Microbiology Lab Exam Master Checklist: Experiments 1–5

Microbiology Lab Exam Master Checklist

Experiments 1–5

This checklist summarizes the essential definitions, concepts, procedures, and exam points for the first five core microbiology laboratory experiments. Use this as a comprehensive study guide to ensure mastery of foundational laboratory skills and concepts.

Experiment 1 – Microscope

Definitions

• Magnification: The process of enlarging the appearance of an object using lenses.

• Resolution (Resolving Power): The ability of a microscope to distinguish two points as separate entities.

• Refractive Index: A measure of how much light bends as it passes through a medium.

• Parfocal: A feature where the specimen remains in focus when switching between objectives.

• Working Distance: The distance between the objective lens and the specimen when in focus.

• Numerical Aperture: A value that indicates the resolving power of a lens; higher values mean better resolution.

• Brightfield Microscope: Uses visible light to illuminate specimens; most common type.

• Darkfield Microscope: Uses a special condenser to scatter light, making specimens appear bright against a dark background.

• Phase-Contrast Microscope: Enhances contrast in transparent specimens without staining.

• Fluorescence Microscope: Uses UV light to excite fluorescent dyes, causing specimens to emit visible light.

• TEM (Transmission Electron Microscope): Passes electrons through thin specimens for high-resolution internal images.

• SEM (Scanning Electron Microscope): Scans the surface with electrons to produce detailed 3D images.

Know

• Microscope parts and their functions

• Light pathway through the microscope

• Total magnification calculations: $\text{Total Magnification} = \text{Objective Magnification} \times \text{Ocular Magnification}$

• Why immersion oil is used: To increase resolution by reducing light refraction

• Why oil is only used with the 100× objective

• Proper microscope care and handling

• Organisms observed and their morphology

Experiment 2 – Ocular Micrometer

Definitions

• Ocular micrometer: A scale in the eyepiece used to measure microscopic objects.

• Stage micrometer: A slide with a precise scale for calibrating the ocular micrometer.

• Calibration factor: The value that converts ocular divisions to micrometers (μm).

• Micrometer (μm): A unit of length equal to one-millionth of a meter.

Know

• Purpose of calibration: Ensures accurate measurement of microorganisms

• Calibration formula: $\text{Calibration Factor} = \frac{\text{Stage Micrometer Units}}{\text{Ocular Divisions}}$

• Calibration factor for each objective lens

• Measuring cocci vs. bacilli: Shape affects measurement approach

• Cell size calculations using the calibration factor

Experiment 3 – Ubiquity of Microorganisms

Definitions

• Ubiquitous: Present everywhere; microorganisms are found in all environments.

• Agar: A gelatinous substance derived from seaweed, used to solidify culture media.

• Culture medium: Nutrient-rich substance used to grow microorganisms.

• TSA (Tryptic Soy Agar): General-purpose medium for bacterial growth.

• Blood agar: TSA supplemented with 5% sheep blood; used to detect hemolysis.

• Hemolysis: The breakdown of red blood cells by bacterial enzymes.

• Bacterial colony: A visible mass of cells derived from a single cell.

• Colony morphology: The physical characteristics of a colony (size, shape, color, etc.).

• Macroscopic appearance: Features visible to the naked eye.

• Aseptic technique: Procedures to prevent contamination by unwanted microorganisms.

Know

• Blood agar contains 5% sheep blood

• Blood agar is a differential medium

• Types of hemolysis:

  • Alpha: Partial hemolysis (greenish)

  • Beta: Complete hemolysis (clear zone)

  • Gamma: No hemolysis

• Colony characteristics:

  • Size

  • Shape

  • Margin

  • Elevation

  • Texture

  • Pigmentation

  • Opacity

Experiment 4 – Preparation of Broth and Agar

Definitions

• Culture medium: Substance containing nutrients for microbial growth.

• Broth medium: Liquid nutrient medium.

• Solid medium: Medium solidified with agar.

• Agar: Solidifying agent; not a nutrient.

• Slant: Solid medium in a tube set at an angle.

• Deep: Solid medium in a tube, not slanted.

• TSB (Tryptic Soy Broth): Liquid version of TSA.

• TSA (Tryptic Soy Agar): Solid medium for general bacterial growth.

• Sterilization: Complete destruction of all forms of microbial life.

• Disinfection: Reduction of microbial load to safe levels.

• Moist heat: Sterilization using steam (e.g., autoclave).

• Dry heat: Sterilization using hot air (e.g., oven).

• Incineration: Sterilization by burning.

• Filtration: Removal of microbes by passing liquid through a filter.

• UV radiation: Non-ionizing radiation causing DNA damage (thymine dimers).

• Ionizing radiation: High-energy radiation causing DNA breaks.

• Thymine dimer: DNA lesion caused by UV light.

Know

• Agar melts at approximately 100°C

• Agar solidifies at approximately 42°C

• Agar is not a nutrient source

• Autoclave conditions: 121°C, 15 psi, 15 minutes

• Dry heat sterilization: 170°C, 2 hours

• UV forms thymine dimers in DNA

• Filtration is used for heat-sensitive solutions

• Incubation temperature: 37°C for most pathogens

• Formula calculations for media preparation

Experiment 5 – Transfer Techniques

Definitions

• Aseptic technique: Methods to prevent contamination by unwanted microorganisms.

• Inoculation: Introduction of microorganisms into a culture medium.

• Inoculum: The sample of microorganisms transferred.

• Culture transfer: Moving microorganisms from one medium to another.

• Contamination: Introduction of unwanted microorganisms.

• Inoculating loop: Wire tool with a looped end for transferring microbes, especially to broths and slants.

• Inoculating needle: Straight wire tool for transferring microbes, especially to deeps.

Know

• Loop vs. needle: Loop for broths/slants/streak plates; needle for deeps

• Broth inoculation: Transfer of inoculum into liquid medium

• Slant inoculation: Transfer onto the surface of solidified slant medium

• Deep inoculation: Stab inoculation into solidified deep medium

• Why flame the tube: Kills microbes at the mouth and creates convection to reduce contamination

• Why flame the loop before and after use: Sterilizes the tool to prevent cross-contamination

• Why cool the loop: Prevents killing the inoculum with residual heat

• Why not set caps down: Prevents contamination of the sterile interior

• Proper transfer sequence: Sterilize loop/needle, open tube, flame tube, transfer, flame tube, close tube, sterilize loop/needle

• Spill procedure: Notify instructor, cover spill with disinfectant, allow contact time, clean up safely

• Serratia marcescens pigment: Red at 20–25°C (room temp), white at 40°C due to temperature-regulated gene expression