micro lab midterm Flashcards
1
Q
Biosafety Level 1
A
Do not typically cause disease in healthy individuals and present a minimal threat to the environment and lab personnel. They can be handled in the open and do not need special containment equipment.
2
Q
Biosafety Level 2
A
Commonly encountered in the community and present a moderate health hazard. They are wide human disease and can usually be treated if identified in a timely manner. They usually cause infection through ingestion, inhalation, or penetration of the skin. You should work with a biosafety cabinet with aerosol generation and splashing.
3
Q
Biosafety Level 3
A
Local or exotic origin and are associated with respiratory transmission. Serious or lethal disease where treatment/vaccine are not available. Must use special ventilation systems to prevent aerosol transmission, and lab access is restricted.
4
Q
Biosafety level 4
A
Great potential for lethal infection. Droplets, aerosol, and autoinoculation are the concerns. The lab should be isolated from other facilities and access is strictly controlled. Ventalation and waste management are regulations to prevent it from being release into the environment. Specially trained professionals are the only ones handling this stuff. Must wear personal, positive pressure, one piece body suits
5
Q
What are examples of organisms at biosafety level 1
A
Bacillus subtilis, Escherichia coli, Rhodospirillum rubrum, and Lactobacillus acidophilus.
6
Q
What are examples of organisms at biosafety level 2
A
Salmonella, Staphylococcus aureus, Clostridium dificile, and Borrelia burgdorferi
7
Q
What are examples of organisms at biosafety level 3
A
Bacillus Anthracis, Mycobacterium tuberculosis, and West Nile virus
8
Q
What are examples of organisms at biosafety level 4
A
Ebola, Marburg, and Lassa Fever
9
Q
Which biosafety levels do we work with in our microbiology lab?
A
BSL-1 and BSL-2
10
Q
Which biosafety level is being described:
§ Organism is associated with a serious disease, for which treatment is available but not always effective. Organism is handled only in a Class II or Class III biosafety cabinet. Access to lab is restricted, and special ventilation systems are used to prevent aerosol transmission out of the laboratory.
A
BSL-3
11
Q
List the procedures we follow when entering and exiting the laboratory in order to reduce the transmission of contaminants to or from the lab.
A
Wash hands before and after each lab, and anytime you leave the room and come back.
At the beginning of lab, the tables are wiped with 75% ethanol and dried, and at the end of class, the tables are wiped with cleaner
12
Q
What are some safety measures we follow during the lab, to reduce the possibility of contamination of self or others?
A
o No food or liquids are brought into the lab
o Appropriate clothing is worn in lab at all times
o Wearing safety goggles or glasses at all times in the lab
o Do not touch face or eyes with hands
o All wounds covered with bandages
o No mouth pipetting
13
Q
How are contaminated glass slides disposed of?
A
They are disposed into the autoclave bag to be disinfected before thrown away.
14
Q
How are bacterial liquid and solid cultures disposed of?
A
Placed in the autoclave bag.
15
Q
What is the procedure for cleaning up a spilled bacterial culture from the lab bench?
A
Cover spills immediate with paper towels. Soak the towels immediately with disinfectant and allow them to stand for 20 minutes. Then place the paper towels in the autoclave bag.
16
Q
What is difference between a bacterial cell and a bacterial colony?
A
A cell is only one bacteria, a colony is a visible mass of cells that arise from one bacterial cell
17
Q
What is a pure culture?
A
Contains only one species or strain of bacteria
18
Q
If you inoculate an LB plate with a cotton swab that you used to sample a door knob, and nothing grows on the plate after 3 days, can you assume that the door knob was sterile? Why/why not?
A
No you cannot assume the doorknob was sterile because some cultures take longer than three days to grow, so there could still be bacterial cells on the agar that are just not yet visible.
19
Q
What terms would you use to describe the shape of the colony as a whole?
A
Round, irregular, punctiform
20
Q
What terms would you use to describe the margin (edges) of the colony?
A
Entire, undulate, lobate, erose, filamentous
21
Q
What terms would you use to describe the surface?
A
Surface- smooths, rough, wrinkled, shiny, dull
22
Q
What terms would you use to describe the texture
A
Texture- moist, mucoid, buytrous, dry
23
Q
What terms would you use to describe the elevation
A
Flat, raised, convex, pulvinate, umbonate, crateriform
24
Q
Will a particular bacterial colony always appear the same color? Why/why not?
A
No they can appear to be different colors because they could be in different stages of growth could result in different colors of the cells.
25
How might you distinguish fungal growth from bacterial growth
Bacterial colonies are tiny, while the fungal colonies are larger. Fungal colonies look “hairy” and grow as powdery mats all over the agar. Fungal colonies arise from a single spore or mycelial fragment.
26
A medium that contains living microbes is called a ?
culture
27
What does it mean to transfer microbes aseptically?
Without contamination of yourself, others, the environment, the source culture, or the medium being inoculated.
28
Describe the steps you would perform when transferring a bacterial culture from one liquid broth medium to another, using an inoculation loop.
Label the sterile broth with name, date, medium, and organism you are inoculating with. Then flame loos from base to tip and make sure the loop becomes orange in color. Loosen the cap of the culture tube, then move the tube to your loop hand. Flame the lip of the tube two or three times. Angle the tube.
29
Why should you hold the tubes close to a horizontal angle, instead of straight up and down when transferring microbes aseptically?
If it is held vertically, it is harder to acquire the culture, and there is a larger chance of creating aerosol contamination from the broth.
30
What is aerosol production and why is it a concern when working with BSL-2 organisms?
It is when the bacteria becomes a droplet suspended in the air, this is worrisome with BSL-2 organisms because they can be spread through inhalation of the bacteria.
31
What is filiform growth?
slant, Dense and opaque with a smooth edge
32
What does friable mean?
slant, dry and crusty
33
What do rhizoid and echinulate describe?
A slant growth that is branched or spiny.
34
How would you describe differences in optical properties on slant?
Color, opaqueness, translucent
35
What is a pellicle (broth)?
Film floating on the surface
36
What is growth that sinks to the bottom of broth called?
sediment
37
What is the difference between uniform fine turbidity and flocculent growth in broth?
Uniform fine turbidity is when the bacteria is broken down throughout the broth, and flocculent growth is when the bacteria grows in flakes inside of the broth.
38
How do you calculate total magnification of a specimen viewed under our light microscopes?
Magnification by the objective lens times the magnification by the ocular lens
39
What is resolution
clarity of an image
40
Resolution improves as the limit of resolution is made....
smaller
41
What does the condenser lens do?
Concentrates the light and makes illumination of the specimen more uniform
42
What is numerical aperture?
The measure of a lens ability to capture light coming from the specimen and use it to make the image. It is marker on the lens
43
Using immersion oil between the specimen and the oil-immersion lens (increases or decreases?) its numerical aperture, which in turn makes its limit of resolution (smaller or larger?).
increases; smaller
44
Wet mount
There is no stain used and the cells are mostly transparent. Can be done to observe motility, cell size, binary fission, arrangement, and morphology
45
Hanging Mount
Longer observation of the specimen because it does not dry out as quickly. Allows for long periods of observation for cell size, morphology, arrangement, binary fission, and motility.
46
What is Brownian motion? How can you distinguish it from true motility?
Motion created by collision between cells and water molecules is Brownian motion, which makes it look like cells are vibrating in place. Non-motile cells still exhibit Brownian motion.
47
How does one prepare a hanging drop slide?
1) Apply light ring of petroleum jelly around the well of the depression with a toothpick 2) Apply a loop of water to a cover of glass (if using broth do not use water). 3) Aseptically transfer a loop of the culture, then flame the loop after transfer. 4) Inver
the depression slide so the drop is centered in the well. Press until the petroleum jelly makes a seal. Then turn the slides right side up and place on the microscope. 5) The drop should look like it is hanging
48
Define and differentiate between bacterial cell morphology and cell arrangements
The cell morphology is the shape of the bacteria, and the arrangement of the bacteria is how many different cells orient themselves to one another
49
What are the 3 basic bacterial cell morphologies?
Spheres (cocci), rods (bacillus), or spirals (spirilla)
50
What is pleomorphism?
The ability of bacteria to alter their morphology depending on the environment they are growing in.
51
A bacterial cell arrangement in which bacilli cells form chains is called ____________.
streptobacilli
52
A bacterial cell arrangement in which cells occur in groups of 4, all in the same plane, is called a _________.
tetrad
53
A bacterial cell arrangement in which cocci cells occur in irregular clusters is called ___________.
staphylococcus
54
What is the bacterial cell arrangement known as sarcina?
8
55
What is the difference between staphylococcus and Staphylococcus?
Staphylococcus is the genus name of a bacteria, and staphylococcus is the arrangement of a bacteria.
56
Because cytoplasm is essentially transparent, viewing cells with the standard light microscope is difficult without stains to provide ___
contrast
57
Stains are solutions consisting of a solvent and a colored molecule called the
chromagen
58
Why do basic stains contain a positive charge
They pick up a hydrogen ion or losing an hydroxide ion.
59
What are some examples of basic stains?
methylene blue, crystal violet, safranin
60
What does heat-fixing accomplish
It kills the bacteria and makes them adhere to the slide, and coagulates cytoplasmic protein to make them more visible. Sometimes it distorts the cell.
61
What are the applications of Negative stain
used to determine morphology and cellular arrangement in bacteria that are too delicate to be heat fixed. It also produces minimal shrinkage so it is good when trying to observe accurate size.
62
What are the applications of Gram stain
Distinguishes between gram + and gram – and is the most important differential stain. It also allows determination of morphology , size, and arrangement.
63
What are the applications of acid fast stain
differential stain used to detect cells capable of retaining primary stain when treated with an acid alcohol. It is used to identify Mycobacterium. Its is imperative in diagnosing tuberculosis
64
What are the applications of a capsule stain
Detects cells capable of producing an extracellular capsule. This capsule increases virulence in microbes
65
What is the difference in cell wall composition of Gram negative versus Gram positive organisms?
Gram negative cells have less peptidoglycan and more lipid than gram
positive cells. The large crystal violet-iodine complexes form in the cytoplasm of both types of cells. The thicker gram positive peptidoglycan layer dehydrates with the decolorizer and “traps” the crystal violet-iodine complexes, thus purple colored cells. The lipid in the gram negative can be dissolved during decolorization and the thin layer of peptidoglycan is not able to “trap” the crystal violet-iodine complexes, thus no purple color is present.
66
List the procedural steps of the Gram stain
1. Apply primary stain (crystal violet) 2. Add mordant (grams iodine) 3. Decolorize with 95% ethyl alcohol 4. Counterstain (safranin).
67
What can happen if you decolorize too long?
Gram positive cells could appear to be gram negative because the cell wall is disrupted and the crystal violet-iodine can escape
68
what can happen if you decolorize not long enough
Gram negative cells can appear to be gram positive because the cell walls were not disrupted and no complexes could escape.
69
What can happen if you forget to heat-fix your slide?
The bacteria smears on the slide can be washed off during the staining steps.
70
Which of the above stains is used to detect cells capable of retaining a primary stain when treated with an acid alcohol?
the acid-fast stain
71
What is a selective media?
Contains components that with either enhance or inhibit the growth of specific organisms
72
What is a differential media?
Contains indicators that will show differences between organisms
73
What are coliform bacteria
A subgroup of Enterobacteriaceae that produce acid and gas from lactose fermentation. Most coliforms are normal inhabitants of the human intestinal tract and their presence in the environment may be evidence of fecal contamination
74
Phenylethyl alcohol agar (PEA):
: undefined, selective: inhibits the growth of many gram negative organisms and some gram positive organisms; it is not differential because no distinction can be made between the organisms that grow on it; digests of casein and soybean meal provide nutrition and sodium chloride provides a stable osmotic environment that is suitable for the addition of sheep blood if desired; selective agent: phenylethyl alcohol which disrupts cytoplasmic membrane of sensitive organisms; used to isolate staphylococci and streptococci from mixtures of bacterial flora: often used to screen out common contaminants E.coli and Proteus species; when prepared with 5% sheep blood, can be used for cultivation of gram positive anaerobes
75
Mannitol salt agar (MSA)
contains carbohydrate mannitol, 7.5% sodium chloride, and phenol red; selective for 7.5% sodium chloride because the high salt concentration can dehydrate and kill most bacteria; differential for fermentation of mannitol (carbohydrate); pH indicator: phenol red; staphylococci thrives on the medium and phenol red indicates if fermentation with an acid end product has taken place by changing the color as the pH changes (fermentation turns yellow on red medium): most staphylococci are able to grow on MSA but do not ferment mannitol, so grow is pink/red. S. aureus ferments mannitol producing acids and lowering pH causing yellow colonies - selects for staphylococci group like staphylococcus aureus and staphylococcus epidermis; used for isolation and differentiation of S. aureus from other staphylococcus species and is NOT used for determining the ability of an isolate to ferment mannitol.
76
MacConkey agar
contains lactose, bile salts, neutral red, and crystal violet; selective for bile salts and crystal violet because these inhibits gram positive organisms; differential for fermentation of lactose: those that ferment lactose to acid-end products lower the pH and colonies turn red/pink; pH indicator: neutral red; occasionally a pink/red precipitate with form in agar due to low pH and the lactose non-fermenters will retain their normal color or the color of the medium; useful for isolating and differentiating members of Enterobacteriaceae family based on ability to ferment lactose; formulations without crystal violet allow growth of gram-positive cocci: Enterococcus and some Staphyloccus which ferment lactose and appear pink
77
Eosin methylene blue agar (EMB)
chemically undefined, selective and differential; contains digest of gelatin (provides nitrogen and carbon), lactose (can be fermented to acid-end products by E.coli, Enterobacteraerogenes NOT Proteus, shigella or salmonella), and the dyes eosin Y and methylene blue; the dyes inhibit most gram positive and react with vigorous lactose fermenters whose acid-end products turn growth dark purple or black (often accompanied with a metallic green sheen) making it differential because lactose is fermented to acid-end products by coliforms such as E.coli and E. aerogenes, and not by pathogens such as Proteus, Shigella, and Salmonella; the less aggressive lactose fermenters Enterobacter or Klebsiella produce pink to dark purple colonies on medium; used for isolation of fecal coliforms
78
Hektoen enteric agar (HE)
contains ferric ammonium citrate for a source of oxidized iron to react with any H2S produced to form black precipitate, the bile salts prevent or moderately selective for bile salts to prevent or inhibit growth of gram positive bacteria and have moderate inhibitory effects on enteric, so high concentrations of animal tissue and yeast extraction are included to offset; differential for fermentation of lactose, sucrose, or salicin to acid-end products; differential: ability to reduce sulfur source (e.g. sodium thiosulfate to H2S; pH indicators: bromothymol blue and acid fuchsin dyes to indicate pH changes; enteric produce acid from fermentation produce yellow to pink colonies and salmonella and shigella do not ferment any sugars yet they break down animal tissue raising the pH and giving colonies a blue-green color; salmonella reduces sulfur to H2S so colonies formed are black; in medical settings used to isolate and differentiate Salmonella and Shigella from other gram negative enteric organisms in a patient’s stool sample; also used in identifying fecal contamination of dairy and poultry products
§ selective for gram negative, isolates Enterobacteriaceae members; differentiates between coliforms (that produce acid from lactose fermentation) and non coliforms; identifies sulfur reducers.
79
Which of the above agar types inhibit gram + bacteria?
Gram positive: MacConkey agar (without crystal violet MacConkey can grow gram positive), EMB
80
which of the above agar types inhibit gram - bacteria
PEA, HE
81
What can you predict about an organism if it does not grow on Phenylethyl alcohol agar?
That it is probably a gram negative organism
82
What does yellow growth on Mannitol Salt agar indicate
Mannitol is fermented and an acid is produced (this happens for S. aureus)
83
An organism grows well on MacConkey agar, but growth is colorless (not red or pink). What can you predict about this organism
The bacteria did not ferment lactose
84
What does dark purple to black growth indicate on Eosin Methylene Blue agar?
Lactose was fermented to acid end-products most likely by a gram negative organism (organism can be E.coli).
85
What does black precipitate indicate on Hektoen Enteric agar
Sulfur was reduced to H2S
86
Which media type’s primary application is the isolation of fecal coliforms
Eosin Methylene Blue
87
Which media type’s primary application is the isolation and differentiation of Salmonella and Shigella species from other Gram-negative enteric organisms in a patient’s stool sample?
Hektoen Enteric
88
What is the purpose of a streak plate isolation? / What is its application?
A mixed culture that produces individual colonies on an agar plate and portion of an isolated colony then may be referred to a sterile medium to start a pure culture
-identification process of an unknown microbe relies on obtaining a pure culture of that organism
89
After touching a culture with your inoculating loop, performing a streak on the first quadrant of your plate, and flame-sterilizing your loop, do you touch the culture again before performing the streak in the second quadrant? Why/why not?
No you do not touch the culture again before streaking the second quadrant because you are trying to spread the bacteria out. If you touch the culture again you would make the bacteria heavily condensed and the streak would not work.
90
What is an isolated colony?
A colony that is well separated from and not making contact with any other colony
91
How specifically does UV exposure harm and/or kill microorganisms?
When DNA absorbs UV radiation, the energy can form new covalent bonds between C-C, C-T, T-C to form pyrimidine dimers – thymine dimers are most common. The dimers distort DNA and interfere with replication and transcription.
92
Which type of UV is the most detrimental to bacteria: UV-A, UV-B, or UV-C
UV-C
93
What can influence the germicidal effect of UV exposure? (Think about the variables we tested in the lab exercises.
time of exposure, lamp intensity, and distance to the target (intensity diminishes by the reciprocal of the square of the distance); there must also be a “line of sight” to the surface being decontaminated.
94
In what applications would UV radiation be a useful germicide?
Disinfect laboratory and health-care environment work surfaces and surrounding air
95
In our lab experiments with UV exposure, why did we test both a 24-hour and 7 day culture of B. subtilis?
After the 7 day mark, endospores (the most resistant form of microbial life) appear
96
Describe light repair (photoreactivation)
DNA photolyase is activated by visible light and monomerizes the dimer and reverses the original reaction à E.coli can do this
97
Describe dark repair (excision repair)
The thymine dimer distorts the sugar-phosphate backbone, which is detected by UvrABC endonuclease that breaks two bonds (one is eight nucleotides in the 5’ direction from the dimer and the other is four nucleotides in the 3’ direction). A helicase (UvrD or helicase II) removes the 13 nucleotide fragment including the dimer and leaves a single-stranded DNA. DNA polymerase I will insert the appropriate complementary nucleotide in a 5’ or 3’ direction to make the molecule double stranded.
98
Which plates, those incubated in the dark box or those incubated on the sunlit windowsill, did we expect to see more growth in the region of the plate exposed to UV radiation? why?
The ones incubated on the sunlit windowsill Why? Because light repair is simpler as it only requires 1 enzyme opposed to the four in dark repair. Light repair is also shorter to happen.
99
Can UV penetrate the plastic petri plate lid? cardboard? paper?
no, no, no
100
Describe the disk diffusion (Kirby-Bauer) method for testing antimicrobials
Antimicrobial-impregnated paper disks are placed on an agar plate inoculated to form a bacterial lawn. The plates are incubated to allow the growth of the bacteria and time for the agent to diffuse into to agar. As the drug moved through the agar, it establishes a concentration gradient. If the organism is susceptible to it, there is a zone of inhibition, then the junction of the zone of inhibition and the growth (minimum inhibitory concentration) indicates how much or little the antibiotic must be. The disk diffusion does not quantify the MIC but uses the size of the zone of inhibition. If the disk kills the bacteria, it is bactericidal, if it stops them from dividing it is bacteriostatic.
101
What is the main application of disk diffusion?
Used to measure the effectiveness of antibiotics and other chemotherapeutic agents on pathogenic microorganisms. It is also essential to prescribe the right treatment for patients.
102
What kind of agar is used in the Kirby-Bauer method?
Mueller-Hinton agar
103
zone of inhibition
The clear zone around the disk where the concentration is high enough to stop growth
104
Define minimum inhibitory concentration (MIC)
The junction where the concentration of antimicrobic has become too low to effectively stop the growth.
105
The zone diameter below which all resistant strains fall is ...?
resistance point
106
The zone diameter above which all susceptible strains fall is the ...?
susceptibility point
107
Does a zone of inhibition around an antibiotic disk always mean that the organism is considered susceptible to that antibiotic?
No, it depends on the size of the zone of inhibition compared to the national standards for antibiotics as to whether the bacteria is susceptible
108
What are some mechanisms by which antibiotics work
The bacterial cell wall * The bacterial plasma membrane * Synthesis of bacterial proteins * Bacterial nucleic acids * Bacterial metabolism
109
What is the difference between broad spectrum and narrow spectrum antibiotics
Broad spectrum treats all kinds of gram + and gram – bacteria, which narrow spectrum treat either one or the other
110
obligate aerobe
organisms that require oxygen for aerobic respiration, the grow at the top where oxygen is plentiful
111
microaerophile
survives only in environments containing lower than atmospheric levels of oxygen.
112
capnophiles
survive only if Co2 is elevated
113
facultative anaerobe
Grow in the presence or absence of oxygen. If they are in oxygen they respire aerobically, if they are not in oxygen they will respire anaerobically (ferment).
114
Aerotolerant anaerobe
do not require oxygen to survive, but are not harmed in the presence of it.
115
obligate anaerobe
Organisms which even small amounts of oxygen can kill them. So they will only grow in the lower region of the medium, depending on where the oxygen has diffused into the medium.
116
Which of the list above would you expect to find growing at only the very surface of your fluid thioglycollate culture tube
obligate aerobe
117
For which of the list above would you expect to find growth throughout the whole tube, which was slightly more robust near the surface of the medium?
facultative anaerobe
118
Which term from the list above would apply to an organism with the growth pattern seen below?
microaerophile
119
Do aerotolerant anaerobes respire oxygen
no
120
What are the purpose of the resazurin in the fluid thioglycollate media?
The resazurin acts as and O2 indicator. It Is pink when oxidized and colorless when reduced
121
What is the primary application of fluid thioglycollate media?
Can be modified to promote growth in a variety of fastidious and non fastidious microorganismss. It can be used to illustrate microbial growth representing all levels of oxygen tolerance. Primary use is cultivation of anaerobic and microaerophilic bacteria
