Microbio Lab Midterm

Exercises 1-15

Fire hazards

Open flames, alcohol near burners

Electrical hazards

Wet hands or cords

Chemical exposure

Stains, disinfectants

Biohazards

Microorganisms treated as potential pathogens

Standard Safety Practices

No eating, drinking, smoking, or mouth pipetting

Handwashing

Wash hands before and after lab, and after spills

Disinfecting benches

Disinfect bench at start and end of lab

Personal Protective Equipment (PPE)

Lab coat, gloves, and safety goggles when handling stains or splashing materials

Face protection

Required when performing aerosol-generating tasks

Proper Disposal of Materials

Contaminated glassware → 'To Be Autoclaved' containers

Broken uncontaminated glass

Dispose in broken glass bin

Pipettes and swabs

Dispose in disinfectant jars or biohazard bins

Slides

Dispose in disinfectant containers

Used towels and disposables

Dispose in biohazard or autoclave waste containers

Ocular lens

Remagnifies image (usually 10×)

Objective lenses

Primary magnifiers (4×, 10×, 40×, 100×)

Stage

Holds slide

Condenser

Focuses light through specimen

Iris diaphragm

Adjusts light/contrast

Coarse & fine knobs

Focus adjustment

Base & arm

Support and carry microscope safely

Total Magnification

Total magnification = Ocular lens × Objective lens

Example of Total Magnification

10× ocular × 40× objective = 400× total

Unit for Microbial Cell Size

Micrometers (µm) or nanometers (nm)

Wet Mount Purpose

Observe living organisms and motility in natural state

Hanging Drop Technique

Drop of specimen hangs from coverslip over a concave depression slide

Brownian Motion

Random vibration due to water molecules hitting cells

True Motility

Directional, purposeful movement (flagellar motion)

Calculating Cells in Field of View

of cells = Field diameter / Cell length

Agar medium

Gel-like nutrient base

Petri dish

Shallow dish for cultures

Agar plate

Petri dish containing solidified medium

Colony

Visible mass of microbial growth from one cell

Pure Culture Purpose

To isolate a single microbial species for identification

Contamination

Presence of unwanted microbes; prevented by aseptic technique (sterilizing loops, using lids, disinfecting surfaces).

Loop

Transfers small liquid culture.

Needle

Transfers from solid media or deep agar.

Smear

Thin layer of cells on a slide for staining.

Fixing

Adheres cells and kills microbes. Heat fixing (flame pass) or chemical fixing (methanol).

Staining

Increases contrast to visualize cells.

Simple stain

One dye (e.g., methylene blue).

Direct stain

Colors cells.

Negative stain

Colors background.

Differential stain

Distinguishes cell types (e.g., Gram, acid-fast).

Gram Stain Steps

Crystal violet (primary stain), Iodine (mordant), Alcohol/acetone (decolorizer), Safranin (counterstain).

Gram-positive

Purple.

Gram-negative

Pink/red.

Clinical use of Gram stain

Guides antibiotic treatment.

Acid-Fast Stain

Detects Mycobacterium species (waxy mycolic acid cell walls).

Endospore Stain

Detects Bacillus/Clostridium spores.

Capsule Stain

Reveals extracellular capsule (virulence factor).

Virulence Factors

Mycolic acid: Prevents digestion; Capsules: Resist phagocytosis; Endospores: Survive extreme conditions.

Mixed Culture

Multiple species.

Pure Culture

One species isolated from colony.

Streak Plate Method

Purpose: Isolate colonies; Advantage: Separation of organisms; Disadvantage: Requires skill, may miss rare species.

Selective Media

Inhibits some microbes (e.g., EMB selects Gram-).

Differential Media

Distinguishes microbes by reaction (e.g., color change).

MSA (Mannitol Salt Agar)

Selective: High salt (Staphylococcus); Differential: Mannitol fermentation → yellow color; Indicator: Phenol red.

EMB (Eosin Methylene Blue)

Selective: Gram-negative bacteria; Differential: Lactose fermentation; Positive: Dark purple or metallic green sheen (E. coli).

Catabolism

Breakdown of molecules for energy.

Starch Hydrolysis

Amylase breaks starch → maltose/glucose; Test: Iodine turns blue-black; clear zone = positive.

Glycolysis

Glucose → 2 pyruvate + 2 ATP + 2 NADH.

O/F Test

Determines oxidation vs. fermentation; Yellow (open & closed tubes): Fermenter; Yellow only in open tube: Oxidizer; Indicator: Bromthymol blue.

Fermentation Test

Indicator: Phenol red; Yellow: Acid (positive); Gas bubble: CO₂ production.

MRVP Test

MR positive (red): Mixed acid fermentation; VP positive (red): Acetoin production.

Citrate Test

Indicator: Bromthymol blue; Blue color: Citrate utilization positive.

MIO Deep

Tests Motility, Indole, Ornithine decarboxylation.

Phenylalanine Slant

Green color: Phenylpyruvic acid formation (ferric chloride indicator).

Peptone Iron Deep

Black precipitate: H₂S production.