Pathogenic Lab

Front 1

Triple Sugar Iron (TSI)

Back 1

• Differentiation of glucose fermenting from organism that ferments lactose or sucrose.
• Also determines H2S production
• Contains 3 sugars:
1. .1% glucose – limiting sugar – allows for differentiation from fermenting lactose or sucrose.
2. 1% lactose
3. 1% sucrose

• Also contain: Phenol red = turns yellow at acidic pH.

Front 2

Indole Test:

Back 2

• Dectects whether organism produces tryptophanse.
• Kovac’s reagent is used
• Incubate in tryptophan broth
• When reagent is added = pink (red) = indole present

Front 3

Methyl Red test:

Back 3

• Determines if organism uses mixed acid fermentation pathway for glucose.
• If yes = strong acids will be produce = MR reagent turns red when added.
• If strong acid, pH of the medium will be below 4.4 = red.

Front 4

Voges-Proskauer Test:

Back 4

• Determines if an organism ferments glucose via 2,3 – butanediol pathway.
• If yes = acid by product = less stable = converted to acetoin and 2,3-butanediol.
• MR test would not work.
• VP test detects acetoin
• VP reagent turns brick red = organisms uses 2,3-butanediol pathway to ferment glucose.

Front 5

Citrate test:

Back 5

• Determines if the organism can use sodium citrate as only carbon sorce.
• Medium contains: ammonium phosphate
• Bromthymol blue is indicator
o Green = pH 6.7
o Blue = pH 7.6
• Turns Blue = positive for using citrate as carbon source, b/c it will convert ammonium phosphate to ammonia and ammonium hydroxide = raising pH and turning medium blue.

Front 6

Decarboxylation of Amino Acids

Back 6

contains glucose, amino acid and bromcresol purple as the pH indicator.
See, if the organism is able to decarboxylate the amino acid (remove a carboxyl group, COOH).
Yes = pH raised and medium turns purple. Then yellow, because fermentation of the glucose can activate the decarboxylase enzyme which will create an alkaline environment.
So, purple starting color  yellow (fermentation, acidic environment to activate decarboxylase)  back to purple = decarboxylation took place (positive result).

Need oil

Read results:
a. Purple = positive result for decarboxylation of amino acid.
b. Yellow = negative result for decarboxylation of amino acid.

Front 7

Phenylalanine Deaminase

Back 7

Determine if the organism can produce phenylalanine deaminase.
If yes, medium will be deaminated (amine group removed) and ammonia and phenylpyruvic acid are produced.
Ferric chloride is the reagent added to the slant medium after incubation and can react with the normally colorless phenylpyruvic acid to produce a green color.
Only Proteus, Providencia and Morganella will have a positive PD test of all the Enterobacteriaceae.

Read result: Add a few drops of 10% ferric chloride, let it run down the slant.
a. Development of green color = positive
b. No green color change/yellow = negative result.

Front 8

Motility

Back 8

Identification of motility.
Contains: tetrozolium salt to aid in the identification of motility by turning red in the presence of an metabolizing organism.

Read results
a. Migration away from inoculation line = positive.
b. No migration away from inoculation line = negative.

Front 9

Fermentation of Inositol and Adonitol

Back 9

To help speciate the members of Providencia.
Bromcresol purple is the pH indicato.
If the organism is able to ferment the sugar, then the medium will turn yellow.
Read result:
a. Yellow = positive.
b. Purple = negative.

Front 10

Urease Test

Back 10

Determine whether the organism can make the enzyme urease.
Contains urea and phenol red as the indicator.
If the organism makes urease, it will be able to hydrolyze urea to ammonia and water.
= turning the phenol red to a cerise (hot pink) color.

Read results
a. Cerise = positive
b. No color change = negative

Front 11

Orthonitrophenyl-Beta-D-Galactopyranoside Test (Beta-Galactosidase Test) – Organisms 1-4
(OPNG)

Back 11

To identify the presence of the enzyme Beta-galactosidase and to determine if organism produces it and if organism is a lactose or non-lactose fermentor.

Read result:
Positive – Yellow color change = -galactosidase produced = lactose fermenter
Negative – No color change = no -galactosidase produced = non-lactose fermenter

Front 12

Deoxyribonuclease (DNase test)

Back 12

Identifies those organisms that produce the enzyme DNase, which degrades DNA.
Media contains green dye.
.
Read result: if the DNA is degraded the dye is released and a clearing around the colony is seen.

Front 13

Arabinose Fermentation

Back 13

S. marcescens is the only enterobacteriaceae that can ferment Arabinose.
Identifying S. marcescens from the other Serrtia as well as Enterobacteriaceae. Bromcresol purple is the pH indicator.


Read results
c. Ferments arabinose and sugar = Yellow = positive.
d. Purple = negative.

Front 14

Salmonella

Back 14

No fermentation of lactose or sucrose, produces H2S, decarboxylates lysine, is motile and urease negative.
Confirm with: urease test. Used to distinguish between salmonella and proteus.

Front 15

Shigella

Back 15

They are non-motile and non-lactose fermenters. To separate S. sonnei from the other Shigella species the ONPG and ornithine tests are used. S. sonnei will be positive for both.

Front 16

Oxidative-Fermentative Medium

Back 16

The typical carbohydrates tested are glucose, maltose and xylose. Two tubes are needed for the OF glucose test, one “open tube” and one “closed tube” (oil overlay ~ 1 cm).
The open tube will detect oxidation while the closed tube will detect fermentation.
A positive result is a yellow color change from the original green.

Front 17

Trypticase Soy Agar (TSA)

Back 17

This is a general purpose medium.
Look for P. aeruginosa to grow and P. fluorescens not to grow.

Front 18

Trypticase Soy Broth (TSB)

Back 18

Used for a wet mount preparation for motility.
Use a microscope to see motility.

Front 19

P Slant Agar

Back 19

Contains magnesium to enhance pyocyanin production.
The only species know to produce pyocyanin is P. aeruginosa.

Front 20

F Slant Agar

Back 20

Medium contains: casein and meat peptone.
This enhances the production of fluorescein, which can be seen under a UV lamp.
Both P. fluorescens and P. aeroginosa produce fluorescein.

Front 21

Creation of an Anaerobic Environment

Back 21

Use: Gas-Pak system to create the anaerobic atmosphere needed in the anaerobic jars. Indicator strip and the catalyst will be placed in the jars.
Distilled water will be added to activate the catalyst and the jar will be sealed.
If the anaerobic environment is created, the indicator strip will turn white within 2 hours and moisture (water) will accumulate in the jar.
The palladium catalyst reacts with the hydrogen and oxygen to form water.
2H2 + 02  H2O

We will be using:
1. Propionibacterium acnes
2. Clostridium sporogenes

Front 22

Enriched Thioglycolate Broth

Back 22

Support growth of most anaerobic bacteria.
Thioglycolate base helps reduces oxygen (except at the top of the broth = pink).
Growth will appear as turbidity.

Front 23

Egg Yolk Agar

Back 23

Contains egg emulsion in a blood agar base. It also contain vitamin K and hemin, helps the growth of anaerobes.
Used for the lecithinase and lipase test. Both of these enzymes break down lipids. The egg yolks provide the lecithin and lipase to test for the enzymes.
A positive test for lecithinase will look like a opaque (whitish) zone under and around the growth of the colony.
A positive lipase test will look like an oily sheen or pearly layer on the surface of the agar (hold plate at an angle to view).

Front 24

Tryptone Broth

Back 24

Used to detect indole production = organism produces tryptophanase.
Add Kovac’s to see result. Pink = positive.

Front 25

Litmus Milk

Back 25

Contains lactose (sugar), casein (protein) and a pH indicator (litmus).
If lactose is fermented, will turn pink (acidic).
If no fermentation, will turn purple or blue (alkaline).

1. Pink – acid
2. Purple or blue – no acid formed
3. Clot – coagulation of milk from fermentation of lactose
4. White – reduction of litmus
5. Proteolysis – clearing of medium