Microbio Lab Final Flashcards

To determine the biochemical properties of isolated
pure bacterial cultures using various enzymatic assays.

So that the results of such tests reflect the biophysical properties of one single species of bacteria and not several. When the results of enough biochemical tests
have been determined, a unique “fingerprint” for each bacterium can be determined that helps to
tell it apart as a unique species or genus compared to other bacteria.

OF Media (with and without mineral oil), upon and within Kligler’s Triple Iron Sugar (TSI) Agar slant, upon a Urea
Agar plate, within a Litmus Milk Broth, upon a BHI Starch Agar plate, upon and within a Nutrient Gelatin slant, within a Nitrate broth, and within a Methyl Red/Voges-Proskauer broth media.

Proteolysis, an enzymatic process, is detected with a nutrient gelatin medium, it is tested for liquefaction as gelatin is solid below 28°C. The incubation can happen at any temperature and after growth, the tubes are placed in an ice bath to test for liquefaction (gelatin breakdown).

Positive test result shows liquefaction of gelatin after cooling to 0 Celsius in an ice bath for 10 min. No color change can be appreciated in this test whatsoever so do not get tricked by that!!!!

Gelatin remains solid after the 10 minute ice bath.

It is inoculated by streaking the surface and then stabbing the agar deep down to the bottom, like this, we inoculate down into the anaerobic-oxygen limited zone as well as on the
aerobic surface zone. The medium contains a complex nitrogen source so most organisms can grow on it. The medium contains 0.1% glucose and 1% lactose and the dye phenol red.

The growth of bacteria within the tube should be observed first. Obligate aerobes will only grow on the surface of the slant whereas facultative organisms will grow both on the
surface and in the deep.

The color of both the slant and butt of the tube should then be analyzed to determine if glucose, lactose, and sucrose have been fermented.

If the bacteria can utilize only glucose (and not lactose), only a small reaction (acid) will be produced around the growth in the deep and the slant should remain pink. For facultative bacteria, the slant is yellow. If the organism can utilize lactose, then the whole tube should turn yellow (acid production from lactose)
because there is ten times as much lactose as glucose. Some bacteria produce alkaline end-products from the metabolism of nitrogenous nutrients, this will be seen as a change in the color from red to pink. Phenol red turns a pink color in alkaline conditions. This reaction generally occurs under
aerobic conditions.

The fermentation that results in gas production should result in a splitting of the agar in the butt of the tube. If the medium turns black, particularly in the butt of the tube, it indicates the production of hydrogen sulfide.

Tests whether glucose is metabolized oxidatively, fermentatively, or both. This test provides only one fermentable sugar and that is glucose.

The OF medium is a semi-solid (agar) medium containing peptone, glucose, and the dye bromo-thymol blue. This dye is yellow in acid conditions and blue in alkaline conditions. It, therefore, appears green (optical illusion) at neutral conditions.

Two test tubes are required, and both are inoculated with the isolate. After inoculation, sterile mineral oil is layered over the medium surface of only one tube. This tube is the anaerobic tube. Oxygen can diffuse into the other. After incubation, the tubes are observed for their color and presence or absence of growth.

If no growth occurs, then this test is invalid.

In addition to the color change, you should be able to detect gas production because, in this semisolid medium, if your unknown produces gas it should “crack” the agar.

The ability of bacteria to produce amylase enzyme can be determined from starch containing agar. After growth, the plate is flooded with Gram's iodine which causes the starch to
turn dark blue or purple in color.

After growth, the plate is flooded with Gram's iodine which causes the starch to turn dark blue or purple in color. If the bacteria produced amylase that has digested the starch, there will be a clear zone around the colonies. So, positive reaction for starch hydrolysis is represented by a clearing zone around
bacterial growth that can extend through entire depth of agar plates. Areas without bacterial growth show as dark
purple due to reaction of iodine with starch and air. The negative results do not have a clearing zone around the bacterial growth.

The ability of bacteria to produce urease is tested with urea agar which contains peptone and glucose with 2% urea. The enzyme urease hydrolyses urea to carbon dioxide and ammonia.

The medium contains phenol red as a pH indicator (yellow in acid, red near pH 7, pink in alkaline conditions). Positive test results in the medium becoming pink after the growth of the bacterium on the agar.

all pink (positive for urease enzyme hydrolyzing Urea and production of alkaline/basic reaction under both aerobic
and anaerobic conditions), pink surface (positive for Urease enzyme hydrolysis only under aerobic conditions), and yellow (no urease enzyme present, but bacterial growth causes acidic conditions and potential gas formation as indicated by yellowing of butt)

Some bacteria can reduce nitrate to nitrite, which is an activity found in situations when oxygen is absent or limiting.

A positive test result is indicated by a color change of the nitrate reagent from a pale yellow or brown to a red or pink
color. If bacteria can only reduce nitrate to nitrite, a color change will occur after the two reagents are added. Therefore, if there is no color change occurring after the addition of nitrite test reagents, zinc powder must be added.

If tested bacteria can reduce nitrate to ammonia, no color change will occur after the addition of zinc, thereby indicating a positive result for the overall test due to the breakdown of nitrate to ammonia. If there is a red or pink color observed after the addition of zinc, nitrates are still present thereby indicating a negative result for the overall test.

These tests can be performed on your bacterial sample immediately and are key indicators used to classify bacteria into broad groups in dichotomous keys or determine which identification test kits to utilize.

A portion of the colony is taken and placed in a drop of 3% H2O2. The generation of bubbles (O2 gas) within a minute is a positive test.

Tests for the presence of cytochrome-C (Cyt-C), presence of the cytochrome-C enzyme in bacterial samples results in a blue/purple color change, while those lacking cyt-C remain colorless.

Detects whether bacteria produce enough fermentation of
glucose to lower the pH of a culture below 4.5 via production of stable acids.

A positive result is a color change to red (or orange) through the methyl red indicator dye.

Just know that is performed alonside the methyl red test, and a positive result is red coloration and a negative result is yellow coloration.

Methyl Red and Voges Proskauer Tests.

To explore the selectivity of the bacteria you isolated
to efficiently use certain carbon and energy sources and to determine more biochemical properties using differential media.

Help to tell apart different bacterial species by causing a color change in the presence of a desired biochemical trait.

Help to confirm the presence of bacteria that can only grow in certain conditions. In this sense, they “select for” certain bacteria. However, selective media can also “select against” the growth of other bacteria

This medium is both selective (contains 7.5% NaCl) and
differential (contains the sugar mannitol and the dye phenol red). Yellow represents acidic fermentation of mannitol sugars. Plates with bacterial growth but the pink color are halophilic or halotolerant but incapable of fermenting mannitol

EMB Agar is mainly used as a differential medium
(due to color changes in the pH indicator dyes eosin-Y and methylene blue), but it is also slightly selective for fecal coliform bacteria. EMB Agar inhibits the growth of most (but not all) Gram positive bacteria. Strong acidity produces a deep purple colony with a green metallic sheen, whereas less acidity may produce a brown-pink coloration of colony. Non-lactose fermenters appear as translucent or pink. Colonies of lactose fermenters will appear very dark purple, or have dark purple centers.

This agar is named for the addition of fresh blood after autoclaving and is used to test the ability of bacteria to break down red blood cells and hemoglobin. Because it contains red blood cells, it is differential in that it can visually show
which bacterial colonies can cause hemolysis and destruction of red blood cells. Alpha hemolysis appears as a greening. Beta hemolysis is the complete destruction of the red blood cells, causing the formation of a zone of clearing around the colony, often as a yellow transparent “halo” surrounding bacterial colonies on blood agar. Bacteria that will grow on blood agar, but which cause no hemolysis of red blood cells, are known as Gamma hemolytic bacteria.

This medium is both selective and differential in nature. It is selective because only Gram negative bacteria can grow on it. Lactose ferments will stain pink while the nonlactose ferments will be clear colonies.

Tests for the ability of an organism to degrade the amino acid tryptophan and produce indole. Indole forms a red ring with the addition of Kovac's reagent indicating the bacterial species is indole positive and that it produces tryptophanase.

This agar is used to determine the ability of bacteria to utilize citrate as a sole carbon source. The test is used to differentiate between different bacterial species based on their
metabolic capabilities. If the organism has the ability to use citrate, the medium usually changes its color from green to blue, though growth on the medium even without colour change is considered a positive result. An observation of no growth is a negative result.

The Analytical Profile Index or API is a system developed for the quick identification of clinically relevant bacteria. Because of this, only known bacteria can be identified.

API20E-Gram negative rods;
Oxidase API20NE-Gram negative rods; Oxidase +
API20Staph-Gram positive cocci
(clusters); Catalase + API20Strep-Gram positive cocci (chains); Catalase API50CH-
Gram positive rods

A) Gram-negative bacillus, pure
B) Gram-positive cocci, pure
C) Gram-positive and negative bacillus, not pure

A) Carbol fuschin

B) Steam/heat

C) Acid alcohol

D) Methylene blue.

VT=(CFU/mL)xDF

CFUs/mL

Bacteria can reduce nitrate to ammonia; positive result for both nitrate and nitrite reductase.