Microbiology Lab 2
What does catalase do in the Catalase Test?
Catalase breaks down H2O2 (hydrogen peroxide) to oxygen and water.
S. epidermidis
Colonies appear small, either white or yellow in color Opportunistic - immunocompromised, catheters Found as a normal commensal of the skin
Differential
Contains factors some bacteria can change in a recognizable way that allow it to be distinguished from other bacteria
Enriched Media
Contains factors which enhance the growth of desired organism For fastidious (fussy) organisms- have complex growth requirements not present in common mediums
What are classical clinical identifications based off of?
Differential biochemical activities -i.e. what the bacteria use for "food" -- the metabolic processes used to generate energy
What is TSI - Triple Sugar Iron Agar?
Differential medium that contains lactose, sucrose, a small amount of glucose (dextrose), ferrous sulfate, and the pH indicator phenol red.
How are carbs broken down?
Exoenzymes & Endoenzymes
What 3 things are we looking for in an TSI - Triple Sugar Iron Agar
Fermentation Sulfur Reduction Gas Production
Pseudomonas Aeruginosa
GRAM NEGATIVE opportunistic pathogen Nosocomial infections -Will grow in distilled water, some disinfectant "Rotted grape" smell associated with growth esp on spoiled foods -ubiquitous, grows at low temps Very antibiotic resistant Cystic fibrosis
Protein Catabolism tests
Gelatin Hydrolysis test Urea hydrolysis Phenylalanine deamination Ornithine decarboxylase broth SIM agar deeps
Escherichia coli
Gut bacterium -lower intestine of warm-blooded animals Coliform -Lactose fermenter indicative of fecal contamination of water Gastroenteritis, UTIs, neonatal meningitis -"jack in the box" outbreaks in California in the 1990's -"Montezuma's revenge" "New Delhi Belly" and "Turkish Trots" -Pathogenesis primarily due to strain specific toxins Appears in an infant's GI tract as soon as 40 hours post birth
Selective/Differential Media tests
PEA agar MSA agar PSP agar MAC agar
S. aureus
"golden cluster seed" On blood agar, colonies appear as large round golden shapes β - hemolytic Furuncles, food poisoning, SSSS (scalded skin syndrome) Commensal of the skin, commonly found in nose of 1/3 of population MRSA - methicillin resistant S. aureus
Results of the MR test
-The MR test measures the end products of the fermentation reaction. If it stays red, acid end products were produced. If it turns yellow, neutral end products were produced -The maintenance of a red color is considered a + test, and the disappearance of a red color is considered a negative result.
Decarboxylation removes _______; Deamination removes ______
Carbon Dioxide; an amino group
What is an MRVP test?
2 tests in one. We grew the organism in two MRVP broths - a simple nutrient broth supplemented with beef extract. In one tube, the MR test is performed, and in the other tube, the VP test is performed **Organisms are usually MR+VP- or MR-VP+. It is rare to get a positive result for both tests.** --Methyl red is a pH indicator that has a baseline of pH 4.4. If the pH of the medium is below pH 4.4, it stays red. If the pH is above pH 4.4 it turns yellow.--
Most decarboxylations involve enzymes that function __________, so we do this...
Anaerobically; so we inoculate our broths with the presence of mineral oil to minimize oxygen exposure. -This favors or chances the decarboxylation reaction!
Exoenzymes
Bacteria have exoenzymes to catabolize carbohydrates for carbon and energy Exo -secreted (i.e. outside the cell) Enzyme - hydrolytic - use water to break down large molecules into smaller molecules
Fermentation
Breakdown of a sugar that results in the generation of acid and can result in gas production (not always)
Spirit Blue Agar
Lipase is an exoenzyme that hydrolyzes lipids into fatty acids and glycerol. agar contains lipids and spirit blue dye. If a microorganism produces lipase, the fats around the streak are decomposed, causing a clear zone to appear The spirit blue dye also migrates through the plate toward the region lacking complete lipids, resulting in the appearance of a dark blue halo around the organism.
In TSI agar-- If the butt of the slant is obscured buy the black precipitate produced by H2S then what?
Look at the top of the slant to determine if the organism could ferment only glucose (RED), or if it could only ferment either lactose and/or sucrose (YELLOW)
MAC Agar
MacConkey Agar -- Selective for BILE SALTS and CRYSTAL VIOLET, (inhibits gram + organisms) Differential: pH indicator and lactose differentiate lactose fermenters -- pH indicator responds to the drop in pH that accompanies the acid produced from fermentation and changes color - Lactose fermenters produce pink/red colonies (POSITIVE RESULT) -White (NEGATIVE RESULT)
MSA Agar
Mannitol Salt Agar -- Selective for GRAM POSITIVE HALOPHILIC BACTERIA (e.g. Staphylococcus spp.) (inhibits gram - and most gram + b/c they are not halophilic e.g. E. Coli) Differential: Presence of mannitol - a common carbohydrate -Both S. aureus and S. epidermidis will grow, but only S. aureus will ferment mannitol - turns yellow b/c pH indicator changes in response to acid production (POSITIVE RESULT) -NEGATIVE RESULT- stays red
Bacteria that are obligate aerobes are generally... Many facultative organisms are usually...
Many facultative organisms are also catalase +. In general, obligate anaerobes are catalase -.
Starch Hydrolysis
Measure of the presence of the exoenzyme amylase -Starch must be broken down before it can be taken into the cell (size factor) Degrades starch present within the media Iodine stains starch within the media + test represented by a clear zone around bacterial growth
Exo- vs. Eno-
Mode of action will occur outside the cell, so you are looking for a visible change in the agar medium surrounding the bacterial growth VS. Remain inside the bacterium, so you are looking for a differential change in the bacterial colonial morphology
Respiration Media tests
Nitrate Reduction Test Catalase Test Oxidase Test
What are carbohydrates?
Organic compounds containing carbon, hydrogen, and oxygen. (CH2O)n
What must you do to preserve the integrity of your results in a Oxidase test?
Oxidase POSITIVE organisms will generally exhibit a brilliant blue color change in the first 10-15 seconds, while those that are oxidase NEGATIVE will change to a blue-brown after 20-30 seconds due to atmospheric oxygen and can be mistaken for a positive result. & use oxidase reagent that is still clear.
In Nitrate Reduction Test --After incubation, add Nitrate A and B reagents...THEN
POSTIVE- The formation of a red color throughout the broth tube is a positive test o A positive test indicates NO3 (nitrate) was reduced to NO2 (nitrite). (Nitrate reagents A and B specifically detect nitrite (NO2). NEGATIVE *FOR FIRST REACTION* -- If no color change occurred after the addition of Nitrate A and B reagents, two possibilities exist. o No reduction occurred and nitrate is still present in the tube. o NO3 (nitrate) was reduced even further, i.e. past NO2 (nitrite) to compounds such as NH3 (ammonia) or NO (nitric oxide).
PEA Agar
PhenylEthyl Alcohol Agar -- Selective for GRAM POSITIVE, (inhibits DNA synthesis in gram -) NOTE: some strains of E. coli are slightly resistant to the phenylethyl alcohol and will exhibit "sickly" or "ghostly" growth
Selective Media
Prevents growth of unwanted bacteria without inhibiting the growth of the desired organism
Ornithine decarboxylase broth test results
Purple - POSITIVE Yellow - NEGATIVE
What does an OF Glucose Medium determine?
READ IN PAIRS!!!! Determines whether an organism is oxidative (O) or fermentative (F) or both (OF), which means it determines if the sugar can be fermented in the presence or the absence of oxygen.
What are the pathogens of enterobacteriaceae?
Salmonella and Shigella spp. AND sometimes...members of the Proteus and Klebsiella genera
3 basic types of media
Selective, Enriched Media, Differential
Pseudomonas P agar
Selective: Irgasan- animicrobial agent in the medium that inhibits gram + and gram - OTHER THAN PSEUDOMONAS SPP. (Only grows this) -MgCl2 and K2SO4 in the medium promote pigment production Differential: Pseudomonas spp. produce pigments (colors) -P. aeruginosa will appear as isolated colonies surrounded by a bluish-green zone (POSITIVE RESULT) others may produce brown, red or orange hues.
Nitrate reduction -- additional info
Some facultative anaerobes are capable of using nitrate as an electron acceptor at the end of the Electron Transport Chain. Enterics that reduce nitrates (NO3) usually produce nitrite (NO2). Other bacteria reduce nitrate and form products such as ammonia (NH3), nitric oxide (NO), and nitrous oxide (NO2).
What does fermentative mean?
Some organisms do not require oxygen to catabolize carbohydrates -Fermentation usually results in the production of acidic organic end products and/or the production of gas
what does oxidative mean?
Some organisms require oxygen to catabolize carbohydrates
Carbohydrate Catabolism tests
Starch Hydrolysis Spirit Blue agar OF Glucose Medium Carbohydrate Fermentation Tubes Simons Citrate Medium MRVP test TSI - Triple Sugar Iron agar
Microaerophiles
Such organisms require limited amounts of oxygen for growth. 7-10% required for optimal growth.
SIM agar deeps tests for what 3 things and is useful in what?
Sulfur reduction Indole production Motility *differentiating enteric organisms*
In TSI agar-- What colors will appear if an organism can only ferment glucose?
The slant will appear red but the butt of the tube will remain yellow
Facultative anaerobes
Thee organisms can grow either in the presence or absence of oxygen. They will preferentially utilize oxygen if it is available and perform aerobic respiration.
Obligate Anaerobes
This class of bacteria will only grow in the absence of oxygen! Exposure to oxygen is lethal for these bacteria
Why must ornithine decarboxylase broth test be incubated for at least 48 hours?
To get a definitive result by: allowing time for the glucose to be exhausted & for the bacteria to have time to break down the ornithine, raise pH and the color can change from yellow to dark purple or red.
On what biochemical basis can many enteric bacteria be identified?
Types of sugars fermented (ex: glucose, lactose, sucrose, etc.) end products produced from that fermentation (ex: gas, acid production, etc.)
Why is the Simmons Citrate Medium used?
Used in the form of a dark green slant to test the utilization of citrate, which some organisms can use as a carbon source
What was the process for the OF Glucose Medium?
We did two tubes per organism. One tube we overlaid with mineral oil (the + tube), and this served to "seal" the media and the inoculated bacteria from atmospheric oxygen exposure. The other tube we did not overlay, and this left the bacteria exposed to oxygen (the - tube).
In Nitrate Reduction Test-- IF no reaction observed in first reaction we add _____ and that confirms which 2 possibilities?
Zinc dust; o Zinc reacts with NO3 (nitrate) and causes a red color change. If this happens, no reduction has occurred since NO3 is still present in the tube, and the reaction is considered negative. o If no color change occurred after adding the zinc dust, then the organism did reduce, but reduction was not to nitrite but to some other nitrogen containing compound, and the reaction is considered positive.
Nitrate Reduction tests what?
ability of organism to reduce nitrate (NO3) to nitrite (NO2) or further reduction to ammonia (NH3) or nitric oxide (NO)
Halophile
able to grow/withstand high salt concentrations -- commonly found as normal flora of the skin (think sweat!)
What is in a Carbohydrate Fermentation Tube?
contains a carbohydrate, a pH indicator, and a Durham tube (little upside down test tube within the big test tube)
What is TSI used to differentiate?
enteric based on the ability to reduce sulfur and ferment carbohydrates.
Endoenzymes
enzymes that are not secreted, but work intracellularly in a similar fashion Caveat: the mode of action of the enzyme may produce a product that gets outside the cell that may change the agar medium surrounding the bacterial growth Ex: fermentation produces acid and gas by-products which can change the agar medium surrounding the bacterial colonies or streak line
Urea Hydrolysis tests for the presence of what?
exoenzyme urease-hydrolysis urea to CO2 and ammonia & "rapid urease +" organisms
What is Enterobacteriaceae?
family of short gram negative none spore-forming bacilli found in the intestinal tract
Nitrate Reduction-- Nitrate-positive
if nitrite (pink color) is detected in the inoculated medium after the addition of reagents A and B or if no color is detected in the medium after the addition of zinc dust.
Nitrate Reduction -- Nitrate-negative
if nitrite is not detected (no color change) after the addition of reagents A and B, or if a pink color develops after the addition of zinc dust to the inoculated medium.
In a Thioglycollate broth Microaerophiles will grow...
in a thin layer below the richly-oxygentated layer, usually in the upper third region of the tube.
In a Thioglycollate broth Aerobes will grow...
in the top portion only
What are normal intestinal flora of enterobacteriaceae?
members of Escherichia and Enterobacter genera -Can also be pathogens under right conditions -Certain toxigenic strains of E. coli are pathogenic
What does the SIM medium contain?
nutrients, iron, and sodium thiosulfate. One of the nutrients is peptone, which contains amino acids, including tryptophan.
Results of the VP test
o The VP test tests for the presence of a specific neutral end product produced from the fermentation process (acetonin) o After adding VP reagent I and VP reagent II, we vortexed the tube and left it exposed to oxygen for 15-30 minutes. A + test is indicated by the formation of a reddish ring at the broth oxygen interface at the top of the tube.
In a Thioglycollate broth Obligate anaerobes will grow...
only in the lower areas of the tube where oxygen is absent or in minimal concentration
Gelatin Hydrolysis tests the presence of what?
the exoenzyme gelatinase- degrades gelatin
Oxidase tests test for?
the presence of cytochrome c, a specific protein in the electron transport chain.
Catalase Tests test for what?
the presence of the enzyme catalase
Obligate Aerobes
these bacteria require the presence of atmospheric oxygen for growth. Will NOT grow in the absence of oxygen.
Aerotolerant anaerobes
these organisms do not utilize oxygen as their terminal electron acceptor, but they are not killed in oxygens presence.
In a Thioglycollate broth Aerotoerant anaerobes will grow...
throughout the medium but will primarily grow from the middle of the tube down, with more turbidity (cloudiness) occurring in the lower regions
In a Thioglycollate broth Facultative will grow...
throughout the tube, yet will appear more cloudy at the upper portion of the broth, due to the increased ATP yield.
How do we test motility in SIM agar deeps?
we can evaluate motility by looking for "spreading growth" from the stab line
Test results for a Urea hydrolysis
• A positive test is indicated by a color change from a light orange to a hot pink color (fuchsia) • Strong fermenters will usually change the orange color of the media to a yellowish hue due to excessive acid production.
Oxidase test results
• A positive test is indicated by the formation of a blue color after the addition of the oxidase reagent to the cotton swab inoculated from one of our plate cultures. • A negative test is indicated by the no color change (the oxidase reagent is colorless without the presence of a cytochrome c protein to react with)
What must you do to / when can you view/judge a Gelatin Hydrolysis test?
• Gelatin liquefies at temperatures above 28°C. Standard incubator temperature is 37°C. Therefore, there are two considerations when doing this test: o Either incubate the tubes at room temperature (~25°C) o If incubation occurs at 37°C, then after incubation, place the tubes in the refrigerator so the gelatinase - tubes can re-solidify. If the organism is gelatinase + it will remain liquid after incubation in the refrigerator. If the organism is gelatinase -, it will re-solidify after refrigerator incubation.
Catalase test results
• If a bacterium is catalase POSITIVE, when we add H2O2 to bacteria growing on a plate we will see "bubbles" o This is the oxygen escaping from the water • If a bacterium is catalase NEGATIVE , no bubbles will form
Results for TSI - Triple Sugar Iron Agar
• If an organism can ferment any of the three sugars present in the medium, the medium will turn yellow. • If an organism can reduce sulfur, the hydrogen sulfide gas which is produced will react with the iron to form iron sulfide, which appears as a black precipitate. • If the fermentation produced gas, you may see fissures in the medium, or the entire slant may be raised above the bottom of the test tube.
How do we test for sulfur reduction in SIM agar deeps?
• If an organism can reduce sulfur to hydrogen sulfide, the hydrogen sulfide will combine with the iron source in the medium to form ferric sulfide, which is a black precipitate. If there is any blackening of the medium, it indicates the reduction of sulfur (the production of H2S) and is a positive result.
Gelatin Hydrolysis test results
• If an organism is gelatinase +, it will liquefy the solid gelatin in the tube -**• Depending upon the inoculum amount, the entire tube may not liquefy. Tilt the tube at a 45°angle, and if any portion of the tube appears to "run", the test is considered a positive.** • If an organism is gelatinase -, the gelatin will remain solid
How do you interpret the results for the OF Glucose Medium?
• If both tubes were still green, the test is negative, as no fermentation of glucose occurred. • If the non-mineral oil tube (the - tube) was yellow, but the mineral oil tube (the + tube) remained green, the organism was considered oxidative (O), meaning it could only break down the sugar in the presence of oxygen. • If the +mineral oil tube was yellow, but the non-mineral oil tube remained green, the organism was considered fermentative (F), meaning it could only break down the sugar in the absence of oxygen. • If both tubes were yellow, the organism was considered to be oxidative/fermentative (OF), meaning it could ferment the sugar in both the presence and absence of oxygen
Results of Simmons Citrate Medium
• If the medium turns blue, the organism is citrate positive. If there is no color change, the organism is citrate negative. Some citrate negative organisms may grow weakly on the surface of the slant, but they will not produce a color change. Usually no color change coincides with the lack of growth on the slant and the inability to use citrate as a carbon source.
What does it mean if the OF Glucose Medium is dark blue?
• OF Glucose Medium in addition to glucose contains a small amount of peptone. Bacteria that cannot ferment can usually break down the proteins in the peptone and this causes the medium to turn from a normal green to a very dark blue, (amino acid deamination that occurs from peptide breakdown is an aerobic alkaline reaction) and this usually occurs in the upper portion of the tube where oxygen levels are highest. • Remember this test does not characterize whether the organism has an aerobic, anaerobic or fermentative metabolism, simply whether it ferments glucose under different conditions of oxygenation.
Results of a Carbohydrate Fermentation Tube?
• Sterile or un-inoculated media appears dark red. If fermentation occurs, it will result in a change from a red color to a yellow color. • If gas is produced during fermentation, a gas bubble will become trapped in the Durham tube. • Bacteria that cannot ferment may grow in the media, but there will be no color change, and is considered a negative reaction. You should check the tube for a cloudy or "turbid" appearance, indicative of bacterial growth. • Sometimes the tube appears a mosaic of yellowish-red. This is an indication of weak fermentation. Sometimes the slight yellow color can be observed at the top or bottom of the tube. This is weak fermentation as well. • You can test any carbohydrate in this manner. We tested 2 different sugars: lactose and sucrose. WEAK RESULT IS STILl POSITIVE!!
How do we test indole production in SIM agar deeps?
• The medium contains tryptophan. When tryptophan is broken down by bacteria, indole is produced. o After incubation of the tubes, we add Kovac's reagent. Kovac's reagent, which is normally yellowish, will react with the indole and produce a cherry red colored ring. ♣ Indole *POSITIVE* - cherry red ring observed at the top of the tube ♣ Indole *NEGATIVE* - no color change occurs after adding Kovac's reagent. (remains yellow)
Process for ornithine decarboxylase broth test
• The medium is a broth that contains glucose, the amino acid ornithine and a pH indicator. Most all bacterial species can ferment the glucose, and the resulting acid that results from fermentation will activate the pH indicator and the broth will turn yellow. - NEGATIVE • At this point, the resulting reaction depends upon the organism. Once the glucose is exhausted, some bacteria can decarboxylate, or remove the CO2 (carbon dioxide) from ornithine as a means of continued metabolic growth. o Removal of CO2 from ornithine leaves an amine by product which raises the pH of the broth. ♣ Ornithine decarboxylase + bacteria will change the broth back to a light purple color. - POSITIVE
Process of Phenylalanine deamination
• This is an aerobic reaction, hence the use of only the slant portion of the tube. • The liberated amino group is converted to ammonia, and you are left with phenyl pyruvic acid. • FeCl3 added to the tubes after incubation and reacts with the phenyl pyruvic acid. This reaction results in a color change of the FeCl3 solution from reddish orange to green. o POSITIVE test is the formation of a green color o NEGATIVE test is no color change after adding FeCl3
What causes the color change in a positive Urea hydrolysis test?
• Urease breakdown of urea generates NH3 (ammonia) as a by-product, which raises the pH of the broth medium. • Urea broth contains phenol red. The pH of prepared urea medium is 6.8, so phenol red is yellowish-orange • As ammonia builds up in the medium, the pH goes up, and the phenol red indicator changes color form yellowish orange to hot pink.
Phenylalanine Deamination test for what?
• the organism's ability to remove an amino (NH2) group from the amino acid phenylalanine.
