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112 Cards in this Set

  • Front
  • Back

What is a CFU?

Colony forming unit; is a single, well-isolated bacteria which grows as pure culture

What is MacConkey's Agar selective for?

MacConkey's agar is selective FOR Gram-negative bacteria.

What is a selective medium?

A selective medium has an inhibitory agent which prevents the growth of a specific type of organism, allowing for the growth of another.



What is MacConkey's agar used to differentiate?

An organisms ability to ferment lactose.

What does it mean if an organism can grow on MacConkey's agar?

They are bile-tolerant and crystal-violet tolerant.

Where is bile found in the human body?

Upper small intestine.

What are the inhibitory agents ("selective factors") in MacConkey's agar?

Bile and Crystal Violet

What is the substrate/differential agent of MacConkey's agar?


Lactose

What is the indicator in MacConkey's agar and how does it indicate? (What is a positive and negative result?)

Neutral red which is a pH indicator



Changes the color of colonies of bacterial growth to a dark red IF acidic conditions result from the fermentation of the sugar lactose.




NLF (non-lactose fermenters will have tan or colorless colonies

What is Columbia CNA agar is to select for?

CNA blood agar is used to select for most gram-positive bacteria.

What is the substrate/differential agent and how is the CNA agar used to differentiate?

Columbia CNA substrate: Blood


Differentiation is based on hemolysis (ability to lyse RBC's)

What are the selective factors (or inhibitory agents) in CNA agar?

Colistin and Naladixic acid (antimicrobials/antibiotics which inhibit many gram-negative bacterias)

What is an exception to the results one may interpret from CNA growth?

Pseudomonas can grow, despite gram-negative.

What are the types of hemolysis?

Gamma = none


alpha = partial


beta = complete

What does the carbohydrate fermentation broth series test? (CHO Series)

The ability to ferment a given carbohydrate into acid; whether their fermentation produces CO2

Describe the set up of the CHO series test?


What are the substrates?


Indicator?

The Carbohydrate Fermentation test consists of a Durham tube apparatus which is a tube full of a given carbohydrate (glucose, sucrose, lactose) and Phenol Red (pH indicator) with an inverted Durham tube inside.

What are the results of the Carbohydrate Fermentation test?

Red= Acid was produced by fermentation of that given carbohydrate


Yellow= Alkaline or neutral molecules produces from fermentation of carbohydrate. Or there was no fermentation.


Bubbles = CO2 produced

What are the results for Pseudomonas of the Carbohydrate Fermentation test?

All negative

What are three characteristics of enteric gram-negative bacilli? (Enterobacteriaceae)

They ferment glucose


They reduce nitrate (as the final electron receptor in anaerobia respiration)


They are oxidase-negative (lack cytochrome oxidase in ETC)

What can enterobacteriaceae be divided into?


What are examples in these groups?

lactose fermenters (coliforms): Escherichia, Enterobacter/Klebsiella.


non-lactose fermenters (non-coliforms): Salmonella and Serratia

What is the difference between Pseudomonas and Enterobacteriaceae?

Pseudomonas does NOT ferment glucose


Pseudomonas is oxidase-positive

What is different about Neisseria vs other Gram negative bacteria?

Neisseria is diplococcus


Neisseria is oxidase positive

What do the letters in the IMViC test stand for?

Indole test, the Methyl red test, Voges-Proskauer test, and the Citrate utilization test.

What are the results from IMViC for Escherichia verse Enterobacter/Klebsiella? Also what is Salmonella entericas?

Escherichia : (++--)


Enterobacter/Klebsiella: (--++)


Salmonella: (-+-+)

What is the substrate for the Indole test?

Indole test: Trypticase Soy Broth (TSB)

What is the Indole test testing?

An organisms ability to hydrolyze the amino acid tryptophan and produce indole. (The presence of Trytophanase)

Indole test process?


Results?

Inoculate TSB broth; allow incubation


Add Kovac's Reagent which combines with Indole to produce a red-colored phase on top of broth.

What is the substrate for MR and VP; what do they test; and what series are they a part of?

Substrate is MRVP broth which contains glucose. They test for the end products after glucose metabolism. They are part of IMViC series.

How does the MR test work and what are possible results? Explain the procedure.

Inoculate TSB broth; incubate

Use Methyl Red reagent to detect presence of acid after fermenation; turns red immediately


How does the VP test work and what are the possible results? Explain the procedure.

Inoculate TSB broth; incubate


Use Barritt's A (droperful) followed by droperful of Barritt's B. Pink results indicates presence of non-acidic compound acetoin which is formed through the butylene glycol pathway.

CItrate Utilization Test


Substrate?


Indicator?


Process/results?

Substrate: Citrate


Indicator: blue pH indicator


Inoculate and incubate. If citrate turns to royal blue, an alkaline end product was produced meaning is can utilize citrate.

What is the substrate for the BEA test?


What is the inhibitory agent?


What is the indicator?


What happens (what are the results?)?



Substrate: Esculin and sulfur


Inhibitory agent: Bile salts


Indicator: ferric citrate (black with esculetin)


Results: Growth = bile-tolerant. Black = positive for esculinase.


What can you determine with a positive growth result on the BEA test? What about a positive esculinase result?


What organisms are positive for growth and esculinase?


What is the exception to expected bacterial growth?

If growth: Organism is bile-tolerant so could be most gram-negative.

If black: Organism can use Esculin for gluocse source (releases esculin when it takes out the sugar)


Organisms: Serratia, Enterobacter/Klebsiella.


Exception: Gram-positive Enterococcus.

What is the exception to the BEA test?

Gram-positive Enterococcus can grow and is esculinase positive.

Which organism can grow on the BEA plate, is positive for esculinase, and is also DNase positive?

Serratia

What is an oxidase test confirmatory for?

Pseudomonas and Neisseria

How does the oxidase test work?

Tests for presence of oxidase (bacteria that perform aerobic respiration have hem-containing proteins called cytochromes which serve as final e- receptor.) is bacteria. Place bacteria on filter paper, then use BactiDrop Oxidase dropper (crush first) and drip on filter paper.

Oxidase test


Substrate?


Indicator?


Positive result?

Substrate: Cytochromes


Indicator: phenylenediamine


Positive result: Pink intermediate color which quickly turns red/purplish, then black. Means they have aerobic respiration.



Which gram-negative bacteria are oxidase positive?

Pseudomonas fluorescens and Neisseria sicca (means they use aerobic respiration)

What is the IMVic used to distiguish between?

Escherichia coli and Enterobacter/Kleibsiella

What does a positive result on the Indole test look like and what does it mean?

Red phase on top of TSB broth; means there is the presence of indole.

What does a positive MR test look like and mean?

Red after adding indicator; means there is acid present after glucose metabolism

What is an extra step that the VP test requires?


What does a positive result look like and what does it mean?

You must incubate VP test after adding both reagents at 37C for 30 minutes.


Pink color means positive for non-acidic products (acetoin) after glucose metabolism.

What is esculin?

A glycoside (sugar bound to alcohol)

Which is a presumptive test for Enterococcus faecalis?

BEA

What is being tested for in a Nitrate reduction test?

Ability to reduce nitrate (NO3) to either nitrite (NO2) or fully to ammonia (NH3) /nitrogen gas(N2).

What type of metabolism reduces inorganic molecules as the final e- acceptors?

anaerobic respiration

Nitrate Reduction Test

Substrate?


Reagents?



Substrate: Tryptic nitrate broth


Reagents: Sulfanili acid and Alpha-naphthylamine; Zinc powder

What are the possible results for the Nitrate reduction test?

If broth turns red after Sulfanilic acid and Alpha-naphthylamine are added, nitrite is present so bacteria has NITRATE REDUCTASE.




If broth does not change color and zinc is added, producing pink color, there is nitrate present, so there are NO enzymes for nitrate present.




If no color is noted there is no nitrate OR nitrite, so it must have Nitrate reductase AND Nitrite reductase; there is only NH3 or N2 left.

What is the Catalase test used to distinguish between microorganisms?

Streptococci/Enterococi and Staphylococcus/Micrococcus

What can be presumed about gram-positive cocci that appear in clusters and are catalase positive?

Staphylococci

What can be presumed about gram-positive cocci that appear in chains and are catalase negative?

Streptococci or enterococci

What is the Catalase test process?

Put Hydrogen Peroxide on a slide


Put loop of bacteria on slide


Produces bubbles? Catalase positive.

What is catalase?

An enzyme that speeds up the breakdown of H2SO2 to H2O and O2. (helps protect bacteria from harmful toxic forms of oxygen like peroxide)

In the lab, what do large bubbles during the catalase test indicate?


No bubbles?

Bubbles mean staphylococcus.


None means streptococcus or enterococcus.

How do we distiguish between Staphylococcus aureus (pathogenic) and Staphylococcus epidermidis?

What tests are used and which is which?



MSA (Mannitol Salt Agar) - S. aureus is positive for mannitol fermentation, S. epidermidis is negative.

What does MSA test for?


Substrate/inhibitory agent?


Differential agent?


Indicator?


Results?


Which bacteria is a positive result presumptive for?

Mannitol fermentation and salt-tolerance


Substrate: 7.5% Sodium chloride


Differential/substrate: Mannitol


Indicator: phenol red pH indicator


Results: Positive fermentation turns plate yellow (acidic)


Staphylococcus can grow but only Staphylococcus aureus is positive for both growth and mannitol fermentation.

What does hemolysis test for? What are the possible results (which bacteria)?

Test for prescence of cytolytic exotoxin


Staphylococcus aureus is beta


S. epidermidis is gamma

What are the results on the DNAse test between S. aureus and S. epidermidis?

Staphylococcus aureus has complete halo


Staphylococcus epidermidis has not complete halo.

What are the TWO reasons one might use DNAse test?

1: To distinguish between S. aureus and S. epidermidis


2: To observe Gram-negative enteric bacilli (Serratia, Salmonella, Pseudomonas)

What is a positive DNAse test?

Zone of clearing around streak line

DNAse test agar


Substrate?


Indicator?

Substrate is DNA


Indicator is pH indicator methyl green

Coagulase test is a confirmatory test for?

Staphylococcus aureus

What does coagulase test for?


Substrate?


What is positive and what is negative results?

Tests for ability of fibrinogin to form fibrin (clot/coagulatation) blood (presence of coagulase).


Substrate is rabbit blood plasma.


Positive results: tube is solid


Negative results: tube is liquid.

What is a common disease caused by Staphylococcus aureus?

Bacteremia


PNA


osteomyelitis

What does halotolerant mean?

Salt tolerant

What is the presumptive test for Staphylococcus aureus? The confirmatory test?

Presumptive: MSA


Confirmatory: Coagulase.

BAP test


Is BAP selective or differential?


Substrate?


Possible results and interpretation?

BAP is differential (has enriched media so not very selective).


Substrate is 5% sheep's blood


Beta hemolysis- complete clearing: Streptococcus pyogenes, staphylococcus aureus


Alpha hemolysis- partial clearing (greenish hue): Streptococcus pneumonia, some Enterobaccoccus faecalis


Gamma hemolysis- no clearing: Enterococcus/streptococcus faecalis

What is the Taxo A and Taxo P refer to? What test are they added to? Which bacteria do they suggest?

Taxo A is Bacitracin; Streptococcus pyogenes is sensitive to this.


Taxo P is Optochin; Streptococcus pneumonia is sensitive to this.



What is the first test used to distinguish between Stretococcus? Which have which results?

BAP:


Streptococcus pneumonia is alpha


Streptococcus pyogenes is beta


Streptococcus faecalis is gamma

What results are useful for determining Enterococcus feacalis?

BEA positive (black plate)


Utilyzes ferric citrate


Esculinase + ferric citrate = black precipitate


Resistant to Taxo A and P

What are throat culture tests?



BAP


MacConkey's

What does a BAP test performed on a throat swab indicate?

Alpha hemolysis indicates Staphylococcus salivarius or streptococcus mitis; most of a healthy person




Beta hemolysis is Streptococcus pyogenes (just a few may still be healthy)



What does a throat culture which has MacConkey growth show?

Ingestion of contaminated food.

What is the maximum lethal wavelength for UV? (Don't forget units!)

265nm

What UV wavelength was used in class?

257.3nm

What bacteria did we use to study UV effects?

Serratia marcessans

How does UV affect DNA?

Creates pyrimidine dimers; thymine dimers and cytosine dimers.

Spell what T stands for (DNA)

Thymine

Spell what C stands for (DNA)

Cytosine

Spell the bacteria we use to study UV radiation in class.

Serratia marcessans

What three things affect UV's lethality on a bacteria?

Distance from light source, length of exposure, and directness of contact.

What are the six methods of disruption for antiseptics/disinfectants?

Inhibition of cell wall synthesis


Inhibition of protein synthesis


Inhibition of pathogenic attachment to or recognition of, host.


Disruption of cytoplasmic membrane


Inhibition of DNA/RNA synthesis


Inhibition of general metabolic pathways

Re: the six methods of action for microbial control, which does heat affect?

Heat inhibits protein synthesis which also inhibits general metabolic pathways.


Re: the six methods of action against microbes, what does UV light do?

Inhibition of DNA synthesis, etc. by forming thymine dimers and cytosine dimers.


What is the irreversible shape change of an enzyme or protein called?

denaturation

Re: the six methods of action against microbials, what do chemical reagents do?

Inhibit cell wall synthesis (alters integrity),


Disrupt cytoplasmic membranes (alters permeability making it leaky or unable to transport across the cell envelope),


Can mess with the DNA, directly affecting metabolism and reproductivity



What is the difference between an antiseptic and a disinfectant?

Antiseptic is safe for human tissue, disinfectant is for nonliving surfaces

What does it mean for a bacterial cell to be viable?

It can survive and reproduce.

Which type of bacteria are more sensitive to chemicals?

Gram positive because they do not have a lipid outer membrane to help protect them.

Why are gram negative cells more resistant to chemicals?

Because they have a lipid outer membrane.

What is the zone of inhibition measured in?

mm

What are the (4) types of disinfectants and what is an example of the ones used in the laboratory?
Alcohol: Ethanol (isopropyl?)

Phenolics: Lysol


Halogen: Sodium hypochlorite (Bleach!)


QUATS: Quaternary ammonium compounds (Zephiran chloride)

What is the definition of TDP?

The temperature at which a bacteria is killed after 10minutes of exposure and 7pH

What is thermoduric?

Resistant to heat

Which gram type is more resistant to heat and why? What does this mean about their TDP?

Gram positive are generally more resistant because of their high peptidoglycan content in their membrane; also they may have endospores which protect even more; this means that their TDP will be higher, on average, than a gram negative MO.

What are gram positive cells more resistant to heat?

They have a high peptidoglycan membrane content.

What is the difference between thermoduric and thermotolerant?

None. They both mean heat tolerant organisms.

What is the definition of an exoenzyme?

An enzyme that functions outside of the cell

What is the difference between a catabolic exoenzyme and a degraditive exoenzyme?

Catabolic exoenzymes break down large molecules into smaller ones that can be used for nutrients. The degradative exoenzyme breaks down large molecules into pieces that can be used as weapons, like virulence factors.

What are the (5) different exoezymes we measured in lab?

Caseinase


Amylase


Lipase


Gelatinase


DNAse

Caseinase:

Substrate?


Enzyme?


Reaction occurring in the media?


How to interpret the results?



Skim Milk Agar


Caseinase


Casein + H2O = Peptides and Amino Acids


(+) means that a zone of clearing is seen surrounding the streak

What reaction does Caseinase promote?

Casein + H2O = peptides and A.A

Lipase:


Substrate?


Enzyme?


Reaction occuring in the media?


How to interpret results?

Spirit Blue Agar (which is olive oil and spirit blue dye)


Lipase


Lipids +H20 = Glycerol and fatty acids


(+) is a zone of clearing around streak; may also see blue precipitate

What is the reaction that lipase catalyzes?

Lipid + H2O = glycerol and fatty acids

Amylase:


Substrate?


Enzyme?


Reaction occurring in the media?


Interpret results?

Starch Agar


Amylase

Starch + H2O= maltose(x2 glucose) + glucose + dextrins

After incubation, plate is flooded with iodine which binds to starch to create a blue/black color. No starch = no color change.


(+) clearing around streak while the rest of the plate turns dark purple




Triglycerides are...

lipids

Starch is a...

polysaccharide

Amylase reaction?

Starch + H20 = maltose, glucose and dextrins

What is a proteolytic enzyme? Which do we examen in class?

One that breaks proteins down into smaller polypeptides or amino acids. Gelatinase and Caseinase.

Gelatinase

Substrate?


Enzyme?


Reaction occurring?


INterpretation?



Substrate: Nutrient gelatin agar deep


Gelatin + H20 = peptides and amino acids


TUBE is inoculated by a tabbing, straight in and out. After 48 HOURS, if gelatin is liquid, organisms is positive for gelatinase.

Reaction for gelatinase?

Gelatin + H20 = peptides and amino acids