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by: Phuong Nguyen


Marketplace > University of North Texas > Biology > BIOL 2041 > REVIEW FOR LAB PRACTICAL
Phuong Nguyen
GPA 3.7
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These are summative notes for the first portions of lab up to Lab 13. You may use this as your review for microbiology lab for the upcoming practical. Best of luck!
Dr. Hyunju Kim
Class Notes




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This 8 page Class Notes was uploaded by Phuong Nguyen on Tuesday October 11, 2016. The Class Notes belongs to BIOL 2041 at University of North Texas taught by Dr. Hyunju Kim in Fall 2016. Since its upload, it has received 41 views. For similar materials see Microbiology in Biology at University of North Texas.




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Date Created: 10/11/16
MICRO LAB QUIZ 1 ● Why do we need nutrients? To live, grow, and reproduce ● Cell Morphology = coccus, bacillus, staphylococcus, streptobacillus, square, star-shaped, spirilla, vibro ● Colony morphology = shape, margin, elevation, size ● Synthetic medium: all compounds kno ​ wn ​ . coli) ● Complex medium: undefined composition (tryptic soy broth, blood, yeast, beef broth) ● Why must we use aseptic technique? o To save work from contamination (to perfect work) o To protect yourself ● Agar: solidifying agent 1. Liquid – 0% agar 2. Semisolid – 0.5-0.6% agar 3. Solid – 1.5-2% agar ● Media o General purpose – allow all different organisms to grow on it (broth, soy) o Selective – only let specific M grow on it, inhibits unwanted o Differential – media that differentiates organisms of the same class based on its biochemical properties (McConkey) o Enrichment – media that provides luxurious growth to M (salmonella) ● Autoclave conditions – to sterilize and kill all living organisms o 121oC o 15 min 2 o 15 psi/lb in​ ● Agar solidifies at 45oC after dissolved, won’t go into solution until broth is heated ● Purpose of sterilizing the loop in between quadrants is to decrease bacterial load ● 4 quadrant streaking is to get a single isolated colony ● Woese’s domains (1978) ● Carolous Linnaeus ​Binomial species o Solid plates for isolation o Liquid for mass multiplication o Semisolid for motility of organisms o Slants – short term storage ● Mixed culture for 2+ colony ● Pure culture for 1 colony ● Gram staining differentiates G+ form G- Lab 7 ● Simple Staining o Purpose: rapid method for staining a bacterial smear, to observe cell morphology o Methylene blue or crystal violet Lab 8 ● Gram Staining o Purpose: to differentiate between G+ and G- o Procedure: 1. Prepare smear 2. Cover smear in crystal violet for 30s-1min 3. Rinse with water 4. Cover in iodine for 1 min 5. Decolorize with 95% ethanol and rinse with water 6. Cover in safranin and stain for 30s-1 min 7. Rinse with water and blot dry with bibulous paper 8. Examine under microscope with immersion oil, G+ will be purple and G- is red/pink Lab 9: Endospore Stain ● We use basic dyes because they have a positive charge and they form and ionic bond attachment to the negatively charged bacterial cell therefore, adhering to the stain to give it pigment (simple stain) ​ ● Endospores form from genera B ​ acillus and ​Clostridium ● Endospores are bacterial cells that are metabolically dormant and can withstand desiccation, heat, and UV radiation ● Have spore coat ● Examples: o C. botulinum o C. tetani o B. antracis ● We use Schaeffer-Fulton method using: Procedure: 1. Place bacterial smear 2. Cover slide with paper towel and place slide over boiling water for 5 min and cover in malachite green,​ continue add stain to prevent drying 3. Discard paper towel and remove from heat to let cool 4. Rinse with water 5. Counterstain with ​safranin​ for 30s-1min 6. Rinse with water and blot with bibulous paper 7. Examine under microscope with immersion oil, endospore will be green, vegetative cells red (vegetative cells are a cell of a bacterium or unicellular alga that is actively growing rather than forming spores) Purpose: To differentiate between cells capable of retaining primary stain after acid alcohol and medically important to identify genus ​Mycobacterium ​ ● Ex. M ● Waxy coat makes it difficult to stain ​ ​ ​ ● Decimal Reduction Rate = 90% of the microorganisms in the broth will be killed @ a given temp ● Inoculate 4 plates: 4°C, 25°C (M (E.c)), 37 (M (E.c)) and 55(T (B.s)) Lab 13: Oxygen Tolerance Purpose: To study role of O2 in growth of microbes Procedure: 1. Strict aerobes – requires O2 2. Obligate anaerobes – killed by O2 ​C. sporogenes 3. Facultative anaerobes – grow with both E. coli and 4. Microaerophilic (peroxidase instead of catalase)​ Enterococcus faecium 5. Aerotolerant – (commensalism) don’t require O2 but don’t mind it ● Fluid thioglycollate is a media that reduces O2 to water ● Resazurin is a redox indicator that turns pink in the presence of O2 (biobags) ● Catalase reduces toxic H2O2 into H2O and O2 ● Biobags allow anaerobes to grow 2O2 + 2H+ → superoxide dismutase → H2O2 + O2 + 2H+ → catalase → 2H2O + O2


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