Microbio Lab 3&4 Exam 1

what is the terminal electron acceptor in aerobic respiration? what is it reduced to?

oxygen (O2)

water (H2O)

what does aerobic respiration generate as byproducts

reactive oxygen species (ROS)

and hydrogen peroxide (H2O2)

what does hydrogen peroxide do as a byproduct

it is a potent oxidizing agent that can harm cellular components like enzymes, DNA, RNA, and lipids by causing oxidative damage

strict (obligate) aerobes

bacteria that require oxygen for growth and rely exclusively on aerobic respiration for energy production

they cannot survive or grow without O2

facultative anaerobes

bacteria that grow optimally with oxygen using aerobic respiration but can switch to fermentation or anaerobic respiration (using alternative electron acceptors) in its absence

allows growth with or without O2

aerotolerant anaerobes

bacteria that do not use oxygen and rely solely on fermentation for energy BUT can tolerate its presence and grow in environments with or without O2

strict (obligate) anaerobes

bacteria that cannot tolerate oxygen as it is toxic to them

they grow only in oxygen-free environments using fermentation or anaerobic respiration

how do bacteria combat oxidative damage from H2O2 and other ROS

through detoxifying enzymes

what do strict aerobes and facultative anaerobes produce to combat oxidative damage

catalase (catalyzes the decomposition of H2O2 into harmless water and oxygen)

protects cells during aerobic metabolism

what do aerotolerant anaerobes produce to combat oxidative damage

non-catalase peroxidases (NADH peroxidase) that reduce H2O2 to water using electron doners so they can survive in oxygenated environments not using O2 for respiration

do strict anaerobes combat oxidative damage?

not really

they lack catalase and peroxidases making them highly sensitive to H2O2 and unable to survive in aerobic conditions as they cannot enutralize effects of ROS

superoxide dismutase (SOD)

converts superoxide radicals into H2O2 and O2 to help aid oxygen tolerance

aerotolerant anaerobes typically have SOD but rely on?

peroxidases rather than catalase for H2O2 breakdown

describe catalase test

involves adding 3%H2O2 t bacterial sample

rapid bubble formation (O2 release) = catalase presence

describe oxidase test

determines if bacterium produces cytochrome C oxidase (enzyme in electron transport chain)

artificial electron acceptor will change from yellow to blue when electrons are transfered from reduced cytochrome C.

positive result means that bacteria are aerobic or facultative

results must be determined before or at 30 seconds

describe KOH test

Gram negative cells hae thin PG layer and will lyse in 3%KOH solution

gram neg = stringy lyse form

gram pos = no lyse present, stay the same

NOT a replacement for Gram stain technique, used to verify Gram stain

selective medium

a media that wil allow only certain groups of microorganisms to grow and will inhibit growth of other microorganisms

ex: incorporation of NaCl into mannitol salt agar (MSA) selects for genus Staphylococcus but inhibits bacteria that cant tolerate salt conc.

differential medium

contains substances that cause some bacteria to take on appearance that distinguishes them from others

ex: when staphylococcus aureus grows on MSA it ferments mannitol making acid and changing pH indicator media from red to yellow

ex:sheep blood agar (SBA) some species lyse RBCs in blood agars

what happens in MSA when there is acid present

changes form red to yellow around colonies

describe lysis in SBA

complete lysis = beta hemolysis

partial lysis and greenish color = alpha hemolysis

no lysis = gamma hemolysis

what is produced if an organism can break down carbs by oxidation or fermentation

acid

describe phenol red carbohydrate tubes

presence of acid is indicated by color change from red 9alkaline) to yellow (acidic)

type of differential media

what does starch macromolecule consist of

amylose: straight chain polymer

amylopectin: larger branched polymer

what do bacteria that hydrolyze starch produce

amylases that degrade starch into maltose, glucose, and dextrins

how can starch hydrolysis be detected

adding Gram’s iodine into starch agar plate (differential medium)

if starch on medium has been degraded by amylase produced, the starch agar will be clear adjacent to the growth after addition of iodine

bile esculin agar

selective and differential medium

used to identify Enterococcus and group D streptococcus on ability to hydrolyze esculin

ex: if organism hydrolyzes eculin a dark brown/black is produced on the medium (more than half has to be this color to be positive)

coagulase test

confirms identification of staphylococcus aureus

inoculate tube of blood plasma with bacteria

plasma coagulates = coagulase postive

this is only valid for Gram positive

describe BHI with 6.5% NaCl

differentiates microorganisms based on ability to grow

inoculate BHI broth and organism for 24-48 hours

positive = growth )turbidity)

negative = no growth (clear)

describe bile esculin hydrolysis

differentiates microorganisms on ablility to hydrolyze esculin

use loop to inoculate media in zig zag pattern up the slant

positive = blackening of tube

negative =. no color change (or less than half is black)

describe catalse test

differentiates based on ability to produce catalase enzyme

use loop to transfer 3-5 colonies onto microscope slide and add H2O2 on top

positive = bubbles

negative = no bubbles

describe the coagulase test

detection of coagulase that is excreted by S, aureus causes fibrin clot

use loop to inoculate loopful of it into coagulase (rabbit plasma)

positive.= coagulation of plasma (clot form)

negative: no coagulation

describe hemolysis on SBA

purpose: differentiates based on hemolytic activity on SBA

incubate for only 24 hours

gamma: no hemolysis or discoloration

alpha: incomplete hemolysis or greening

beta: complete hemolysis or clearing

describe KOH tst

confrims gram stain

Gram positive: String absent

Gram negative: strings present

describe MSA test

selects microorganism on ability to grow in this agar

positive: growth +/ acid + yellow colonies

negative: growth +/acid - or growth -/acid 0 colorless (red)

describe nitrate reduction test

differentiates on ability to reduce nitrate to nitrite

incomplete positive: red coloration after the addition Nitriate A and B reagents

complete positive: no color change after addition

negative: red coloration after addition of zinc

describe oxidase test

Differentiates microorganisms based on the presence of the enzyme cytochrome C oxidase in bacteria using oxidase reagent containing N,N,N′,N′- tetramethyl-p-phenylenediamine

Positive: Any blue to purple color change within 30 seconds

Negative: No color change or blue color within 30 seconds

describe phenol red carb test

differentiates organisms based on carb fermentation

positive: yellow color (fermentation)

negative: red color

describe starch hydrolysis test

differentiates based on ability to hydrolyze starch

positive: ddark agar with clearing around line

negative; dark agar to the streak line