Lab mid term
on what plate does gram negative organism grow on?
Levine Eosin Methylene Blue (EMB)
is a differential medium used to detect the presence of fluorescein-producing Pseudomonas species.
Luisetti's
what color is a strong lactose fermenter
Metallic green
is a simple broth that contains peptone, buffers, and glucose. Following growth of the organism
Methyl Red and Voges-Proskauer broth
what pH indicator turns yellow at neutral pH and red acidic pH, is added to the broth
Methyl-Red
stain the background, but not the cells. The unstained cells can be visualized as a silhouette against a dark background. The background should appear grey-black; the capsule colorless, and the cells gray to red in color. Note the size of the capsule relative to the size of the bacterium.
Negative Stain
determined by detecting the production of indole using Kovac's reagent
Tryptophan digestion
mutagenic (causes DNA mutations) but at high levels or for extended periods of time it can be microbicidal (kill microbes). helpful for sterilization and disinfection purposes, but its use is limited because it does not penetrate deeply into proteinaceous matter (such as blood or sputum) and it does not pass through glass or plastic.
Ultraviolet radiation
purpose: tool in the diagnosis of diseases caused by these bacteria. Mycobacterium and Nocardiaare resistant to the dye and can only be visualized by
acid-fast stain
what stain is -Mycobacterium sp. (fuchsia) and non- acid-fast bacterium Staphylococcus sp. (blue/purple).
acid-fast stain
able to survive but not multiply in acid
acidoduric
Many fungi and some bacteria grow optimally in acidic environments
acidophiles
Solutes that decrease the pH of a solution
acids
what is pink on top of thioglycollate agar deep
aerobic
final electron acceptor but some microbes can respire anaerobically by using alternative final electron acceptors such as nitrate (NO3-), ferric iron (Fe3+), sulfate (SO42-), or carbonate (CO32-).
aerobic respiration
useful for growing microbes with low or zero oxygen requirements (anaerobes) deeply within the agar.
agar deep
Petri plate containing nutrient agar that provides the largest surface area for growth of microorganisms.
agar plate
test tube of nutrient agar that was allowed to solidify at an angle creating a larger flat surface for growth at the top of the agar.
agar slant
Third step: dissolves the outer membrane of gram-negative cells causing them to lose the violet crystals but has no effect on the gram-positive cells.
alcohol, decolorize
solutions with low [H+] and high [OH-] have high pH
alkaline or basic.
Microbes that prefer to grow at high pH
alkalophiles
why streak plate is important?
allow bacteria and fungi to grow on a semi-solid surface to produce discrete colonies and helps identify organism
Microorganisms that can grow in the absence of oxygen
anaerobes
Color description: Thioglycollate agar deep
anaerobic
1. Clean the microscope slide with a Kimwipe®. Label the frosted edge of the slide with the name of the organism and your name or initials using a pencil or sharpie pen. 2. Light the Bunsen burner. 3. Place several drops of water onto the slide using the glass dropper bottle. 4. Hold the inoculation loop in your dominant hand like you would hold a pencil. Flame the inoculation loop by holding it in the flame until the wire turns red. Wait for it to cool slightly. 5. Using the sterile inoculation loop obtain ONE isolated colony from the agar plate and transfer the bacteria onto the water on the slide. Spread the bacteria around the entire slide. 6. Allow the smear on the slide to briefly air dry on the bench, don't wait more than 2 or 3 minutes for this. If they are not totally dry after 2-3 minutes, just go on to the next step. 7. Heat-fix the cells onto the slide by setting the slide with the bacteria side facing up on the slide warmer (set at 65-75°C) for 5 minutes. Heat fixing causes the cells to stick to the slide (so that they do not get washed off during staining), kills the cells and makes them more permeable to the dye. But be careful because overheating them causes them to distort and lyse. 8. Allow the slide to cool before going on to the staining procedure.
bacteria smear
What type is Spirllium: spiral shaped bacteria stained pink?
bacterial morphological
increase the pH of a solution
bases
In Simmons citrate agar slant the pH indicator bromothymol blue- high pH color
blue
In an Acid-fast stain, what is staphylococcus epidermis color and shape?
blue and spherical
used when we want large numbers of cells for use in a single application.
broth deep
In a positive catalase test, you will see
bubbles
The oxygen that is produced by the catalase reacting with added H2O2 is visible as
bubbles
produces the electron carriers, NADH and FADH2.
citric acid cycle
After Kovacs reagent is added what is a negative test produces
clear
what color indicate gram-negative lactose non-fermenters
colorless
converges or focuses the light ray onto a single point on the specimen, have greatly contributed to an increase in the resolving power of modern compound microscopes
condenser lens
first step: basic and is therefore attracted to the cell cytoplasm by virtue of its charge. and is purple,
crystal violet, Primary stain
In Cytochrome Oxidase, what color will the spot be if positive?
deep blue or black
are useful in distinguishing between groups of bacteria
differential
# of viable bacterial cells or colony forming units in the original sample (cfu/ml) = # of colonies on the plate X dilution factor / ml of dilution plated
dilution factor
final stage of respiration, the electrons of NADH and FADH2 are fed into
electron transport chain
what stain is red/ rod and green and sphere
endospore staining
highly resistant to hostile physical and chemical conditions, these are a dormant form of the bacterium that allows it to survive sub-optimal environmental conditions, resistant to heat, radiation, disinfectants, and desiccation, they are difficult to eliminate from medical and pharmaceutical materials and are a frequent cause of contamination.Only a few genera of bacteria such as Bacillus and Clostridiumare capable of forming endospores.
endospores
produces ATP from glucose without utilizing O2 through a biochemical pathway known as can be described as anaerobic catabolism of glucose.
fermentation
Escherichia coli is
gram-negative bacilli, single cells, pink
shorter the wavelength
greater resolution
In Simmons citrate agar slant the pH indicator bromothymol blue- Low pH color
green
grows optimally in high salt concentrations but a halotolerant organism simply survives those conditions but does not grow in them very well.
halophile
microbes that grow in high salt concentrations
halophiles
solution that has higher solute concentration than the inside of a cell
hypertonic
A solution that has lower solute concentration than the inside of a cell
hypotonic
As the resolution decreases
images blend and the objects are indistinguishable from one another.
spread the cells from the mixed culture over the surface of an agar plate.
inoculation loop
distance between the pupils of the eyes.
interpupillary distance
adjusts the amount light that hits the specimen
iris diaphragm
water concentration is equal inside and outside of the cell and there is no net movement of water
isotonic
lower the magnification power
larger the working distance
Solutions with a high concentration of H+ ions have low concentration of OH- ions
low pH acidic
specimen is placed to hold the object being viewed
mechanical stage
The material that microbiologists use to grow microorganisms
medium
If the organism produced acids through mixed-acid fermentation of the glucose, the pH indicator will turn
medium red
Microbes that grow best at intermediate temperatures, between 20C and 40C,
mesophiles
microbes that grow best in low concentrations of oxygen
microaerophilic
Contains 2 or more different bacteria
mixed culture
Organisms that only grow along the stab line are red pigment only on th stab line
negative
perform a capsule stain
negative stain
Most microbes prefer to grow at pH near neutral
neutralophiles
In a negative catalase test, you will see
no bubbles
What happens if the organism does not produce Cytochrome Oxidase? (negative)
no color
mathematical constant that describes the relative efficiency of a lens in bending rays of light.
numerical aperture
lens closest to the specimen
objective lens
killed in the presence of oxygen and can only grow in its absence.
obligate anaerobes
The lens that you look through
ocular lens
f a solution is determined by the concentration of H+ ions and is defined as the negative log to the base 10 of the H+ ion concentration.
pH
keeps an image in focus even when changing from one objective lens to another provided that the object is in the center of the field of view.
parfocal
1. Malachite green stain is forced into the spore by heating the cells. 2. Vegetative cells are then decolorized with water and stained red/pink with spore safranin counterstain.
perform Endospores stain
organisms that grow optimally at the specific conditions
phile
Neisseria sicca
pink gram-negative
what color indicate lactose fermentation but not as strong
pink, burgundy
Organisms that grow away from the stab, especially those that grow all around the tube, are red pigment NOT isolated to stab line
positive
what is microbial isolation technique that microbes are put into the agar while it is still semi-liquid but not so hot that it kills them.
pour-plate technique
Contains 1 single species of organism
pure culture
contain only one species of microbe
pure culture
In flagella stain, what is peritrichous flagella color and shape?
purple and rode
Bacillus cereus
purple gram-positive
a differential stain which selectively stains external capsules surrounding bacterial cells.
purpose of capsule stain
In an Acid-fast stain, what is mycobacterium segmatis shape and color?
red and rod
What color is the production of acid
red to yellow
After Kovacs reagent is added what is a positive test produces
red, fuchsia
what happens when dye is absorbed into the bacterial cells?
reduced to the insoluble red colored pigment formazan.
used as an indicator of the presence of oxygen.
resazurin
smallest distance between two nearby objects that can be clearly distinguished and also indicates the diameter of the smallest object that can be clearly seen with that lens system.
resolution
resolving power formula
resolving power (RP)= wavelength of light in nm/ 2x numerical aperture
After incubating bacteria in tryptone broth, the reagent is added and if indole is present it reacts to produce a red dye called
rosindole
used to estimate the number of viable bacterial cells present in the original sample.
serial dilution
bacteria in the original sample are serially diluted by sequentially transferring a constant volume of cells into a constant volume of broth medium. 100ml of cells is then spread onto agar plates and the plates are incubated to allow bacterial colonies to grow.
serial dilution-spread plate technique
two techniques that use agar medium for isolating various species of bacteria from a mixed culture
serial dilutions and streak plate
allow microbiologists to determine cellular morphology (shape), size and cell arrangement.
simple stain
three general types of bacterial staining techniques
simple, differential, structural
The better the resolving power, indicated by
smallest value for RP, the greater the ability of the microscope to produce a very clear image.
useful for determining if the bacteria possess certain important structures such as flagella, capsules and endospores.
structural
Where can liquid (broth) be placed in?
test tubes
The production of acid will be indicated by phenol red, a pH indicator which turns yellow under acidic conditions. The production of gas will be determined by looking for gas bubbles inside a small, inverted tube, called a Durham tube that will be at the bottom of the broth
test unknown microbe
the higher the value for NA
the better the resolution.
Microbes that grow best at relatively high temperatures, between 40c and 90C,
thermophiles
determine the oxygen requirement of your microbe
thioglycollate agar deep
why acid-fast bacteria are resistant to gram-staining.
to the presence of large amount of lipoidal material in their cell wall which prevents the penetration of decolorizing solution.
organisms that can survive those specific conditions but do to grow well or do not grow at all in them.
tolerant or duric
how to calculate total magnification
total magnification = ocular lens x objective lens
e semi-solid motility agar medium contains_____, which is colorless in its oxidized form.
triphenyltetrazolium chloride (TTC)
distance between the specimen and the objective lens when the lens is focused on the object
working distance
If there was no acid production, the medium will remain_____ after adding methyl-red
yellow
acid production with gas red to yellow and bubble/air in durham tube
+A/+G
Acid production without gas change of color from red to yellow and no bubble
+A/-G
no acid production, no gas color remains red and no bubbles
-A/-G
what is ml of dilution plated
0.1
P2 pipette =
0.2 and 2 microliters
How to perform acid-fast stain?
1) Carbolfuchsin stain 2) Decolorizing agent 3) Counterstain is methylene blue.
Light can be adjusted in three ways
1) rheostat or dimmer switch 2) adjusting the height of the condenser using the condenser height adjustment knob 3) opening and closing the iris diaphragm with the diaphragm adjustment lever.
What does it mean if p2 is set to 152?
1.52 microliters
what is ml in the original culture
10
p1000 =
100 and 1000 microliters
0.1 ml =
100 microliters
1 millimeter =
1000 microliters
What object does oil immersion use?
1000x
What does it mean if p20 is set to 152?
15.2 microliters
What does it mean if p200 is set to 152?
152 microliters
p20=
2 and 20 microliters
p200 =
20 and 200 microliters
final ATP yield from the complete aerobic oxidation of a single molecule of glucose is
32 ATP molecules.
What does it mean if p1000 is set to 052?
520 microliters
microorganisms that use oxygen for their respiration.
Aerobes
perform anaerobic metabolism but they are not killed in the presence of oxygen.
Aerotolerant anaerobes
1) protects your cultures from being contaminated with unwanted organisms and 2) protects you and the environment around you from being contaminated with the microbes that you are working with.
Aseptic technique
what reagents are used in Voges-Proskauer
Barritt's A and B
what stain is- Streptococcus pyogenes
Capsule stain
group of microbial cells growing on a solid surface that is visible to the naked eye
Colony
The vegetative cells stain red/pink and the spores stain green.
Endospore stain
What bacteria in lab used for EMB, lactose fermenter pink
Enterobacter aerogenes
What bacteria in lab used for EMB, strong lactose fermenter metallic green
Eschericha coli
capable of both aerobic and anaerobic metabolism
Facultative anaerobes
bacteria are motile, motility allows the bacterium the ability to find nutrients or move away from toxic compounds, consist of a single protein filament with a diameter of about 15 nm.
Flagella
Proteus mirabilis showing the peritrichous positioning of flagella. Stained light purple.
Flagella stain
is a pigment that fluoresces yellow-green when placed under UV light
Fluorescein
first biochemical pathway in the oxidation of glucose, is composed of 10 chemical reactions which convert glucose to pyruvate. The reactions of glycolysis do not directly require oxygen, so they are carried out by aerobic and anaerobic organisms.
Glycolysis
What do you use to perform flagella stain
Gram idoine, mordant
What stain is pink/rod or spherical bacteria?
Gram stain -gram negative
what stain is purple stained rod/spherical bacteria
Gram stain -gram positive
second step: mordant is a stabilizer that causes the primary dye to form large crystals in the peptidoglycan meshwork of the cell wall. purple
Gram's iodine, mordant
presence of catalase is detected by adding
H2O2
Where can solid (agar) be placed?
Petri dish or test tubes
what species will grow in fluoresce yellowish grenn
Pseudomonas
microbes that can grow best at relatively low temperatures, between 0oC and 20oC.
Psychrophiles
the colorless gram-negative cells are counterstained with a secondary dye_______, order to make them visible once again. basic dye which stains the cells pink, but________ can only stain cells that are not already stained. Therefore, at the end of the gram-staining procedure, gram-positive cells look purple and gram-negative cells look pink.
Safranin, counterstained
two important ways of making microorganisms visible:
Staining and culturing
gram-positive cocci, irregular clusters, violet
Staphylococcus epidermidis
