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Bacterial Identification Tests
Some tests may be absent from this ppt presentation. These pictures are f rom students. If you see an error,please email me at fester@unlv.nevada.edu . Most of these pictures were given to me by Austin McDonald,from 351 Fall 2007. Thanks Austin!!
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eno e - ermen a onglucose, sucrose, lactose for
Escherichia coli Lac (left) gas+
Glu( middle) gas + Suc (right) no gas
Phenol red indicator used to see if fermentation has occurred. Durhamtubes are red before anyfermentation has occurred.
Fermentation produces gas and/oracid from the breadkdown of carbohydrates
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Phenol Red (PR) Fermentation
glucose, sucrose, lactose for Alcaligenes faecalis
Suc (left) Lac (middle)
Glu (right)
Think about why A. faecaliscould not breakdownglu,suc, or lac?
This is a negativeThis is a negativeresult, must haveresult, must havefull yellow to befull yellow to bepositive. Donpositive. Don ttworry the examworry the examones will be moreones will be moreobviousobvious !!
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Phenol Red (PR) Fermentation
glucose, sucrose, lactose forSaccharomyces cerevisiae
Lac (left) Glu (middle) gas Suc (right)
Why did S. cerevisiaeNOT change color?
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Starch hydrolysis
Zone of clearing +
No zone Bacillus subtillis +, Alcaligenes faecalis
Escherichia coli (Clockwise) Iodine must be on the plate to
visualize the zone of clearing
surrounding the bacteria. Thiszone indicates starch wasbroken down to dextrins,maltose, and glucose/alpha-amylase
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Lipid Hydrolysis Dark blue precipitant zone /
clearer blue zone + No color change Bacillus subtilis &
Staphylococcus epidermidis +w / clearer blue zone aroundbacterial growth
Spirit blue agar w/3%Bactolipase reagent is used to see if
triglycerides are hydrolyzed intoglycerol and free fattyacids/lipase
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Lipid Hydrolysis
For the Egg Yolk agar,the growth must havea white halo aroundthe colony growth if itutilizes the lipidstherefore having theenzyme lipase (hardto see in pics!).Bacillus spp. +Escherichia coli
Alcaligenes faecalis
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Casein hydrolysis Zone of clearing + No zone Test used to see If
casein is degraded
into amino acids foruse as a carbonsource/proteolytic
enzymes Escherichia coli ,
Alcaligenes faecalis Bacillus subtilis +
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Gelatin hydrolysis
Liquid on gelatin +
No liquid Hydrolysis of gelatin
into amino acids to beused asnutrients/gelatinase
Escherichia coli (top) Bacillus spp. +
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Catalase Bubbles + No bubbles Reagents 3% H 2O2Tests for the ability to break down toxic O 2 products/superoxide
dismutase (catalyzes the destruction of superoxide) & catalaseperoxidase (catalyzes the destruction of hydrogen peroxide)
2 O2
-+ 2 H + ---superstable dismutate O 2 + H 2O2
2 H 2O2 ---catalase 2 H 2O + O 2
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Oxidase
Blue (30 sec) +
No color change Tests done on Oxidase strips Tests for the oxidation of reduced cytochrome c to form
water and reduced cytochrome c / Cytochrome oxidase
Oxidized cyt C + reagent Wursters blue + red cyt C
clear dark purpleoxidized
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Sulfur reduction test, Indole
production, Motility (SIM) deepsall 3 tests done w/SIM deeps just add Kovacs reagent for Indole test Alcaligenes faecalis (left) - Escherichia coli (middle) Proteus vulgaris (black
precipitate) +
Reagent: Ferrous ammonium
sulfate-indicator. H 2S reacts w/ferrous sulfate forming theblack precipitate Sodium
thiosulfate is reduced tosulfite/thiosulfate
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Motility Spreading growth +(Spreading growth looks like a
mascara brush in the deep)Escherichia coli ( right)Proteus vulgaris (left)
Linear growth Staphylococcus epidermidis(middle)
To test for the ability of bacteriumto migrate in solid agar deep
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Indole (IMViC tests)
Enterobacter aerogenes
Escherichia coli (pink/red) + Kovacs reagent detects if
tryptophan has beenhydrolyzed toindol/tryptophanase
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Methyl Red (MR) (IMViC tests)
Enterobacter
aerogenes (left)
E. coli (bright red) +
Reagent: Methyl red
indicator identifies pHchange due to mixedacid fermentation
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Voges Proskauer (VP)
(IMViC tests) Enterobacter aerogenes +
E. coli (left)
Barritts reagent Tests foracetoin, precursor to 2,3
butanediol fermentation Addition of alpha-napthol
and KOH
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Citrate (IMViC tests)
E. coli (left green)
Enterobacter aerogenes(right royal blue) +
Reagent: Bromothymolblue indicator tests forability to use citrate assole carbon source/citratepermease
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B-galactosidase
E. coli (yellow) + no color change clear
(not shown)
Reagent ONPG Hydrolysis of lactose toglucose and use of lactose as sole carbonsource / B-galactosidase
We use ONPG disks forthis test
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Nitrate Red color after reagents/no color after
zinc +Escherichia coli (right)
No color change after zinc is a + for
denitrification to nitrogen gas orammoniaSoil- (not pictured, would have a gas
bubble in durhamtube)
Color change after Zn added will be for nitrate reductaseMicrococcus luteus (left)
Alcaligenes faecalis (middle)
Reduction of nitrate to nitrite to beused as a final electronacceptor/Nitrate reductase
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Coagulase Results:+ clotting in thebottom of the broth Reagents:Plasma
Reason/EnzymesClots plasma to avoid attack byhosts defenses/Coagulase
Staphylococcus aureus +;Staphylococcus epidermidis
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Mannitol salt Mannitol salt agar is a
selective and differential
medium used fordifferentiating betweendifferent stapylococci
Staphylococcus aureuschanges medium to yellow Staphylococcus epidermidis
will not change the medium Why does S. aureus changethe color of this medium?
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Hemolysis Check which bacteria are
capable of lysing red blood cells(RBCs) by using blood agar(sheep blood).
= partial lysis of red bloodcells blood looks greenish
= complete lysis of bloodclearing = no lysing
Clockwise starting from the left:Staphylococcus aureus ,Staphylococcus epidermidis ,
teeth
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Antibiotic Ability of antibiotics to inhibit
growth on Mueller-Hinton agar
plates (Whether bacteria aresusceptible, intermediate, orresistant depends on the amount
of antibiotic and the diameter of zone of inhibition, check table43.1 of your lab manual )
N l Mi bi h
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Normal Microbiota on the
human body Both pictures
show examples of normal microbiotathat grow on the
ear,arm, palm,and feet. What bacteria do
you think thesecoloniesrepresent?
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Temperature Escherichia coli ,Serratia
marcesens , Bacillusstearothermophilus are on all threeplates
At 4 C (top) no bacteria grew At 30 C (middle) both Serratia
marcesens and Escherichia coligrew At 60 C (bottom) only Bacillus
stearothermophilus grows
What range of temperaturescategorizes bacteria intopsychrophiles, mesophiles, andthermophiles?
Why isnt Serratia marcesens red?
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pH
Bacterial tolerance to
different pH varies muchmore than humantolerance.
Can you remember thepH ranges foracidophiles,neutrophiles, andalkalophiles?
No Demos will not
be a station
Osmotic Pressure
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Osmotic PressureNaCl%= 0 on TSA plate
Detects ability of an
organisms salttolerance
Staphylococcusepidermidis +
Escherichia coli + Halobacterium
salinarium --
O ti P
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Osmotic Pressure
NaCl%= 0.5 on TSA plate
Detects ability of anorganisms salttolerance
Staphylococcusepidermidis +
Escherichia coli ++ Halobacterium
salinarium --
O ti P
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Osmotic Pressure
NaCl%= 5 on TSA plate
Detects ability of anorganisms salttolerance
Staphylococcusepidermidis ++
Escherichia coli ++ Halobacterium
salinarium --
O ti P
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Osmotic Pressure
NaCl%= 10 on TSA plate
Detects ability of anorganisms salttolerance
Staphylococcusepidermidis +
Escherichia coli -- Halobacterium
salinarium --
Osmotic Pressure
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Osmotic Pressure
NaCl%= 20 on TSA plate Detects ability of an
organisms salttolerance
Staphylococcusepidermidis --
Escherichia coli -- Halobacterium
salinarium ++
Compare how well the 3 bacteria grow on
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p geach plate of different NaCl conc. Is the
streak thickness the same on all of them?
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