BIOL 2614 Microbiology Lab Final

GloGerm

a product that was developed to illustrate the effects of unclean and unsanitary conditions

Agar slants

used to store/maintain a culture

Stock cultures

used as a source of inoculum for other cultures; used for the growth and the refrigerated storage of pure microbial cultures

Agar deeps

made in either a solid or a semisolid; inoculate either form by carefully insertion a culture-laden needle down the enter to the bottom

Stab culture

culture created in agar deep

What does a stab culture show?

exhibits growth either along the entire length of the stab or only at certain places, depending on the microbes' need for tolerance of oxygen

Broth

liquid medium used for growing microorganisms

Static incubation

a certain amount of oxygen is absorbed into the surfaces of broth in test tubes sitting in an undisturbed rack on the incubator shelf

To increase the oxygen than found in static broth tubes...

place the culture at an angle to increase the surface area and rotate them on a roller drum aparatus

Aeration

providing oxygen to broth cultures

Fermenters

For larger volumes of broth, air is filter sterilized and pumped into large broth-filled vessels

Turbidity

Some microbes uniforms increase in milkiness or cloudiness of the broth as their numbers increase

Pellicle

Microorganisms growing only at the surface of a static broth culture; mat of cells; require a considerable amount of oxygen for growth

Sediment/button

Microbial species that settle to the bottom of the tube to form this

Mycelia

may form clumps of filamentous cells have the appearance of small cotton balls floating in otherwise clear broth. Characteristic of fungi

Types of microscopes

light and electron

Light microscopes

use photons of visible light to form images

Why gram stain?

Most microbial cells are either colorless or they have so little pigment they cannot easily be seen with the brightfieqld microscope. One way to increase the contrast between microbes and the surrounding fluid is to stain the cells

Why must you heat fix the cells before gram staining?

Dyes do not readily penetrate living cells; so microorganisms are usually killed before dye is applied.

Simple stain

color the cell or the background in a way that enables you to observe whether the cell is a straight or curved rod or a coci and whether the cells are arranged in pairs or clusters

differential stains

use a combination of dyes that take advantage of chemical differences among cells.

Structural stains

preferentially cool only one part of the cell so that it can be distinguished from the rest of the microbe

Types of simple stains

Basic dyes

Acidic dyes

Types of differential stains

Gram stain

Acid-fast stain

Type of structural stains

endospore stain

flagella stain

capsule stain

A dye base with a positive charge

Crystal violet; attracted to the negatively charged surfaces of the cells, and some of it binds to components on their surfaces. If the cells are dead, the dye are penetrates the cell envelope and binds with negatively charged interior parts.

Morphology

the study of the size, shape, and arrangement of cels.

Primary stain

Crystal violet

Mordant

Gram's iodine; increases affinity between the primary stain and the reactive substances of the cell

Decolorizing agent

any chemical txt is a good lipid solvent and a good dehydrating agent

Counterstain

any dye as long as it is a color that contrasts with the deep blue-violet of the gram-positive

Standards in staining

One smear of a known gram-negative organism and another smear of a gram-positive organisms with unknown

Gram positive standard

E. coli

Gram negative standard

P. fluorescens

Structural staining allows a researcher to observe what?

Different microbial structures such as endospores, capsules, flagella, and carbon storage molecules

Endospore

a structure that is formed inside certain types of bacteria

Vegetative cell

lacks an endospore

Sporangium

once a mature endospore is formed, this is what the rest of the cell is called

After forming the endospore, the sporangium may what?

Die and eventually disintegrate (cell lysis

Free spore

If the resulting spore retains no visible evidence of the sporangium

What does it mean if you see no endospores on the microscope?

It may mean that the culture is too young and has not yet formed endospores, or it may mean thet you have not provided the correct nutrition for endospore formation

What do cells need before they can produce endospores?

The correct nutrients; if critical nutrients are lacking, endospores are not formed

As an endospore matures inside the sporangium, it becomes _________________.

desiccated (loses water) so mature endospores contain much less water than vegetative cells

The most significant endospore characteristic is its resistance to?

High temperatures

The mature endospore has a _______ ___________ tan the surrounding sporangium or vegetative cells

Greater density

The mature endospore having a greater density than the sporangium makes the endospores look very ____________ when cultures containing endospores are observed using a wet-mount preparation and a brightfield microscope

Bright (refractive)

What can you tell from a differential stain when looking at a sporangium and its endospore?

The presence of endospores are seen (noticeable because of the dye exclusion) and the gram characteristics of the sporangium or vegetative cell

Which dye can force into the spore by raisin the dye temperature to about 100c?

Malachite green

When you do a malachite green stain, what do you see?

Endospores in green and sporangium cells clear

To be able to see a malachite green stained endospore clearly inside a sporangiam with a bright field microscope, the sporangium must be what?

counterstained with a dye of contrasting color (fuschian)

Many bacteria produce ________________ ______________ that accumulate around the outer surface of the cell wall

extra cellular polymers; can be polysaccharides or proteins

Capsule

a structured mass of extracellular polysaccharide that tightly adheres to the cel wall

Slime layer

If the extracellular polysaccharide is less organized and not os closely bound to the cell surface

How are capsules best observed?

Negative staining

Capsule detection method we used

crystal violet with drops of copper sulfate on the top (Anthony's capsule stain)

How does Anthony's capsule stain work?

Crystal violet is used as the primary stain, interacting iwht the protein material in the culture broth, and copper sulfate serves as the modern. No addition of a negative stain . At the completion of the stain, the bacterial cells and the background will be stained by the crystal violet, while the unstained capsule will appear white.

What do capsules do?

Protect the cell from desiccation and act as buffers or osmotic barriers between the cell and its environment

Living cells can be easily studied with which microscope?

Phase-contrast

Main differences between a phase contrast and brightfield microscope

The placement of an annular stop below the condenser lens

The phase plate inside the objective lens

Motile organism

moves rapidly in one direction or appears to be tumbling rapidly; caused by the cell's flagella or cilia, which propel the cell throughout the fluid

even if cells are killed or nonmotile, you will observe some movement when they are suspended in a fluid. This appears as random wiggling. All colloidal particles exhibit this motion, called ____________

Brownian movement

Wet-mounts help determine what

Whether the cell is motile

One way to prevent movement of cells in a liquid environment is to?

Sandwich them between a coverslip and an agar-covered microscope slide

Prokaryotes average about __________ smaller than eukaryotes, and there internal structures appear _______ __________

five to ten times smaller, appear less complex.

What is the function of the flagella for prokaryotes?

Propel the cell through its aqueous environment just as the cilia and flagella do for eukaryotic cells

Motility

the movement caused by the action of the flagella

Positive chemotaxis

Motility toward a favorable environment

Negative chemotaxis

movement away from an environment that is less favorable or possible harmful

Chemoreceptors

specific proteins within the plasma membrane that cause motility

Tannic acid

Used in motility observation - precipitates around flagella

Polar flagellation

cells with flagella inserted only at the ends of a cell

Peritrichous flagellation

cells with flagella inserted all over the surface

Flagellin

the protein all bacterial flagella are made of

Swarmer

A very actively motile bacterial culture may rapidly spread itself across the surface of agar plates

Turbidity: what is it and what is it caused by?

The effect of light scattering by a colloidal suspension. They do not settle out of suspension and they scatter light

Spectrophotometer

Measure changes in turbidity - measure the amount of light that passes through a liquid medium

How is growth reported in turbidity

optical density (OD) with time. This is easier to deal with because OD is directly proportional to cell concentration. As cell numbers increase, so does the optical density

How can you give rise to a colony

If you can spread cells o a streak plate so that you separate cells from each other by a few millimeters or more, each cell that grows will give rise to a colony

If deposited cells are too close together, the colonies grow together. What is this called?

Confluent growth

Steps to a proper t-streak

1) Sterilize inoculating loop

2) Touch the loop to the agar surface

3) Position yourself so you can see the faint indentations left by the loop as it glides across the surface

4) sterilize loop between streaking on area of the plate and the next

5) to prevent contaminate-laden dust from falling onto you surface, hold the petri dish cover so that it partially covers the open plate

Viable number

a number o cells capable of disown on a solid medium

CFU

colony forming units

What is the best method available for determining viable numbers?

Serial dilution and plating

Sampling error

usually occurs because of an unequal distribution of cells in the culture or dilution fluid; the goal is to distribute the cells evenly by thorough mixing and then to obtain a representative sample in the pipette

Technical error

Most often due to some inaccuracy in preparing dilution blanks or in pipetting.

Serial dilution

the method of sequentially diluting a culture through a series of sterile dilution blanks

One should dilute microorganisms in a fluid that is osmotically similar to the environment from which they were transferred

Dilution factor

the denominator of the final dilution

Spread-plate method

involves spreading a small, known volume of a cell suspension onto the surface of a pre-poured agar plate

Pour-plate method

a known volume of cell suspension is inoculated into a tempered agar deep.

Tempered

When an agar deep is changed to a liquid sol and then cooled and held at temperature just above that which the agar solidifies (about 40c)

What is the advantage of the pour-plate method?

Satisfactory for growing either facultative or the microaerophilic banter (those that do not grow in the absence of oxygen OR in 20% oxygen levels)

You may prefer the pour-plate technique if you want only to determine the viable number of cells in a pure culture in which the cells are not strict aerobes.

General/all-purpose media

contain a variety of nutrients and can therefore supportt the growth of a variety of microorganisms with different nutrient requirements

Selective miedium

contain at least one ingredient that can INHIBIT the growth of unwanted microorganisms without preventing the growth of the desired type

Selective agents

ingredients that inhibit the growth of unwanted organisms (antibiotics, inhibitory chemicals, types of dyes)

Coliform bacteria

residents of the human intestine, and their presence in water indicates that the water is contaminated with fecal material

Differential medica

designed to to distinguish one type of microorganism from all others in a mixed cultures

Differential media contain a special ingredient that changes during gate growth of only certain types of microorganisms, causing their colonies or the agar around their colonies to take on a distinctive appearance

Differential media can be...

selective or nonselective