Chapter 2 Tools of the Laboratory

Inoculation

-producing a culture.

Isolation

-seperating microbes from one another.

Incubation

-placed in a temperature controlled chamber.

Inspection

-colonies or broth are observed macroscopically for growth characteristics (color, texture, size)

Identification

-Information used can include relevant data already taken during initial inspection and additional tests that further describe and differentiate the microbes.

If an individual bacterial cell is _______ from other cells and has space on a nutrient surface, it will grow into a mound of -_______.

-seperated -cells-colony

A colony consists of __________.

-one species.

Isolation techniques include:

-streak plate technique -pour plate technique -spread plate technique

Media can be classified according to three properties:

-Physical state -Chemical composition -Functional type

Physical state

-liquid,semisolid, and solid.

Chemical composition

-synthetic(chemically defined) and nonsynthetic (complex).

Functional type

• general purpose, enriched, selective, differental, anaerobic growth, specimen transport, assay, Enumeration

Liquid Media Broth

-water based solutions that do not solidify at temperatures above freezing.

Semisolid Media

-exhibit a clotlike consistency because they contain an amount of solidifying agent (agar or gelatin) that thickens them but does not produce a firm substance.

Solid Media

-provide a firm surface on which cells can form discrete colonies.

Most commonly used media

-Nutrient Broth -Nutrient Agar

Nutrient Broth

-liquid medium containing beef extract and peptone disolved in water.

Nutrient Agar

-solid medium containing beef extract and peptone, and agar.

Most commonly used solidifying agent is____, which is a polysaccharide isolated from______.

-agar. -red alga "Gelidium.

Chemical Content of Synthetic Media

• contains pure organic and inorganic compounds in an exact chemical formula.

Chemical Content of Complex or Nonsynthetic Media

• contains at least one ingredient that is not chemically definable.

Chemical Content of General Purpose Media

-grows a broad range of microbes that do not have a special growth requirements.

Chemical Content of Enriched Media

• contains complex organic substances such as blood, serum, hemoglobin, or special growth factors that certain species must be provided in order to grow.

Selective media

• contains one or more agents that inhibit the growth of some microbes and encourage the growth of desired microbes.

Differential media

• allows growth of several types of microorganisms but are designed to bring out visible difference among those microorganisms.

Reducing medium

• contains a substance that absorbs oxygen or slows penetration of oxygen into medium; used for growing anaerobic bacteria.

Carbohydrate fermentation medium

• contains sugars that can be fermentation, converted to acid, and a pH indicator to show this reaction.

Key characteristics of a reliable microscope are:

-Magnification - ability to enlarge. -Resolving power - ability to show detail.

Magnification in most microscopes results from interaction between.....

-light waves and curvature of lens

Angle of light passing through convex surface of glass changes -

-refraction.

Depending on the _______________ of the lens, the image appears enlarged

-size

Extent of enlargement -

-magnification.

Magnification occurs in two phases -

-The objective lens forms the magnified real image. -The real image is projected to the ocular where it is magnified again to form the virtual image.

Total magnification of the final image is a product of the...

-seperate magnifying powers.

Power of Objective X Power of Ocular =

-Total Magnification.

Power of Objective X Power of Ocular = 4X scanning objective 10X =

-Total Magnification. 40X

Power of Objective X Power of Ocular = 10X low power objective 10X =

-Total Magnification. 100X

Power of Objective X Power of Ocular = 40X high dry objective 10X =

-Total Magnification. 400X

Power of Objective X Power of Ocular = 100X oil immersion objective 10X =

-Total Magnification. 1,000X

eyepiece

This is the part you look through that allows you to view the image. It contains the ocular lens.

nosepiece

This part holds the objective and rotates to change the magnification.

objective lenses

They are found on the nosepiece and range from low to high power.

stage clips

These are used to hold a slide in place on the stage.

light source

Sends light upwards through the diaphram to allow you to see the specimen.

arm

This is the part that supports nosepiece and eyepiece. We also use it when the microscope is carried.

coarse adjustment knob

This is the larger of the two knobs. It is used to move the stage so the you can focus the microscope.

fine adjustment knob

This is the smaller knob. It is used when you need to sharpen the focus or fine tune the view of your specimen

diaphragm

This part is used to adjust the amount of light that reaches the specimen.

base

This is the bottom part of the microscope.

stage

The part that holds the slide and the stage clips.

slide

Small glass plate that holds the specimen.

power

Refers to the magnification or strength of the lens.

specimen

The object or organism that is on the slide.

magnify

To make something look bigger.

field of view

The area you see when looking through a microscope.

magnification

How much larger an objected appears. For example; four hundred times larger or 400x.

simple microscope

It uses one lens to make things look bigger; an example would be a hand lens.

compound microscope

A microscope with two sets of lens.

temporary slide

It is used once and cleaned off. It is useful for studying living things.

permanent slide

It is used over and over. It can only be used to study nonliving things.

Resolution

• defines the capacity of an optical system to distinguish or seperate two adjacent objects.

Bright-field

-most used. specimen is darker than surrounding feild; live and preserved specimen.

Dark-field

-brightly illuminated specimen.

Phase-contrast

-transforms subtle changes in light waves passing through the specimen into difference in light intensity. -best for observing intercellular structures.

Variations on the Optical Microscope

-Bright-field -Dark-field -Phase-contrast

Specimen Preparation for Optical Microscopes, slides depends on....

-condition of specimen -aims of the examiner --type of microscope avaliable.

Specimen Preparation for Optical Microscopes, Wet mounts and hanging drop mounts

-allow examination of characteristics of live cells-size,motility, shape, and arrangement.

Specimen Preparation for Optical Microscopes, Fixed mounts are made by

-drying or heating a film of specimen. -A smear is stained by the dyes.

Dyes create contrast by imparting a ____ to cells or cell parts.

-color

Basic dyes

--cationic dyes that have a positive (

Acidic dyes

-anionic dyes that have a negative (-) charge.

Positive staining

-the dye sticks to cells and gives them color. -surface of microbes are negatively (-) charged and attract basic dyes. -background Not stained (generally white)

Negative staining

-does not stick to specimen but dries around its outer boundry, forming a silhouette. -microbes repels dye, the dye stains the background (dark gray or black).

Simple stain

-one dye is used to reveal shape, shape, size, and arrangement.

Differential stains

-use two differnt-colored dyes, called the "primary dye" and the "counterstain". (example: Gram stain, Acid-fast stain and endospore stain)

Structural stains

-use to reveal certain cell parts that are not revealed by conventional staining methods.

Dyes used for Positive (

) Staining

Dyes used for Negative (-) Staining

-Acidic dyes: -Nigrosin -Indian ink

Subtypes of Positive (

) Stains

Subtypes of Negative (-) Stains

-Few types: -Capsule -Spore

gram stain

-permits ready differentiation of major categories based on the color reaction of the cells: "gram-positive", which stain purple, and "gram-negative", which stain red.

Which of the following is not part of the Five I's? -inspection -identification -induction -incubation -inoculation

-induction

The term "culture" refers to the____growth of microorganisms in ____.

-macroscopic -media

A mixed culture is____.

-one that contains two or more known species.

Agar is superior to gelatin as a solidifying agent because agar....

-does not melt at room temperature. -is not usually decomposed by microorganisms.

The process that most accounts for magnification is____.

-refraction of light rays.

A subculture is a____.

-culture made from an isolated colony.

Resolution is____with a longer wavelength of light.

-worsened

A real image is produced by the____.

-objective.

A microscope that has a total magnification of 1,500X when using the oil immersion objective has an ocular of what power?

-15X

The specimen for an electron microscope is always____.

-killed.

Motility is best observed with a____.

-hanging drop preparation.

Bacteria tend to stain more readily with cationic (psitively(

)charged) dyes because bacteria____.

The primary difference between TEM and SEM is in____.

-preparation of the specimen.

A fastidious organism must be grown on what type of medium?

-enriched medium.

What type of medium is used to maintain and preserve specimens before clinical analysis?

-transport medium.

Which of the following is NOT an optical microscope? -dark-field -confocal -atomic force -fluorescent

-atomic force

For the selected type medium, select all descriptions that fit.

-mannitol salt agar (MSA):

-selective medium (contains one or more agents that inhibits the growth of a certain microbe or microbes)

-differential medium (contains a dye (phenol red) that changes colors under variations of pH, and manitol, a sugar that can be converted to acid)

-complex medium

For the selected type medium, select all descriptions that fit.

-chocolate agar:

-enriched medium (contains complex organic substances such as blood, serum, hemoglobin, or special growth factors)

-complex medium

For the selected type medium, select all descriptions that fit.

-MacConkey agar (MAC):

-selective medium (contains one or more agents that inhibits the growth of a certain microbe or microbes)

-differential medium (contains a dye (phenol red) that changes colors under variations of pH, and manitol, a sugar that can be converted to acid)

-complex medium