Lab Quiz 2 Review
Week 4-Lab 3: Examine Mixed Fluorescent Plates & Streak/Spread/Serial Dilute Unknown Culture
• Being familiar with examples of how a colony on a plate can be characterized? (Hint: What can you and have you looked for in describing colonies in lab?)
by size, color, texture, elevation, form, and margin
• In terms of serial dilutions, understanding the colony range for a good CFU/ml determination?
CFU/mL = (# colonies/mL plate) * dilution factor
choose a plate that has between 30-200 colonies
mL plated = 0.1mL(100 μL)
dilution factor = 10^-5 , 10^-6 , 10^-7, 10^-8
• Be familiar with how to determine CFU/ml when given the following variables: number of colonies, number of ml plated, and dilution factor
CFU/mL = (# colonies/mL plate) * dilution factor
• Why is Chromagar considered selective and differential media? What grows on Chromagar?
Chromagar is a selective and differential media because it selects for the growth of a particular microorganism, through the use of antibiotics(bacteria that are resistant survive). It selects for gram + bacteria, which show up as pink colonies, while gram - bacteria show up as colorless or blue
it helps differentiate between staph a and other staph species because staph a uniquely ferments the media, generating lactic acid
• Understand the importance of fermentation leading to lactic acid and how that impacts bacteria characteristics on Chromagar? What do pink/purple colonies mean? What would colorless or blue colonies indicate?
staph a can go through fermentation creating lactic acid which a pH indicator in the chromagar recognizes, leads to pink color. Pink and purple colonies are gram positive while blue and colorless colonies are gram negative
• Why is Blood Agar considered a differential media? What grows on Blood Agar?
because it differentiates between microbes based on their production of hemolysins, so hemolytic bacteria grows on blood agar
• What are hemolysins? What do they do? How do they impact infection? Understanding the different types of hemolysis and what does that indicate?
hemolysins are enzymes that attack and breakdown the cell membranes of cells like red blood cells
they aid in infection because, they cause the destruction of immune cells (because red blood cells begin to release iron for pathogens to use).
complete hemolysis is called beta-hemolysis
partial is alpha and none is gamma
• Why is MacConkey Agar considered a selective and differential media? What grows on MacConkey Agar? Understand how lactose fermentation influences results. What do pink colonies mean? What do off-white/opaque colonies indicate?
macconkey agar is used for the isolation and differentiation of gram-negative rods
only gram negative bacterial species grow on macconkey
if the microobe is a lactose fermenter the colonies are pink, if they are a non-lactose fermenter the colonies are off-white or opaque
Week 5-Lab 4: Examine Unknown Culture Plates from Lab 3/Restreak Them; Gram Staining of Control Bacteria: S. aureus/ E. coli
• Understanding the importance of re-streaking plates
it’s important to re-streak plates in order to obtain a pure culture; re-streaking plates allows for isolation of individual colonies, which each originate from a single bacterial cell, resulting in pure cultures. Pure cultures are essential for microbe identification and studying the behaviors of an individual bacterial species
• What type of staining is Gram Staining? How do the Basic Dyes used in this procedure work?
gram staining is a type of staining that allows one to differentiate between bacterial cells based on differences in cell wall structure and composition.
it uses basic dyes that carry a positive charge (POSITIVE STAINING)
type of positive staining that attracts negatively charged cellular components
Gram (+) bacteria’s cell walls have a THICK layer of peptidoglycan while gram (-) bacteria’s cell walls have a think layer of peptidoglycan as well as an outer membrane (the thin layer of peptidoglycan is sandwiched between the outer membrane and the cell membrane)
gram + bacteria retain the crystal violet stain and stain purple while gram - bacteria lose the crystal violet and stain red/pink from the safranin counter stain
• Understand what cellular structure is the main determinant in Gram Staining?
peptidoglycan - gram (+) has thick layer, gram (-) has thin layer
• Understand the sequence of events of Gram Staining? How is a smear prepared? What are the Dyes, Mordant, Decolorizer, Times for each step?
smear preparation-
place a small drop of water on slide
place innoculating loop, with bacteria, into water and stir
allow the smear to dry
now fix the smear by passing it over fire 2-3 times
allow it to cool then start gram staining
gram staining process
first put crystal violet on for a minute then wash it off (dye)
then put iodine on for a minute, it makes the dye less soluble so it will adhere better to the cell walls (mordant)
then alcohol which washes away the stain from gram-negative cell walls (decolorizer) 10-20 seconds
add safranin which adheres to gram-negative cells (counterstain) for 1 minute
• Following successful Gram Staining, what results should be expected for Gram Positive and Gram
Negative bacteria?
gram + bacteria would appear violet/blue while gram - would stain red/pink
• What are the expected Chromagar, Blood Agar, MacConkey Agar results for Controls:
Staphylococcus aureus, Streptococcus pneumoniae, and E. coli?
• What were the results of Chromagar, Blood Agar, and MacConkey Agar using Unknowns Culture?
• What are the expected Gram Staining, Cell Shape/Morphology, and Arrangement results for
Controls: Staphylococcus aureus, Streptococcus pneumoniae, and E. coli?
• What were the expected Gram Staining, Cell Shape/Morphology, and Arrangement results for
Unknowns Culture?
Week 6-Lab 5: Gram Staining/Staphaurex/Catalase on Unknown Bacteria from Lab 4 Plates; Set Up Mueller Hinton Plates
• Understanding the Staphaurex Test knowing the background, purpose, and what is being looked at?
the text detects the presence of the clumping factor produced by staph A
staph A produces coagulase or the clumping factor which interacts with and enzymatically converts fibrinogen to fibrin, an insoluble protein (key in blood clots)
this causes bacterial cells to clump
protein A released by staph A binds to Fc portion of antibodies, preventing phagocytosis by immune cells
• What would be the expected Staphaurex Test results for the Control Bacteria: Staphylococcus aureus, Streptococcus pneumoniae, and E. coli?
for staph A it would be positive for strep and ecoli. it would be negative
• Understanding the Catalase Test knowing the background, purpose, and what is being looked at?
• What would be the expected Catalase Test results for the Control Bacteria: Staphylococcus
aureus, Streptococcus pneumoniae, and E. coli?
catalase breaks down hydrogen peroxide into water and oxygen and catalase also protects pathogens from ROS (reactive oxygen species) generated by the immune system
Staph A and Ecoli are catalase (+) while strep is catalase (-)
causes bubbling in solution because of the oxygen released
• Understanding the Kirby-Bauer Method knowing the background, purpose, and what is being looked at? Know what is meant by the “Zone of Inhibition”
standardized method for disk diffusion tests
the sensitivity and resistance of bacteria to certain drugs are determined using tables that relate zone diameters to microbial resistance (measured in mm)
zone of inhibition - clear zones where bacteria is not growing or is dead
• Knowing the purpose of the MacFarland Standard for the Kirby-Bauer Method
the macfarland standard is essential in order to compare the turbidity of our bacterial inocuulum with that of the standard. this standardized inoculum density helps ensure consistency in the distribution of bacteria on the agar surface
inoculum can be adjusted by either adding more bacterial colonies or adding saline
• Be familiar with the bacteria targets of Penicillin, Cefoxitin, and Erythromycin
the disks used
control -no antibiotic
penicillin - targets peptidoglycan cell wall
cefoxitin targets peptidoglycan cell wall
erythromycin - targets ribosome inhibiting protein synthesis
Week 4-Lab 3: Examine Mixed Fluorescent Plates & Streak/Spread/Serial Dilute Unknown Culture
• Being familiar with examples of how a colony on a plate can be characterized? (Hint: What can you and have you looked for in describing colonies in lab?)
by size, color, texture, elevation, form, and margin
• In terms of serial dilutions, understanding the colony range for a good CFU/ml determination?
CFU/mL = (# colonies/mL plate) * dilution factor
choose a plate that has between 30-200 colonies
mL plated = 0.1mL(100 μL)
dilution factor = 10^-5 , 10^-6 , 10^-7, 10^-8
• Be familiar with how to determine CFU/ml when given the following variables: number of colonies, number of ml plated, and dilution factor
CFU/mL = (# colonies/mL plate) * dilution factor
• Why is Chromagar considered selective and differential media? What grows on Chromagar?
Chromagar is a selective and differential media because it selects for the growth of a particular microorganism, through the use of antibiotics(bacteria that are resistant survive). It selects for gram + bacteria, which show up as pink colonies, while gram - bacteria show up as colorless or blue
it helps differentiate between staph a and other staph species because staph a uniquely ferments the media, generating lactic acid
• Understand the importance of fermentation leading to lactic acid and how that impacts bacteria characteristics on Chromagar? What do pink/purple colonies mean? What would colorless or blue colonies indicate?
staph a can go through fermentation creating lactic acid which a pH indicator in the chromagar recognizes, leads to pink color. Pink and purple colonies are gram positive while blue and colorless colonies are gram negative
• Why is Blood Agar considered a differential media? What grows on Blood Agar?
because it differentiates between microbes based on their production of hemolysins, so hemolytic bacteria grows on blood agar
• What are hemolysins? What do they do? How do they impact infection? Understanding the different types of hemolysis and what does that indicate?
hemolysins are enzymes that attack and breakdown the cell membranes of cells like red blood cells
they aid in infection because, they cause the destruction of immune cells (because red blood cells begin to release iron for pathogens to use).
complete hemolysis is called beta-hemolysis
partial is alpha and none is gamma
• Why is MacConkey Agar considered a selective and differential media? What grows on MacConkey Agar? Understand how lactose fermentation influences results. What do pink colonies mean? What do off-white/opaque colonies indicate?
macconkey agar is used for the isolation and differentiation of gram-negative rods
only gram negative bacterial species grow on macconkey
if the microobe is a lactose fermenter the colonies are pink, if they are a non-lactose fermenter the colonies are off-white or opaque
Week 5-Lab 4: Examine Unknown Culture Plates from Lab 3/Restreak Them; Gram Staining of Control Bacteria: S. aureus/ E. coli
• Understanding the importance of re-streaking plates
it’s important to re-streak plates in order to obtain a pure culture; re-streaking plates allows for isolation of individual colonies, which each originate from a single bacterial cell, resulting in pure cultures. Pure cultures are essential for microbe identification and studying the behaviors of an individual bacterial species
• What type of staining is Gram Staining? How do the Basic Dyes used in this procedure work?
gram staining is a type of staining that allows one to differentiate between bacterial cells based on differences in cell wall structure and composition.
it uses basic dyes that carry a positive charge (POSITIVE STAINING)
type of positive staining that attracts negatively charged cellular components
Gram (+) bacteria’s cell walls have a THICK layer of peptidoglycan while gram (-) bacteria’s cell walls have a think layer of peptidoglycan as well as an outer membrane (the thin layer of peptidoglycan is sandwiched between the outer membrane and the cell membrane)
gram + bacteria retain the crystal violet stain and stain purple while gram - bacteria lose the crystal violet and stain red/pink from the safranin counter stain
• Understand what cellular structure is the main determinant in Gram Staining?
peptidoglycan - gram (+) has thick layer, gram (-) has thin layer
• Understand the sequence of events of Gram Staining? How is a smear prepared? What are the Dyes, Mordant, Decolorizer, Times for each step?
smear preparation-
place a small drop of water on slide
place innoculating loop, with bacteria, into water and stir
allow the smear to dry
now fix the smear by passing it over fire 2-3 times
allow it to cool then start gram staining
gram staining process
first put crystal violet on for a minute then wash it off (dye)
then put iodine on for a minute, it makes the dye less soluble so it will adhere better to the cell walls (mordant)
then alcohol which washes away the stain from gram-negative cell walls (decolorizer) 10-20 seconds
add safranin which adheres to gram-negative cells (counterstain) for 1 minute
• Following successful Gram Staining, what results should be expected for Gram Positive and Gram
Negative bacteria?
gram + bacteria would appear violet/blue while gram - would stain red/pink
• What are the expected Chromagar, Blood Agar, MacConkey Agar results for Controls:
Staphylococcus aureus, Streptococcus pneumoniae, and E. coli?
• What were the results of Chromagar, Blood Agar, and MacConkey Agar using Unknowns Culture?
• What are the expected Gram Staining, Cell Shape/Morphology, and Arrangement results for
Controls: Staphylococcus aureus, Streptococcus pneumoniae, and E. coli?
• What were the expected Gram Staining, Cell Shape/Morphology, and Arrangement results for
Unknowns Culture?
Week 6-Lab 5: Gram Staining/Staphaurex/Catalase on Unknown Bacteria from Lab 4 Plates; Set Up Mueller Hinton Plates
• Understanding the Staphaurex Test knowing the background, purpose, and what is being looked at?
the text detects the presence of the clumping factor produced by staph A
staph A produces coagulase or the clumping factor which interacts with and enzymatically converts fibrinogen to fibrin, an insoluble protein (key in blood clots)
this causes bacterial cells to clump
protein A released by staph A binds to Fc portion of antibodies, preventing phagocytosis by immune cells
• What would be the expected Staphaurex Test results for the Control Bacteria: Staphylococcus aureus, Streptococcus pneumoniae, and E. coli?
for staph A it would be positive for strep and ecoli. it would be negative
• Understanding the Catalase Test knowing the background, purpose, and what is being looked at?
• What would be the expected Catalase Test results for the Control Bacteria: Staphylococcus
aureus, Streptococcus pneumoniae, and E. coli?
catalase breaks down hydrogen peroxide into water and oxygen and catalase also protects pathogens from ROS (reactive oxygen species) generated by the immune system
Staph A and Ecoli are catalase (+) while strep is catalase (-)
causes bubbling in solution because of the oxygen released
• Understanding the Kirby-Bauer Method knowing the background, purpose, and what is being looked at? Know what is meant by the “Zone of Inhibition”
standardized method for disk diffusion tests
the sensitivity and resistance of bacteria to certain drugs are determined using tables that relate zone diameters to microbial resistance (measured in mm)
zone of inhibition - clear zones where bacteria is not growing or is dead
• Knowing the purpose of the MacFarland Standard for the Kirby-Bauer Method
the macfarland standard is essential in order to compare the turbidity of our bacterial inocuulum with that of the standard. this standardized inoculum density helps ensure consistency in the distribution of bacteria on the agar surface
inoculum can be adjusted by either adding more bacterial colonies or adding saline
• Be familiar with the bacteria targets of Penicillin, Cefoxitin, and Erythromycin
the disks used
control -no antibiotic
penicillin - targets peptidoglycan cell wall
cefoxitin targets peptidoglycan cell wall
erythromycin - targets ribosome inhibiting protein synthesis
