Tools of Lab

Microbial culture

growing microorganisms in or on a nutrient medium under controlled laboratory conditions

• Remains an important tool for

identification, diagnosis, and research

Culture-dependent methods

• Require growth of the microorganism

in the laboratory

• Examples: culture, isolation,

biochemical testing

Culture-independent methods

• Detect or characterize microbes

without first growing them in culture

• Examples: PCR and DNA sequencing

Inoculum

a sample containing microorganisms used to start a culture
may come from many sources:

• Environmental samples

• Food or water

• Clinical specimens

Inoculation

introducing the inoculum into a growth medium

Enrichment culture

uses specific growth conditions to

favor the growth of particular microorganisms

Enrichment bias

growth conditions may favor certain microorganisms over others

• Fast-growing or easily cultured organisms can become dominant

• These organisms may not have been the most abundant or important microbes in

the original sample

• Therefore, what grows in the lab does not always represent the entire microbial

community

Culture-independent methods

have revealed much of the microbial diversity missed by cultivation

Many microbes don’t grow under standard laboratory conditions, so growing them (cultivation) misses a lot of the different kinds present.

Growth

the increase in cell number as a

result of cell division

Medium

nutrients for the

growth of microbes

Loop

transferring or streaking cultures

Needle

inoculating deep or semisolid

media

Pipette

transferring liquid cultures

Swab

inoculating surfaces or agar

plates

Aseptic technique

prevents contamination of the

culture, the environment, and the individual

performing the procedure.

Liquid

Broth, no agar, grow large numbers of MO, used in tubes/flasks

Semisolid

low agar concentration, often used to test motility, used in tubes

solid

Agar, Higher agar concentration, provides a surface for microbial growth, used to isolate colonies and observe colony characteristics, used in plates and tubes

Agar

a polysaccharide used to solidify many microbiology media

Defined (Synthetic)

• You know every chemical and its exact amount.

• Composition is precisely chemically defined

• Contain pure organic and inorganic compounds that vary little from one source to another

• Molecular content specified by means of an exact formula

Complex (Nonsynthetic)

• At least one ingredient is a mixture whose exact chemical composition isn’t fully known, such as yeast extract or peptone.

• One or more components is not chemically defined

• Contains extracts of animals, plants, or yeasts

• Blood, serum, meat extracts or infusions, milk, yeast

extract, soybean digests, and peptone

Enriched media

General-purpose media supplemented with

additional nutrients to support the growth of

fastidious microorganisms

Fastidious microbes

Have complex nutritional requirements

• May not grow well on standard media

Selective media

contain one or more agents that inhibit

some microorganisms while allowing others to grow.

• Useful for isolating a desired microorganism from a

  mixed sample

• Suppress unwanted background organisms, making the

  desired organisms easier to recover

Selective agents may include

• Antibiotics

• Salts

• Dyes

• Acidic or basic pH

Differential media

allow multiple types of microorganisms to grow but produce visible differences that help

distinguish them. Contain specific substrates and/or indicators that reveal

differences in microbial metabolism

differential media reactions:

• Color changes in the medium

• Changes in colony color or appearance

• Gas production

• Precipitation or clearing around growth

Mannitol Salt Agar

• Selective: The presence of salt (inhibits

many organisms)

• Differential: Fermentation of the sugar

mannitol (media turns yellow) vs non-

fermentation (media remains red)

Incubation

Exposing the inoculated medium to optimal growth

conditions, generally for a few hours to days

Incubator

a temperature-controlled chamber to encourage the

multiplication of microbe

Temperatures used in laboratory propagation of microorganisms

20 to 25°C (room temperature – environmental samples)

• 37°C (human body temperature – many pathogens

Biosafety Levels

describe increasing levels of laboratory containment based on the risks of the agents and procedures being performed.

BSL-1

(Basic Safety)

• Agents: nonpathogenic E. coli K-12, B.

subtilis

• Risk: none in healthy adults

• Practices: open bench work, hand

washing, disinfect surfaces

• Gear: lab coat, gloves, goggles if

needed

• Example: teaching labs

BSL-2

(Moderate Containment)

• Agents: Staph. aureus, Salmonella,

Hepatitis A virus

• Risk: human disease, moderate

hazards

• Practices: restricted access, biohazard

signs, waste autoclaved

• Gear: lab coat, gloves, goggles;

biosafety cabinet for aerosols

• Example: clinical & hospital labs

BSL-3

Agents: M. tuberculosis, Y. pestis

• Risk: Serious or lethal by inhalation

• Practices:

• All work in biosafety cabinets (Class

II/III)

• Negative air pressure, double-door

entry

• Exhaust air passes through one HEPA

filter

• Gear: Lab coat, gloves, goggles; respirators

sometimes required

BSL-4

(Maximum Containment)

• Agents: Ebola, Marburg, Lassa fever viruses

• Risk: Frequently fatal, no

vaccines/treatments

• Practices:

• Fully isolated labs or “hot zones”

• Entry/exit with chemical showers,

vacuum rooms, security checks

• Exhaust air passes through two HEPA

filters

• Gear: Positive-pressure suits with own air

supply; work done in sealed cabinets or by

robotics

hepa filters

a type of pleated mechanical air filter that captures 99.97% of tiny particles down to 0.3 microns in size

May 2021: 42 operational BSL-4 labs plus 17 planned in 23

countries

May 2025: 110 BSL-4 labs in 34 countries

Where Are BSL-4

Laboratories

Located?

Refrigeration

short-term storage of cultures

Deep-freezing:

Culture placed in a suspending liquid and frozen at -80oC

• Can be thawed and grown several years later

Lyophilization

Frozen (-54 to -72oC) and dehydrated in a vacuum

• Can be stored for years/decades and revived via liquid culture

media

Colony

a visible mass of microbial cells growing on solid media, typically arising from a single cell or colony-forming unit (CFU)

Streak plate

Where are cells diluted?
through successive
How is samples added
inoculum is streaked across the agar surface with a sterile loop
Where do colnies grow
surface only
common use
isolation/obtating a pure culture
Dilution is ON plate
streak + Spread = surface colnies

spread plate

where are cells diluted?
Before plating using serial dilution in tubes
How is smaple collected?
A measured volume of diluted sample is placed on the agar surface and spread with sterile spreader
where do colnies grow?
surface only
common use
isolation and viable cell count (CFU/mL)
dilutiion occurs before plating

pour plate

where are cells diluted?
before plating using serial dilution in tubes,
how is sampe added?
diltued smaple is mixed with molten agar (45-50c) and poured into a plate
Where do colnies grow?
within and on surface
common use
isolation and viable cell counts
pour plate= surface + subsurface

Magnification

the ratio of an object’s image size to its real

size

• Enlarges objects

Resolution

the measure of the clarity of the image, or the

minimum distance between two distinguishable points

• Distinguishes fine detail

• Resolving power of the human eye: 0.2 mm

• Resolving power of the light microscope using the oil

immersion lens: 0.2 μm (1000x smaller!!

Contrast

the difference in brightness between the light

and dark areas of the image

• Specimen stands out from background

Light microscopes

Use visible light

• Can view living or preserved

specimens

• Commonly used for routine

microbiology

Electron microscopes

Use a beam of electrons

• Provide much greater resolution

• Require specially prepared,

nonliving specimens

• Used to visualize much smaller

structures and fine cellular det

Images are always in black and white, but they may be

artificially colored

Brightfeild

specimens are viewed against a white background

darkfeild

specimen appears white against a balck background
improve visualization without staining

phase-contrast

allows greater differentiation of internal structures

improve visualization without staining

Lenses

refract (bend) the light, so that the image is magnified

subcellular

most ___ structures are too small to be resolved by light microscopy

Scanning electron microscopes (SEMs)

focus a beam of electrons onto the

surface of a specimen, producing images that look three-dimensional

Differential interference contrast microscopy (DIC)

similar to phase-contrast, but uses two beams of light

instead of one

• Prisms split beams, adding color contrast

• Produces a 3D-like image with enhanced detail

• Great for viewing live, unstained specimens

Fluorescence microscopy

uses specific wavelengths (often

ultraviolet light) of light to excite fluorescent molecules

(fluorophores)

Microorganisms or cellular structures can be labeled with

fluorescent dyes

Fluorophores

absorb light and emit light at a longer

wavelength

Fluorescent antibody (FA) tests

use labeled antibodies to detect

specific microbial antigens

• Can be used directly with cells, tissues, or clinical specimens

Nucleic Acid Stains

fluorescent dye, Bind to DNA or RNA

• Make microbial cells easier to detect and count

• Can be used with environmental, food, or clinical

samples

viability stains

Help distinguish cells based on membrane integrity

• Often displayed as:

• Intact membranes

• Damaged membranes

fluorescent dye

Fluorescent staining

can detect and characterize microbes

without first culturing them

transmission electron microscopes (TEMs)

focus a beam of electrons

through a specimen

• TEM is used mainly to study the internal structure of cells

slide 31

1 st colums and last in 2nd column

Wet mount

drop of culture on slide, covered

with slip

Hanging drop

drop suspended from coverslip,

reduces drying, better for motility

Why Stain Microorganisms?

Staining increases contrast, making cells

and cellular structures easier to see.

• Bacterial cell surfaces generally carry a

net negative charge.

Basic Dyes

Positively charged (+)

• Attracted to negatively charged

components of bacterial cells

• Typically stain the cells

Acidic Dyes

Negatively charged (−)

• Repelled by the negatively charged

bacterial surface

• Typically stain the background

Simple stains

only require a single dye and an

uncomplicated procedure:

• Cause all the cells in the smear to appear

more or less the same color, regardless of

type

• Reveal shape, size, and arrangement

Positive stain:

dye sticks to the specimen and

gives it color

Negative stain

does not stick to the specimen

but settles some distance from its outer

boundary, forming a silhouette:

Differential stains

Use two differently colored dyes: the primary dye and the counterstain

• Distinguish cell types or parts

• More complex and require additional chemical reagents to produce the desired

reaction

• Examples: Gram stain and Acid-fast stain

Special stains:

used to stain specific parts of

microorganisms such as endospores, flagella, or capsules

negative special stain

prescence of a capsule aidsin determining an organism’s ability to cause disease, capsules do not accept most dyes and appear as halos surrounding each stained basterial cell

endospore staining

An _____ is a special resistant,

dormant structure formed within a cell

that protects a bacterium from adverse

environmental conditions. Endospores

cannot be stained by ordinary

methods, because the dyes don’t

penetrate the endospore’s thick wall.

flagellum staining

Bacterial flagella are structures of

locomotion too small to be seen with a

light microscope without staining. The

number and arrangement of flagella can

be used as diagnostic aids.

obligate intracellular organisms

must

reproduce within host cells

Metagenomics

Studying All

Genes in a

Community

next-generation sequencing (NGS)

ead

millions of DNA fragments at once

Multi-Omics

individual organisms or lab cultures)

• Combines multiple “omics” layers for one microbe or

defined system:

• Goal: link genes → expression → function in a single

organism or controlled system

Meta-Omics

(complex microbial communities)

• Applies multi-omics approaches to mixed, whole

communities

• Examples: soil, ocean water, gut microbiome

• Focuses on:

• Who is there (community composition)

• What they are doing (functional activity at the

community level)

• Goal: understand interactions, diversity, and ecosystem

function

Culture-independent tools reveal diversity, but

Detection ≠ Expression (genes may not be active)

• Culturing remains essential to fully test properties and functions