Micro Lab Quiz 4
What are the substrates of sucrase?
(Fig 5-3, Pg 246) sucrose
What are the products of beta-galactosidase?
(Fig 5-3, Pg 246) glucose and galactose
How does amylose and amylopectin differ?
Amylopectin (branched) contains polysaccharide side chains connected to approx. every 30th glucose in the main chain and a glucose is branched creating 1,6-alpha-glycosidic linkage (FIG 5-39)
What is a lactose fermenter?
An organism that splits the disaccharide lactose into the monosaccharides glucose and galactose and then ferments monosaccharides
[Citrate Test] With the presence of citrate-permease enzyme, what happens?
Citrate enters the cell and is converted to pyruvate
What does catalase do?
Converts hydrogen peroxide into water and gaseous oxygen
When flavoprotein transfers electrons directly to the final electron acceptor, hydrogen peroxide is produced. What other consequences might result from electron carriers in the ETC being passed?
Creates highly potent cellular toxin: superoxide radical Less H+ being pushed to create a sufficient gradient and decreases production of ATP
[Oxidase test] No color change to blue within 20 seconds? Bacteria example?
Cytochrome c oxidase is not present (-) Example: Enterobacteriaceae
[Oxidase test] Dark blue within 20 seconds interprets? Bactera example?
Cytochrome c oxidase is present (+) Example: Neisserria and Pseudomonadaceae
What is Urea a product of?
Decarboxylation of certain amino acids
PR glucose broth: pink broth, no bubble in tube
Degradation of peptone; alkaline end products (K)
What is the SIM medium used for?
Determination of three bacterial activities: sulfur reduction, indole production from tryptophan, and motility
What is the use of the Citrate Test?
Differentiates members of Enterobacteriaceae, all of which are facultative anaerobes They have the ability to ferment carbs, but use CAC to respire
What type of sugar is sucrose?
Disaccharide derived ffrom glucose and fructose
What type of sugar is lactose?
Disaccharide sugar derived from galactose and glucose
What is the purpose of MR-VP?
Dual-purpose medium that tests an organism's ability to follow either or both of the two specific fermentation pathways
[SIM] How would you expect this result to change if you eliminated the ferrous ammonium sulfate?
Eliminating ferrous ammonium sulfate can result in a false negative
What is the purpose of Snyder Test medium? pH of medium?
Favor growth of oral bacteria and discourage growth of other bacteria medium pH is 4.8
What is Phenol-Red broth general purpose?
Fermentation of medium containing on of several carbohydrates (glucose, lactose, sucrose) and a pH indicator to detect acid formation
No color change (or development of copper color) of a VP test indicates?
Negative
Starch hydrolysis + iodine = not clear, means?
Negative
What does no clearing of iodine around the organism in a starch hydrolysis test indicate?
Negative for amylase and starch hydrolysis
If the control is solid and a tube is also solid, is it acceptable to read the result before the complete incubation time has elapsed? Why?
No, because the inoculated tube may need more incubation time to break down
How can gelatinases be detected in gelatin hydrolysis test?
Nurtrient gelatin which is composed of gelatin, peptone, and beef extract
[(Fig. 5-5) MR-VP]: What are three acids that can be produced by fermentation of pyruvate?
Acid, gas, alcohol ***CORRECT?***
What kind of pH does oral bacteria prefer?
Acidic (around 4)
What two enzymes break the glycosidic linkages between sugar subunits?
Alpha-amylase (breaks down linear form) Oligo-1,6-glucosidase (breaks down branched form)
Suppose you could selectively prevent production of α-amylase or oligo-1,6-glucosidase in an organism that normally hydrolyzes starch. Which enzyme would the organism miss the most and why?
Alpha-amylase because it causes hydrolyzation and is responsible for breaking down food
What can Urea be hydrolyzed into? Bacteria example?
Ammonia and CO2 by bacteria containing the enzyme urease Example: Most enteric bacteria possess the ability to metabolize urea
What is the products of urease?
ammonia (2NH3) and CO2
[SIM] What does the enzyme cysteine desulfurase do? And what does it produce?
catalyzes putrefaction of the amino acid cysteine to pyruvate (Fig 5-54, Pg 323) Produces: H2S (hydrogen sulfide)
[SIM] When H2S combines with ferrous ammonium sulfate (iron), what color is produced for negative result?
no blackening/color change no sulfur reduction
What is an indole-test negative result?
no color change/clear at the top of medium
What does a urease-negative result look like?
orange or yellow
What does a urease-positive result look like?
pink
What is an indole-test positive result?
pink at the top of medium
What color is phenol-red above pH 7.4?
pink to magenta
What indicates a VP(+) result?
red color
What is the substrate of urease?
urea
What does the SIM medium include as source of amino acids?
casein and animal tissue
The only nutrients in Urea Broth:
Urea and a small trace of yeast extract
What enzyme is used for gelatin hydrolysis test?
gelatinases
What are the substrates of beta-galactosidase? (Fig 5-3, Pg 246)
lactose
PR glucose broth: yellow broth, bubble in tube
- Fermentation with acid and gas end products (A/G)
PR glucose broth: yellow broth, no bubble in tube
- Fermentation with acid end products; no gas produced (A/-)
PR glucose broth: red broth, no bubble in tube
- No fermentation (-/-)
What was the VP test designed for?
- Organisms that are capable of fermenting glucose, but quickly convert their acid products to acetoin and 2,3-butanediol
What does the MR test designed to detect and how?
- Organisms that are capable of performing a mixed acid fermentation - They overcome the phosphate bufferr in the medium and lower the pH
What does MR-VP contain? (ingredients)
- Peptone (protein) - Glucose (carbs) - Dipotassium Phosphate (buffer)
What does microbial fermentation produce?
- gas (CO2) - acid (lactic) - alcohol (ethanol)
What is the reason for indicator dye and what are examples?
- phenol red - bromcresol purple - bromthymol green They indicate color change when pH changes and each dye operates best in specific pH range (helps in identifying type of organism)
What is starch hydrolysis?
- presence (+) and absence(-) of amylase
[Skin and Throat handout] List three anatomical sites that normal flora is found
1. skin 2. mouth/teeth 3. eye conjunctiva
Catalase mediated conversion of H2O formula
2H2O2 --catalse--> 2H2O + O2(g)
[Gelatin Hydrolysis Test] How long of an incubation period is usually sufficient to see liquefaction of the medium?
7 days
How is a positive result of catalase slide test determined?
Bubbles are formed with the use of hydrogen peroxide
How is mixed acid fermentation verified?
By adding methyl red as an indicator dye following incubation
[SIM] How can hydrolysis of tryptophan be detected?
By the addition of Kovac's reagent DMABA. The formation of red in the reagent layer indicates positive reaction (+ of tryptophanase)
[SIM] How is indole production in the medium made possible?
By the presence of tryptophan (contained in casein and animal protein)
If given the Simmons Citrate agar medium recipe, determine the carbon and nitrogen sources in the media
Carbon source: sodium citrate Nitrogen: ammonium dihydrogen phosphate
[Snyder] What are the carbon and nitrogen sources?
Carbon: yeast extract, casein, and dextrose Nitrogen: yeast extract and casein
[SIM] What does the enzyme thiosulfate reductase do? And what does it produce?(Fig 5-55, Pg324)
Catalyzes the reduction of sulfur (in the form of sulfate) at the end of the anaerobic respiratory ETC Produces: H2S (hydrogen sulfide)
Some microbiologists recommend reincubating organisms producing methyl red negative results for an additional 2-3 days. Why do you think this i done?
Gives the organism time to ferment and prevents a false negative result
[SIM] What do bacteria possessing the enzyme tyrptophanase do?
Hydrolyze tryptophan to pruvate, ammonia, and indole (Fig 5-58, Pg 324)
What does the VP test identify?
Identifies bacteria that are capable of producing acetoin which will produce diacetyl giving a red color (+)
[Skin and Throat handout] Explain what blood agar tests are used for
Inoculated to demonstrate the alpha-hemolytic and beta-hemolytic reactions of some steptococci and staphylococci
[Skin and Throat handout] Analyze why sabaroud agar is selective media
Inoculated to detect yeasts and molds Yeast: develop pigmented/non-pigmented colonies that are elevated, moist, and glistening Mold: appear fuzzy, powdery growths arising from a mycelial mat in the agar medium
What is the copper color in VP test a result of?
Interactions between reagents and should not be confused for a positive result
What is gelatin and what is derived from?
It is a protein derived from collagen- a component of vertebrate connective tissues
What color is methyl red at pH 4.4 and what does it indicate?
It is red; indicates positive result
What is the purpose of the Oxidase test?
It is used to identify bacteria containing respiratory enzyme cytochrome c oxidase
What is a slight disadvantage of nutrient gelatin?
It melts at 28C, therefore inoculated stabs are typically incubated at 25C
Why were you told to shake the VP tubes after the reagents were added?
It requires oxygen and gives a better indicator of color change
In many tests it is acceptable to read a positive result before the incubation time is completed. Why is this not the case with Starch agar?
It takes time for the starch to breakdown if amylase is present
Why is the MR test read immediately?
It tells us if it is basic of acid
[Snyder] What bacteria survive these harsh conditions and what do they do?
Lactobacilli and oral steptococci, ferment glucose and lower the pH even further
What two forms does starch exist as?
Linear (amylose) and branched (amylopectin)
What does gelatinase-positive organisms do to medium? Bacteria example?
Liquefy the medium Example: Staphylococcus aureus, Serratia, and Proteus
How is alcohol indicated with fermentation?
Liquid and colorless. They do not ionize in H2O and have minimal effect on pH of medium
What is carbohydrate fermentation?
Metabolic process by which an organic molecule acts as an e- donor (oxidized) and one or more of its organic products act as the final (or terminal) e- acceptor
Considering, again, the Snyder Test, what kinds of dietary items would likely increase the number of lactobacilli in saliva?
Milk products like cheese and yogurt
What does the MR test detect?
Mixed acid fermentation
What kind of pH do enteric bacteria prefer?
More neutral environment around 7
[Urea] Typically, this medium is filter-sterilized because autoclaving breaks down the urea. Even unsterilized broth, however, rarely produces false-positive results. Why do you think this is true?
Only a few species produce urease
Starch hydrolysis + iodine = clear, means?
Positive
What does clearing of iodine around the organism in a starch hydrolysis test indicate?
Positive result for amylase and starch hyrdolysis
What are considered rapid urease-positive organisms? What color indicates and how fast?
Proteus Morganella Providencia Pink color in less than 24 hours indicates rapid urease-positive organism
What is a VP positive result?
Red
[Starch Hydrolysis] How would you expect the results of this exercise to change if you were to add glucose to the medium.
Since few bacteria can rely on starch as a carbon source to grow, adding glucose will make it hard to differentiate between bacteria since most bacteria can use glucose as a carbon source
What is starch and what is it made up of?
Starch is a polysaccharide and made up of alpha-D-glucose subunits
[Starch Hydrolysis] Suppose you had poured iodine on your plate and noticed clearings in the uninoculated area, as well as around both of your transferred cultures. What are some possible explanations for this occurrence? Was integrity of the exercise compromised? What kind of things might be done to avoid this problem in future exercises?
The plate could have been contaminated before the experiment. Also the starch could not have been mixed well on the plate. You can conclude that the experiment is invalid and needs to be redone
How can you indicate a gas is created with fermentation?
The use of a Durham tube in broth media (bubbled within tube)
What does gelatinase-negative organisms do to medium? Bacteria example?
They do not secrete gelatinase and do not liquefy the medium Example: Staphylococcus epidermidis
What is reagent iodine used for in starch hydrolysis?
Used as an indicator for the presence or absence of starch in the vicinity around the bacterial growth
Why is the VP test read after 60 minutes?
VP takes time for a chemical to react; looking for acetoin production
What color is negative in urea test?
Yellow or orange
What color is methyl red at pH 6.2 and what does it indicate? What about the color orange?
Yellow; indicates negative result Orange indicates negative as well
[Gelatin Test] If the control is solid and an inoculated tube is liquid, is it acceptable to read the result before the complete incubation time has elapsed? Why or why not?
Yes because gelatinase was present to liquefy the inoculated tube and it wasn't the temperature that liquefied it
[SIM] When H2S combines with ferrous ammonium sulfate (iron), what color is produced for positive result?
black
What does the SIM medium have as iron-containing?
ferrous ammonium sulfate
What are the products of sucrase? (Fig 5-3, Pg 246)
glucose and fructose
[Snyder] What color is the medium at or above 4.8?
green
What indicates an MR(+) result?
the color red
Why is it advisable to perform the catalse test on a known catalase-positive organism along with the organism you are testing?
the known catalase-positive organism serves as a positive control to ensure the peroxide reagent is good
What color is phenol-red below pH 6.8?
yellow
[Snyder] What is the color of the medium below 4.8? What does it indicate?
yellow; indicates fermentation and highly suggestive of presence of dental decay-causing bacteria
