Lab Practical #1 Flashcards
(99 cards)
0
Q
Aseptic
A
Without contamination
1
Q
Inoculation
A
Transfer of bacteria or other organisms into or onto a suitable growth medium (media = lab food)
2
Q
Differential medium
A
Snyder agar
3
Q
Normal flora
A
Organisms that live in or on body surfaces that do not normally cause diseases
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.
5
Q
Resident flora
A
Well-established species that thrive in or on the body. Will re-establish after disruption
6
Q
Pathogens
A
Organisms that cause disease
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
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
9
Q
Nutrient broth (NB)
A
- Sterile H2O
- yeast extract
- beef peptones
- NaCl
- pH buffers
10
Q
TSA/B
A
Trypticase soy agar/ broth
11
Q
Boiling water
A
100* C or 212* F
12
Q
Freezing
A
0* C or 32* F
13
Q
Body temp
A
37* C or 98.6* F
14
Q
Room temp (RT)
A
25* C or 68-72* F
15
Q
Aliquot
A
Measured amt
16
Q
Meniscus
A
Curve of the liquid
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
18
Q
Isolated colonies
A
Visible areas of growth, distinct from other colonies
19
Q
Pure culture
A
- subculturing isolated colonies
- only one type of bacteria growing in or on a culture medium
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
21
Q
Magnification
A
Making an object appear larger
22
Q
Resolution
A
Ability to see that 2 objects are separate
23
Q
Brightfield Microscope Magnification
Of objective lens
A
(4x) red = scanning
(10x) yellow = low power
(40x) blue = high power
(100x) white = oil immersion
24
Total Magnification
Ocular X Objective
4x X 10x = 40x
10x X 10x = 100x
40x X 10x = 400x
100x X 10x = 1000x
25
Rheostat
Varies the amt of voltage of light obtained
| - scales from 1-5 (dark to light)
26
Parfocal
Equal focus
27
Working distance
Between the objective lens and the slide
28
Depth of Field
How many layers you can see
29
Revolving Nosepiece
Houses the objective lenses
30
Condenser
Focuses all light to the stage
- raise the light, lower the contrast
- houses the iris diaphragm
31
Iris Diaphragm
Changes the amt of light going from the lamp to the stage.
32
Size of Field
Area of a slide being viewed through the ocular lens
| - round circle of light
33
Refraction
Bending or scattering of light
34
Refractive Index
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)
35
Steps to alter lighting of the microscope
A) adjust the iris diaphragm
B) move the condenser up or down
C) change the rheostat
36
Simple Unstained Wet Mount
- 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
37
Recite smear prep using both broth cultures and solid media
:)
38
Chromophore
Part of the dye that contains the color
39
Basic Dyes
- 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
Simple Stain
- 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
Salt
Chemical compound made of two parts
- positively charged part (cation)
- negatively charged part (anion)
Attraction of the two components forms the ionic bond
42
Acidic Dye
(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
Negative Stain
- one drop of India ink at one end of slide
- stir organism in drop of India ink
- make a push smear
44
Spirochetes
Free-living, non-pathogenic organisms that are very rare in microbiology
45
Gram Stain
```
-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
Target circles
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
Subculture (sub)
Taking organisms from an original "primary plate" or broth and placing then onto or into another plate or broth
48
Problem: organisms aren't blue enough
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
Problem: gram + appear -
Reason: over decolorizing
| - decrease time of ethyl alcohol
50
Problem: gram - appear +
Reason: under decolorizing
| - either increase decolorizer time or make thinner smear
51
Problem: organisms not red enough to see
Reason: safranin not left on long enough
| - increase timing of safranin
52
Problem: organisms stain unevenly due to state of cell wall
Reason: orgs are too old
| - use fresh cultures (18-24 hours old)
53
Special stains
Used to see structures like flagella, endospores, and capsules
- acid-fast stain
- Shaeffer Fulton Endospore stain
- capsule stain
54
Acid-Fast Stain
```
-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
Dual smear
Two bacterial species on one slide
56
AFB
Acid Fast Bacilli
57
Endospore Stain
-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
Endospores
Only made by a few genera of bacteria
| -notably Bacillus and Clostridium
59
Sporulation
Spores are not fully extruded from the cells
60
Glycocalyx
Bacterial capsule
61
Capsule Stain
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
Prepared slides viewed in class
- Treponema pallidum
- syphilis
- spirochetes
- Neisseria gonorrhoeae
- gram - intercellular diplococci
- Flagella
63
Turbidity
Cloudiness
64
Counting Colonies (NAP)
```
0 colonies = NG
1-10 colonies = 1+
11-50 colonies = 2+
51-100 colonies = 3+
Over 100 colonies = 4+
```
65
Counting Colonies (NB)
```
No turbidity = NG
Slightly turbid = 1+
Moderately turbid = 2+
Mod to heavy turbid = 3+
Heavy turbidity = 4+
```
66
CFU
Colony-forming units
- gram if solid material
- ml if liquid material
67
Nutritional requirements
```
Carbon: autotrophs or heterotrophs?
Nitrogen
Minerals
Vitamins
Growth factors: fastidious or not?
Energy source: phototrophs or chemotrophs?
Water
```
68
Physical requirements
Temperature: mesophilic, thermophilic, or psychrophilic?
pH: acidophilic or alkalophilic?
Atmosphere: aerobic or capneic?
69
Agar
- solidifying agent
- polysaccharide derived from algae
(Rhodophyta)
- thickens liquid
- complex carb
- bacteria do not use as a nutritional source
70
Serial dilution
Series of repeated steps
71
Titer (concentration)
___ # CFU/ml or gram
72
Dilution
Amount transferred/ new total volume
73
Bacterial population counts
>300 TNTC (too numerous to count)
<30 record #
Inbetween find the titer
74
Direct microscopic counts
- 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
Turbidometric Methods
```
# of orgs vs spectrophotometer reading, then tubes are plotted on the standard curve
- both dead and alive orgs
```
76
Standard Plate Counts (SPC)
- serial tube dilutions
- played into solid media
- DF on each plate used to determine CFU per ml
- only counts living orgs
77
Quebec Counter
Aids in counting colonies by magnifying and back-lighting the agar plates
78
Metabolism
- aerobic respiration (38 ATP)
- anaerobic respiration (32-34 ATP)
- fermentation (2 ATP)
(ATP/glucose molecule)
79
Thioglycollate Broth
- resazurin is the O2 indicator
80
Anaerobic Culture Ex 18
Orgs used: Alcaligenes faecalis, Staphylococcus aureus, Escherichia coli, Clostridium sporogenes
Media used: thioglycollate broth, NB, NA slant
-37* plate is the control
81
Capneic
Orgs that like CO2
82
Disinfectants
Decrease # of vegetative cells on an inanimate surface
83
Antiseptics
Decrease # of vegetative cells on living tissues such as skin
84
Chemotherapy
They are taken into the body to control infection
| - antibiotics and synthetics
85
Antibiotics
Substance against life
| - penicillin
86
Synthetics
Drugs that do not come from a living source
87
Broad spectrum
Chemical agent that works against both gram types
88
Narrow spectrum
Chemical agent that is effective against either gram + or gram - orgs
89
Sensitive vs Resistant
:)
90
Cidal
Totally killing organism
91
Static
Inhibition of growth of org
92
KB Test
Kirby Bauer Antibiotic Sensitivity Test
| - standardized procedure to determine best antibiotic to use for treatment of a patient isolate
93
Agar Diffusion Method
Saturating paper discs with agent and allowing the discs to incubate on the surface of agar plates previously inoculated with the test organism
94
Zone of Inhibition
Clear area of "no growth" around a specific disc
95
Chemical Control Agents Ex 21
Orgs used: Staphylococcus aureus, Escherichia coli, and Bacillus subtilis endospores
96
ATCC
American Type Culture Collection
| - where we get our cultures for lab
97
Factors Standardized for KB Test
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
KB Test Ex 22
Orgs used: E. Coli, Staph. aureus, enterococcus faecalis, Serratia marcescens, Pseudomonas aeruginosa, Baccilus megaterium
- 8 pre-saturated discs of antibiotics
