Practical 2 Tests 251

7/30/2019 Practical 2 Tests 251

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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 [email protected] . Most of these pictures were given to me by Austin McDonald,from 351 Fall 2007. Thanks Austin!!
mailto:[email protected]:[email protected]:[email protected]


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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


Escherichia coli ++ Halobacterium

salinarium --

O ti P


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Osmotic Pressure

NaCl%= 5 on TSA plate


Staphylococcusepidermidis ++

Escherichia coli ++ Halobacterium

salinarium --

O ti P


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Osmotic Pressure

NaCl%= 10 on TSA plate



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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Practical 2 Tests 251