Microbiology Chapter 3 (Cowan)

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1. The Five I's Inoculation

Incubation
Isolation
Inspection
Identification

2. Culture Cultivation of microorganisms

Any growth that appears in or on
the medium after incubation

3. Medium (plural: media): A nutrient-containing environment

in which microbes can multiply

4. Incubation: Usual temperatures: 20 - 40°C

(temperature it normally occurs at, Incubators also control atmospher-
growth on liquid vs solid medium) ic gases such as oxygen and car-
bon dioxide
Microbial growth in liquid medium:
Cloudiness, sediment, scum, color
Microbial growth on solid medium:
Colonies: visible masses of
piled-up cells

5. Media is classified according to three Physical state

properties: Chemical composition
Functional type (purpose)

6. Liquid media Water-based solutions that do not

solidify at temperatures above
freezing and flow freely
Broths, milks, infusions

7. Semisolid media: Exhibits a clot-like consistency at

room temperature
Contains enough gelatin or agar to
thicken but not produce a firm sur-
face
Used to determine motility of bac-
teria

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Also used to localize a reaction to a
specific site

8. Solid media: Provides a firm surface upon which

cells can form discrete colonies
Used to isolate bacteria and fungi

9. Agar Complex polysaccharide isolated

from the red alga Gelidium
Solid at room temperature, lique-
fies at 100oC, and once liquefied,
does not begin to solidify until it
reaches 42oC
Any medium containing 1% - 5%
agar usually has the word "agar" in
its name

10. Defined (synthetic) media: Media whose chemical composi-

tions are precisely chemically de-
fined
May contain pure organic or inor-
ganic compounds that do not vary
from one source to another
Have a molecular content defined
by means of an exact formula
Useful in research

11. Complex media: Contains at least one component

that is not chemically definable
Contains extracts of animals,
plants, or yeasts
May contain ground-up cells, tis-
sues, or secretions
Examples: blood, serum, meat ex-
tracts or infusions, milk, yeast ex-
tract, soybean digest, peptone

12. General purpose media: Grows as broad a spectrum of bac-

teria as possible
Complex media that contains a
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mixture of ingredients that support
a wide variety of microbial life

13. Enriched media: Contains complex organic sub-

stances that fastidious bacteria re-
quire for growth
Contain growth factors: specific vit-
amins or amino acids

14. Differential Media Allow multiple types of microorgan-

isms to grow, but display visible dif-
ferences between colonies.
Differences in colony size or color,
media changes, or formation of gas
bubbles or precipitates
Variations may be due to metab-
olism of certain ingredients that
cause a color change

15. Selective Media Contains one or more agents that

inhibit the growth of certain mi-
crobes
Encourage a select microbe to
grow
Important in primary isolation of a
certain type of microorganism from
a mixed sample

16. Blood Agar: Used as an enrichment medium for

fastidious microbes as well as dif-
ferential media

17. Hemolysins enzymes that lyse red blood cells to

release iron-rich hemoglobin

18. Beta hemolysis complete lysis of red blood cells

19. Alpha hemolysis incomplete lysis of red blood cells

20. Gamma hemolysis no hemolysis

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21. Media can be both selective and differ- True

ential (T/F)

22. Misc. Media Reducing media:

Grow anaerobic bacteria
Carbohydrate fermentation media:
Contains sugars and pH indicators
to show fermentation
Transport media:
Maintains and preserves speci-
mens
Assay media:
Used to test the effectiveness of
antimicrobial drugs
Enumeration media:
Used in industrial and environmen-
tal microbiology

23. Colony A discrete mound of cells formed

on solid nutrient surface
Consists of just one species and no
other if formed from a single cell

24. Streak plate method: A small droplet of culture or sample

is spread across the surface of a
medium with an inoculating loop
The pattern used to inoculate grad-
ually thins out the sample and sep-
arates the cells in order to encour-
age the growth of discrete colonies

25. Loop Dilution or Pour Plate Method Sample is diluted serially into
cooled, but still liquid agar tubes
Inoculated tubes are poured into
sterile Petri dishes and allowed to
harden
Diluted cells have enough space to
grow into separate colonies
Some colonies may grow deep in
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the medium rather than just on the
surface

26. Spread Plate Technique: A small volume of sample is pipet-

ted onto the surface of the plate
A sterile spreading tool or "hockey
stick" is used to spread the sam-
ple around evenly on the surface to
form individual colonies

27. Pure culture: A container of medium that con-

tains only a single known species
or type of microorganism
Used most frequently for laboratory
study

28. Anexic free of other living things except for

the one being studied

29. Subculture a second-level culture from a

well-isolated colony

30. Mixed culture: A container that holds two or

more identified, easily differentiat-
ed species of microorganisms

31. Contaminated culture: A culture that was once pure or

mixed that now contains contami-
nants, or unwanted microbes of un-
certain identity

32. Biochemical tests can determine Nutrient requirements

fundamental chemical characteristics Products given off during growth
such as: Presence of enzymes
Mechanisms for deriving energy

33. Genotypic testing detects microbes based on their

DNA

34. Immunologic testing

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testing the isolate against known
antibodies

35. Microbial profiles are determined Phenotypic testing

through combining: Genotypic testing
Immunologic testing
Macroscopic analysis
Microscopic analysis

36. American Type Culture Collection Maintains the largest stock of live
(ATCC): cultures for study and experimenta-
tion
Freeze dried fungal, bacterial, viral,
and algal cultures

37. Biohazardous material: Cultures and specimens collected

from patients or the environment
Steam sterilizing (autoclaving) or
incineration are used to destroy mi-
croorganisms

38. What are two definitions of the term Culture can mean to grow a medi-
"culture"? um (verb)
Culture can mean a growth (noun)
There are also different kinds of
cultures (pure cultures, mixed cul-
tures, subculture)

39. What are the three physical states of Liquid media, semisolid media, and
media? solid media

40. What are the differences between se- Selective media is made to only
lective and differential media? grow one kind of colony where as
differential media allows all kinds
of growths but is made to tell the
difference between growths (could
be different colors)

41. List and describe two types of media MacConkey Agar- MacConkey
that are both selective and differential. agar is a selective and differ-
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ential culture medium for bacte-
ria designed to selectively isolate
Gram-negative and enteric bacil-
li and differentiate them based on
lactose fermentation.

Mannitol Salt Agar- Mannitol salt

agar or MSA is a commonly used
selective and differential growth
medium in microbiology. It encour-
ages the growth of a group of cer-
tain bacteria while inhibiting the
growth of others

42. What dimensions are macroscopic or- meters (m) and centimeters (cm).
ganisms measure in?

43. What dimensions are microscopic or- measured from millimeters (mm)
ganisms measured in? to micrometers (¼
m) to nanometers
(nm).

44. Measurement of smallest bacteria: 200 nm

45. measurement of protozoa and algae: 3-4 mm

46. Viruses measurement 20-800 nm

47. Eukaryotes vs. Prokaryotes Eukaryotes have a nucleus and

prokaryotes do not

48. First microscopes vs. later micro- first had a simple magnifying lens
scopes and a few working parts and the
later ones had two magnifying lens-
es, a lamp in the base to illuminate
specimen, and a condenser to fo-
cus the light

49. Three properties of an effective micro- Magnification

scope: Resolution
Contrast
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50. Objective lens the lens closest to the specimen.

Forms the real image

51. Ocular lens: closest to the eye, also known as

the eyepiece.
Forms the virtual image, received
by the eye and converted into a
retinal or visual image

Virtual image is upside down and

inverted!

52. How to determine the magnifying pow- Magnifying power of the micro-
er of a microscope: scope is calculated by multiplying
the power of the ocular lens by the
power of the objective lens.
4x scanning objective x 10x = 40x
10x low power objective x 10x =
100x
40x high dry objective x 10x = 400x
100x oil immersion objective x 10x
= 1000x

53. Resolution and Resolving Power The capacity of an optical system

to distinguish or separate two ad-
jacent objects or points from one
another.
Human eye: 0.2 millimeters
Microorganisms cannot be re-
solved by the naked eye

Determined by a combination of
characteristics of the objective lens
and the wavelength of light being
used to illuminate the sample.
Optical microscopes use visible
light

54. Oil Immersion Lens:

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Oil prevents the scattering of light
rays and increases the numerical
aperture and resolution

55. Refractive index: A measurement of the degree of

contrast of an image
Refers to the degree of bending
that light undergoes as it passes
from one medium to another
The higher the refractive index, the
greater the contrast

56. Four types of light microscopes: Bright-field

Dark-field
Phase-contrast
Interference

57. Fluorescence microscope uses ultraviolet radiation as the illu-

minating source.

58. Confocal microscope uses a laser beam as the illuminat-

ing source

59. Wet mounts or hanging drop mounts: Cells are suspended in a suitable
fluid: water, broth, or saline
Fluid maintains viability and pro-
vides a medium for movement
Provide a true assessment of size,
shape, arrangement, color and
motility of cells

60. Wet Mounts Consist of a drop or two of culture

placed on a slide overlaid with a
coverslip
Advantages: quick and easy to pre-
pare
Disadvantages:
Can damage larger cells
Susceptible to drying

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Can contaminate the handler's fin-
gers

61. Hanging Drop preparation: Prepared with concave (depres-

sion) slide, Vaseline adhesive or
sealant, and a coverslip
Overcomes the disadvantages of
wet mounts

62. Dyes used in microbial staining: have a positive charge, attracted to

Basic (cationic) acidic, negatively charged compo-
nents on bacterial cell walls

63. Acidic (anionic): have a negative charge, repelled by

acidic, negatively charged compo-
nents on bacterial cell walls

64. Positive stain: Positively charged stain is attracted

to negatively charged cell walls
Stick to the cell and give it color

65. Negative stain: Negatively charged dye is repelled

by negatively charged bacterial cell
walls
Produces a black background
around the cells

66. Simple stains: Require a single dye

Uncomplicated procedure

67. Differential stains: Use two differently colored stains

to clearly contrast cell types or cell
parts
Complex staining technique

68. Types of Differential Stains Gram stain

Acid-fast stain
Endospore stain
Capsule stain

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69. Gram staining Developed a century ago, it re-
-what color does gram positive bac- mains the universal diagnostic
teria stain and what color does gram staining technique for bacteria.
negative bacteria stain? Permits ready differentiation of ma-
jor categories based on the color
reaction of cells:
Gram-positive bacteria stain purple
Gram-negative bacteria stain pink

70. The Acid-Fast Stain Differentiates acid-fast bacteria

from non-acid-fast bacteria:
Acid-fast bacteria stain pink
Non-acid-fast bacteria stain blue
Detects the agents of tuberculosis
and leprosy

71. The Endospore Stain Used to distinguish endospores

from vegetative cells.
Detects endospore-forming mem-
bers of the genera Bacillus and
Clostridium.

72. Capsule: An unstructured protective layer

surrounding the cells of some bac-
teria and fungi.
Visualized through negative stain-
ing with India ink or special positive
stains.
Useful in identifying pathogens
such as Cryptococcus.

73. Flagella: Tiny, slender filaments used by

bacteria for locomotion

74. Flagellar staining: Flagella must be enlarged in order

to be seen
A coating is deposited on the
outside of the filament and then
stained

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75. What is the total magnification of a 400
specimen viewed with the 40x objec-
tive?

76. What is the importance of resolving Determines how much detail can
power? be seen

77. Describe the difference between sim- Simple stain is one color and in dif-
ple and differential stain. ferential staining you use two colors
to create a contrast. Simple stain-
ing is only going to show you basic
information regarding shape and
arrangement but differential stain-
ing will show you more about the
structure.

78. What is the purpose of the Gram stain? Gram staining differentiates bacte-
ria based on the structure of their
cell walls. If the cell wall is positive
is will stain purple and negative it
will stain pink.

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