Lab Exam 1 Flashcards
(163 cards)
1
Q
What are two important factors that influence bacterial growth?
A
Incubation time and temperature
2
Q
medium that contains living organism is called?
A
a Culture
3
Q
If a culture contains a single species it is said to be?
A
A Pure culture
4
Q
What does Aseptically mean?
A
Without contaminating yourself, others, the environment, the source culture, or the medium being inoculated.
5
Q
Medium used to grow microbes when fresh cultures or large numbers of cells are required
A
Broth
6
Q
Medium typically used for obtaining isolation in species
A
Plated media
7
Q
A culture that contains two or more species is
A
a mixed culture
8
Q
What is a commonly used isolation technique?
A
Quadrant or T streak - streak plate techniques
9
Q
What does CFU stand for
A
Colony-forming-unit
10
Q
T streak vs. quadrant streak
A
T streak involves three streaks with the inoculating loop, while a quadrant streak involves four streaks with the inoculating loop
11
Q
How do you hold the inoculating loop
A
Like a pencil, at a 45 degree angle
12
Q
Most colonies on streak plates grow from isolated colony-forming units (CFUs). On rare occasions, however, a colony may be a mixture of two different organisms. If a culture is started from this colony (thinking it is pure), correct identification will be next to impossible because the extra organism could confound the identifying test results. How could you verify the purity of a colony? (The answer may vary depending on what experience you have had prior to performing this exercise) if you found the colony to be a mixture of organisms, what could you do to purify it?
A
One way to attempt to purify a colony is by the quadrant streak plate technique. Individual colonies should form in quadrants three or four. The individual colonies can then be transferred to a pure media and be tested further to see if the colony has been purified.
13
Q
What is the primary negative consequence of not spreading the inoculum evenly over the agar surface?
A
Bacteria would be uneven resulting in difficulty with examination because the bacteria are tightly clustered in some areas.
14
Q
To get isolated colonies on a plate, only about 300 to 50 cfus can be in the inoculum. What will happen if the cell density of the inoculum significantly exceeds this number?
A
If the cell density of the inoculum results in more than 300 cells being deposited on the plate, then it becomes too technically difficult to differentiate and count more than 300 isolated colonies.
15
Q
Suppose you have two organisms in a mixture and Organism A is 1,000 times more abundant than Organism B. will you be able to isolate Organism B using the spread plate technique?
A
It would be difficult to isolate organism A from organism B using the spread plate technique as the culture is 1000 times more concentrated.
16
Q
Organism that do not reside on or in a specific plant or animal host and are not known to cause disease?
A
Are free living and non-pathogenic
Frequently called saprophytes
17
Q
Saprophytes preform an import ecological role, what is it?
A
Decomposition of organic matter
18
Q
Conversion of inorganic compounds into usable compounds by what cycle?
A
Biogeochemical cycles
19
Q
Organisms that cause damage to their host are?
A
Pathogens
20
Q
If a microbe benefits its host what it that an example of?
A
Mutualism
21
Q
If a microbe benefits from its host but has no negative effect on the host, that microbe is?
A
commensal
22
Q
What are opportunistic pathogens?
A
Bacteria are capable of becoming pathogenic or disease-causing if introduced into a suitable part of the body.
23
Q
A reservoir is?
A
Any area, even outside of the host organism, where a microbe resides, and serves as a potential source of infection
24
Q
What are the Colony morphology terms usually used to describe size?
A
- Diameter, if circular in shape
- width and length if shaped
otherwise
25
List all colony morphology Characteristics.
Size, Shape, margin, Surface, Texture, Elevations, Color/density
26
A visible mass of cells
Colony
27
What are the Colony morphology terms usually used to describe shape?
- round(circular)
- irregular
- punctiform(tiny pinpoint)
28
What are the Colony morphology terms usually used to describe Margin?
- entire (smooth with no
irregularities)
- undulate(wavey
- lobate(lobed
- filamentous (unbranched
strands)
- Rhizoid(branched like roots
29
What are the Colony morphology terms usually used to describe Surface?
- smooth
- rough
- wrinkled(rugose)
- shiny
- dull
30
What are the Colony morphology terms usually used to describe Elevations?
- Flat
- Raised
- Convex
- Pulvinate (very convex)
- Umbonate (raised in the
center)
31
What are the Colony morphology terms usually used to describe Color?
- Opaque - can't see through it
- Translucent - light passes
through it.
32
What are the Colony morphology terms usually used to describe Texture?
- moist
- mucoid(sticky)
- butyrous(buttery
- dry
33
Three critical aspects of a description of bacterial growth are colony size, color, and shape. At least three other important factors -- not descriptions of the organism itself -- typically are included when describing bacterial growth. What are they and why are they important?
1. Margin: Important to identify the border of an organism, and identify like organisms in a culture.
2. Elevation: Helps display where the colony is thickest and growth pattern.
3. Describes how quickly the colony will grow
34
What is a filiform?
growth, dense and opaque with a smooth edge
35
What does friable mean?
Dry, crusty (described texture)
36
What does echinulate mean?
Spiny (described margin)
37
Matching:
____ Filiform
____ Spreading edge
____ Transparent
____ Friable
____ Pigmented
Options:
1. Produces colored growth
2. smooth texture with solid edge
3. Solid growth, seeming to radiate outward
4. almost invisible, or easy to see light through
5. Rough texture with crusty appearance
__2__ Filiform
__3__ Spreading edge
__4__ Transparent
__5__ Friable
__1__ Pigmented
38
List some reasons why growth characteristics are more useful on agar plates then agar slants.
Much easier to see isolation on an agar plate than in an agar slant, also it is easier to look at a colony in an agar plate than in an agar slant. Typically agar plates are used to isolate a bacterial colony so studying morphology is more appropriate when observing isolated CFU's.
39
Why are agar slants better suited than agar plates to maintain stock cultures?
Agar slants are easier to keep free of air contaminants. Also because the agar is thick, it will not dry out as quickly. They also are relatively easy to store in test tube stock racks.
40
Suggest possible reasons for how temperature affects pigment production
Higher temperatures decrease the amount of pigmentation. Higher temps may deactivate the mechanism within the organism that leads to pigmentation.
41
A surface membrane is called a?
Pellicle
42
Bacteria at the bottom of the broth tube are typically referred to as?
Sediment
43
Growth throughout the broth tube
Uniform Fine turbidity
44
Define Flocculent
Suspended chunks and pieces
45
What factors besides the physical growth characteristics of the organism are important when recording data? Why?
Growth medium, incubation temperature, and atmosphere (aerobic or anaerobic).
46
Mathing:
___ Flocculent
___ Sediment
___ Ring
___ Pellicle
___ Uniform fine turbidity
Options:
1. Evenly cloudy throughout
2. Growth at top around the edge
3. Growth on the bottom
4. Membrane at the top
5. Suspended chunks or pieces
_5_ Flocculent
_3_ Sediment
_2_ Ring
_4_ Pellicle
_1_ Uniform fine turbidity
47
Minimum and optimum temperatures also know as?
Cardinal Temperature
48
Organisms the grow below 20 degrees Celsius are?
Psychrophiles
49
Organisms that grow between 0- 30 degrees Celsius are?
Psychrotrophs
50
Organisms the grow between 15 - 45 degrees Celsius are?
Mesophiles
51
Organisms the grow above 40 degrees Celsius are?
Thermophiles
52
Organisms the grow between 65 - 110 degrees Celsius are?
Extreme Thermophiles
53
There are obligate and facultative thermophiles, what does this mean?
Some thermophiles can grow below 40 degrees Celsius(facultative thermophiles)
Others cannot grow below 40 degrees Celsius (obligate thermophiles)
54
Why do different temperatures produce different growth rates?
Different temperatures can affect growth rates because they influence the metabolic rates of organisms.
55
In what ways could you adjust the incubation temperature to grow an organism at less than its optimal growth rate?
By adjusting the temperature up or down you can either surpass or go below the bacteria's optimal growth zone, resulting in a decrease in replication speed as the metabolism slows.
56
Plates with more than 300 CFU are?
Very difficult to count
57
Plates with less then 30 CFU are
not statistically reliable
58
150CFU
-------- x 10,000 = 3x10^6 CFU/ml
0.5ml
CFU
-------------- x Dilution factor =
Amount Plated CFU per ml
59
What are the different types of Light Microscopy and how do they differ?
Bright-field: can observe the levels of the organisms by adjusting focus
Dark-field: see a three-dimensional image
Phase contrast: emphasizes different part then in bright and dark field microscopy.
Fluorescence: uses a fluorescent dye that emits fluorescence when illuminating with ultraviolet radiation.
60
What does Bright-field Microscopy do to the image?
It produces an image made from light that is transmitted through a specimen. This can produce contrast and with the addition of stain visuals can become even more contrast providing great viewing of the bacteria.
61
Total magnification = Magnification by the objective lens x Magnification by the Ocular Lens.
:) Cool Fact!
62
What does the condenser lens do?
concentrates light and makes illumination of the specimen more uniform
63
What is refraction?
The bending of light
64
What is the Objective lens?
an objective is an optical element that gathers light from an object being observed and focuses the light rays from it to produce a real image of the object.
65
What is the real image?
A compound microscope uses an objective lens close to the object being viewed to collect light, which focuses a real image (image 1) of the object inside the microscope tube. That image is then magnified by the eyepiece lens, which creates an enlarged, inverted virtual image of the object (image 2)
66
What is the ocular lens?
The part of the microscope that magnifies the image produced by the microscope's objective so that it can be seen by the human eye.
67
What is a virtual image?
The inverted image, seen by the human eye.
68
The clarity of image is?
Resolution
69
The limit of resolution or resoling power is?
The actual measurement of how far apart two points must be for the microscope to view them as being separate. Notice that resolution improves as the limit of resolution is made smaller.
70
How do you calculate the limit of resolution?
` λ
D = ----------------------------------
NA condenser + NA objective
NA: stands for numerical apertures
D: stands for minimum distance at which two points can be resolved
71
What is Numerical Aperture?
The measure of a lens's ability to capture light coming from the specimen and use it to make the image.
72
What is Dark-Field microscopy?
Only the Light reflected off the specimen enters the objective. The appearance is of a brightly lit specimen against a dark background, often with better resolution than that of a bright-field microscope
73
What is Phase Contrast Microscopy?
exploits subtle differences in the refractive indices of water and cytoplasm components to produce contrast
74
What is Fluorescence Microscopy?
It uses a fluorescent dye that emits fluorescence when illuminated with ultraviolet radiation.
75
What is the meaning of Parfocal?
objectives that can be changed with minimal or no refocusing
76
Why aren't the magnifications of both ocular lenses of a binocular microscope used to calculate total magnification?
Because the image actually reaches via one ocular lens only and then travel to both eyes.
77
What is the total magnification for each lens setting on a microscope with 15X oculars and 4X, 10X, 45X, and 97X objective lenses?
Apply total magnification formula: Magnification of objective lens x Magnification of ocular lens:
1. 15x ocular x 4x objective = 60x
2. 15x ocular x 10x objective = 150x
3. 15x ocular x 45x objective = 675x
4. 15x ocular x 97x objective = 1455x
78
Assuming that all other variables remain constant, why will light of shorter wavelengths produce a clearer image than light of longer wavelengths?
The shorter the wavelength, the easier it is to identify smaller areas and details. Longer wavelengths have a harder time passing through small details, making the image seem blurry
79
Why is wavelength the main limiting factor on limit of resolution in light microscopy?
The image of two particles cannot be seen individually if it is smaller than the wavelength.
80
On a given microscope, the numerical apertures of the condenser and low-power objective lens are 1.25 and 0.25, respectively. You were supplied with a filter that selects A wavelength of 520 nm.
A. What is the limit of resolution
on this microscope?
B. Will you be able to
distinguish 2 points that are
300nm apart as being
separate, or will they blur
into one?
A. Apply limit of resolution
formula:
` λ
D = ----------------------------------
NA condenser + NA objective
In this case:
` 520nm
D = --------------------------------------
1.25+ 0.25
D = 346.67nm
B. No, because the limit of
resolution is 346.67, and 300
is smaller than 346.67
therefore, the points will
blur together.
81
Why should closing the iris diaphragm improve your ability to determine thread order?
It focuses light on the point of interest.
82
What is the ocular micrometer?
A type of ruler installed in the microscope eyepiece, composed of uniform but unspecified graduations.
83
The device used to calibrate ocular micrometers is called?
Stage micrometer
84
The value of ocular micrometers _____ as magnification increases.
Decreases
85
What are the three important features of microscope microscopy?
- Magnification
- Resolution
- Contrast
86
What particular thing is used to help stain set on flagella?
a mordant
87
What are the different terms for flagellar positions?? And what do they mean?
Polar Bacteria with a single flagella
Monotrichous: singular flagella at one end of the cell
amphitrichous: flagella at both ends of the cell
Lophotrichous: tufts of flagella at the end of the cell
Peritrichous: with flagella emerging from the entire cell surface.
88
Amphitrichous
Singular flagella at both ends of the cell
89
Lophotrichous
Tufts of flagella at the end of the cell
90
Peritrichous
With flagella emerging from the entire cell surface.
91
Monotrichous
Singular flagella at one end of the cell
92
Why can't flagella be observed in action?
The flagella's diameter is below the resolution limit - to see them in a light microscope, the flagella are coated with stains that allow them to be visualized, but this kills the cells.
93
Flagella have a diameter of about 1 nm. A hypothetical question: to resolve flagella, what is the maximum wavelength of the electromagnetic spectrum that would have to be used to create the image (given numerical apertures of 1.25 for both the condenser and the oil lens[objective])?
Use the limit of resolution formula, but lace λ as your unknown.
The formula:
` λ
D = ----------------------------------
NA condenser + NA objective
Becomes:
λ = (NA condenser + NA objective) x D
Therefore:
(1.25nm + 1.25nm) x 1nm = 2.5nm
94
Explain BSL levels 1 precautions
Mechanical pipetting (no mouth pipetting allowed)
Safe sharps handling
Avoidance of splashes or aerosols
Daily decontamination of all work surfaces when work is complete
Regular handwashing
Prohibition of food, drink, and smoking materials
The use of personal protective equipment (PPE), such as goggles, gloves, and a lab coat or gown
Biohazard signs
95
Explain BSL levels 2 precautions
In addition to the safety protocols established for BSL-1 labs:
All procedures that could cause infection from aerosols or splashes must be performed within a biological safety cabinet.
Decontamination of infectious materials prior to disposal, generally through the use of an autoclave
Self-closing, lockable doors
Access to a sink and eyewash station
Biohazard warning signs
96
Explain BSL levels 3 precautions
In addition to the safety protocols established for BSL-1&2 labs:
The use of PPE, including goggles and gloves; respirators may also be required
The use of solid-front wraparound gowns, scrub suits, and/or coveralls is often required
Access to a hands-free sink and eyewash station available near the exit
Sustained directional airflow to draw air into the laboratory from clean areas toward potentially contaminated areas (exhaust air cannot be recirculated)
Self-closing set of locking doors with access away from general building corridors
Restricted and controlled access to the lab at all times.
97
Explain BSL levels 4 precautions
In addition to the safety protocols established for BSL-1,2,&3 labs:
Personnel must change clothing before entering the facility and shower upon exiting
All materials must be decontaminated before leaving the facility
Personnel must wear the PPE from lower BSL levels, as well as a full-body, air-supplied, positive pressure suit.
Access to a Class III biological safety cabinet
98
Cocci or coccus
spherical
99
Bacilli or bacillus
rods
100
Spirilla or spirillum
spirals
101
for two coccus
diplococcus
102
for two bacillus
diplobacillus
103
for a chain of coccus
streptococcus
104
for a chain of bacillus
streptobacillus
105
irregular clusters
staphylo-
106
Tetrads
clusters of four cocci arranged within the same plane
107
Sarina
Tetrads stacked on top of each other a total of eight cells
108
Palisade and angular
Palisades: The bacilli bend at the points of division following the cell divisions, resulting in a palisade arrangement resembling a picket fence and angular patterns that look like Chinese letters.
109
Staphylococcus
irregular cluster of coccus-shaped cells
110
What is a chromogen?
the solvent containing a colored molecule used in stains
111
What is a chromophore?
The portion of the chromogen that gives it its color
112
Auxochrome
The charged portion of a chromogen that allows it to act as a dye through ionic or covalent bonds between the chromogen and the cell
113
What is a basic stain?
Basic stains are attracted to negatively charged molecules in the cell including nucleic acids (DNA and RNA) and some proteins.
114
Examples of basic stains:
Methylene blue, crystal violet, safranin
115
What is heat fixing?
Heat fixing kills the bacteria but makes them adhere to the slide, and coagulates cytoplasmic proteins to make them adhere to the slide.
116
What stains are used in a simple stain?
crystal violet
safranin
methylene blue
117
What are the consequences of overstaining?
floods the cell, making the image become supersaturated and hard to discern.
118
What are the consequences of understaining?
The stain will not be thoroughly absorbed. Leaving a cell with no or little color differentiation. Prevents proper analysis.
119
Consider a coccus and a rod of equal volume:
A. Which is more likely to survive in a dry environment?
B. Which is better adapted to a moist environment?
A. A coccus-shaped bacteria is more likely to survive in a dry environment because it has a lower surface-to-volume ratio, meaning it won't dry out as quickly as a rod-shaped bacterium would.
B. A rod-shaped bacterium would be better adapted for a wet environment because it has a greater surface-to-volume ratio allowing it to transfer more H2O and nutrients into the cell at a faster rate.
120
Structural vs. Differential stains
Differential stains use more than one stain, and cells will have a different appearance based on their chemical or structural properties, they allow for differentiation between individual characteristics of the cell.
A structural stain simply helps visualize the cell's overall structure
121
Explain the dyes used in a gram stain; what order are they used in, and what purpose they serve in the staining process? Be specific and detailed.
A gram stain uses
1. Crystal violet - which stains both gram-negative and gram-positive cells
2. Gram iodine, which acts as a mordant and creates a crystal violet-iodine complex
3. An alcohol decolorizer is used to remove the crystal violet and grams iodine stain from the gram-negative cell.
4. Safranin acts as a counter stain, making Gram-negative cells visible again
122
Define decolorization's purpose in Gram staining.
Decolorization is used to rinse the crystal violet iodine complex out of the gram-negative cell.
123
Define a counterstain. What purpose does it have in staining techniques?
A counterstain introduces color to specific cellular structures to provide contrast to the colored enzyme substrate.
124
Explain the results of over-decolorization and under-decolorization
You will get gram-variable results.
Over-decolorizing will lead to an erroneous result where gram-positive cells may stain pink to red, indicating a gram-negative result.
Under-decolorizing will lead to an erroneous result where gram-negative cells may appear blue to purple, indicating a gram-positive result.
125
Predict the effect on Gram-positive and Gram-negative cells of the following "mistakes" made when performing a Gram stain. Consider each mistake independently.
A. Failure to add the iodine
B. Failure to add the decolorizer
C. Failure to apply safranin
D. Reversal of crystal violet and
safranin stains
A. Could cause decolorization of gram positives and no noticeable effects on gram negatives
B. All cells will appear purple since the crystal violet stains both gram-positive and gram-negative cells, and the safranin is not likely to be seen over the crystal violet
C. Gram-positives will be purple, and the gram-negative will be colorless since they have been decolorized but not counterstained
D. Safranin will be washed out during decolorization, so both gram-positive and gram-negative will be colorized by the counterstain crystal violet
126
Both crystal violet and Safranin are basic stains and may be used to do simple stains on Gram-positive and Gram-negative cells. This being the case, explain how they end up staining Gram-positive and Gram-negative differently in the Gram stain.
Gram-positive cell possesses a thick peptidoglycan in its cell wall which helps retain the purple color of the crystal violet, while Gram-negative cells will loose out this color when flushed with alcohol but takes up the red or pink color when counter-stained with safranin.
127
One of your Lab partners has followed the recommended procedures of running Gram-positive and Gram-negative control organisms on her Gram stain of an unknown species. Her choices of controls were E. coli and B. Sub. She tries several times, and each time concludes she is decolorizing too long because both controls have pink cells (one more than the other). What might you suggest she try and why?
I would suggest that she make sure she is using new cultures, because if she is using old cultures, then they lose their ability to retain the Crystal Violet.
128
What are the ZN and K methods of staining? How do they differ?
ZN method also called the Ziehl-Neelsen (ZN) uses steaming to help Carbolfushsin dye enter the cell wall.
K method also known as the Kinyoun method, lets the dye sit for a longer time to make up for not steaming as in the ZN method. It also typically uses a more concentrated and lipid soluble carbolfuchsin dye.
129
What are Fast Acid stains useful in identifying
Acid-Fast Bacilli
(AFB)
130
Why do we run Acid Fast Tests? How does the cell structure play a role in this staining technique?
Acid-fast stains are used to differentiate acid-fast organisms such as mycobacteria. Acid-fast bacteria have a high content of mycolic acids in their cell walls. Acid-fast bacteria will be red, while nonacid-fast bacteria will be stained blue with the counterstain methylene blue.
131
How does the cell structure play a role in the staining technique for Gram stains?
The basic principle of gram staining involves the ability of the bacterial cell wall to retain the crystal violet dye during solvent treatment. Gram-positive microorganisms have higher peptidoglycan content, whereas gram-negative organisms have higher lipid content.
The gram-negative will lose the crystal violet stain when decolorizing because of its low peptidoglycan content. While gram positive won't because of their high peptidoglycan content.
132
Why might you get a gram-variable cell with gram staining?
Old cultures tend to lose the peptidoglycan cell walls, which predisposes gram-positive cells to be gram-negative or gram-variable.
133
Explain the purpose of the different staining techniques
- Simply stain
- Gram stain
- Acid-fast stain
- Capsule stain
- Endospore stain
- A simple stain is used to improve the cell's contrast against the slide.
- A gram stain is used to differentiate between gram-negative and gram-positive cells
- An acid-fast cell is used to identify cells with Mycolic acid in their cell walls. Helps you identify acid-fast bacilli. (AFB)
- A capsule stain is used to identify whether or not a cell has an exterior capsule layer
- An Endospore stain is used to identify endospores within a bacterial culture.
134
What color is a gram-negative cell? What color is a gram-positive cell? What causes the difference in color?
A gram-negative cell is red because the crystal violet and grams iodine complex get rinsed out of the cell during decolorization.
A gram-positive cell is purple because the crystal violet and grams iodine stain do not get washed out of the cell during decolorization.
135
What color is an Acid-Fast + cell?
What color is an Acid Fast - cell?
What causes the color differences?
An Acid Fast + cell is red because it doesn't absorb the methylene blue stain the Acid Fast - cells do
An Acid Fast - cell is Blue because it absorbs the counter stain methylene blue.
136
What dyes are used in an Acid-fast stain? What order are they used in? What is their purpose in the staining process?
1. Carbolfuchsin is used to stain the body of the cell
2. Methylene blue is added as a counter stain. Acid-fast + cells do not absorb it, which results in the color difference.
137
How does heating the bacteria smear during a ZN stain promote entry of carbolfuchsin into the Acid-fast cell wall?
Heating melts the mycolic acid and allows the stain to penetrate the cell walls.
138
What parts of the cell are stained in an endospore stain? Which dye(s) is responsible?
Is the slide itself stained for added contrast?
The endospore is stained by malachite green, while the cell body is counter-stained by safranin.
The slide itself is not stained.
139
What parts of a cell are stained in a Gram stain?
Which dye(s) is responsible?
Is the slide itself stained for added contrast?
The cell body of Gram-positive is stained by crystal violet. Grams iodine acts as a mordant. The gram-positive absorbs the counterstain safranin but is hidden by the crystal violet.
The cell body of Gram-negative is stained by Safranin (acting as a counterstain.
The slide is not stained for added contrast
140
What parts of a cell are stained in a simple stain?
Which dye(s) is responsible?
Is the slide itself stained for added contrast?
The cell body, the basic stain used (methylene blue, safranin, crystal violet).
The slide is not stained for added contrast.
141
What parts of a cell are stained in an Acid-fast stain?
Which dye(s) is responsible?
Is the slide itself stained for added contrast?
The cell body. Both acid-fast negative and positive are stained by carbolfuchsin. But only Acid-fast-negatives are stained by Methylene blue.
The slide is not stained for added contrast
142
What parts of a cell are stained in a capsule stain?
Which dye(s) is responsible?
Is the slide itself stained for added contrast?
The cell body, the capsule is left unstained
Congo red stains the body of the cell
Maneval stain is used to stain the slide for added contrast against the capsules
143
Are acid-fast negative cells stained by carbolfuchsin? If so, how can this be a differential stain?
The waxy cell wall of acid-fast cells repels typical aqueous stains. Most acid-fast positive organisms are only weakly gram positive. Acid-fast negative cells are stained by carbolfuchsin to be the primary stain, but acid alcohol is used to decolorize nonacid-fast cells; the acid-fast stain is a differential stain used to detect cells capable of retaining a primary stain when treated with an acid alcohol.
144
Why do you suppose the acid-fast stain is less widely used than the gram stain? When is it more useful than the gram stain?
- Very few bacteria are acid-fast positive, so the test is less useful than a gram stain, which separates organisms into two large groups.
-acid-fast is useful when acid-fast positive bacteria are suspected
145
What is a capsule? What role do they play in the cell functionality?
Capsules are comprised of mucoid polysaccharides or polypeptides that repel most stains because they are neutrally charged. They help protect the cell against phagocytosis.
146
Why can't you heat-fix a capsule stain?
Because heat fixing causes cells to shrink, which then leaves a white halo around the cell that could easily be interpreted as an capsule.
147
What dye(s) are used in a capsule stain? What order are they used in and what is their purpose in the staining process?
1. Congo red is used to stain the cell bodies inside of the capsules
2. Maneval's stain is used as a counter stain to stain the slide itself. Only the capsules will remain transparent and uncolored, allowing for their identification.
148
Capsules are neutrally charged. This being the case, What is the purpose of emulsifying the sample in serum in this staining procedure
The purpose is to help the bacterium adhere to the slide because we cannot heat fix when doing a capsule stain as it will tamper with the results.
149
Some oral bacteria produce an extracellular "capsule." Of what benefit is a capsule to these cells?
For Protection.
They prevent being phagocytized by macrophages by the capsules smooth negatively charged outer surface so that macrophages cannot adhere to it.
150
Why is the endospore resistant to staining? How do we overcome this when staining?
- tough outer layer of keratin
- Dehydrated state
- DNA protective proteins.
By steaming the bacterium while applying the malachite green stain or by letting the malachite green sit for an extended period of time on the slide.
151
What dye(s) are used in an endospore stain? What order are they used in and what is their purpose in the staining process?
1. Malachite Green is used to stain the endospore. This dye is typically applied while steaming the bacterium, which allows for easier entry, or the dye is left to sit for an extended amount of time allowing the dye to enter the spore.
2. The slide is rinsed with water and then Safranin is added as a counter stain to provide good contrast for observing the endospores against the cell body.
152
Define a vegetative cell
A cell of a bacterium or unicellular alga that is actively growing rather than forming spores.
153
Spore mother cells do what?
They are the cells that produce the endospore.
154
What are the three locations of endospores within the cell?
Central - Middle
terminal - End
Subterminal - between middle and end
155
Spores are either _____ or _____ shaped
spherical and elliptical
156
What is a saprophyte?
Perform important ecosystem role of decomposing organic matter.
157
Consider the possible results of an endospore stain:
A. What does a positive result for the endospore stain indicate about the organism?
B. What does a negative result for the endospore stain indicate about the organism?
A. A positive result indicates that the bacterium is in a hostile environment
B. A negative result indicates that the Bacterium is in a healthy environment.
158
Why is it unnecessary to include a control for the endospore stain procedure?
Endospore stain is a differential staining procedure that allows to see both spores and vegetative cells, including separate control consisting of only vegetative cells not required
159
Endospores do not stain easily. Perhaps you have seen them unstained as white objects inside Bacillus species in other staining procedures. if they are visible as unstained objects in other stains, of what use is the endospore stain?
When the structure is an unstained object (white), it might be a spore, or might be a spore granule or other cellular inclusion. The spore stain technique provides good evidence that the structure is a spore.
160
Explain the different procedural steps for all stain types:
- Simple stain
- Gram stain
- Acid Fast stain
- Capsule stain
- Endospore stain
Simple Stain:
1. Heat fix bacterium to slide
2. to the heat-fixed slide,
add the desired dye (safranin, methylene blue, or crystal violet). Let rest for the designated time.
3. Once the stain has set rinse with distilled water until all dye has been removed from the slide.
4. gently blot dry with bibulous paper
Gram Stain:
1. With the heat-fixed slide, cover the smear with crystal violet and let stain for 1 min.
2. Rinse the slide with distilled water to remove the crystal violet stain.
3. Add Grams iodine stain and let rest for 1 min.
5. Grasp the slide with the slide holder and rinse with distilled water to remove grams of iodine stain.
6. Then add Gram decolorizer (typically a diluted ethanol). Decolorize until runoff is clear
7. Immediately rinse the slide with distilled water
8. Add the counter stain safranin and let rest for 1 min
9. Rinse with distilled water
10. Blot dry with bibulous paper.
Acid-Fast Stain:
1. to a heat-fixed slide, add carbolfuchsin stain and let rest for 5-10 min.
2. rinse with distilled water
3. Decolorize with Acid Alcohol
4. Rinse with distilled water
5. counter stain with either methylene blue, or brilliant green stain for 1 min.
6. rinse with distilled water
7. blot dry with bibulous paper
Capsule stain:
1. Place a loopful of sheep serum to the end of the slide, then add a drop of Congo red stain to the slide.
2. Aseptically inoculate your sheep serum and Congo red mixture on the slide with the bacterium.
3. then, using another slide held at a 30-degree angle, slide it along the slide into the stain and then push back to the end of the slide.
4. Let air dry
5. Next, stain slide with Maneval's stain for 1 min.
6. dip slide in a beaker of water two to three times to rinse off stain.
7. gently tap the edge of the slide on a paper towel to remove excess water.
8. let air dry
Endospore stain:
1. On a heat-fixed slide, place a piece of bibulous paper over the slide and add a malachite green stain. Then steam for 5 to 7 min. Ensure that the paper remains moist with dye throughout the steaming process
2. rinse with distilled water
3. apply Safranin counter stain and let sit for 1 min.
4. rinse with distilled water
5. blot dry with bibulous paper.
161
Explain the procedural steps for serial dilutions.
1. Label microtubes 1:10 -
1:10,000,000 (10^-1 - 10^-7)
2. Using a sterile pipette man aliquot 450uL of nutrient broth into each of your labeled microtubes
3. Then using the Pipettman aliquot 50uL of the E. coli culture into the tube labeled 10 ^-1
then, from the 10^-1, transfer 50 uL to the microtubule labeled 10^-2
4. repeat the process till all of microtubules have been inoculated
162
What is a complex media?
A media whose exact composition is unknown or undefined
163
What is an active ingredient?
The ingredient that kills or stops bacteria
If the active ingredient kills, it is bactericidal
If the active ingredient stops bacterial growth it is bacteriostatic.
