Microbiology Lab Practical #1

Microbiology Lab Practical #1 This Lab Practical covers Exercises 1-7, safety, and handouts. Safety & Exercise "1: Introduction to Microbiology Lab Microbiology Laboratory Rules • Come prepared. Read lab exercise before lab. Complete all take home work. Safety is priority "1. Report accidents immediately to instructor. Do not bring food, drinks, or use cosmetics in the lab. Be organized and be aware of what is happening around you. • Disinfect benchtop and wash hands before and after every lab. Handle every specimen as if it were a patient sample. Label! Remove any marks and tape before placing glassware in cart for cleaning. • Clean and handle microscopes carefully. Report all incidents to the instructor immediately. Read the Lab Manual and come to lab prepared. Follow instructions closely Do not attempt a procedure is you are not sure how to do it. Exercise "2: Microscopy Know the parts of the microscope and their functions. Magnification is increase in apparent size of an object. Staining is generally needed to increase the contrast of bacteria with the background. Resolution is the ability to distinguish objects as distinct and separate. Resolving power (RP) depends on: quality of the objective lens, wavelength of light used and refractive index. =)/2 x NA (2).wavelength, NA numerical aperture) Scope storage: 1. Light turned off, rheostat onl 2. Low power objective (4x) in position 3. Stage down 4. Condenser down 5. Clean off oil with lens paper and methanol 6. Cord coiled and stowed in pocket. 7. Push in mechanical stage gear so it is flush with stage 8. Carry with 2 hands Exercise 3: Biomolecules A Biomolecule is any molecule present in living organisms. 4 classes of macromolecules: • Carbohydrate: (CH₂O), energy storage and structure • Lipid: mostly hydrocarbon, membranes, energy storage, steroid hormones • Protein: polymers of amino acids, many functions including enzymes Nucleic Acid: DNA & RN storage and expression of genetic information Class Macromolecule Carbohydrate Polysaccharides Lipid Phospholipids, triglycerides, sterols Protein Protein Subunit Monosaccharides Fatty acids, sterols Amino acids Ivucleic Acid DNA, RNA nucleotides Bond Glycosidic Ester Peptide Sugar-phosphate backbone Examples Starch, cellulose, glycogen Membranes, fat storage, steroid hormones Enzymes, hemoglobin, antibodies, actin Nucleotides are adenine, guanine, cytosine, thymine, uracil pll is a measure of the II concentration. It is an inverse log scale. pH = -log[H] Be able to recognize which class of macromolecules a model belongs to 1. Phospholipids 2. Protein 3. DNA 4. Carbohydrates a. Starch b. Glycogen Cellulose 5. Water ice Microbiology Lab Practical #1 Exercise 4: Aseptic Transfers and Smear Aseptic technique is used anytime microbes are taken out of test tubes or other containers Prevent contamination of both the pure microbial culture and the surroundings. Staining adds colored dyes to bacteria to increase contrast. Inoculating loop or needle is used to aseptically transfer microbes. Heat fixation: • kills bacteria adheres them to the slide denatures proteins for better staining Exercise 5: Simple and Negative Stains Chromophore is the colored ion of a dye. Basic dyes have a positive-charged chromophore, which binds to the negative charges on cells. This is direct staining. Acidic dyes have a negative-charged chromophore. Acidic dyes color the background in negative staining. Exercise "6: Gram and Capsule Stains Gram stain is a differential stain based on cell wall structure of bacteria. Know Gram stain dyes and procedure. Gram positive cells have thick cell walis, Gram-negative cells have a thin wall plus an outer membrane. Capsules are external cell structures that do not stain. Exercise #7: Endospore and Acid-Fast Stains Endospores are survival structures and difficult to stain Schaeffer-Fulton endospore sai Ziehl-Neelson acid-fast stain Stain Review Table STAIN PROCEDURE Simple Stain: Methylene blue Negative Stain: Congo Red or Nigrosin Gram Stain: Crystal Violet (primary) Jodine (mordant) 95% Ethanol (decolorizer) Safranin (counterstain) Endospore Stain: Malachite Green (primary) Water (decolorizer) Safranin (ccunter stain) Acid-Fast Stain Carbol Fuchsin m acid-Alcohol RESULTS Simple Stain: Cells stain clue Negative Stain: Cells are unstained (clear) Background is stained Gram Stain: Gram (+violet/Blue Grare (Red/Pink Endospore Stain: Endospores Green Vegetative Cells Red Acid-Fast Stain: Pritive Rec Najative Blue COMMENTS Methylene Blue: Simple Stain Acid-Fast Stain Safranin: Gram Stain Endospore Stain Decolorizing agents: 95% Ethanol in Gram Stain Acid-Alcohol in Acid-Fast Stain Alternate Names: Endospore Stain = Schaeffer-Fulton Acid-FastZiehl-Neelson Counter Stains: Safranin: Gram & Endospore Methylene Blue: Acid-Fast ethylene Blo