Microbiology Lab Quiz #4
To be used as an indicator of possible fecal pollution, an organism should be:
(1) Present in the intestine in large numbers; (2) Nonpathogenic to humans; (3) Easily detectable in the laboratory A good example is Escherichia coli
m-FC Medium
Coliforms like E. coli form blue colonies; Rosolic Acid is added to inhibit Gram-positive organisms
Levine EMB Agar
Coliforms like E. coli form colonies with a green sheen; Gram-positive bacteria are inhibited
Colilert-18 Reagent
Contains 2 substrates for enzymes commonly found in coliforms - ONPG and MUG
Endo Agar
E. coli forms dark colonies with a metallic sheen; Enterobacter forms pink colonies
Coliform Density
Expressed in terms of the number of coliforms per 100ml of water Coliform colonies/100 ml = (Coliform colonies counted x 100) / (ml of water sample filtered)
MacConkey's Agar
Lactose fermenters form red colonies, non-fermenters form white colonies
Bleach (Sodium Hypochlorite)
TSB Plates: 100, 50, 25, 20, 5 Transfer 5 ml of the stock E. coli suspension to each of 5 sterile 13 x 100 tubes, which you have also labeled. A stock solution of 1% bleach is provided. Add 100 ul of the bleach to the 100 tube, etc.. Mix the solutions carefully and allow them to sit for 10 minutes. Then remove a 100 ul portion of each suspension, add it to the center of the corresponding agar plate, and spread the liquid evenly over the surface with sterilized glass beads.
KF-Streptococcus Medium
Fecal streptococci form red colonies
B-Galactosidase
Formed by E. coli and by other fecal coliforms that can degrade lactose
Doubtful Presumptive Test
Gas found after 24 hours in any tube
Positive Presumptive Test
Gas found within 24 hours in any tube
Non-Fecal Coliforms
Gram-negative - Enterobacter, Klebsiella, Citrobacter
Fecal Coliforms
Gram-negative - Escherichia, Shigella, Salmonella Gram-positive - Fecal streptococci, Streptococcus and Enterococcus
O-Nitrophenol
Yellow in color under alkaline conditions
ONPG
o-nitrophenyl-B-D-galactopyranoside Can be hydrolyzed by B-galactosidase to D-galactose and o-nitrophenol
pH
6.2 - 8.4
Free Chlorine
0 - 10 mg/L
Total Chlorine
0 - 10 mg/L
MUG
4-methylumbelliferyl-B-G-glucuronide Can be hydrolyzed by a B-Glucuronidase to glucuronate and a MUG product that shows a blue fluorescence under UV light; The B-glucuornidase is made specifically by E. coli
Chemical Tablets
5 TSB plates: 0, 1, 2, 5, 10 Coleman water purification tablets will be available and each tablet contains 16.7% tetraglycine hydroperiodide. At the designated time points, remove a 100 ul potion of the suspension, add it to the center of the corresponding agar plate, and spread the liquid evenly over the surface with sterilized glass beads.
API 20E Strip
API 20E Strip is used rather than running an extended set of of identification tests in separate glass tubes or slants. Multi-test systems do several tests at once on a small scale. API 20E is designed primarily for the identification of Enteric Bacteria that are associated with GI infections. Suspend the bacteria in 5ml of 0.85% NaCl (saline) and vortex the solution and look at the relative turbidity (should be faint). Use P-1000 micropipetter. Scoring: Negative reaction is given a 0; Positive reaction is given 1, 2, or 4 depending on the test
Escherichia coli
Ability to breakdown the disaccharide Lactose, using the enzyme B-Galactosidase and its potential to use the resulting monosaccharides (D-Glucose and G-Galactose) as substrates for fermentation Fermentation of lactose typically leads to the formation of both organic acids and gas. While some strains of E. coli do cause gastroenteritis, most do not. Use a simple colorimetric/fluorometric assay for B-Galactosidase as an indication of the presence or absence of coliform bacteria in a water sample
Simple Colorimetric / Fluorometric Assay
Add 50mL of your water sample to an IDEXX plastic container. Add one snap-back of Colilert-18 reagent. Screw the lid on tightly and mix thoroughly by inverting the container 5 times. Place the container in the 37C incubator to incubate for 24 hours. The container then will be moved to the refrigerator for storage until next lab. Results: A particular sample may contain both products; This assay is qualitative and only indicates the presence or absence of coliforms or E. coli
Coliform Bacterium
Any bacterium that is commonly found in the large intestine or colon
Total Alkalinity
As CaCO3, 0 - 240 mg/L
Total Hardness
As CaCO3, 0 - 25 gpg and 0 - 425 mg/L
Testing a Water Sample for Coliforms with a Simple Presence/Absence Assay
Because it is not easy to test for every possible pathogen, it is common to test instead for the presence of Fecal Coliforms; Coliform Bacterium; Fecal Coliform Bacterium
Membrane Filters
Can be placed on several types of media to detect different types of bacteria M-Endo Broth, M-FC Broth, KF-Streptococcus Broth, m-Coli Blue 24
m-Coli Blue 24
Detect total and fecal coliforms; Non-fecal coliforms form Red Colonies; E. coli form Blue Colonies
Concentrations of Nitrate and Nitrite
Dip a test strip for these compounds into water sample; Compare the colors to those on the side of the container
Concentration of Phosphate
Dip phosphate test strip into water sample; Compare the color to those on the side of the container
m-FC Agar Results
E. Coli - Blue small dots Enterobacter aerogenes - Clear/cream colonies; Spreading growth Klebsiella pnemonia - Light blue continuous globs Serratia marcescens - Tiny pink dots Shigella sonnei - Continuous blue dots Streptococcus mutants - 3 cells, all white circles Enterococcus faecalis - Tiny white dots; Continuous Bacillus subtilis - Tiny white distinct round colonies
MacConkey's Agar Results
E. Coli - Dark, pink circles, spreading growth Enterobacter aerogenes - Clear continuous dots; Pink on edges Klebsiella pnemonia - Pink globs; Continuous Serratia marcescens - Tiny clear continuous growth Shigella sonnei - Tiny white distinct colonies; Dots Streptococcus mutants - No growth Enterococcus faecalis - 1 pink colony Bacillus subtilis - 1 pink colony
Endo Agar Results
E. Coli - Metallic gold; Continuous growth; Streak Enterobacter aerogenes - Continuous pink growth; Dots Klebsiella pnemonia - Globular pink growth Serratia marcescens - Tiny pink dots Shigella sonnei - Small pink dots in a line Streptococcus mutants - Distinct large pink colonies Enterococcus faecalis - Slight metallic gold; Spreading growth Bacillus subtilis - Small distinct pink dots
EMB Agar Results
E. Coli - Metallic green growth; Continuous dots Enterobacter aerogenes - Pink spreading growth Klebsiella pnemonia - Brown globs; Continuous Serratia marcescens - Tiny continuous clear dots Shigella sonnei - Brown continous dots Streptococcus mutants - Pink circles, spreading Enterococcus faecalis - Small pink dots Bacillus subtilis - Small distinct pink dots
Streptococcus Agar Results
E. Coli - No growth Enterobacter aerogenes - No growth Klebsiella pnemonia - No growth Serratia marcescens - No growth Shigella sonnei - Small brown dots; Continuous Streptococcus mutants - Tiny distinct brown dots Enterococcus faecalis - Continuous tiny brown dots Bacillus subtilis - Tiny, distinct, brown dots
TSB Agar Results
E. Coli - Spreading cream colonies; Tiny Enterobacter aerogenes - Spreading cream colonies Klebsiella pnemonia - Spreading cream colonies; Globs Serratia marcescens - Spreading cream colonies Shigella sonnei - Spreading cream colonies Streptococcus mutants - Spreading cream colonies Enterococcus faecalis - Tiny, distinct cream colonies Bacillus subtilis - Golbular cream colonies
Series of Fermentation Tubes containing inverted Durham Tubes
Inoculated with different volumes of a water sample; 3-5 tubes of double-strength medium are inoculated with 10ml, 3-5 tubes of single-strength medium are inoculated with 1ml, and 3-5 tubes of single strength medium are inoculated with 0.1ml Several different media are commonly used, including lactose broth, lauryl tryptose broth, and brilliant green bile broth, and these media vary in their selectivity for lactose fermenters or E. coli and in their ability to select against Gram-positive organisms After incubation, the tubes are scores for the presence of acid and/or gas. The number of positive tubes in each set can be used to estimate the most probable number of coliforms in the water sample.
Fecal Coliform Bacterium
Might be released into the environment from human or animal feces
Negative Presumptive Test
No gas after 48 hours in any tube
Most Probable Number Method
Obtain 3 tubes of double-strength lactose broth and 6 tubes of single strength lactose broth. This medium does not contain a pH indicator but the tubes will have an inverted Durham tube for the collection of gas. Add 10.0ml of the environmental water sample to the three tubes of double-strength broth. Then add 1.0ml or 0.1 ml to the tubes of single-strength broth as shown in the figure (pg. 59). Place the tubes in the rack for incubation at 37C for 24 hours. The tubes will be refrigerated until the next lab. The tubes that have gas in the Durham tubes are scored as positive and are a presumptive indication that coliforms are present. The tubes that show no gas or take longer to develop are presumed to be negative. On the basis of the results, use the following table to estimate the most probable number in a 100 ml sample.
Testing a Sample Quantitatively for Coliforms Using a Membrane Filtration Assay
Set up a quantiative assay for coliforms by filtering a water sample through a fine pore (0.45 um) membrane filter. The filter will then be placed on an agar medium designed to detect coliform bacteria.
Testing a Water Sample Quantitatively for Coliforms Using the Most Probably Number Method
Set up a simple assay that will allow you to estimate the most probable number of coliforms in the water sample; Most traditional method of water analysis and is still used today in some places
Membrane Filter Technique
The purpose is to filter a water sample and place it in a petri dish on top of a filter paper pad saturated with m-Coli Blue24 Broth. (1) Obtain 2 small sterile plastic petri dishes with filter paper pads and m-Coli Blue 24 broth (2) Add 2ml of broth to each filter pad; Label one plate 20 ml and one plate 10ml (3) Assemble a filtration system by placing a Nalgene test filter funnel on top of an adaptor, which has been inserted into a large filtering flask; The flask should be connected to a vacuum pump (4) Measure out 20ml of your water sample and filter it through the system (5) Carefully remove the filter funnel from the adaptor (6) Unscrew the top from the bottom and remove the membrane filter; Place it in the corresponding small petri dish on top of a filter paper pad that has been soaked with m-Coli Blue24 broth (7) Repeat the whole process with a separate 10ml volume of your water sample; This is because you do not know how many microbes are in your sample and there may be too many colonies to count easily with the larger 20ml sample (8) Place this filter in the other small petri dish on top of a filter paper pad that has been soaked with m-Coli Blue24 broth (9) Place both of the petri dishes in the designated tray for incubation at 37C for 24 hours; The plates will then be saved int he refrigerator until the next lab period when the number of colonies on the filters will be determined
Boiling
To test the effectiveness of boiling on the bacterial suspension, obtain 5 plates of tryptic soy broth agar and label them 0, 0.5, 1, 2, and 3 minutes. Transfer 10ml of the bacteria suspension to a sterile tube. Place the tube in a beaker of boiling water. At each designated time point, remove 1.0 ml (1000 ul) portion of the suspension and add it to a sterile microcentrifuge tube. Once all the samples have been collected and allowed to cool to room temperature, remove 100 ul of each sample and add it to the center of the corresponding agar plate. Spread the liquid evenly over the surface for sterilized glass beads.
M-FC Broth
Used for detecting fecal coliforms
KF-Streptococcus Broth
Used for detecting fecal streptococci
M-Endo Broth
Used for detecting total coliforms
5-in-1 Test Strip from Hach Corporation
Used to determine Free Chlorine, Total Chlorine, Total Hardness, Total Alkalinity, and pH. Dip the strip into stock bottle of water and then compare the colors to those on the side of the container; Use the colors appropriate to each test and record the results
Cirtrate Utilization Test
Used to determine if a bacterium can utilize citrate as its sole source of carbon and energy. Bacteria that can utilize citrate as the sole source of carbon and energy must have the membrane-associated transporter Citrate Permease. If the bacteria utilize citrate, the byproduct CO2 combines with Na+ in the medium to form NaCO3, an alkaline compound. The pH indicator bromothylmol blue turns blue in an alkaline pH. A change in color of the medium from green to blue denotes a positive test. Indicates presence of CO2 as the CO2 dissolves in the aqueous phase of the agar to form HCO3- or CO32-, which raises the pH and turns the pH indicator from green to blue
Hydrogen Sulfide Production Test
Used to determine if a bacterium is able to dismantle Cysteine or to reduce Thiosulfate and form Hydrogen Sulfide as a byproduct via Cysteine Desulfurase. Hydrogen Sulfide (H2S) is a by product not used by the bacteria. Other bacteria reduce thiosulfate in energy-generating pathways and also release H2S. To detect H2S production, Ferrous (Fe2+) ion are included in growth media that contain protein and thiosulfate. Fe2+ combines with S-2 to form a black precipitate (FeS). Black Agar = Positive Yellow Agar = Negative Plate on Peptone Iron Agar or SIM Medium Look for blackening of the agar as this is indicative of the presence of Cysteine Desulfurase, an enzyme that breaks down cysteine with the formation of H2S, which can then react with the iron in the medium to form FeS
Oxidation-Fermentation (OF) Glucose Test
Used to determine if a gram-negative bacterium uses fermentation or aerobic respiration in utilizing a particular sugar for energy production. To distinguish between bacteria that obtain their energy by fermentation or by aerobic respiration of sugars, an Oxidation-Fermentation (OF) Medium contains a high concentration of a specific sugar and a low concentration of Peptone (an enzymatic digest of protein). The small amount of peptone supports the growth of bacteria unable to utilize the sugar. To detect the production of organic acids during fermentation, the pH indicator bromthymol blue is included in the medium. If acids are present, the medium appears yellow: the bacteria have fermented the sugar in the tube. If no acids are present, the medium appears green: fermentation of the sugar did not take place. Two tubes of OF medium are used to test a single bacterium: one tube is open to the air (aerobic conditions) and the other is covered with a layer of sterile mineral oil or melted paraffin to prevent diffusion of atmospheric oxygen into the medium (anaerobic conditions). Bacteria that ferment the sugar will produce organic acids in both tubes, and both tubes will appear yellow throughout. This result will also be seen for bacteria that have enzymatic pathways for both fermentation and aerobic respiration. Bacteria that use an aerobic respiration pathway for extracting energy from the sugar will produce acid only in the tube open to the air. Only the medium in the unsealed tube will turn yellow at the top; the medium in the sealed tube will remain green. If the top of the medium in both tubes appears slightly yellow, it indicates either that the bacterium is only a slow fermenter, or is both an aerobic oxidizer and a slow fermenter. If the color of the medium in both tubes remains green after incubation, the bacterium is considered to be a nonfermenter and anonoxidizer of the sugar. If the bacterium lacks the enzyme to digest lactise, maltose, or sucrose, both OF tubes will also remain green after incubation. An OF medium can also be used to detect motility because it contains semi-solid agar. Bacteria with flagella will swim away from the stab used to inoculate the medium, resulting in a "cloud" of bacterial growth around the stab. A yellow color indicates the fermentation of the substrate (D-glucose of lactose) while a green color indicates that the substrate was metabolized only by respiration (or not at all)
Spectrophotometer
Used to determine if your sample contains dissolved solids or other particles. Set the wavelength to 600nm and use a 3ml cuvette filled with filtered deionized water to set the instrument to 100% transmittance. Remove the cuvette and replace the pure water with 3ml of your environmental sample; Put the cuvette back in the instrument and measure the percent transmittance. A value less than 100% would indicate the presence of solids in the sample that give it a distinct turbidity
Phenylalanine Deamination Test
Used to determine the ability of a bacterium to remove the amino group (Deaminate) from the AA Phenylalanine. Members of the genera Proteus and Providencia used the enzyme Phenylalanine Deaminase to catalyze the removal of the amino group from Phe. In addition to NH3, phenylpyruvic acid is the major organic product. The presence of phenylpyruvic acid is detected by the addition of a solution of Ferric Chloride (FeCl3). A dark green color appears in a positive test. If no phenylpyruvic acid is present, the medium appears yellow, the color of FeCl3. Green = Positive Yellow = Negative Plate on Phenylalanine Agar Add 3 - 5 drops of a 10% FeCl3 solution to the slant surface; The formation of a dark green color is indicative of the presence of Phenylalanine Deaminse, which converted the phenylalanine to phenylpyruvic acid
Amino Acid Decarboxylase Test
Used to determine the ability of a bacterium to remove the carboxyl group from an AA (Decarboxylation). Members of the bacterial family Enterobacteriaceae can be distinguished by their production of a specific Decarboxylase that removes the carboxyl group from particular AA. These AA include the AA Lysine, Ornithine, and Arginine. The reaction is assessed by changes in the color of the pH indicator in the medium. Fermentation of glucose in the medium results in accumulation of acids (purple to yellow). However, with decarboxylation of the AA, the resulting amine causes an increase in pH (yellow to purple). A positive result for an AA decarboxylase is purple in the medium; a negative result is yellow. Purple = Positive Yellow = Negative Plate on Lysine Decarboxylase Medium Note the presence of growth and the color of the medium; A purple color is indicative of the presence of a decarboxylating enzyme that removes the carboxylic acid group from the amino acid, causing an increase in pH
Fermentation of Carbohydrates (Durham Tubes)
Used to determine the ability of some bacteria to ferment a particular carbohydrate. In this test, acid production is identified by a change in the color of the Phenol Red, a pH indicator that is included in the medium. In acid pH, phenol red is yellow. To collect gases, an inverted smaller tube (a Durham tube) is place in the medium. A yellow color indicates the formation of organic acids as a by-product of fermentation. These cause the phenol red pH indicator to change from red to yellow. A bubble in the Durham tube indicates the formation of a gas such as CO2 or H2 as a by-product of fermentation.
Voges-Proskauer Test (Butanediol Fermentation)
Used to determine the ability of some bacteria to ferment glucose via butanediol fermentation. Some bacteria ferment glucose via the Butanediol Fermentation pathway. The precursor for the neutral alcohol 2,3 butanediol is acetylmethylcarbinol, also known as Acetoin. Voges-Proskauer (VP) reagents react with acetonin in the presence of O2 to form a red product. This red color denotes a positive VP test. No color change after addition of the VP reagents denotes a negative test. Add 10 drops of Voges-Proskauer (Barritt's) Reagent A and 5 drops of Voges-Proskauer (Barritt's) Reagnet B to the other tube; Mix the solution vigorously; Allow the tube to sit for up to 30 minutes; The formation of a pink or red color at the top is indicative of the formation of 2,3-butanediol as a fermentation product.
Methyl red Test (Mixed Fermentation)
Used to determine the ability of some bacteria to ferment glucose via mixed-acid fermentation. Products of Mixed-Acid Fermentation of glucose include significant amounts of organic acids. These acids lower the pH of the medium to <5. When the pH indicator Methyl Red is added to the medium and the pH is 4.5 and below, the methyl red reagent remains red in color. This is a positive test. At higher pH values (less acid present), the color may be orange or yellow. These colors denote a negative test. Use MR-VP Medium Methyl red reagent is a pH indicator and will turn red if the pH is acidic; This is indicative of the formation of organic acids as by-products of fermentation
Indole (Tryptophan Degradation) Test
Used to determine the ability of some bacteria to split the AA Tryptophan into Indole and Pyruvic Acid. Red = Positive Yellow = Negative Use Tryptone Broth Add 5 drops of Indole (Kovac's) Reagent to the top of the Tryptone Broth; The formation of a red layer on top of the medium indicates that the tryptophan in the medium was converted to indole by the enzyme Tryptophanase.
