Learn: Microbiology Techniques, Media, and Microscopy: Key Concepts and Definitions

Aseptic technique

Technique used to prevent contamination of yourself, the environment, and the experiment during culturing.

Aseptic

Free of contamination.

Pure culture

Culture containing only one species of microorganism.

Mixed culture

Culture containing two or more species of microorganisms.

Subculture

Transfer of microorganisms from one medium to another.

PPE — 3 always-worn items

Lab coat, goggles, and gloves.

Biohazard bin

Container used to dispose of contaminated biological materials.

Agar

Solidifying agent extracted from seaweed; has no nutritional value for bacteria.

Agar solidification temperature

40°C.

Agar melting temperature

100°C.

General-purpose medium

Medium that supports the growth of non-fastidious organisms.

Fastidious organism

Organism that requires a heavy supply of ready-made organic compounds and is considered a "picky eater."

Non-fastidious organism

Organism that requires fewer nutrients and is easier to grow.

Enriched medium

Medium containing additional nutrients to support fastidious organisms.

Defined medium

Medium in which the exact proportions and chemical composition of the ingredients are known.

Undefined medium

Medium containing ingredients whose exact chemical composition/proportions are not known.

Blood agar

Tryptic Soy Agar enriched with 5% sheep blood; supports fastidious and non-fastidious organisms and is differential based on hemolysis.

Differential medium

Medium that allows differences between organisms to be observed.

Hemolysis

Lysis or breakdown of red blood cells.

Hemolysin

Exotoxin responsible for hemolysis.

Beta (β) hemolysis

Complete destruction of red blood cells; produces total clearing of agar around growth.

Alpha (α) hemolysis

Partial destruction of red blood cells, producing a greenish discoloration around growth.

Gamma (γ) hemolysis

No visible hemolysis or destruction of red blood cells.

Magnification

Enlargement of an image.

Resolution

Ability to see fine details and distinguish two close objects as separate.

Contrast

Ability to differentiate the specimen from the background.

Microscope ocular magnification

10X.

Scanning objective

4X.

Low-power objective

10X.

High-dry objective

40X.

Oil-immersion objective

100X.

Total magnification

Objective magnification × ocular magnification.

Total magnification with 4X objective

40X.

Total magnification with 10X objective

100X.

Total magnification with 40X objective

400X.

Total magnification with 100X objective

1000X.

Immersion oil

Oil with the same refractive index as glass; used with the 100X objective to improve resolution.

Numerical aperture — 4X

0.10.

Numerical aperture — 10X

0.25.

Numerical aperture — 40X

0.65.

Numerical aperture — 100X oil

1.25.

Condenser numerical aperture without oil

0.9.

Condenser numerical aperture with oil

1.25.

Limit of resolution formula

d = wavelength of light ÷ (NA condenser + NA objective).

Maximum microscope resolution setup

500 nm wavelength with the 100X oil-immersion objective and 1.25 NA condenser/objective.

Resolution calculation from Wayground

500 nm ÷ (0.9 + 1.25) = approximately 233 nm.

Koehler illumination

Method of specimen illumination that produces even illumination and prevents the light source from being visible in the image.

Objective used to set up Koehler illumination

10X low-power objective.

Koehler illumination frequency

Set up Koehler illumination every time the microscope is used.

Parfocal

Microscope remains in focus when switching to a higher-power objective.

Parpositional

Image remains in the same position when switching to a higher-power objective.

Bacillus

Rod-shaped bacterial cell.

Coccus

Spherical or ovoid bacterial cell.

Spirillum

Rigid, helical bacterial cell.

Spirochete

Flexible, helical bacterial cell.

Vibrio

Bacterial cell with one curve.

Diplococcus

Pair of cocci.

Streptococcus

Chain of cocci.

Staphylococcus

Cluster of cocci.

Tetrad

Four cocci arranged together.

Sarcina

Cubical packet of eight cocci.

Bacterial morphology

When asked for morphology, give both the cell shape and arrangement; use the arrangement seen most commonly on the slide.

Fluid thioglycollate broth

Medium that creates an oxygen gradient and allows different oxygen requirements to be observed.

Resazurin

Redox indicator; faint pink when oxidized/oxygen is present and colorless when reduced/no oxygen is present.

Sodium thioglycollate and L-cysteine

Chemicals that reduce oxygen in fluid thioglycollate broth.

Agar in thioglycollate broth

Small amount of agar slows oxygen diffusion and helps create the oxygen gradient.

Obligate aerobe

Requires oxygen to grow.

Obligate anaerobe

Requires complete absence of oxygen; oxygen is lethal.

Facultative anaerobe

Grows with or without oxygen and can use fermentation and respiration.

Aerotolerant anaerobe

Grows in oxygen but does not use oxygen metabolically.

Microaerophile

Requires oxygen but cannot tolerate atmospheric oxygen levels.

Sediment growth pattern

Growth concentrated at the bottom of the tube.

Uniform fine turbidity growth pattern

Growth distributed evenly throughout the broth.

Pellicle growth pattern

Growth concentrated at the top/surface of the broth.

Flocculent growth pattern

Visible clumps or flakes of growth suspended throughout the broth.

Phenol Red Carbohydrate Broth

Medium used to determine whether an organism ferments a carbohydrate.

Phenol Red substrate

Carbohydrate/sugar being tested, such as glucose, lactose, mannitol, or sucrose.

Phenol Red

Acid-base indicator that is yellow in acidic conditions and pink in basic conditions.

Durham tube

Small inverted tube used to capture gas produced during fermentation.

Yellow broth + bubble

Positive acid and gas production (A/G); carbohydrate was fermented with acid and gas production.

Yellow broth + no bubble

Positive acid production without gas (A/-); carbohydrate was fermented.

Red broth + no bubble

Negative fermentation (-/-); carbohydrate was not fermented.

Pink broth + no bubble

K result; carbohydrate was not fermented and peptones were degraded.

Why pink Phenol Red broth matters

Peptone degradation produces an alkaline/basic condition, causing Phenol Red to become pink.

Phenol Red over-incubation problem

Over-incubation can cause a false negative because the organism can consume the carbohydrate and then degrade peptones, changing the broth from yellow to pink.

Urea Hydrolysis Test

Determines whether an organism can hydrolyze urea.

Urea hydrolysis substrate

Urea.

Urea hydrolysis enzyme

Urease.

Urea hydrolysis end product

Ammonia (NH₃).

Urea hydrolysis indicator

Phenol Red.

Positive urea test

Pink/fuchsia medium; urea was hydrolyzed and urease was produced.

Negative urea test

No color change or yellow medium; urea was not hydrolyzed and urease was not produced.

IMViC purpose

Used to differentiate members of Enterobacteriaceae from other Gram-negative rods.

IMViC — I

Indole.

IMViC — M

Methyl Red.

IMViC — V

Voges-Proskauer.

IMViC — C

Citrate.

Enterobacteriaceae

Typically Gram-negative rods, non-spore-forming, glucose fermenters, simple nitrate reducers, oxidase negative, and usually citrate positive.

Methyl Red test

Tests for glucose fermentation through the mixed-acid fermentation pathway.

Methyl Red substrate

Glucose.