question
zone of inhibiton
answer
concentration of antibiotic is high enough to stop grwoth
question
minimum inhibitory capacity
answer
junction where the concentration of the antibiotics is too low to effectviely stop growth
question
why does the Mueller Hinton agar in KB use epth of 4mm
answer
the thick agar slows lateral diffusion and this produces smaller zones than plates held to 4mm.
question
what phase is used for antibiotic test
answer
exponential phase
question
resistance brealkpoint
answer
the zone diameter below which all resistant strains fall
question
the susceptibility breakpoint
answer
the zone diameter above which all susceptible strains fall
question
what might be the consequence of pouring the plates 2 mm deep instead of 4 mm deep
answer
thin agar, increases lateral diffusion and produces larger zones of inhibiton than 4mm. This would lead you to believe that the bacteria is more susceptible than it is
question
What negative effect would moisture have on the Kirby Bauer test?
answer
moist plates would increase diffusion and this would cause larger zones of inhibiton. You think that the bacteria is more susceptible than it is
question
`why must the diluted cultures be used within 30 minutes for KB
answer
30 minutes is the standard generation time , past 30 minutes and your broth would be too turbid and no longer eq. to the standard
question
E coli
answer
gram -
question
Staph A
answer
gram +
question
is there a diff in susceptibility between the gram + and gram - bacteria for a antibiotic
answer
Yes drugs that target PG cell wall are less effective on gram (-) cells; cannot get past outer membrane
question
does the agar have antibiotic beyond the zone of inhibiton?
answer
Yes, edge of zone of inhibition represents the MIC - minimum inhibitory concentration, but not the end of diffusion
question
chemotherapeutic drugs
answer
The treatment of a disease with a chemical substance is called chemotherapy and the chemical substance used for the purpose is known as a chemotherapeutic drug/agent (generally called therapeutic drug/agent).
question
selective toxicity
answer
A drug that kills harmful microbes without damaging the host
question
bacterial lawn
answer
Bacterial lawn is a term used by microbiologists to describe the appearance of bacterial colonies when all the individual colonies on a petri-dish agar plate merge to form a field or mat of bacteria.
question
antimicrobials
answer
agents that are used to treat bacterial infections
question
KB Test is used to:
answer
measure the effectivess of antibiotics and other chemotherapeutic agents on pathogenic microorganisms. tool used for rx of tx
question
variables in the KB method of antibiotic testing (controls)
answer
1. use of mueller-hinton agar- 7.2-7.4 PH
2. Depth of agar used 4mm in 150mm or 100mm Petri dishes
3. use of 0.5 McFarland turbidity Standard for broth culture
4. Disks dispensed onto inoculated plate
5. Temperature at which incubated 35C
6. Time incubated 16-18 hours
7. Clear zones measured after incubation and compared to a standardized table of results
2. Depth of agar used 4mm in 150mm or 100mm Petri dishes
3. use of 0.5 McFarland turbidity Standard for broth culture
4. Disks dispensed onto inoculated plate
5. Temperature at which incubated 35C
6. Time incubated 16-18 hours
7. Clear zones measured after incubation and compared to a standardized table of results
question
identify bacterial and fungal species which are known for production of antibiotics. list the three genera that produce most antibiotics and one antibiotics originally isolated from each of the genera
answer
penivillium (fungal) - penicillin
streptomyces (fungal) - streptomycin
bacillus (bacterial) - bacitracin
streptomyces (fungal) - streptomycin
bacillus (bacterial) - bacitracin
question
summarize and give examples of how most antibiotics accomplish selective toxicity
answer
Selective toxicity relies upon differences between the structure and metabolism of the patient's cells and the structure of the microbe being targeted.
They interfere with pathway with cell wall synthesis. since mammals don't have cell walls; they also interfere with the nucleic acid and protein synthesi
They interfere with pathway with cell wall synthesis. since mammals don't have cell walls; they also interfere with the nucleic acid and protein synthesi
question
broad spectrum antibiotics
answer
effective against many types of microbes and tend to exchibit higher toxicity to the host
question
narrow spectrum antibiotcs
answer
effective against limited froup of microbes and tend to exhibit lower toxcitiy to the host
question
Examine colonies within a zone of inhibition and classify them as resistant mutants or contaminants.
answer
A resistance mutation is a mutation in a virus gene that allows the virus to become resistant to treatment with a particular antiviral drug.
The main source of antibiotic contamination is wastewater from antibiotic manufacturer, large scale animal feeding operations, households, and hospitals
The main source of antibiotic contamination is wastewater from antibiotic manufacturer, large scale animal feeding operations, households, and hospitals
question
chloramphenicol
answer
prevents peptide bond formation during translation (broad or narrow)
question
ciprofloxacin
answer
interferes with DNA rep (broad or narrow)
question
penicillin
answer
inhibits cross linking of cell walls to PG (broad or narrow)
question
streptomycin
answer
bliock initiation complex formation in pro synth
question
tetracycline
answer
blocks attachment of aminocyl tRNA to A site on ribosome
question
trimethoprim
answer
inhibits puring and pyrimidine syth
question
defined media
answer
Defined - Totally made up of specific amounts of chemicals
-Composed of inorganic salts and usually a carbon source such as glucose
Glucose Salts Agar, only organisms that can make all their cellular components from glucose and inorganic salts can grow on this medium
-Composed of inorganic salts and usually a carbon source such as glucose
Glucose Salts Agar, only organisms that can make all their cellular components from glucose and inorganic salts can grow on this medium
question
undefined media
answer
Undefined - Contains such mixtures as extract or enzymatic digests of protein
Sometimes called "rich medium"Contain more preformed nutrients, and the organisms do not have to use energy and materials to synthesize the compounds supplied in the medium
Some bacteria can ONLY grow on this type of medium because they cannot synthesize necessary components needed for growth and must be provided with preformed amino acids, vitamins, and other growth factors
Bacteria that require many growth factors are termed "fastidious"
Example: Trypticase soy agar (TSA), can support the growth of organisms that require vitamins or other growth factors
Sometimes called "rich medium"Contain more preformed nutrients, and the organisms do not have to use energy and materials to synthesize the compounds supplied in the medium
Some bacteria can ONLY grow on this type of medium because they cannot synthesize necessary components needed for growth and must be provided with preformed amino acids, vitamins, and other growth factors
Bacteria that require many growth factors are termed "fastidious"
Example: Trypticase soy agar (TSA), can support the growth of organisms that require vitamins or other growth factors
question
differential media
answer
- a media which differentiates or distinguishes between different type sof microorganisms based on differences in appearance of growth or color changes; the option for these tests are either/or (either ferment lactose or don't )
o Ex: starch hydrolysis (amylase test ) - either hydrolyse starch or don't
o Ex: starch hydrolysis (amylase test ) - either hydrolyse starch or don't
question
All-purpose medium
answer
This media that support the growth of a variety of microbes
" Ex: nutrient agar and tryptic soy agar
" Ex: nutrient agar and tryptic soy agar
question
why no universal medium
answer
" Due to the differences in bacteria... whether they are gram pos gram neg etc.
" There is no such thing as a universal medium ; any set of nutrients is capable of inhibiting some microorganism while providing the growth of others
" There is no such thing as a universal medium ; any set of nutrients is capable of inhibiting some microorganism while providing the growth of others
question
five types of all purpose media
answer
TSA
blood agar
meuller hinton agar
brain heart infusion agar
blood agar
meuller hinton agar
brain heart infusion agar
question
alpha hemolysis
answer
A greenish zome appears around the colonies; this means that there was incomplete lysis of RBCs
question
beta hemolysis
answer
there is clearing around the colonies and in the agar; this is a result of complete lysis of RBCs
question
gamma hemolysis
answer
Appears as only growth and no change to the medium; this results from no hemolysis
question
Understand why, when streaking a blood agar plate, the medium was stabbed with the loop upon completion of the streaking
answer
the stabs encourage streptolysin (hemolysins produced by streptococci) activity because of the reduced oxygen concentration of the subsurface environment
question
Water soluble pigments
answer
will diffuse through the agar
question
insoluble bacterial pigments
answer
will stay with the culture itself
question
Differentiate the two media Pseudomonas Agar F and Pseudomonas Agar P.
answer
...
question
-exoenzyme this is a digestive enzyme exreted from cells to then be phagocytized for nutrient absorption within the cell
answer
this is a digestive enzyme exreted from cells to then be phagocytized for nutrient absorption within the cell
question
-endoenzyme this is a non-digestive enzyme that functions within the cell; most enzymes fall under this category
answer
this is a non-digestive enzyme that functions within the cell; most enzymes fall under this category
question
hydrolyze
answer
add water to break bond
question
how are exoenzymes beneficial for bacteria
answer
-exowenzymes can breakdown macromolecules (ex: starch ) into smaller molecules that then can be taken though the cells membrane and be used as individual glucose molecules for metabolic value
question
selective medium
answer
" this media that contains dyes or toxic substances which inhibit the growth of certain microbes, but support the growth of others
question
Demonstrate the advantages of a medium that is both selective and differential
answer
Both are used individually to narrow down possible identities of microbes
-So both would further narrow increasing the likelihood of properly identifying an unknown organism
-So both would further narrow increasing the likelihood of properly identifying an unknown organism
question
Mannitol Salts Agar
answer
Differential - provides the substrate for fermentation
Selective - sodium chloride makes the medium selective because its concentration is high enough to dehydrate and kill most bacteria
-Staphylococci thrive
-Phenol red indicator
Used to isolate coliforms
Selective - sodium chloride makes the medium selective because its concentration is high enough to dehydrate and kill most bacteria
-Staphylococci thrive
-Phenol red indicator
Used to isolate coliforms
question
eosin methylene blue
answer
differenial - acid produced by fermentation changes the color of dyes (ph indicator)
the dyes are inhibitors and indicators
selecticve - for gram negative organisms becuase of the concentration of sugars and dyes
the dyes are inhibitors and indicators
selecticve - for gram negative organisms becuase of the concentration of sugars and dyes
question
columbiana CNA
answer
differnential - blood which allows hemolysios to occur
sheeps blood [provides the .x factor - heme and yeast provides B12]
selective - antibiotics colistin and nalidixic acid (CAN) interfere with DNA
sheeps blood [provides the .x factor - heme and yeast provides B12]
selective - antibiotics colistin and nalidixic acid (CAN) interfere with DNA
question
why is a colony on selective not considered pure
answer
selective media can still grow more than one species of bacteria
question
how to get a pure culture on selective media
answer
remove several of what appear sot be the same species using a inoculum loop
-must do another streak for better isolation
-must do another streak for better isolation
question
CNA what is selected for
answer
staphylococci, streptococci, enterococci
(gram +)
(gram +)
question
mannitol what is selected for
answer
staphylococci spp
question
CNA inhibits
answer
klebsiella, proteus, pseudomonas (gram -)
question
emb selects for:
answer
coliforms (gram -)
question
emb inhibits
answer
gram +
question
Green on EMB agar plate
No color change: gram negative, no fermentation
No color change: gram negative, no fermentation
answer
acidic and vigorous fermemnter
question
Purple results EMB
answer
: slightly acidic and moderate fermenter
question
No color change: EMB
answer
gram negative, no fermentation
question
Distinguish the appearance of Staphylococci spp. on Mannitol Salt Agar.
answer
All staphylococci grow but staphylococcus aureus turns yellow to show mannitol fermentation
question
Why are bacteria, other than Staphylococci spp. unable to grow on Mannitol Salt Agar.
answer
-the sodium chloride concentration is too high - salt causes most bacteria to shrink and die
question
Understand the benefits and limitations of using the two antibiotics in Columbia CNA agar.
answer
it contains two antibiotics, colistin and naladoxic acid, which inhibit growth of gram neg bacteria , thus selecting for gram pos
question
-specificity
answer
is making sure that everything is the same , otherwise a false positive could result
question
sensitivity
answer
getting false negatives
question
colisitn
answer
affects membrane integrity of gram neg
question
nalidixic acid
answer
acts as a selective agent against gram neg by interfereing with DNA rep
question
sheep blood make possible differentiation of gram + based on
answer
hemolytic rxn
question
what is columbia CNa used for
answer
to isolate staphylocci, streptococci, and enterococci , sheep blood allows for differentiation of hemolytic rxn
question
PG or NG CNA
answer
organism is inhibited by colistin and nalidixic acid --> probably a gram negative
question
GG with clearing
answer
b hemolysis --> organism is not inhibited by colisitin and nalidixic acid and is completely hemolyses HBC
probably B hemolytic G pos
probably B hemolytic G pos
question
GG with greening of medium q
answer
a hemolysis--> org is not inhib by colisitn and naladixic and partially hemolyses RBC
prob a hemolytic gram +
prob a hemolytic gram +
question
GG with no change in the medi
answer
gamma hemolysis--> organism is not inhibited by colisistn and NA, and does not hemolyze RBC
prob gamm hemolytic gram+
prob gamm hemolytic gram+
question
what ingredient in CNA supplies carbon
answer
casein, digest of animal tissue, beef extract, yeast extract, corn starch, and sheep blood
question
which ingredients in CNA supply N
answer
beef extract and peptones
question
is Columbia CNA agar a defined or an undefined medium? Provide the reasoning behind your choice and explain why this formulation is desirable.
answer
it is an undefined, differential, and selective medium that allows growth of Gram-positive organisms (especially staphylococci, streptococci, and enterococci) and stops or inhibits most Gram-negative organisms
question
in your own words, what are the roles of colistin and nalidixic acid in CNA and how does each work?
answer
colistin and nalidixic acid are antibiotics that act as selective agents AGAINST Gram negative organisms
C = colistin contains many polycationic regions that can insert into the outer membrane of the Gram negative bacterial cell wall the insertion of colistin into the outer membrane disrupts the integrity of the outer membrane, which can lead to bacterial lysis
NA = nalidixic acid inhibits DNA gyrase/topoisomerase
DNA gyrase/topoisomerase allows supercoiled DNA to be relaxed and reformed and is necessary for DNA replication. Therefore, nalidixic acid inhibits DNA synthesis Gram negative bacteria are more sensitive to nalidixic acid than Gram positive bacteria
C = colistin contains many polycationic regions that can insert into the outer membrane of the Gram negative bacterial cell wall the insertion of colistin into the outer membrane disrupts the integrity of the outer membrane, which can lead to bacterial lysis
NA = nalidixic acid inhibits DNA gyrase/topoisomerase
DNA gyrase/topoisomerase allows supercoiled DNA to be relaxed and reformed and is necessary for DNA replication. Therefore, nalidixic acid inhibits DNA synthesis Gram negative bacteria are more sensitive to nalidixic acid than Gram positive bacteria
question
what is the role of sheep blood in CNA
answer
sheep blood makes possible differentiation of gram + orga based on their hemolytic rxn
question
why wouldn't it be advisable to compare growth of the organisms on each plate to each other? there are at least two answers to this question.
answer
You do not know how the inhibitors may have affected the growth of the different organisms. So you can't compare growth on selective media. Even on TSA the growth of different organisms is likely different. One may grow really well and one less well (thinner growth). You might also inadvertently add different amounts of organism, but hopefully you are getting more consistent.
question
would removing colisitin and NA from CNA alter the medias sensitivity or specificituy
answer
specificity
question
why might colisitin affect Gram - more than G +
answer
cNA affect the membrane . Gram + have thicker PG wall
question
compare the recipes of NA and CNA . which would grow better
answer
Columbia CNA agar is much more nutritious than nutrient agar. Both have the same amount of beef extract, so there is no difference there. The only additional nutrient source in NA is peptone, but Columbia CNA agar has casein, animal tissue, yeast extract and corn starch. Columbia CNA agar inhibits Gram-negative organisms, but those that aren't inhibited are treated to a diverse menu.
question
EMB what provides C
answer
gelatin provides C and N, also lactose
question
what is the purpose of the dyes in EMB
answer
they inhibit the growth of most gram + and they react with vigourous alctose fermenters whose acid end products turn the growth purple or black
question
what does ecoli look like with EMB
answer
dark growth with metallic sheen
question
what do less aggressive fermenters look like with EMB
answer
pink to purple
question
what do non lactose fermenters look like on EMB
answer
retain their normal color or take . on the color of the medium
question
what is EMB used for
answer
for the isolation of fecal coliforms
question
PG/NG on EMB
answer
organism is inhibited by eosin and methy blue
gram +
gram +
question
GG EMB
answer
org is not inhibit by eosin and meth blue
gram -
gram -
question
growth is pink EMB
answer
org ferments lactose with little acid prdcion
possible coliform
possible coliform
question
growth is dark with or without sheen EMB
answer
org ferments lactose and or sucrose with acid prdction
probable coliform
probable coliform
question
growth is colorless EMB
answer
org does not ferment lactose or sucrose
noncoliform
noncoliform
question
EMB Test.
In your own words, what is the application (purpose) of EMB agar?
In your own words, what is the application (purpose) of EMB agar?
answer
EMB agar is used to isolate Gram negatives from clinical samples and to subsequently differentiate between lactose-fermenting coliform and lactose-nonfermenting coliforms
question
EMB Test.
Is EMB agar a define or an undefined medium? Provide the reasoning behind your choice and explain why this formulation is desirable
Is EMB agar a define or an undefined medium? Provide the reasoning behind your choice and explain why this formulation is desirable
answer
It is undefined due to the gelatin in the mediu. Gelatin is a family of related biochemicals, and the exact chemical formula of the gelatin used in making Levine EMB is not necessarily known. An undefine medium is desirable to support growth of a wide variety of the organisms being selected for.
question
EMB Test.
In your own words, what are the roles of eosin Y and methylene blue in EMB agar?
In your own words, what are the roles of eosin Y and methylene blue in EMB agar?
answer
Both act as selective agents against Gram positives. They also are responsible for the color changes produced by lactose fermenters
question
EMB Test.
In your own words, what is the role of lactose in EMB agar
In your own words, what is the role of lactose in EMB agar
answer
EMB agar is a differential medium based on the ability of an organism to ferment lactose to acid end products; lactose is the substrate of the fermentation. Coliforms perform this fermentation, whereas noncoliforms do not.
question
EMB Test
Growth on the EMB agar and NA plates was recorded as "good growth,' "poor growth," or "no growth." These are qualitative and at least for the first two, subjective terms. What did you use to establish what constituted "good growth?"
Growth on the EMB agar and NA plates was recorded as "good growth,' "poor growth," or "no growth." These are qualitative and at least for the first two, subjective terms. What did you use to establish what constituted "good growth?"
answer
The nutrient agar plate inoculated with the same organisms provided examples of what "good growth" for each organism looks like on a nonselective media. Without these for comparison, it wouldn't be possible to tell if sparse growth on EMB was due to inhibition or was just normal growth for that species.
question
EMB Test.
Why wouldn't it be advisable to compare growth of the organisms on each plate to each other? There are at least two answers to this question.
Why wouldn't it be advisable to compare growth of the organisms on each plate to each other? There are at least two answers to this question.
answer
On the NA plate, "good growth" for one organism may be very dense, whereas "good growth" for another might be fairly thin. In the context of this exercise you want to compare the amount of growth of each organism on the selective medium against its growth on NA to see if inhibition occurred. You wouldn't want to compare growth of the organisms on the EMB plate to each other because you wouldn't know which ones were inhibited and which were normal
question
EMB Test.
Would removing eosin Y and/or methylene blue from EMB agar alter the medium's sensitivity or specificity? Explain your answer.
Would removing eosin Y and/or methylene blue from EMB agar alter the medium's sensitivity or specificity? Explain your answer.
answer
Removal of one or both of these would alter specificity because organisms that "shouldn't" grow on EMB would, and additionally some of those might be able to ferment lactose to acid end products. It also would alter sensitivity. Although you would still probably be able to detect growth of organisms that "should" grow on EMB, your ability to detect color changes associated with lactose fermentation would either be lost or hampered, depending on whether both or only one are removed.
question
EMB Test
Suppose a mistake is made in preparing a batch of EMB agar and the starting pH is 7.6 instead of 6.9-7.3. Would that affect the medium's sensitivity or specificity? Explain your answe
Suppose a mistake is made in preparing a batch of EMB agar and the starting pH is 7.6 instead of 6.9-7.3. Would that affect the medium's sensitivity or specificity? Explain your answe
answer
If a batch of EMB were made at a higher than normal pH, it would take more acid production from lactose fermentation to produce the dark purple/green color of a positive. It is possible then, that a true positive for lactose fermentation might not change the medium's color in the allotted incubation time and would be incorrectly recorded as a negative. This would affect the medium's sensitivity.
question
EMB Test
Compare the recipes of nutrient agar and EMB agar. If an organism can grow on both media, on which would you expect it to grow better?
Compare the recipes of nutrient agar and EMB agar. If an organism can grow on both media, on which would you expect it to grow better?
answer
It would probably grow better on NA, which has beef extract (a rich mix of biochemicals) and peptone. Levine EMB supplies one carbohydrate and one protein derivative (gelatin) for carbon and nitrogen sources.
question
what is the ph indicator in mannitol sugar agar
answer
phenol red
question
phenol red colors
answer
yellow <6.8
red 7.4-8.4
pink at 8.4 and above
red 7.4-8.4
pink at 8.4 and above
question
what makes MSA differential
answer
mannitol because it provides the substrate for fermentation
question
what makes the MSA selective
answer
NA CL , bc it is concentrated high enough to kill most bacteria . but staphylococci thrive
question
what does phenol red indicate in MSA
answer
it indicates whether fermatation with acid end products take place
question
what is MSA used for
answer
isolation and differentiaion of staph A from pother staphylococcis
*not used to det. ability to isolate or ferment mannitol
*not used to det. ability to isolate or ferment mannitol
question
PG or NG MSA
answer
organism is inhibited by NACL - not staphylococcus
question
GG MSA
answer
org not inihibited by NACL
staphylococcus
staphylococcus
question
YG or halo MSA
answer
org produces acid from mannital ferm
possible pathogenic - SA
possible pathogenic - SA
question
red growth (no halo) MSA
answer
org does not ferment mannitol (not SA but a staph)
question
which ingredients in MSA supply C
answer
mannitol
question
which ingredients in MSA supply N
answer
beef extract
question
is MSA defined or undefined
answer
There are defined media. Be sure to know the difference. I think all we used were undefined. What makes a medium undefined is the presence of an ingredient that is itself a mix of nutrients, growth factors and the like. As Jane says, from one batch to another the amounts within those ingredients will change. They are really asking you to identify the undefined ingredients.
question
what is the role of sodium chloride in MSA and how does it work?
answer
sodium chloride makes the medium selective because its high concentration to dehydrate and kill most bacteria
question
what is the main fn of mannitol in MSA
answer
mannitol provides the substrate for fermentation and makes the medium differiential
question
what is the fn of phenol red in MSA
answer
phenol red indicates whether fermentation has taken place by changing colors as the pH changes
question
would removal of sodium chloride from MSA alter the medium's sensitivity or specificity? Explain your answer.
answer
It would alter the medium's specificity because you could not select for the Staphylococcus
question
Suppose a mistake is made in preparing a batch of MSA and the stating pH is 7.4 instead of 7.0-7.2. Would that affect the medium's sensitivity or specificity? Explain.
answer
Sensitivity is getting false negatives. We are looking for a color change due to acid production. If you start at a higher pH, more alkaline, then it will take longer (more acid) to change the color.
question
OF test purpose
answer
to differentiate bacteria on the basis of fermentation or oxidative metab of carbs
question
what does the OF test have a high sugar to peptone ration
answer
to reduce the possibility that alkaline products from peptone will neutralize the weak acids
question
bromothymol blue OF
answer
yellow 6.0
green 7.1
blue 7.6
green 7.1
blue 7.6
question
what is low agar concentration for OF
answer
...
question
what is low agar concentration for OF
answer
low agar cconcentration makes it a semisolid medium that also allows determination of motility
question
what is oil used in the OF test
answer
to promote anaerobic growth and fermentation -
question
Prepare samples and interpret the results of O-F Medium to test the oxidative/fermentative abilities of organisms.
answer
Sealed: green or blue = oxidation
Unsealed: any yellow = oxidation
Both: yellow throughout = oxidation and fermentation, slightly yellow at top = slow fermentation, green or blue = no fermentation
Unsealed: any yellow = oxidation
Both: yellow throughout = oxidation and fermentation, slightly yellow at top = slow fermentation, green or blue = no fermentation
question
OF Tubes that are (1) sealed and (2) unsealed and show yellow throughout
answer
1. O and F
2. F only
2. F only
question
OF Tubes sealed: show green
Tubes unsealed: show yellow on top/ green on bottom
Tubes unsealed: show yellow on top/ green on bottom
answer
ox only
question
OF sealed: yellow on top/ green bottom
Unsealed: yellow on top/ green bottom
Unsealed: yellow on top/ green bottom
answer
slow fermentors
question
tubes that show bliue or show no change in color for OF
answer
are not able to metabolize sigar
question
ox organisms ox the carb to
answer
CO2, H2o, E
question
Prepare samples and interpret results of the selective/differential medium MacConkey Agar/ what is the purpose of the MacConkey Agar?
answer
it is used to isolate and differentiate members of the Enterobacteriaceae based on the ability to ferment lactose
question
Understand the selective and differential aspects of MacConkey Agar.
answer
bile salts and crystal violet make it selective (inhibit Gram-positive bacteria)
neutral red makes it differential. lactose fermentation builds up acid, turning the fermenters red. Used to differentiate Enterobacteriacaea.
neutral red makes it differential. lactose fermentation builds up acid, turning the fermenters red. Used to differentiate Enterobacteriacaea.
question
How is MacConkey Agar modified to allow the study of Enterococcus spp. and Staphylococcus spp.?
answer
By not adding crystal violet. The colonies ferment lactose and appear pink.
question
What is the complete definition of a coliform?
What defines the family Enterobacteriaceae?
What defines the family Enterobacteriaceae?
answer
a coliform is a Gram-negative, non spore forming rod that can ferment lactose; is a member of the Enterobacteriaceae
a family of gram-negative, facultatively anaerobic, rod-shaped bacteria, usually motile; found in soil, water, and plants and in animals from insects to humans; Escherichia, Klebsiella, Enterobacter, Proteus,Providencia, and Serratia
a family of gram-negative, facultatively anaerobic, rod-shaped bacteria, usually motile; found in soil, water, and plants and in animals from insects to humans; Escherichia, Klebsiella, Enterobacter, Proteus,Providencia, and Serratia
question
which ingredient(s) in MacConkey agar supply(ies)
a) carbon
b) nitrogen
a) carbon
b) nitrogen
answer
a) lactose: fermentable carbohydrate providing carbon
b) casein and animal tissue: provides nitrogen
b) casein and animal tissue: provides nitrogen
question
what are the roles of crystal violet and bile salts in mackonkey
answer
Bile salts and crystal violet inhibit the growth of Gram positive organisms
question
what are the roles of neutral red and lactose in MacConkey agar?
answer
Lactose provides the sugar substrate for fermentation
- Neutral red dye is a pH indicator which turns red when acidic and is colorless when basic, which indicates the fermenters from non fermenters
- Neutral red dye is a pH indicator which turns red when acidic and is colorless when basic, which indicates the fermenters from non fermenters
question
growth on the MacConkey agar plates was recorded as "good growth," "poor growth," or "no growth." These are qualitative and, at least for the first two subjective terms. What did you use to establish what constituted "good growth?"
answer
good growth: organism is not inhibited by crystal violet or bile (Gram negative)
poor growth or no growth: organism is inhibited by crystal violet and/or bile (Gram positive)
pink to red growth with or without bile precipitate: organism produces acid from lactose fermentation (probably coliform)
growth is "colorless" (not red or pink): organism does not ferment lactose (noncoliform)
poor growth or no growth: organism is inhibited by crystal violet and/or bile (Gram positive)
pink to red growth with or without bile precipitate: organism produces acid from lactose fermentation (probably coliform)
growth is "colorless" (not red or pink): organism does not ferment lactose (noncoliform)
question
would removing bile salts and/or crystal violet from MacConkey agar alter the medium's sensitivity or specificity? Explain.
answer
Removing makes it no longer selective and reduces specificity; allows growth of gram positive
question
Nutrient Agar vs MacConkey
answer
Would grow faster on Nutrient Agar -- it contains both beef extract and peptone. Organism does not have to overcome nutrient sources
question
consider the controls of the oxidase-fermentation test:
a) what is the purpose of the uninoculated control tubes used in this test?
a) what is the purpose of the uninoculated control tubes used in this test?
answer
Two uninoculated control tubes are needed to show the results of the medium in both aerobic and anaerobic environments. It is used to show that the medium remains green under both conditions, showing it is sterile and also as a color comparison
question
consider the controls of the oxidase-fermentation test:
b) are the controls positive or negative controls? Explain.
b) are the controls positive or negative controls? Explain.
answer
negative.. a positive result would either turn the media yellow, indicating fermentation and an acidic environment, or it would turn blue, indicating deamination and an alkaline environment
question
consider the controls of the oxidase-fermentation test:
c) why is it necessary to use two controls rather than just one? what are the specific purposes of each?
c) why is it necessary to use two controls rather than just one? what are the specific purposes of each?
answer
one of the controls exhibits an anaerobic environment, and the other an aerobic environment
question
some microbiologists recommend inoculating a pair of OF basal media (without carbohydrate) along with the carbohydrate media. Why do you think this is done?
answer
Basal media (without carbs) allows you to see what the organism does with medium without the presence of the carbohydrate. Allows you to have an "active" negative control.
question
all enterics (Enterobacteriaceae) are facultative anaerobes; that is, they have both respiratory and fermentative enzymes. What color results would you expect for organism in O-F glucose media inoculated with an enteric? Remember to describe both sealed and unsealed tubes.
answer
Enteric bacteria are facultative anaerobes, meaning they are capable of both aerobic and fermentative metabolism. What results would you expect to see in both sealed and unsealed OF tubes with such a bacterium?
question
Suppose that when you examined your tubes after incubating them, you noticed that the unsealed control contained slight yellow at the top. Suppose further that pair 1 showed complete yellow of both tubes and pairs 2 and 3 showed slight yellowing of the unsealed tube. Assuming all other tubes were green, what conclusions could you safely make?
Which results, if any, are reliable? Why
Which results, if any, are unreliable? Why not
Which results, if any, are reliable? Why
Which results, if any, are unreliable? Why not
answer
Probably the inoculations made according to the directions in the procedure contained in the lab book. The procedure calls for three organisms to be inoculated. The slight yellowing in the unsealed control tube suggests there might be a contaminant (aerobic) in the media. This would make the slight change in the sets 2 and 3 questionable and probably the tests should be done again. Pair 1 shows that the organism is fermentative and possibly oxidative, due to the complete yellowing of both tubes. We found that the oxidative only organisms tended to show much more than "slight" yellowing, so having the control suggests that pairs 2 and 3 are negative, from our experience.
question
why is it important to perform the reagent tests last in the API20E test?
answer
Because you need to look for spontaneous reactions first. Also, if you splash reagents into adjacent cups you could change their color
question
spontaneous rxn
answer
doesnt require reagents
question
suppose after 24 hours incubation, you notice no growth in the tubes containing mineral oil. Assuming that it is behaving properly under these conditions, what do you know about the organism and what predictions can you safely make about its performance in the decarboxylase tests, fermentation tests, and nitrate reduction tests? Is it a member of Enterbacteriaceae?
answer
o, because Enterbacteriaceae are facultative anaerobes; therefore, it could behave under anaerobic conditions
question
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answer
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question
0
Understand why one tube of O-F medium is layered with sterile mineral oil and another left unsealed for each organism tested
Understand why one tube of O-F medium is layered with sterile mineral oil and another left unsealed for each organism tested
answer
test anaerobic vs aerobic
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