Misc. Flashcards
What are the 8 characteristics used to describe macroscopic morphology?
- Size = colony diameter (mm)
- Shape = whole colony
- Margin
- Pigmentation
- Texture
- Appearance
- Optical properties
- Elevation
How is size recorded in macroscopic morphology?
Usually colony diameter is measured in millimeters (mm)
How is a shape of a bacterial colony described?
Circular/round; irregular; filamentous/rhizoid; spindle
How is the margin of a bacterial colony described?
Outer edge of colony is:
Entire/smooth; undulate/wavy; filamentous
How is pigmentation of a bacterial colony described?
= colour of colony; as specific as possible
How is texture of a bacterial colony described?
Smooth; wrinkled; rough; concentric rings (choose one)
How is the appearance of a bacterial culture described?
Dull/matte; shiny/glistening
How are the optical properties of a bacterial colony described?
translucent; opaque
Light passes through colony; light does not pass through colony
How is elevation of a bacterial colony described?
Flat; raised; convex/dome-shaped; umbonate; Craterform
Gram-positive cells stain […]
Gram-positive cells stain purple (may be a bluish-purple)
Gram-negative cells stain […]
Gram-negative cells stain pink (may be a reddish-pink)
How is the shape of an organism described?
- Cocci
- Shape? Round; oval; kidney-shape
- Rod
- Length to width ratio (e.g., 2:1 vs 5:1)
- Shape of ends? Rounded, squared, pointed
How are spores described?
- If present, describe and/or diagram location (central; sub-terminal; terminal)
- If absent, record ‘none observed’
How is the arrangement of cells in the microscope field described?
- Order of most prevalent to least prevalent arrangement; record at least two
- e.g., clusters > chains, singles > pairs
Describe best microbiology lab practices.
- Organization
- Lab and personal safety
- Aseptic technique
- Control (organisms, media, equipment etc.)
- Record keeping
- Working independently and with others
- Keeping and leaving your lab area clear, clean and organized
- Being a responsible lab citizen
What is a biological agent?
A biological agent includes:
* Bacteria
* Viruses
* Fungi
* Parasites
* Prions
* and even toxins produced by microorganisms.
Describe Risk Group 1.
Unlikely to cause disease in healthy individuals
These agents represent no or little risk to an individual and no or little risk to the community.
Describe risk group 2.
Any pathogen that can cause human disease but, under normal circumstances, is unlikely to be a serious hazard to laboratory workers, the community, livestock, or the environment.
These agents represent a moderate risk to an individual but a low risk to the community.
Describe risk group 3.
Indigenous or exotic agents with potential for aerosol transmission, disease may have serious or lethal consequences.
These agents represent a high risk to an individual but a low risk to the community.
Describe risk group 4.
Dangerous/exotic agents which pose high risk of life-threatening disease, aerosol- transmitted lab infections; or related agents with unknown risk of transmission.
These agents represent a high risk to the individual and a high risk to the community.
Compare biosafety level 1 labs and level 2 labs.
Compare biosafety level 3 and level 4 labs.
List some Lab safety DOs.
Name some lab safety DON’Ts.
If 4 ml of a bacterial sample is added to 94 ml of a diluent, the dilution of this bacterial suspension is:
4 x 10 ^-2
Starting from an unknown sample, it is important to prepare multiple plates from multiple dilutions.
True or False?
True.
Starting from an unknown sample, it is important to prepare multiple plates from multiple dilutions in order to more accurately determine the number of CFU/ml in the original sample.
This is because different dilution levels will result in different numbers of colonies, allowing for more accurate estimations.
Additionally, multiple plates can provide a more representative sample of the bacteria present in the original sample.
A 10^-7 dilution was prepared from a bacterial sample and 1 ml was spread on an agar plate. If you counted 72 colonies on this plate, then the original number of CFU/ml would be:
7.2 x 10^8
In the standard plate count, the purpose of using serial dilutions is: [3]
- To reduce the cell population by a known amount
- To obtain plates that can be counted
- To generate isolated colonies (…)
The key difference between the spread plating and pour plating techniques is:
the method of distributing the sample
The key difference between the spread plating and pour plating techniques is that spread plating involves spreading a sample of bacteria onto a plate and allowing it to dry, while pour plating involves pouring a liquid sample of bacteria directly onto a plate.
Spread plating is typically used to count and isolate colonies of bacteria, while pour plating is often used to measure the concentration of bacteria in a sample.
Both methods allow for the cultivation of bacteria in plate cultures and can be used for various types of testing and research.
CFU’s from pour plates appear:
on the surface and in the agar.
CFU’s from spread plates appear:
on the surface of the agar
What would you predict as the outcome of a Gram-stained slide that has been incubated too long by ethanol during the Gram stain procedure? Assume that the counter stain has not be used.
Both Gram positive and Gram negative cells would not be stained (clear).
What type of microscope is used to view a Gram-stained slide?
Light microscope
The overall magnification of the Gram stained slide using a 100X objective would be:
(100X) objective lens * (10X) eyepiece = 1000X
In spread plating, why is the glass spreader sterilized with alcohol and the bunsen burner flame? [2]
- To avoid contamination
- To ensure there is no carry-over of bacteria between samples
When performing a restriction enzyme digest, what other components would be added to the tube? [4]
- Buffer
- Water
- DNA
- Enzyme
In DNA eletrophoresis, a 0.7% agar gel percentage in a gel of 70mL volume is made by:
0.49 grams agarose + 70 mL buffer
Which component allows one to visualize the expected migration of the DNA bands during electrophoresis?
Bromophenol blue & Xylene cyanol
You have loaded your DNA sample into the well of an agarose gel, and apply the electric field. In which direction do you expect the DNA to migrate, and why?
Towards the positive electrode because the DNA is negatively charged.
You have loaded a sample of undigested plasmid DNA into the well of an agarose gel. Even though you were careful during the isolation protocol, you know that some DNA will be cut. This may result from shear forces that act on DNA (and DNA is a relatively fragile molecule).
Starting from the top of the gel where the wells are towards the bottom of the gel), what is the order of DNA bands you expect to see after running the DNA in the electric field?
Open circle, linear, supercoiled
Both the standard plate count method and optical density measure the exact number of cells in a sample.
True or False?
False.
You have a bacterial broth culture sample and you want to determine the concentration of this original sample. You perform the dilution scheme shown below. As you are unsure of how much to dilute the sample, you prepare a variety of plates . Use the figure to answer the next set of questions.
What is the final dilution of tubes #3, #4 and #5?
If 4 ml of a bacterial sample is added to 94 ml of a diluent, the dilution of this bacterial suspension is:
4x10^-2
Starting from an unknown sample, it is important to prepare multiple plates from multiple dilutions. True or False?
True.
A 10-7 dilution was prepared from a bacterial sample and 1 ml was spread on an agar plate. If you counted 72 colonies on this plate, then the original number of CFU/ml would be:
7.2 x 10^8
In the standard plate count, the purpose of using serial dilutions is: [3]
- To reduce the cell population by a known amount
- To obtain plates that can be counted
- To generate isolated colonies
The key difference between the spread plating and pour plating techniques is:
The method of distributing sample
CFU’s from pour plates appear:
On the surface and in the agar.
What would you predict as the outcome of a Gram-stained slide that has been incubated too long by ethanol during the Gram stain procedure? Assume that the counter stain has not be used.
Both Gram positive and Gram negative cells would not be stained (clear).
What type of microscope is used to view a Gram-stained slide?
Light microscope
The overall magnification of the Gram stained slide using a 100X objective would be:
1000X
You have a tube that contains a bacterial culture that has 10,000 colony forming units (cfu)/ml.
i) If you did not perform any dilutions, how many colonies should grow if you plate:
a) 1mL
b) 10uL
a) 10,000
b) 100
You have a tube that contains a bacterial culture that has 10,000 colony forming units (cfu)/ml.
i) You do a 1 in 100 dilution.
a) What is the concentration of the culture in the blank?
b) How many colonies will grow if you plate 1 mL?
c) How many colonies will grow if you plate 200 uL?
a) 100
b) 100
c) 20
A student prepares a 10–6 dilution of a broth culture and plates 0.1 ml in a spread plate and 0.2 ml in a pour plate. After 48 hours of incubation, the student counted 42 colonies on the spread plate and 124 colonies in the pour plate. The student thinks there might be something wrong with their assay. Given that the same dilution blank was used for both plates, the student expected to see approximately 84 colonies in the pour plate since twice the volume was plated relative to the spread plate.
Suggest two reasons why the numbers don’t seem to match up
- Some bacteria may have been killed by a hot spreader.
- Some bacteria may have been killed by ethanol if the spreader was not flamed.
- Some bacteria were still stuck to the spreader and not transferred to the plate.
- Pipetting errors.
- Contamination
.
- Add resuspension buffer to resuspend cells
- Add NaOH and SDS to lyse cells
- Potassium acetate and ethanol solution to neutralize the mixture
- Centrifuge to pellet cell debris and chromosomal DNA
- Put supernatant through the column; plasmid binds to resin
- Use the column to wash solution
- Add TE to solution to elute plasmid
What is the critical step that allows separation of plasmid DNA from chromosomal DNA and why?
- Addition of potassium acetate to neutralize the NaOH
- Chromosomal DNA, due to its large size, cannot re-anneal and becomes trapped in the K/SDS complex and can be centrifuged out.
- The plasmid DNA is smaller and easily renatures with addition of KOAc.
- Renatured plasmids remain soluble in the solution and can be found in the supernatant
A student isolated a plasmid from E. coli and performed a restriction enzyme digest using three different restriction enzymes (as single digests). After the digest, the student used gel electrophoresis to separate the DNA fragments. She used a linear DNA ladder so that she could compare the sizes of the DNA fragments. The DNA ladder is in Lane 5 and the uncut plasmid is Lane 1. Lanes 2 – 4 represent one of the bands from Lane 1. What are the three forms that a plasmid that exist in once it has been isolated from the bacterial cells and indicate which of Lanes 2 – 4 represents that form of the plasmid.
2. Approximately how big is the plasmid?
3. Where on the gel (relative to the wells) would the positive and negative electrodes be, why?
.
