Exploring Microbes Flashcards
1
Q
What is a microbe
A
Simple, tiny microorganisms that cannot be seen with the naked eye
2
Q
What is a microbiological medium
A
It is a synthetic rich environment in which bacteria, algae, yeasts, specific cells or tissue types are grown in a laboratory environment.
3
Q
What % of microbes are successfully cultivated in an artificial media
A
10%
4
Q
What are the different physical classifications of medias
A
Liquid media; broths
Solid media; agar plates, agar slants, agar deeps
Semi solid media; agar deeps (can be solid or semi solid)
5
Q
What are the different media classifications based on functional form
A
General
Selective
Enriched
Differential
6
Q
What is general purpose media
A
Contain all nutrients that allows most bacteria to grow, exceptions are bacteria that need special food like growth factors or vitamins. Good for looking at diversity of organisms
7
Q
Examples of general purpose media
A
TSA: Trypticase Soy Agar
NA: Nutrient Agar
8
Q
TSA
A
Plant based. Contains soy bean and casein, pH 7.3, general purpose media
9
Q
NA
A
Animal based. Contains gelatin, peptones and beef extract, pH 6.8, general purpose media
10
Q
Selective media
A
Contain selective agents that will allow only certain types of organisms to grow while inhibiting growth of unwanted organisms
11
Q
Examples of selective media
A
PEA
SabDex
EMB
MAC
12
Q
PEA
A
Phenyl Ethyl Alcohol agar. Selective media. Contains Phenyl Ethyl Alcohol that inhibits most gram negative bacteria and fungi. Allowing gram positive bacteria to grow, particularly cocci
13
Q
SabDex
A
Sabouraud dextrose agar. Selective media. Contains high dextrose concentration, Chloramphenical, and low pH around 5.6 inhibiting most bacteria and allowing growth of most fungi.
14
Q
EMB
A
Eosin Methylene Blue agar. Selective and differential medium. Used for isolation and differentiation of gram negative bacteria by inhibiting most gram positive bacteria.
15
Q
What are the selective/inhibitory agents of EMB
A
The dyes eosin Y and methylene blue. Methylene blue inhibits the gram positive bacteria. Eosin changes color to a dark purple when the medium around the colony becomes acidic due to lactose fermenting bacteria
16
Q
MAC
A
MacConkey agar. Selective and differential. For isolation and differentiation of gram negative bacteria.
17
Q
How is MAC selective and differential
A
It contains crystal violet and bile salts inhibiting most gram positive bacteria and allowing growth of gram negative bacteria. Contains a lactose sugar and indicator dye for differentiation of lactose fermenters from non fermenters.
18
Q
Enriched media
A
Contains additional substances that are needed by fastidious bacteria that cannot grow on general purpose media
19
Q
Examples of enriched media
A
Blood agar and chocolate agar
20
Q
Blood agar
A
Enriched media, can also be used for differentiation based on hemolytic properties. Contains sheep blood that has GF not found in general media. Allows growth of fastidious bacteria that cannot grow without these specific GF.
21
Q
Chocolate agar
A
Contains lysed blood that releases growth factors that certain fastidious bacteria cannot grow without
22
Q
Differet types of differential media
A
- EMB
- MAC
- BAP
23
Q
What does a differential media do
A
Contain various substances that cause certain bacteria to look different from other species
24
Q
Function of differential EMB
A
Contains lactose sugar and indicator dyes used for differentiating lactose fermenters from non lactose fermenters. Eosin dye and methylene blue respond to changes in pH, going colorless to black in acidic conditions. Vigorous fermenters will appear dark purple to black. Slow or weak fermenters will produce mucoid pink colonies. Colorless or normal colored indicate non lactose fermenters.
25
What is the differentiating purpose of MAC
It contains a lactose sugar and indicator dye used to differentiate lactose fermenters (red colonies--> pH<6.8) from lactose non-fermenters (colorless--> pH>6.8)
26
What is the differentiating purpose of BAP
It contains sheep blood that can be lysed by certain bacteria that possess hemolytic enzymes. Different species contain different classes of hemolysins.
27
What are the different hemolysins produced by fastidious bacteria
1. Alpha hemolysis is partial and can appear green around the colony.
2. Beta hemolysis is complete and will appear transparent around the colony.
3. Gamma will show no change and has no hemolysis.
28
What is the difference between a differential medium and a selective medium
A selective medium will allow only certain types of microorganisms to grow, while inhibiting other types. A differential medium has different dyes and/or chemicals that allow the identification of closely related or groups of bacteria by observing their different characteristics or growth patterns.
29
When would you use an enriched medium over a general purpose medium
An enriched medium would be used when one is trying to grow fastidious bacteria that cannot grow on a general medium.
30
What is an example of a selective and differential medium
EMB
| MAC
31
What types of mediums grow Pseudomonas fluorescens?
What does it look like?
Gram +/-?
1. TSA,EMB,MAC,BAP
2. Translucent to opaque, circular, shiny, smooth, raised, beige,
flat and turned TSA medium green, pink on EMB-->weak fermenter,
3. Gram negative
32
What types of mediums grow Staphylococcus aureus?
What does it look like?
Gram +/-?
1. TSA, PEA, BAP
2. circular, flat, glistening, beige, translucent on TSA, convex on PEA, raised and grey and beta hemolysis on BAP
3. Gram positive
33
What types of mediums grow E. Coli?
What does it look like?
Gram +/-?
1. TSA, EMB, MAC, BAP
2. Circular to irregular, undulate margin, flat opaque, smooth to glistening
Beige on TSA and BAP, black& metallic green on EMB, pink to red on MAC= fermenting
3. Gram negative
34
What types of mediums grow Bacillus subtilis?
What does it look like?
Gram +/-?
1. BAP, PEA, TSA
2. Beige, circular to irregular, undulate, flat, opaque
Yellow and dull on PEA, beta hemolysis and wrinkled surface on BAP
3. Gram positive
35
What are the different descriptions of bacterial forms/shapes on agar plates
Circular, filamentous (hairy), rhizoid (root-like), irregular, punctiform, spindle
36
What are the different descriptions of bacterial elevations agar plates
Raised, convex, flat, umbonate, crateriform
37
What are the different descriptions of bacterial margins on agar plates
Entire, undulate, filiform, curled, concentric, lobate
38
What are the different descriptions of bacterial surface and opacity on agar plates
Surface: rough, dull, rugose, smooth, glistening
Opacity: transparent, opaque, translucent, iridescent (changing colors in reflected light)
39
What is microscopy
The technology of making very small things visible to the human eye
40
What are different types if light interactions in microscopy
Reflection, transmission, absorption, refraction
41
Wavelength
Length of a light ray
42
Resolution
Refers to ability to see two items as separate and discrete units
43
Types of microscopy use in lab
BF and Phase contrast
44
Scanning objective lens
4x
| NA 0.10
45
10x objective power
Low power
| NA 0.25
46
High power objective
40x
NA 0.65
AKA dry power
47
100x objective lens
Oil immersion
| NA 1.25
48
Ocular lens is what magnification
10x
49
How does oil immersion help view objects under the microscope
It has same refractory index of glass and allows unrefracted light to enter lens. When lens touches the oil it will eliminate any air that could cause refraction of light. This will prevent loss of resolution.
50
Ocular scale distance
Scanning-->25um
Low--> 10um
High-->2.5um
Oil-->1um
51
What does the iris diaphragm do
It controls the intensity of light onto the stage
52
To Calculate resolution power
Wavelength of light [550nm (or given)] over 2x NA
53
What is meant by parafocal lenses
Lenses that stay in focus when changing to another magnification.Results in minimal focusing when moving between objects
54
What is a compound microscope
A light microscope that has a 2 lens system
1. Objectives
2. Eye piece
55
In order to use microscopy effectively, what 3 important aspects are important to understand
1. RP
2. Working distance
3. Refractive index
56
What is working distance
The distance between the object on the slide and the objective lens, when the object is on focus
57
Refractive index
Pertains to the refraction or light bending property of the different media the light passes through, namely, glass,air,oil
58
How does the phase shifting plate and condenser help get a better resolution
They cause some light to strike out of phase with the other which causes the specimen to have different degrees of brightness and darkness. In a phase-contrast microscope, these phase shifts are converted into changes in amplitude, which can be observed as differences in image contrast.
59
What is microscopic morphology
Shape, size, shape, and arrangement
60
What color are bacteria normally under the microscope
Colorless, require staining
61
Positive staining technique
It provides contrast with the surroundings. It's done by using basic dyes which carry a positive charge. They are attracted to the negatively charged cell and thus stain the cell. The bacteria appear colored against a white background.
62
What happens if the bacteria have nonionic structures that prevent staining with basic dyes
You can use acidic dyes which are negatively charged. They are repelled by negatively charged cell and stain the background. The bacteria appear clear against a colored/dark background. (Negative staining)
63
What is simple staining commonly used for
To study bacterial morphology. Only 1 dye is used, all bacteria are stained the same color
64
What are the 3 common shapes of bacteria
Coccus
Bacillus
Spiral
65
Coccus characteristics
Spherical or oval, elongated or flattened on one side, approximately 0.5 to 1.0 um in diameter
66
Describe basic functions of different types of dyes
►Most dyes are ionic.
►Basic: Positive charges, microbes have net negative charge so attract basic stains.
►Acidic: Negative charge, repelled by cells, stain background.
►Mordant: Increase cells affinity for the dye.
67
What does a simple stain do
►Uses a single dye to observe cellular morphology and arrangement.
►Highlights entire organism.
•Basic shape.
•Basic size.
►Stains everything the same.
68
Why is a Differential Stain used
.
►Uses multiple dyes to differentiate between two different cells, or two different parts of the same cell.
►Different cells, chemicals or structures can be distinguished.
►Gram stain differentiates between cell types.
►Capsule stain differentiates between a cell and its capsule
69
Cationic Dyes
►These stains are positively charged.
►Bind to the negative cell wall.
►Methylene blue, crystal violet and safranin are all examples.
70
Gram Stain
►MOST important differential stain in Bacteriology.
►Reveals differences in the nature of bacterial cell walls based on the amount of peptidoglycan.
►Positive cells appear purple: LARGE amounts of peptidoglycan and NO lipopolysaccharide.
►Negative cells appear pink: SMALL amounts of peptidoglycan and, characteristically, lipopolysaccharide (LPS).
71
Gram Stain Procedure
►Prepare a smear, dry, heat fix.
►Flood with Crystal Violet 1 minute
►Rinse with water.
►Flood with Gram's Iodine let stand 1 minute
►Pour off iodine, rinse with water and decolorize with ethanol/acetone for no more than 15 seconds
►Rinse with water.
►Flood with Safranin for 1 minute.
►Rinse with water.
►Drain slide and Blot dry.
►Observe under 10x and 100x oil immersion.
72
Purpose of Heating Fixing
►Kills the bacteria.
►Helps the bacteria adhere to the slide.
►Makes the bacteria more permeable to stains.
73
What is the difference between true motility and Brownian movement?
Brownian motility is movement caused by the molecules in a liquid striking/hitting against the particles of an object (eg. Microorganisms) leading to shaking and vibration , whereas True motility is the deliberate/ self-propelled movement of the organism using its own features such as flagella,pili/fimbriae. They will move in a directional, independent movement over greater distances.
74
What is microscopic morphology
1. Preliminary methods to study microbes
| 2. Includes size, shape, and arrangements of bacteria
75
What technique would play an essential role in the study bacteria under the microscope
Staining them
76
Why is positive staining used
To provide contrast of the bacterial cell with its surroundings
77
How is basic staining achieved
By using basic dyes which carry positive charged ions that are attracted to the negatively charged cell and result in stained cells. The bacteria are colored against a white background
78
What are bacterisl nonionic structures
Structures that do not stain or are damaged by dyes
79
What are the different bacterial shapes
1. Coccus
2. Bacillus
3. Spiral
80
What is a coccus- shaped bacteria
It is spherical, some appear oval, elongated, or flattened on one side. Size is approximately 0.5 um to 1.0 um in diamenter. They have a tendency to remain attached after replication
81
What are the different arrangements of Cocci
1 plane-->Coccus, diplococcus, streptococcus
2 planes-->Tetras
3 planes-->Sarcina
Random-->Staphylococcus
82
What are the different arrangements of bacillus
1. Bacillus -->single (most)
2. Diplobacillus--> 2
3. Streptobacillus--> chain
4. Coccobacilus--> single cocci shaped bacilli
5. Palisades--seperate "picket fences
83
What are the different types of spiral bacteria
1. Vibrio
2. Spirillum
Spirochete
84
Spirals range from what size
5um-40um, some measure over 100um in length
85
Describe what yeast can look like under the microscope
appear spherical, diameter of 3-5um
86
Yeasts are
Unicellular, commonly reproduce asexually by budding, eukaryotic
87
Simple stain procedure
1. Collect sample and mix in 1 drop of water. Spread suspension over third if slide.
2. Let air dry completely
3. Heat fix.
4. Flood with crystal violet stain, let sit for 1 minute
5. Wash with water.
6. Blot with bibulous paper
88
What will E coli look like under microscope
Small bacilli that have just divided look like cocci, non divided ones are rod shaped
89
What do spirochete look like under the microscope
They are the thinnest of bacteria, they resemble extremely thin and wavy pencil lines
90
What would happen to the bacteria if you heat fix the smear before they are completely dry
Cellular proteins may coagulate, cells may get distorted and splatter. This would make it hard to see the morphology of the bacteria under the microscope
91
What is the scientific method
A process used to make observations, solve problems and test hypotheses
92
What is a hypothesis
An explanation proposed on the basis of limited evidence as a starting point for further investigation. It can include making a prediction of the desired results
93
What is an experiment
A test under controlled conditions that examines the validity of a hypothesis. It includes recorded data that is analyzed and a conclusion.
94
What things should a hypothesis contain
1. If and then
2. State a tentative relationship
3. Dependent and independent variable
95
What is independent variable
That which is manipulated or changed by the experimenter. There is only one.
96
What is a dependent variable
That which is measured or observed
97
What are possible controlled variables in an experiment
Ingredients in medium, incubation temperature, pH, etc. These can effect the observations and results. They must be held constant to eliminate the possibility that they will affect the outcome of the experiment.
98
Experimental control
It is set up to demonstrate that the dependent variable is a result of the independent variable being tested. A control is set up by eliminating the independent variable or setting it to a standard value.
99
What 2 things di you need to set up an experimental control
Negative control
| Positive control
100
Positive control
An experiment whose result is already known
101
Negative control
An experiment whose result is set to be negative
102
An experimental control is referred to as
Control
103
Bacterial growth is affected by temperature
| Rewrite as a hypothesis
If temperature affects growth, then increasing the temperature will cause more bacteria to grow, while decreasing the temperature will cause little to no bacterial growth
104
What is one thing that can help bacteia withstand environmental stresses
Their bacterial cell wall maintains the shape and protects the cell from osmotic pressure
105
What are the 4 divisions of prokaryotes based on their cell wall
1. Gram+ bacteria
2. Gram- bacteria
3. Archaea--> pseudopeptidoglycan
4. Bacteria lacking cell walls
106
What are the strengths of gram+ bacterial cells wall
Their cell walls are physically stronger due to the many layers of peptidoglycan that reinforce the cell structure
107
What are the strengths of Gram- bacteria
They are made of 2 layers of peptidoglycan but are chemically stronger due to the outer membrane that prevents many harmful chemicals from penetrating the cell
108
What are the strengths of archaea
Pseudopeptidoglycan walls are very resistant to extreme environments such as temperature and acidity
109
Are their strengths of bacteria lacking cell walls
These have cell membranes reinforced with sterols but are not rigid enough to maintain their shape and protect the from osmotic pressure
110
How is stain held by gram + bacteria when performing Gram Staining
Their cell walls consist of a thick layer of peptidoglycan and smaller pores. The cell wall serves as a net that holds the stains within the cell walls. When Gram's iodine is added, it forms CV-I complex that is bigger than the pores if the peptidoglycan.
111
What role does the alcohol have in gram staining g+ bacteria
It dehydrates and shrinks the peptidoglycan. The CV-I crystals do not leave the cell and it remains purple.
112
What role does the alcohol have in gram staining g- bacteria
They are made of a thin layer of peptidoglycan and an outer membrane containing lipopolysaccharide. They also have large pores. The alcohol dissolves the outer membrane leaving the thin peptidoglycan with larger pores exposed. The CV-I washes out and the G- take up the counter stain and turn pink.
113
What is Gram Type
* Refers to cell wall
* Gram type positive-->thick peptidoglycan layer, smaller pores
* Gram type negative-->thin peptidoglycan layer, larger pores, outer membrane
114
Gram Reaction
* Refers to color of the cell after gran stain procedure
* Gram reaction positive--> when cells are purple
* Gram reaction negative--> when cells are pink
115
KOH test
1. Faster and simpler to determine type of gram bacteria
| 2. String indicates gram negative organism
116
What happens when gram negative bacteria is placed in 3% KOH
Its alkalinic solution destroys the cell walls and this releases the cell contents, including DNA. Gram positive bacteria are not lysed.
117
KOH test procedure
1. Place a drop of 3% KOH onto glass slide
2. Emulsify a loopful of culture in the KOH
3. MIX for 60 seconds
118
What is a colony
A mass of cells originating from a single cell, a clone of bacteria all genetically alike
119
What determines/influences microbial growth
Genetic make up/environmental factors; nutrient availability, temperature, incubation time
120
What are colony morphological characteristics
```
Colony size
Shape
Margin
Surface
Texture
Elevations
Color and optical properties
```
121
Why are agar plates incubated upside down
Storing plates in the refrigerator can cause condensation. By storing them upside down you prevent the water from dripping onto to the agar. This will prevent colonies from migrating and mixing with one another. It will also reduce the chances of contamination.
122
What is the hanging drop method
Hanging drop slides are useful in observing the general shape of living bacteria and the arrangement of bacterial cells when they associate together. Organisms are observed in a drop that is suspended under a cover glass in a concave depression slide.
123
What is Lysozyme
It is an enzyme present in animal serum, tissues,and in cytoplasmic granules of phagocytes. It is released in mucosal secretions like tears, sweat, saliva, and mucus. It is a type of defense mechanism against bacterial infections. Also known as body's own antibiotic.
124
How does Lysozyme work
It catalyzes hydrolysis of the bonds between the sugars in the repeating disacharride "backbone" of peptidoglycan. In other words it breaks the sugar bonds that attaches NAM and NAG of the glycan molecule in peptidoglycan.
125
How is the cell vulnerable after exposed to Lysozyme
It destroys the cell wall and leaves it vulnerable osmotic pressure which could cause it to burst/destruction of the cell (lysing).
126
Would Lysozyme affect gram positive bacteria
Yes because its is outermost layer is made up of peptidoglycan and Lysozyme destroys the sugar bonds of the peptidoglycan.
127
Would Lysozyme affect gram negative bacteria
No because its outermost layer is made up of lipid bilayer that would prevent Lysozyme from reaching the peptidoglycan layer.
128
Which bacteria in lab were sensitive to Lysozyme
The gram positive bacteria: Micrococcus luteus, Staphylococcus epidermis, Bacillus subtilis.
129
Bacillus subtilis under microscope shape and arrangements
Rod shaped, short chains or single, gram positive
130
Micrococcus luteus under microscope shape and arrangements
Spherical, pairs and tetrads, gram positive
131
Staphylococcus epidermidis
Spherical, single, double and clusters, gram positive
132
What is Ampicillin
It is a derivative of penicillin, an antibiotic and a close relative of amoxicillin. It can penetrate and prevent the growth of most gram negative bacteria.
133
What is the mechanism of action of penicillin
It inhibits the transpeptidation reaction of peptidoglycan. It does this by binding to the enzyme transpeptidase. This enzyme is responsible for the transpeptidation of peptidoglycan.The B-lactam ring of penicillin imitates the D-alanine peptide of the peptidoglycan side chains. This is how it is able to bind to transpeptidase and prevent the new wall synthesis. It also activates endogenous enzymes that degrade the peptidoglycan.
134
How would penicillin affect gram positive bacteria
It would be effective because the outer layer of this bacteria is peptidoglycan which is what penicillin attacks by interfering with the formation of peptide crossbridges .
135
How would penicillin affect gram negative bacteria
It would not be very effective since gram negative bacteria has a lipid bilayer that prevents the entry of penicillin.
136
Which bacteria frkm lab would be more sensitive to penicillin
Micrococcus luteus, Staphylococcus epidermidis, Bacillus subtilis
137
How does temperature effect Gram negative bacteria
An increased temperature would cause a decreased production of LPS. This would make the cell wall more fragile and allow substances to get in that don't normally get in.
138
How does temperature effect Gram positive bacteria
It may effect some part of the cell wall synthesis process. This could cause the wall to become thin and fragile.
139
What formation does penicillin inhibit
Formation of peptides alanine and lysine
140
What happens to cell wall after penicillin prevents transpeptidation
Cell wall weakens, becomes more porous, leaves cells membrane exposed. This will cause osmotic pressures to destroy the cell.
141
Does penicillin work on bacteria that that's no longer multiplying or growing
It only works on bacteria that is growing and works to reduce the population to allow immune system to take over
142
How would an older culture effect gram staining
As the culture ages, cells start to die or weaken. This will cause a breakdown of the cell wall. If it is stained, it will react like a gram negative stain. The alcohol will wash away the primary stain because it won't have the thick peptidoglycan layer to hold the color in. Therefore when the counter stain is used, the final result will be pink.
143
What is gram variable
Some organisms are Gram-variable (that means, they may stain either negative or positive)
144
How does an Endospore appear when Gram stained?
Although endospores themselves are resistant to the Gram stain technique, bacterial cells captured in the process of creating these structures can be stained. In this case, the endospores are seen as clear oval or spherical areas within the stained cell.
145
Escherichia coli arrangement and morphology under microscope
Rod shaped, single, sometimes coccobacillus, gram negative
146
Serratia marcescens morphology and arrangement under microscope
Rod shaped, single, gram negative
147
Saccharomyces cerevisiae morphology and cell arrangement
single with budding, ovoid in shape, with some spherical and elongated cells as well.
