MICRO LAB EXAM 1
Why should long hair be controlled in a microbiology laboratory? How does this apply to a patient care situation?
So that it doesn't drag through a culture, contaminate media, or catch on fire in a Bunsen burner. Long hair should also be controlled in a patient care situation so that you don't drag it through a patient specimen, wound, etc. and so that you don't contaminate media or medical equipment or supplies with it.
Staphylococcus, Micrococcus, Streptococcus, Bacillus, Rhodospirillum
Staphylococcus, Micrococcus, Streptococcus, Bacillus, Rhodospirillum
Why is location of an endospore important?
The location and an endospore develops in a vegetative cell helps us to determine the identity of the organism. Different bacteria will form endospores in different locations within the vegetative cells (i.e., Bacillus will form them in the middle of the cell and Clostridium will form them at the end of the cell - then the remaining vegetative cell degrades, leaving just the endospore at the end of the sporulation process).
When an agar plate is inoculated, why is the loop flamed between quadrants?
The loop is flamed between quadrants to dilute the bacteria (i.e., to spread them thin), so that you get individual colonies by the 4th quadrant.
Why do you flame the neck of the culture tube when it is opened?
To sterilize it briefly in case it has dust or other contamination on it, or if you accidentally touch it with your finger - to prevent contamination.
Ocular Lens
eyepiece (magnifies 10x)
How would you determine if your culture media were sterile?
Examine solid media to be sure that there are no microorganisms growing on the surface and examine liquid media to be sure that it is clear and not turbid.
NUTRIENT BROTH
General purpose complex liquid medium for growing microorganisms
NUTRIENT AGAR SLANT
General purpose complex solid medium for growing microorganisms in a tube (on a slanted surface).
Condenser
Condenses or focuses the diffuse cone of light from the light source into a narrow beam through a hole in the stage.
Iris Diaphragm
Controls the amount of light passing through the specimen
How do you cool the inoculating loop after it has been sterilized and just prior to recovering live bacterial cells from a slant culture and from a plate culture?
Hold it still; never wave or blow on it. You can also sizzle the loop in the medium in an non-inoculated spot on the medium before obtaining a colony from a plate or some growth from a slant.
What is the function of the Iodine solution in the Gram stain?
It acts as a mordant, setting the crystal violet stain in the Gram + bacteria.
What is the function of the iris diaphragm?
It controls the amount of light passing through the specimen.
What is the function of the 95% ethanol in the Gram stain?
It is a decolorizer and decolorizes the Gram - bacteria when it is applied and left on for only around 15 sec. (If it stays on longer, it can decolorize the Gram + bacteria as well.)
What is a bacterial colony?
It is a visible mound of cells growing on a solid surface, typically an agar plate.
What is the value of a capsule to a microorganism?
It is protective (against drying, etc. and against the white blood cells of the immune system).
What counterstain is used in the Gram stain and why is it necessary?
Safranin. It is necessary to stain the Gram - bacteria because they are colorless at this point in the process.
Name two types of solutions that may be administered to a patient by intravenous injection and therefore must be sterile? From what we've learned in this laboratory what characteristics would indicate that the solution is not sterile?
Any type of solution that is injectable, ex. IV solutions, insulin, allergy antigens, etc. You must inspect injectables for turbidity to make sure that they are not contaminated.
Monotrichous (single hair), amphitrichous (at both ends), lophotrichous (in a tuft), peritrichous (all around)
Monotrichous (single hair), amphitrichous (at both ends), lophotrichous (in a tuft), peritrichous (all around)
Objective Lens
One of 3 or 4 lenses that can be rotated into place for viewing specimens at different magnifications (4X, 10X, 40X, 100X).
Heat fixation
Passing an air-dried smear of bacteria on a slide through a Bunsen burner several times so that the smear will be fixed to the slide (and the bacteria are also killed). We heat fix slides to be stained so that the smear doesn't wash off in the water rinse step.
What is a pure culture?
One species of bacteria growing in a container.
. What is a mixed culture? What is a pure culture?
A mixed culture contains 2 or more species of microorganisms. A pure culture contains only 1 species of microorganism.
DIFFERENTIAL STAINING
A process of staining that facilitates differentiation of various structures in a specimen.
What is the importance of the capsule stain, the flagella stain, and the endospore stain?
All of these staining processes help to identify the organism, which is necessary in order to determine treatment for the patient's infection.
. Should we Gram stain after we isolate the pathogen on nutrient media or is it useful to Gram stain a clinical specimen directly from the patient? Or both? Explain. It depends on the type of specimen collected. We won't focus on explaining why at this point in the course - the explanation will become clearer later in the course
. Should we Gram stain after we isolate the pathogen on nutrient media or is it useful to Gram stain a clinical specimen directly from the patient? Or both? Explain. It depends on the type of specimen collected. We won't focus on explaining why at this point in the course - the explanation will become clearer later in the course
Describe two conditions in which an organism might stain gram variable?
(1) If the culture is old (over 24 hrs. old), it will not stain properly in the Gram staining process. (2) If you mess up the process by leaving the ethanol decolorizer on too long, then all cells will stain as if they were Gram -.
CULTURE CULTURE
(Noun) A container of living, growing microorganisms. (Verb) To cultivate microorganisms in a container in the lab.
100x oil immersion objective because bacteria are so small
100x oil immersion objective because bacteria are so small
Why is immersion oil used with the 100X objective?
Because the oil has the same angle of refraction as the glass in the slide and in the objective lens and too much light would be lost if air was between the slide and lens instead of oil. See oil immersion definition above and text illustration below:
Why is it necessary to isolate individual colonies from a mixture in the clinical lab?
Because, it is too difficult to read biochemical and other tests from a mixture.
Motility
Capable of or demonstrating movement by independent means
Cell grouping aids in identification of the organism
Cell grouping aids in identification of the organism
What are the signs of growth in a liquid medium?
Cloudiness or turbidity
How would you differentiate colonies of E. coli from M. luteus?
Colonies of M. luteus are bright yellow in color and are punctiform (small, pinpoint colonies), whereas colonies of E. coli are off-white in color and are larger than those of M. luteus.
What is colonial morphology?
Colony shape, form, appearance, etc.
Micrococcus luteus Nutrient Agar Slant Escherichia coli Nutrient Agar Slant
Micrococcus luteus Nutrient Agar Slant Escherichia coli Nutrient Agar Slant
Acidic dyes
Negatively charged, repelled by bacteria, purpose is the stain the background so that you can see surface features of the bacteria.
Is bacterial endosporulation a reproductive mechanism? Explain.
No, because there is no increase in the number of cells: 1 vegetative cell produces 1 endospore when conditions are harsh, and then when conditions improve, that 1 endospore will become 1 vegetative cell again.
What is the advantage of the parfocal lenses?
Once you get your specimen in focus on one objective, then you should be able to switch to any other objective and the specimen should still be in focus (with possible minor adjustment of the fine focus knob only - our microscope repairman has the microscopes parfocal to within a quarter-turn of the fine focus knob each year in August).
Basic dyes
Positively charged, stick to bacteria, purpose is to stain the bacteria so that you can see them.
Protozoa are eukaryotes and, therefore, their flagella are larger and more complex than those of bacteria, which are prokaryotes. Cilia are only found in eukaryotes.
Protozoa are eukaryotes and, therefore, their flagella are larger and more complex than those of bacteria, which are prokaryotes. Cilia are only found in eukaryotes.
Oil immersion
The 100X objective (total magnification when used is 1000X) that is used with immersion oil. The oil immersion (100X) objective is immersed in optical grade oil because this oil refracts light at the same angle as the glass in the slide and the glass in the microscope lenses, so you don't lose light that is bent at other angles through air. Optical oil is only useful at higher magnifications such as this (1000X total mag.). You will not detect the loss of light through the air at the lower magnifications (using the less powerful objectives, such as 40X and 10X). *Note that only the oil immersion objective is specially sealed to be immersed in oil - the 40X and 10X objectives are NOT sealed and will actually be ruined by immersion in oil. *Also note that the oil must be removed from the microscope lens after each lab period's use because it will thicken and become yellow if left on the microscope and you will not be able to see very well or remove it as easily next time.
What is the advantage of the Gram stain over the simple stain?
The Gram stain is differential, so that it stains different cells differently (i.e., bacteria with a Gram + wall structure stain purple and bacteria with a Gram - wall structure stain red). The simple stain stains all cells one color so that you can see the cells, but you can't tell which has which type of wall structure.
Resolving Power
The ability to distinguish between two adjacent (side-by-side) objects or the ability to see one object. Resolving power is calculated by a formula that considers the wavelength of light (shorter blue wavelengths have greater resolving power than longer red wavelengths), and the size of the lens. The resolving power value will tell you how far apart two things have to be in order to know that they are 2 separate things and not view them as one (because they appear to overlap). This value also tells you how big something must be in order to be able to distinguish it from the background (an example of this would be if you have had trouble resolving a dim and distant star in the night sky).
Working Distance
The distance between the specimen on the stage and the objective lens (how much room you have to move the stage up and down = how much room you have to focus on the image of the specimen).
Total Magnification
The total amount that an image is magnified, calculated by multiplying the magnification of individual lenses together, ex. 10X ocular x 40X objective = 400X total magnification.
The value of a wet-mount prep in the clinical lab is being able to see a living specimen and look for motility, which aids in identity of the organism.
The value of a wet-mount prep in the clinical lab is being able to see a living specimen and look for motility, which aids in identity of the organism.
Why are culture tubes held somewhat horizontally when transferring cells, rather than vertically?
To prevent contamination of the tubes from airborne microbes, such as those attached to dust particles, falling into the tubes due to gravity
Why are test tube caps and Petri dish lids never placed on the bench top?
To prevent them from getting contaminated, since the bench top is not sterile and airborne microbes could also contaminate the media and/or the caps/lids. Also, to prevent the bench top from being contaminated by microbes in a tube or plate culture.
Trichomonas vaginalis causes an STD.
Trichomonas vaginalis causes an STD.
True motility is intentional movement of living organisms, Brownian movement is nonspecific vibrational movement.
True motility is intentional movement of living organisms, Brownian movement is nonspecific vibrational movement.
COLONY
Visible mound of cells, presumably all descended from one single cell or colony forming unit.
Why do we disinfect the bench top before and after our laboratory period?
We disinfect the bench top before lab to get rid of dust and dirt that can contaminate our cultures and to be sure that the last class didn't leave contaminants. We disinfect afterwards to clean up any inadvertent contamination for the next class.