Lecture 2

Question 1

What are the 5 techniques performed in the lab?

Answer 1

innoculation, incubation, isolation, inspection, identification

Question 2

What is inoculation?

Answer 2

The placement of microorganisms into or onto culture media

Question 3

What is a culture?

Answer 3

The growth of microorganisms with various media

Question 4

What is a medium?

Answer 4

a nutrient used to grow microorganisms outside their natural habitat

Question 5

What is an incubator?

Answer 5

chamber that is temperature-controlled (and sometimes control of other environmental factors), use for growth of microbes

Question 6

What temperature is used for human pathogens?

Answer 6

temperatures fall between 20°C and 40°C for human pathogens

Question 7

What happens during incubation?

Answer 7

During incubation, microbes grow and multiply, producing visible growth in the media; *growth indicates increase in NUMBER rather than increase in SIZE

Question 8

What is a pure culture

Answer 8

just the one microorganism in the container

Question 9

What is a mixed culture?

Answer 9

2 or more identified organisms in a container

Question 10

What is minimal media?

Answer 10

media that only contains a few components

Question 11

What is complex media?

Answer 11

contain at least one ingredient that is not chemically definable

Question 12

What is selective media?

Answer 12

contains one or more components that inhibit the growth of a certain microbes but not others

Question 13

What is differential media?

Answer 13

allows multiple types of microorganisms to grow but are designed to display differences among those microorganisms so that they can be distinguished from one another

Question 14

What is reducing media?

Answer 14

contains a substance (i.e. thioglycollate) that absorbs oxygen or slows the penetration of oxygen

Question 15

What is fermenation media?

Answer 15

contain sugars that can be fermented and a pH indicator that shows acid production as the
result of this reaction

Question 16

what is transport media?

Answer 16

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

Question 17

What is a colony?

Answer 17

a visible cluster of microbes on a solid medium

Question 18

What is isolation?

Answer 18

Requires solid media with enough surface area to spread a culture for separation

Allows for identification of multiple organisms in mixed cultures

Question 19

What is inspection and indentification?

Answer 19

Microbes can be identified through a combination of

microscopic appearance – wet mounts or staining

use of selective and differential media
characterization of cellular metabolism and products produced during growth, presence of certain enzymes

genetic and immunological characteristics

*A combination of these methods is used as usually one alone is not sufficient for positive identification

Question 20

What is resolution?

Answer 20

Ability to distinguish detail
Higher the resolution, clearer the picture
Resolving power – closest 2 points can be to each other and still be distinguished

Question 21

What is resolving power?

Answer 21

1) size of objective lens (larger the lens the greater the resolving power)
2) wavelength of light passing through specimen (shorter wavelengths tend to give better resolution)
3) refractive index of the material between the objective lens and specimen – oil, air…

Question 22

What is magnification?

Answer 22

Enlargement of an image

Requires a convex lens (thicker in center than at the edge)

Question 23

What is total magnification?

Answer 23

Number of times larger the image seen appears compared to the actual sample

Question 24

What is a wet mount?

Answer 24

Drop of liquid containing microorganisms on a slide with a coverslip on top
Allows you to see the true size and shape, motility

Question 25

What is a stain?

Answer 25

Increase contrast, bind to certain parts of microorganisms, allow them to be visible Vital stains – stain living cells, can be added directly to a wet mount Some staining requires fixation of the cells

Question 26

What is fixation?

Answer 26

Getting the organism to stay on the slide

Question 27

What are the types of dyes?

Answer 27

Basic dyes – positively charged (safranin, basic fuchsin, crystal violet, methylene blue) Acidic dyes – negatively charged (Eosin, acid fuchsin, congo red) Mordants – intensify staining, increase the cell’s affinity for a dye

Question 28

What are the types of stains?

Answer 28

Simple Differential Special

Question 29

What are simple stains?

Answer 29

Basic dyes that allow the cell to be visible, all cells are stained the same color

Question 30

What are differential stains?

Answer 30

Distinguish between types of organisms based on how they stain

Question 31

What are the 3 steps of differential staining?

Answer 31

Three steps: primary staining – same as simple staining destaining – removes stain in certain cells counterstaining – stains parts of cells that were destained to allow them to be seen

Question 32

What is Gram's Stain?

Answer 32

Divides bacteria into 2 groups, Gram positive and Gram negative

Question 33

What is acid fast stain?

Answer 33

Used to identify Mycobacterium tuberculosis, only stains mycobacteria and some actinomycetes (called acid fast bacteria) These bacteria resist destaining because of a waxy material in their envelope Acid fast bacteria stain red, non-acid fast stain blue

Question 34

What are structural stains?

Answer 34

cell wall, flagella, | capsule

Question 35

What is an example of a hard to stain bacteria?

Answer 35

spirochetes

Question 36

What is negative stain?

Answer 36

stains everything except structure you’re looking for (capsule stain)

Question 37

What are the types of microscopy?

Answer 37

Brightfield, Darkfield, Phase contrast, Differential, | Fluroscent, confocal, electron, transmission, and scanning

Question 38

What is Brightfield Microscopy?

Answer 38

Brightfield is a stain and the light waves move in sync. | You have to have stain in this type and you have to set the right settings

Question 39

What is darkfield microscopy?

Answer 39

``` Live organisms (wet mount) Makes the field dark and the organisms glow ```

Question 40

What is phase contrast microscopy?

Answer 40

Allows visualization of live, unstained organisms (wet mount) Phase rings in objective lens and condenser that send some light rays through specimen, some around it (where as brightfield sends rays straight through the specimen), allowing for visualization)

Question 41

What is differential microscopy?

Answer 41

Like the phase-contrast microscope, the differential interference contrast (DIC) microscope provides a detailed view of unstained, live specimens by manipulating the light. two prisms that add contrasting colors

Question 42

What is fluorescence microscopy?

Answer 42

Used in research and diagnostically. Stain only binds to a certain organism so when you had the stain to the sample and something glows then that is what you are looking for. You are using dyes that glow under certain lights. Different wavelengths cause different things to glow in different colors

Question 43

What is confocal microscopy?

Answer 43

Really nice clean images. | Z stacks take a bunch of pictures and stack them to make a 3d image

Question 44

What is electron microscopy?

Answer 44

Used for examining objects smaller than 2uM. | Uses a beam of electrons instead of light (much shorter wavelength, so get much higher resolution)

Question 45

What is transmission microscopy?

Answer 45

Fixed samples that are dead

Question 46

What is scanning microscopy?

Answer 46

Beam of electrons is directed over the surface of the specimen, knocking electrons out of the surface of the specimen. These secondary electrons are transmitted to an electron collector, amplified, and used to produce a 3-D image