Microbiology Lab Review Exercise Points

Laboratory Safety and Guidelines (Exercise 1)

  • Personal Protective Equipment (PPE):
        * The three main pieces of PPE used throughout the semester are lab coats, goggles, and gloves.
        * Goggles were of high importance during lab procedures.
        * Gloves must be worn when working with organisms or touching anything contaminated with bacteria.
        * Microscope Exception: Gloves must NOT be worn while using microscopes; they must be kept clean with no glove contact.
        * Additional Precautions: Long hair must be tied back at all times.

  • Workplace Sanitation:
        * Benches are wiped down with disinfectant at the beginning and the end of every class.
        * The primary disinfectant used in the lab is 10%10\% Clorox solution.

  • Biosafety Levels (BSL):
        * BSL-1: Handling organisms like Escherichia coli and Bacillus megaterium. Only BSL-1 and BSL-2 were utilized in the laboratory this semester.
        * BSL-2: Handling organisms like Staphylococcus aureus.
        * BSL-3: Handling respiratory pathogens like Mycobacterium tuberculosis. These are handled in dedicated clinical labs, such as the one on Bailey Road in Rochester.
        * BSL-4: Reserved for highly dangerous viruses like Ebola and Marburg virus. To the instructor's knowledge, there is no BSL-4 facility in Rochester.

  • Aseptic Technique:
        * The core goal is to ensure materials are transferred aseptically to avoid any type of contamination.

Waste Disposal and Glassware

  • Disposal Categories:
        * Glass Waste: Used for dead/fixed material. Smears that have been heat-fixed can be placed in the gray tin containers located at the front or back of the room.
        * Clorox Beakers: Any slide containing live bacteria must be placed in a Clorox beaker to kill the organisms before disposal.
        * Orange Biohazard Bags: All plates and items with live bacteria go into the orange biohazard bags at individual benches. These are eventually emptied into the main orange biohazard bag in the back.
        * Prohibited Items in Biohazard Bags: Gloves, paper towels, and glass must NEVER be placed in the orange biohazard bags.

Principles and Application of Microscopy

  • Equipment Type: The lab uses compound light microscopes.

  • Lens Systems: These microscopes utilize three lens systems: the ocular lenses, the objective lenses, and the condenser lenses.

  • Image Properties: Images are inverted and backwards due to the lens system. Moving a slide to the right causes the image to move left; moving it up causes it to move down. A common example is the letter "e," which appears upside down and backwards.

  • Magnification Calculations:
        * Total magnification is calculated as:
         Total×Magnification=Ocular×Magnification×Objective×MagnificationTotal \times Magnification = Ocular \times Magnification \times Objective \times Magnification
        * Ocular lenses are always 10×10 \times.
        * Objective lenses range from 4×4 \times (scanning) to 100×100 \times (oil immersion).
        * Total magnification range: 40×40 \times to 1000×1000 \times.
        * This range is suitable for bacteria and eukaryotic cells but not viruses.

  • Key Terminology:
        * Resolving Power: The minimum distance two objects can be apart and still be seen as distinct. For these microscopes, it is 0.2 μm0.2 \,\mu m.
        * Parfocal: This means that once the specimen is focused under the scanning (4×4 \times) objective, it remains nearly in focus when moving to higher powers, requiring only the fine adjustment knob.
        * Control Adjustments: Includes fine adjustment, coarse adjustment (used only for the 4×4 \times objective), and light adjustment.

Aseptic Transfer and Microbial Handling (Exercise 6)

  • Sterilization: Loops are sterilized using a Bincinerator.

  • Safety Warning: Loops should never be "parked" inside the Bincinerator. Doing so makes the metal frail (causing it to crack or break) and causes the handle to heat up, potentially burning the user's hand.

  • Cooling Period: After sterilization, the loop must cool for 20 to 3020 \text{ to } 30 seconds before touching live bacteria to avoid killing the sample.

Bacterial Staining and Morphology (Exercise 3, 4, 5)

  • Morphology Shapes:
        * Cocci: Circles.
        * Bacillus: Rods.
        * Spirobacteria/Spirochetes: Spiral-shaped.
        * Vibrio: Comma-shaped (e.g., Vibrio cholerae).
        * Corynebacterium: Club-shaped or resembling a baseball bat.

  • Arrangements:
        * Staphylo-: Clusters (e.g., Staphylococcus).
        * Strepto-: Chains (e.g., Streptococcus).

  • Smear Preparation Steps:
        1. Label slide with a wax pencil circle.
        2. Place a loopful of water on the slide.
        3. Aseptically mix bacteria into the water.
        4. Heat-fix on a slide warmer to gel the phospholipids in the membrane, which glues the cells to the slide.

  • Stain Types and Charges:
        * Simple Stains: Use cationic (positively charged) stains that are taken up by the negatively charged bacterial membrane. Examples: Safranin, Crystal Violet, Methylene Blue.
        * Negative Stains: No heat fixation required. Uses anionic (negatively charged) stains like Nigrosin or India ink. These repel the cell membrane, staining only the background.
        * Comparison: Negative staining is superior for determining true morphology as it avoids heat distortion of proteins.

  • The Gram Stain (Differential Staining):
        * Gram-Positive: Possess a thick layer of peptidoglycan (protein-sugar mix) that retains Crystal Violet.
        * Gram-Negative: Possess a thin layer of peptidoglycan and an outer membrane composed of Lipopolysaccharide (LPS). LPS makes them more antibiotic-resistant.
        * Process: Acetone-alcohol (decolorizer) removes the lipid outer membrane of Gram-negatives, causing the Crystal Violet to fall out. Safranin is then used as a counterstain so Gram-negatives appear red/pink, while positives remain purple.
        * Common Errors: Poor washing or insufficient decolorization results in mixed pink and purple cells.
        * Examples: Gram-positive (Staphylococcus, Streptococcus, Bacillus); Gram-negative (E. coli, Salmonella).

Isolation of Bacteria and Culture Media

  • Streak Plate Method: Used to isolate individual colonies from mixed cultures. The loop must be incinerated between each of the four quadrants to dilute the bacteria.
        * Example: A mixture of E. coli, S. aureus, and Provinciae (which grew in fat, unusual shapes).

  • Culture Media Types:
        * Blood Agar: Differential media. Distinguishes bacteria based on hemolysis.
            * Beta ($\beta$): Complete hemolysis; clear/yellowish zone (e.g., Strep pyogenes).
            * Alpha ($\alpha$): Partial hemolysis; greenish hue.
            * Gamma ($\gamma$): No hemolysis; growth but no color change.
        * PEA (Phenylethyl Alcohol Agar): Selective only. Favors Gram-positives. Phenylethyl alcohol acts similarly to Acetone-alcohol, disrupting the Gram-negative outer membrane and preventing their growth.
        * MAC (MacConkey Agar): Selective for Gram-negatives and differential for lactose fermentation. Lactose fermenters produce a bright pink color (e.g., E. coli).
        * MSA (Mannitol Salt Agar): Selective for Gram-positives (due to high salt) and differential for mannitol fermentation.
            * Positive Result: Media turns yellow (e.g., Staph aureus).
            * Negative Result: Growth but no color change (e.g., Staph epidermidis).
        * TSA (Tryptic Soy Agar) and Nutrient Agar: General purpose media; grow both pathogens and non-pathogens.

Biochemical Testing and Susceptibility

  • Catalase Test: Distinguishes Staphylococci (positive) from Streptococci (negative). Positive results involve bubbling when hydrogen peroxide is added, as the catalase enzyme converts H2O2→H2O+O2H_2O_2 \rightarrow H_2O + O_2.

  • Coagulase Test: Distinguishes Staph aureus (positive) from other staph species. Staph aureus possesses Protein A on its surface, which flips antibodies to evade phagocytosis.

  • Kirby-Bauer Method (Antimicrobial Susceptibility):
        * A lawn of bacteria is created, and antibiotic discs are applied.
        * Zone of Inhibition (ZOI): Measured across the diameter in millimeters (mmmm). If only a radius is visible, multiply by two.
        * Implications: ZOI size determines if an antibiotic should be used in a patient, though it doesn't account for human metabolic variables.

  • Specific Sensitivities:
        * Bacitracin: Streptococcus pyogenes (Group A Strep) is uniquely susceptible.
        * Optochin: Used to identify Streptococcus pneumoniae.

  • Bile Esculin Test: Used to identify Enterococcus faecalis (Gram-positive). A positive result is indicated by black growth on the slant agar. Groups that observed this included Mike and Monica, Kelly and Brina (Tuesday), and Katie, Julia, and Trevor (Thursday).

HIV ELISA Exercise (Exercise 15)

  • Methodology: This was an Indirect ELISA, which detects antibodies in patient serum, not the antigen itself.

  • Procedural Steps:
        1. Coat plate with HIV proteins (antigen).
        2. Add patient serum (contains primary antibodies if exposed).
        3. Add secondary antibody: An anti-anti-HIV antibody conjugated with the enzyme Horseradish Peroxidase (HRP).
        4. Add substrate: Interacts with HRP to produce a color change (bright red in this lab).

  • Controls: Performed in duplicate or triplicate for reproducibility. Included positive and negative control samples.

  • Confirmatory Testing: If an ELISA is positive, a Western Blot (using polyacrylamide gel) is performed for qualitative and quantitative accuracy. Genetic testing is now the fastest detection method.

Respiratory, Skin, and Dental Microbiology

  • Skin Flora: Frequently includes Staph epidermidis, Micrococcus, Corynebacterium, and Cutibacterium. Some individuals are transient or permanent carriers of Staph aureus.

  • Respiratory Flora: Many people carry Strep pyogenes (Group A Strep) as normal flora due to microbial antagonism. Strep pyogenes uses M Protein to resist phagocytosis.

  • Snyder Test (Dental): Measures susceptibility to tooth decay by evaluating bacteria that ferment sucrose (e.g., Lactobacillus and Streptococcus mutans).
        * Indicator: Color change from blue-green to yellow.
        * Protocol: Agar is kept at 65 ∘C65 \, ^\circ C to stay liquid; after adding saliva, the tube is rolled between hands to distribute bacteria.

API 20 and Enterobacteriaceae

  • Purpose: Identifies Gram-negative rods (Enterobacteriaceae), which are common hospital-acquired infections (e.g., Klebsiella, Proteus).

  • Format: 20 biochemical tests performed simultaneously in cupules.

  • Atmospheric Requirements:
        * Humid Environment: Created to prevent the strip from drying.
        * Anaerobic Reactions: Required mineral oil to block oxygen.
        * Aerobic Reactions: Cupules were filled to the top to ensure oxygen access.

  • Oxidase Test: All Enterobacteriaceae test negative, as they are mostly anaerobic and lack the specific electron transport chain enzyme.