Tools of the Lab

What are the 5 I’s of Microbiology

1.) Inoculation

2.) Incubation

3.) Isolation

4.) Inspection

5.) Identification

Inoculation

means to introduce or implant the microbes onto a culture medium

Inoculum

the material used to inoculate a medium

Culture

propagation of microbes on a medium

Medium

a solid or liquid nutrient in which microorganisms have what is needed for growth

Examples of where clinical specimens/samples can come from to inoculate

• blood

• cerebral spinal fluid (CSF)

• urine

• sputum

• fecal matter

• etc.

Name three inoculating tools

1.) Inoculating loop

2.) Inoculating needle

3.) Sterile swab

Streaking a plate

the procedure involves flaming the loop, picking up the sample, streaking the first quadrant, sterilizing the loop again, then dragging it into part of the previous section to dilute the microbes into subsequent quadrants, repeating until the plate is streaked

• the principle: gradual dilution of microorganisms across the agar surface, so that single cells grow into visible, isolated colonies

Pouring a sample plate method

1.) pipette bacterial sample onto petri dish

2.) pour liquid nutrient agar

3.) swirl to mix

4.) colonies grow on agar surface and subsurface (some need oxygen and some do not need oxygen to grow)

Aseptic technique

method of handling microbes and material in a way that minimized contamination (this way you know you are growing exactly what you want)

The three conditions of cultures

1.) pure culture

2.) mixed culture

3.) contaminated culture

pure culture

a container of medium that grows only a single known species or type of microorganism

• most frequently used for laboratory study because it allows for the systematic examination and control of one microorganism by itself

mixed culture

container that holds two or more identified, easily differentiated species of microorganisms

contaminated culture

was once pure or mixed but has since had contaminants (unwanted microbes of uncertain identity) introduced into it

What are three different physical forms of media?

1.) Liquid

2.) Semisolid

3.) Solid/reversible to liquid

Liquid media

water-based solutions that do not solidify at temperatures above freezing and that tend to flow freely when the container is tilted; growth occurs throughout the container and can then present a dispersed, cloudy, or particulate appearance

ex. broth

Semisolid media

have more body than liquid media but less body than solid media; they do not flow freely and have a soft, clot-like consistency at room temperature; used to determine the motility of bacteria and to localize a reaction at a specific site

Solid/reversible to liquid

Media containing 1-5% agar are solid enough to remain in place when containers are tilted or inverted; they are reversibly solid and can be liquefied with heat, poured into a different container, and resolidified; provide a firm surface on which cells can form discrete colonies

Media test tubes

Broth = liquid

Slant/deep = solid

Nutrient agar

• complex polysaccharide from algae

• flexible and moldable; can hold moisture and nutrients

• BUT it is not a digestible nutrient for microorganisms

When does nutrient agar liquify?

100 degrees Celsius

When does nutrient agar solidify?

42 degrees Celsius

Selective media

• contains one or more agents that inhibit the growth of certain microbes, but not others

• important in the primary isolation of a specific type of microorganism from samples containing dozens of species

Differential media

• allow multiple types of microorganisms to grow but are designed to display differences among those microorganisms

• differentiation shows as variations in colony size or color, media color changes, formation of gas bubbles or precipitates

Can media be both selective and differential?

Yes

_____ are used as pH indicators that change colors in response to the production of an acid or base

dyes

blood agar media

• Appearance: opaque, cherry-red, solid agar plate

• Differential growth medium: tests for bacterial enzymes that destroy red blood cells, classified as beta (clear), alpha (green), or gamma (no change) hemolysis

Mannitol salt media

• Appearance: bright red/pink color, opaque, and solid

• Selective and differential growth medium: the high salt concentration selects for salt-tolerant organisms, while the phenol red indicator turns yellow if the bacterium ferments mannitol to produce acid

Eosin Methylene Blue Agar (EMB) Media

• Appearance: dark red to maroon-purple, usually slightly hazy, and may have a fine precipitate

• Selective and differential growth medium: used to detect and isolate gram-negative enteric bacteria (coliforms) and inhibits gram-positive bacteria; differentiates lactose fermenters from non-fermenters based on colony color

MacConkey agar media

• Appearance: pinkish-beige to reddish tinge

• Selective and differential medium: used to detect and isolate gram-negative enteric bacteria and inhibits gram-positive bacteria; can further differentiate the gram-negative organisms based on their lactose metabolism

Thioglycolate broth media

• Appearance: translucent, light amber, or golden-yellow liquid

• Differential medium: creates an oxygen gradient from top (aerobic) to bottom (anaerobic), allowing for the differentiation of five major groups of bacteria based on where they grow

Phenol Red broth media

• Appearance: it appears as a clear, red-orange liquid with an inverted Durham tube to capture gas

• Differential medium: used to determine an organism’s ability to ferment a specific carbohydrate and produce gas, commonly identifying Gram-negative enteric bacteria

Triple-Sugar Iron agar media

• Appearance: initially red (alkaline), it turns yellow (acidic) when fermented, and blackens if hydrogen sulfide is produced, and shows cracks or bubbles if gas is produced

• Differential medium: primarily used to identify and distinguish between different types of enteric bacteria based on their metabolic activities

Incubation (Incubator)

A temperature-controlled chamber in which inoculated media are placed

Usual laboratory propagation temperatures fall between what range?

20-40 degrees Celsius

Atmospheric gases such as _____ and ______ may be required for the growht of certain microbes

Oxygen; carbon dioxide

During incubation, microbes grow and multiply, producing visible ____ in the media

growth

Isolation

based on the concept that if an individual cell is separated from other cells on a nutrient surface, it will form a colony

What is a colony?

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

What are the requirements for a colony to form?

• a medium with a firm surface

• a Petri dish

• inoculating tools

Microbes can be identified through what means?

• microscopic appearance when stained

• characterization of cellular metabolism

• determination of products given off during growth, presence of enzymes, and mechanisms for deriving energy

• genetic and immunological characteristics

If microscopes are to be used, specimens must be _____

prepared

What is needed to identify microbes?

• glass slide

• flame

• inoculation loop

• distilled sterile water

• a wax pencil

• cover slip

What are the steps to identify microbes?

1.) draw a wax circle on the slide and add a drop of water inside the circle

2.) flame the loop and, after cooling it, just barely touch one colony on the plate

3.) mix the loop in the drop of water spreading it to the margin of the wax circle

4.) pass the slide through the flame to evaporate the water

5.) now it is ready for staining and a cover slip!

Metric units

used to express the sizes of microbes

The basic unit of length in the metric system is the _____; it is equivalent to ____ inches

meters (m); 39.4 inches

The sizes of bacteria and protozoa are usually expressed in terms of _________; it is one millionth of a meter

micrometers (um)

A typical spherical bacterium (coccus) is approximately ___ um in diameter

1 um

A typical rod-shaped bacterium (bacillus) is approximately ____ um wide by ____ 3 um long

1 X 3 um

The sizes of viruses are expressed in terms of _____; this is equal to one billionth of a meter

nanometers (nm)

Most of the viruses that cause human diseases range in size from ____ to ____

10 to 300 nm

Ebola virus, a cause of viral hemorrhagic fever, can be as long as ______

1000 nm (1 um)

When using a microscope, the sizes of microorganisms are measured using an ______ ________

ocular micrometer

Microscopes

an optical instrument that is used to observe tiny objects, objects so small that they cannot be seen with the unaided human eye

Each optical instrument has a limit as to what can be seen using that instrument; this limit is referred to as what?

resolving power or resolution

The resolving power of the unaided eye is approximately ____ mm

0.2 mm

Simple microscope

contains only one magnifying lens

Why can a magnifying glass be considered a simple microscope?

it has a lens that magnifies objects 3 to 20 times larger than the object’s actual size

Anton van Leeuwenhoek’s simple microscopes had a maximum magnifying power of about _____ times

300

Compound microscopes

contains more than one lens

Compound light microscope

one lens with visible light used as the source of illumination

Compound light microscopes usually magnify objects about ______ times

1000

The resolving power of a compound light microscope is approximately ___ um

0.2 um (about 1000 times better than the resolving power of the unaided human eye)

What do our microscopes have for lenses?

• Rotating lenses (4x, 10x, and 40x) 

• Lens, mirror, and another lens so as light passes through the specimen, enlarged through first lens, then hits the mirror, and a reflection is sent through the second lens that enlarges it even more (10*10=100x magnification because magnifying the result of the lens each time)

Oil immersion lens

• uses oil to capture light that would otherwise be lost to scatter

• reducing scatter increases resolution

• oil immersion lens can resolve images that are at least 0.2 um in diameter and at least 0.2 um apart

Refractive index

a measurement of the degree of bending that light undergoes as it passes from one medium to another

The higher the difference in refractive indexes, the _____ the contrast

greater

The _____ ______ can control the amount of light entering the condenser and increase constrast

iris diaphragm

Special lenses and dyes are also used to ______ contrast

increase

Because objects are observed against a bright background or “bright field,” the compound light microscope is sometimes referred to as a __________ __________

brightfield microscope

If the condenser is replaced with what is known as a darkfield condenser, illuminated objects are seen against a dark background or “dark field”; the microscope is then called what?

a darkfield microscope

Phase-contrast microscopes

used to observe unstained living microorganisms

• organisms are more easily seen because the light refracted by living cells is different from the light refracted by the surrounding medium

Fluorescence microscopes

contain a built-in ultraviolet (UV) light source

• when the UV light strikes certain dyes and pigments, these substances emit a longer-wavelength light, causing them to glow against a dark background

Electron microscopes

enable us to see extremely small microbes such as rabies and smallpox viruses as they have much higher resolving power than compound microscopes

Living organisms cannot be observed using an electron microscope. Why?

The processing procedures kill the organisms

In electron microscopes, an electron ____ is used as the source of illumination, and ____ are used to focus the ____

beam; magnets; beam

Two types of electron microscopes

1.) transmission

2.) scanning

Transmission electron microscope

• uses an electron gun to fire a beam of electrons through an extremely thin specimen (<1 um thick)

• an image of the specimen is produced on a phosphor-coated screen

• magnification is approximately 1000 times greater than with the compound light microscope

• resolving power is approximately 0.2 nm

Scanning electron microscope

• electrons are bounced off the surface of a specimen and the image appears on the monitor (this is used to observe the outer surfaces of specimens)

• resolving power of this microscope is about 100 times less than that of transmission electron microscopes

TEMs and SEMs are ___________ images

black-and-white

Why do we stain bacteria?

• preserve morphology and fix bacteria to the slide

• colors the bacteria so that we are able to visualize them

• kills the bacteria in the process!

Simple stains

one dye is used and everything is the same color

Differential stains

two dyes that take differently to different types of bacteria

• especially important if we have a mixed culture

Types of stains

• Negative Staining

• Gram Staining

• Acid-Fast Staining

• Spore Staining

• Capsule Staining

Negative Staining

• main purpose: size and shape

• key result: clear cells on dark background

Gram Staining

• main purpose: cell wall type

• key result: Purple is Gram-Positive & pink is Gram-Negative

Acid-Fast Staining

• main purpose: waxy cell wall

• key result: Red=acid-fast

Spore Staining

• main purpose: endospore presence

• key result: green spores

Capsule Staining

• main purpose: capsule detection

• key result: clear halo

Why is culturing, growing, and staining microorganisms from samples rally important in differential diagnosis of patients?

Informs doctors/practitioners:

• of treatment options (antibiotics vs. antivirals, etc.)

• tells them the severity of what they are working with

• course of the disease and other treatment options