Lab Test Flashcards
When and how do you disinfect the lab station?
Before and after each session, wet counter with disinfectant and wipe over entire surface with a paper towel.
How long do you wash your hands?
15 seconds
What do you do when equipment is missing or not working?
Alert instructor immediately.
What happens if an accident or injury happens in lab?
Alert lab instructor, potentially fill out green notice of injury form, and give to environmental health and safety officer.
What’s the difference between risk group 1 and risk group 2?
Risk group 1: unlikely to cause illness and pose low risk. no safety measures.
Risk group 2: organisms that may cause illness in otherwise healthy individuals. Wear disposable gloves when handling, do not use open flame, and use disposable plastic inoculating loops.
Where do you put petri plates, gloves, glass slides, broken glass, pasteur pipettes, swabs, toothpicks, pipette tips, plastic pipettes, glassware when you’ve finished?
petri plates: biohaz
gloves: biohaz
glass slides: sharps
broken glass: sharps
pasteur pipettes: sharps
swabs: sharps
toothpicks: sharps
pipette tips: sharps
plastic pipettes: sharps
glassware: removed of labels, then wash trolley
What happens when you spill a bacterial culture?
- cover spill with paper towel
- squeeze disinfectant onto outer edges of the spill and work towards the centre
- let sit for 1 minute
- gather soaked towels and discard in garbage
- disinfect area again with fresh paper towels
What do you do if culture is splashed on skin, eyes, mouth, and clothes?
Skin: wash with alcohol, then soap and water
eyes: rinse thoroughly with water from tap or eyewash station
mouth: rinse with copious amounts of water, and report to instructor
clothes: disinfect with small amount of disinfectant and dried completely. wash clothes separately with detergent and bleach.
What does the WHMIS gas cylinder symbolize?
Gas under pressure
What does the WHMIS flame symbolize?
fire hazards
what does the WHMIS flame over circle symbolize?
oxidizing hazards
what does the WHMIS skull and crossbones symbolize?
cause death or toxicity with short exposure to small amounts
what does the WHMIS silhouette with branching heart symbolize?
may cause/suspected of causing serious health effects (carcinogen)
what does the WHMIS exclamation mark symbolize?
May cause less serious health effects, or damage the ozone layer.
What does the WHMIS biohazard symbol mean?
organisms or toxins that can causes diseases in people or animals
what does the WHMIS test tube spilling on metal and hand mean?
corrosive damage to metals as well as skin and eyes
what does the WHMIS explosion mean?
explosion or reactivity hazards
What are the magnifications of the ocular lenses and the objective lenses?
ocular: 10x
objective: 10x, 40x, 100x
what does parfocal lenses mean?
the user does not have to adjust the focus when changing the power of magnification
what does the iris diaphragm and condenser do?
Iris diaphragm: controls width of beam. open to see colour through moving a L to R lever.
Condenser: focuses light into concentrated beam. move condenser up and down using lever beside focus knobs
What differentiates prokaryote structure from eukaryote structure?
Prokaryotes are generally very small and have no visible organelles, whereas eukaryotes are larger and have a visible nucleus as well as visible cell components.
Can you tell the difference between bacteria and archaea on a microscope?
No, you can only differentiate them based on molecular and chemical makeup.
What are heterocysts?
specialized cells that carry out nitrogen fixation in cyanobacteria
How do you prepare a wet mount?
- obtain a clean slide and draw a circle in the middle with grease pencil
- place drop of culture onto slide within grease pencil circle
- place cover slip onto the drop of culture
What is the difference between fungi and bacteria on an agar plate?
fungi have long filamentous hyphae that appear fuzzy/powdery, whereas bacteria are small, and shiny.
How do you report size of a colony?
Measure diameter in milimeters
What are the types of colony shapes?
circular, irregular, filamentous, wrinkled, or punctiform
what are the types of colony elevations?
flat, raised, convex, pulvinate (semi circle), and umbonate (top of acorn)
what are the types of colony margins?
entire (smooth line), undulate (squiggly), lobate (lobe like), erose (sharp and pointy)
what are the types of colony textures?
brittle (crumbly), butyrous (like butter), membraneous (thin and cohesive), viscid (slimy)
Why do we use immersion oil?
oil has the same refractive index as glass and prevents it from bending to increase the amount of light and therefore resolution of the image.
What are the types of shapes of bacteria?
bacillus, coccus, spirillum, spirochete, appendaged, pleomorphic
What are the steps to perform the streak plate technique with liquid broth culture?
- Flame loop
- open tube and waft over pilot flame
- dip loop into tube
- waft over pilot flame and close tube
- lift petri plate lid
- streak agar at one angle 3 times
- flame loop
- turn plate and streak 3 lines
- flame loop
- turn plate and streak 3 lines
- flame loop
- drag loop once through third section and streak remaining space.
- flame loop.
What are the steps to perform the streak plate technique with colony from plate?
- Flame loop
- open petri plate and touch an isolated colony
- lift new petri plate lid
- streak agar at one angle 3 times
- flame loop
- turn plate and streak 3 lines
- flame loop
- turn plate and streak 3 lines
- flame loop
- drag loop once through third section and streak remaining space.
- flame loop.
Why do we perform the streak plate technique?
Diluting a sample as you spread it allows single cells on the surface of the agar. Single cells multiply and grow into isolated colonies, whose morphology can aid in identification.
What is the purpose of the standard plate count?
To estimate the bacterial population in the original sample.
What is the purpose of a serial dilution?
To dilute a bacterial culture to obtain a plate with a countable number of colonies.
what are the limits of the gibson mechanical pipette?
1000uL - 100uL OR 1mL - 0.1 mL
How to do a serial dilution + spread plate?
- Aseptically put a sterile tip onto a pipette
- Aseptically transfer 1mL of bacterial culture into a 9mL water blank
- Discard the tip and repeat step 2-3 until you reach desired dilution
- With fresh tip, take 0.1 mL desired dilution and place onto an agar plate.
- Sterilize glass hockey stick by dipping into alcohol and passing it quickly through flame
- Lift lid halfway and spread 0.1mL with hockey stick until it has been absorbed into the plate
- Immediately put hockey stick into alcohol and incubate plate.
How to do the standard plate count?
- count colonies (<30 and >300 is insignificant)
- calculate average titre using # colonies/(volume)(dilution)
- If there is more than one significant count, take the titres of all significant counts and average them.
How to prepare a bacterial smear from liquid culture?
- mix culture by tapping
- flame loop
- open tube and take a loopful of broth
- place loopful onto clean slide and flame loop
- repeat step 2 and 3 until culture is size of a nickel when spread out.
- flame loop and allow slide to air dry
- heat fix after air drying to get ready for staining.
What is the gram stain? How does each gram wall work?
Differential stain that differentiates bacteria into groups based on cell wall (gram -ve, gram +ve).
Gram +ve: alcohol dehydrates thick peptidoglycan and decreases pores, trapping the CV-I complex inside
Gram -ve: alcohol disrupts outer cell membrane which allows CV-I to be washed out of the thin peptidoglycan.
How does the KOH string test work?
3% KOH is added to a sample of bacteria. Gram negative cell walls are disrupted by the alkalinity of KOH and form strings of released DNA, while Gram positives have no effect.
What are the roles of each of the stains in the gram stain?
Crystal Violet: basic stain that is trapped in the thick peptidoglycan of gram positive, and is attracted to the negative outer surface of the cell of the gram negative.
Iodine: mordant; forms complex with CV to increase the intensity of the stain
Ethanol: shrinks pores of gram positive cell wall to trap CV-I, disrupts outer membrane of gram negative to allow CV-I to be washed out
Safranin: counterstain to colour the bacteria that were destained.
How to make a smear from solid media?
- Flame loop and place small loopful of tap water onto the surface of a glass slide
- Flame loop and take small amount of bacteria from plate and mix into the water, spreading it out into a dime shape
- flame loop and air dry the slide
- heat fix the slide over the pilot flame after the slide is completely dry
What are the steps of a gram stain?
- Add drops of crystal violet to smear and let sit for 1 minute
- wash off with tap water on the sides of the slide and blot dry
- add drops of iodine and let sit for 1 minute
- wash off without blotting dry
- Destain with alcohol (MAX 7 drops) and rinse with water
- counterstain with safranin for at least 10 seconds
- rinse and blot dry
What is the difference between gliding and flagellar motility?
Gliding: on a surface and involves pili/slime layer
Flagellar: due to chemo taxis; CCW rotation = run, CW rotation = tumble. More running, less tumbling upon attractant, more tumbling, less running upon repellent
What are methyl-accpeting chemotaxis protiens?
Sensors on the cytoplasmic membrane that are responsible for turning the environmental signals into flagellar rotation
What are the types of arrangement of flagella?
Monotrichous: singular flagella polar on one end, nonpolar anywhere else
Amphitrichous: two flagella on either end of the bacteria (spirillum)
Lophotrichous: Tufts of flagella; polar on one end, non polar anywhere else (spirillum)
Peritrichous: flagella that surround the cell (proteus genus)
How to view flagella under the microscope?
apply mordant to thicken flagella (usually tannic acid) and then add silver nitrate or methylene blue
How to prepare a hanging drop slide?
- Pick a colony from plate and suspend in tube of broth, placing it in 28ºC incubator for 45 mins for it to form flagella
- Draw circle on cover slip with grease pencil and add vaseline to 4 corners
- flame loop and transfer 1 loopful of broth in the middle of the circle
- Invert hanging drop slide over the cover slip so the drop is within the depression
- when finished, clean with disinfectant
How does a motility tube differentiate between non motile cells?
Motile bacteria grow outwards from the stab, whereas non-motile bacteria grow within the stab. Growth can be seen due to electron acceptor dye that turns red in presence of metabolically active bacteria
What is a capsule?
polysaccharide/protein layer with varying thickness and rigidity that assists in attachment to surfaces, protection against phagocytosis and resistance to desiccation
What are the steps of the capsule stain?
- place slide on double layer of paper towel
- aseptically transfer loopful of bacteria from plate to one end of a clean glass slide
- add 1 drop of crystal violet and use the loop to create a homogenous mixture
- add one drop of nigrosin and mix the two stains together with the loop
- use a second clean glass slide to scrape the stains across the surface of the slide, letting excess stain collect on the paper towel and let air dry.
What is an endospore?
Highly differentiated cells that are resistant to heat, harsh chemicals and radiation. They appear in the dormant stage of bacterial life cycle.
How do you perform a simple endospore stain?
- Prepare a smear of an old culture of bacteria
- simple stain the slide with safranin
- draw black lines across the slide with permanent marker
How to perform classic endospore stain?
- prepare smear of old culture of bacteria
- place paper towel on slide and flood with malachite green
- pass over flame
- rinse with water
- apply safranin as counter stain
What is an example of a bacterium that produces a capsule, what is its contribution to virulence?
Streptococcus pneumonia is a virulent, capsule-producing bacteria, which causes diesase. Their capsule prevents digestion from white blood cells.
How do cavities form?
Bacteria found within plaque produces acid as a result of carbohydrate fermentation. The acid removes minerals in the outer enamel creating holes that allow the bacteria and acid to reach the dentin of the tooth.
What are the microbiota of the mouth and their gram reactions and morphologies?
- streptococcus: gram positive, cocci in chains
- Fusobacterium: negative, single rods
- Borrelia: negative, spirochetes
- Actinomyces: positive, branched filaments
- Lactobacillus: positive, rods in chains
Why is the skin an unfavourable environment for bacteria?
It is dry and has a low pH
What are the two types of normal biota of the skin?
transient microbes: skin picks up from environment and unable to establish residence
resident microbes:thrive on skin and are mostly gram positive, which are better adapted to dry conidtions. They convert oils to antimicrobial unsaturated fatty acids to prevent gram negative and fungi to take over.
What is the main genus of gram positive bacteria found on skin and give two examples.
Staphylococcus epidermis: non-pigmented bacterium
Staphylococcus aureus: yellow colony and associated with pimples, boils and food poisoning. Antibiotic resistant S. aureus is rampant in hospital personnel and cause nonsocomial infections.
What is the purpose of mannitol salt agar?
Selective and differential medium used to find halotolerant staphylococci such as staphylococcus aureus, which turns bright yellow as it ferments mannitol.
What is the purpose of blood agar and an example of a bacteria that has a zone of clearing?
Enriched and differential medium used to differentiate hemolytic bacteria. Can either be gamma hemolytic (no clearing), alpha hemolytic (green zone of clearing) or beta hemolytic (yellow zone of clearing). Staphylococcus aureus is an example of a betahemolytic bacteria.
What are examples of members of the family enterobacteriaceae? What can they do?
escherichia coli, enterobacter, bacteroides, clostridium, enterococcus. Produce Vitamin K, stimulate immune system via competition, ferment lactose (coliforms)
Why do we use MacConkey Agar?
Differentiates between gram negative (can grow) and gram positives (cannot grow) and produces pigment when fermenting lactose. Extra strong if the bile salts spread.
What are the 10 macronutrients? Which make up the macromolecules? Which make up cofactors?
CHOPKNSCaFeMg
Macromolecules: CHONPS
Cofactors: KMgFeCa
What are the three classes of growth factors?
- amino acids (to make protein)
- purines and pyrimidines (to make nucleic acids)
- vitamins (organic cofactors for metabolism)
Why is agar added into the milk bottle separately from the solution?
Does not dissolve at room temperature.
How much is the volume of the milk jar?
approx. 100mL
What agar ingredient is added after autoclaving and why? How is it sterilized?
MgSO4 is added after autoclaving because it is heat sensitive and will precipitate at high temps. It is sterilized separately through membrane filtration.
How do you know how much of a solution of certain concentration you need for an agar recipe?
C1V1 = C2V2
C1: concentration of stock
V1: volume of stock needed
C2: concentration required
V2: volume of medium
concentrations must be in the same units, volumes must be in the same units
How does the autoclave work?
water is heated to boiling temp and steam is injected into chamber containing materials. Chamber is under pressure (15 pounds per square inch) which brings temperature of steam to 121ºC, killing cells and endospores within 20 minutes. Bottles with screwcaps must be loose and cannot be filled with liquids that boil over.
What is the laminar flow hood?
Wipe interior surfaces with disinfectant and flow of air from back of hood pushes out any contamination. Air is filtered with HEPA filter, which as 99.97% efficiency at removing particles 0.3um or higher.
What is the difference between defined/synthetic medium and a complex medium?
Defined: Medium where all elements are of known quantities (e.g. glucose salts agar)
Complex: exact components of the medium are not known (e.g. peptone agar, tomato juice agar)
What is an enriched medium?
Complex media with an added nutrient source to encourage the growth of fastidious heterotrophs (E.g. blood agar, chocolate agar)
What is a selective medium and two examples?
Has an ingredient that inhibits a type of bacteria, and supports the growth of the wanted organism
(e.g. Burks agar is nitrogen less, and contains molybdenum to support the growth of nitrogen-fixing bacteria. Mannitol salt agar has a high salt concentration which inhibits all bacteria except those that exhibit halotolerance)
What is a differential medium?
a medium that may or may not be selective that allows different kinds of organisms to grow but allows you to visually discriminate between different bacteria. This is based on the appearance of the colony, or the presence of a zone around the colony.
What are the requirements for a medium? What else can you add?
- MUST contain carbon source (unless autotrophic)
- MUST contain nitrogen source (unless nitrogen fixer)
- MUST contain inorganic salts.
- may or may not have agar for solid media
- may or may not be enriched with growth factors (e.g. peptone)
- may or may not have a selective agent
- may or may not have a pH indicator for differential medium.
What are the phases of growth on a bacterial growth curve?
- Lag phase: Bacteria are adjusting to their environment
- Log phase: exponential increase in number of cells, growth rate > death rate
- Stationary phase: growth rate = death rate; cell division is equal to cell death as nutrients and oxygen begin to deplete and toxic wastes begin to build up
- Death phase: initiates when growth rate < death rate due to complete depletion of nutrients and oxygen
Why do you use a semi-log scale on a growth curve?
Y axis is graphed as a log and x axis is graphed as arithmetic because growth rate is calculated linearly rather than from raw exponential data.
How do you use a spectrophotrometer?
- before using, make sure that correct wavelength for measurement is selected
- Take a blank cuvette and zero the machine
- Take a cuvette of culture and measure after zeroing it
What is the difference between the growth rate (k) and the generation time (g)?
growth rate (k): the number of generations that occur per minute/hour
generation time (g): amount of time it takes for one generation to occur
What are the formulas for growth constant and generation time?
k = (logA2-logA1)/0.301(delta T)
g = 1/k
What are the types of antimicrobials?
Alcohols, Quarternary Ammonium compounds (QUATs), Chlorine, Triclosan
How is alcohol an antimicrobial agent?
Strongly antimicrobial but do not destroy endospores. They act quickly and evaporate, but do not penetrate organic matter well. (E.g. isopropanol and ethanol)
How are Quarternary Ammonium Compounds antimicrobial agents?
Aka cationic detergents, strongly microbial against gram positive bacteria but not endospores. PEnetrate organic matter well and leave a film of disinfectant that can maintain killing power for hours after application. (e.g. benzalkonium chloride, benzethonium chloride, methylbenzethonium chloride, and cetylpyridinium chloride)
How is chlorine an antimicrobial agent?
Destroys bacteria, endospores, and viruses with excellent penetrating power, though they break down over time. (E.g. sodium hypochlorite)
How is triclosan an antimicrobial agent?
Phenol containing antimicrobial that has been doubted in value over the years.
Why is the disk diffusion assay unreliable to make comparisons between the antimicrobial effect of different agents?
Zone of inhibition is influenced by other factors besides the ability of the product to inhibit growth of bacteria. (e.g. hand creams dont diffuse into the agar well due to hydrophobic nature)
How does the pour plate method work?
- Perform a serial dilution until you have a concentration you want
- Aseptically take 1mL of the concentration and pipette into the petri plate
- Pour molten agar into the petri plate with the dilution inside and swirl on the counter with the lid closed
What are the digestive enzymes of bacteria?
- amylase: starch to maltose
- cellulase: cellulose to glucose
- gelatinase: protease that can hydrolyze gelatin
- caseinase: protease that can hydrolyze casein
What is the starch plate?
Agar plate with high concentration of starch that can be used to determine if bacteria produce amylase. Iodine binds to starch but not to maltose, so zones of clearing indicate production of amylase.
