Lab Exam Flashcards
3 types of hazards in the lab:
- Microbial hazards
- Chemical hazards
- Physical/mechanical hazards
Define “biohazard”
microbial infectious agents or other biological materials that present a risk or potential risk to the health of humans or animals
Describe a microorganism that is classified under Risk Group 1.
One that is unlikely to cause human/animal disease
Describe a microorganism that is classified under Risk Group 2.
One that MIGHT cause human/animal disease, but is unlikely to be a serious hazard.
Lab exposure may cause mild to moderate illness > effective treatments and preventative measures are available
Our lab is a Containment Level ___ lab
2
What is one of the greatest risks of working in a microbio lab?
Accidental exposure to aerosolized suspension of microorganisms.
Define “sterilization”
Complete destruction/removal of ALL microorganisms by chemical or physical means
Define “disinfection”/”decontamination”
The destruction of specific types of organisms, usually by chemical means
Diff b/w sterilization and disinfection?
Sterilization: complete destruction of ALL microorganisms
Disinfection: reduces the amt of microorganisms on a surface
Purpose of WHMIS?
To reduce the incidence of illness and injury caused by hazardous materials in the workplace
3 key elements of WHMIS?
- Labels
- MSDS’s
- Worker Education
What is brightfield microscopy?
Microscopy where a dark image is observed on a bright background
Two types of lens are utilized in compound microscopes:
- ocular lens
2. objective lens
What’re the three objective lens magnifications? Which lens can be used w/ oil immersion?
10x, 40x, and 100x
100x = oil immersion
Total magnification = ?
Total magnification =
ocular magnification x objective magnification
Define resolution (microscopy).
The ability of a microscope to distinguish two objects as being separate and distinct if they are v. close together.
Essentially, it’s a measure of how clearly objects can be seen.
How does oil immersion improve resolution?
It reduces light from scattering > more light passes through specimen > improves resolution
Gram stain rxn, cellular morphology, and cellular arrangement of Staphylococcus aureus?
gram positive, coccus, and clusters
Gram stain rxn, cellular morphology, and cellular arrangement of E. coli?
gram negative, bacillus, and singles
Gram stain rxn, cellular morphology, and cellular arrangement of Bacillus cereus?
gram positive, bacillus, chains
Gram stain rxn, cellular morphology, and cellular arrangement of Streptococcus pyogenes?
gram positive, coccus, chains
What colour do gram positive bacteria stain?
blue to purple
What colour do gram negative bacteria stain?
pink to red
What type of stain is used for Mycobacterium sp.?
acid-fast stain
Are E. coli motile?
Yes, E. coli are motile (they have a true direction of motion)
What’s the purpose of a hanging drop?
To observe living bacteria in their natural habitats, and to observe features of living microbes (such as motility)
Purpose of staining bacteria before observation?
increase contrast b/w bacteria and the background > allows for easier observation of bacteria and special structural features
Purpose of air-drying and heat fixing of bacteria on slide? (3)
- Kill bacteria > no infection risk
- Bacterial surface accepts stain more readily
- Bacteria become firmly stuck to glass surface > less likely to be washed away
More gram positive or negative cells found in oral cavity?
More gram positive bacteria
When gram staining an oral cavity specimen, large, flat, pink-staining material are also seen. What could these be?
- Plaque
2. Epithelial cells
Why’s staining needed in brightfield microscopy?
Microscopic organisms = transparent > staining contrasts bacteria w/ the background of the slide
When would you use a simple stain (methylene blue)? What’s an advantage of the simple stain?
When you only want to know whether organisms are present, and their shapes
Advantage of simple stain: It’s simple!
When would you use a gram stain?
When you want to know whether there’re gram positive or gram negative bacteria present
What can be seen in a hanging-drop that can’t be seen in a stained prep?
- Living organisms
2. Motility
Why do we discard stained slides and hanging drops in a disinfectant soln?
They contain biohazardous material > potential danger to lab demonstrators
Why don’t we perform a spore stain on E. coli or S. aureus?
These two bacteria do not form spores
What bacterium forms spores?
Bacillus cereus
Which microscope objective is most satisfactory for studying bacteria? Why?
100x oil immersion bc it allows for the best visualization of bacteria
Purpose of heating and steaming during the spore stain?
Drives spore stain into spore coat (which is usually v. impermeable under normal conditions)
What is agar?
solidifying agent which provides a physical support for bacterial growth - it’s not used nutritionally by bacteria
In order to grow bacteria, what must be understood about them?
Their nutritional needs
Define autotroph bacteria.
Those who obtain their carbon from inorganic sources
Define heterotroph bacteria.
Those who obtain their carbon from organic sources.
4 categories of culture media:
- Basic media
- Enriched media
- Selective media
- Differential media
This category of media contains additional components (e.g. blood, serum, or meat infusions) due to the need to support bacteria who have special nutritional needs.
Enriched media
This category of media has substances added to it in order to inhibit certain bacteria, while still allowing the growth of others.
Selective media
This category of media allows all bacteria to grow, but has certain substances added to it which allows the differentiation of one species of bacteria from another.
Differential media
Mannitol salt agar has a high salt conc, and inhibits the growth of most bacteria except _____
Staphylococci
What is MacConkey’s agar used for?
To isolate Enterobacteriaceae away from other bacteria in mixed pops (e.g. stool samples), and to differentiate b/w lactose fermenters from non-fermenters (thus, MacConkey’s agar = selective AND differential)
In nitrogen-free agar, where do bacteria get their nitrogen from?
The air
Define “pure culture”
a pop of bacteria consisting of only a single bacterial species
How can you tell whether or not there’s bacterial growth in a tube of nutrient broth?
If it’s cloudy, then there’s growth
Why must the loop be cooled before touching it to a culture of bacteria? (2)
- Avoid aerosol formation
2. Avoid killing the bacteria
Why should we invert the petri plates during incubation?
Less chance of plates drying out
How could you tell whether a colony on a streaked plate was a contaminant?
- If the colony grows outside of streak lines
2. For pure cultures, each colony should be the same > if diff colony appears, then it’s a contaminant
What would happen if the streak plates had been incubated for a week at 37ºC instead of only 24 h?
The bacteria would’ve grown over that time into larger colonies (though not indefinitely)
Two ways of measuring bacterial numbers?
- Bacterial suspension turbidity (absorbance)
2. Plate count method
T or F: Higher turbidity and absorbance is correlated w/ lower bacterial cell numbers.
F
It’s correlated w/ HIGHER bacterial cell numbers
Disadvantage of measuring the turbidity of bacterial suspensions w/ a spectrophotometer?
Can’t differentiate b/w living and dead cells
If 0.1 mL of dilution is plated and 35 bacteria are counted on the plate, how many organisms were in the original solution (assuming a final dilution factor of 10^-6)?
35 * 1/0.1 * 1/(10^-6) = 3.5*10^8 cells/mL
Higher absorbance = ?
higher cell #
T or F: It’s possible for a suspension of bacteria to have a high absorbance value yet give zero CFU/mL on a plate count
T
Dead bacteria can block light
Why might pour and spread plating give diff CFU/mL values?
Pour plating allows anaerobes to grow in addition to aerobes, hence it’s expected to result in higher CFU/mL values
What’s plate count agar? What’s it usually used for?
It’s a non-selective nutrient media used for enumerating aerobic bacteria
What’s MacConkey agar? What can it be used for?
Selective media for gram negative bacteria (due to bile salts and crystal violet).
Differential media as well > lactose fermenters (pink or purple colonies) from non-fermenters (no colour change)
It can be used for determining a coliform count
What does a high coliform count indicate?
Fecal contamination of meat
What’s an “indicator organism”?
It indicates that there’s bacterial contamination of food (e.g. coliforms indicate fecal contamination)
Equation for calculating the number of organisms per mL in the original sample?
= # of colonies on plate * 1/vol of sample plated * 1/final dilution factor
In a soil envir, how might the production of an antibiotic benefit the producer organism?
Less competition > Uninhibited growth
Why was it necessary to use a 10-day old culture (as opposed to a fresh 24h culture) of the Penicillium, Bacillus polymyxa, and Streptomyces in order to demonstrate the production of antibiotics?
Antibiotic production is a secondary metabolism that occurs only when access to nutrients is reduced
Thus, as cultures get older, antibiotic production increases
What vars might alter the zone of inhibition around an antibiotic disc in the disc diffusion assay?
- Too few or too many bacteria plated
2. Different incubation temperatures
What does it mean when colonies are observed growing within the zone of inhibition in a disc diffusion assay?
It means that these cells are spontaneous mutants that’re resistant to the antibiotic
Despite the tube dilution test for antibiotic sensitivity being more accurate than the diffusion method, what is a major advantage of the disc diffusion method?
It’s MUCH easier to do!
What does it mean when the minimum bactericidal and minimum inhibitory concentrations are the same?
The antibiotic is bactericidal
What does it mean when the minimum bactericidal and minimum inhibitory concentrations are different?
The antibiotic is bacteriostatic (at lower concentrations)
Is tetracycline bactericidal or bacteriostatic?
Bacteriostatic
Is penicillin bactericidal or bacteriostatic?
Bactericidal
Compare and contrast b/w sterilization, disinfection, and antisepsis.
All three = reducing viable microbes on/in s.th.
Sterilization: destroy ALL microbial life and spores
Disinfection: reduce level of microorganisms on an inanimate surface (maybe spores too)
Antisepsis: reduce level of microorganisms on living tissue
Which is more effective at killing bacteria and spores - autoclaving (moist) or dry heat?
Autoclaving (moist heat)
How does pasteurization affect bacteria?
How would the effects of pasteurization differ b/w pure culture broths and milk?
Pasteurization doesn’t completely kill all the bacteria.
In milk, there’re organic materials that can protect bacteria, and so even less would be killed in milk compared to a pure culture broth.
Would B. cereus or E. coli be more sensitive to heat?
E. coli would be. B. cereus is more resistant due to it being a spore-forming bacterium.
How does the length of time an organism is exposed to the disinfectant affect how readily it’s killed?
Longer = more killing, but more toxic
Are disinfectants designed to be more or less potent than antiseptics? Why?
They are designed to be MORE potent bc they’re meant to be used on non-biological surfaces
Why is a glass rod that is coated with nutrient broth bacteria not a good representation of what might be encountered in real-life settings, and hence not represent a disinfectant’t true effectiveness in the real-world?
Bc in the real-world, bacteria are usually present along with other organic matter (e.g. in feces, blood, etc.)
Why does the Use-Dilution test used for disinfectants NOT mimic real-life conditions?
Bc we exposed the bacteria to disinfectants for a much longer duration than usual
Define “oligodynamic action”
The ability of small amts of heavy metals to exert a lethal effect on bacteria
Which metals are effective at inhibiting bacterial growth?
Zn and Ag
Which metals are ineffective at inhibiting bacterial growth?
Fe, Mg, Cu, Pb
Does oligodynamic action also apply to fungi or viruses? Why or why not?
Yes bc heavy metals act against proteins > viruses and fungi also have proteins that can be disrupted/denatured
Resident flora are…
bacterial species that’re permanently colonized in or on some part of the body
Transient flora are…
bacterial species that’re present for only hours or days in or on the body
Why might washed hands have MORE bacteria than unwashed ones?
Warm water brings resident bacteria to the surface from the pores
Are antimicrobial soaps or Isagel more effective at removing bacteria than reg soap?
They should be due to added chemicals being used
Can our experiments that dealt with hand washing’s effects upon bacteria tell us anything about how effective these treatments might be against other microbes, such as fungi or viruses?
No
How would increased time exposed to UV light affect bacteria?
It would be more lethal
Which species of bacteria are more sensitive to UV?
Which are less sensitive? Why?
More sensitive: E.coli and Staph. aureus
Less sensitive:
Mycobacterium (due to its mycolic acid that prevents penetration of UV rays)
B. cereus (due to being a spore-forming bacterium)
Why’s it necessary to remove the lid from the plate when exposing it to UV?
UV cannot pass through ordinary plastic or glass
A solution that’s been passed through a bacterial filter is “sterile” (in quotes). What does this mean?
The solution is free from bacteria, but NOT from smaller organisms and viruses
Describe an experiment, using only bacterial filters and nutrient agar plates, that would allow you to estimate the approximate size of a bacterial cell.
- Have different filters of diff sizes
- Filter bacteria through each of them
- Plate each of the filtrates
- If there’s no growth, then we would conclude that the bacteria are larger than the diameter of the particular filter used
Predominant organisms in throat cultures?
Streptococcal species
What’s the purpose of the “Catalase Test”?
To tell the difference b/w Staphylococcus and Streptococcus species of bacteria
What are the 3 possible hemolysis rxns in blood agar? Describe each.
- alpha hemolysis: partial breakdown of RBCs in the agar > media turns greenish around the colonies
- beta hemolysis: complete breakdown of the RBCs > complete clearing of media around the colonies
- gamma hemolysis: no change/no rxn on the media
Which species is catalase negative? Which species is catalase positive?
Catalase negative: Streptococcus species (no gas production)
Catalase positive: Staphylococcus species (gas production)
Why would a gram stain not be useful for stool samples - why?
There’re thousands of bacterial species in feces > impossible to tell the diff b/w pathogenic and normal bacteria under a normal gram stain
What type of stain is used on blood to visualize white blood cells? What’s it composed of?
Wright’s Giemsa stain
It’s made up of methylene blue (basic) and eosin (acidic)
5 types of white blood cells?
- Basophils
- Eosinophils
- Monocytes
- Lymphocytes
- Neutrophils
When observing the 5 diff types of white blood cells, some of them weren’t seen (or not seen as much). Why?
Some of the WBCs are lower in percentage in the blood compared to others
Define serology
Study of antigen and antibody rxns in vitro
Describe agglutination rxns
A rxn used to demonstrate an antibody-antigen interactions
It involves antibodies interacting w/ antigens, forming an immune complex consisting of many cells cross-linked to e/o by antibody molecules > eventually, this cross-linking is able to be seen w/ the naked eye
Suppose you suspected that a pt had a Salmonella infection. How would you test for this?
Use an agglutination rxn by using the pt’s serum and anti-Salmonella antibodies
Media most commonly used to isolate and grow fungi? What specific characteristics allow it to select for fungi?
Sabouraud’s agar
It has reduced pH (inhibits bacterial growth)
It has a high level of dextrose (a sugar that’s readily fermented by fungi)
What dye is used for fungi?
Lactophenol cotton blue dye
If a fungus w/ hyphae is observed, we’re dealing w/ a _____
mold
If a fungus reproduces by budding, then it’s a _____.
yeast
