microbio lab quiz lab 16-27 Flashcards
1
Q
Cytochrome oxidase test (Lab 16)
A
- The heme containing enzyme Cytochrome oxidase is the terminal redox protein used in the electroon transport chain of microorganisms that use oxygen as their terminal electron acceptor
- used a chromogenic reducing agent as an indicator to detect bacteria that produce cytochrome oxidase
- indicator = p-aminodimethylaniline oxalate
- used to differentiate strict aerobic respiratory microorganisms from facultative or fermentative microorganims
- used to differentiate the oxidase negative enterbacteriaceae from the oxidase positive pseudomonadaceae
2
Q
catalase test (Lab 17)
A
- distinguish members of the catalase-postive staphlococcaceae and micrococcaceae from the catalase-negative members of the streptococcaceae
- the chemical superoxide dismutase is used to convert superoxides to peroxides and the chemical catalase is able to convert peroxides to water and molecular oxygen
- The catalase test is used to detect the presence of catalase enzyme by the decomposition of hydrogen peroxide to release oxygen and water as shown by the following reaction: 2 H2O2 → 2H2O + O2 The catalase reaction is evident by the rapid formation of bubbles
- flavoprotein
- add H2O2 to slide smear from a single colony
- H2O2 is broken into water and molecular oxygen if bubbling occurs. this is good b/c H2O2 is toxic to cells
- Bubbling on slide = catalase +
- No bubbling on slide = catalase -
3
Q
Indole production from tryptophan hydrolysis (Lab 18)
A
- measures bacteria’s ability to break down tryptophan to indole
- indole test is considered an enzyme assay b/c enzyme assay’s measure enzyme activity and the indole test is measuring tryptophan (an enzyme / amino acid)
- indole is a waste product produced when tryptophan is broken down
- add Kovac’s reagent, mix. Kovac’s reagent reacts w/ indole to produce a red/pink color
- No color change = indole -
- Pink line on top of tube = indole +
- differetiate members of the enterobacteriaceae
4
Q
Sulfide production in SIM medium (Lab 19)
A
- used in the detection of the enzymes involved in H2S gas production by certain members of the family Enterobacteriaceae
- also used to check for the presence of motility through the Semi-solid agar
- Pathway 1
reduction of amino acids that contain sulfur like cysteine (found in peptone) using cysteine desulfurase (bacteria capable of decomposing cysteine are present in anaerobic environments) - Pathway 2
reduction of inorganic sulfur (sodium thiosulfate) compounds using thiosulfate reductase - FeSO4 combines w/ the gas to form black sulfide precipitate
- Note: black precipitate is seen at the bottom of the test tube b/c sulfur reduction is an anaerobic process
Black precipitate = hydrogen sulfide +
No black precipitate = hydrogen sulfide -
5
Q
Gelatin Hydrolysis (Lab 20)
A
- Gelatin is a polymer gel composed of interlocking strands of collagen.
- Gelatinases are members of a group of extracellualr enzymes that degrade collagen into individual amino acids that can then be taken into the cell and used as a source of energy or used for biosynthesis
- when gelatinase positive bacteria are stab inoculated into this medium they decompose the gelatin and cause the medium to liquefy
- This reaction is a two step process in which the gelatin is first de-polymerized, yielding isolated peptides. The peptides are then hydrolyzed to yield individual amino acids
- the reaction is slow and may take 7 days at 37C to complete liquify the medium
- Liquifaction = gelatinase + (aka hydrolysis occurred)
- Solidification = gelatinase - (aka hydrolysis DID NOT occur)
6
Q
Urease Test (Lab 21)
A
- determines an organism’s ability to degrade urea using urease (ammonia is basic, released when urea is degraded)
- indirect test for the presence of an enzyme (urease) b/c it is examining pH change, indicating whether or not a large amount of ammonia is present, indicating urea concentration.
- urease broth contains phenol red indicator (only large pH changes are observed b/c of buffering)
- observe color change of broth
- organism can degrade urea = dark pink, basic
- organism CANNOT degrade urea = yellow, acidic
7
Q
Amylase Test (Lab 22)
A
- used to detect positive members of the genera Bacillus, Clostridium, Bacteroides and the group D streptococci
- tests an organisms ability to produce amylase, which is broken down by starch
- Starch: polymer of D-glucose. broken down by the enzyme amylase
- flood starch agar plate w/ iodine for 30 secs (iodine stains starch)
- starch agar breaks down if amylase is present
- clearing around bacterial growth = amylase +
- NO clearing around bacterial growth = amylase -
8
Q
Nitrate reduction Test (Lab 23)
A
- performed to assess the ability of certain, primarily facultative anaerobes to utilize various nitrogen compounds in the generation of energy
- scenario 1
1. Tubes incubated
Results:
A. tube shows gas production/bubble: Nitrate reduced to N2O, NO, or N2 (aka nitrogen gas is the end product - make sure this is the case aka verify that the gas is not actually another end product of fermentation. do this by adding solutions A+B and adding Zinc and if there is no change for either then it is verified)
B. tube shows no gas production/no bubble
- Scenario 2
2. Nitrate reagents added to tube (A+B added)
Results:
A. tube turns red: Nitrite produced (aka Nitrate was converted to Nitrite)
B. tube DOES NOT turn red (aka Nitrate was NOT converted to Nitrite)
- Scenario 3
3. Zinc added to tube
Results:
A. tube turns red: no Nitrate reduced
B. tube DOES NOT turn red: Ammonia produced
9
Q
Carbohydrate Fermentation (Lab 24)
A
- buffers are used which will buffer the medium against a transition to the pH of a positive test result. Only those organisms that produce enough acid or base to overcome the buffering capacity of the medium will be able to alter the medium pH and change the color of the indicator
- Reversion (this is the production of a base after acid has been produced by fermentation.
- use a phenyl red broth (Lactose, glucose and mannitol)
- phenyl red, the indicator in this medium will turn the medium yellow when acid production by fermentative bacteria reduces the pH below 6.4
10
Q
Methyl Red Vogus-Proskauer Test (Lab 25)
A
- used when discriminating members of the family Enterobactericeae, such as Escherichia and the closely related enteric genera klebsiella and enterobacter
- This test is used to determine which fermentation pathway is used to utilize glucose
- Methyl red - determines an organisms ability to oxidize glucose to make acid end products
- Voges Proskauer - identified organisms able to produce acetoin from the degradation of glucose from 2,3-butanedoil fermentation
11
Q
Citrate Utilization (Lab 26)
A
- ability to take citrate into the cell and use it as a sole source of organic carbon
- sodium citrate as the sole carbon source
- ammonium phosphate as the sole nitrogen source
- determines organism’s ability to use the enzyme citrate to break down citrate as a sole carbon source
- growth + blue = citrate utilized as a carbon source
- no growth + green = citrate NOT utilized as a carbon source
12
Q
Litmus milk test (Lab 27)
A
- makes use of this complexity to provide the opportunity for multiple biochemical evaluations with a single medium
- differentiate between organisms that can transform milk substrates into various end products
* There are four basic reactions: lactose fermentation, litmus reduction, casein coagulation and casein hydrolysis - Litmus: pH indicator in the litmus milk medium
- litmus reduction: fermentation requires an e- acceptor, litmus acts as the acceptor
- litmus fermentation: organisms that use B-galactosidase to degrade lactose for use as a carbon source (results in lactic acid production, which turns litmus pink)
- gas production: fermentation may produce gaseous end products seen in the tube as cracks or fissures
