Lab Pract. 2 Flashcards
1
Q
Explain the function of CARBOHYDRATES in broth media.
A
1.) They are substrates for fermentation (yellow) ( A for acid).
2
Q
Explain the function of PEPTONES in broth media.
A
- ) Nutrient source
- ) When peptides break down, ammonia is released and will change color of the broth
- ) Alternative substrate for non-fermenters (hot pink) ( K for alkaline)
3
Q
Explain the function of PHENOL RED in broth media. What is it made of?
A
A pH indicator:
- ) Orange/pink at neutral pH 7
- ) Hot pink above 7.4
- ) Yellow bellow pH 6.8
- ) Allows us to determine whether or not an acid has been formed as an end product of fermentation.
5.) Made of peptone (hydrolyzed proteins), carbohydrate, water, phenol red
4
Q
Explain the function of DURHAM TUBE in broth media.
A
- ) Turned upside down in broth
2. ) Used to collect gas that is being released by the organism during fermentation
5
Q
What are the end products of aerobic respiration? Are any of these products acidic? How do they know if the cultures are using aerobic respiration or fermentation for their energy production?
A
End products:
- ) Water
- ) ATP
- ) Carbon Dioxide
1.) Fermentation pH will drop below 6.8 and turn broth yellow , Aerobic respiration is orange
6
Q
Do all the organisms use pyruvic acid in the same way for fermentation?
A
1.) No, metabolism of pyruvate is not the same for all microorganisms. An assortment of end products define their fermentative capabilities
7
Q
How would you interpret your results if you inoculated a carbohydrate broth tube and after incubating the tube for several days, the broth had turned hot pink in color?
A
1.) Broth is negative for fermentation and are “K” (alkaline).
8
Q
How do pH indicators work?
A
- ) Weak acids that reflect a particular wavelength of light
- ) Placed in solutions of different pH’s, the loss or gain of electrons causes them to absorb a different wavelength of light
- ) Slight chemical change causes them to reflect different wavelength (color) of light then previous
9
Q
If a particular bacterium is forming acids from a specific carbohydrate during fermentation, the pH will?
A
- ) Drop below 6.8 and turn broth yellow
2. ) Report this as A (acid)
10
Q
Some bacteria genera do ferment carbohydrates to form acids, but can utilize the peptone present in the broth as a nutrient source. When these peptides are broken down?
A
1.) Ammonia is released
11
Q
Ammonia, which has a pH of 10 will turn a broth what color?
A
1.) Hot pink (negative for fermentation/reported as K for alkaline)
12
Q
Broths that remain orange/pink are considered?
A
1.) Negative for fermentation
13
Q
Many phenol red broths will have a small tube turned upside down in the broth. What is this called and what does it do?
A
- ) Durham tube
2. ) Used to collect gas that’s being released from organism during fermentation
14
Q
When inoculating broth that contain Durham tubes, you should not swish your loops violently b/c this introduces air bubbles into the tube. If your’e careful when inoculating your broth any glass that accumulates in the tube will be interpreted as?
A
- ) Positive result
2. ) Reported as (G for gas)
15
Q
An isolate that is turning phenol red broth yellow and releasing gas, which is collecting gas in the Durham tube will be reported as?
A
1.) AG
16
Q
(Fermentation pathways)
Escherichia/Shigella begins with and ends with what products?
A
- ) Begins w/ pyruvate as final electron acceptor
2. ) Ends w/ mixed acids
17
Q
(Fermentation pathways)
Proteus begins w/ and ends w/ what products?
A
- ) Begins w/ pyruvate as final electron acceptor
2. ) Ends w/ CO2 and H2
18
Q
(Fermentation pathways)
Acetobacter begins w/ and ends w/ what products?
A
- ) Begins w/ pyruvate as final electron acceptor
2. ) Ends w/ Acetic acid
19
Q
(Fermentation pathways)
Propionibacterium begins w/ and ends w/ what products?
A
- ) Begins w/ pyruvate as final electron acceptor
2. ) Ends w/ Proprionic acid
20
Q
(Fermentation pathways)
Enterobacter/Klebsiella/Serratia begins w/ and ends w/ what products?
A
- ) Begins w/ pyruvate as final electron acceptor
2. ) Ends w/ Butanediol (an alcohol)
21
Q
Describe reversion.
A
- ) Due to incubation time, once a bacteria has exhausted the supply of available carbohydrate, they will sometimes start digesting peptone
- ) The medium will reflect an increase in pH w/ a color change from yellow back through orange to hot pink
22
Q
What would happen if powdered zinc was added to an uninoculated nitrate broth tube?
A
- ) Catalyze any remaining NO3 (nitrate) to NO2 (nitrite)
2. ) NO2 (nitrite) will combine w/ sulfanilic acid and will form red precipitate
23
Q
How does nitrogen reducing soil bacteria participate in the nitrogen biogeochemical cycle?
A
- ) “Fix” nitrogen and convert nitrogen to ammonia NH3
- ) Helps plants get nutrients from air and soil
3) Plants don’t have enzymes to convert Nitrogen into a biologically useful form
24
Q
In step 2, your P. aeruginosa tube remained clear, indicating that neither NO3- nor NO2- were present in the tube. What happened to the NO3- that was present in the tube before you inoculated your tube?
A
1.) It gassed out of the tube in the form of NH4+ or N2
25
Why is it advantageous for enteric bacteria to perform anaerobic respiration?
1.) Allows them to live in environments where the oxygen is low to nonexistent
26
(Motility of bacteria)
| S. typhimurium
Motile (+)or(++) or Non-motile (-):
1.)
Using an ETC? (+) (-):
1.)
27
(Motility of bacteria)
| P. Vulgaris
Motile (+)or(++) or Non-motile (-):
1.)
Using an ETC? (+) (-):
1.)
28
(Motility of bacteria)
| S. aureus
Motile (+)or(++) or Non-motile (-):
1.)
Using an ETC? (+) (-):
1.)
29
(Motility of bacteria)
| M. smegmatis
Motile (+)or(++) or Non-motile (-):
1.)
Using an ETC? (+) (-):
1.)
30
When observing the results of the motility test, why do aerobes sometimes show inconclusive results?
1. ) They are obligate aerobes
2. ) Can only survive near the surface of the tube
3. ) Inconclusive for motility, positive for aerobic respiration
31
What is indicated when the bacteria growing along the stab do not turn red?
1.) Bacteria are producing ATP via fermentation
32
List two findings that can be determined about your unknown from the results of this medium.
1. ) Ability to move through watery medium
| 2. ) Respiratory status
33
During aerobic respiration________serves as the final electron acceptor? In this catabolic pathway, oxygen bonds w/ two hydrogen ions to form water, an event which is catalyzed by?
1. ) Oxygen (catabolic)
| 2. ) Cytochrome oxidase
34
Describe cytochrome oxidase.
1. ) An enzyme also known as Complex IV
2. ) Large transmembrane protein complex found in bacteria and the mitochondrion of eukaryotes.
3. ) Last enzyme in the respiratory electron transport chain of mitochondria or bacteria located in the mitochondrial or bacterial membrane.
35
What is the key difference between aerobic and anaerobic respiration during anaerobic respiration?
1.) An inorganic molecule such as nitrate, nitrite or sulfate serves as an alternate final electron acceptor, rather than oxygen (which is also inorganic)
36
What is the benefit of using anaerobic respiration as an alternate final electron acceptor even though it generates less ATP for microorganisms?
1. ) Allows them to survive in a greater diversity of environments (dry and waterlogged soils)
2. ) Environments w/ O2 diffusion may be slow or nil
37
What's the benefits of using the motility test?
1. ) Allows us to differentiate between species
2. ) Its ability to move through a watery medium
3. ) Respiratory status of the isolate (is the bacteria using a respiratory chain when producing ATP
38
As the nutrient level drops,__________bacteria will remain in place, but the___________bacteria will remove away from the stab line in search of nutrients.
1. ) Non-motile bacteria
| 2. ) Motile bacteria
39
In many motility media, an indicator dye-Triphenyltetrazolium trichloride (TTC) is incorporated into the medium to?
1. ) Allow for easier visibility of motile bacteria
| 2. ) Gives info regarding respirator status of organism
40
TTC (Triphenyltetrazolium trichloride) is a colorless molecule that is a?
1.) Competitive inhibitor of Complex 1 of the ETC (also called NADH dehydrogenase).
41
During respiration in the bacterial cell, NADH donates_______to Complex I?
1) Electrons
42
If TTC is present in the bacterial cell, NADH can donate an electron pair to TTC instead of donating them to?
1.) Complex I
43
What happens when NADH donates an electron pair to TTC? What happens to the color of the bacteria using this electron transport chain?
1. ) TTC is reduced to formazan, a red colored molecule.
| 2. ) Bacteria turn red
44
Bacteria that are producing ATP via fermentation will remain?
1.) Colorless
45
When bacteria are producing ATP by cellular respiration the tube will appear______in the tube?
1.) Red
46
Bacterial Species: S. aureus
Carbohydrate:
Glucose-
Lactose-
Sucrose-
Growth:
Final Color of broth:
Acids Produced:
Gas Produced:
How Reported (A,AG,K,or -):
47
Bacterial Species: E. coli
Carbohydrate:
Glucose-
Lactose-
Sucrose-
Growth:
Final Color of broth:
Acids Produced:
Gas Produced:
How Reported (A,AG,K,or -):
48
Bacterial Species: P. vulgaris
Carbohydrate:
Glucose-
Lactose-
Sucrose-
Growth:
Final Color of broth:
Acids Produced:
Gas Produced:
How Reported (A,AG,K,or -):
49
Bacterial Species: P. aeruginosa
Carbohydrate:
Glucose-
Lactose-
Sucrose-
Growth:
Final Color of broth:
Acids Produced:
Gas Produced:
How Reported (A,AG,K,or -):
50
Describe the structure and composition of a cell wall of gram-positive bacteria
1. ) Cell wall-composed of 60-90% peptidoglycan
2. ) Multiple layers of peptidoglycan
3. ) Negatively charged polyalcohols (teichoic acids) extend throughout cell wall
4. ) Lipoteichoic acids, teichoic acids covalently linked to lipids, help anchor peptidoglycan layer to the cytoplasmic membrane
51
Describe the structure and composition of a cell wall of gram-negative bacteria
1. ) Cell wall contains only a few layers of peptidoglycan (10-20%)
2. ) No teichoic acids
3. ) Have an outer membrane (asymmetrical bilayer)
4. ) Inner leaflet of outer membrane composed of phospholipids and lipoproteins (link outer membrane to peptidoglycan)
5. ) Outer leaflet composed of lipopolysaccharide (LPS), consist of sugars and Lipid-A
6. ) LPS embedded in outer membrane and sugars extend outward from the surface
7. ) Thinner than gram-positive but more complex
52
The gram stain is a differential staining technique. Explain what this means
A staining process which uses more than one chemical stain. Using multiple stains can better differentiate between different microorganisms or structures/cellular components of a single organism.
53
Identify the cell structures that react differently to the stains and reagents used in gram staining.
```
Gram-Positive:
Primary stain-violet
Mordant-violet
Acetone/alcohol-violet
Counterstain-violet
```
```
Gram-Negative
Primary stain-violet
Mordant-violet
Acetone/alcohol-colorless
Safranin-red
```
54
List the basic steps in the gram stain procedure
1. ) Crystal violet
2. ) Iodine (mordant)
3. ) Alcohol/acetone (decolorization)
4. ) Safranin (counterstain)
55
What would happen if a student forgot to add the mordant during the gram stain?
Iodine is used as a mordant - to form a complex with crystal violet - the formation of this complex prevents the dye from being removed easily - that is it fixes the dye.
if iodine is not added then the dye will leach out readily and will not help in differentiating between Gram positive and Gram negative organisms
56
Could bacteria that do not contain cell walls be stained using the gram stain?
The purpose of a gram stain is to determine the type of cell wall a certain bacteria has. It can be thick or thin resulting in positive or negative bacteria types.
The gram stain sticks to the wall turning thick walls purple and thin walls pink. So if a cell doesn't have a wall it won't work. Even if it did it would be pointless.
57
Could a counterstain other than safranin be used?
Basic Fuchsin can be used as a counter stain to give decolorized gram-negative bacteria a pink color (more distinct than sasafranin).
58
What is the substrate of the catalase reaction?
1.) H2O2
59
Explain what results you would see if you performed a catalase test w/ bacteria taken from a medium containing blood.
1.) You would see nothing b/c blood cells contain catalase and trace contamination of a medium can lead to false positive results.
60
Any microbe that lives in the presence of O2 must produce the enzyme?
1.) Superoxide dismutase
61
How is oxygen and its by-products toxic to cells?
1.) Forms of O2 produced during aerobic respiration is toxic
62
The presence of catalase is detected by adding a drop of the substrate, H202, to bacterial cells, what does it mean if it bubbles? What does it mean if it no bubbling appears?
Bubbling-indicates the liberation of molecular oxygen gas
No bubbling-organism is either an anaerobe or breaks down hydrogen peroxide w/ peroxidase
63
What two enzymes rapidly convert hydrogen peroxide into non-toxic end products?
1. ) Catalase
| 2. ) Peroxidase
64
Why would the use of selective and differential media decrease the time necessary for identification of clinical specimens or environmental samples?
.
65
What are the ingredients that make MSA a selective medium?
1.) MSA is selective because it contains 7.5% salt–a high salt concentration that promotes the growth of some organisms while discouraging the growth of others. MSA is a differential medium because it contains the sugar mannitol and the pH indicator phenol red.
66
What do these ingredients identified in #2 select for?
1. ) MSA-growth of halophilic bacteria due to the presence of 7.5% salt.
2. ) Mannitol-whether gram-positive, halophilic bacteria growing on the medium can or cannot ferment this carbohydrate
3. ) Phenol red pH indicator-detects acid (pink to yellow)
67
Define differential media.
1. ) Select for the growth of specific organisms
2. ) Due to additional ingredients in the media, differentiate between the organisms capable of growing under those selective conditions.
68
How can you differentiate between S. aureus and S. epidermidis growing on MSA?
1.) S. aureus can ferment mannitol and its production of the enzyme coagulase will clot plasma. S. epidermidis cannot ferment mannitol.
69
You inoculated a plate of MSA w/ a mixture of S. epidermis and Micrococcus luteus. After incubating the plate for 24 hrs at 37 degrees C, explain what would you observe?
.
70
Staphylococcus saprophyticus is second to E. Coli as a cause of?
1. ) Urinary tract infections in women
2. ) Able to ferment mannitol which can be used to differentiate it from S. epidermidis as well as its resistance to novobiocin.
71
Micrococcaceae include?
1.) Staphylococci and the micrococcus species
72
M. roseus and M. luteus are?
1. ) Gram-positive
2. ) Catalase-positive cocci that are not salt tolerant
3. ) Found in air and can be part of normal human cutaneous flora
4. ) May cause diseases in immunocompromised patient
73
Staphylococci are?
1. ) Gram-positive
2. ) Catalase-positive
3. ) Halophilic cocci that can be selected for inoculating them on MSA
74
What are the three main human species of Staphylococci?
1. ) Staphylococcus epidermidis
2. ) Staphylococcus aureus
3. ) Staphylococcus saprophyticus
75
Staphylococcus epidermidis
1. ) Normal flora of skin and respiratory tract
2. ) Recent years recognized as pathogen in infections of prosthetic devices and neurosurgical shunts.
3. ) Cannot ferment mannitol
76
Staphylococcus aureus
1. ) Pathogen responsible for a variety of infections
2. ) Boils, carbuncles, erysipelas, impetigo, scalded skin syndrome, toxic shock syndrome, and pneumonia
3. ) Can ferment mannitol
4. ) Produces enzyme coagulase, will clot plasma
5. ) Also part of normal flora of the skin or respiratory tract
77
Explain why the coagulase enzyme is a virulence factor for those bacteria that can synthesize it?
When fibrin is synthesized, a clot is formed that surrounds S. aureus in the tissues. The clot protects, temporarily, from the detection and destruction by immune system cells such as neutrophils. (considered virulence factor & contributes to pathogenicity)
78
Describe the basis for the Coagulase test in terms of bacteria that are coagulase positive and those that are coagulase negative.
.
79
In the human and animal body, clot formation is caused by?
1. ) The chemical conversion of fibrinogen, a soluble protein found in blood and body tissue, into fibrin.
2. ) Fibrin is stringy, sticky protein that when synthesized, precipitates out of solution. Forms a sticky mesh that catches blood cells, thus forming a clot
80
Staphylococcus aureus produces________that is both bound to the cell membrane and secreted unbound from the cell.
1.) Coagulase
81
Coagulase-positive staphylococci include?
1. ) Staphylococcus aureus subsp. anaerobius
2. ) Staphylococcus aureus subsp. aureus
3. ) Staphylococcus hyicus
4. ) Staphylococcus intermedius
5. ) Staphylococcus schleiferi subsp. coagulans
82
Coagulase-negative staphylococci include?q
1. ) S. saprophyticus
2. ) S. cohnii subsp. urealyticum
3. ) S. capitus subsp. capitus
4. ) S. warneri
5. ) S. hominis
6. ) S. epidermis
7. ) S. caprae
83
Are micrococci also coagulase negative? T or F?
True, they are coagulase negative
84
What is the most common staphylococcal pathogen?
1.) S. aureus
85
How does the coagulase test allow us to differentiate the pathogenic S. aureus form other species?
1.) B/c of the common staphylococcal bacteria, only S. aureus is coagulase positive.
86
The presence of bound coagulase (clumping factor) will react with prothrombin in rabbit plasma to catalyze the formation of fibrin from fibrinogen. As fibrin is formed on the surface of the cells, it causes the cells to stick together, forming visible climbs. Bacteria that are coagulase negative will?
1. ) Not clump in plasma
| 2. ) Rabbit plasma will take on "milky" look
87
Staphylococcus epidermidis
1. ) Benign under normal conditions
2. ) A mutualist growing on the surface of skin
3. ) Staph infections in health care settings have been encountered on prosthetic devices and indwelling catheters.
4. ) Coagulase negative
88
Staphylococcus
1. ) One of the most common causes of urinary tract infections
2. ) Coagulase negative
89
What are hemolysins? How are they useful?
1. ) Toxins produce by certain bacterial organisms that destroy red blood cells and damage human tissues
2. ) Useful method of differentiating gram positive cocci
90
Describe the blood agar plate (BAP)
1.) Tryptic soy agar supplemented w/ 5% ovine or bovine blood
91
Complete destruction of the red blood cells in the agar is referred to as? How would this appear in the agar?
1. ) Beta hemolysis (B-hemolysis) or complete hemolysis
2. ) Produces lightened area in the blood agar around and underneath the colonies that will appear slightly yellow and transparent.
92
Incomplete destruction of the red blood cells is referred to as?
1. ) Alpha hemolysis (a-hemoysis) or partial or incomplete hemolysis and is the result of partial breakdown of hemoglobin in the red blood cells
2. ) Pattern can be distinguished by observing a green discoloration around or under the colonies
3. ) No change occurs in BAP, then organisms is said to be non hemolytic or to display gamma hemolysis
93
If no change occurs in BAP, then organisms is said to be?non hemolytic or to display gamma hemolysis
1.) Non hemolytic or to display gamma hemolysis
94
In addition to being a differential medium, blood agar is also an example of?
1.) Enriched medium and can be used to culture fastidious organisms
95
Virulence factors are structures or molecules that allow pathogens to successfully invade and colonize a host. Why are hemolysisn considered virulence factors?
.
96
Why is beta hemolysis also called complete hemolysis?
.
97
Why is alpha hemolysis also called partial hemolysis?
.
98
Streptococcal bacteria are?
1.) Gram-positive and usually arranged in chains or pairs.
99
Group A Streptococcus progenies is sensitive to low concentrations of the antibiotic________while other beta hemolytic streptococci, such as S. agalactiae, are not.
1.) Bacitracin
100
Sensitivity to bacitracin is demonstrated by a zone of inhibition with a?
1.) Diameter of 10mm or more.
101
Resistance to bacitracin is demonstrated by?
1.) No zone of inhibition, in which the bacteria grow right up to the antibiotic disc, or the zone of inhibition has a diameter less than 10mm.
102
What are the three medically important Lancefield groups?
1. ) Group A including streptococcus pyogenes
2. ) Group B including Streptococcus agalactiae
3. ) Group D including streptococci and enterococci
103
Lancefield Group A (S. pyogenes) and Lancefield group B (S. agalactiae) are?
1. ) Both beta hemolytic and pathogenic
2. ) Group A (S. pyogenes) causes a variety of infections, including acute streptococcal pharyngitis, scarlet fever, rheumatic fever, glomerulonephritis, and certain skin infections.
104
Lancefield Group D includes enterococci.
1. ) Includes medically important enterococci, such as E. feacalis along w/ certain species of streptococci such as S. bovid, S. sues, and S. equines.
2. ) Usually display alpha or gamma hemolysis, are all capable of growing in bile and can hydrolyze esculin.
3. ) Ability to grow in 6.5% Saline solution
4. ) Enterococci-are halophiles, more commonly associated w/ human disease, and are acquired through fecal contamination.
5. ) Non-enterococci-can't grow in high salt concentrations
6. ) Found in intestines of humans and animals, occasionally contaminate food.
105
Name a nosocomial infection caused by enterococci thats of rising concern?
1.) Vancomycin resistant enterococci (VRE)
106
How can group D be differentiated from other streptococci?
1.) Bile esculin agar (BEA)
107
Describe BEA or bile esculin agar.
1. ) Selective medium b/c it contains bile which is inhibitory to gram-positive organisms but not to enteric streptococci and enterococci
2. ) BEA is also differential for Group D streptococci and enterococci which can hydrolyze esculin into glucose and esculetin in the presence of bile.
108
What happens when esculetin is formed? Describe its appearance.
1. ) It will bind w/ the color indicator, ferric citrate, present in BEA to produce a dark brown-black iron (Fe3+) precipitate.
2. ) Observatoin of an inoculated BEA slant in which at least half of the agar is darkened indicates a positive reaction. No growth or no color change in the agar indicates a negative reaction.
109
How can separation of the Group D streptococci into enterococci and non-enterococci be determined through the use of a 6.5% saline Tryptic soy broth?
1. ) High concentrations of sodium chloride (NaCl) are inhibitory to non-enterococci such as S. bovis but selective for enterococci such as E. faecalis.
2. ) 6.5% NaCl broth is a selective only medium, a positive result is observed as turbid or sedimentary growth in the broth. A broth that remains clear, w/ no growth, indicates a negative result.
110
The Streptococcus viridans group includes?
1. ) Alpha-hemolytic streptococci that are normal flora in the respiratory tract and oral cavity such as S. mutans, S. mitts, and S. salivarus.
2. ) Number one cause of dental caries is S. mutans-synthesizes a dextran capsule from the breakdown of dietary sucrose.
3. ) S. mutan's capsule contributes to formation of a biofilm, allowing it to attach to tooth surface. Once attach S. mutans and other bacteria ferment the fructose from sucrose hydrolysis into lactic acid which destroys tooth enamel
111
Streptococcus pneumonia is the leading cause of?
1. ) Bacterial pneumonia, otits media, and meningitis.
2. ) Virulent strains possess a prominent polysaccharide capsule which results in a cultural characteristics of mucoid growth on agar media.
3. ) Considered an opportunistic pathogen often existing as normal flora in healthy individuals
4. ) 2 Vaccines available-peadiatric and adult form, protects against most strains
5. ) Can be distinguished from other streptococci by its alpha hemolytic reaction on blood agar and its sensitivity to optochin.
112
Definition of optochin?
1. ) Surface reactant that will lyse the cells of S. pneumonia but not other alpha-hemolytic streptococci.
2. ) Sensitivity is demonstrated by a zone diameter of 10mm or more.
3. ) Resistance demonstrated by no zone of inhibition.
