Diagnostic Tests

Front 1

What test would you use to determine if the organism is capable of breaking down starch to maltose? What enzyme accomplishes this?

Back 1

Amylase/Starch Hydrolysis

Enzyme- alpha amylase

Front 2

What is the general procedure for the amylase test?

Back 2

Streak plate down center line, incubate, add iodine and watch for clearing

Clearing = positive

Front 3

What is the bile esculin test? What percent bile is the medium?

Back 3

Determines organism's ability to hydrolize the glycoside esculin to esculetin and glucose. Medium is 40% bile.

Front 4

Why is the bile esculin test basically two tests?

Back 4

1. Growth on bile

2. Hydrolysis of esculin

*some streptococci that can split esculin can't tolerate such high bile

Front 5

General bile esculin test procedure?

Back 5

Bile esculin slant. Light inoculum of colony, Insert loop to bottom of agar. Zig zag on the way out.

Pos- half+ tube is black/brown

Neg- less than half is black brown

Front 6

What test determines ability of an organism to degrade a specific carbohydrate in basal medium? What does it produce? ( 2 things)

Back 6

Carbohydrate Fermentation Test

1. Acid (pos, color change)

2. Acid with visible gas

Front 7

Carbohydrate Test procedure?

Back 7

Stab to 1/4" of bottom of tube with needle. Incubate 48hrs.

Fermentation pos: any yellow color, neg: red, pink, orange

Gas: pos: significant bubbling in semi solid medium.

neg: no excessive gas bubbles.

Front 8

Catalase test- what does it test for, what is a positive?

Back 8

Tests for enzyme catalase. Positive is bubbling,

Front 9

Catalase test procedure?

Back 9

Pick loopful of colonies, place on slide. **NOT FROM BLOOD AGAR PLATE! (blood contains catalase). Add hydrogen peroxide.

*Nitrochrome wire

Front 10

What tests for the hydrolysis of DNA?

Back 10

DNAse Test

Front 11

DNAse Test: Methyl Green indicator is stable at what pH? Does the pH of the agar increase or decrease when hydrolysis of DNA occurs?

Back 11

Stable at pHs >7.5

Hydrolysis causes drop in pH

Front 12

DNAse Test procedure?

Back 12

Sterile loop, inoculate DNA+Methyl Green agar plate with culture. Heavy streak line. Incubate.

Pos- clearing around areas of growth

Neg- color of agar is the same, no hydrolysis

Front 13

What tests for hydrolysis of gelatin? What is pos and what is neg result?

Back 13

Gelatinase Test. Pos = gelatin liquidizes
Neg= gelatin stays the same

Front 14

Gelatinase Test Procedure?

Back 14

Obtain two solid gelatin butts. Heavy inoculum, stab to depth of 2". Use other tube as control.

**DO NOT SHAKE, if gelatin and medium mix when warm, may get false pos.

Front 15

What type of plate would you use for a Hemolytic Reaction Test?

Back 15

Sheep Blood Agar (SBA)

Front 16

What enzymes are you testing for in Hemolytic Reaction Test? What do they do?

Back 16

Hemolysins. Lyse red blood cells.

Front 17

What 3 types of hemolytic activity can you see in the hemolytic reaction test? What does each type indicate?

Back 17

Alpha - green/brown zone. Loss of K from RBCs

Beta- complete lysis, reduction of released hemoglobin. Clear zone around colonies.

Gamma- no hemolytic reaction, no change of medium

**Check only around isolated colonies.

Front 18

What test imitates the environment of the skin? What bacteria does it typically test for?

Back 18

Mannitol Salt Agar Test. Staphylococcus species- indicated by any significant growth.

Front 19

Mannitol Salt Agar Test procedure? Pos/neg indicators?

Back 19

Streak MSA plate with light line of inoculum from pure culture. Incubate.

Pos- growth; yellow color. yellow indicates acidic pH, product of fermentation

Negative- growth/no growth; red or orange

Front 20

What tests for an organism's ability to reduce nitrate to nitrite, then to free nitrogen gas? What serves as an electron acceptor?

Back 20

Nitratase Test (Nitrate Reduction; Denitrification)

Nitrogen in nitrate serves as electron acceptor

Front 21

Test procedure of Nitratase test?

Back 21

Innoculate nitrate agar slant with pure culture, heavy inoculum. Add 2-3 drops of both Reagent A and B. Can add small amount of zinc powder if no color occurs right away.

Front 22

Pos/Neg results in Nitratase Test?

Back 22

Positive: Red color develops quickly

Negative: pink to dark red after adding zinc (nitrate is present, but not reduced by organism

Positive: no color (reduced all nitrate to nitrite and then to N2 gas, which escaped the tube

Front 23

Which reagent in the Nitratase Test is potentially carcinogenic?

Back 23

Reagent B: 0.6% N, N-dimethyl-alpha-napthylamine in 30% acetic acid

Front 24

What is Novobiocin? What does the Novobiocin Disc Test detect?

Back 24

Novobiocin is an antibiotic. Tests detects organism's susceptibility.

Front 25

Novobiocin Test procedure?

Back 25

Streak BHI plate, turn 90 degrees and streak again. Place disk in middle. Incubate, observe.

Front 26

What test would you use to determine if bacterium makes enzyme betagalactosidase?

Back 26

1% ONPG Test

Front 27

Procedure for 1% ONPG Test?

(ortho-nitrophenyl-galagopyranoside)

Back 27

Inoculate 1% ONPG tube with unknown., Incubate for 24hrs. ONPG contained in medium is hydrolyzed, produce B-galactosidase, get yellow color. Negative = colorless

Front 28

What part of the bacterial cells does the Optochinin Disc Test target? What bacteria is it used mainly for?>

Back 28

Fragility of bacterial cell wall. Streptococcus pneumoniae (sens) and other Strep species (nonsens)

Front 29

Optochin Test procedure?

Back 29

Single pure colony. Innoculate a SBA plate., Streak entire plate.. MUST USE SBA because colony is fastidious. Apply disc, incubate, observe.

Front 30

What test determines the presence of oxidase enzymes? What is an example of an oxidase enzyme?

Back 30

Oxidase Test. Cytochrome c oxidase.

Front 31

Oxidase Test procedure?

Back 31

Grow on BHI plate. Warm plate to 20-37dC. Pick up chunk and rub onto DrySlide card.

pos- purple color in 20seconds.

Saturate slide with Kovac's oxidase reagent.

Neg- do not change color in 20seconds.

Front 32

What type of bacteria are oxidase negative?

Back 32

1. Most gram + bacteria

2. All Enterobacteriaceae

Front 33

Why can't you do the oxidase test on bacteria on media with glucose

Back 33

Glucose fermentation inhibits oxidase enzyme activity, could give false negative.

Front 34

What is the procedure for oxygen requirement test?

Back 34

Inoculate 5ml of BHI broth. Incubate. *Broth is better. Light inoculum otherwise.

Use thioglycolate tube, use need to stab culture down to the bottom. Incubate.

Front 35

What type of growth will a facultative anaerobe produce? A microaerophile?

Back 35

Facultative Anaerobe- Growth throughout, but slightly more at top.

Microaerophile- Band of growth near top, but not touching surface.

Front 36

Penicillin Test

Back 36

Same as other disc tests

Front 37

What tests an organism's ability to tolerate various osmotic concentrations?

Back 37

Salt Tolerance Test

Front 38

Test procedure for Salt Tolerance Test?

Back 38

Sterile loop/needle, inoculate broth tubes with different salt concentrations. Incubate.

pos- turbidity

negative-no turbidity

Front 39

Urease test determines organism's ability to break urea down into what? (3 things)

Back 39

Carbon dioxide, water, ammonia

Front 40

What is the urease test primarily used to distinguish between?

Back 40

Urease+ proteeae from other Enterobacteraceae

Front 41

Urea Test procedure?

Back 41

Use heavy fresh inoculum. Streak entire surface of slant. Incubate. Observe color change at 6hrs, 24 hrs, and every day up to 6 days.

pos- bright pink, any pink