BIO6 lab exam #2

A differential stain used to distinguish between metabolically active vegetative cells and doorman, resistant spores. Like the gram stain and acid-fast stain, the spore stain requires the use of 2 contrasting stains - a primary stain followed by a counterstain of a different color. Spores will appear green & vegetative cells will appear orange-red.

Agar slant culture of Bacillus cereus
Malachite Green Stain
Safranin Stain
Hot Plate
Beaker set-up in fume hood

Prepare slides from a solid medium.
Heat-fix on heating block.
Cover slide with bibulous paper.
Flood slide with malachite green for 5 minutes.
Remove slide, cool and wash with water.
Apply safranin counterstain
Wash off counterstain.
Blot with bibulous paper.

Distinguishes bacteria and helps identify mycobacteria.
Cells that retain the primary stain in the presence of acid are referred to as acid fast.
Acid-fast cells appear cherry red or magenta. Non acid-fast bacteria appear blue.

In acid-fast staining, a wax in the cell wall that makes it possible to differentially stain organisms for diagnostic purposes. Heat must be used to melt the waxy substance.

Used as the primary stain in acid-fast staining because it is lipid-soluble and penetrates the waxy cell wall (mycolic acid). Steam heat allows the wax to melt, letting the stain move into the cell.

Distinguished in acid-fast staining. Associated with TB and Leprosy.

In acid-fast staining, it's used to decolorize non acid-fast cells after using Carbol Fuchsin. Acid-fast cells resist this decolorization.

In acid-fast staining, a counterstain

Growth medium that favors growth of one group of microorganisms and inhibits or prevents growth of others.

Growth media that contains an indicator (usually color) to detect the presence of absence of a specific metabolic activity.

Mannitol Salts Agar - Contains the carbohydrate mannitol, 7.5% NaCl, and the pH indicator phenol red.

In the MSA test, mannitol provides the substrate for fermentation and makes the medium differential. Yellow is positive for mannitol fermentation.

In the MSA test, NaCl makes the medium selective because its concentration is high enough to dehydrate and kill most bacteria. Staphylococci thrive in the medium because of their adaptation to salty habitats like human skin.

In the MSA test, Phenol red indicates whether fermentation has taken place by changing color as the pH changes. Phenol red is yellow below pH 6.8, red at pH7.4-8.4 and pink at pH 8.4 and above.

Microorganism that grows best at 3% or higher in NaCl

MacConkey Agar - used to isolate and differentiate members of the Enterobacteriaceae based on the ability to ferment lactose - revealed by turning pink.

MAC - a selective and differential medium containing lactose, bile salts, neutral red and crystal violet. Inhibits Gram-positive.

In the MAC test, lactose fermenters turn red.

In the MAC test, neutral red dye is a pH indicator that is colorless above a pH of 6.8 and red at a pH less than 6.8. Acid accumulating from lactose fermentation turns the dye red.

In the MAC test, bile salts and crystal violet inhibit growth of Gram-positive bacteria. Formulations without crystal violet allow growth of Enterococcus and some species of Staphylococcus, which ferment the lactose and appear pink on the medium.

In the phenol red broth test, a small inverted test tube used in some liquid media to trap gas bubbles and indicate gas production.

Test the capacity of bacteria to oxidize various carbohydrates via a fermentative pathway. A differential test medium prepared as a base to which a carbohydrate is added. PR broth is used to differentiate members of Enterobacteriaceae and to distinguish them from other Gram-negative rods. Ingredients are sugar (lactose, glucose or sucrose).

Is yellow below pH 6.8, pink to magenta above pH 7.4 and red in between.

In the phenol red broth test, fermentation to gas gives an air gap in the durham tube.

In the phenol red broth test, growth means sugar utilization.

Used to indicate whether a microorganism produces the enzyme catalase that breaks down hydrogen peroxide to water and oxygen. This test is used to differentiate between Streptococcus (negative) and Staphylococcus (positive) species.

This enzyme is important to aerobic organisms because it detoxifies hydrogen peroxide, turning to water and oxygen. Catalase positive organisms will exhibit bubbling, catalase negative will not.

Detects the presence of the enzyme, cytochrome oxidase, which transfers electrons from cytochrome c to molecular oxygen in the electron transport chain. This test is used to differentiate between enteric (facultative anaerobic) and non-enteric (aerobic) gram-negative bacteria.

In the Oxidase test, Oxistrips have and artificial substrate (TPPD) that turns purple when oxidase oxidizes it. No color means the organism is negative for cytochrome oxidase. Aerobic organisms are positive, while enterics are negative.

Oxistrips
Tetramethyl-p-phenylenediamine dihydrochloride

Exoenzymes are enzymes secreted from the organism to catalyze reactions outside the cell. AKA extracellular enzymes. These enzymes are hydrolytic and break down large biomolecules that are too large to be easily transported into the cell.

In exoenzyme test, hydrolytic reactions that use water to split complex molecules.

Starch must be broken down into glucose; protein into amino acids; triglycerides into fatty acids and glycerol.

Amylase exoenzyme breaks down starch into glucose. A clear zone around the inoculum indicates a positive reaction - the starch has been broken down (hydrolyzed). Dark color up to the edge of the inoculum indicates the absence of starch hydrolysis.

A way to read reactions on plates by seeing a clear zone around the inoculation

Breaks down the milk protein, casein, into amino acids. A clear zone surrounding the innoculum is indicative of casein hydrolysis.

Tests for the presence of the gelatinase exoenzyme, which breaks down the protein, gelatin, to amino acids. Liquifaction is positive for this test.

For the gelatinase test, liquefaction represents a positive for the breakdown of gelatin into amino acids.

Tests for lipase exoenzymes that breakdown lipids to glycerol and fatty acids. A clearing of the agar around the inoculum indicates the breakdown of lipids in the medium.

Methyl Red & Voges Proskauer Test; main ingredient is GLUCOSE.

added to detect acid byproducts & used to identify bacteria that produce mixed acids (lactic, acetic, or formic) as a result of glucose fermentation. The acid end products lower the pH of medium to 5.0 or <. Indicator turns the medium red in presence of acid from glucose fermentation.

Added to detect acid byproducts (acetoin)
Barritt's A - 5% alpha naphthol
Barritt' B - 40% KOH
Red ring @ top = positive

Main ingredient is nitrate. The process whereby nitrate is reduced to nitrite, ammonia, or molecular nitrogen.

In nitrate reduction test, zinc turns nitrate to nitrite. If red, nitrate exists. If colorless, nitrogen gas or ammonia exists.

Ingredients: Simmon's citrate agar, bromthymol blue. Determines whether a bacterium can use citric acid as its sole source of organic carbon. Stab - streak process.

The enzyme citrase breaks down citrate. Blue tube is postive for alkaline product.

Ingredients: urea, phenol red. Determines whether an organism has urease that breaks down urea to CO2, ammonia and H20.

The enzyme urease breaks down urea. Hot pink is positive for the alkaline product ammonia.

Ingredients: ferrous sulfate, tryptophan, agar deep. Determines whether an organism produces hydrogen sulfide (S); whether it can metabolize the amino acid tryptophane to indole (I), pyruvic acid and ammonia, and whether or not the organism is motile (M).

Sulpher - if hydrogen sulfide gas is produced, it combines with ferrous sulfate to give black precipitate. This is BEFORE testing for indole production.

Indole is produced if tryptophan is metabolized. After adding Kovac's reagent, a thin band of magenta color above the agar surface is produced - a positive result.

After adding - cloudiness away from the stab line is postive. Done BEFORE testing for indole production.