Lab EXAM 1 Flashcards
1
Q
Total Magnification
A
objective lens x ocular lens
2
Q
Magnification
A
ability of a lens to enlarge the image of an object when compared to the real object - 10x means that the object appears 10 times bigger
3
Q
Resolution
A
ability to tell that 2 separate points are separate
- a low resolution image appears fuzzy whereas a high resolution image appears sharp
4
Q
Resolution is affected by…
A
- the wavelength: shorter wavelengths = better resolution
- the numerical aperture (lenses ability to gather light): higher numerical aperture = better resolution
5
Q
Stains
A
used to increase contrast between the cells and the background, making them easier to see under the microscope
6
Q
Oil immersion
A
used to improve resolution by reducing the refraction of light
7
Q
Eye piece or ocular lense
A
1st set of lenses, this is the lense which we look through
8
Q
Revolving nosepiece
A
used to hold objective lenses
9
Q
Objective Lenses
A
2nd set of lenses, used to magnify the image of the specimen
10
Q
Coarse Focus Knob:
A
used for large-scale movements with 4⨯ and 10⨯ objective lenses
11
Q
Fine Focus Knob
A
used for small-scale movements, especially with 40⨯ or 100⨯ objective lenses
12
Q
Stage
A
where you keep the specimen on the slide in position
13
Q
Illuminator
A
light source, typically a high-intensity bulb below the stage
14
Q
Diaphragm
A
reduces or magnifies intensity of light
15
Q
Condenser Lens
A
3rd set of lenses, focuses all of the light rays on the specimen to maximize illumination
16
Q
X-Y Mechanical Stage Knobs
A
move the slide that is on the stage
17
Q
Rheostat
A
a dimmer switch that controls the intensity of the illuminator
18
Q
Agar
A
solidifying agent extracted from seaweeds commercially available as a coarse white power
- liquefies at 100C & solidifies @ 42C
19
Q
All culture media are sterilized using…
A
autoclave
20
Q
Cultures
A
concentrated microorganisms in broth or agar
21
Q
Loops & Needles
A
used to inoculate fresh media with the given cultures (are sterilized by incineration or flaming)
22
Q
Deeps
A
agar is solidified in upright position in test tube, used to store anaerobic cultures or to test oxygen requirements
- for stabbing with loops/needles
23
Q
Slants
A
agar is soldier in a slanted position in test tube, used to store pure cultures
- for streaking (zigzag) with loops/needles
24
Q
Normal or Resident Microbiota
A
Microbes which are normally present in and on the human body
- these are mostly bacteria which protect the skin and our body by producing inhibitory chemicals and acids which can kill pathogenic (disease causing) bacteria
25
Broths
liquid media, used to obtain large number of bacteria
26
Transient Bacteria:
Microbes which we come into contact on a daily basis and are present for a short time on our skin
- Transient bacteria do not usually colonize the skin and are easily removed with the use of soap and water
27
What is the purpose of heat fixing?
To kill the cells and make them adhere onto the slide
28
Chromophore
colored part of the stain
29
If the chromophore is positively charged, the stain is called a ___ stain?
Basic stain, it enters the cell giving a color to the cell
30
If the chromophore is negatively charged, the stain is call a ___ stain?
Acidic stain, it stains the background and leaves the cell colorless
31
Three types of stains:
simple, differential, special
32
Simple Stains:
uses 1 stain (basic or acidic) on 1 slide
- positive/direct and negative stains
33
Differential Stains:
uses multiple stains on 1 slide
- gram staining and acid-fast staining
34
Special Stains
staining special structures
- endospore, flagella, capsule
35
Smear
thin film of a solution of microbes on a slide
- a smear is usually fixed to attach the microbes to the slide and to kill the microbes
36
Fixation
kills a specimen, preserving its integrity and stopping it from moving & attaches the specimen to the slide
37
Types of Fixation:
- heat fixation: where you spread specimen on the slide (called a smear) and briefly heat it over a heat source
- chemical fixation
38
Basic Dyes used in Positive Staining:
Crystal Violet OR Methylene Blue
39
The most accurate staining technique used to get the morphology and arrangement of bacterial cells?
Negative Stains
40
Why is negative staining technique considered the most accurate method?
- no heat is involved
- use acidic stains, which do not penetrate the cells therefore retaining their original size
41
Acidic Dyes used in Negative Staining
Nigrosin
42
Gram Stain was discovered by
Hans Christian Gram in 1884
43
What is Gram Stain
a differential staining technique that uses a primary stain and a secondary stain to distinguish between gram positive and gram negative bacteria
44
Considered the most important staining technique for bacteria?
Gram stain
45
Gram stain classifies bacteria into:
gram positive & gram negative bacteria
- gram positive bacteria tends to be killed by penicillin and detergents
- gram negative bacteria are more resistant to antibiotics
46
Structure of Gram Positive bacteria
* more simple
* thick layer of peptidoglycan with embedded teichoic acid
47
Structure of Gram Negative Bacteria
* more complex structure
* contains three layers: inner membrane, thin layer of peptidoglycan, and an outer membrane containing LIPOPOLYSACCHARIDE
48
Staphylococcus aureus
gram positive bacteria (remains blue)
49
Escherichia coli:
gram negative bacteria (becomes decolorized and becomes pink)
50
Bacillus subtilis
gram positive bacteria (remains blue)
51
Gram Stain Process
- Crystal Violet: the primary stain; stains cell purple
- Iodine: the mordant; binds with primary stain to form insoluble complex
- Alcohol: the decolorizer; decolonize gram negative cells but gram positive retain the primary stain
- Safranin: the secondary stain; stains gram negative cells red/pink
52
Why are the gram native cells stained pink and not the gram positive?
Because of their thin peptidoglycan layer
53
Flagella
cellular structures that facilitate movement in liquid environments
- made up of flagellin protein subunits
- has a basal body that is anchored to the plasma membrane, it acts as the engine
- has a hook region that links basal body to the filament
- basal body differs between gram+ and gram-
54
Monotrichous
bacteria with a single flagellum, usually at one end of the cell
55
Amphitrichous
bacteria with a flagellum or clusters of flagellum at both ends
56
Lophotrichous
bacteria which have clusters of flagellum at one end of the cell
57
Peritrichous
bacteria with flagella distributed over the entire cell surface of the cell
58
Capsules
sticky gelatinous covering of uncharged polysaccharides or polypeptides
- do not bind with basic stains
- are anti-phagocytic (examples: B. anthracis, S. pneumoniae
- increase pathogenicity (ability to cause disease) & adherence
59
Dyes/stains used in Capsule Staining:
Capsule staining technique:
- does not use heat fix bc it distorts cell and damages capsule
- both basic and acidic stains are used in this type of staining
- acidic stains stain the background
- basic stains stain the cell
60
Results of Capsule Staining:
- results: appear as colorless halos around the cells since capsules do not have any charge and therefore do not pick up any stains
61
Examples of pathogenic bacteria with capsules:
- Bacillus anthracis, Streptococcus pneumoniae, Neisseria meningitidis
62
Characteristics of Endospores:
- are produced when water & nutrients are used up
- can be dormant for thousands of years
- have thick layers of peptidoglycan & protein coats
63
Positions of Endospores:
terminal, subterminal, & central
64
Christmas Tree Stain
Endospore stain;
- uses malachite green which stains the spores green
- uses Safranin which stains the cells red
65
Importance of spores:
help cells survive adverse conditions
66
Serial Dilution & Bacterial Counts
used to estimate the number of bacteria in a given sample
67
Direct Bacterial Counts
involve counting live cells which give rise to colony forming units (cfu)
68
Indirect Bacterial Counts
depend on the measurement of cell presence or metabolic activity of the cells without counting individual cells
69
Serial Dilution
involves diluting a fixed volume of cells mixed with dilution solution using the previous dilution inoculum. The rest is dilution of the original culture by an exponentially growing factor
70
Spread Plate Method:
sample is poured into solid agar and then spread using a sterile spreader. This process is repeated for each serial dilution prepared. The resulting colonies are counted and provide an estimate of the number of cells in the original volume
71
Countable Plates, TFTC, TNTC:
* countable: 30 to 300 colony forming units
* TFTC: less than 30 colonies
* TNTN: greater than 300 colonies
72
Formula to calculate an estimate of the number of cells per ml of a given sample:
CFU/ml = number of colonies counted/(Dilution*amount plated)
73
Pure culture:
contains only one species or strain
74
Colony
: a population of cells arising from a single cell or spore or from a group of attached cells
75
What method is used to isolate pure cultures?
Streak plate method
76
Neutrophiles
- grow best at neutral pH (around 7.0)
- most bacteria fall in this category
77
Acidophiles
- grow optimally at pH near 3.0
- struggle to grow near neutral pH
78
Alkaliphiles
- grow optimally between 8.5 and 10.5
- extreme Alkaliphiles also struggle to grow near neutral pH
79
Spectrophotometer
powerful method for measuring bacterial growth indirectly by assessing the turbidity of a broth culture
80
Turbidity in spectrophotometer:
indicates bacterial growth, making the solution cloudy
81
Light transmittance in spectrophotometer
a specific wavelength of light is passed through the bacterial suspension
82
Measurements of Spectrophotometer
measures either the percentage of light transmitted or the absorbance (optical density)
83
Correlation of Spectrophotometer:
as bacterial numbers increase, turbidity increases, resulting in lower transmittance and higher absorbance
- high absorbance or OD = less % transmittance = heavy growth
- low absorbance or OD = high % transmittance = low growth
84
Obligate aerobes or strict aerobes
can grow only in the presence of oxygen
- depend on aerobic respiration and use oxygen as terminal electron acceptor in cellular respiration
- growth at top
- example: alcaligenes faecalis
85
Obligate anaerobes or strict anaerobes
cannot grow in the presence of oxygen
- depend on anaerobic respiration using molecules rather than oxygen
- growth is at bottom
- example: clostridium sporogenes
86
Facultative anaerobes
grow best in the presence of oxygen but will also growth without it
- capable of both aerobic and anaerobic respiration
- most of the growth is a top but somewhat throughout
- example: e.coli
87
Aerotolerant anaerobes
do not require oxygen but they can grow in the presence of oxygen without any harm
- growth is equally distributed
88
Microaerophiles
need oxygen to grow, but at concentrations of 1% to 10% which is lower than 21% oxygen in air
89
Thioglycollate Media
reducing media used to determine the oxygen requirements for bacteria
- contains sulphur as the reducing agent
- contains resazurin as the oxygen indicator (gives pinkish color wherever there is oxygen)
90
Enzymes
proteins with 3D structures
91
What affects enzymes?
- increase in temp: disrupts the 3D structure
- decrease in temp: reduces the activity of enzymes
92
Thermophiles
40 to 72C
93
Mesophiles
10 to 50C
94
Psychrophiles
-10 to 20C
95
Obligate Thermophiles:
only above 50C
96
Thermal Death Point (TDP)
lowest temperature at which all cells in a culture are killed
- time is constant & temp is varied to determine the minimum temp required to kill an organism
97
Thermal Death Time (TDT)
time during which all cells in a culture are killed
- temp is held constant & time is varied to determine how long an organism can survive at a given temp
98
Autoclave temp, psi, minutes
121C and 15psi pressure for 15 minutes
99
Pasteurization
kills pathogens and reduces the number of spoilage-causing microbes while maintaining food quality
100
Methods of Pasteurization:
- HTST : 72C for 15 secs
- UHT: 138C for 2 secs
101
Radiation
physical method of microbial control
- radiation in various forms, from high energy radiation to sunlight, can be used to kill microbes or inhibit their growth
- sunlight has a broad spectrum of radiation that includes visible light and ultraviolet rays (UV)
102
Ionizing radiation
includes x-rays, gamma, rays, and high energy electron beams
- introduces double strand breaks in DNA molecules
- it causes DNA mutations to occur
- as these mutations accumulate, they eventually lead to cell death
103
Non-ionizing radiation:
example: UV, can damage DNA by causing the formation of thymine dimers (bonds between adjacent pyrimidines) with a single strand of DNA
104
UV
Ultraviolet radiation, which causes the formation of thymine dimers in DNA, leading to lethal mutations
105
DNA polymerase
A repair enzyme of non-ionizing radiation
106
Light travels in the form of…?
Waves
107
Wavelength
The distance between any two adjacent peaks or crests of a wave
- expressed in nanometers
- Shorter wavelength = higher energy (UV)
- longer wavelength = lower energy
108
Three categories of UV radiation
UVC
UVB
UVA
109
UVC
- ranging from 200 nm to 290 nm
- is biocidal and the most dangerous in living cells
110
UVB
- ranging from 290 nm to 320 nm
111
UVA
- ranging from 320 nm to 400 nm
112
