Nt1330 Unit 2 Lab Summary
Lab Practicum, Micro 200, study guide
When you did your hand wash experiment, you decided a variable to test, say, hot vs cold water, you called this variable an “experimental variable” since it was the variable you were testing experimentally. In order to get meaningful data, you need to keep other things the same, for example the washing time had to be the same for both, you called the washing time a _Control_______ variable.
Through the semester, you have used two bacteria that produce colorful colonies: one produce yellow colonies, another one pink. What are the names of these two bacteria? Are they Gram+ or Gram-?
Micrococcus Luteus, yellow colonies, gram +
Serratia Marcescens, pink colonies, gram -
There was one bacterial …show more content…
species for which the colonies were waxy, thus you had to be very patient when making smears to do staining, what is the name of this bacteria?
Mycobacterium
Different types of agar plates are used to culture different types of microbes: What (microbes) is Sabourand agar used for? NA,TSA and Blood agar plates are all used to culture bacteria, but the costs are very different, when do you use the cheapest one (NA), the more expensive one (TSA), the most expensive one (Blood agar)?
Sabourand agar: fungus, collects bacteria from the air, selective medium.
NA: Bacteria in things (eg. table, floor, door knob)
TSA: Bacteria outside our bodies (hands, fingernails)
Blood agar: Bacteria inside our bodies ( throat, blood)
Plates are used according to bacterias natural habitat.
The temperature at which you incubate your plates is important, what temp would you incubate microbes harvested from your desk top? From your tongue?
Desk top: room temp, 25-30 celsius
Tongue: body temp, 37 celsius
What is a wet mount? What is a hanging drop? When do you do the former, when the later?
Wet mount: a drop of bacteria covered with a plastic slip to observe the shape of the bacteria (no staining)
Hanging drop: observes motility of bacteria.
Different types of microscope are used for different purposes, what microscope is used to observe the motility of bacteria?
Which to observe the cell membrane/nuclear membrane of a cell (microbe)?
Dark field: Motility
Electron Microscope: cell membrane and nuclear membrane.
On each lens of your microscope, there is a # showing the magnification power (4X, 10X, 40X, 100X), there is another # (_____numerical_____ aperture) you use to calculate the resolution power of that lens, do you know the difference between “magnification” and “resolution” power?
Magnification: Makes image bigger to see detail.
Resolution: Clarity of an object, resolution is more important than magnification.
You have learnt many staining techniques through the lab exercises:
What dye did you use in simple stain? Crystal Violet
What dyes did you used in Gram stain? Crystal violet, gram’s iodine, and safranin violet.
Why did you have to heat up the slide when you did the acid fast stain and the spore stain?
To open the spores and allow the dye to enter and stain. Once cooled down the spores closed trapping the dye inside.
What color is a “+” in acid fast stain? Which bacteria are “+” for acid fast stain? What color is a “+” in spore stain? Which bacteria are “+” for acid fast
stain?
green, Mycobacterium
purple/red, Bacillus
Why did you have to do “negative stain” to look at bacterial capsules? What is “negative stain”?
Negative stain: Staining of the background of the slide.
The bacterial capsule is not stain and the capsule appears as a halo.
In lab exercise #8, you were trying to determine the “generation time” (= doubling time) of a bacterial culture. Knowing the different phases of a bacterial culture, which phase the bacterial culture should be in when you did the experiment?
Lag phase, log phase, stationary phase, death phase.
Log phase
When you used all those paper pieces to construct the molecular interactions in a commercial pregnancy test, you used two antibodies against HCG, why two antibodies? Do they recognize the same site on a HCG molecule? These sites are called antigenic ____Epitopes_____
anti HCG beta
Anti HCG alpha
They do not recognize the same site.
In a clinical trial, one doesn’t want to give away what each patient is getting, to avoid the placebo effect. For this reason, FDA asks for double-blinded study, who are kept blind in the trials?
The patient/customer and the Doctor/vendor
If I give you one Gram+ and one Gram- bacteria to test for their sensitivity to lysozymes in your tears, which one would you predict to be more sensitive to your tears, i.e., having a bigger ring of inhibition?
Gram + due to the peptidoglycan
Gram stain on an old culture of Gram+ bacteria can produce confusing results, since old culture may have cells that stain pink (looks like Gram_). Do you know why?
Pink stains of a gram + are old culture because they did not have the ability to hold the dye, therefore causing the stain to appear pink.
Healthy stain of a gram + are purple (eg. Staph)
To demonstrate the difference between Gram+ and Gram- bacteria, you were often given Staphylococcus aureus and E. coli for comparison in a lab exercises, I hope you know the Gram stain pattern and the shape of these two bacteria.
Gram -, rod, pink color
Gram +, purple, cocci
To demonstrate how spores are hard to kill, you were given Bacillus to compare with other bacteria in the anti-microbial experiments (hot water, UV), hopefully you understood why your results were the way they were.
The spores open up.
You need to know how to convert: g (gram) into mg (milligram)
l (liter) into ml (milliliter), µl (microliter)
m (meter) into mm (millimeter)
You need to be able to write in the power of tens for numbers such as 1/100,000,
100,000,
5/1000,000
5,000,000
You flame your inoculating loop to sterilize it, but you should let the loop cool down before you pick up your bacteria, why? When you incubate your plates, why do you incubate your plates upside down?
Aerosol, bacteria does not go into the air.
To sterilize things we used in the lab, the lab staff did autoclaving which uses high temp and high pressure. We all understand high temp, do you know why high pressure?
Temperature and pressure are proportional, the pressure will allow the temperature to rise making it more efficient in killing the bacteria.
To avoid the increase in antibiotic resistant bacteria, we are advised not to overuse antibiotics to avoid the “natural selection” of these drug resistant bacteria. Can you explain how the process of natural selection promotes the spread of the antibiotic resistant bacteria?
bacteria adapts to the antibiotic making it less effective and causing mutations allowing the bacteria to spread.
You would hope your Snyder agar color stay green when you inoculate your mouth bacteria, why?
Less harmful bacteria, no or minimal cavities.
In your bacterial transformation experiment to obtain those beautiful fluorescent colonies, I said that the bacteria you were using were already made “competent” (able to pick up the plasmids) by the lab staff, do you know what she did to make these bacteria competent?
The cell wall has been treated to open membrane in cell wall.
Some of you would get Staphylococcus as one of your unknowns, the task is to differentiate the two Staphs on the list, what plate would you use to do this?
MSA plate
When you did your hand wash experiment, you decided a variable to test, say, hot vs cold water, you called this variable an “experimental variable” since it was the variable you were testing experimentally. In order to get meaningful data, you need to keep other things the same, for example the washing time had to be the same for both, you called the washing time a _Control_______ variable.
Through the semester, you have used two bacteria that produce colorful colonies: one produce yellow colonies, another one pink. What are the names of these two bacteria? Are they Gram+ or Gram-?
Micrococcus Luteus, yellow colonies, gram +
Serratia Marcescens, pink colonies, gram -
There was one bacterial …show more content…
species for which the colonies were waxy, thus you had to be very patient when making smears to do staining, what is the name of this bacteria?
Mycobacterium
Different types of agar plates are used to culture different types of microbes: What (microbes) is Sabourand agar used for? NA,TSA and Blood agar plates are all used to culture bacteria, but the costs are very different, when do you use the cheapest one (NA), the more expensive one (TSA), the most expensive one (Blood agar)?
Sabourand agar: fungus, collects bacteria from the air, selective medium.
NA: Bacteria in things (eg. table, floor, door knob)
TSA: Bacteria outside our bodies (hands, fingernails)
Blood agar: Bacteria inside our bodies ( throat, blood)
Plates are used according to bacterias natural habitat.
The temperature at which you incubate your plates is important, what temp would you incubate microbes harvested from your desk top? From your tongue?
Desk top: room temp, 25-30 celsius
Tongue: body temp, 37 celsius
What is a wet mount? What is a hanging drop? When do you do the former, when the later?
Wet mount: a drop of bacteria covered with a plastic slip to observe the shape of the bacteria (no staining)
Hanging drop: observes motility of bacteria.
Different types of microscope are used for different purposes, what microscope is used to observe the motility of bacteria?
Which to observe the cell membrane/nuclear membrane of a cell (microbe)?
Dark field: Motility
Electron Microscope: cell membrane and nuclear membrane.
On each lens of your microscope, there is a # showing the magnification power (4X, 10X, 40X, 100X), there is another # (_____numerical_____ aperture) you use to calculate the resolution power of that lens, do you know the difference between “magnification” and “resolution” power?
Magnification: Makes image bigger to see detail.
Resolution: Clarity of an object, resolution is more important than magnification.
You have learnt many staining techniques through the lab exercises:
What dye did you use in simple stain? Crystal Violet
What dyes did you used in Gram stain? Crystal violet, gram’s iodine, and safranin violet.
Why did you have to heat up the slide when you did the acid fast stain and the spore stain?
To open the spores and allow the dye to enter and stain. Once cooled down the spores closed trapping the dye inside.
What color is a “+” in acid fast stain? Which bacteria are “+” for acid fast stain? What color is a “+” in spore stain? Which bacteria are “+” for acid fast
stain?
green, Mycobacterium
purple/red, Bacillus
Why did you have to do “negative stain” to look at bacterial capsules? What is “negative stain”?
Negative stain: Staining of the background of the slide.
The bacterial capsule is not stain and the capsule appears as a halo.
In lab exercise #8, you were trying to determine the “generation time” (= doubling time) of a bacterial culture. Knowing the different phases of a bacterial culture, which phase the bacterial culture should be in when you did the experiment?
Lag phase, log phase, stationary phase, death phase.
Log phase
When you used all those paper pieces to construct the molecular interactions in a commercial pregnancy test, you used two antibodies against HCG, why two antibodies? Do they recognize the same site on a HCG molecule? These sites are called antigenic ____Epitopes_____
anti HCG beta
Anti HCG alpha
They do not recognize the same site.
In a clinical trial, one doesn’t want to give away what each patient is getting, to avoid the placebo effect. For this reason, FDA asks for double-blinded study, who are kept blind in the trials?
The patient/customer and the Doctor/vendor
If I give you one Gram+ and one Gram- bacteria to test for their sensitivity to lysozymes in your tears, which one would you predict to be more sensitive to your tears, i.e., having a bigger ring of inhibition?
Gram + due to the peptidoglycan
Gram stain on an old culture of Gram+ bacteria can produce confusing results, since old culture may have cells that stain pink (looks like Gram_). Do you know why?
Pink stains of a gram + are old culture because they did not have the ability to hold the dye, therefore causing the stain to appear pink.
Healthy stain of a gram + are purple (eg. Staph)
To demonstrate the difference between Gram+ and Gram- bacteria, you were often given Staphylococcus aureus and E. coli for comparison in a lab exercises, I hope you know the Gram stain pattern and the shape of these two bacteria.
Gram -, rod, pink color
Gram +, purple, cocci
To demonstrate how spores are hard to kill, you were given Bacillus to compare with other bacteria in the anti-microbial experiments (hot water, UV), hopefully you understood why your results were the way they were.
The spores open up.
You need to know how to convert: g (gram) into mg (milligram)
l (liter) into ml (milliliter), µl (microliter)
m (meter) into mm (millimeter)
You need to be able to write in the power of tens for numbers such as 1/100,000,
100,000,
5/1000,000
5,000,000
You flame your inoculating loop to sterilize it, but you should let the loop cool down before you pick up your bacteria, why? When you incubate your plates, why do you incubate your plates upside down?
Aerosol, bacteria does not go into the air.
To sterilize things we used in the lab, the lab staff did autoclaving which uses high temp and high pressure. We all understand high temp, do you know why high pressure?
Temperature and pressure are proportional, the pressure will allow the temperature to rise making it more efficient in killing the bacteria.
To avoid the increase in antibiotic resistant bacteria, we are advised not to overuse antibiotics to avoid the “natural selection” of these drug resistant bacteria. Can you explain how the process of natural selection promotes the spread of the antibiotic resistant bacteria?
bacteria adapts to the antibiotic making it less effective and causing mutations allowing the bacteria to spread.
You would hope your Snyder agar color stay green when you inoculate your mouth bacteria, why?
Less harmful bacteria, no or minimal cavities.
In your bacterial transformation experiment to obtain those beautiful fluorescent colonies, I said that the bacteria you were using were already made “competent” (able to pick up the plasmids) by the lab staff, do you know what she did to make these bacteria competent?
The cell wall has been treated to open membrane in cell wall.
Some of you would get Staphylococcus as one of your unknowns, the task is to differentiate the two Staphs on the list, what plate would you use to do this?
MSA plate