose Sugar Fermentation ( w/ Durham tube) | To determine if bacteria can ferment glucose | | Color change from yellow to red – acid produced AND/OR Media displacement (bubble) in Durham tube. – gas produced | No color change – no acid productionAND OR No media displacement – no gas production
| Lactose Sugar Fermentation ( w/ Durham tube) | To determine if bacteria can ferment lactose. | | Color change from yellow to red – acid produced AND/OR Media displacement (bubble) in Durham tube. – gas produced | No color change – no acid productionAND OR No media displacement – no gas production | Methyl Red Test | To determine if bacteria can ferment glucose into several organic acids | Methyl Red | Color change from yellow to red- glucose fermented into several acids | No color change, media remains yellow – glucose not feremented | Voges- Proskauer test | To determine bacteria’s ability to ferment sugars to 2,3 Butanediol | Alpha – naphthol & Potassium hydroxide | Color change from clear to pink/red- 2,3 Butanediol produced | No color change media remains clear. – no 2,3 Butanediol produced | proTryptophan Degradation | To determine bacteria’s ability to degrade Tryptophan to indole, ammonia & pyruvic acid | = Indole | Deep red color on top of media- indole produced | No color change in media- no indole produced | Mac-Conkey Agar Plate | Determines if bacteria can ferment media AND produce acid | | Agar changed color: red yellow- yellow means acid produced. Red colonies mean lactose is fermented. | No color change - no acid productionNo growth – bacteria is gram positiveColorless colonies – bacteria cannot ferment lactose | Hydrogen Sulfide Test | TO determine if bacteria can produce H2S from the amino acid cysteine. | | Formation of black precipitate- H2S is produced | No color change – No Hydrogen Sulfide produced | Citrate Utilization | To determine if bacteria can utilize citrate as a source of carbon | | Color change of the media from dark green to deep blue. – Citrate can be used a source of carbon | No color change, media remains dark green- citrate is not a useable source of carbon | Phenylalanine Test | To determine if bacteria can deaminizes the amino acid phenylalanine. | 10% Ferric Chloride/TDA | Deep green color – the enzyme phenylalanine deamniase is present | No color change – enzyme is not present | FTM- Fluid Thioglycollate media | To determine the oxygen requirement for the unknown bacteria | Aerobes – bacteria will grow within the top portion of the tube. Facultative anaerobe – bacteria will grow unevenly through out media. | Microaerophile – bacteria will grow ideally right under the most aerated portion of the media | Anaerobe – bacteria will grow at the bottom of the tube. Aerotolerant – bacteria will grow evenly through out tube. | Nitrate Test | To determine if the bacteria can reduce Nitrate | Sulfanilic acid & N,N-dimethyl-1-naphthylamine | Formation of a red precipitate – Bacteria can reduce Nitrate | No color change – Bacteria cannot reduce Nitrate | Urea Hydrolysis | To determine if the bacteria can hydrolyze Urea. | | Color change from yellow to read. – Urea can be hydrolyzed by bacteria | No color change in media –Urea cannot be hydrolyzed | Motility | TO determine if bacteria is motile or non- motile | | Motile bacteria- growth within and outside of the stab site in the media. | Non- Motile bacteria – growth only occurring with the needle stab and on top of the media. |