Microbio Lab Exam

Why is handwashing critical?

Removing microbes and preventing transmission

Safety with live organisms

Wearing PPE, avoid aerosols, disinfect surfaces, proper disposal

Why decontaminate counters with 10% bleach?

Kills microbes and prevents contamination

Total magnification

Ocular (10x) × Objective

Resolving power

Ability to distinguish two close points; determines clarity

Brightfield

General, stained specimens

Phase‑contrast

Live, unstained cells; internal detail.

Fluorescence

Detect fluorescent‑tagged microbes.

TEM vs SEM

TEM= Internal SEM=Surface

Simple vs Gram vs Acid‑fast stain — Simple

One dye

Simple vs Gram vs Acid‑fast stain — Gram

Cell wall differences

Simple vs Gram vs Acid‑fast stain — Acid‑fast

Mycolic acid detection

Why coverslip on wet mount?

Protects specimen, prevents drying, improves viewing.

Focusing steps

Start at 4x → 10x → 40x; center and focus each time.

Yeast vs bacteria size

Yeast are much larger (5–10× bigger).

Wet Preps & Motility — Two types of movement

Brownian motion and true motility

Brownian motion

Vibration

True motility

Directional

Structure for motility

Flagella

Why discard wet preps in biohazard?

Contain live organisms

Typical magnification range?

40x-1000x

Best magnification for bacterial morphology

1000x (oil immersion)

Microscope for fluorescent dyes

Fluorescent Microscope

Microscope for viruses

Electron microscope (TEM)

Morphology of E. coli vs S. aureus — E. coli

Gram negative rods

Morphology of E. coli vs S. aureus — S. aureus

Gram positive cocci in clusters

Why increase light at higher magnification?

Higher magnification=reduced light at the eye

Aseptic technique

Prevents contamination of cultures and environment

Safety with live cultures

PPE, flame tools, disinfect surfaces, proper disposal

How bacteria obtain nutrients

Absorb nutrients from media

Broth vs agar — Broth

Liquid

Broth vs agar — Agar

Solid

Why agar?

Solidifies media, not metabolized by bacteria

Signs of growth in broth

Turbidity, pellicle, sediment

Inoculating Liquid Media Steps:

Flame loop → cool → open tube → flame neck → inoculate → flame neck → close

Why flame loop?

Sterilizes before/after use

Why flame tube neck?

Creates convection to reduce contamination

Purpose of streaking?

Isolate colonies

Isolated vs confluent growth — Confluent

lawn, covers entire surface

Isolated vs confluent growth — Isolated

Single colonies

Troubleshooting failed isolation

Reduce inoculum, streak properly, flame loop between quadrants

Importance of pure culture

Accurate identification

Pure vs mixed — Pure

One species

Pure vs mixed — Mixed

Multiple

Examples of sterile solutions

IV, injectable drugs, saline

How to identify non‑sterile IV solution

Cloudiness, particles

Why never use non‑sterile solutions

Risk of infection

If cultures become contaminated

Results invalid, must repeat

Role of pH

Affect enzyme activity, media buffers maintain it

Improper aseptic technique

Leads to contamination and unreliable data

What is Gram stain?

Differential stain developed by Hans Christian Gram

Gram+ vs Gram– cell walls - Gram +

thick peptidoglycan

Gram+ vs Gram– cell walls - Gram -

thin PG + outer membrane.

Three basic shapes

Cocci, bacilli, spirilla; various arrangements

Cocci

spherical bacteria

Bacilli

rod-shaped bacteria

Spirilla

Rigid, spiral shaped bacteria

Smear preparation step

Add bacteria → air dry → heat fix

Why heat‑fix?

Adheres cells and kills bacteria.

Reagent roles - CV

primary stain

Reagent roles - iodine

mordant

Reagent roles - alcohol

decolorize

Reagent roles - safranin

counterstain

Omitting iodine

Week CV bind → false gram -

Omitting alcohol

No decolorization → false Gram+

Why safranin?

Stains gram so they are visible

Differentiate Gram+ vs Gram color - Gram+

Purple

Differentiate Gram+ vs Gram color - Gram -

pink

Gram reactions - E. coli

Gram– rods

Gram reactions - S. aureus

Gram+ cocci clusters.

S. aureus vs S. pneumoniae

both Gram+, but different arrangements

S. aureus vs N. meningitidi

Gram+ vs Gram–.

E. coli vs P. vulgaris

both Gram– rods; Gram stain cannot distinguish.

Applications - Why essential?

First step in ID; guides treatment.

Applications - Helps antibiotic planning

Gram– often more resistant.

Applications - Advantages over simple stain

Differentiates cell wall types

Applications - Urgent clinical need

Meningitis, sepsis, pneumonia

Causes of errors

Old cultures, over/under‑decolorizing, thick smear

Mixed results

Mixed culture or contamination

Why fresh cultures?

Old Gram+ may appear Gram–.

Four types of media

Supportive, enriched, selective, differential

TSA vs BAP/CHOC

TSA = general growth; BAP/CHOC = enriched for fastidious organisms.

Role of sheep blood

Nutrient + hemolysis detection

Types of hemolysis - Alpha

green

Types of hemolysis - Beta

Clear

Types of hemolysis - Gamma

None

Organisms on BAP

Most bacteria; hemolysis indicates enzyme activity

TSA - Why supportive?

Supports many non‑fastidious organisms.

TSA - Who grows?

Broad range of bacteria