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:

• 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:

• 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