Triple Sugar Iron Agar, Oxidase Test, Malonate Utilization Test, MacConkey Agar, Gram Staining Test, Urea Hydrolysis, Citrate Utilization Test, MIO Test, Catalase Test, Lysine Iron Agar, Blood Agar, 10% Tryptone Test, Carbohydrate Fermentation Test

TSIA Testing For

• Glucose Fermentation • Sucrose/Lactose Fermentation • Gas Production • Sulfur Reduction

TSIA Glucose Fermentation Reagent

Phenol Red

TSIA Sucrose/Lactose Fermentation Reagent

Phenol Red

TSIA Gas Production Reagent

N/A

TSIA Sulfur Reduction Reagent

Iron in Ferrous Sulfate

TSIA Glucose Fermentation Postive Result

Red Slant/Yellow Butt

TSIA Glucose Fermentation Negative Result

Red Slant/Red Butt

TSIA Sucrose/Lactose Fermentation Positive Result

Yellow Slant/Yellow Butt

TSIA Sucrose/Lactose Fermentation Negative Result

• Red Slant/Red Butt
• Red Slant/Yellow Butt

TSIA Gas Production Positive Result

Bubbles Present

TSIA Gas Production Negative Result

No Bubbles Present

TSIA Sulfur Reduction Positive Result

Black Precipitate

TSIA Sulfur Reduction Negative Result

No Black Precipitate

TSIA Test: How to read the interpretation.

• Slant/Butt/Gas/H2S
• K - Alkaline
• A - Acidic
• Bubbles or cracks in the tube indicate gas production
• Black precipitate indicates H2S production

TSIA Test: Be able to describe the meaning of K/A, A/A, A/K, and K/K.

-A/A (yellow butt and slant): glucose and lactose/sucrose fermenter with acid accumulation
-K/A (red slant/yellow butt): glucose fermenter only, alkaline products due to reversion
-K/K (red slant and butt): no fermentation; peptone catabolized, not enteric
-A/K (yellow slant red butt?): You should never get, if you do, improper inoculation
-H2S (black precipitate in agar): sulfur reduction and fermentation

TSIA Test: Can you get a A/K reaction?

No, if you do you did not inoculate correctly. You must stab AND streak.

TSIA Test: What causes the change in color of the butt of the tube?

Acid Production and Alkaline Products

TSIA Test: What causes the change in color in the slant?

glucose, lactose, and sucrose fermenter

TSIA Test: What butt reaction will you see for all enterics?

Yellow Butt

TSIA Test: What are the three sugars and what is the purpose of their concentrations?

• Glucose
• Sucrose
• Lactose

TSIA

​Oxidase Testing For:

Presence of Cytochrome C oxidase

Oxidase Reagent Used:

Tetramethyl-p-phenylenediamine (Oxidase)

Oxidase Positive Result:

Dark blue or purple in 30 seconds

Oxidase Negative Result:

No color change within color changes

Oxidase Test: How is the test performed?

1. Place culture on filter paper
2. Add drops of reagent
3. View color change in 30 secs.

Oxidase Test: Why should you read the results within 30 seconds?

Reagents used for this test are unstable and may oxidize independently shortly after they become noise.

Oxidase look like

Malonate Testing For:

Utilization of Malonate as a carbon source

Malonate Reagent:

Bromothymol Bue

Malonate Positive Result:

Dark Blue

Malonate Negative Result:

No color change or slightly yellow

Malonate Test: Determine the chemical reaction that takes place.

competitive inhibition of succinate dehydrogenase.

Malonate Test: What causes the color change?

use of malonate alkalinizes the medium

Malonate Look Like

MAC Testing For:

• Growth of Gram Negative
• Bacteria Lactose Fermentation

MAC Growth of Gram Negative Bacteria Reagent:

Cyrstal Violet Bile Salts

MAC Lactose Fermentation Reagent

Neutral Red

Growth of Gram Negative Bacteria Positive Result:

Growth

Growth of Gram Negative Bacteria Negative Result:

No Growth

Lactose Fermentation Positive Result:

Red or Pink

Lactose Fermentation Negative Result:

Colorless (color of agar)

What does the bacteria look like on the media?

• Streak line visible but no colonies - Negative for growth
• Streak line visible positive for growth but not purple - colonies but negative for lactose
• Streak line purple and growth - positive for growth and lactose

MAC: Is this test selective or differential? Why?

• This test is selective because inhibits growth for gram-positive bacteria.
• Differential for lactose fermentation

Testing For

Gram-negative or gram-positive cells based on their peptidoglycan layer and composition of the cell wall.

Reagent/Indicator

• Crystal Violet (Dye)
• Iodine (mordant)
• Ethanol (decolorizer)
• Safranin (Dye)

Gram Postive Results

Purple

Gram Negative Results

Pink

Differential Stain: (ex. 3-7)

Decolorization step is added between two basic stains. Using multiple stains can better differentiate between different microorganisms or structures/cellular components of a single organism.

Distinguishing characteristics of Gram-positive cell wall: (ex. 3-7)

• Peptidoglycan = 90% of cell wall
• Teichoic Acid inserted within the peptidoglycan layer
• A greater degree of cross-linking
• No outer membrane

Distinguishing characteristics of Gram-negative cell wall: (ex. 3-7)

• Thin layer of peptidoglycan (10%)
• Outer membrane layer = lipid bilayer
• Innermost layer = phospholipid bilayer
• Outermost layer = lipopolysaccharides (lipids and sugars)

How and why the stains interact with a gram-positive cell. (ex. 3-7)

• Crystal violet and the iodine turn stain purple
• Decolorization keeps stain purple
• Adding safranin keeps stain purple
• Teichoic Acid helps Trap crystal violet - iodine complex more effectively (helps keep the purple color)
• Less susceptible to decolorization so the stain doesn't turn clear

How and why the stains interact with a gram negative cell. (ex. 3-7)

• Crystal violet and the iodine turn stain purple
• Decolorization turns stain clear
• Safranin turns stain pink
• Decolorization extracts the lipid
• D/C makes it more porous, making wall incapable to retain crystal violet - iodine complex (turns clear)
• Because stain is clear safranin turns stain Pink

Gram Staining: Physically repeating this procedure. (ex. 3-7)

• Heat fixed smears (after stain has dried)
• Cover smear with crystal violet (1 minute)
• Rinse with distilled water
• Cover smear with iodine (1 minute)
• Rinse with distilled water
• Rinse smear with decolorization (ethanol) until clear
• When clear, rinse with distilled water
• Cover smear with safranin (1 minute)
• Rinse with distilled water

Urea Testing For:

Ability to hydrolyze urea

Urea Reagent:

Phenol Red

Urea Positive Result:

Bright Pink

Urea Negative Result:

Orange/Yellow

Determine the chemical reaction that takes place.

catalyzes the hydrolysis of urea into carbon dioxide and ammonia. The reaction occurs as follows: (NH2)2CO + H2O → CO2 + 2NH.

Urea

Citrate Testing For:

Utilization of citrate as a carbon source

Citrate Reagent:

Bromothymol Blue

Citrate Positive Result:

Blue or no color change with growth

Citrate Negative Result:

No color change with no growth

Citrate Test: Determine the chemical reaction that takes place.

Citrate Fermentation

Citrate Test: What causes the color change?

bromomethyl blue is used which is green when acidic and blue when basic. conversion of green to blue is positive because pH goes up. If no color change or no growth, citrate is being utilized and it is positive.

Citrate

MIO Testing For:

• Motility
• Indole Production
• Ornithine Decarboxylation

MIO: Motility Reagent

Semi-Solid Agar

MIO: Indole Production Reagent

Kovac's Reagent

MIO: Ornithine Decarboxylation Reagent

Bromocresol Purple

MIO Motility Positive Result:

Medium Appears Cloudy

MIO Motility Negative Result:

Original stab line is visible

MIO Indole Production Positive Result:

Red Layer on top of border

MIO Indole Production Negative Result:

No Color Change

MIO Ornithine Decarboxylation Positive Result:

Purple

MIO Ornithine Decarboxylation Negative Result:

Yellow

MIO Test: Why is this a semisolid medium?

To allow culture to move around easier if motile

MIO Test: In what order should the tests be read?

• After incubation, read motility and ornithine decarboxylation first.
• After reading motility and ornithine results, add 5 drops of Kovac's reagent to test for indole production.

MIO Test: What causes the color change?

If the organism can decarboxylate ornithine then there is an increase in ph - turns purple

MIO Test: Understand the biochemical reactions that are taking place.

First the organism must ferment glucose. This will create acid turning the media yellow. If present, Ornithine and the acid will initiate decarboxylation yielding putrescine, which increases pH and turning bromocresol purple back to purple.

MIO

Catalase Testing For:

Catalase Production

Catalase Reagent:

Hydrogen Peroxide

Catalase Positive Result:

Bubbles

Catalase Negative Result:

No Bubbles

Catalase Test: How is the catalase test performed?

1. Place culture on slide
2. Add 1 drop of hydrogen peroxide
3. Look for bubbles

Catalase Look Like

Lysine Iron Agar Testing For

• Lysine Deaminase
• Lysine Decarboxylate

Lysine Reagent used for Deaminase

Ferric Ammonium Citrate

Lysine Reagent used for Decarboxylate

Bromcresol Purple

Lysine Positive Result for Deaminase

Red Slant/Yellow Butt

Lysine Negative Result for Deaminase

Purple Slant/Yellow Butt

Lysine Positive Result for Decarboxylate

Purple Slant/Purple Butt

Lysine Negative Result for Decarboxylate

Purple Slant/Yellow Butt

Lysine Test: How is the burgundy slant helpful diagnostically?

This result is interpreted to mean that deamination is happening

Lysine Test: What causes a burgundy slant and what three genera is this indicative of?

The beginning of decarboxylation process
Proteus, Morganella, Providencia

Lysine Test look like

Blood Agar Testing For:

Hemolysis of RBC's

Blood Agar Reagent:

5% sheep blood in blood agar