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Micro1 > Lab Practical #1 > Flashcards

Lab Practical #1 Flashcards

0
Q

Aseptic

A

Without contamination

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1
Q

Inoculation

A

Transfer of bacteria or other organisms into or onto a suitable growth medium (media = lab food)

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2
Q

Differential medium

A

Snyder agar

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3
Q

Normal flora

A

Organisms that live in or on body surfaces that do not normally cause diseases

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4
Q

Transient flora

A

Organisms that colonize the body for short periods of time due to environmental exposure. Easily disrupted by hand washing, antibiotics, etc.

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5
Q

Resident flora

A

Well-established species that thrive in or on the body. Will re-establish after disruption

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6
Q

Pathogens

A

Organisms that cause disease

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7
Q

Opportunists

A

Organisms that are generally non-pathogenic, but will take the opportunity to do so under certain conditions

  • after antibiotic therapy
  • in the very young or old
  • immunocompromised individuals
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8
Q

Nutrient agar (NAP)

A
  • seaweed carbohydrate
  • rhodophyta
  • bacteria do not use agar as energy
  • agar melts at 100* C, re solidifies at 45* C
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9
Q

Nutrient broth (NB)

A
  • Sterile H2O
  • yeast extract
  • beef peptones
  • NaCl
  • pH buffers
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10
Q

TSA/B

A

Trypticase soy agar/ broth

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11
Q

Boiling water

A

100* C or 212* F

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12
Q

Freezing

A

0* C or 32* F

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13
Q

Body temp

A

37* C or 98.6* F

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14
Q

Room temp (RT)

A

25* C or 68-72* F

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15
Q

Aliquot

A

Measured amt

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16
Q

Meniscus

A

Curve of the liquid

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17
Q

Dental Caries Exp

A 
Org. = Streptococcus mutans (GPC)
- turns sucrose into sticky carb called dextran
- colonization forms a biofilm on teeth
- biofilm becomes "dental plaque"
- leads to tooth decay and causes cavities, or "caries"
Media used = Snyder agar
- pH indicator: bromcresol green (4.8)
- alkaline color = green
- acid color = yellow
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18
Q

Isolated colonies

A

Visible areas of growth, distinct from other colonies

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19
Q

Pure culture

A
  • subculturing isolated colonies

- only one type of bacteria growing in or on a culture medium

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20
Q

Quadrant Streak Plate Exp.

A 
- made to separate individual organisms from a mixed culture
Mix 1:
- Eschrichia coli (GNR)
- Micrococcus luteus (GPC)
Mix 2:
- Escherichia coli (GNR)
- Serratia marscecens (GNR)
- Staphylococcus epidermidis (GPC)
NAP marked with an X and 1/2" streak
3 quadrants plus a lazy S for 4th quadrant
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21
Q

Magnification

A

Making an object appear larger

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22
Q

Resolution

A

Ability to see that 2 objects are separate

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23
Q

Brightfield Microscope Magnification

Of objective lens

A

(4x) red = scanning
(10x) yellow = low power
(40x) blue = high power
(100x) white = oil immersion

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24
Q

Total Magnification

A

Ocular X Objective

4x X 10x = 40x
10x X 10x = 100x
40x X 10x = 400x
100x X 10x = 1000x

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25
Q

Rheostat

A

Varies the amt of voltage of light obtained

- scales from 1-5 (dark to light)

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26
Q

Parfocal

A

Equal focus

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27
Q

Working distance

A

Between the objective lens and the slide

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28
Q

Depth of Field

A

How many layers you can see

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29
Q

Revolving Nosepiece

A

Houses the objective lenses

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30
Q

Condenser

A

Focuses all light to the stage

  • raise the light, lower the contrast
  • houses the iris diaphragm
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31
Q

Iris Diaphragm

A

Changes the amt of light going from the lamp to the stage.

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32
Q

Size of Field

A

Area of a slide being viewed through the ocular lens

- round circle of light

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33
Q

Refraction

A

Bending or scattering of light

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34
Q

Refractive Index

A

The degree that light bends
- a measure of the bending power of a medium
(Ex. Refractive index of immersion oil is the same as glass)

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35
Q

Steps to alter lighting of the microscope

A

A) adjust the iris diaphragm
B) move the condenser up or down
C) change the rheostat

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36
Q

Simple Unstained Wet Mount

A
  • place one drop of liquid material in center of clean slide
  • place cover slip @ 45* angle over slide and let go so it covers entire drop of liquid
  • view slide in Brightfield microscope
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37
Q

Recite smear prep using both broth cultures and solid media

A

:)

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38
Q

Chromophore

A

Part of the dye that contains the color

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39
Q

Basic Dyes

A
  • methylene blue
  • malachite green
  • crystal violet
  • safranin
  • basic dyes stain the cytoplasm of the cell
  • *best for bacterial smear
  • **positively charged chromophores stain negatively charged materials
40
Q

Simple Stain

A
  • make a heat fixed smear
  • place slide on staining rack
  • flood slide with methylene blue or other basic dye
  • allow dye to sit for 1 min.
  • rinse off excess dye with tap water
  • allow slide to dry
  • view!
41
Q

Salt

A

Chemical compound made of two parts
- positively charged part (cation)
- negatively charged part (anion)
Attraction of the two components forms the ionic bond

42
Q

Acidic Dye

A

(Ex. India ink)

  • used in a negative stain
  • used making a push smear
  • negatively charged chromophores stain positively charged materials
  • faster
  • done in one step
  • morphology is more accurate
  • no heat fixing
43
Q

Negative Stain

A
  • one drop of India ink at one end of slide
  • stir organism in drop of India ink
  • make a push smear
44
Q

Spirochetes

A

Free-living, non-pathogenic organisms that are very rare in microbiology

45
Q

Gram Stain

A 
-differential stain
Primary stain: Crystal Violet (1 min)
Mordant: Gram's Iodine (1 min)
Decolorizer: 95% Ethyl Alcohol (3-5 sec)
Counterstain: Safranin (2 min)

Blue cells = gram positive
Red cells = gram negative
colors the cell wall
**originally used to distinguish Streptococcus pneumoniae and Escherichia coli
**we tested Staphylococcus epidermidis and Escherichia coli

46
Q

Target circles

A

Sharpie pen used to make a small circle in the center of the slide. This is where the organism will be placed onto the slide.

47
Q

Subculture (sub)

A

Taking organisms from an original “primary plate” or broth and placing then onto or into another plate or broth

48
Q

Problem: organisms aren’t blue enough

A

Reason 1: crystal violet not left on long enough
- increase timing of primary dye
Reason 2: iodine not left on long enough
- leave iodine on same amt of time as cv

49
Q

Problem: gram + appear -

A

Reason: over decolorizing

- decrease time of ethyl alcohol

50
Q

Problem: gram - appear +

A

Reason: under decolorizing

- either increase decolorizer time or make thinner smear

51
Q

Problem: organisms not red enough to see

A

Reason: safranin not left on long enough

- increase timing of safranin

52
Q

Problem: organisms stain unevenly due to state of cell wall

A

Reason: orgs are too old

- use fresh cultures (18-24 hours old)

53
Q

Special stains

A

Used to see structures like flagella, endospores, and capsules

  • acid-fast stain
  • Shaeffer Fulton Endospore stain
  • capsule stain
54
Q

Acid-Fast Stain

A 
-dual smear
Primary Dye: carbolfuchsin (5-7 min)
Decolorizer: acid alcohol (10-30 sec)
Counter stain: methylene blue (2 min)
-slide is steamed with bibulous paper and carbolfuchsin over tea light

AFB’s are bright fuchsia pink
Non-AFB’s are blue
Orgs. used: Mycobacterium smegmatis (AFB) and Micrococcus luteus (non-AFB)

55
Q

Dual smear

A

Two bacterial species on one slide

56
Q

AFB

A

Acid Fast Bacilli

57
Q

Endospore Stain

A

-Schaeffer Fulton Endospore stain
Primary dye: malachite green (5 min)
Decolorizer: long rinse w tap water (30 sec)
Counterstain: safranin (2 min)
- steamed with bibulous paper and malachite green dye

Green endospores and red vegetative cells
Orgs used: Bacillus subtilis (old and young)

58
Q

Endospores

A

Only made by a few genera of bacteria

-notably Bacillus and Clostridium

59
Q

Sporulation

A

Spores are not fully extruded from the cells

60
Q

Glycocalyx

A

Bacterial capsule

61
Q

Capsule Stain

A

Primary dye: India ink (push smear)
Decolorizer/fixative: 0.1N HCl (30 sec)
Counterstain: safranin (1 min)
-make a push smear with the India ink, let air dry and flood smear with HCl. Then poor off excess and stain with safranin.

Red colored rods or yeast cells (vegetative cell = red)
Orgs used: Klebsiella pneumoniae, Bacillus megaterium, and Saccharomyces cerevisiae (yeast)

62
Q

Prepared slides viewed in class

A
  • Treponema pallidum
  • syphilis
  • spirochetes
  • Neisseria gonorrhoeae
  • gram - intercellular diplococci
  • Flagella
63
Q

Turbidity

A

Cloudiness

64
Q

Counting Colonies (NAP)

A 
0 colonies = NG
1-10 colonies = 1+
11-50 colonies = 2+
51-100 colonies = 3+
Over 100 colonies = 4+
65
Q

Counting Colonies (NB)

A 
No turbidity = NG
Slightly turbid = 1+
Moderately turbid = 2+
Mod to heavy turbid = 3+
Heavy turbidity = 4+
66
Q

CFU

A

Colony-forming units

  • gram if solid material
  • ml if liquid material
67
Q

Nutritional requirements

A 
Carbon: autotrophs or heterotrophs?
Nitrogen
Minerals
Vitamins
Growth factors: fastidious or not?
Energy source: phototrophs or chemotrophs?
Water
68
Q

Physical requirements

A

Temperature: mesophilic, thermophilic, or psychrophilic?
pH: acidophilic or alkalophilic?
Atmosphere: aerobic or capneic?

69
Q

Agar

A
  • solidifying agent
  • polysaccharide derived from algae
    (Rhodophyta)
  • thickens liquid
  • complex carb
  • bacteria do not use as a nutritional source
70
Q

Serial dilution

A

Series of repeated steps

71
Q

Titer (concentration)

A

___ # CFU/ml or gram

72
Q

Dilution

A

Amount transferred/ new total volume

73
Q

Bacterial population counts

A

> 300 TNTC (too numerous to count)
<30 record #
Inbetween find the titer

74
Q

Direct microscopic counts

A
  • diluting out # of organisms and placing in calibrated fields on a slide
  • # of orgs counted is X by dilution factor (DF)
  • good for evaluating safety of milk
  • counts dead and alive orgs
75
Q

Turbidometric Methods

A 
# of orgs vs spectrophotometer reading, then tubes are plotted on the standard curve
- both dead and alive orgs
76
Q

Standard Plate Counts (SPC)

A
  • serial tube dilutions
  • played into solid media
  • DF on each plate used to determine CFU per ml
  • only counts living orgs
77
Q

Quebec Counter

A

Aids in counting colonies by magnifying and back-lighting the agar plates

78
Q

Metabolism

A
  • aerobic respiration (38 ATP)
  • anaerobic respiration (32-34 ATP)
  • fermentation (2 ATP)

(ATP/glucose molecule)

79
Q

Thioglycollate Broth

A
  • resazurin is the O2 indicator
80
Q

Anaerobic Culture Ex 18

A

Orgs used: Alcaligenes faecalis, Staphylococcus aureus, Escherichia coli, Clostridium sporogenes

Media used: thioglycollate broth, NB, NA slant

-37* plate is the control

81
Q

Capneic

A

Orgs that like CO2

82
Q

Disinfectants

A

Decrease # of vegetative cells on an inanimate surface

83
Q

Antiseptics

A

Decrease # of vegetative cells on living tissues such as skin

84
Q

Chemotherapy

A

They are taken into the body to control infection

- antibiotics and synthetics

85
Q

Antibiotics

A

Substance against life

- penicillin

86
Q

Synthetics

A

Drugs that do not come from a living source

87
Q

Broad spectrum

A

Chemical agent that works against both gram types

88
Q

Narrow spectrum

A

Chemical agent that is effective against either gram + or gram - orgs

89
Q

Sensitive vs Resistant

A

:)

90
Q

Cidal

A

Totally killing organism

91
Q

Static

A

Inhibition of growth of org

92
Q

KB Test

A

Kirby Bauer Antibiotic Sensitivity Test

- standardized procedure to determine best antibiotic to use for treatment of a patient isolate

93
Q

Agar Diffusion Method

A

Saturating paper discs with agent and allowing the discs to incubate on the surface of agar plates previously inoculated with the test organism

94
Q

Zone of Inhibition

A

Clear area of “no growth” around a specific disc

95
Q

Chemical Control Agents Ex 21

A

Orgs used: Staphylococcus aureus, Escherichia coli, and Bacillus subtilis endospores

96
Q

ATCC

A

American Type Culture Collection

- where we get our cultures for lab

97
Q

Factors Standardized for KB Test

A
  1. Swab technique (confluent growth)
  2. Discs- size, spacing, pre-labeled, pre-saturated
  3. Mueller Hinton agar plate- plate size, depth= 4mm, pH= 7.2-7.4
  4. Temp.= 35+-2*C
  5. # of orgs- MacFarland standards 0.5
  6. Age of orgs- 18-24 hours
  7. Incubated 16-18 hours
  8. QC orgs
98
Q

KB Test Ex 22

A

Orgs used: E. Coli, Staph. aureus, enterococcus faecalis, Serratia marcescens, Pseudomonas aeruginosa, Baccilus megaterium

  • 8 pre-saturated discs of antibiotics

Micro1 (2 decks)

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