exam 1 information Flashcards
1
Q
what factors is resolving power based off of
A
- wavelength of light
- numerical aperture of the lens
2
Q
what microscope do we use in class
A
compound light-field
- resolution limit is 0.2 micrometers
3
Q
how to determine total magnification
A
(magnification of objective lens) x (magnification of ocular lens)
4
Q
resolving power
A
how well it allows two objects to be seen as distinct and separate
5
Q
resolution of oil lens
A
typically better because refractive index is very close to glass
6
Q
why can’t some lenses be used with oil
A
- degrades portions of the lens
- must wipe and clean off immediately
7
Q
electron microscopy
A
smaller wavelength than light, much better resolution power
- uses electrons and measures that wavelength rather than light, uses magnets
8
Q
bacterial morphology
A
cocci: diplococci, cocci clusters, cocci chains
bacilli: flagellate rods, bacilli chains, spore formers
spiral: spirochaetes, spirilla, vibrios
- use this when writing observations in lab
9
Q
how are petri dishes always incubated
A
in an inverted position– this prevents condensation that forms on the cover during solidification
10
Q
aseptic technique
A
practices and procedures to prevent contamination from pathogens and minimize the risk of infection
- includes: handwashing. sterilization, keeping flasks and plates closed
11
Q
when is loop used over needle
A
in a broth, slant, and plate
- needle is only used in agar deep
12
Q
what to label nutrient broth tube with
A
organism, name, section number, date
13
Q
inoculation
A
adding a microbe to the tube
14
Q
agar slant
A
stick loop down to the bottom of the slanted portion
- use zig-zag motion and drag loop toward top of tube
15
Q
starting with a mixed culture, isolation of different species may be obtained by…
A
streaking
16
Q
stain (dye)
A
an organic compound containing a benzene ring plus a chromophore and auxochrome group
17
Q
acidic stain
A
sodium, potassium, calcium, or ammonium salts of colored acids ionize to give a negatively charged chromogen
18
Q
basic stain
A
the chloride or sulfate salts of colored bases ionize to give a positively charged chromogen
19
Q
difference between gram-positive and gram-negative
A
gram pos:
- thick peptidoglycan layer
gram neg:
- peptidoglycan layer is much thinner, surrounded by outer lipid-containing layers
20
Q
lysozyme or penicillin
A
has the ability to denude the Gram-positive cell wall
21
Q
differential stain
A
different microbes will give different results when stained by the same procedure
- e.g. gram stain
22
Q
acid-fast stain (Ziehl-Neelson method)
A
differential stain used to detect bacteria from the genus Mycobacterium
uses three different reagents:
1. primary: carbolfuchsin
- a red phenolic stain that is soluble in the lipoidal materials that constitute the major portion of the mycobacterial cell wall, can penetrate mycobacterial wall, STRONG STAINN
- further enhanced using heat
- makes all cells appear red
- decolorizing agent: acid-alcohol (3% HCl + 95% ethanol)
- prior to decolorizing, the smear is cooled (allows waxy cell substance to harden)
- primary stain is more easily removed from non-acid fast cells during decolorization, leaving these cells colorless or unstained - counterstain: methylene blue
- this is used as the final reagent to stain previously decolorized cells
- only non-acid-fast cells undergo decolorization, may now absorb the counterstain and take on its blue color,
- acid-fast cells retain red color of the primary stain
23
Q
mycolic acid
A
waxy polymer that makes the cell envelope of Mycobacteria significantly less permeable compared to other groups of bacteria
- makes Mycobacteria resistant to staining, helps with differentiation
24
Q
spore stain (Schaeffer-Fulton method)
A
a differential stain used to detect bacterial endospores formed by the members of the genus Bacillus and Clostridium
- primary stain: malachite green
- spore does not accept primary stain easily– requires heat
- after the primary stain is applied and the smear is heated, both the vegetative cell and spore will appear green - decolorizing agent: water
- when the spore is green, it cannot be decolorized by tap water
- the water will remove coloring from vegetative cell components (because of a low affinity) –> return colorless - counterstain: safranin
- colors the decolorized vegetative cells –> turns them red
- spores retain green color of the primary stain
25
Bacillus and Clostridium
bacteria of these genus are significant because they include some of the most important human and animal pathogens, causing diseases such as anthrax, tetanus, botulism, and gangrene
- Clostridium typically have a slight bulge around the spore, giving the appearance of a drumstick
- Bacillus does not show this bulge --> can differentiate
26
bacterial endospore
complex structure with a coat composed of highly condensed proteins that is resistant to penetration by most chemicals
- the resistance of bacterial endospores makes
them highly refractory to both staining and destaining
- most commonly used spore staining procedure is the Schaeffer-Fulton method
27
vegetative cells
growing cells
- spores usually germinate back into vegetative cells when conditions are more favorable
28
three basic types of growth media
- general growth media
- selective media
- differential media
29
general growth media
non-specific media configured to culture a wide range of microorganisms without particular restrictions
- e.g. nutrient agar and Luria-Bertani Broth (LB broth)
30
selective media
specifically configured to restrict the growth of particular organisms while allowing others to grow
- typically will have a growth-inhibiting additive (e.g. bile salts, crystal violet or an antibiotic, which will limit growth on the medium to only those organisms that are desired
- e.g. Pseudomonas Isolation Agar (PIA), which is used int he isolation and cultivation of Pseudomonas, while restricting the growth of most common contaminating organisms
- selective media are used in the identification of particular microorganisms and when it is desirable to restrict the growth of contaminants
31
differential media
specifically configured to yield an observable characteristic associated with the growth a particular organism or group of organisms that are able to grow on the medium
- e.g. blood agar, which will support the growth of a wide range of organisms, but is used to detect the ability of particular organisms to lyse red blood cells, which is observed as a zone of complete or partial clearing around the colony
32
selective and differential media
allows only particular organisms to grow and yielding an observable diagnostic characteristic such as a color change, which is associated only with the growth of a subset of these organisms
33
the more selective the medium...
the more stressful it is to the organisms that grow on it
- individual organisms that are already stressed may not grow on a particular medium even when the medium is specifically configured for the growth of members of that group of organism
- the problem of stressed or injured microorganisms typically results in false negative results (viable but non-culturable, VBMN)
34
viable but non-culturable (VBMN)
- gives the impression that an organism is not present in a particular sample when the reverse is actually true
- many bacteria that are not able to be cultivated on
artificial media are still able to carry out an infection and cause disease
35
mannitol salt agar
representative of both selective and differential media:
SELECTIVE for Staphylococcus sp.
- high salt (NaCl) concentration (7.5%)
- inhibits the growth of most bacteria, except staphylococci
DIFFERENTIAL for Staphylococcus aureus
- the carbohydrate mannitol is fermented by S. aureus, resulting in acidic end products which turn the phenol red indicator yellow around S. aureus growth
- for other Staphylococcus species, there is no change in the medium
36
blood agar
tends to be used to recover fastidious bacteria, often Streptococcus sp. (pathogens)
- mixture of Tryptic soy agar and whole sheep blood
DIFFERENTIAL: bacteria are differentiated on their ability to cause hemolysis (lysis of red blood cells (RBCs))
- the most important of these pathogens are the hemolytic streptococci and certain members of the genera Entercoccus and Aerococcus
types of hemolysis: alpha, beta, gamma
37
alpha hemolysis
partial/incomplete lysis of RBCs
- zone of partial clearing = green halo around bacterial colonies
- e.g. Streptococcus pneumoniae
38
beta hemolysis
complete hemolysis of RBCs
- complete zone of clearing around colonies
- e.g. Streptococcus pyogenes
39
gamma hemolysis
no lysis of RBCs
- no clearing of the medium surrounding colonies, no color change
- e.g. usually non-pathogenic bacteria
40
MacConkey agar
SELECTIVE for Gram-negative enterics (Enterobacteriaceae):
- bile salts and crystal violet in the medium inhibit growth of Gram+ organisms which are sensitive to these agents due to the lack of an outer membrane to protect the cell wall, allowing for isolation of Gram+ bacteria
DIFFERENTIAL: distinguishes coliforms (usually colon bacteria) which ferment lactose, from non-coliforms that do not ferment lactose (pathogenic bacteria)
Lactose and neutral red pH indicator permit differentiation between different enteric bacteria based on their ability or inability to ferment lactose
- neutral red is an indicator dye that is colorless above a pH of 6.8 and turns red at pH values below 6.8
- the production of acid by lactose fermentation leads to a lowering of the pH of the medium and causes a change in the indicator to red
- LACTOSE FERMENTERS/COLIFORMS: bacteria exhibit red/pink coloration on their surface
- NON-LACTOSE FERMENTERS/PATHOGENS: no change; colonies appear uncolored
(colonies are often transparent and will appear to be the same color as the medium
E. coli produces more acid than other coliforms so the medium surrounding E.coli will also turn red
P. aeruginosa does not ferment lactose but does grow on the plate.
S. epidermidis does not grow in the presence of bile salts and crystal violet, both of which inhibit the
growth of gram-positive bacteria.
41
eosin-methylene blue (EMB) agar
SELECTIVE for Gram-negative enterics (Enterobacteriaceae): eosin and methylene blue dyes inhibit growth of most Gram+ organisms, allowing for isolation of Gram- bacteria
DIFFERENTIAL: distinguishes enteric coliforms from non-coliforms
- lactose and the dyes, eosin and methylene blue, allow for differentiation:
(1) lactose fermenters/coliforms: thick, mucoid purple/pink colonies due to the decreased pH from lactose fermentation
(2) E. coli: colonies are blue-black with a metallic green sheen due to the large quantity of acid made by lactose fermentation that precipitates the dyes onto the colon surface
(3) non-lactose fermenters/pathogens: no change, colorless colonies-- may appear purple due to color of the medium
EMB agar is used for the isolation and presumptive identification of fecal coliform bacteria
- contains peptone, lactose, sucrose, and the dyes eosin Y and methylene blue
- the sugars act as fermentable substrates, which yield acid by-products
- the dyes inhibit growth of Gram-positive bacteria and also act as pH indicators
- eosin Y and methylene blue will precipitate in the presence of high amounts of acid and will impart a green color
- S. epidermidis fails to grow in the presence of eosin and methylene blue
- K. aerogenes, is able to carry out limited fermentation of the sugars in the medium, turning the methylene blue indicator red and imparting a pink color to the colonies
- E. coli produces acid by-products from
mixed acid fermentation of lactose which causes precipitation of eosin and methylene blue and
imparts a green metallic sheen to the colonies.
42
phenylethyl alcohol agar
SELECTIVE: phenylethyl alcohol is partially inhibitory to Gram-negative organisms
- gram-negative organisms may form visibile colonies but the sie and number are smaller than on other media
- gram-positive organisms will predominate
- S. aureus and E. faecium grow well on both plates
- E.coli and K. pneumoniae are inhhibited by PEA, but E. coli is not completely stopped from growing
43
how to get large macromolecules through the cell membrane
they must first be hydrolyzed by specific extracellular enzymes into their respective building blocks
- low molecular-weight substances can then be transported into the cells
44
starch
a high molecular-weight branching polymer composed of glucose molecules linked together by glycosidic bonds
- amylase is a common enzyme involved for the hydrolysis into ultimately maltose molecules
- the final hydrolysis of maltose yields low molecular weight, soluble glucose molecules that can be transported into the cell and useful for energy production through the process of glycolysis
45
starch agar
used to demonstrate the hydrolytic activity of amylase
- the medium is composed of nutrient agar supplemented with starch, which serves as the polysaccharide substrate
46
starch test (amylase test for starch hydrolysis)
following growth, the detection of the hydrolytic activity is made by this test to determine the presence or absence of starch in the medium
- starch in the presence of iodine will impart a blue-black color to the medium, indicating the absence of starch-splitting enzymes and representing a negative result
- if the starch has been hydrolyzed, a clear zone of hydrolysis will surround the growth of the organism, this is a positive result
47
lipids (triglycerides)
high molecular weight compounds possessing large amounts of energy that may serve as a bacterial carbon and energy source but are too large to enter the cell
- some bacteria produce and secrete exoenzymes called lipases (esterases)
48
lipases (esterases)
hydrolyzes triglycerides
49
lipid hydrolysis
lipases cleave the ester bonds in triglycerides by the addition of water to form the building blocks glycerol (an alcohol) and fatty acids
following the inoculation and incubation of the agar plate cultures:
(1) POSITIVE: organisms excreting lipase will show a zone of lipolysis, which is demonstrated by a clear area surrounding the bacterial growth
- this loss of opacity is the result of the hydrolytic reaction yielding soluble glycerol and fatty acids
(2) NEGATIVE: the bacterial culture does not have the exoenzyme, there will be no clearing
50
tributryin agar
used to demonstrate the hydrolytic activities of the exoenzyme lipase
- the medium is composed of nutrient agar supplemented of the triglyceride tributyrin as the lipid substrate
- tributyrin forms an emulsion when dispersed in the agar, producing an opaque medium that is necessary for observing exoenzymatic activity
positive reaction: organisms excreting lipase will show a zone of lipolysis (clear area surrounding the bacterial growth) due to hydrolytic reaction yielding soluble glycerol and fatty acids
negative reaction: no clearing (due to no exoenzyme)
51
gelatin
a polymer gel composed of interlocking strands of collagen
- below 25 degrees C: solid
- above 25 degrees C: liquid
51
collagen
a protein polymer that forms the principal component of connective tissues in vertebrate animals
52
gelatinase/collagenase
type of proteolytic extracellular enzyme, can degrade collagen into individual amino acids that can the be taken into the cell and used as a source of energy for biosynthesis
- once this degradation occurs, even low temperatures (e.g. 4 degrees C) cannot restore solidification
53
use gelatinase test on what microorganisms
gelatinase producing microorganisms:
- pathogenic members of the genera Pseudomonas and Clostridium
microorganisms that can be differentiated with the gelatinase test:
- members of the Enterobacteriaceae such as Serratia and Proteus
54
how to determine ability of microorganisms to degrade and ferment carbohydrates
production of an acid OR acid and gas
55
carbohydrate fermentation test used to determine...
the ability of an organism to ferment various simple carbohydrates (sugars)
fermentation characteristics are used in identification of specifically the Gram negative enteric (gut) bacteria
56
how do most microorganisms get their energy?
1. GLYCOLYSIS (Embden-Meyerhof Pathway)
glucose (1 molecule) --> pyruvate (2 molecules)
- followed by:
2a. AEROBIC pathway (+O2)
pyruvate oxidation --> citric acid cycle --> respiratory chain
**OR**
2b. ANAEROBIC pathway (-O2)
fermentation --> lactate or alcohol, acidic waste products
- organisms use carbohydrates differently depending on their enzyme makeup
57
what type of organisms can use both aerobic and anaerobic pathways
facultative anaerobes
58
alternative name for glucose
dextrose
59
fermentation
substrates such as carbohydrates and alcohols undergo anaerobic dissimilation --> produce an organic acid accompanied by gases
- facultative anaerobes usually ferment carbohydrates
organic acid ex: lactic, formic, acetic acid
gas product ex: hydrogen, carbon dioxide
60
what steps precede the fermentation process
1. degradation of glucose by the Embden-Meyerhof pathway (i.e. glycolytic pathway)
- 1 mole glucose --> 2 moles of pyruvic acid
subsequent metabolism of pyruvate is not the same for all organisms
- variety of end products result that define their different fermentative capabilities
61
aerobic respiration
bio-oxidations in which molecular oxygen can serve as the final electron acceptor
- type of cellular respiration
62
anaerobic respiration
bio-oxidations in which inorganic ions other than oxygen, such as NO3- or SO4^2-, which can serve as the final electron acceptors
- type of cellular respiration
63
fermentation definition
a bio-oxidative process not requiring oxygen in which an organic substrate serves as the final electron acceptor
64
acid-base indicator dyes
can be used to determine the pH range of a medium in which an unknown microbe has been cultured
- over a particular range of pH values, the indicator dye will turn the medium one color and over another range of pH values the indicator will turn the medium a different color
by knowing what indicator has been used, the microbiologist can determine the range within the pH of the medium must fall
65
methyl red acid-base indicator dye
RED: below pH 4.4
ORANGE: pH 4.4-6.2
YELLOW: above pH 6.2
66
phenol red acid-base indicator dye
YELLOW: below pH 6.4
ORANGE: pH 6.4-8.5
RED: above pH 8.2
67
bromcresol purple acid-base indicator dye
YELLOW: below pH 5.4
MAGENTA: pH 5.4-7.0
PURPLE: above pH 7.0
68
bromphenol blue acid-base indicator dye
YELLOW: below pH 3.0
GREEN: pH 3.0-4.6
BLUE: above pH 4.6
69
bromthymol blue acid-base indicator dye
YELLOW: below pH 6.0
GREEN: pH 6.0-7.6
BLUE: above pH 7.6
70
azolitmin acid-base indicator dye
PINK: below pH 4.5
PURPLE: pH 4.5-8.3
BLUE: above pH 8.3
71
carbohydrate fermentation medium components
carried out in a fermentation broth tube containing a Durham tube
- Durham tube: inverted inner vial for the detection of gas production
typical carbohydrate fermentation medium contains:
1. nutrient broth for the support of the growth of all organisms
2. a specific carbohydrate that serves as the substrate for determining fermentative capabilities
3. pH indicator, phenol red
- phenol red is red at a pH of 7.0, and changes to yellow at a slightly acidic pH of 6.8
- indicates that slight amounts of acid will cause a color change
72
carbohydrate fermentation test results (lactose & glucose)
Positive results
**acid**: yellow color
- acid production produces a color change from red to yellow, indicating that the organism can metabolize the sugar in the tube
**acid, gas**: yellow plus gas bubble
- fermentation indicated by a color change to yellow
- gas is trapped in the Durham tube, replacing the medium and forming a bubble indicating gas production
Negative results: indicated in two ways
1. **no color change**:
- indicates that sugar was not utilized by the organism
2. **color change to a dark pinkish-red**
- indicates an alkaline or basic metabolic product due to the utilization of the peptone, rather than the sugar
73
what are IMViC tests used for
it is one of the standard sets of analyses performed to differentiate members of the Enterobacteriaceae
the Enterobacteriaceae are short, Gram-negative, nonspore-forming bacilli including:
1. pathogens such as members of the genera Salmonella and Shigellaa
2. occasional pathogens such as members of the genra Proteus and Klebsiella
3. normal intestial microbiotaa (referred to as normal flora) such as members of the genera Escherichia and Enterobacter, which are saprophytic inhabitants of the intestinal tract
74
why are IMViC tests important
many of the Enterobacteria are hard to tell apart based on one test alone
- IMViC analysis can differentiate them
- e.g. E. coli is dangerous to humans, Klebsiella aerogenes is not but they are very similar in terms of staining, cultural, and morphological properties
75
what tests are found in the IMViC tests
- indole test (along w/ H2S production)
- methyl red test
- motility test
- Voges-Proskauer test
- citrate utilization test
76
indole production test used for
to assay for the presence of bacteria able to hydrolyze the amino acid tryptophan
the indole test involves the inoculation of SIM medium with a pure culture of bacteria suspected of being positive for indole production
- tryptophanase positive bacteria will de-aminate tryptophan producing indole
- indole is the nitrogen containing heterocyclic ring that forms the R-group of tryptophan
- when Kovak’s reagent (DMABA, HCl, and butanol) is added to the tube, indole is extracted from the medium into the reagent by the acidified butyl alcohol component and forms a complex with DMABA, *yielding a cherry red color in the ring around the top of the medium*
77
what genera of bacteria can produce tryptophan
can produce: Escherichia, Morganella, and Edwardsiella
cannot produce: Klebsiella, Enterobacter, Proteus, and Salmonella
all of these are members of the family Enterobacteriaceae
- important for indole test (part of IMViC tests)
78
positive versus negative result for indole test (IMViC)
Indole positive: cultures producing a red reagent layer following addition of Kovak’s reagent
Indole negative: the absence of red coloration demonstrates that the substrate tryptophan was not hydrolyzed
79
Purpose of methyl red test
To determine the ability of microorganisms to oxidize glucose with the production and stabilization of high concentrations of acid end products
- used to identify bacteria that produce stable acid end products by means of mixed acid fermentation of glucose
In this test, the pH indicator methyl red detects the presence of large concentrations of acidic end products
- although all enteric microorganisms ferment glucose with the production of organic acids, **this test is of value in the separation of E. Coli and K. aerogenes**
80
How does the methyl red test (IMViC test) differentiate between the two species of interest?
Species of interest: E. coli and K. aerogenes
Both E. coli and K. aerogenes produce organic acid end products during the early incubation period
- the low acidic pH (4) is maintained by E. coli at the end of incubation
- during the later incubation period, K. aerogenes enzymatically converts these acids to non-acidic end products, resulting in an elevated pH of 6
81
Type of results from the methyl red test (IMViC test)
Positive: red color (occurs at pH less than 4.4)
Negative: yellow (occurs at pH greater than 6.0)
An orange color indicates a pH between the two, requires further incubation
82
Voges-Proskauer test purpose
To identify organisms able to produce acetone from the degradation of glucose during a 2,3-butanediol fermentation
- especially useful to differentiate members of the Enterobacteriaceae
The test allows for further differentiation among enteric organisms such as E. coli, K. aerogenes (both same as MR test), and K. pneumoniae
83
What IMViC tests can be performed in the same medium
Methyl red and Voges-Proskauer
84
VG test (IMViC) how does it work
The test determines the capability of some organisms to produce non acidic or neutral end products such as acetylmethylcarbinol from the organic acids that result from glucose metabolism in organisms such as K. aerogenes
- the reagent used in this test is Barritt’s reagent
- detection of acetylmethylcarbinol requires this end product to be oxidized to a diacetyl compound, this reaction will occur in the presence of the alpha naphthol catalyst and a guanidine group that is present in the peptone of the MR-VP medium
- as a result, a pink complex is formed, imparting a rose color to the medium
