Exam 1 Micro Lab
any magnifying object using only one lens
Uses multiple lenses, has ocular lens as well as multiple objective lenses through which it compounds light.
Explain how immersion oil allows a microscopist to get better resolution
Every time light moves through slide and hits specimen, it bends and refracts. As it goes from glass back into air, some of that light will bend so that it no longer is captured by our magnification lens. We have lost that information. If we eliminate that transition between the glass and the air, by adding immersion oil, we are going to gather as much light as possible up into the mag lens and we have that much better a picture. Increases the numerical aperture.
Explain the connection between resolving power, numerical aperture, and wavelength of light
Resolving power - how far apart can two dots be and you be able to distinguish them as two separate dots and not fuzzy
Wavelength of light - One of the easiest ways to improve microscope resolution is to improve quality and type of light you are using. Light moves as a wave and how tight that wave is depends on the energy level of the light. If we want improved resolution, use short wavelength because light moving in tight pattern and will hit spots tight together and you can distinguish between those spots.
Numerical aperture - the ability of the lens to capture light. Oil immersion increases this ability and thus increases resolution
why is a blue filter used on the microscope?
Blue light has the shortest wavelength of the visible spectrum. The shorter the wavelength, the higher the resolution one can achieve with a light microscope.
The resolution using only blue light is about 4 times better than with using all the wavelengths of visible light.
Increases contrast between a specimen and its background. Better higher resolution.
Describe the change in working distance as magnification increases. What does this mean in regards to observing samples at higher magnification?
In most compound microscopes, as magnification increases, working distance decreases dramatically. It is important to be aware of this, as changing magnifications without being mindful of the change in working distance can damage the lenses.
Evaluate the advantage to the microscopist of parfocality. (What does parfocal mean?)
Parfocal: having corresponding focal points all in the same plane.
Advantage: allows more accurate focusing at maximum focal length and then zooming back to a shorter focal length to compose the image.
Recognize how the optics of the microscope change the orientation of the image seen when compared to the orientation on the slide.
The optics of microscope inverts the image (Example was the letter "e" slide).
What happens to the size of the field of view as magnification increases? How does this apply to looking at a specimen on a slide?
It decreases. The field of view is how much of your subject you can see through a lens. As you magnify the subject or 'zoom in' you aren't able to see as much of the subject, but zoomed in.
As you increase the magnification, the lens gets closer to the specimen. Increasing the magnification on a light microscope will decrease the diameter of the field of view. You are essentially looking closer and closer at the objects.
Compare the size of human blood cells with yeast cells and typical bacilli and cocci
Blood cells 6-7. yeast cells are smaller. bacteria are think and even smaller.
What is depth of field?
the distance between the nearest and farthest objects in a scene that appear acceptably sharp in an image
Distinguish between prokaryotic and eukaryotic cells.
Eukaryotes have a distinct nucleus as well as additional organelles present
Observe, identify, and draw the common shapes (morphologies) and arrangements of procaryotic
Understand the relationship between the arrangement of cells and the number of planes of division which occur.
(Remember: When determining morphology and arrangement look for the most common morphology and the most complex arrangement.)
SHAPES: Cocci (spheres) [coccus—singular], bacilli
(rods) [bacillus—singular], spirilla (spirals) [spirillum—singular] ARRANGEMENTS: diplo or strepto
form in a line, tetrad forms in two lines(an x and y axis), sarcina forms in an x, y, and z axis, staph forms in a grape cluster effect.
Differentiate spirilla and spirochetes by description and observation.
Both are spiral but spirilla are more rigid and use polar flagella for motility whereas spirochetes are flexible and motility through axial filaments.
(Know the following species: diplococcus—Neisseria gonorrhoeae and spirochete—Treponema pallidum)
Understand what "pleomorphic" means. Observe and draw pleomorphic cells.
Organisms that exhibit growth in a variety of shapes (If you see a slide w/multiple shapes that you cannot necessarily identify think pleomorphic)
Characteristics of cyanobacteria:
=do not have chloroplasts
--do not have a nucleus
any bacterium of a genus that includes the diphtheria bacillus
Diphtheria is a serious and highly contagious disease caused by bacteria called Corynebacterium diptheriae. This disease affects the upper respiratory tract (nose, throat, voice box and upper windpipe) and occasionally, the skin. It can affect anyone, at any age and can be fatal.
Palisade arrangement is said to be like "chinese letters". It often includes bacteria cells at weird angles to one another resulting from the type of division they have. They divide by snapping apart their outer cell wall so this results in their unique arrangement.
(in bacteria) a rounded resistant form adopted by a bacterial cell in adverse conditions
a bacterium of a large group typically having simple cells with rigid cell walls and often flagella for movement. The group comprises the “true” bacteria and cyanobacteria, as distinct from archaebacteria
Differentiate between akinetes and heterocyst. What is the function of each?
Akinete - These resting cells, which do not grow or expend much energy on biological functions, have evolved to survive poor growth conditions. An akinete is one type of resting cell, with a characteristically thick wall and a store of nutrients inside. When favorable conditions develop in the environment around the akinete, then this resting cell bursts and releases multiple new cells, which begin growing as normal.
Heterocyst - Some plants use special cyanobacteria, called heterocysts, in order to convert nitrogen in the air to a biologically available form
lab culture containing a single species of organism.
Lab culture containing two or more species of organism
without contamination of the culture, the sterile medium, or the surroundings
killing all forms of microbe life
the unintended introduction of microbes in areas or on surfaces they should not be
a growth medium in a test tube; slanting the medium provides more surface area for microbial growth
When a liquid is in a cylinder the liquid will be higher than in the middle creating a concave. This is a meniscus.
Identify times when a microbiologist must use a pure culture.
To look for a particular pathogen that caused a disease
To look for antibiotic susceptibilities on that pathogen
To inhibit evolutionary change
Describe characteristics of a properly adjusted Bunsen burner flame.
When properly adjusted, Bunsen burner produces a flame with two cones - an outer cone that is relatively colorless and an inner cone that is blue in color and should be about 1 inch in height.
Describe instruments microbiologists most often use to transfer microorganisms.
· Inoculating loop
· Inoculating needle
· Nutrient agar slants
Describe aseptic technique procedures that are performed before, during and after working with
· Disinfect work area and wash hands properly
· Put lab coat and goggles on
· Position work items in an organized way on work table
· Loosen caps off slants and broths
· Label test tubes
· Turn on flame
· Sterilze inoculating loop by burning in flame. Start to flame loop away from tip.
· Flame the lip of slant culture
· Place sterilized loop in slant culture and collect sample of Sm
· Touch loop to inside of slant to get rid of excess Sm
· Flame lip of slant and replace cap
· Flame lip of new slant
· Insert loop with Sm into new slant
· Swerve loop on top of agar
· Flame lip of new Sm slant
· Flame loop
· Tighten cap of original cultures and place in biohazard
· Put all materials away and disinfect work area.
Explain how to protect yourself from a laboratory-acquired infection.
· Always disinfect work area before and after working
· Avoid creating aerosols
· Wear googles and lab coat
· No electronic devices in lab
· No eating in lab
· Always use aseptic techniques properly
· Put stools out of the way to avoid tripping over them
Understand the purpose of stock cultures and why a stock culture is made before using a new culture for other purposes.
A stock culture is made to have microbes to work with in the future. A stock culture is prepared before using culture for other purposes to decrease any chance of contaminating the stock culture and keep it as pure as possible.
be careful not to confuse with serologic pipette (which is calibrated through the delivery tip)
calibration lines of MOHR end above delivery tip, so the volume is delivered between 2 lines and the pipette is NEVER allowed to drain completely or blown out
the tip is not as accurate as the stem
variable volumes may be delivered between 2 calibration lines, or if the delivery tip is involved in the measurement, the pipette is allowed to drain freely and then the last drop is blown out of the tip
List four characteristics of microbial morphology as identified in the lab book.
positively charged ion
negatively charged ion
a substance that can be readily converted into a dye or other colored compound
the portion of the chromogen that gives it its color
Differentiate between basic and acidic dyes and how they work.
An acidic dye carries a negative charge because it is missing hydrogen.
A basic dye carries a positive charge: it has either gained hydrogen or lost a hydroxide.
Understand why, when using solid media, the inoculum is serially diluted on the slide.
The concentration of the bacteria is too high, serial dilution takes place to help isolate the bacteria's
on the slide better.
Know the purpose of air-drying (or gently warming) smears
Excess water left on the slide will boil during the fixing stage, causing most microbes present to
rupture and become airborne
Know the purpose and effects of heat fixing a slide.
Basic stains are heat fixed. Heat fixing kills bacteria, makes them adhere to the slide, and coagulates cytoplasmic proteins to make them more visible under the microscope.
Identify two common negative stains.
Congo red and Nigrosin (India Ink)
Explain how a negative stain interacts with bacteria and the result obtained.
the negative stain uses a dye solution in which the chromogen is acidic and carries a negative charge.
The negative charge on the bacterial surface repels the negatively charged chromogen, so the cell remains unstained against a colored background.
Understand why slides prepared with negative stains are not heat fixed.
Heat fixing negative stains will distort the bacteria cells so the cell's size can be determined and be able
realize that the slides are not sterile.
Identify two reasons for using a negative stain.
The negative staining technique is used to determine morphology and cellular arrangement in bacteria that
are too delicate to withstand heat-fixing and to avoid distorting of the cell.
What are bacterial capsules and how do you identify them?
Excreted by cell to form clear, gelatinous, protective layer. Background will appear darker (negative stain), then a clear barrier surrounding the stained cell.