Lab 7

Objectives:	Reading:
1. Growth Requirements	1. Tortora, Chapter 6
2. Selective and Differential Media	2. P: pages 22; 24-25; 54-58; 60-61; 66;
3. Perform Gram stain	71-73; 76; 80; 97
	3. L: pages 5; 7-8; 13-16; 40-42; 44-46
	53; 71-72

A Review of Basic, Selective, and Differential Media

Media	Ingredients	Action
TBAB	Nutrients and agar; Basic	Most organisms will grow
Rodac	Nutrients and agar; Basic	Most organisms will grow
Blood Agar (BAP, TSA, Tryptic Soy Agar)	5% Sheep blood Basic, Enrichment, Differential	Most organisms will grow; Alpha, beta, and gamma hemolysis
Mannitol Salt Agar (MSA)	7.5% NaCl, mannitol, phenol red pH indicator; Selective, Differential	Selects for halophiles; Differentiates between S. aureus and S. epidermidis
MacConkey Agar	Bile salts, crystal violet, lactose, neutral red indicator; Selective, Differential	Inhibits Gram positives; Lactose fermenters turn red/pink
Phenlyethyl alcohol (PEA)	Phenylethanol, 5% sheep blood; Selective, Differential	Inhibits facultative anaerobic Gram negative bacilli; supports Gram positives
Hektoen	Bile salts, lactose, sucrose, salicin, ferric ammonium citrate, bromthymol blue and acid fuchsin indicators; Selective, Differential	Selects for enteric organisms; Differentiates between Salmonella and Shigella
Bile Esculin (BE)	Bile salts, esculin, ferric citrate; Selective, Differential	Selects for enteric organisms; those that can hydrolyze esculin in the presence of bile turn the medium black (Enterococcus)
Simmon's Citrate	Citrate, bromthymol blue indicator; Differential	Differentiates between bacteria that can utilize citrate as sole carbon source and those that cannot

Growth of the bacterial colony is an indicator of selective media processes.

Color changes and hemolysis due to bacterial colony growth are indicators of differential media processes.

There is a difference between growth and color changes.

There is a difference between selective and differential processes.

Use the table below to help you understand the difference.

Note: Whereas a medium can never be both basic and selective, it can be both selective and differential

Write "G" for growth if the organism grew on the medium. Write "NG" for no growth if the organism did not grow (or grew poorly) on the medium.

Describe the color change that took place as a result of differential characteristics (for example, "a hemolysis" or "colony is pink" or "medium is yellow").

Organism	BAP	MSA	Mac	PEA	Hektoen	BE	Citrate
Staphylococcus aureus
Staphylococcus epidermidis
Entrococcus faecalis
Streptococcus sanguis
Escherichia coli
Pseudomonas aeruginosa
Salmonella enteriditis

CITRATE TEST

The citrate test is used to determine whether or not a bacterium can metabolize citrate when it is the only source of carbon. Citrate utilization is an aerobic process.

Only those bacteria whose DNA codes for the protein enzyme citrase have this ability.

Besides citrate, this medium also contains the pH indicator bromthymol blue.

Bromythymol blue is green at a pH lower than 7.6 (it is formulated at a pH of 6.9 and appears green when it is freestanding)
Bromthymol blue is blue at a pH higher than 7.6

When organisms metabolize citrate in aerobic respiration, the end product (CO₂) combines with other compounds to produce an alkaline product

This alkaline product causes the medium to turn blue

If the medium remains green, this means the organism was not able to metabolize citrate

The green medium on the left is indicative of a negative citrate reaction; the blue medium on the right is a positive citrate reaction.

Carbohydrate Broths and Durham Tubes

Anaerobic or facultatively anaerobic bacteria often ferment carbohydrates to produce organic acids and gases. This characteristic is useful is distinguishing Gram negative bacilli.

The fermentation end products can be observed through the use of a pH indicator in the liquid medium (broth) and a small, inverted test tube (for capturing gas bubbles).

A variety of carbohydrates can be used; in this lab, we are using glucose, lactose, and sucrose.

The presence of an acid product causes a color change to yellow to occur.
No acid present causes no change in color.
The presence of a gas product causes the glass tube to become filled with air, displacing the liquid broth.
No gas present is observed by the liquid broth remaining inside the glass tube.

Thus, this is a differential test medium in that it differentiates between organisms that produce acid only, acid and gas, and gas only during fermentation of specific sugars and those organisms which show growth only (but no acid or gas production)..

When observing these tubes, there are three observations to be made:

Is there growth?
Is there gas production?
Is there fermentation?

Note that growth may occur in the absence of fermentation and gas production.

Note that growth may occur in the absence of fermentation, but in the presence of gas production (in this instance, the gas production would not be a product of fermentation, of course).

Note that growth may occur in the absence of gas production, but in the presence of fermentation.

Note that growth may occur with fermentation and gas production.

To summarize, look for:

Growth only
Growth with gas only
Growth with fermentation only (color change)
Growth with fermentation and gas

The broth tube on the upper left is an example of no growth, no fermentation, and no gas.

The broth tube on the upper right is an example of growth, but no fermentation, with production of gas.

The broth to the left here demonstrates growth, with both fermentation and gas production.

Oxidase Test

The oxidase test is one of the first biochemical tests performed on Gram negative organisms.

This test determines the presence of cytochrome oxidase.

An artificial electron donor is used to reduce cytochrome oxidase
If present, cytochrome oxidase oxidizes the artificial electron donor, which is a colorless reagent, and turns it dark purple or blue.
If cytochrome oxidase is not present, the reagent remains colorless.
Note the redox relationship: the artificial electron donor (the colorless reagent) reduces cytochrome oxidase; in the process, cytochrome oxidase oxidizes the artificial electron donor.

Neisseria species are oxidase positive.

The Enterobacteriaceae are oxidase negative.

Non-enterobacteriaceae, such as Pseudomonas species, are oxidase positive.

: The glass slides above have been set on black paper so that the positive, dark purple reaction on the right is more easily disinguished from the negative reaction on the left.

CATALASE TEST

This test identifies organisms that are capable of producing the enzyme catalase.

Organisms that produce catalase can break down hydrogen peroxide into water and oxygen gas.

When a drop of 3% hydrogen peroxide is added to a glass slide (or petri dish) that contains catalase positive bacteria on it, bubbles of oxygen gas become clearly visible in the mixture of hydrogen peroxide and bacteria.

No bubbles is a negative result and means that the bacteria on the slide (or petri dish) could not produce catalase.

Controls must always be run, because hydrogen peroxide is unstable and its integrity must be confirmed in order to rule out a false negative result.

Streptococcus are catalase negative, whereas Staphylococcus are catalase positive.

The colonies on the right, above, clearly show evidence of catalase production because of the bubbles of oxygen produced after hydrogen peroxide was added. No bubbles of oxygen were produced by the colonies on the left. These organisms are catalase negative.

Coagulase

This test distinguishes between Staphylococcus aureus and Staphylococcus epidermidis.

Recall that S. aureus is a pathogen and S. epidermidis is considered to be normal skin flora

Thus, it is performed on Gram positive, catalase positive cocci.

It detects the ability of S. aureus to clot plasma.

It involves incubating the bacteria with plasma that has been treated to prevent normal clotting from occurring (at 37^o C.).
If S. aureus is present, the plasma will clot within 2-4 hours (although, some may take as long as 24 hours).
If S. aureus is not present, the plasma will remain clot-free.
Positive and negative controls, using a known S. aureus (for the positive) and a known S. epidermidis (for the negative) are required.

This test is specific to S. aureus

Thus, just as growth on MSA that causes the medium to turn yellow (due to fermentation of the mannitol) is specific to S. aureus, so is a coagulase positive test of a Gram positive, catalase positive coccus specific for S. aureus.

The tube on the left, above, contains freely flowing plasma, indicative of a coagulase negative result. The tube on the right contains clotted plasma, indicative of a coagulase positive result.

Gram Negative Dichotomous Scheme

Gram Positive Dichotomous Scheme

Gram Stain

Instructions for Gram staining are re-printed below. Select one Gram positive colony and one Gram negative colony for staining. Have instructor check both. Because the identity of the organisms is known, today's Gram staining procedure will check your technique!

Instructions for Gram Stain:

Materials:

Inoculating loop

Glass slide

Slide holder

Glass marking pen

Bunsen burner

Flint lighter

Bibulous paper

1. Label your slide, if you plan on keeping it.

2. Place a small drop of .85% saline on the slide.

3. Sterilize your inoculating loop and select a colony. Pick it up and mix it with the saline.

4. Allow the saline/colony solution to dry fully.

5. Heat fix it.

6. Gram stain it:

a. Crystal violet, one minute. Rinse with water.

b. Iodine, one minute. Rinse with water.

c. Alcohol, until runoff is clear OR 20 seconds, whichever is shorter. Rinse with water.

d. Saffranin, one minute. Rinse with water.

7. Blot dry, using bibulous paper.

8. Coarse focus on 10x; fine focus, using oil, on 100 x.

Take Out Food for the Brain:

Anthrax has been in the news a lot lately. According to the Centers for Disease Control and Prevention (CDC), it is considered to be a potential agent for use in biological warfare. Thus, the Department of Defense recently announced that "systematic vaccination of all U.S. military personnel" would begin. Up until recently, only individuals who received workplace exposure to imported animal hides, furs, bonemeal, wool, animal hair and bristles, or who were involved in diagnostic or investigational activities bringing them into contact with anthrax spores were recommended for the vaccination series.

Anthrax is an acute infectious disease that is caused by Bacillus anthracis, a large Gram positive, facultatively anaerobic spore-forming bacillus. A common pathogen of herbivores, it was the first organism to be proven as the causative agent of a disease by Koch's postulates. In humans, it can take one of three forms:

Cutaneous anthrax occurs when the spore enters a break in the skin.

The skin infection spreads to the lymph glands and may lead to septicemia,

and about 20% of untreated cases result in death. However, antimicrobial

therapy is very successful at containing the infection.

Intestinal anthrax follows the consumption of spores in contaminated meat.

There is inflammation of the intestinal tract with subsequent vomiting of blood

and severe diarrhea. The death rate is 25% to 60% of cases.

Inhalation anthrax, which occurs when B. anthracis spores are inhaled,

resembles the common cold in the beginning. It progresses to a severe

pneumonia and shock, resulting in death within one or two days of acute

symptoms.

Treatment for anthrax involves the use of penicillin, erythromycin, tetracycline, and/or chloramphenicol. These antimicrobials are most effective when begun early in the course of the disease, primarily because much of the damage is caused by an exotoxin produced by the bacterium.

The vaccine that is used to prevent anthrax is prepared from a cell-free filtrate of an avirulent, nonencapsulated strain. A cell-free filtrate means that a filter was used to sterilize the solution--there are no bacterial cells in the final product, only antigens that promote immunity by stimulating the production of specific antibodies. A drawback to the vaccine is that it is only known to be effective against cutaneous anthrax. It has never been tested in humans for its efficacy in preventing intestinal or inhalation anthrax. The vaccine has been shown to effectively prevent these types of anthrax in rhesus monkeys, however.

Should a vaccine that is promoted as preventing one type of disease in humans be required for certain high-risk populations, when there is not scientific proof that it can actually prevent that disease? You tell me.

Robert Koch's original micrographs of Bacillus anthracis

Take Home Thought

War is a proving ground.

CITRATE TEST

CATALASE TEST

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