Week 1 Lecture 2 Flashcards
the differences between microscopes is
Resolution. the ability to see two objects as separate.
light microscopes are used for
they are used to look at whole cells. they have a resolution limit of 0.2µm = 200nm This limit of resolution mean that objects closer than 0.2 micrometres together will not be seen as separate. light microscopes can magnify an image up to 1000x
electron microscopes are used for
looking at internal cell structures and viruses. they have a resolution limit of 0.2nm meaning they have x1000 more magnification than light microscopes
1000 µm equals
1mm
Why does bacteria have both a cytoplasmic membrane and a cell wall?
the cell wall stops bacteria from bursting and gives shape and rigidity to the cell.
what does peptidoglycan (have to memorise) do?
it traps the purple colour in gram-positive rods
What are the differences between gram-negative and gram-positive cell walls
gram-negative cell walls have a complex, multilayered structure while gram-positive cell walls have a thicker layer of peptidoglycan. they also have an outer membrane in addition to their peptidoglycan. this outer membrane is made up of phospholipid, protein and polysaccharide, often called the lipopolysaccharide layer, or LPS.
Main function is to provide cell structure, LPS is often toxic to animals – known as endotoxin
The cell wall of both gram-positive and gram-negative cells contains peptidoglycan - a combination of polysaccharides and amino acids. strands of peptidoglycan are joined together by tetrapeptide cross-links. these cross-links differ between gram-positive and gram-negative bacteria
Why might a pharmaceutical product made from sterile river water be toxic?
it may have dead bacteria even if its sterile because endotoxins are still present such as e. coli or salmonella
Gram-negative bacteria that are toxic to humans include Salmonella, Shigella, Escherichia
Why might it be hard to detect that a cell culture was contaminated with mycoplasmas?
they have no peptidoglycan therefore no dye gets trapped by their cell wall even when they have two membranes
How would you make blood agar?
blood agars are bright red. bacteria love to grow on them. how do we add blood to an agar without putting it in a steriliser? we add blood to the agar without putting it in the steriliser. the jelly is made in the steriliser first and then its put in a water bath to cool it down. there is a bottle of blood that is pre warmed and we inject a bit of the blood in the agar. The sterile media is then inoculated with the bacteria of interest. Must be done using aseptic technique to prevent air-borne contaminants from entering cultures
Work next to bunsen burner flame, be careful where you place objects such as lids of tubes, lids of agar plates, the loop, etc
The width of a staphylococcus cell is
1 micrometre. a coccus is one micrometre wide. viruses are nanometres wide
Viruses require what to replicate?
Cells. Some viruses could use DNA to replicate but some are RNA viruses. But what they all need is cells to replicate
The most important greenhouse gas is
Methane is more important than CO2, because it is 25x more polluting
Explain why immersion oil is used for light power light microscopy
Oil immersion must be used with the 100x objective - increases the light-gathering ability of the lens
Name the parts on a light microscope
Eyepiece Circular nose piece Objective lens, usually a x4 (scanning lens), x10, low power, x40 high dry, x100 oil immersion Colour coded Course focus knob Fine focus knob Stage Illumination source, some sources have a field diaphragm Condenser with iris diaphragm Vernier scale
Outline basic steps in setting up a light microscope for good resolution
Focus on the specimen using x 10 objective.
Close the field diaphragm to its most closed state such that you can see the edges of the diaphragm (may be blurry) in the field of view.
Use the condenser focus knob to bring the edges of the field diaphragm into the best focus possible (look for purple red edge).
Use the condenser-centering screws to center the image of the closed field diaphragm in the field of view.
Open the field diaphragm just enough so that its edges are just beyond the field of view.
Adjust the condenser diaphragm lever to introduce the proper amount of contrast into your sample. The amount of contrast added will depend on the sample, however too much contrast can introduce artefacts into your images.
Explain what is meant by ‘heat fixation’?
Heat-fixed smears of bacterial cells are used – heat fixing causes the cells to adhere to the glass slide, the heat kills most of the cells
Describe the Gram stain
In the Gram stain an insoluble crystal violet-iodine complex forms
Acetone will remove this complex from gram-negative but not gram-positive bacteria
Gram-positive cells have a thick cell wall consisting of several layers of peptidoglycan
Acetone dehydrates the cells, closing the pores in the cell wall and trapping the crystal violet-iodine complex inside
In gram-negative cells, acetone penetrates the lipid-rich outer membrane (LPS), the complex is removed from the cells
Identify bacterial cell morphologies and arrangements
Bacteria that are round in shape are called cocci (singular: coccus, double: diplococcus)
Bacteria that are cylindrical are called rods (bacilli)
Some rods are curved or spiral
During cell division, some bacteria remain in groups, form specific cell arrangements such as chains and clusters (mostly important in differentiating staph and strep)
Some bacteria are filamentous, form long, thin cells or have tapering ends (fusiform), some can be branching
Which of the following cannot be viewed with a light microscope?
a. Influenza virions
b. E. coli
c. Giardia cysts
d. Yeast cells
e. Bacillus endospores
A. Influenza Virions
What part of the microscope collects the light and focuses it on the slide?
The condenser
Where should the condenser be when you start to focus for a gram stain?
Right to the the top of the stage and then back 2mm.
With which lens do you start to focus on your slide?
The 10X objective.
What should the light intensity be set at?
The light intensity should be set at 8
