Lab Practical

Microbiology Lab Practical Study Guide

Know media name, selectivity, differential properties, what is tested, organisms, and expected results (color, growth, zones)

Brightfield microscopy

Used for dead, stained cells with low contrast

Phase-contrast microscopy

Used for live, unstained cells with high contrast

Telescopic eyepiece

Aligns phase rings in phase-contrast microscopy

True motility

Directional movement of bacteria

Brownian motion

Random vibration of particles, not true movement

Streaming

Movement caused by fluid currents

Tetrazolium dye

Turns red where bacteria grow in motility media

Motility positive result

Growth spreads outward from stab line

Motility negative result

Growth stays along stab line

Gram stain results

Gram-positive = purple, Gram-negative = pink

Endospore stain

Detects bacterial spores

Acid-fast stain

Identifies Mycobacterium species

Capsule stain

Detects capsules, indicates virulence

Negative stain

Outlines cells without staining them

MSA (Mannitol Salt Agar)

Selective for Staphylococcus (high salt), differential for mannitol fermentation

MacConkey agar

Selective for Gram-negative bacteria, differential for lactose fermentation

EMB agar

Selective for Gram-negative, lactose fermentation; E. coli shows green metallic sheen

Endo agar

Differentiates lactose fermenters

TSI test

Tests sugar fermentation and hydrogen sulfide (H2S) production

Urease test

Tests urea breakdown; positive = pink

Indole test

Tests tryptophan breakdown; positive = red ring

Citrate test

Tests ability to use citrate as carbon source; positive = blue

Oxidase test

Tests for cytochrome c oxidase; positive = purple

Coagulase test

Identifies Staphylococcus aureus; positive = clot formation

Blood agar

Differentiates hemolysis: alpha (green), beta (clear), gamma (none)

Kirby-Bauer test

Measures antibiotic sensitivity via zones of inhibition

MPN method

Estimates bacterial count using dilution series

Anaerobic jar

Grows obligate anaerobes

Pipette types

Know different pipettes used in lab

T2 bacteriophage

Lytic phage that infects E. coli

Plaques

Clear zones showing bacterial lysis by phage

PFU/mL calculation

Plaques × dilution ÷ volume

E. coli

Gram-negative, lactose fermenter

Pseudomonas

Gram-negative, non-lactose fermenter

Staphylococcus aureus

Gram-positive, MSA positive, coagulase positive

Staphylococcus epidermidis

Gram-positive, MSA negative, coagulase negative

Klebsiella

Gram-negative, citrate positive

Proteus

Gram-negative, urease positive

Catalase Test

What you look for:

• Bubbles = Positive result
  

  • Oxygen gas is being released

  • The bacteria have catalase

• No bubbles = Negative result

  • No oxygen produced

  • The bacteria do NOT have catalase

Staphylococcus (like S. aureus) → Catalase positive
You will see bubbling

Streptococcus → Catalase negative
You will see no bubbling

Thermal death point (TDP)

the lowest temperature at which a

bacterial population ties in 10 min.

Thermal death time (TDT)

the shortest time to kill bacteria at a

specific temperature.

• Antibiotics

compounds that kill or inhibit bacteria.

Bacteriostatic

inhibits growth of bacterial cells but does not kill them.

Bactericidal (cidal = kill)

Agents that kill

bacterial cells

Disinfectants –

Kills viruses and

bacteria on a surface, much

stronger than sanitizers.

Antiseptics

Lowers the number

of germs on a surface to a safer

level.

K/A (red slant / yellow butt)

Glucose fermentation only; slant reverts to alkaline (red) after glucose is used, butt stays acidic (yellow)

A/A (yellow slant / yellow butt)

Glucose + lactose and/or sucrose fermented; large acid production keeps entire tube yellow

K/no change (red slant / red butt)

No fermentation; organism uses peptones only (alkaline)

K/H₂S (red slant / black butt)

No lactose/sucrose fermentation; glucose fermented + H₂S produced

Cracked or lifted agar

Gas production from fermentation

Bacterial Morphology

Coccus

Bacteria Morphology

Bacillus

Bacteria Morphology

Cocci Arrangement

Coccus (single-celled)

Cocci Arrangement

Diplococci (occur in pairs)

Cocci Arrangement

Tetrad (group of 4 cocci)

Cocci Arrangement

Streptococci (chain-like morphology)

Cocci Arrangement

Staphylococci (grape-like cluster)

aerobes

respire (O2)

microaerophiles

respire (decreased O2)

faculatives

respire if O2 is available, ferment if not

aerotolerants

ferment only (tolerate O2)

anaerobes

ferment (often killed by O2)

psychrophiles

5-15, arctic waters

psychrotrophs

mesophiles that can grow at refrigerator temps

mesophiles

25-45, most bacteria, including human pathogens

thermophiles

45-70, soil, compost piles

hyperthermophiles

>75, hot springs

thermodurics

survive but do not grow at high temperatures

Isotonic

solute conc. same inside and outside the cell, water moves equally in both directions

Hypotonic

solute conc. higher inside the cell, water moves into cell

Hypertonic

solute conc. higher outside the cell, water moves out