Lab exam 2 Flashcards
(121 cards)
1
Q
Radiation
A
- physical method of microbial growth
- radiation in various forms, from higher-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)
2
Q
Light travels in forms of …
A
waves
3
Q
Wavelength
A
- the distance between any two adjacent peaks or crests of a wave
- expressed in nanometers
- shorter wavelength = higher
- longer wavelengths = lower energy
4
Q
Shorter wavelength/higher energy to longer wavelength/lower energy
A
- gamma rays
- x-rays
- UV
- Infrared
- Micro-waves
- Radio waves
5
Q
Ionizing radiation
A
- includes X-rays, gamma rays, and high-energy electron beams
- introduces doubles strand breaks in DNA molecules
- causes DNA mutations to occur
- as these mutations accumulate, they eventually lead to cell death
6
Q
Non ionizing radiations
A
- like UV can damage DNA by causing the formation of thymine dimers (bonds between adjacent pyrimidines) within a single strand of DNA
- This can lead to mutations which will ultimately kill the cells
7
Q
UV Radiation
A
- causes the formation of thymine diners in DNA, leading to lethal mutations in the exposed microbes
- germicidal lamps that emit UV light are commonly used in the laboratory to sterilize equipment
8
Q
3 Categories of UV Radiation
A
- UVC ranging from 200 nm to 290 nm is Biocidal and the most dangerous to living cells
- UVB ranging from 290nm to 320nm
- UVA ranging from 320nm to 400nm
9
Q
Cell systems can repair the damage with the help of…
A
repair enzymes like DNA polymerases, which can repair the altered pairing
10
Q
Antibiotics
A
- are chemicals produced by microorganisms that can kill (bactericidal) or curb the metabolic activities and thereby cause reversible inhibition of growth (Bacteriostatic)
11
Q
Bactericidal
A
microorganisms that can kill
12
Q
Bacteriostatic
A
curb the metabolic activities causing reversible inhibition of growth
13
Q
Broad Spectrum
A
antibiotics target multiple groups or species of organisms including both gram + and gram - bacteria
14
Q
Narrow Spectrum:
A
antibiotics are specific and target either gram negative or gram positive bacteria
15
Q
Examples of Bacteriostatic antibiotics
A
- Tetracyclines
- Chloramphenicol
- Trimethoprim
16
Q
Examples of Bactericidal antibiotics
A
- Penicillins
- Cephalosporins
- Ciprofloxacin
- Vancomycin
17
Q
Method used to test the effectiveness of antibiotics on bacteria and determine whether the bacteria are susceptible (killed by) or resistant (not killed by) to the antibiotic being tested?
A
Kirby Bauer disc diffusion method
18
Q
If the bacteria cannot grow in the presence of a compound /antibiotic there will be…
A
a clear area (aka zone of inhibition)
19
Q
Disinfectants
A
chemicals used on non-living surfaces to kill microbes
Examples: Clorox, bleach, ammonia
20
Q
Antiseptics
A
chemical used on living tissue to kill microbes
Examples: Iodine, hydrogen peroxide
21
Q
What is used to detect fecal contamination of water?
A
Indicator coliforms like E. coli
22
Q
E.coli is …
A
- facultative anaerobe
- gram negative
- no endospore
- rod shaped
- ferment lactose with acid & gas production
23
Q
Fermentation Detection and enumeration method
A
- multiple tube technique
- performed as 3 tests
- MPN (finding most probable number)
24
Q
Presumptive test
A
- day one test, first test
- various amount of water samples added to lactose fermentation broth and incubated
- positive tubes are counted and compared to statistical tables
- estimation is called most probable number of MPN method
25
Confirmatory Test
* Day two test
* Positive samples from presumptive test are examined for coliforms by testing on selective media
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Completed Test
* day three test
* positive from confirmatory test are inoculated on agar slants and lactose fermentation broth
27
Negative Carbohydrate Fermentation broth will be:
pink/red
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Positive carbohydrate fermentation broth will be:
Yellow
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Fermentation & Gas Production broth will be
Yellow
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Motility
the ability of an organism to move independently using metabolic energy
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Zone of inhibition:
marked by a clear area around the disc where the bacteria cannot grow
* measured in millimeter and checked against the known antibiotic susceptibility table
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Susceptible
killed by
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Resistant
no killed by
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Based on this data which antibiotics are broad spectrum?
* look to see if both gram + and gram - are are susceptible (killed by) the antibiotics to determine this
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Lactose fermentation test results
* color changing media from red to yellow indicates coliform (+)
* gas production is assessed by bubbles
36
What type of organisms can grow on EMB plates:
only G-
37
How to determine motility?
Motility Media which contains the chemical TTC , tri phenyl tetrazolium chloride. TTC incorporates into the moving bacterial cell and gets reduced to a red pigment called formazan
38
Motility Test:
* with a deep, just stab with bacteria and incubate
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Motile (how will it look like)
thick reddish tornado like line
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Non-Motile (how will it look like)
thin reddish line in the media
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Flagella
structures of locomotive for bacteria
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Monotrichous
one flagellum at one end of the cell
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Lophotrichous
A tuft of flagella at one end of the cell
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Amphitrichous
Flagella at both ends of cell
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Peritrichous
Flagella over the entire surface of the cell
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No flagella:
non-motile bacteria
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Biochemical activities of microorganisms are based on
enzymatic reactions
48
Two major categories of hydrolytic enzymes seen in bacteria:
Exoenzymes/extracellular enzymes and Endoenzymes/intracellular enzymes
49
Exoenzymes/Extracellular enzymes:
secreted by the bacteria to the outside environment, where the substrate polymers will be broken down into smaller monomers
* presence of amylase: starch acids —> glucose
* presence of lipase: triglycerides —> glycerol & fatty
* presence of casease: casein —> amino acids
* presence of gelatinase: gelatin —> amino acids
50
Endoenzymes/intracellular enzymes (examples):
work inside the cell. The substrate molecules are transported into the cell, and the Endo enzymes exercise their activities inside the so
* presence of catalase: hydrogen peroxide —> water & oxygen
* presence of nitrate reductase: nitrates —> nitrites
* presence of urease: urea —> ammonia & CO2
Oxidase & IMVIC tests
51
Carbohydrates are…
Complex macromolecules made of carbon, hydrogen, and oxygen
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Bacteria are capable of a fermenting carbohydrates with the production of…
acids or acids with gas
53
How can carbohydrate fermentation be tested?
With 4 fermentation broths containing the sugar (carbohydrates) with phenol red as pH indicator and small inverted tube called Durham tube for detecting the presence of gas. Incubate at 37*C
* glucose (monosaccharide)
* lactose (disaccharide)
* sucrose (disaccharide)
* mannitol (sugar alcohol)
54
Positive in Carbohydrate Fermentation:
broth will change from red to orange-yellow (change in color indicates the change in pH from neutral to acidic)
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Negative in Carbohydrate Fermentation
broth will remain red
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The presence of a bubble inside the Durham tube will indicate …
the production of gaseous product as a result of fermentation
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Hydrolytic exoenzymes
break down macromolecules into smaller subunits that can then be transported into the cell
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Extracellular Enzymes
* aka extracellular enzymes
* amylase (starch hydrolysis)
* lipase (lipid hydrolysis)
* casease (casein hydrolysis)
* getinase (gelatin hydrolysis)
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Endoenzymes
do their activities inside the cell and they further degrade the smaller subunits which are absorbed by the cell.
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Intracellular enzymes
* aka endoenzymes
* catalase test
* oxidase test
* urea broth
* IMVic tests
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IMVIC tests
used for differentiating gram negative enteric organisms in the family Enterobacteriaceae
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IMVIC includes
four separate tests; indole tryptophan production test, methyl red test, voges-proskauer test, and citrate test
63
Tubes for IMVIC tests should be incubated at
37 degrees Celsius
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What indicator is used in indole test?
kovac’s reagent
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Positive indole test
* Postive cherry red color
* positive result means that it can degrade tryptophan with the enzyme tryptophanase
66
Indole Production Test
used to check if bacteria has the enzyme tryptophanase which can degrade tryptophan to indole & pyruvic acid
* after growing the bacteria in tryptophan broth, the presence of indole can be detected by the indicator Kovac’s reagent which acts on the indole to produce a cherry red color
67
What indicator is used in the methyl red test?
Methyl red indicator
68
Positive Methyl Red Test
* methyl indicator turns red
* and positive means that it is a glucose fermenter
69
Methyl Red Test
used to identify glucose fermenters with large quantities of acid production.
* After bacterial growth in MRVP medium (contains peptone, glucose, and buffers) methyl red indicator is added to the broth
* if positive, then it will turn red
* if negative, it will remain yellow
70
What indicator is used in the Voges-proskauer test:
Barrett’s reagents (A & B)
71
Positive VP Test:
* forms a red layer of yellow broth
* positive means that it can ferment glucose to acids to non-acidic products
* has AMC
72
voges-proskauer test
Some organisms can ferment glucose to acids and then to non-acidic products called AMC (acetyl methyl carbinol) or acetoin.
* Presence of AMC can be detected by adding equal amounts of Barritt's reagents (A & B) to the broth after bacterial growth in MRVP medium. After adding the reagents, tubes should be kept at 37°C for about 30 minutes.
* Formation of a red layer indicates a positive test result (it has AMC)
73
What indicator is used in Citrate test?
Simmons Citrate Agar
74
Positive Citrate Test:
* turns a blue color
* positive test means that organisms can use citrate as the carbon source
75
Citrate Test
used to determine if bacteria have the intracellular enzyme citrase or citrate lyase.
* streak and stab
* Simmons Citrate Agar color change from green to blue means its positive for citrate
* Nitrogen source: ammonium phosphate
* pH indicator: bromothymol blue
* green color indicates pH is neutral (is positive for citrate)
76
What indicator is used for Nitrate Test?
Diphenylamine & zinc
77
Positive Nitrate Test
* when adding first indicator, if it turns red then it indicates nitrate has been reduced
* after adding zinc, if there is no color change that nitrate was reduced
78
Negative Nitrate Test
* if there is a red color change after adding a pinch of zinc
* zinc has reduced to nitrate (nitrate was not reduced therefore it’s a negative test)
79
Nitrate Test:
Many bacteria can use nitrates as the final electron acceptor. Some bacteria can reduce nitrates to nitrites. These bacteria have the intracellular enzyme nitrate reductase. Some can reduce it further down to nitric oxide, nitrous oxide or nitrogen gas. These bacteria have nitrite reductase and other nitrite reducing enzymes in addition to nitrate reductase.
* if red develops, this means that nitrate has been reduced to nitrate, indicating a positive test result for nitrate reduction
* if no red color develops check if nitrate is still present in the media by adding a pinch of zinc powder, if red color develops after adding zinc then you know that the nitrate was not reduced by a microbes enzyme, indicating a negative test result for nitrate reduction
* if no color change is observed after the addition of zinc, this indicates a positive result for nitrate reduction
80
Some aerobic & anaerobic bacteria are able to reduce Nitrates to the following:
Nitrites
Ammonia
Nitric oxide
Nitrous oxide
Nitrogen
81
What indicator is used in Urease Test?
Phenol red
82
Positive Urease Test
* color changed to bright pink or fuchsia
* positive means bacteria produces urease since it has hydrolyzed urea to produce ammonia giving the pink color
83
Urease Test:
Urea is a waste product of protein digestion. Bacteria with the endoenzyme urease can hydrolyze urea to produce ammonia.
* Urea broth which contains peptone, glucose, urea and phenol red (pH6.8) is used to detect the presence of this enzyme.
* Color change to a bright pink - fuchsia indicates pH change to basic ammonia with pH 8.4
84
Urea
intracellular enzyme
* If the bacteria produces urease it can hydrolyze urea to produce basic ammonia
*Color changed to bright pink - fushcia
85
What indicator is used in Catalase Test?
Hydrogen peroxide
86
Positive Catalase Test
* produces bubbles
* this means it has the enzyme catalase
87
Catalase Test
Toxic hydrogen peroxide is produced inside cells as a result of metabolic activities.
* Catalase is toxic to cells
* Endo enzyme catalase degrades hydrogen peroxide to water & oxygen.
* used to differentiate catalyst positive and catalyst negative organisms (Staphylococcus catalase positive from Streptococcus (catalase negative))
* All catalase positive bacteria will have the enzyme catalase which react with hydrogen peroxide to give oxygen bubbles.
88
Catalase
intracellular enzyme
89
What indicator is used in oxidase test?
There is no indicator, a dry slide is used instead
90
Positive Oxidase Test?
* dry slide will turn blue
* indicates that it has the enzyme oxidase
91
Oxidase Test
In aerobic bacteria, cytochromes in the plasma membranes carry electrons to oxygen. Oxidase test is used to determine the presence of cytochrome c in some bacteria. Oxidase reagent in the media is Tetramethyl -p- phenylenediamine TMPD.
* If the organism has cytochrome c the reagent TMPD will change color to DARK BLUE (should be read in 15s to 20s)
92
Why should the oxidase test be read in 15s to 20s?
Because oxidase reagent is unstable and it will oxidize in the presence of atmospheric oxygen
93
Oxidase
intracellular enzyme
94
Starch
polysaccharide made up of glucose monomers
95
How is starch hydrolysis tested and what is the indicator?
* presence of Amylase.
* After making a streak of organisms on the plate, add several drops of iodine to starch plate on the plate
96
Starch hydrolysis positive result
* clear halo around culture
* positive test result means that the organism has the enzyme amylase to hydrolyze starch
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Starch hydrolysis negative result
* in areas with starch iodine will bind with starch giving a dark brown/purple color
* negative result means that no starch hydrolysis was done
98
Enzyme Amylase
is extracellular, and can hydrolyzed starch outside the cell
99
Positive test result for the production of Amylase
absence of blue black color around the culture, thus creating a colorless halo surrounding the culture (positive, has the enzyme analyses to hydrolyze starch)
100
Lipase
can breakdown lipids to monomers and use those as sources of carbon and energy
101
Lipid hydrolysis:
* tests for lipase
* bacteria which have the enzyme lipase, can break down lipids to monomers and use them as sources of carbon and energy.
* Spirit blue agar contains a combination of emulsified oils and spirit blue dye. If the bacteria can ingest the oils with lipase the blue plates will display colorless halos around the bacterial colonies (positive)
102
Positive Lipase Test
* on the spirit blue plate, the bacteria which produce lipase will hydrolyze the olive oil in the media, giving a colorless halo
* positive result indicates that lipids are broken down into glycerol and fatty acids
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Negative Lipase Test
* remains a blue color
* and negative test result means that the there is no lipase activity, so lipids are not broken down
104
Selective media
Curb the growth of unwanted bacteria and encourage the growth of desired ones
* example: there are selective media which facilitate the growth of gram-negative bacteria, but inhibit the growth of gram-positive vice versa)
105
Differential Media
Based on the unique enzymatic properties of bacteria (bacteria grow on this media, but show difference in appearance)
106
Mannitol salts agar plate
* This media contains 7.5% Sodium chloride
* The saltiness of this media makes it selective by inhibiting the growth of many bacteria
* The sugar is mannitol and the pH indicator is phenol red
* Species of the genus staphylococcus can be identified using this media.
* Pathogenic Staphylococcus aureus can ferment the mannitol releasing acids which will turn the media yellow around the colonies
* non-pathogenic staphylococcus epidermis is can grow but do not change the color of the media
* media is both selective and differential
107
What organism can grow on the Mannitol Salt Sugar plate but does not change the color
Non-pathogenic staphylococcus epidermidis
108
Positive Mannitol Salt Agar Result:
Media goes from pink to yellow indicating fermentation of mannitol
* opH changes to acidic
109
Negative EMB results
No color change on the plate
110
Positive EMB results
* metallic green (indicating e.coli)
* some other color change
* positive means that it contains lactose
111
Eosin Methylene Blue Agar Plate (EMB)
* contains gram + inhibitors, eosin & methylene blue, which make it selective for gram -
* The sugar source lactose allows the differtiation of gram negative enteric bacteria into coliforms and non-coliform
* will show e.coli very easy
* EMB are selective (g-) and differential (lactose fermentation)
112
E.coli can be identified the quicker by the
* Eosin Methylene Blue (EMB)
* shows metallic green color
113
Positive result of MAC
Pink color developing on the streak
114
Negative result of MAC
No change of streak color in plate
115
MacConkley Agar Plate (mac)
* Bile salts and crystal violet incorporated in this media are gram positive inhibitors which make the media selective for gram negative enteric bacteria
* Lactose is the sugar source
* Phenol red is the pH indicator
* Lactose fermenter/coliforms will appear pink in this media as opposed to the non-fermenters/non-coliforms which appear colorless
* are both selective (g-) and differential (lactose fermenters)
116
Blood Agar/Rose Agar Plate
* differential media
* most organisms can grow
* differentiates hemolysis activity (alpha, beta, gamma)
* some bacteria produce hemolysis which can break down the red blood cells
117
Alpha hemolysis
Aka partial hemolysis
* causes partial hemolysis of red blood cells resulting in a dark blackish green halo around the culture termed as Alpha hemolysis
118
Beta hemolysis
Aka true hemolysis
* causes complete lysis of blood cells which produces a clear halo around the culture termed
119
If the organism is not capable of any lysis but can grow on the blood plate, then it is termed as …
Gamma hemolysis
120
Coliforms
Aka Lactose fermenter, can ferment lactose
121
Non-coliforms
* can not ferment lactose
