Microbio 302 Quiz 1
What is the size of the microscope pointer used to determine the size of objects under the microscope?
10X pointer: 25um 40X pointer: 6um 100X pointer: 2.5um
Where is the closest fire extinguisher?
Inner lab by the inner hallway door
Why do you invert a petri dish when you incubate it?
It stops condensation from dripping onto the Petri lid and onto the agar surface. If it drips on the surface of the agar the bacteria move in the pool of liquid and won't form distinct colonies
Do slides that have been stained need to be autoclaved?
No, because the staining process kills the organisms
What is epidemiology?
The branch of public health concerned with identifying the factors that can influence the occurrence and spread of communicable diseases
What don't simple stains tell us?
There is no info on the cell wall structure of the organism
Why is it dangerous to create an aerosol by flaming a big loopful of bacteria?
This can cause the bacteria to become airborne
What is the equation to calculate the number of organisms per volume (conventionally 1mL) that were in the original sample?
# organisms/mL in original sample = (# colonies/plate) x (1/amount plated) x (1/total dilution) example: 219 colonies were counted on the plate made from 0.1 mL of the 10^-6 dilution: 219 x 1/0.1 x 1/10^-6
What are some examples of the benefits of bacteria?
- Bacteria degrade and recycle materials in the environment - add desirable flavors to our food - produce commercially valuable compounds such as antibiotics and other chemicals
What two mediums are used to grow bacteria typically?
- broth: a nutritious liquid - gelatin-like medium, which has been solidified by the addition of agar
What are the different forms of colonies based on their position in agar?
- surface of the agar: round and raised - imbedded into agar: lens-shaped - between bottom of the agar and Petri dish: thin, flat, and spreading
1 micrometer (um) is what in meters and inches?
10^-6 meters (one millionth of a meter) and 1/25,600 inch
What is the range of colonies for a countable plate?
30 to 300 colonies
Which lens should be in place when the microscope is stored?
4X
What temperature does agar liquify?
95-100 degrees
Why might it be more difficult to locate organisms from a smear that was created using a broth?
A broth is generally less concentrated than one from a solid culture so it may be difficult to locate organisms
What is another name for a gram stain and why?
A gram stain is also called a differential stain because it shows the differences in the cell wall structure of bacteria, aka gram positive and gram negative cell walls
Euglena
A motile alga Observations: Saw green formations that were changing between blogs and narrow shapes. Found them to be around 5um
Why is agar used as part of a medium for growing bacteria?
Agar doesn't provide any nutritents, but it is a great solidifying agent due to its low melting temperature and a relatively low solidifying temperature.
What is the temperature you want to cool agar?
Around 50 degrees. If the temperature drops to less than 45 degrees the agar will solidify.
Why must you wipe the oil off the 100X lens at the end of each lab period?
Because the oil would get cakey and is not good long term on the lens
Brownian motion:
Brownian motion is exhibited by all small objects in liquid media - motion due to bombardment by atoms and looks like random vibration
Why do you melt agar to do a plate count?
By doing so, the bacteria being counted are trapped in position on or in the agar medium and can grow into a visible colony
What does a darkfield stop do?
Darkfield illuminates the object from the side like a sunbeam illuminating dust. The brightness needs to be all the way up and the iris diaphragm needs to be completely open
What is the first step in identification of an unknown bacterium?
Determine the shape and Gram stain reaction of the organism. However, this is not enough info to tell you the identity of a bacteria
Aseptive technique: transferring broth with a wire loop and pipette
Draw loop into flame by starting near the handle then finally to the tip --> Let loop cool for 12 seconds --> open up broth 1 without putting down the cap --> then put loop into broth without touching the edges and pick up a little circle of broth --> close the bottle and then open up the broth 2 without putting down the cap --> with limited loop touching any surfaces gently tap the loop against the side to release the drop and place broth 1 contents into broth 2 --> cap broth 2 and put the loop through the flames like you did in the beginning. Do the same thing but instead with pipette
What is the possible effects of evaporating water from the wet mount?
Evaporating water could cause currents which can look like motile bacteria, except everything would be moving in the same direction
Do you think there will be more organisms on your finger or in your mouth?
I think there will be more organisms in your mouth due to the body temperature and moisture within your mouth. Your finger is a skin layer that can be a harsher environment with a dryer environment and less nutrients.
If more than 300 colonies are present why do you not utilize this plate count?
If more than 300 colonies are present, errors are more likely and it was too crowded for optimum growth conditions and all organisms plated may not have formed visible colonies. If less than 30 colonies, the sampling error is too great
What are two reasons why proper aseptic technique should be used at all times when handling microorganisms
It maintains sterility of the specimen and limits contamination in the specimen the the outside environment
What is one big message of the ubiquity of microbes that is relevant to lab?
It shows how easy it is to contaminate sterilized media, sterile instruments, or your laboratory bench with unwanted bacteria
Will there be the same kinds of bacteria on the washed hand as on the unwashed hand?
No, there will be differences and possibly microbes from deeper in your pores after washing because washing can remove the microbes closer to the surface
Why are plastic Petri dishes discarded into an autoclave bag, while glass tubes with plastic caps are discarded directly into the bin?
Petri dishes are not reusable and will be thrown away while the glass materials are reusable (?)
What are the different sizes of Protozoa, Yeast, Bacteria, and Viruses
Protozoa: 20-100 um Yeast: 4-10um Bacteria: 1um Virus: 0.1 um (too small to be seen with a light microscope)
What temperature were the aseptic technique tubes incubated?
Room temperature (22 degrees celsius)
Steps of Period 1 isolation and identification of pure cultures from mixtures of bacteria
Select a broth and record the broth number --> swirl the turbid suspension and do a gram stain --> streak the broth suspension onto each of the 2 TSY plates (each partner does two so in total you will have 4 as a group) --> invert and incubate at 37 degrees (body temperature) --> examine gram stain under oil immersion --> if time permits, prepare a wet mount of the suspension and examine for motility
Why do you need to expose the plate to air for 30 to 60 minutes in the ubiquity lab?
Some bacteria that are aerobic may need oxygen to survive
Why is it important to mix a culture before taking a sample?
Some organisms have a tendency to sink in the broth
What is the main purpose of stains and special stains
Stains make microbe shape and grouping more visible. Special stains are used to demonstrate the presence and/or localization of specific structures
plate count steps for Saliva
Steps while agar is melting: Properly label all dilution tubes and plates before beginning and make sure to thoroughly mix samples before the next dilution --> spit into a Petri dish until you have about 1mL amount --> Put 1mL into a 9mL blank (this is a 10^-1 dilution) and mix --> Put next 0.1mL into a 9.9 mL blank (this is a 10^-3 dilution because 10^-1 x 10^-2=10^-3) --> After mixing put the 0.1mL into another 9.9mL blank (this is a dilution of 10^-3 x 10^-2 = 10^-5) --> plate 0.1mL and 1mL of the solution into two different Petri dish after each 9.9mL blank dilution --> pour a tube of melted agar into each of the four Petri dishes, mix, and wait for it to harden --> incubate plates at 37 degrees (body temperature)
How do you know if bacteria has grown in a broth?
The broth would look cloudy, also called turbidity
What is the purpose of the epidemiology experiment?
The experiment is supposed to demonstrate how an epidemiologist might track the spread of a communicable disease back to its original source
define parfocal
The lenses of the microscope can stay in focus even if the magnification changes. This is why usually only a fine focus change will be needed between magnifications
objective lenses
There are four of these, 4X, 10X, 40X, 100X - These magnify 4-fold, 10-fold, 40-fold, and 100-fold respectively
Hay infusions
These were obtained by collecting water from the Montlake cut or other sources and adding a few grains of barley and some hay. The samples contain bacteria and various protozoa, which are great fun to observe Observations made: Most likely protozoa around 12um that had flagella and moved with direction and in circles. We also noticed Brownian vibrations
Why are wet mounts useful?
They are a quick way of observing organisms to determine size, morphology, grouping, and motility.
What must be done to all contaminated materials before they are discarded into the regular garbage
They have to be kept separate and be autoclaved
Is it possible to design a medium that will grow all bacteria in a mixed sample?
This is very unlikely. It is difficult to meet all the nutrient requirements and other requirements such as temperature and oxygen. Therefore, plate counts don't necessarily count the actual complete population of bacteria in a sample
What is the benefit of closer together streak lines in a streak plate?
This means the inoculum will be more thoroughly diluted
What is the purpose of a streak plate?
To obtain pure cultures and can use it to differentiate isolates and begin to identify them
What is TSY?
Trypticase soy yeast: made up of trypticase peptone (a digest of casein), an enzymatic digest of soybeans, and yeast extract. They provide nutrients for growth of a lot of different microorganisms
How do you prepare a culture from a broth medium?
Use sterile loop that's been cooled to transfer a droop of culture to the slide --> don't spread the drop. If you add tap water that will make the culture too dilute --> slide warmer
If you have multiple plates, how do you use both colony counts?
Use the average of the number of colonies on the plates for the calculation
How should the microscope be carried
Use two hands and hold the bottom and hand hold. Hold it away from your body
ocular lenses
What your eyes look through like binoculars - magnify the object 10-fold
What do you write if you count over 300 colonies in a plate count?
Write that there are Too Numerous to Count (TNTC)
Will the freshness of food influence the number of bacteria on its surface?
Yes, because food that has been processed has had more chemicals and has been at higher heats which both can kill of certain microbes
How do you determine the identity fully of a bacteria?
You determine the metabolic capabilities of the isolate through a series of biochemical tests
Steps of the epidemiology experiment:
You will get a pencil with an ID, write down the ID number on your data sheet --> Put a latex glove on your non-writing hand --> only touch the pencil with the latex glove and make thorough contact with the pencil and the latex glove --> Put the pencil back in its tube --> one of the student's pencil is contaminated with Serratia marcescens --> shake hands with 4 different people --> keep track of their names, pencil ID numbers, and the order in which you came in contact with each individual (wait for TA's go ahead to shake each person's hand) --> After shaking hands with 4 people rub the palm and fingers of your glove on the surface of a TSY agar, don't break agar surface --> remove glove without touching the outside and place in biohazard waste container by the door --> return pencil and wash your hands --> incubate plate inverted at 22 degrees (room temperature)
why is gelatin no longer used as commonly as a solidifying agent?
because of gelatins low melting temperature, it is liquid at the common incubation temperature of 37 degrees celsius (body temperature)
Where do you disposed of plastic pipettes and swabs?
biohazard waste buckets
Where is the laboratory safety information kept
by the inner hallway door of the main lab (?)
Describe the cord and the stage on a microscope properly prepared for storage
cord is fully wrapped up in the back of the microscope and the stage is all the way down
Simple stain methods
cover the surface of a dried fixed smear with any basic aniline dyes (crystal violet, methylene blue, safranin, and malachite green). --> wait for 30-60 seconds --> wash off stain with tap water --> blot gently with paper towel --> wait for it to fully dry --> examine under oil immersion
Equation for finding the dilution
dilution = amount added / amount added plus the amount of diluent examples: diluent: 9mL blank amount added: 1mL 1/1+9 = 1/10 = 10^-1
The most common repair is to fix bent or out-of-alignment stage mechanisms. What then is the most important consideration when putting away your microscope?
don't bump the slide holder/stage mechanism on the locker
Where do the bacteria on your hair originate?
from your environment (?)
How do you adjust the light so that objects in the fluid are observable?
increase contrast by closing the iris diaphragm then lower the light intensity. The iris diaphragm controls the contrast
Where is the closest shower
inner hallway to the right, outside the inner lab
Where is the closest eyewash
main sink in the front
Yeast
microbes used to make bread and alcoholic beverages. Look for bud formation, which characterizes yeast
How do you find the actual magnification of an object?
multiple the ocular lens power to the power of the objective lens at use. For example, the maximum is 1000X (10X x 100X)
How do you determine the total dilution?
multiply each individual dilution (aka add the exponents) to obtain the total dilution. Example: dilutions are 10^-2, 10^-1, and 10^-1 10^-2 x 10^-1 x 10^-1 = 10^-4
What are some good things to look at when observing bacteria in a petri dish?
size, color, consistency, edge, and elevation
How do you prepare a culture from a solid medium?
sterilize loop, cool it --> place drop of tap water on slide --> sterilize loop, cool it --> lightly touch culture to remove small amount --> disperse in water so it is faintly turbid --> spread out about the size of a dime --> mark on the other side with sharpie where your smear is --> place smear on slide warmer (56 degrees celsius)
What are the steps to a gram stain?
sterilized/cool loop used to drop tap water onto slide --> sterilize/cool loop and place specimens of interest into the tap water --> dry on slide warmer --> Once dried cover smear with crystal violet and leave for 30-60 seconds --> wash dye off with tap water --> gram positive and negative bacteria should now be purple --> Then rinse with Gram's iodine and cover the smear in it for a few seconds to a minute or longer --> wash off with water --> the iodine is a mordant (fixer) that reacts with the crystal violet --> both gram positive and negative bacteria should appear purples --> Now critical timing piece is adding 95% ethanol for 20-30 seconds --> the ethanol is an alcohol that dehydrates bacteria. However, the G+ have lots more peptidoglycan that traps in the color but G- only have a small layer so color is released. --> rinse slide in water --> gram positive bacterial should stay purple and gram negative bacteria should be colorless --> counterstain with safranin for 20+ seconds --> the gram+ organisms stay purple and gram-negative turn pink --> wash off with tap water --> blot and let dry
How to made a streak plate
swirl broth culturally slightly to mix it --> get a loopful with a sterilized/cooled loop --> streak loop over 1/3 of the agar plate --> sterilize/cool loop --> streak through the first 1/3 several times and then without going back through the first 1/3 streak through another new 1/3 of the plate --> do the same thing for the final 1/3 of the dish. Incubate the plate at 37 degrees (body temperature)
why shouldn't the 40X lens be passed through the oil?
the 40x lens is not made to be placed in the oil immersion and does not need it
coarse focus
the bigger dial that changes focus
iris diaphragm
the dial right under the stage that limits the light entering
condensor lens
the glass directly under the slide
fine focus
the smaller dial that changes focus more incrementally than the coars focuse
What is the cotton for at the top of the plastic pipettes?
to filter incoming air and to partially prevent material from flowing over the top
Why should you disconnect the hose at the end of the lab period?
to make sure gas isn't leaking. The gas will smell like sulfur so you should notice if it's leaking
where do you dispose of the disinfectant-soaked paper towels that you use to swab your desk?
trash
where do you dispose of the wrappers from pipettes and swabs?
trash
when should you use the pilot light rather than full flame?
when you are not currently using the flame but will need to keep using the Bunsen Burner
Can you place your finger on agar to test the microbes on the agar?
yes
