Gram Negative Rods

Gram Negative RodsEnterobacteriaceae (Coliforms)

Tests Used to ID Unknown Organisms

HOW TO ID AN UNKNOWN ORGANISM

• Your goal with the enteric unknown is to determine the genus of the organism you have.

• Individual tests can be performed in separate tubes (conventional method) or all the tests can be performed in one specially designed Enterotube (rapid identification method). Comparisons between rapid identification methods and conventional methods show that they are equally accurate.

• NOTE: Be able to match tests and media to their reagents, pH indicators, substrates, products, enzymes, and results, as indicated in the table at the end of the lecture transcript and flashcards.

• Complete chart is on flashcards

GETTING STARTED• Perform a Gram stain. Since the longest step is air drying, make 3-

4 slides and allow them to air dry at the same time, but only use one to perform your Gram stain. That way, if your culture is not decolorized properly, you have several slides ready to go so you can perform another stain quickly. When you observe your organism under the microscope, check to make sure your culture is pure. Sometimes, the Gram stain becomes contaminated or your culture may be contaminated. If you see more than one organism, you need to go back to the original pure culture and start again. If you only see one organism, the next step is to do a Negative stain with India ink, which is the best way to see the arrangements of the organism. Record the shape (rods or cocci?) and arrangement (singles, clusters, or chains?).

METHOD OVERVIEW• Determine the morphological characteristics of your unknown

organism by performing a Gram stain, motility stab, capsule stain, and negative stain. If you have a Gram positive rod, you will also need to do a spore stain, since only Gram positive rods make endospores (only certain species).

•  Next, determine the cultural characteristics of your organism by observing the growth patterns in broth and on agar slants and plates. Determine the optimal temperature and oxygen requirements, and determine what type of hemolysis your organism displays.

•  Next, determine the physiological characteristics of your organism. This will require about 18-20 individual tests to find out what enzymes your organism makes, its fermentation pathway, etc.

MORPHOLOGICAL CHARACTERISTICS: Gram stain, size determination, motility, capsule stain, spore stain

• SIZE DETERMINATION• If you have a Gram + organism, mix a

loopful of it with a loopful of a Gram neg organism whose size is known. If you have a large organism, pick a large organism to compare it with. If you have a small organism, pick a small organism. Estimate the size of yours compared to a known organism.

Controls• When performing tests on unknown organisms, control organisms

are also used.• A “control” is a test media that is inoculated with a pure culture of a

known organism. • One tube is inoculated with an organism that is known to be positive

for that test, another tube is inoculated with an organism that is known to be negative for that test, and another tube is inoculated with the unknown organism.

• When you see the results of the unknown organism, it is compared to the positive and negative control tubes to determine if the unknown is positive or negative for that test.

• If the positive control or negative control did not turn the expected color, we know something is wrong with the media.

MOTILITY TEST • Inoculate a motility stab. Use a needle to obtain the inoculum. Stab

the needle into the motility medium, almost all the way to the bottom, then pull the needle back out in a straight line, backing the needle out of the same stab line you made going in. Remember, these need to be incubated at room temperature (25°C). If they are placed at room temperature, the flagella will detach, giving a false negative result for motile organisms. Also remember that motility media uses TTC as a terminal electron acceptor. If the organism can use it, the media will turn red, meaning the TTC has been reduced. If there is no red color at all, you will need to do a wet mount or hanging drop to observe the organism directly to determine if it is motile. NOTE: an Enterotube can test for MRVP, citrate, indole, carbohydrate and protein catabolism, but a motility test cannot be performed in an Enterotube.

MOTILITY TEST • HANGING DROP PREPARATION• Supplies: Toothpick, petroleum jelly (Vaseline), cover slip, depression slide, gloves• Spank the bottom of the nutrient broth to mix it.• Remove one loop of broth and touch it lightly to the center of the coverslip, which is

sitting on a paper towel. Try to get the smallest drop possible.• Use the toothpick to apply one small dab of Vaseline to each corner of the coverslip.

This keeps the coverslip secure when it is upside down.• Clean the depression slide and press the open well down gently onto the coverslip.• Gently flip the slide over so the drop hangs in the depression well.• Since we will look under the microscope at live cells, there will be no contrast, so turn

the iris down to increase the contrast.• Observe under scanning (40x); position the slide so that the edge of the drop is in the

center of the field. Focus on the edge of the drop.• Increase to LP then HP, but do NOT go to 1000x because the depth of field at 1000x

is too small to observe the depth of the droplet.• Look for tiny specks that are moving and record what type of motility is present.

Motility Test

Positive

Motility Test

• Hanging Drop Method

• http://www.youtube.com/watch?v=ujzSmsmg7ok

MOTILITY TEST • Problems with hanging drop slides:• False positive results: the organism appears to move, but it

cannot. This can happen if you are observing it too long under the hot light, and the liquid starts to evaporate and recede.

• False negative results: the organism is capable of motility, but it does not appear to be moving.

• No motility could be observed if the drop falls into the well and you don’t realize it, or if the iris diaphragm is opened too wide. Motility could be observed without false results if the culture is too concentrated.

MOTILITY TYPES• TUMBLES: the bacterium seems to be rolling over itself

like a rolling stone.• RUNS: the bacterium moves from point A to point B• JIGGLES: the bacterium jiggles like it is in an

earthquake, but it does not move from one part of the slide to another. THIS IS BROWNIAN MOTION, WHICH IS NOT TRUE MOTILITY.

• BROWNIAN MOTION is caused by water molecules hitting a cell with low mass. It causes movement of low mass cells by the inertia created by molecular bombardment of water molecules.

NOTE: • After a person has identified if their

organism is Gram positive, the next test is to do a spore stain. If it has no spores, then you need to do an Acid Fast stain to see if it is Mycobacteria.

• After a person has identified if their organism is Gram negative, the next test to do is to use a thioglycollate broth to determine oxygen requirements.

CULTURAL CHARACTERISTICS: Growth patterns, temperature, hemolysis, and oxygen requirements

• GROWTH PATTERNS• Use your working stock and reserve stock to observe the

growth patterns of your organisms and record that information in your journal. The terminology to use is in your lab manual. When you have recorded the morphology on your reserve stock, it will be kept in the refrigerator. You will not use it except in emergency. Also use your TSB tubes from your optimum temperature experiment to determine the pattern of grown in broth.

OPTIMUM TEMPERATURE • Inoculate one loop-full of your organism into 3 TSB

tubes. Label one tube 25 °C, one tube 30 °C, and one tube 38°C. Make sure your name is on the tube. These tubes will be used to determine the optimal temperature for your organism. Use the spectrophotometer to calculate their optical density at the next lab period. The tube with the most growth (highest OD) is the temperature they prefer. Organisms that grow well in room temperature as well as body temperature might be opportunistic pathogens. These tubes can also be used to determine their pattern of growth in broth.

SODIUM THIOGLYCOLATE TUBES (OXYGEN REQUIREMENT)

• This medium has an oxygen gradient, which means that most of the oxygen is at the top of the tube, and the least amount of oxygen is at the bottom of the tube. To prepare this medium, a reducing agent called Sodium thioglycolate was added, which removes the free oxygen by chemically binding with it. Therefore, thioglycolate broth is called a REDUCING MEDIUM. It gets rid of the oxygen. There is also a pink indicator dye called rasazarin that shows you where the oxygen is. Notice that the pink color is only at the top of the tube. We have to be careful not to shake the tube, or we will aerate it (add more oxygen). We need the oxygen gradient to be maintained for a successful test. The results of this test determine what oxygen requirements your organism has.

SODIUM THIOGLYCOLATE TUBES (OXYGEN REQUIREMENT)

• Procedure: • Hold the thioglycolate tube carefully, taking care to move it gently

without shaking, jiggling, or stirring them (which introduces oxygen into the medium).

• Label the tube with your name, the date, the organism, and “Thio” for Thioglycolate.

• Put some of your unknown bacteria on a sterile loop and gently push the loop straight down to almost the bottom of your tube.  Do not touch the bottom as this may ruin the loop, and do not introduce air by stirring or shaking the tube! 

• Gently pull the loop straight out of the tube and sterilize it. 

SODIUM THIOGLYCOLATE TUBES (OXYGEN REQUIREMENT)

• STRICT AEROBES require oxygen to grow. There will only be growth on the surface of the thio broth tube (pseudomonas and Bacillus megatarium)

• STRICT ANAEROBES require the absence of all oxygen. There will only be growth at the butt (bottom) of the tube (clostridium).

• FACULTATIVE ANAEROBES grow best aerobically but do not require it. Growth is throughout the tube, but is best at the top and decreases as one descends. (E.coli, staph aureus)

Thio Broth: Determines oxygen requirements

This test is done to see if the organism is aerobic (growth only at the top), anaerobic (growth only at the butt), or facultative (growth throughout). A facultative organism can live with or without air, so that is considered a virulence factor.

Thio Broth

Aerobic Facultative

Not shown: anaerobic; growth only at the butt

CHARACTERISTICS OF ALL ENTEROBACTERACEAE (Gram neg rods in colon)

• Gram negative rods• Oxidase negative• Catalase positive• Fermentation of glucose (positive)• Reduce nitrate (NO3; an inorganic substrate in the API

tube. Reduction = positive)• Facultative anaerobes• If they are motile, they have peritrichous flagella, so they

can run and tumble.

Amino Acids

• All proteins are made of amino acids.• There are only about 22 different types of

amino acids in our bodies.• Each amino acid has an amine group (NH2)

and a carboxyl (acid) attached to it.

Amine group: NH2

Carboxyl group: COOH

“R” refers to the rest of the molecule that makes it a particular amino acid.

Amino Acids• The amino acids we will be working with in lab are these:• Tryptophan (breaks down into ammonia, pyruvic acid,

and indole; causes feces smell)• Arginine (breaks down into ornithine)• Lysine (breaks down into cadaverine)• Ornithine (breaks down into putrescine)• Phenylalanine (breaks down into ammonia, pyruvic

acid, and hydrogen sulfide)

Enzymes

• An enzyme is a protein that cuts a substance into smaller parts.

• Enzymes that cleave proteins are called protease enzymes.

• Enzymes that cut an NH2 group off are called deaminases.

• Enzymes that cut the carboxyl group off are called decarboxylases.

Enzymes• The substrate is the item that is cut by

the enzyme.• The products are the pieces that remain

after the enzyme cuts the substrate.• The pH indicator is what changes color to

show us the presence of the products created by enzymes.

• You need to know the substrates, products, enzymes, and pH indicators used in the tests we will discuss in lab.

PHYSIOLOGICAL TESTS FOR GRAM NEGATIVE RODS

• NOTE: Know which tests have what color of a positive test: Brown, Orange or Red, Blue, Yellow, Diffused black pigment, Pink ring on top, etc.

• LACTASE: this tests for an enzyme that breaks down milk sugar (lactose) into glucose and galactose. If the bacteria have this enzyme, the test is positive (yellow), but the organism is not pathogenic. Only pathogens are missing this enzyme (negative is clear). The medium is MaConkey’s agar or a lactose fermentation tube.

Lactase Test

• MacConkey’s agar

Lactase Test

• Fermentation Broths with Durham tubes

FERMENTATION BROTHS • If an organism has the ability to ferment sugars, the end

products of the fermentation process are acids. We are looking for fermentation with acid (A) or acid + gas (AG). If there is fermentation, it will be yellow. If there is gas, the inverted miniature tube inside the media will fill with a gas bubble. If there is no fermentation, it is red, so record it as no change (NC) or Alk (protein digestion).

• The medium has a Durham tube (a miniature tube that is inverted on the inside of the test tube). If gas is produced, it will form a bubble inside the inverted tube. It also has phenol red as an indicator. Phenol red turns yellow if acid is present, and red if bases are present.

FERMENTATION BROTHS • Inoculate one each of the following tubes: glucose, lactose, and

sucrose. • These are different carbohydrates. After 24 hours, if the inoculated

medium is yellow, it fermented the sugar in that tube. It may or may not have produced gas. Gas is produced during sugar fermentation, so when gas is present, fermentation is present as well, but not all organisms ferment with gas. If it is yellow, record it as (A). If it has gas in the Durham tube (a bubble that take up 10% of the tube, not a little bubble), record it as (AG). If it did not turn yellow (stays red), you have to look at it again in another 24 hours. After 48 hours, if the media is still red, the organism is negative for fermentation of that sugar. These tubes must be read in 24 hours, because in 48 hours, any change in color will revert to the original color.

FERMENTATION BROTHS

• This is what happens: • Some organisms that ferment sugars can also

digest proteins. When these organisms begin to ferment a sugar, the media becomes acidic (yellow in 24 hours), which enables them to begin digestion of the proteins which are in the media. When proteins are digested, the media becomes alkaline, and the media will turn back to red. If you want to know if it fermented the sugar, you need to read the tube in 24 hours.

FERMENTATION BROTHS

• Suppose a student did not observe their tube right away, and then they see that it is red but it has gas. Since the gas is present, that indicates that it probably fermented the glucose (turned yellow at 24 hours, but he missed it), and then the organism proceeded to digest the protein, turning the media alkaline (back to red again). That would explain why it was red, but has gas (gas is produced during the fermentation process).

If Lactose Negative

• Perform an Indole test (using SIM media or IMViC test):– Positive

• Proteus vulgaris (H2S negative)• Providencia stuartii (H2S positive)• E. coli (Citrate negative)• Citrobacter freundii (H2S positive, Citrate positive)

– Negative• Perform a urease test (all should be negative)• Perform the MR-VP test

IMViC• This stands for a series of tests:• 1) Indole (this test is also used in SIM Media)• 2) Methyl Red• 3) Voges-Proskauer• 4) Citrate• The small “i” does not stand for anything; it

just makes pronunciation easier.

IMViC• The IMViC tests were developed as a means of

separating members of the Enterobacteriaceae, particularly the coliforms, to determine if drinking water is contaminated with sewage. A coliform is a gram negative, aerobic or facultative anaerobic rod which produces gas from lactose within 48 hours. The presence of some coliforms indicates fecal contamination. Coliforms are only found in the GI tract of warm-blooded animals. We will perform the indole test as part of the SIM media. We performed the citrate test in the Simmon’s Citrate media. Now we need to perform the MR-VP test to complete the IMViC series.

Indole Test• The enzyme tryptophan deaminase breaks an amine group

off of tryptophan (an amino acid) down into indole (which contributes to the smell of feces), pyruvic acid, and ammonia. If tryptophan deaminase is present, the indole end product reacts with Kovac’s reagent. If a red ring forms at the top of the tube, it is positive for indole, so the organism makes tryptophan deaminase. Kovac’s reagent has alcohol in it. Alcohol is lighter than water, so when the test is positive and turns red, the red ring floats to the top of the tube. (Control = E. coli). The substrate is tryptophan (an amino acid; also found in turkey). This amino acid might encourage sleep, which is why you might feel sleepy after a Thanksgiving turkey meal.

Tryptophan Deaminase (Indole) Test

Tryptophan deaminase is an enzyme that slices tryptophan into indole and pyruvic acid.Add Kovac’s reagent. Red ring is positive.

MR-VP TEST (Methyl Red/Voges-Proskauer)

• We do two tests with this medium: The MR test and VP test. We will inoculate one MR-VP tube today, let the culture grow until the next lab period, and then add 5 drops of Methyl Red to perform the MR test. In the next lab period, we will inoculate a new MR-VP tube, let the culture grow, and then add alpha-naphthol and potassium hydroxide reagents to perform the VP test.

MR-VP TEST• Anaerobic respiration is called fermentation. We are

looking for the ability of the organism to perform glucose fermentation. Bacteria convert glucose to pyruvate using different metabolic pathways. One pathway produces unstable acidic products which quickly convert to neutral compounds. Another pathway (the butylene glycol pathway) produces neutral end products, including acetoin and 2,3-butanediol. A third pathway is the mixed acid pathway, which produces stable acidic end products which remain acidic.

MR-VP TEST• If an organism produces a lot of acid from the

fermentation of sugars, it can override the buffer in the test media. If this happens, the amber media will turn red. MR-VP broth differentiates organisms that are single acid fermenters from organisms that are mixed acid fermenters because it contains over-riding buffers that affect organisms that are single acid fermenters.

MR-VP TEST• An organism that produces only one type of acid after

sugar fermentation will not produce much acid, so the buffer blocks the media from changing color. But if the organism produces many different kinds of acids, it overrides the buffer and causes the color to change.

MR = METHYL RED TEST• A positive MRVP broth will be red.• Methyl Red is a yellow colored pH indicator which turns

red if the organism uses the mixed acid fermentation pathway, which is that pathway that produces stable acidic end-products. If positive, the enzyme present is formic hydrogenylase. The acids will overcome the buffers in the medium and produce an acidic pH. When methyl red is added, it will go from yellow to red, which is positive for an organism that uses the mixed acid fermentation pathway.

MR Test: pH indicator

Positive(acid present)

Negative

MR = METHYL RED TEST• NOTE: Methyl red differs from Phenol red • Methyl Red: starts off yellow, turns red

when acids are present (indicating glucose fermentation). Used in MR-VP test (the first part of the test) for mixed acid fermentation

• Phenol Red: starts off red, turns yellow when acids are present (indicating glucose fermentation). Used in Urea broth and in the Fermentation broths

VP (Voges-Proskauer) TEST • The VP test is an indirect method of testing for an

organism that ferments glucose using the butylene glycol pathway. Glycolysis forms pyruvic acid, which undergoes fermentation and produces acetoin, which can then be taken into one of several different fermentation pathways, depending on the organism. In the butylene glycol pathway, the end product is 2, 3 butanediol. We cannot test for that, but we can test for acetoin. If acetoin is present, it turns red (positive) and colorless if negative. A positive test means the organism uses that particular pathway for fermentation.

VP Test

PositiveNegative

VP (Voges-Proskauer) TEST • The VP reagents are called Barritts’s A

(alpha napthol, a carcinogen!) and Barrett’s B (potassium hydroxide; KOH, a very caustic base, found in draino). If acetoin is present, it will turn a rust or red color (Gram negatives tend to do this). Therefore, red is a positive result, colorless or brown is negative.

IMViC (+ + - -)E. coli

IndolePositive

MRPositive

VPNegative

CitrateNegative

IMViC (- - + +)Enterobacter

CitratePositive

MRNegative

IndoleNegative

VPPositive

IMViC (+ - - -)

IndolePositive

MRNegative

VPNegative

CitrateNegative

MR-VP Tests

+/-Citrobacter freundii

-/+Klebsiella pneumoniae (non-motile)Enterobacter aerogenes (motile)

CITRATE TEST • Citrate is a salt of citric acid. It is a part of the Kreb’s

cycle. In this medium, citrate is the sole carbon source. If the organism can use citrate as its only carbon source, the medium will become basic (blue) because an acid (citric acid) is breaking down. The medium starts out green and turns blue (basic pH) if it is a positive test. The pH indicator is Bromthymol blue, which is green when acids are present. It may only be blue at the top, which is still positive. Acid = green (negative) and base = blue (positive). A negative tube will also show no growth.

Citrate

NegativePositive

Amylase Test with Starch Agar• This media has starch. Some molecules, such as starch, cannot be

taken into a bacterial cell because the molecules are so large. The organism can only use starch if it has an enzyme, called amylase, which can hydrolyze (break down) the starch into simple sugars that can be absorbed into the cell. We will flood the plate with iodine, which reacts with starch and turns it black. If the organism has the enzyme, there will be no more starch left, so there is nothing for the iodine to react with. Therefore, the presence of amylase will show up as a halo (area of clearing) around the organism (positive test). If the organism could not use the starch, the starch forms a complex with iodine to give a black precipitate around the organism. That means the organism is negative for amylase. NOTE: the black color only lasts a few minutes, so you have to read the test right away before the color disappears.

Amylase Test with Starch Agar• After incubation, iodine is poured on the plate, which turns starch black. If

there is a clear zone around the inoculation, the bacteria already digested the starch. That means it has the enzyme called Amylase.

Oxidative Fermentation (O-F) Test• We are looking for the ability to ferment or oxidize

glucose. The pH indicator is Bromthymol blue, which is yellow when acid is present. You will STAB two O-F tubes of glucose. One tube will need a layer of sterile oil to create an anaerobic environment so we can check for fermentation. The other tube will not have oil, so we can have an aerobic environment to check for oxidation. Next time, you will see if it turns yellow. If it is yellow, record it as “A “ (acid present). If there is gas in the tube, also record “G”. If there was no change (stayed green), write “NC”.

Oxidative Fermentation (O-F) Test

• Yellow means it fermented glucose. If it can only do that in the tube exposed to air, it is a positive O-F test.

Oxidative Fermentation (O-F) Test

Urease Test• When protein is digested, the pH becomes a base. This

test checks for the enzyme called urease, which breaks urea down into ammonium (a base) and carbon dioxide (water is not a product of this reaction). The ammonium will increase the pH. The medium has a pH indicator called phenol red. When pH goes up, it will turn bright pink (positive), negative is yellow. Urea is made when proteins are broken down. The kidneys are supposed to filter the urea and put it into the urine for elimination. If the kidneys malfunction, urea can build up in the blood and be very toxic. The smell of OLD urine is from ammonia.

Urease Test

Positive Negative Negative

Urease Test

• Pink is positive

If Urease Negative

• Perform a motility test– Positive

• Salmonella enterica (H2S positive)• Serratia marcescens (H2S negative)

– Negative• Shigella somnei

Gelatinase

• Some organisms produce an enzyme called gelatinase, which breaks down gelatin. If the gelatin is broken down, it becomes liquefied, and can no longer solidify, even when cooled in the refrigerator. Gelatin is a protein, so gelatinase is a protease. Gelatin is the only thing making the media solid. If it remains liquid, even after refrigeration, it is positive. Solid is negative.

Gelatinase

• Being able to liquefy gelatin is positive.

Phenylalanine Deaminase Test• We are looking for the enzyme, phenylalanine

deaminase, which removes an NH2 group from the amino acid, phenylalanine, to produce pyruvic acid, ammonia, and hydrogen sulfide. When 5 drops of ferric chloride is added to this, it will turn green, indicating a positive test. A negative test stays yellow. Don’t get this mixed up with the SIM media, where ferrous sulfate turns the media black.

Phenylalanine Deaminase Test

• When 5 drops of ferric chloride are added, green color is positive.

SIM Media• Check for three things on this one tube.•  H2S PRODUCTION (Hydrogen sulfide): Some organisms use sulfur as their terminal

electron acceptor in the electron transport chain. These bacteria have the enzyme thiosulfate reductase, which reduces thiosulfate (the substrate in the medium) and produces H2S gas (a rotten egg smell). When the H2S reacts with iron in the medium, a dark precipitate of iron sulfide is produced and the media will turn black (positive for H2S production). (Control = Proteus vulgaris). Don’t get this mixed up with the phenylalanine test, where the addition of ferric chloride turns the media green.

•  • INDOLE PRODUCTION: The enzyme tryptophan deaminase breaks an amine

group off of tryptophan (an amino acid) down into indole (which contributes to the smell of feces), pyruvic acid, and ammonia. If tryptophan deaminase is present, the indole end product reacts with Kovac’s reagent. If a red ring forms at the top of the tube, it is positive for indole, so the organism makes tryptophan deaminase. Kovac’s reagent has alcohol in it. Alcohol is lighter than water, so when the test is positive and turns red, the red ring floats to the top of the tube. (Control = E. coli). The substrate is tryptophan (an amino acid; also found in turkey). This amino acid might encourage sleep, which is why you might feel sleepy after a Thanksgiving turkey meal.

•  Motility: This media will show you if your organism is motile.

SIM Media• Purpose:  To differentiate between bacteria based on three tests: sulfur reduction

(cysteine desulfurase), indole production (tryptophanase), and motility.

A. Indole positive and hydrogen sulfide positive

B. Hydrogen sulfide positive

C. Indole positive and motility positive (note fuzzy growth away from stab line)

D. Negative Control

Decarboxylase Broths

• This tests for the presence of the enzyme decarboxylase. This test is useful for differentiating the Enterobacteriaceae. This enzyme removes and digests the acidic carboxyl group (COOH) from amino acids, plus cleaves off NH3, which will raise the pH. The pH indicator is bromcresal purple. The media is made to start out slightly acidic (pH 6). Bromcresal purple is yellow when acids are present and purple when bases are present.

Decarboxylase Broths• Three tubes are inoculated. Each tube contains

glucose plus one amino acid; either lysine, arginine, or ornithine. The carboxylase reaction requires an anaerobic environment, so each tube needs to be covered will a layer of sterile mineral oil to prevent air from reaching the culture.

•  NOTE: PICK THESE TUBES UP FROM THE RACKS ONE AT A TIME AND LABEL THE TUBE BEFORE YOU PICK UP THE NEXT TUBE. THEY ARE ALL THE SAME COLOR AND YOU MIGHT GET THEM MIXED UP!

Decarboxylase Broths• Each decarboxylase enzyme produced by an

organism is specific to the amino acid on which it acts. Therefore, we test the ability of organisms to produce arginine decarboxylase, lysine decarboxylase, and ornithine decarboxylase using three different but very similar media.

• If an organism is able to decarboxylate the amino acid present in the medium, alkaline byproducts are then produced and it will turn purple (positive).

Decarboxylase BrothsADC, LDC, and ODC

Media turns red-purple if it can digest the particular protein in the tube.

This is a photo of a lysine decarboxylase broth, so it only contains the amino acid, lysine (a protein).

ADC = Arginine decarboxylaseLDC = Lysine decarboxylaseODC = Ornithine decarboxylase

Positive Negative

Decarboxylase Broths• ADC: the substrate is arginine. The indicator is bromcresal purple. If

the organism has the enzyme arginine decarboxylase, it can break down arginine (an amino acid), test is positive (purple). Negative is yellow. When ADC breaks down argentine, it becomes ornithine, which is more basic. If the pH goes down, it is acid (yellow), and if the pH goes up, it is basic (purple). The substrate of the ADC test is arginine, because that is what is broken down. The product is ornithine. If the pH becomes alkaline because the organism has the decarboxylase enzyme, the media will turn purple in 48 hours (pos).

Decarboxylase Broths• LDC: Lysine decarboxylase. The substrate is lysine, another amino acid. If

the organism has the enzyme lysine decarboxylase, it will take lysine and break it down into cadaverine (rotten flesh smell). Purple is positive and yellow is negative. When an animal dies, there is a lot of protein breakdown, called putrefaction, and give that stink of “death”. Cadaverine contributes to that smell. Lysine breaks down into cadaverine, which is more basic, so it turns purple. If you check it in 24 hours, you might see that it is yellow because it fermented the glucose in the medium, but that does not mean it is a negative test. You have to check it in 48 hours to allow the decarboxylase activity to occur. If the pH becomes alkaline because the organism has the decarboxylase enzyme, the media will turn purple in 48 hours (pos).

Decarboxylase Broths

• ODC: Ornithine decarboxylase. This tests for decarboxylation of ornithine (an amino acid) into putricine (putrid smell). Purple is positive and yellow is negative. Ornithine was the PRODUCT of the ADC well, but in the ODC well, it is the SUBSTRATE. The product here is putricine, which is also a product of putrefaction and has that death smell. If the pH becomes alkaline because the organism has the decarboxylase enzyme, the media will turn purple in 48 hours (pos).

Triple Sugar Iron (TSI) Slants• This medium contains three sugars (below), iron (Fe ++),

thiosulfate (oxidized sulfur), and phenol red (indicator where acid is yellow and basic is red).

• 10x Sucrose (1%) (sucrose is a plant sugar made of glucose and fructose)

• 10x Lactose (1%)• 1x Glucose (0.1%)• Note that there is 10x more Sucrose and Lactose than

there is glucose.

Triple Sugar Iron (TSI) Slants• NOTE: When protein is digested, the pH becomes basic

because the byproducts are amino acids, which break down into CO2 and ammonia. When sugars are fermented, the pH turns acidic because the byproducts are acids.

• Know how to record the results of this test, and know what each of the following looks like: A/A,G, K/A,G K/A,G, H2S K/A, H2S A/A, H2S A/A,G, H2S

Triple Sugar Iron (TSI) Slants• Test results are recorded for the slant and the butt. “A” is

for acid, and “K” is for base.• A TSI slant that is all yellow is recorded as A/A• A TSI slant that is red at the slant and yellow at the butt

is recorded as K/A

K/AG K/K K/K K/A H2S

A/AG

Triple Sugar Iron (TSI) Slants

A/AG

A = acid (sugar was fermented)K = negative resultG = gas formationH2S = sulfur was used, iron was reduced to black color

Triple Sugar Iron (TSI) Slants• If either sucrose or lactose is fermented, slant will be A/A

because there is so much acid present, it overwhelms the little bit of ammonia that was there.

• If just glucose is fermented, more protein was degraded, so the ammonia will be stronger and show up basic, K/A

• If the tube has black in it, the iron was reduced and H2S formed.• If the tube has produced enough gas to separate the agar, it is

positive for gas.• An A/A tube with black is recorded as A/A + H2S.• The same tube that also has gas is recorded as A/A + H2S + G

Symbols that indicate test results:

• A: acid has formed. This usually indicates a sugar nutrient was broken down.

• ALK: means the media is alkaline after the test. This usually also indicates a negative test, so we just abbreviate it to “K”. Therefore:

• K: means the test was negative• G: means gas formed

• The rest of the following tests we will not perform, but they are on the exam.

Catalase Test• Some facultative aerobes have the enzyme called

catalase, which breaks down hydrogen peroxide (H2O2) into harmless water plus oxygen. Having this enzyme protects organisms from being destroyed by the H2O2 in the lysosomes of a white blood cell. Your instructor will lift the lid on your agar plate next lab period, and put one drop of H2O2 onto the colony. A positive test will show the oxygen bubbles rising up from the plate. That means the organism has the enzyme, so it is catalase +. NOTE: do not get catalase mixed up with oxidase. Catalase breaks down into oxygen, but is it not the oxidase test!

Catalase Test

Oxidase Test• Some aerobes have the enzyme called cytochrome

oxidase, which is a molecule that is a terminal electron acceptor in the electron transport chain. On a piece of paper, place one drop of the oxidase reagent Dimethyl-p-phenylene diaminic hydrochloride (this substance is carcinogenic). Then use a toothpick to obtain the organism from your TSA plate, and scrape the sample onto the drop of reagent on the paper. The test should be done in comparison to a positive control, because time is essential in the development of the test results. Count the number of seconds it takes to turn purple and record the time in your journal. If purple is observed at any time, it is positive for oxidase. If there is no color change, it is negative.

Oxidase Test

Caseinase Test (Skim Milk Agar)

• Some organisms produce an enzyme called caseinase (a protease), which breaks down the protein that makes milk white. It breaks the protein down into small peptides that can be absorbed into the cell. Do a heavy streak in the center. Positive is a clearing (halo) around the area of growth of the organism because the milk is broken down and the white color disappears. Casein is what makes milk look cheesy when it is left unrefrigerated.

Caseinase Test (Skim Milk Agar)

• A and B are positive because of the clearing (digestion of casein)

• C is negative

Litmus milk• This is done to see if the organism has the enzyme

lactase. If it does, it will ferment lactose, the milk sugar. The waste product will be an acid, so it will change the pH indicator from bluish to pink.

• If gas is produced during this process, gas bubble will also appear. Some organisms ferment lactose and produce gas, and some ferment lactose without making gas.

• Since milk also contains a protein called casein, this test will also determine if the organism has the enzyme caseinase to break that protein down. If it breaks the protein down only partially, curds (solid milk) will form. If it can break the milk protein all the way down to amino acids, a brown ring appears at the top.

Litmus milk• Know how to record the results of this test, and know what each

of the following looks like: AR, ACR, AGCR.• Litmus is a pH indicator that turns color only when oxidized.

Reduced litmus is colorless. If litmus is reduced, it will be colorless, so the white milk shows through. This is recorded as “R”.

• If it turned pink, it fermented lactose and is acidic. Record this as “A”

• If it is bluish, the pH is basic, record it as “K”• If it formed an acid curd (tilt the tube and the milk doesn’t spill),

record it as “C”• If you see tracks of gas in a curd, also record “G”.• If the casein protein in the milk was broken down and it is clear

on the top and has a brown ring in the tube, that is proteolysis, recorded as “P”.

Litmus Milk Test

White (R) means litmus was reduced and became clear.Pink (A) means it fermented lactose and is acidic. Bluish (K) means the pH is basic, so lactose was not fermentedCurds formed (C) Gas in the curd (G)Brown ring (P) means the casein protein in the milk was broken down (proteolysis)

P A A/C A/CG

A A K

Litmus Milk

A/CG

Lipase Test

• The Spirit Blue media has lipids. If the organism has the enzyme lipase, fatty acids will be released, and pH will decrease (become acidic). The indicator dye is called Spirit blue. When the pH decreases, a positive result is a dark blue streak in the center of the plate where you inoculated it. If lipase if produced, the concentration of the blue will increase where it was inoculated. Having clearing is NOT a positive test; it should be darker blue.

Lipase Test (Spirit Blue Agar)

DNAse Test

• This tests for the presence of the enzyme, DNAse. It contains the indicator dye, Methyl green complexed with DNA. Digestion of DNA releases the dye, so in otherwise green agar, a clear halo formed around the growth indicates a positive test.

DNAse Test

• Plate is flooded with toluidine blue, positive is pink area surrounding colony.

Nitrate Reduction Test

• Add one drop of Nitrate A and then one drop of Nitrate B. Red color is positive. If there is no color change after adding the two reagents, add zinc powder. If it does NOT turn red it is positive. If the zinc powder makes it turn red, it is negative.

Nitrate Reduction Test• Nitrate is reduced to nitrite, which can be further reduced to

nitrogen. If nitrate (NO3) has an oxygen molecule removed, it has been reduced. The new molecule is nitrite (NO2). Nitrite can also be reduced to nitrogen gas (N2) if it loses oxygen. The reactions look like this:

NO3 (nitrate) NO2 (nitrite) N2 (nitrogen gas)

• If an organism has the enzyme called nitrate reductase, it can reduce nitrate like this:

NO3 (nitrate) NO2 (nitrite)

• Is this enzyme clinically important? Not really. Some of you have brown eyes and some of you do not have brown eyes. It serves as a way of classifying organisms on a flow chart.

Nitrate Reduction Test• After you add the first two reagents, if it turns red, the test is positive

for nitrite, so that means the organism reduced NO3 to NO2. If it does not turn red, it might be because there is no nitrate present for the organism to reduce, so we cannot say it is a negative test yet.

• To find out if nitrate is present, add zinc powder. That will reduces nitrate to nitrite and produce a red color. This time, if it turns red, it means there was nitrate present during the first part of the test and the organism did not reduce it, which means the organism is unable to reduce nitrate. Therefore, the nitrate reduction test is negative. If there is NO color change after adding zinc powder, the organism already reduced the nitrate, so the test is positive for nitrate reduction.

Nitrate Reduction Test

• The nitrate broth we started with contains NO3. If the organism has nitrate reductase, it will reduce NO3 to NO2 so there will be no more NO3 present , just NO2. First, we add reagent A to the tube. Reagent A will bind to NO2 , forming a complex. However, this complex is clear, so it does not tell us anything. Then we add Reagent B, which turns the complex a red color. If you add reagents A + B and the tube turns red, the organism has nitrate reductase.

Nitrate Reduction Test

• However, some organisms with that enzyme reduce NO3 all the way to N2. They take all of the nitrate and reduce it all the way to nitrogen gas. In this case, there will be no NO3 or NO2 in the tube, so there is nothing for reagent A to react with, and reagent B will not turn the tube red, even though the organism has the nitrate reductase enzyme. So, although a red color is a positive test, a colorless tube is NOT a negative test.

Nitrate Reduction Test• When the tube is colorless, there are two possibilities:• The organism does not have nitrate reductase, and there is still

NO3 in the tube

• The organism has nitrate reductase, and there is no NO3 or NO2 in the tube.

• If a tube is colorless after adding Reagents A + B, we need to test the tube to see if there is NO3 in the tube. We do this by adding a little zinc powder by scooping some on the flat end of a toothpick and adding it to the tube. Zinc will react with NO3 if it is present (reduces any residual nitrate to nitrite) and it will turn red. Zinc is used to confirm a negative test.

Nitrate Reduction Test

The tubes above have been treated with dimethyl 1-naptholamine and sulfanilic acid.  The tube on the right indicates that the organism reduced nitrate to nitrite.  In order to further determine positive/negative nitrate reduction, zinc powder must be added to detect the presence of nitrate.

A tube will turn a deep red color on the addition of zinc if nitrate is present.  The uninoculated control turned red indicating that nitrate is still present in the tube, so if it turns red after adding zinc, it is negative.

Step 1: add naptholamine Step 2: add zinc powder

Nitrate Reduction Test

API 20E Test Strip

• You could perform each of those tests in individual test tubes or plates, as shown in the previous slides.

• Or you could do many of those tests at once with an API-20E Test Strip.

API 20E Test Strip

All Negative

All Positive

The fast way to run many tests on an organism is to inoculate either an APT 20E test strip ($12 each) or an Enterotube ($45 each). The organism is placed into each well and the results are read two days later.

API 20E Test Record Sheets

Enterotube: more expensive than API-20E media

Common Enteric Organisms• Citrobacter freundii• Escherichia coli• Enterobacter aerogenes• Klebsiella pneumoniae• Proteus vulgaris• Providencia stuartii• Salmonella enterica• Shigella somnei• Serratia marcescens

Citrobacter freundii

Citrobacter freundii

• Uses citrate as their sole source of carbon.• These bacteria can be found almost

everywhere in soil, water, wastewater, etc. It can also be found in the human intestine.

• They are rarely the source of illnesses, except for infections of the urinary tract and infant meningitis.

Citrobacter freundii

+

Motility: negative

MR: positive

--+ +- -

Escherichia coli

Motile speciesNon-motile species

Escherichia coli

Escherichia coli• One of the main species of bacteria living in the lower

intestines of mammals, known as gut flora. • There are about 1 trillion excreted in the feces daily.• However, the bacteria are not confined to this

environment, and specimens have also been located, for example, on the edge of hot springs.

• The E. coli strain O157:H7 is one of hundreds of strains of the bacterium that causes illness in humans.

• E. coli are unable to sporulate. Thus, treatments which kill all active bacteria, such as pasteurization or simple boiling, are effective for their eradication, without requiring the more rigorous sterilization which also deactivates spores.

Escherichia coli• E. coli can generally cause several intestinal and extra-

intestinal infections such as urinary tract infections, meningitis, peritonitis, septicemia and pneumonia.

• Although it is more common in females due to the shorter urinary tract, urinary tract infection is seen in both males and females. Poor toilet habits can predispose to infection (doctors often advise women to "wipe front to back, not back to front") but other factors are also important (pregnancy in women, prostate enlargement in men).

• If E. coli bacteria escape the intestinal tract through a perforation (a hole or tear, for example from an ulcer, a ruptured appendix, or a surgical error) and enter the abdomen, they usually cause peritonitis that can be fatal without prompt treatment. However, E. coli are extremely sensitive to such antibiotics as streptomycin or gentamycin.

Escherichia coli• Certain strains of E. coli, such as Escherichia

coli O157:H7, produce a toxin and can cause food poisoning usually associated with eating cheese and contaminated meat (contaminated during or shortly after slaughter or during storage or display).

• The usual countermeasure is cooking suspect meat "well done"; the alternative of careful inspection of slaughtering and butchering methods (to make sure that the animal's colon is removed and not punctured).

Escherichia coli• Escherichia coli O157:H7 is the strain is believed to be

associated with the 2006 United States E. coli outbreak linked to fresh spinach. Severity of the illness varies considerably; it can be fatal, particularly to young children, the elderly or the immunocompromised

• E. coli are susceptable to amoxicillin, cephalosporins, aminoglycosides, streptomycin, or gentamycin.

• Antibiotic resistance is a growing problem. Some of this is due to overuse of antibiotics in humans, but some of it is probably due to the use of antibiotics as growth promoters in food animals.

Escherichia coli

- -

Enterobacter aerogenes

Enterobacter aerogenes• Several strains of the these bacteria are

pathogenic and cause opportunistic infections in immunocompromised (usually hospitalized) hosts.

• The urinary and respiratory tract are the most common sites of infection.

• Enterobacter can be distinguished from other Gram neg rods by virtue of being a 'fast fermenter' of lactose (as are E. coli and Klebsiella).

Enterobacter

+

Methyl Red: negative

+

Klebsiella pneumoniae

Klebsiella pneumoniae• This is a nonmotile, encapsulated, lactose-

fermenting, and facultatively anaerobic organism.

• It is found in the normal flora of the mouth, skin, and intestines.

• It naturally occurs in the soil and about 30% of strains can fix nitrogen in anaerobic conditions.

Klebsiella pneumoniae• K. pneumoniae can cause bacterial pneumonia, typically

due to aspiration by alcoholics, though it is more commonly implicated in hospital-acquired urinary tract and wound infections, particularly in immunocompromised individuals.

• Klebsiella ranks second to E. coli for urinary tract infections in older persons.

• Feces are the most significant source of patient infection, followed by contact with contaminated instruments.

• Klebsiella is resistant to ampicillin but susceptible to aminoglycosides and cephalosporins.

Klebsiella pneumoniae

Proteus vulgaris

Proteus vulgaris• Responsible for many human urinary tract

infections.• Proteus species do not ferment lactose,

are oxidase negative, and urease positive; some species are motile.

Proteus vulgaris

Providencia stuartii

Providencia stuartii• This organism is motile.• They are opportunistic pathogens in

humans and can cause urinary tract infections, particularly in patients with long-term indwelling urinary catheters or extensive severe burns.

• Some strains are sensitive to ampicillin.

Providencia stuartii

IMViC: + + - +

ODC: -

ONPG: +

H2S: -

Mannitol: -

Salmonella enterica

Salmonella enterica

Salmonella enterica• This organism causes typhoid fever and

foodborne illness.• Salmonella species are motile and

produce hydrogen sulfide.• They usually do not ferment lactose.

Salmonella enterica• Food poisoning from Salmonella is usually from

poultry and raw eggs and from food that has been cooked or frozen, and not eaten straight away.

• The overuse of antibiotics in both the poultry and beef industries have created a strain of salmonella which is potentially resistant to antibiotics.

• It can also be caught by handling reptiles, such as iguanas.

Salmonella enterica• The prevention of Salmonella as a food

illness involves effective sanitizing of food contact surfaces.

• Alcohol has proven to be an effective topical sanitizer against Salmonella.

Salmonella enterica

Shigella somnei

Shigella somnei• This organism is non-motile, non-spore

forming. It is H2S positive.• It is the causative agent of human

shigellosis. • Shigella infection is typically via ingestion

(fecal–oral contamination).• 10 bacterial cells can be enough to cause

an infection.

Shigella somnei• The stool may contain blood, mucus, or pus (eg

dysentery). • Shigella cause dysentery that results in the

destruction of the epithelial cells of the intestine.• Some strains produce an enterotoxin similar to

E. coli O157:H7• Symptoms usually begin two to four days after

ingestion. Symptoms usually last for several days, but can last for weeks.

• Severe dysentery can be treated with ampicillin.

Shigella somnei• IMViC: - + - - or + + - -• ODC: + or -• LDC: -• Urease: -• H2S: -• Sucrose: -• TSI: K/A

Serratia marcescens

Serratia marcescens • This organism is involved in nosocomial

infections, particularly urinary tract infections and wound infections.

• It is motile.• Due to its preference for damp conditions, it is

commonly found growing in bathrooms (especially on tile grout), where it manifests as a pink discoloration.

• Once established, complete eradication of the organism is often difficult, but can be accomplished by application of a bleach-based disinfectant.

Serratia marcescens• S. marcescens can cause conjunctivitis

and tear duct infections. • It is common in the respiratory and urinary

tracts of adults and the gastrointestinal system of children.

Serratia marcescens• IMViC: - + + + or - - + +• LDC: +• ADH: -• ODC: +