Lab Quiz 1 Flashcards
What shape is bacillus megaterium
rod
what shape is staphylococcus epidermidis
round
Of what value is a simple stain?
to determine cell morphology, size, and arrangement
What is the purpose of heat-fixing the smear?
it denatures bacterial enzymes, preventing them from digesting cell parts. it also preserves the microbes
Another method of fixing smears is to use methanol instead of heat. How does alcohol chemically fix the bacteria?
the alcohol dehydrates the cell, causing them to denature and to be preserved onto the slide
In heat-fixing, what would happen if too much heat were applied?
cells will be distorted and unusable
Methylene blue can be prepared as a basic stain or an acidic stain. How would the pH of the stain affect the staining of bacteria?
the pH of the stain affects characteristics and transformation of bacteria
Can dyes other than methylene blue be used for direct staining?
Yes. crystal violet is catonic, and can directly stain positively charged microbes
Bacteria can be seen without staining. Why, then, was Koch’s recommendation of fixing and staining important for the discovery of the bacterial causes of diseases?
How bacteria stain is very important in medical diagnostics and treatment with the correct antibiotic. Also, staining bacteria can make small characteristics in the bacteria visible that otherwise would not be.
Quality control staff in a sterilization unit of a hospital used a simple stain to determine whether bacteria were present in sterilized materials. A simple stain of sterile saline used for respiratory therapy revealed the presence of bacteria. Is the saline contaminated?
It is possible. Negative controls and proper aseptic technique are needed to ensure the dye itself and immersion oil are not contaminated. These controls will help narrow down the source of the bacteria. However, staining does not show whether or not the cells were alive.
What color will a gram-negative cell stain?
red
What color will a gram-positive cell stain?
blue
What shape is bacillus subtilis?
rods
what shape is escherichia coli?
rods
How does staphylococcus epidermidis gram stain?
gram positive (blue)
How does bacillus subtilis gram stain?
gram positive (blue)
How does escherichia coli gram stian?
gram negative (red)
Why do gram-positive cells more than 24 hours old stain gram-negative?
The cells can no longer hold the primary stain.
Can iodine be added before the primary stain in a gram stain?
No, it will prevent the crystal violet from washing.
List the steps of the gram staining procedure in order (omit washings), followed by the color of gram-positive cells and gram-negative cells after each step.
STEP: GRAM+/GRAM-
- crystal violet: blue/blue
- iodine: blue/blue
- ethanol or acetone: blue/clear
- safranin: blue/red
Which step can you omit without affecting determination of the gram reaction?
safranin
Suppose you performed a gram stain on a sample from a pure culture of bacteria and observed a field of red and purple cocci. Adjacent cells were not always the same color. What would you conclude?
The sample is old and some of the cells are losing their ability to hold the primary stain.
Suppose you are viewing a gram-stained field of red rods and purple cocci through the microscope. What do you conclude?
the sample has a mixed culture
Human cells can be stained with crystal violet and safranin, so why can’t human cells be gram stained?
the only cells that stain gram positive are those that have a thick cell wall, so the primary would be easily removed by alcohol. safranin would bind to remaining structures with a negative charge.
Considering that it isn’t possible to identify bacteria from a gram stain, why might a physician perform a gram stain on a sample before prescribing an antibiotic?
to determine which antibiotic the bacteria will be sensitive to
What color will an acid-fast organism stain in the acid-fast stain?
fuchsia/red
What color will a non-acid-fast cell stain in the acid-fast stain?
blue
What shape is mycobacterium smegmatis?
rods
What will mycobacterium smegmatis stain with acid-fast stain?
acid-fast positive (red)
What will escherichia coli stain with acid-fast stain?
acid-fast negative (blue)
Did the acid-fast stained sputum slide indicate a possible positive test for tuberculosis?
yes
What are the large blue-stained areas on the sputum slide for an acid-fast stain?
WBCs and non acid-fast organisms
What is the decolorizing agent in the gram stain?
ethanol
What is the decolorizing agen in the acid-fast stain?
acid alcohol
What diseases are diagnosed using the acid-fast procedure?
tuberculosis, leprosy, and nacordiosis
What is phenol (carbolic acid), and what is its usual application?
Phenol is an alcohol with a benzene ring attached. It’s an aromatic compound and it is used as a disinfectant because it controls the growth of microorganisms.
The original acid-fast stain required heating the smear to force the carbolfuchsin into the wall. Why can heat be eliminated in the Kinyoun modification that you used?
Because by adding tergitol to the stain, it reduces the surface tension between the cell wall of the mycobacterium and the stain.
How might the acid-fast characteristic of Mycobacterium anhance the organism’s ability to cause disease?
Thy have a waxy coating around their cell, that makes it difficult for antibiotics to penetrate the cell wall, making the chances of disease higher.
In 1882, after experimenting with staining Mycobacterium, Paul Ehrlich wrote that only alkaline disinfectants would be effective against Mycobacterium. How did he reach this conclusion without testing the disinfectants?
Mycobacterium are resistant to acid so the bacterium does not decolorize when exposed to acid alcohol.
Clinical specimens suspected of containing Mycobacterium are digested with sodium hydroxide (NaOH) for 30 minutes prior to staining. Why is this technique used? Why isn’t this technique used for staining other bacteria?
Digestion removes unwanted bacteria, sputum, and human cells. It is not used for staining others because it kills other bacteria.
The acid-fast stain is used to detect Cryptosporidium protozoa oocysts in fecal samples. Which of the following would you expect to be major component of the oocyst wall: carbohydrates, lipids, or proteins?
lipids
What disease is caused by Cryptosporidium?
cryptosporidiosis, or crypto
When a medium is selected for culturing bacteria, what 3 things must be provided?
macronutrients, an energy source, and any necessary growth factors
a medium whose exact chemical composition is known
chemically defined medium
media for which the exact chemical composition varies slightly from batch to batch
complex media
What type of bacteria are routinely grown on complex media?
chemoheterotrophic bacteria
commonly used liquid complex medium
nutrient broth
when agar is added to nutrient broth and it becomes a solid medium
nutrient agar
an extract from marine red algae, has some unique properties that make it useful in culture media. few microbes can degrade this substance, so it remain solid during microbial growth.
agar
most common method of sterilizing culture media that are heat stable by using stem under pressure
steam sterilization, or autoclaving
contain solid media that provide a large surface area for examination of colonies
petri plates
microbes that are intentionally introduces
inoculated
bacteria that are inoculated into culture media increase in number during this time period
incubation period
after suitable incubation, liquid media become _____, or cloudy, as a result of bacterial growth
turbid
a population of cells that arises from a single bacterial cell
colony
a colony that arises from a group of the same microbes attached to one another
colony-forming unit
each colony that appears different is usually what?
a different species
clumps of microbial cells
flocculent
membrane across the surface of the broth
pellicle
microbial cells that have settled on the bottom of the tube
sediment
unwanted microbes
contaminants
used in microbiology to exclude contaminants
aseptic technique
rendered free of all life
sterilized
provide large numbers of bacteria in a small space and are easily trasported
broth cultures
test tubes containing solid culture media that were left at an angle while the agar solidified. provide a solid growth surface, but are easier to store and transport than petri plates.
agar slants
agar is allowed to solidify in the bottom of a test tube. often used to grow bacteria that require less oxygen than is present on the surface of the medium.
an agar deep
used to determine whether a bacterium is motile. motile bacteria will move away from the point of inoculation giving the appearance of an inverted pine tree
semisolid agar deeps containing 0.5-0.7% agar instead of the usual 1.5% agar
end of the wire is bent into a loop
inoculating loop
end of wire is straight
inoculating needle
Why are mixed cultures of little use in studying microorganisms?
it’s difficult to determine which organism is responsible for an observed activity
name the three dilution methods for isolating bacteria
streak plate, spread plate, and pour plate
isolation technique where a loop is used to streak the mixed sample many times over the surface of a solid culture medium in a petri plate. this causes the bacteria to fall off the loop one by one an ultimately to be distributed over the agar surface, where each cell or group of the same bacteria as a pair or chain develops into a colony. most common technique
streak plate technique
quantitative techniques to determine the number of bacteria
spread plate and pour plate
the number of bacteria in a sample can be determined by counting what?
the number of colony-forming units
small amount of previously diluted specimen is spread over the surface of a solid medium using a spreading rod
spread plate technique
a small amount of diluted sample is mixed with melted agar and poured into empty, sterile petri dishes. after incubation, bacterial growth is visible as colonies in and on the agar
pour plate technique
how many colonies must be in the plate to calculate the number in the original sample of bacteria?
25-250
colony-forming units per ml=?
number of colonies/(dilution X amount plated)
What is the advantage of using petri plates rather than test tubes in microbiology?
gives room for different colonies to grow
What are bacteria using for nutrients in nutrient agar?
carbon, nitrogen, phosphorus, sulfur, protein
What is the purpose of the agar?
to support growth on the surface of a petri plate and it also is used as a solidifying agent
How could you determine whether the turbidity in your nutrient broth tube was from a mixture of different microbes or from the growth of only one kind of microbe?
you could transfer the sample to a petri plate with agar and see if different types of colonies grow
What other methods can be used to determine motility?
hang drop and wet mount
What is the primary use of slants?
a solid growth site that is easier to store and transport
What is the primary use of deeps?
biochemical (oxygen and motility)
What is the primary use of broths?
transport, arrangement, large number of bacteria
What is the purpose of flaming the loop before use? After use?
Sterilization. To kill any previous bacteria on the loop before exposed to preferred bacteria. Afterwards it’s to kill any bacteria it was exposed to.
Why must the loop be cool before you touch it to a culture? Would you set down the loop to let it cool? How do you determine when the loop is cool?
If it is not cool before you touch it to a culture, the heat will kill the bacteria. You should not set down the loop to let it cool. It will be cool after approximately 20-30 seconds.
Why is aseptic technique important?
It involves certain procedures that have to be performed carefully in order to minimize contamination by the pathogen. It is crucial in order to identify the bacteria cultured.
Why was the arrangement of Lactococcus in the broth culture different from the arrangement on the slant culture in the second period?
There is more room for binary fission in the broth
For a bacterium, what evolutionary advantage is associated with forming a pellicle in a liquid medium?
motility and a greater chance of survival (protection)
How can you tell that the media provided for this exercise were sterile?
There were no indicators of growth
Describe the growth pattern of a motile organism in a semisolid deep.
There will be lateral growth. It will move away from point of an inoculation and will look like an inverted pine tree.
How will the arrangement of the Lactococcus Lactis in broth differ from the arrangement in the slant culture?
They will be more packed together in the broth, and more spread out in the slant.
abhorescent pattern of growth
branched
echinulate pattern of growth
pointed
filiform pattern of growth
even
rhizoid pattern of growth
rootlike
consisted of biconvex lenses and were essentially magnifying glasses
simple microscopes
has two lenses between the eye and the object, which magnifies and illuminates the object
compound microscope
shows dark objects in a bright field, used most often
brightfield compound microscope
holds the slide
the stage
transmits the magnified image
body tube
holds the light source in the microscope
the base
is above the light source and consists of several lenses that concentrate light on the slide by focusing it into a cone
condenser
controls the angle and size of the cone of light. this ability to control the amount of light ensures that optimal light will reach the slide
iris diaphragm
microscope with only one ocular lens
monocular microsope
microscope with two ocular lenses
binocular microscope
larger knob used for focusing with the low-power objectives (4X and 10X)
coarse adjustment
smaller knob used for focusing with the high-power and oil immersion lenses
fine adjustment
the area seen through a microscope
field of vision
depends on the type of objective lens used with the ocular lens
magnification
objective with 4X
scanning lens
objective with 10X
low-power lens
40X-45X objective
high-dry lens
objective with 97X-100X
oil immersion lens
most important lens in microbiology
oil immersion
why don’t they make microscopes with higher magnification?
the resolution would be poor
can be adjusted with a wheel that regulates the amount of current to the bulb
intensity of the light
usually requires more light
higher magnification
the ability of a lens to reveal fine detail or two points distinctly separated
resolution, or resolving power
the resolving power is a function of the wavelength of light used and a characteristic of what lens system?
numerical aperture
smaller wavelengths of light _____ resolving power
improve
the amount the light bends
refractive index
using _____ minimizes light loss, and the lens focuses very close to the slide
oil
as light rays pass through a lens, they are bent to converge at the ______, where an image is formed
focal point
multiple focal points create a fussy periphery
spherical aberration
how can you minimize spherical aberration
iris diaphragm
when a multitude of colors is seen in the field
chromatic aberration
light source of one wavelength. most logical, but expensive method of eliminating chromatic aberrations
monochromatic light
when a subject is in focus with one lens, it will be in focus with all the lenses
parfocal
the distance between the objective lens and the specimen
working distance
What 2 plants were used to remove phosphorous and ammonia and the benson sewage plant?
cattails and bullrush
In the final treatment of effluent, what chemical did they use?
chlorine
