Lab Exam Flashcards
Define ubiquitous
Always present, can be found everywhere
Briefly list the most important rules of aseptic technique
- Wear proper PPE (lab coats, shoes, goggles)
- Don’t work over your notes
- Clean bench before and after work
- Wash hands before and after work
- Properly label plates
- Discard lab equipment in their designated places
Where should agar plates be discarded?
Autoclave bucket at the front of the lab
Where should culture tubes be discarded?
Test tube rack near the sink
Where should microscope slides be discarded?
Used slide container near the sink
Where should micropipette tips and glass pipettes be discarded?
Biohazard disposal and pipette bucket, respectively
Where should clean broken glass and plastic be discarded?
Blue bucket
Where should contaminated glass and plastic be discarded?
Yellow bucket
Describe how you should label and prepare an agar plate for storage
Label agar side with:
- name/initials
- date
- treatment
- lab section
Wrap once with masking tape and place agar side up
List the things you must include on a scaled diagram
- use pencil on white paper
- do not shade
- drawing should be large and should only include one representative cell in detail
- provide simple drawings of surrounding cells to provide the cellular arrangement
- label identifiable structures to the right of the diagram using straight lines
- draw a scale bar in μm
What does one ocular division calculate to with 10x, 40x, and 100x?
10x: 1 ocular division = 10μm
40x: 1 ocular division = 2.5μm
100x: 1 ocular division = 1μm
How do you calculate the scale bar and magnification for a scaled diagram if your specimen is 5 ocular divisions under 100x and your drawing is 73mm?
5od = 5μm (label the scale bar 5μm)
Total Mag = 1000x
Mag of drawing = 73000μm/5μm = 14600x
What features must a diagram caption include?
- Figure #.
- shape
- full Latin name, if given
- gram reaction, if known
Should provide as much information as possible
Define microbial nutrition
Involves nutrient acquisition, transformation, and polymerization of complex macromolecules
What are macronutrients? List them
Nutrients required in large amounts; oxygen, carbon, nitrogen, and minerals (P, S, K, Mg)
What are micronutrients? List them
Nutrients required in small amounts; metal ions (Fe, Cu, Ni) and vitamins (thiamine, biotin, etc)
What is chemically defined media?
Consists of a basic salt solution (supplied inorganic nutrients), and organic compounds (energy and carbon) in which the exact chemical composition is known
What is complex media?
Consists of substances rich in inorganic and organic nutrients (meat/veggie infusions, blood, hormones) in which the exact chemical composition is not known
What is enriched media?
Loaded with nutrients and macromolecular monomers to promote growth of fastidious organisms
What is minimal media?
Contains only basic nutrients
What is autoclaving?
A sterilization technique that kills cells by putting them under conditions such as high pressure and temperature (15psi and 121°C)
Liquid media is called:
Broth
Solid media is called:
Agar
How do bacteria grow on agar vs. broth?
Colonies and turbidity, respectively
When one colony morphology is present on agar, this is called:
A pure culture
How many defining terms do we use for colony morphology? What are they?
6; shape/form, surface, elevation, size, pigment, opacity
What shapes may bacterial colonies come in?
Punctiform, circular, irregular, filamentous, and rhizoid
What surfaces may bacterial colonies come in?
Smooth, rough, mucoid, moist, dry
What elevations may bacterial colonies come in?
Flat, raised, convex, umbonate, crateriform
How is bacterial colony size measured?
In mm with a ruler
What pigments may bacterial colonies come in?
Non-pigmented (cream, white, beige), purple, red, pink, yellow, etc. May be water-soluble (dissolves into media)
What opacities may bacterial colonies come in?
Transparent or opaque
How many defining terms do we use for cell morphology? What are they?
4; cell shape, size, arrangement, and gram reaction
What shapes may bacterial cells come in?
Spherical (cocci) and rod-shaped (bacilli)
How is cell size measured?
Cocci: diameter in μm under 100x
Bacilli: length in μm under 100x
What arrangements may bacterial cells come in?
Random, pairs, tetrads, chains, clusters
How does a gram-positive bacteria stain?
Purple
How does a gram-negative bacteria stain?
Pink
What is a colony forming unit (CFU)? What number should you aim for when calculating the concentration of the stock culture?
Each colony on a plate is a CFU. You should aim for 30-300
What is selective media?
Media that contains compounds that selectively enrich/repress growth of certain organisms while not affecting the growth of others
What is differential media?
Media that contains an indicator that differentiates the occurrence of specific chemical reactions such that groups of bacteria can be differentiated from each other based on their ability to carry out that reaction
What are endospores?
Differentiated bacterial cells that form due to changes in the nutritional and physical environment of the organism
What are vegetative cells?
Undifferentiated bacterial cells (before it forms an endospore)
What is the Ames test?
A test used to screen chemicals to determine if they are mutagenic and potentially carcinogenic via measuring the rate of back mutations in auxotrophs
Describe how the Ames test works
A histidine auxotroph lacking DNA repair enzymes is exposed to a chemical agent and inoculated on histidine-deficient media. The rate of reversion to prototrophy is calculated by counting the number of colonies that grow where each colony represents a his—> his+ revertant
What does a positive result of the Ames test look like?
When there is more growth on the treatment plate than the negative control plate (negative control is spontaneous revertants)
What are the selective, differential, and macronutrient ingredients found in MAC agar? What colour is the agar?
Selective: crystal violet and bile salts
Differential: lactose
Macronutrients: peptones and yeast extract
Colour: darkish-red
What does MAC agar select for?
Gram-negative bacteria
How does MAC agar differentiate?
Lactose-fermenting (pink-brick red colonies) and lactose-nonfermenting (colourless)
What are the selective, differential, and macronutrient ingredients found in SF agar? What colour is the agar?
Selective: sodium azide
Differential: dextrose and bromescol purple
Macronutrients: peptones
Colour: purple
What does SF agar select for?
Gram-negative bacteria
How does SF agar differentiate?
Enterococcus (yellow) and Streptococcus (purple), where yellow indicates presence of acid (dextrose fermentation)
What are the selective, differential, and macronutrient ingredients found in MSA agar? What colour is the agar?
Selective: high [NaCl]
Differential: mannitol
Macronutrients: peptones and beef extract
Colour: bright red
What does MSA agar select for?
Gram-positive Staphylococcus
How does MSA agar differentiate?
Mannitol-fermenting (coagulase positive) turns media and colonies yellow. Negative results show small, red colonies and red medium. Micrococcus makes large, white-orange colonies and red medium
When acid-fast staining, what chemicals are used? What do positive and negative results look like?
Carbolfuchsin is used as the initial stain, then Brilliant Green is used as a counterstain. Positive organisms are red and negative are blue/green
When endospore staining, what chemicals are used? What do the results look like?
Malachite Green is used as the initial stain, then Safranin is used as a counterstain. Endospores are green and are located within the bacteria, and the rest of the cell is pink
When can we see bacterial growth in relation to their incubation temperatures?
When the bacteria are incubated within their cardinal temperature range
If you rank an organism “0” for growth, what does this mean?
No growth
If you rank an organism “1” for growth, what does this mean?
A few individual colonies or light growth throughout streak
If you rank an organism “2” for growth, what does this mean?
Broken/incomplete growth throughout streak
If you rank an organism “3” for growth, what does this mean?
Solid growth throughout streak
What are the three cardinal temperatures?
Minimum, optimum, and maximum
What is a psychrophile?
A bacterium that grows best at cold temperatures (<15°C)
What is a mesophile?
A bacterium that grows best at medium temperatures (15-45°C)
What is a thermophile?
A bacterium that grows best at high temperatures (45-80°C)
What is a hyperthermophile?
A bacterium that grows best at extremely high temperatures (>80°C)
What is an alkaliphile?
A bacterium that prefers high pH (>8), turbidity can be observed at high pH
What is a neutrophile?
A bacterium that prefers neutral pH (5.5-7.9), turbidity around neutral pHs
What is an acidophile?
A bacterium that prefers low pH (<5.5), turbidity at low pH
How do bacteria gain heavy metal resistance?
By acquiring plasmids that encode for enzymes that pump ions out of the cell or alter the ion’s oxidation state
What is a zone of inhibition?
A disk where no bacterial growth can be observed due to some agent killing them. The highest concentration the bacteria can withstand is located around the circumference
What is copper sulphate and its affect on bacteria?
Copper sulphate contains a heavy metal (Cu2+) that inhibits cell growth by inhibiting essential enzymes and binding proteins and DNA
What is the purpose of an oxidase test? Explain positive and negative results
Cyt c oxidase is an enzyme within the aerobic ETC that oxidizes cyt c. An oxidase test determines if oxidase is present (positive) or absent (negative) by using TMPD, an agent that turns blue when oxidized. A positive result is defined by the TMPD strip turning blue (oxidized) and a negative result is defined by the strip staying colourless (reduced)
What is the purpose of a catalase test? Explain positive and negative results
Catalase converts H2O2 into water and O2 gas. If the bacteria are catalase positive, they will produce bubbles when exposed to H2O2. If it is negative, no change will be observed. Sometimes, the catalase may take a while to produce bubbles, but if it does, then it’s positive
What are the four basic features of fermentation?
- occurs in the absence of oxygen
- no ETC
- less energy efficient than respiration
- metabolic intermediates are produced
What is assimilative nitrate reduction?
When nitrate is used as the final e- acceptor, the products (NO2) may be incorporated into amino acids
What is dissimilative nitrate reduction?
When nitrate is used as the final e- acceptor, the products (NO2) are expelled from the bacteria as gas
What is denitrification?
The ability of some bacteria to convert nitrate to N2
What is a deep shake culture used for? Describe the results you may see
Used to identify gas production at different oxygen levels. If gas is produced in the absence of oxygen (agar is cracked at the bottom), it is anaerobic. If it is produced in the presence of oxygen (cracked near the top or no cracks at all), its aerobic. If cracks in the agar are observed throughout the agar, the organism is facultative or aerotolerant
What is thioglycolate medium? Describe the results you may see
Used to identify the respiratory pathway of organisms. Aerobes will be located at the top, anaerobes at the bottom, facultative throughout but mostly at the top, and aerotolerant is located at a consistent concentration throughout
What colour is H&L medium? What colour does it change into when an organism is utilizing the glucose/lactose?
Green; yellow
What causes the change in colour in an H&L test?
Acid production from the metabolization of the provided sugar
If an H&L medium is only yellow at the top, and entirely green when sealed, what does this mean?
Obligate aerobe
If an H&L medium is only yellow if the medium is sealed, what does this mean?
Obligate anaerobe
If an H&L medium is yellow in both covered and uncovered tubes, what does this mean?
Facultative aerobe/anaerobe of aerotolerant
What do positive and negative H&L tubes look like?
Positive: yellow, negative: blue or green
If your litmus milk turns pink, what does this mean?
Acid production via lactose fermentation
If your litmus milk turns blue, what does this mean?
Alkaline production via partial casein digestion (makes ammonia)
If your litmus milk turns white, what does this mean?
Litmus is reduced by a fermentation reductase enzyme
If your litmus milk shows coagulation and a pink layer on top, what does this mean?
Media acidification (lactose fermentation) precipitates casein, and litmus reduction, as the casein is white
If your litmus milk loses opacity, turns yellow, and forms layers, what does this mean?
Peptonisation via casein digestion, leaving whey
Describe an MR test and what its positive and negative results look like
3-4 drops of methyl red are added to inoculated broth. If mixed acids are present, the low pH will result in a red colour (positive). If they’re not present, the high pH will result in a yellow/orange colour (negative)
Describe a VP test and what its positive and negative results look like
15 drops of a-naphthol followed by 3 drops KOH are added to the inoculated broth. If acetoin is present, it will become red (positive). If it is absent, the reagents will turn brown/copper (negative)
Explain why organisms can be MR + but not VP +, or vice versa
An MR + organism uses mixed acid fermentation, so it cannot use butanediol fermentation (must be VP -), and vice versa
What does a positive nitrate reduction test look like?
A bubble will form in the Durham vial and media may become turbid
What does a negative nitrate reduction test look like?
The Durham vial will not contain a bubble as the nitrates were not reduced. The media may still be turbid
Antimicrobial substances can be categorized into which groups?
- cidal (kill)
- static (growth inhibiting)
- lytic (lysing)
Antiseptics fall under which microbial substance category?
Can be all three (cidal, static, or lytic) but are safe to use on living tissue
Disinfectants fall under which microbial substance category?
Potent cidal or lytics that should be used on inanimate material due to their toxicity
What is the phenol coefficient?
The ratio of the test agent’s effectiveness to phenol’s effectiveness. The reciprocal effective dilution of test disinfectant / reciprocal effective dilution of phenol
If the phenol coefficient is >1, what does this mean? <1?
If greater than 1, it’s more effective than phenol. If less than one, it’s less effective
What is the effective dilution?
The dilution of agent that completely inhibits growth at 10 minutes exposure, but not at 5 minutes exposure
What is a Kirby Bauer Disc?
A disc containing an antibiotic used to determine the effectiveness of the antibiotic by measuring the zone of inhibition
What is a transposon (Tn)?
A genetic element which can move from one site on a DNA molecule to another site either on the same or on a different DNA molecule. May encode for antibiotic resistance
When is a transposon mutagenic?
When the insertion site is within a gene, which disrupts the linear continuity
Tn5 encodes for resistance to which antibiotic?
Gentamicin
Which three properties does plasmid pSUP102::Tn5-B22 contain?
- gentamicin resistance gene
- oriT
- E. coli specific origin of replication (unable to replicate after conjugated)
What is a suicide vector?
A vector that can only replicate within a specific cell. Once conjugated into a different species, it cannot be passed on
What is the multiplicity of infection (MOI)?
The ratio of phage particles to bacterial cells in a culture at a given point in time
What does having a low MOI assure?
Near 100% chance that every phage adsorbs to a different host cell
What is synchronized infection?
Adsorption and penetration occur simultaneously
What are the periods of phage infection?
Adsorption period, eclipse, maturation
Why is the MOI at 0 during the latent phase?
The phage progeny are within the host cell and are not planktonic. It increases when the cell lyses
What is the burst size?
The average number of new virions released from each infected cell
What is theoretical burst size? Why is this number almost always lower than observed burst size?
It is the number of phages we expect to be made from the bacterial DNA, calculated as bacterial genome size/page genome size. Almost always lower because the DNA isn’t the only source of nucleotides in the cell available for phage genome assembly
What is a plaque forming unit (PFU)?
The number of plaques on a bacterial lawn. Represents the number of phages within the sample
How do you calculate the frequency of transposition?
- Determine the average number of cells that underwent conjugation and transposition per mL
- Calculate the number of transposed cells in the total cell culture using the number calculated in step 1
- Calculate the frequency of transposition by dividing the total number of recipient cells (stock culture concentration) by the number calculated in step 2
- The number that you get tells you that 1 cell in every ______________ potential hosts is transposed
What is the frequency of transposition?
The amount of which cells are both conjugated and transposed
How do you calculate phenol coefficients?
- Determine the effective dilution of the test agent
- Determine the effective dilution of phenol
- Divide the reciprocal of the effective dilution of the test agent over the reciprocal of the effective dilution of the phenol
How do you enumerate a bacterial culture?
- Choose a plate with 30-300 colonies, count them
- Multiply by 1 (if 1 mL was used), 10 (if 0.1 mL was used), 100 (if 0.01mL was used), or 1000 (if 0.001mL was used)
- Multiply by the reciprocal of the plate dilution concentration
What does the natural lysis give us?
Generation time and burst size
What does premature lysis give us?
Eclipse, maturation, and burst size
What does a combination of natural lysis and premature lysis give us?
Generation time, eclipse phase, maturation phase, latent phase, and burst size
What is the eclipse phase? How do you determine its start and finish?
The end of adsorption and beginning of assembly. Can only be found by using premature lysis. When determining the end of adsorption, use the time point that last has bacteriophages present. Additionally, when determining the beginning of assembly, use the time point that first has phages present
What is the latent phase? How do you determine its start and finish?
The end of adsorption to the end of cell lysis. Using both PL and NL, you take the end of adsorption from PL and the end of cell lysis by NL, which is where the NL line plateaus
What is generation time? How do you determine its start and finish?
The beginning of infection (t = 0) to the end of cell lysis using NL
How do you calculate the assembly/maturation phase?
Latent phase - eclipse phase
What are the most important factors when writing the results from a test?
- observations for each treatment
- where it is in the tube
- what kind of result is this? (+ve or -ve)
- what does it indicate?
- what can you interpret from it? (respiration and metabolism of organism, denitrification if bubble)
If an organism is both catalase and oxidase positive, this means:
The organism is aerobic
If an organism is both catalase and oxidase negative, this means:
The organism is anaerobic
If an organism is positive for catalase and negative for oxidase, or vice versa, this means:
The organism is facultative or aerotolerant
