Tools of the Lab

Microbial culture

method of multiplying microbial
organisms by letting them reproduce in a culture
medium under controlled laboratory conditions

• Foundational diagnostic method used as a
  research tool in molecular biology

Culture-dependent and culture-independent
methods are _________ .

combined to evaluate the diversity and distribution of bacterial communities

Culture dependent

traditional culture and enrichment methods

Culture independent

molecular methods (absence of laboratory culture)

To fully characterize a microbe ___________ .

isolating in a pure culture is best

Enrichment culture

a culture that employs highly selective
laboratory methods for obtaining microbes from natural
samples

• begins with collecting a sample from the
  appropriate habitat to serve as the inoculum

Successful enrichment requires a proper ________ .

inoculum (sample) containing the organism of interest

Inoculation

placing sample in media

• Makes it possible to handle and manage microbes in an artificial environment and begin to analyze what the sample may contain

  
  

enrichment bias

a problem with enrichment cultures in which
“weed” species tend to dominate in the enrichment, often to the exclusion of the most abundant or ecologically significant organisms in the inoculum

Culture independent molecular methods reveal ______ .

how what we grow is not representative of diverse microbial environments

Culture

to grow microorganisms

Growth

increase in cell number as a result of cell division

Medium

nutrients for growth of microbes

Inoculum

small sample of microbes

Inoculation

introduction of an inoculum into media to
culture microbes

• can come from clinical specimens obtained from body fluids, discharges, anatomical sites, or diseased tissue. Among other sources.

Culture media can be contained in ____________ .

test tubes, flasks, and petri dishes

Media may be inoculated with ___________ .

loops, needles, pipettes, and cotton swabs

Aseptic technique

not introducing outside organisms and protecting ourselves

Agar

• Complex polysaccharide added to solid media

• not digestible for most microbes

• flexible and moldable (Liquefies at 100°C; solidifies at 42°C or below)

What media condition would have the highest agar %?

solid

Defined (synthetic)

• Composition is precisely chemically defined; can identify components

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

• specified by means of an exact formula

Complex (nonsynthetic)

• one or more components is vague or not chemically defined

• Contains extracts of animals, plants, or yeasts
  • Blood, serum, meat extracts or infusions, milk, yeast
  extract, soybean digests, and peptone

General purpose media

grows a broad spectrum of microbes; generally complex

Enumeration media

used for counting microbes

Ex: microbes in food, water, soil, or milk

Assay media

Test the effectiveness of something or a product

Ex: antimicrobial drugs, disinfectants, antiseptics, cosmetics, and preservatives

Reducing medium

Contains ingredients that remove dissolved oxygen from the medium to allow the growth of anaerobes

transport media

Used to maintain and preserve specimens that have to be held for a period of time before clinical analysis

Enriched media

Contains complex organic substances such as blood, serum, hemoglobin, or special growth factors for the growth of fastidious microbes (need something special to grow)

Ex: blood agar and chocolate agar

Selective media

Contains one or more agents that inhibit the growth of a certain microbe or microbes (selects by inhibiting other organism)

• helpful in isolating one type of microbe from a sample containing many

• suppressing unwanted background organisms and favoring the growth of the desired ones

• examples of agents: pH, antibiotics, salts, dyes

Differential media

Allow multiple types of organisms to grow but display visible differences in how they grow

Variations often come from chemicals in the media with which microbes react:
1. Variations in colony size or color
2. Media color changes
3. Production of gas bubbles

A medium can be both -_________ .

selective and differential

Ex: mannitol salt agar

Dyes are used as differential agents because _________ .

many are pH indicators that change color in
response to the production of an acid or a
base

Incubation

a temperature-controlled chamber to encourage the
multiplication of microbes that is seeable without a microscope

Temperature for incubating environmental samples

20 to 25 degrees celsuis

Temperature for incubating pathogens

37 degrees celsuis

BSL-1

lab setting with low risk microbes

• no threat to healthy adults

• no special precautions; basic teaching labs

BSL-2

labs that work with moderate risk microbes

• lab coat, goggles, gloves

• follow these procedures in our micro lab

BSL-3

labs that work on exotic or indigenous microbes that can cause serious health risks through inhalation (biosafety cabinets prevent airborne transmission)

BSL-4

labs that work with highly dangerous and fatal microbes like ebola (have no treatments)

• uses negative pressure which sucks back microbes into lab (prevents escape)

• exhaust air filtered twice with HEPA filters

refrigeration

short-term shortage of cultures (around 4 degrees Celsius)

deep freezing

Culture placed in a suspending liquid and frozen around -80 degrees celsuis

• Can be thawed and grown several years later

• can be used out in the field by using liquid nitrogen

Lyophilization (freeze-drying)

Frozen (-54 to -72 degrees Celsius) and dehydrated in a vacuum
• Can be stored for years and rehydrated via liquid culture media

colony

a macroscopic cluster of cells appearing on a solid
medium arising from the multiplication of a single cell

Isolation requirements

1. A medium with a firm surface
2. A Petri dish
3. An inoculating loop (streak plate method)

The microbial basis of disease and biochemical diversity has relied on ________ .

the ability to grow and isolate microorganisms in the laboratory

colony morphology

visible characteristics of a colony

Colony morphology can be used to determine if a culture is _________ .

1. Pure (only one microbe is present)
2. Contaminated (undesired organisms co-occur with a desired organism)
3. Mixed (many microbes are present)

Propagation of microbial cultures requires _______ .

aseptic technique

• a series of steps by which microbes are
  transferred between growth media without contamination

streak plate

Inoculum is streaked over the agar surface to “thin
out” the bacteria. Some individual bacterial cells are separated and well-spaced from each other.
• Primary technique

Pour plate

Mixing bacteria (of various dilutions) in melted
medium and pouring the medium into a petri dish to solidify
• Colonies grow on and in solidified medium

Spread plate

Dilutions of bacteria are added to the surface of
pre-poured, solidified medium
• Colonies grow only on the surface of solidified medium

microbes can be identified through:

-Microscopic appearance
• Characterization of cellular metabolism
• Determination of nutrient requirements, products given off during growth, presence of enzymes, and mechanisms for deriving energy
• Genetic and immunologic characteristics
• Testing of cultures using procedures that analyze biochemical and
enzyme characteristics, immunologic reactions, drug sensitivity, and genetic makeup

Three important aspects of microscopy

• magnification

• resolution

• contrast

magnification

looks bigger than it actually is; ratio of object’s image to its actual size

resolution (resolving power)

clarity; the minimum distance between two points; distinguishes fine detail and structures

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

difference in brightness between light and dark areas of an image

Compound light microscopy

has a series of lenses and uses visible light as its source of illumination

• allows us to see things in fine detail

• observes unstained cells easier

light microscope

visible light is passed through a specimen and then through glass lenses
• Lenses refract (bend) the light, so that the image is magnified

• most subcellular structures are too small to be viewed by this

brightfield

specimens are viewed against a white background; use in lab

darkfield

specimen appears white against a black background

phase contrast

allows greater differentiation of internal structures

differential interference contrast microscopy

uses two beams of light instead of one
• Prisms split each light beam, adding contrasting
colors to the specimen
• Appears three-dimensional

fluorescence microscopy

uses an ultraviolet light source to illuminate specimens that will fluoresce
• If the specimen does not naturally fluoresce, it is stained with a fluorescent dyes called a fluorochromes

fluorescent antibody technique can _______ .

detect bacteria or other pathogenic microorganisms, even within cells, tissues, or
other clinical specimens

electron microscopy

Objects smaller than about 0.2μm , such as viruses or the internal structures of cells, must be examined with an electron microscope.
• Uses electrons instead of light
• Used to examine structures too small to be resolved with light microscopes
• Images are always in black and white, but they may be artificially colored

Light microscopy allows us to ________ .

see living and motile organisms

One advantage of light microscopy over electron microscopy is that ________ .

it allows you to view dynamic processes in living cells

Fluorescence microscopy uses an ______ source to illuminate specimens that will fluorescence.

UV light

Type of medium used to maintain and preserve specimens before clinical analysis

transport medium

A mixed culture is ________ .

one that contains two or more species

Selective cultures are designed to ________ .

encourage the growth of specific types of bacteria while inhibiting others

Mannitol in mannitol salt agar is a carbon source that some bacteria may ferment when this happens the media will turn yellow. What does this make the mannitol?

differential

Scanning electron microscopes (SEMs)

focus a beam of electrons onto the surface of a specimen, producing images that look three-dimensional

Transmission electron microscopes (TEMs)

focus a beam of electrons through a specimen
• mainly to study the internal structure of cells

The manner in which a specimen is prepared depends on _________ .

• The condition of the specimen: living or dead

• The aims of the examiner: observation of overall structure, identification, or movement

• The type of microscopy available

Cells are suspended in water, broth, or
saline to maintain viability and provide a
medium for ________ .

locomotion

wet mount

Consists of a drop or two of culture
placed on a slide and overlaid with a
cover slip

• cells are alive

• provide a medium for locomotion

  
  

staining

any procedure that applies colored chemicals (dyes) to specimens

• basic and acidic dyes

• makes cells easier to see

• Bacteria have numerous negatively charged substances and attract basic dyes, but repel acidic dyes

Basic dyes have a _______ .

positive charge; cells attract it; used for positive staining (direct staining)

Acidic dyes have a __________ .

negative charge; cells repel this dye; used for negative/ indirect staining

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 staining

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 dyes

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

• most common: gram stain (gram positive or gram negative) and acid-fast stain

  
  

acid-fast stain

Used to identify bacteria that are not decolorized
by acid-alcohol; can be used to differentiate between cell walls

• non acid-fast bacteria = when stained with methylene blue they appear blue

• acid-fast bacteria = retain carbofulschin stain and remain pink/red

special stains

used to stain specific parts of microorganisms such as endospores, flagella, or capsules

negative staining

• use to identify presence of capsules

• capsules do not accept most stains so they appear as unstained halos around bacterial cells

• aids in determining whether an organism can cause disease (the presence of capsules)

endospore staining

• detects presence of endospores in bacteria

• malachite green penetrates and stains endospores green

• safranin (red) is applied and stains the remainder of the cells red or pink

endospore

a special resistant, dormant structure formed within a cell that protects a bacterium from adverse
environmental conditions.

• cannot be stained by ordinary
  methods, because the dyes don’t
  penetrate the endospore’s thick wall

flagella staining

• used to determine presence of flagella (structures of locomotion, can’t be seen without staining)

• mordant is used to build up the diameters of the flagella until they become visible microscopically when stained with carbolfuchsin

• number and arrangement of flagella can be used as diagnostic aids

Gram-positive bacteria

retain the crystal violent stain and appear purple

Gram-negative bacteria

do not retain crystal violent, remain colorless until counterstained with safranin and then turn pink

identification

Analysis of collected data to help support a final
determination of the types of microbes present in the original sample

• Lays the groundwork for further research into the nature and roles of these microbes; it can also provide numerous applications in infection diagnosis,
  food safety, biotechnology, and microbial ecology.

Obligate parasites

microbes that only grow on live cells or animals; must use alternative growth methods

Ex: viruses and some bacteria

Viable but non-culturable (VBNC)

bacteria that are alive but cannot be cultured using standard microbiological methods; in a resting dormant state but still have active metabolic pathways

• Upwards of 99% of microbes in the environment

• Genetic technology aids identification

Great Plate count anomaly

We can only successfully cultivate in the lab a few microbes of the many that we can see
under the microscope in the original sample.

• Estimates are that we can cultivate roughly one out of every 100 microbes.

• Bypass the growth step altogether and develop approaches to directly sequence the genomes
  of the so-called “unculturable” microbes from the environment.
  • Learn a lot from the sequence information, but our knowledge is still limited by our inability to
  grow these microbes in the lab

Flourescent dyes

stain microorganisms from virtually any microbial habitat. Can make all microorganisms, including viruses, fluoresce.

Dyes that stain DNA are used for __________ .

enumeration (counting) of microorganisms in food, clinical, and environmental samples

Viability stains yield both ________ .

abundance and viability data at the same time
• The basis of differentiating between live and dead
cells lies not with a cell’s DNA but whether its
cytoplasmic membrane is intact or not

An encompassing approach to the genetic characterization of microbial communities is _________ .

metagenomics

• all genes in a given microbial community can be sampled and, the information obtained can support a much deeper understanding of the structure and function of the community than can single-
  gene analyses

• goal is to use next-generation data sequencing to identify as many genes as possible from an environmental DNA sample and determine the phylogeny
  of the organism(s) to which the genes belong