Microbio LAB 3 – Flashcards

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thick slime layer of a bacteria is called what? 2

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capsule or
glycocalyx
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Why are Special techniques have to be used to stain a capsule?
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b/c capsule is damaged with heat fixxing,  capsules also repel most stains
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capsule staining begins with what? cells are spread in a film with what? and are not What?

 

 

what kind of dye are used for capsule staining?

 

what do they appear like on microscope?

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capsule stain- negative stain

 

on acidic film-no heat fixing

 

indian dye

 

halo

 

afterwards. similar neg staining protocol

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resting stages of genera bacillus and clostridun, it's function
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;resting stages called endospores when they exhaust essential
nutrients.
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what function does endospre serve
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dorment stage of in bacterial help protect against enviromental conditions not suitable for growth

;

--> making stains hard to penetrate keratin of cell wall

 

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terminal
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end of cell
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central
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middle
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subterminal
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b/w terminal and middle
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two different kinds of shapes
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Spherical or elliptical (oval)
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method using to stain endospores
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Schaeffer-Fulton

 

"futon shaver"

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stain used for endospore

 

stain used for vegetative portion of cell 

 

color of each

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malachite green to stain
the endospore

 

safranin to stain the vegetative portion
? Endospores will be green
? Vegetative cells and spore
mother cells will be pink

 

GIVES

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function of streak plate  method
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isolate mixed colonies to produce COLONIES (one type of cell) or colony-forming unit (CFU)

 

mixed--> isolated

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2 types of streak methods
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T-streak & quadrant streak

 

T quadrant

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function of streak plating
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Purpose is to reduce the number of cells being spread with each successive series
of streaks thus diluting the concentration of cells.
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wat should we not do when preparing streaks
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Do NOT Gouge the Agar!! Try to make all streaks as close as
possible
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lab 4 FLASHCARDS

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DEFINE AEROTOLERANCE
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ABILITY TO LIVE WITH OXY PRESENT
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OBLIGATE AEROBES
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REQUIRE HIGH O2
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AEROBES
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REQ O2 - LOWER CONCEN
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MICROAERIPHILES
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REQ REDUCED  O2
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MICROAEROPHILES
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REQ REDUCED O2
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AEROTOLERANT ANAEROBES/ FACULATIVE ANAEROBES
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DO NOT REQUIRE AND ARE NOT NEG AFFECTED BY O2
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AEROTOLERANT ANAEROBES
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DO NOT REQUIRE O2

BUT DOES NOT MEAN IT IS HARMFUL EITHER

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FACULATIVIVE ANAEROBES
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FLEXIBLE, NOT ADVERSELY AFFECTED BY O2
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OBLIGATE ANAEROBES
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hates O2- DOES NOT WANT IT AT ALL
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WHERE DOES OBLIGATE GROW (STRICT)
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TOPWHERE OXYGEN IN ABUNDANT
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FACULATIVE ANAEROBES
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FLEXIBLE, GROWTH IS MORE RAPID WITH, BUT WILL DO WITHOUT
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Aerotolerant anaerobes
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Grow equally well whether oxygen is present or not
 Do not require oxygen and are not adversely affected by it.

 

"IMMUNE"

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Microaerophiles

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- Survive only in environments containing lower than atmospheric levels of oxygen.

 

- DESIRE SMALL AMOUNTS

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HABITAT OF Obligate (strict) anaerobes
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DEEP MUD, LAKES,OCEANS AND SOILS
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WHAT FUNCTION DOES INOCULATING FLUID THIOGLUCOLLATE MEDIA DO?

 

what kind of agent is thio and why?

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IT TEST FOR OXYGEN REQUIREMENTS OF AN ORG.

 

-SUPPORTS GROWTH OF BOTH AERIOBIC AND ANAEROBIC BACTERIA

 

thioglco- is a reducing agent which reduces oxygen to water

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WHAT IS THE DYE IN THIOGLYCOLLATE, WHAT FUNCTION DOES IT SERVE?

 

what color does it change, what does this represent

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DYE: RESAZURIN- INDICATOR FOR OXYGEN --> BECOMES PINK

 

res-a-zur-in turns pink in the pressence of oxygen

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DYE RESAZURIN BECOMES W/ COLOR IN THE PRESENCE OF OXYGEN
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AFTER BOILING FTM BROTH AND IS PINK,(GETTING RID OF OXY) DEPENDING ON OXYGEN REQUIREMENTS WHERE WILL AEROBIC AND ANAEROBIC ORG BE LOCATED IN CONTAINER?
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AEROBIC ORG- AT THE TOP

 

ANAEROBIC- BOTTOM

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FUNCTION OF ANAEROBIC JAR
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CREATE A ANAEROBIC ENVIROMENT FOR THE CULTURE OF STRICT ANAEROBES
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WHAT IS USED TO CONVERT H2 AND 02 --> WATER IN ANAEROBIC JAR
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PLADDIUM CATALYST
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KEY METABOLITES IN METABLOISM

 

2

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ACETYL CoA

 

PYRUVIC ACID

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ARE METABLOITES LINEAR
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NO SEVERAL DIFFERENT PRODUCTS FOR DURING REACTION
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PYRUVIC ACID IS FORMED IN WHICH PROCESS
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GLYCOLYSIS (GLUCOSE)
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DEFINE FERMINTATION
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process of deriving energy from the
oxidation of organic compounds, such as carbohydrates.
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Carbohydrate fermentation is the metabolic process by which
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an organic molecule acts as an electron donor
(becoming oxidized) and one or more of its organic products
acts as the final electron acceptor.

 

OXYGEN IS NOT THE FINAL E- ACEPTOR

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PRODUCTS OF FERMENTATION
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ACIDS AND ALCOHOL
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MICROORG PRESENT IN FERMEN
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ANAEROBIC BACT/ EUKARYOTES/ YEASTS
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F OF GLUCOSE TEST
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determine if an organism is able to ferment
glucose
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IF ORG ABLE TO FERMENT GLUCOSE W/ IS THE BYPRODUCT
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acid will be produced and gas
may be produced.
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IF ACID IS INDEED FORMED IN GLUCOSE TEST WHAT COLOR WILL OF TUBE B AND WHY?
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YELLOW-->PHENOL RED INDICATOR

positive fermentation of glucose

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IN METHY RED AND VOGES PROSKAUER TESTS

HOW MANY MEDIAS ARE YOU USING

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1-SAME
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FUN OF METHYL RED TESTS
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TEST FOR PRODUCTION OF MIXED ACIDS FROM GLUCOSE FERMENTATION
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HOW MANY DROPS OF METHYL RED INDICATOR ARE PLACED IN OVERNIGHT CULTURE
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5-6
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RED COLOR MEANS
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POSITVE ACID
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ORANGE
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INCONCLUSIVE
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NO COLOR CHANGE
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NEH RESULT
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F OF METHYL RED AGAIN
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MIXED ACID FERMENTATION PRODUCT
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FUNCTION OF VOGUES MAGAZINE
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to see if the organism
produces 2,3 butanediol instead of acids from glucose
fermentation
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THERE IS NO WAY TO DIRECTLY TEST FOR 2,3 BUTANEDIOL SO WHAT IS THE VOGUES REALLY TESTING FOR??
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ACETONIN- PRECURSOR TO 2,3 BUTANEDIOL
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ACETONIN IMPORTANCE
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no way to test the presence of 2,3 butanediol directly.
Therefore, the VP test is actually testing for the presence of
acetoin, which is a precursor of 2,3 butanediol
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AFTER ADDING BARRITS REAGENT a (ALPHA NAPTHOL) And BRB + KOH(s/p 3 days)

 

if reagents cause acetonin to oxidize to diacetyl which further reacts with guanidine nuclei from peptone what color will be produced?

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red or pink, indicates a positive reaction for acetoin, which
tells you that the organism is a butanediol fermenter
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what does a positive reaction of acetoin indicate( red or pink)
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org is a butanediol fermenter (remember can't direct test for this) GOal is this!
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recap: function of VP
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Tests for the precusor of acetoin, which is a precursor of
2,3 butanediol fermentation product

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in respiration final electron acceptor is always....
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inorganic molecule
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function of catalase
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converts hydrogen peroxide into oxygen and wawter

 

h2o2--> h2 + o2

 

Mainly used to distinguish between staphylococcus and streptococcus**

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function of nitrate test
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test for presence of enzyme nitrate reductase
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what is enzyme nitrate reductase
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-produced by faculative bacteria to carry out anaerobic respitation

-enzyme catalyzes the transfer of electrons from
cytochrome b to nitrate reducing it to nitrite

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red or pink color change reprsents what for nitrate reduction
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represents positive for indicating that org capable of anaerobic respiration
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function of nitrate reduction test
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indicates org capable of anaerobic respiration
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if nitrate red test has no color change that means it's neg--> if you suspect this to be the case add some zinc dust if it turns red what does this represent
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confirmed to be negative-

 

there is no color change
after adding the zinc, then this indicates that the nitrate was reduced a different nitrogenous compound, not nitrite

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define oxidation

 

define reduction

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  • Oxidation is the loss of electrons

 

  • Reduction is the gain of electrons
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lab 5 flash cards
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function of media
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to differentiate org based on ability to grow when esstential nutriens are limited
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function of citrate slant
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some organisms can utilize citrate as ONLY carbon source
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function of CITRATE PERMEASE
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enzyme some org that only use carbon to transport citrate into the cell producing pyruvate from it
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what is contained in medium for utilization test
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medium contains:

 

- sodium nitrate--C source

- ammonium phosphate--N source

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what is the indicator dye used for citrate test
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bromthymol blue due - indicator
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as the pH increase medium color changes to what
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green-----> Blue
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bacteria using citrate and survive in media convert ______ into ____ and ____ ____ causing media to become more ____
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ammonium phsphate into ammonia and ammonium hydroxide, alkaline

 

bacteria using citrate and survive in media convert ammonium phosphate into ammonia and ammonium hydroxyde causing media to become more alkaline

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Blue color represents

 

green color change

 

can organism use citrate as sole source of carbon

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blue-- org uses citrate as sole Carbon source

 

green- NO media stay green

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function of hydrolytic enzymes
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perform hydrolytic reactions - using water to split compelx molecules
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intraceulluar vs exoenzymes
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intracellular enzymes- catalyze the reaction inside the cell

 

exoenzymes- secreed from the cell to catalyze the reactions outside the cell

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3 different test for hydrolytic exoenzymes
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1. starch hydrolysis

2. casease

3. gelatinase

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test for intracellular enzyme
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urease

 

 

urine comes from the inside

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2 glucose subunits
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 amylose (linear) -losing streak


amylopectin (branched)- birds peck on trees with variety of directions

 

 

alpha-amylase and


oligo 1,6-glucosidase

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what are we using to test wheather an org can produce alpha-amylase and oligo 1,6 glucosidase?
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Starch agar- simple plated medium of beef extract soluble starch, and agar
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if test proves positive for alpha amylase/ or oligo what does this mena
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will hydrolyze starch
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indicator for starch
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iodine- turns blue with positive starch
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when starch indicator reacts with starch the media should change what color?
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dark brownish color
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for a positive result of exoenzymes what should petri look like?
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clear zone around bacteria
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negative result
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no clear zone
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when do you add iodine to starch hydrolysis ( testing for exoenzymes)
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24 hours
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function of urease enzyme
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hydrolyzes urea into ammonia and CO2
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what will be using to determine urease prod
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urea broth
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what is the ingrediants of urea broth
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- yease extract and urea

- has phenol red as pH indicator***

 

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what is the pH indicator in urea broth
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phenol red
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if org grown in urea broth is urease,  pH and color is effected how?
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ph will increase, phenol red will change to yellow to red to PINK
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how long does urease phenol red take to complete
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8 days
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 function of casein hydrolysis test
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to test if bacteria requires protein as a source of amino acid.
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function of CASEASE enzyme??
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breakes down large proteins into subunits that can be brough into the cell
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for casein hydrolysis test how do you interpret result??

 

pos

neg

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positive- clear zone

negative result- no clear zone

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gelainase test function
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test to see if organism has enzyme that allows some organisms to hydrolyze gelatin
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gelatin
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Gelatin is a protein derived from collagen – a component of
vertebrate connective tissue.
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what is a important step in gelatinase test
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Chill media for (4°C) ~30 mins before looking at results.
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positve or neg result of gelatinase test
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positive- gel liquid

negative- solid gel

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? Starch Hydrolysis:
? Polysaccharide catabolism, exoenzyme catalyzed
? Casein Hydrolysis:
? Polypeptide catabolism, exoenzyme catalyzed
? Urea Hydrolysis:
? Protein catabolism, intracellular enzyme catalyzed
? Gelatin Hydrolysis:
? Polysaccharide catabolism, exoenzyme catalyzed
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function of combination differential media
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- combination differential media combine compoenents of several compatible tests into one med

 

- include core test to differentiate members of specific bacterial group

 

- can be used as a follow up to selective media

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function of SIM, what does it stand for
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sem-solid media

 

tests: sulfur reduction/ indole prod from tryptophan/ motility

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sulfur reaction: function of cysteine desulfurase/ thisulfate reductase
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bacteria can reduce sulfur to hydrogen sulfide using either of 2 enzymes both produce hydrogen sulfide gas
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If the organism can reduce sulfur___will be seen
in the tube.
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, a black color
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indole production:

 

function of tryptophanase

 

(4)

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bacteria that produces enzyme hydrolyze trypophan to pyruvate, ammonia, and indole
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how to tet for indole production
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kovak's reagent is used on an inoculated, incubated culture
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kovac's reagent will react with indole to produce what?
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red color
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what indicates a positive result for enzyme tryptophanase?
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red color

 

brown means neg

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motility 3rd test function
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stabbing technique is used, if cloudiness is seen in the media away from stab line, motility is indicated
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you want to maintain the needle how during sim motility test
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straight
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what does the SIM motility resemble
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gelain media
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how far in do you want to in, what position do you want to keep the
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~2/3, upward

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TSIA - tripple sugar iron agar

3 tests it provides

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sugar utilization

gas production

hydrogen sulfide production

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3 sugars agar contains
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lactose, sucrose, glucose small amt
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what is the indicator for TSIA
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phenol red
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what indicates ferrous sulfate
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hydrogen sulfide

 

no color change- no sugars fermented

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page 25
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chapter 1
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Fastidious heterotrophs


Nonfastidious heterotrophs

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Fastidious heterotrophs – rely heavily on the environment to supply ready-made organic compounds

 


Nonfastidious heterotrophs – do not rely as heavily on
environment. Can make some of their own compounds.
     ex. Autotrophs are less fastidious than the most nonfastidious heterotrophs.

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Never use what with oil immersion??
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Never use the coarse adjustment with the oil immersion lens!
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total magnification =
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Total magnification = Magnification of Objective X Magnification Of Ocular
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LAB 2
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[image]pellicle coat
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• some float on top of medium and produce a type of surface membrane
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sediment
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• sink to bottom
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Uniform fine turbidity (UFT)
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?? look up
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flocculent
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clumping growth
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describe bacteria
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Bacteria are:

 

Simple
 Lack a nuclear membrane

 

 Have circular DNA

 

Cell surrounded by peptidoglycan

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Spirillum (spirilli
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a rigid curved or spiral shaped cylinder
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vibrio
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Gently curved rods

 

part of spirilli

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spirochetes

 

part of spirilli

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Slender flexible spirals are called spirochete (spirochetes) which is greek
for coil of long hair
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gram positive
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Gram positive (+) cells have a thicker peptidoglycan
layer.

 

retain the primary stain of crystal
violet because they have a thick outer layer of peptidoglycan
that traps the dye among its high degree of teichoic acid
crosslinks.

 

purple

pos dark

counter-intuitive

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crystal violet

water

iodine

ethanol

safranin

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