biochemical tests.pdf

8/10/2019 biochemical tests.pdf

1/11

MMBB255 Week 6 1

Biochemical and Metabolic Properties of Microbes I. Objective

The goal of this experiment is to characterize an organism based on its ability to produce proteins that allowit to carry out a set of unique reactions, its motility, and oxygen requirements. You will also follow up on thegrowth curve and unknown two. The Photographic Atlas for the Microbiology Laboratory will be useful.

II. Background

Microorganisms can be classified, or distinguished from one another, by the ability to (1) grow on differentsubstrates and/or production of different end products, (2) produce specific enzymes, (3) use oxygen, or (4) bemotile. For example, certain microbes can use different carbohydrates as sources of energy and/or carbon.Because such variability exists in carbohydrate utilization between different microbes, this can aid in the group,genus, or species identification. We will examine the following methods for classification:

A. Carbohydrate utilization: acid and/or gas productionB. Enzyme productionC. Aerobic or anaerobic mode of growthD. Motility

A. Carbohydrate utilizationCarbohydrates can be fermented or oxidized via aerobic or anaerobic respiration. When a carbohydrate

is fermented, acid , and sometimes gas, is produced. The most common end product of carbohydratefermentation is lactic acid (lactate). Other acids include formic and acetic acid. Microbes includingStreptococcus and Lactobacillus can produce lactic acid, formic acid, and/or ethanol. Enteric organisms (see

below) can produce lactic acid, formic acid, succinic acid, as well as ethanol and gasses CO 2 and H 2.Acid production can be detected by addition of a pH indicator, such as phenol red or brom cresol purple

to the medium. Phenol red (PR) turns yellow in acidic conditions (slightly under pH 7.0) while at neutral or basic pH it is red. Gas production can be monitored by addition of a small tube, called a Durham tube ,which has been inverted in the carbohydrate-containing growth medium to trap gas bubbles. If gas is

produced, it typically is CO 2 or H 2.

Carbohydrates that are used in microbial classification include the following classes:a) Monosaccharides are simple sugars with 1 to 6 carbons. For example, tetroses are 4 carbon sugars,

such as erythrose. Pentoses are 5 carbon sugars and include ribose, xylose, arabinose, and ribulose.Hexoses are 6 carbon sugars and include glucose (also called dextrose), galactose, mannose, andfructose (also called levulose).

b) Polysaccharides are poly mers of monosaccharides. For example, a disaccharide (di = two) containstwo monosaccharide units. Some examples include sucrose (also called saccharose) composed of thehexoses glucose and fructose, maltose composed of two units of the hexose glucose, and lactosecomposed of the hexoses glucose and galactose.

c) Alcohol sugars are polyhydric alcohols that are reduction products of a monosaccharide. Someexamples include adonitol, dulcitol, mannitol, and sorbitol.

Carbohydrate utilization is important in distinguishingamong the members of the family Enterobacteriaceae(the enteric bacteria) . The Enterobacteriaceae is a familyof organisms that are facultatively anaerobic, oxidasenegative, Gram negative rods that ferment glucose. Thisfamily includes the genera Escherichia, Salmonella,

Proteus, Enterobacter, Serratia, Yersinia, Edwardsiella, Providencia, Hafnia, Citrobacter, Shigella, and

Klebsiella, to name a few. Of the above genera the only lactose fermenters are Escherichia coli, Klebsiella,Citrobacter, and Enterobacter .

B. Enzyme productionListed below are some brief descriptions of enzymes you might find in a microbe. The ability of an

organism to produce or express a particular enzyme can be assayed (tested) in the laboratory using simple biochemical reagents and substrates. Several of these tests require only small amounts of cellular materialand may take only a few minutes to interpret. Subsets of microbes can then be classified into groups basedon their ability to produce a positive or negative reaction. The results of biochemical tests are used withother data, including the Gram reaction, to identify an organism by name. These tests play critical roles inthe swift identification of bacteria causing infection.

Enterobacteriaceae (enteric bacteria) arefacultatively anaerobic, oxidase negative,Gram negative rods that ferment glucose .


2/11

MMBB255 Week 6 2 1. Catalase: H2O2 (hydrogen

peroxide) is produced as anoxidation product during growthon carbohydrates in the presenceof oxygen and is toxic if allowedto accumulate. Many organismsmake the enzyme catalase toremove hydrogen peroxide. The catalase test

measures the ability of an organism to producethe enzyme catalase that degrades H 2O2 to H 2Oand O 2. Active catalase is a homotetramer (fouridentical subunits) with two heme groupscontaining trivalent iron (ferric, Fe 3+). To see if an organism makes catalase, H 2O2 is added to cells. Ifcatalase is present, dioxygen gas is liberated and bubbles will be produced. Note that two molecules ofH2O2 are needed because this is an oxidation-reduction reaction in which one molecule of H 2O2 serves asthe substrate and the other serves as a donor. Water is the reduced product and O 2 is the oxidized product.

2. Oxidase : The enzyme oxidase is part of the electron transfer system used by some organisms that usemolecular oxygen as aterminal electron acceptor.Oxidase interacts with themembrane boundcytochromes and deliverselectrons from thecytochromes to O 2. As a result, H 2O2 orH2O is generated. Strict anaerobes do notuse O 2 and hence do not possess theoxidase enzyme. Most Gram positive

bacteria are oxidase negative as well as themembers of the family Enterobacteriaceae.

3. Gelatinase (an extracellularenzyme) : This test determineswhether an organism producesa proteolytic-like enzyme thatcan liquefy or digest gelatin, a

protein produced by the

hydrolysis of collagen. Proteinsare too large to be taken up bymicrobes, so gelatinases (and other proteases) are secreted into the environment where they break

proteins into peptides and amino acids, which can be taken up by the cell.4. Amylase (an extracellular enzyme): This test measures whether an organism produces amylase, an

enzyme that breaks down starch, a glucose polymer. Amylase hydrolyses starch into dextrins (smaller polysaccharides) and maltose (disaccharides), which now can be imported into the cell.

5. Lipase, lecithinase, proteases (extracellular enzymes): Lipases are enzymes that break downtriglycerides (lipids) into their constituent glycerol and fatty acids. Lecithinase breaks down lecithinwhich is a modified phospholipid; one of the fatty acids has a special modification. Cells can use thesecomponents as energy or to produce other cellular components. Proteases are enzymes that break down(hydrolyze) proteins and peptides.

6. Urease: The enzyme urease is an

amidase produced by certain eukaryotesand prokaryotes. It plays an importantrole in the decomposition of organiccompounds. Because not all microbes

produce this enzyme, a simple test forthis enzyme can be used to distinguish

between various microbes. For example,urease activity is very characteristic of all

Proteus spp. This test aids in rapiddetection of lactose-nonfermentingenteric organisms such as Proteus vulgaris, an opportunistic pathogen.

A Few Examples Catalase Positive Catalase Negative

Micrococcus and Staphylococcus Streptococcus Bacillus Clostridium

Listeria monocytogenes and someCorynebacterium sp. Erysipelothrix

A Few Examples Oxidase Positive Oxidase Negative

Neisseria, Moraxella Acinetobacter Aeromonas, Alcaligenes,

Flavobacterium, Pseudomonas, Vibrio Enterobacteriaceae

A Few Examples Gelatinase Positive Gelatinase Negative

Staphylococcus aureus Staphylococcus epidermidis + slow Micrococcus + slow

Corynebacterium sp. (most) Listeria monocytogenes

A Few Examples Urease Positive Urease Negative

Klebsiella Escherichia Proteus Providencia

Bordetella bronchiseptica Bordetella pertussis

Yersinia pseudotuberculosis, Y. enterocolitica Yersinia pestis

The oxidase test is a useful test for distinguishingbetween the Gram negative rods Pseudomonas

and the Enterobacteriaceae.

The catalase test is useful to differentiatebetween the Staphylococci/Micrococci and the

Streptococci (see the table above).


3/11

MMBB255 Week 6 3

C

H 2 N

H 2 N

O 2 HOH+ CO 2 H 2O 2 NH 3++

Urea Water Carbon

Dioxide AmmoniaWater

urease

The urease test measures the ability of a microorganism to split, or hydrolyze, a molecule of urea intotwo molecules of ammonia using the enzyme urease. A heavy inoculum of a young culture is added tourea broth (this media is designed specifically to differentiate Proteus from other enterics). The criticalingredients are urea and the pH indicator, phenol red. The initial pH of the medium is 6.8 and is slightlybuffered (there is less buffer if you use a urease assay like urea agar - designed for organisms other than

Proteus ). Phenol red is yellow at pH 6.8 (slightly acidic) and red at pH 8.4. Microbes that can makeurease , will hydrolyze urea as shown in the box, resulting in a shift in the pH of the medium towardalkaline pH. This

changes the phenol redindicator to pink (redor cerise). Urea is adiamide of carbonicacid that is split intocarbon dioxide andammonia by the ureaseenzyme as shown in the box.

C. Aerobic or anaerobic mode of growth (Oxygen Requirements)A very important requirement of cellular growth and metabolism is the requirement for oxygen.

Microbes can be grouped into five different categories based on their requirement for molecular oxygen.Strict or obligate aerobes must have O 2 for growth because O 2 is used as the terminal electron acceptor foroxidative phosphorylation. In contrast, strict or obligate anaerobes cannot grow in the presence of O 2 and

may be killed by trace amounts of O 2. Microaerophilic organisms need a small amount of O 2 for growth but too much O 2 will kill them. A subset of anaerobes can tolerate O 2 but they do not use it. They are calledaerotolerant anaerobes . Facultative anaerobes grow best when O 2 is available, but they can also grow,though not as well, if O 2 is not present. We will test several organisms for their growth pattern in medium todetermine if they prefer aerobic or anaerobic conditions. Note that the handling of very strict anaerobes is

beyond the scope of this class.

D. Motility: swimming, swarming, twitching, and glidingMicrobes move in a variety of

ways that are roughly analogous toour walking, running, swimming,or crawling movements. Manyorganisms can swim in liquid (suchas lakes or oceans, blood system).

Most swimmers move by rotationof one or more external appendagescalled flagella . This is a complex

process that is very well studied.The helical shape of the flagellumis reminiscent of a motorboat

propeller; movement results fromrotating one or more flagella. Flagella can rotate very fast when the cell is in liquid medium, allowing forrapid swimming. When cells with flagella are in more viscous (thicker) medium, such as broth containingsome agar, the rate of swimming is reduced. Some examples of swimmers include Escherichia coli,Salmonella Typhimurium, and Bordetella pertussis .

Some swimmers can also swarm . Swarming occurs when a swimmer cell enters a viscous environmentor a solid surface. The swimmer then increases in length and makes a large number of flagella. Someexamples include Proteus mirabilis and Serrratia marcescens .

Twitching motility is a slow movement of cells over a surface via type IV pili. The cell throws out a pilus which attaches to a surface and is subsequently depolymerized so that the cell moves toward theattachment point. Pathogens, such a Neisseria and Pseudomonas , use twitching motility, as does the soil

bacterium Myxococcus xanthus .Some organisms, particularly soil organisms, move by gliding . Gliding is a translocation over a solid

surface (soil, agar). Gliders do not have flagella or cilia but appear to move by secretion of a material. Someexamples of gliders are Myxococcus xanthus and Beggiotoa spp.

Motility can be used to distinguish between certain genera and to aid in species identification. Note thatmotility can vary as a function of temperature. Therefore it is important to measure motility at thetemperature indicated for each organism.

A Few Examples Temp. Motile Nonmotile

37C

Vibrio spp. Acintobacillus spp. Enterobacter spp. Klebsiella spp.

Aerogenic Escherichia coli Anaerogenic Escherichia coli

Other Bacillus spp. Bacillus anthracis Pseudomonas spp. Pseudomonas mallei

22C Listeria spp. Corynebacterium spp.(C. aquaticum is positive)

Yersinia pseudotuberculosisY. enterocolitica Yersinia pestis


4/11

MMBB255 Week 6 4

Tuesdays Procedures:A. Carbohydrate fermentation and enzymes in carbohydrate catabolism Work in Pairs.

1. You will be assigned E.coli plus one other organism from the listto the right. These will be provided as broth cultures. You willneed to record the results for the other organisms you do not use .

2. Obtain and label the following media: (Note that the extra twotubes are for you and your partner to inoculate your secondunknown)

4 tubes of PR-lactose with Durham tubes4 tubes of PR-glucose with Durham tubes4 tubes of PR-sucrose with Durham tubes

3. Inoculate each tube aseptically with your loop. Label each tubewith the name of the organism, the sugar, the date, your name and lab section. The instructor will keepuninoculated carbohydrate media as negative controls and incubate them along with the inoculated tubes.

4. Incubate all tubes at 37C for 24 hrs. and examine.Note: The nomenclature for the Salmonella species has been changed. There are now only three species of

Salmonella, enterica (used to be Salmonella choloraesuis ) , bongori, and subterranean. Most human pathogens are serovars of S. enterica subspecies enterica (there are 6 subspecies total). For exampleSalmonella Typhimurium is short for S. enterica subspecies enterica serovar Typhimurium; similarly

there is Salmonella Typhi, Paratyphi, Enteritidis short names for the subsp. enterica . Please note howthey are formatted. If you were to hand write them you would underline all italics.

B. Enzymes: Work in Pairs.i. Extracellular enzyme gelatinase:

1. The bacterial culture s. For these tests, we will use the following microorganisms:Staphylococcus aureus

Bacillus cereusStaphylococcus epidermidis

Pseudomonas aeruginosa 2. Inoculate . Using an inoculating needle or loop aseptically stab each organism into a labeled nutrient

gelatin tube. Incubate at 37C for 48 hrs.

ii. Extracellular enzyme amylase:1. The bacterial cultures . For these tests, you will use the following organisms:

(you may also test your second unknown if you want, you will need sixsectors)

Staphylococcus aureus Bacillus cereusStaphylococcus epidermidis

Pseudomonas aeruginosa2. Inoculate . Using your inoculating loop aseptically make a line in the

quadrant of a starch agar plate with each of the test organisms (see thediagram). Label the back and incubate at 37C for 48 hrs.

iii. Extracellular enzymes lipase, lecithinase, and protease:1. The bacterial cultures . For these tests, you will use the following organisms:

(you may also test your second unknown if you want, you will need sixsectors)

Staphylococcus aureus Bacillus cereusStaphylococcus epidermidis

Pseudomonas aeruginosa2. Inoculate. Obtain one plates of egg yolk nutrient agar (EY-NA) from the

instructor. The addition of egg yolk at 5-10% (v/v) makes the medium lookmilky. Aseptically inoculate a single line for each organism on the plate (see thediagram). Incubate at 37C for 24-48 hrs.

Enterobacter aerogenesCitrobacter freundii

Enterococcus faecalis Escherichia coli

Klebsiella pneumoniae Proteus mirabilis Pseudomonas aeruginosaSalmonella Typhimurium-see note


5/11

MMBB255 Week 6 5

iv. The urease test1. Work with the following bacterial cultures.

Escherichia coli (plate stock) Enterobacter aerogenes (plate stock) Klebsiella pneumoniae (plate stock) Proteus mirabilis (plate stock) Your unknown (plate stock) Your partners unknown (plate stock)

2. Obtain and label : 6 tubes of phenol red urea medium (urea broth).3. Inoculate . Using your inoculating loop, aseptically transfer a heavy inoculum (loopful) of each

organism into a separate tube of urea broth. The lab instructor will keep aside one tube uninoculatedand incubate it at 37C as a control.

4. Incubate at 37C. Check your reactions after about 4-6 hours if possible . Fast positive organisms, suchas Proteus , may turn pink in a few hours. Check for a positive test at 24 hr . Date and time that tubes were inoculated: _____________ time________________

C. Oxygen requirements Work in Pairs. 1. The bacterial cultures .

Escherichia coli Pseudomonas aeruginosa

Clostridium sporogenesStreptococcus agalactiae2. Three methods will be used to monitor the oxygen needs of the four bacterial strains:

i. Method 1. Agar deeps.Obtain four tubes of TSA (trypticase soy agar) deeps, tempered at 55C. Remove one tube from the55C bath and aseptically add two drops of an organism into the tube using a sterile pasteur pipette.Immediately and vigorously roll the tube between the palms of your hands to evenly distribute the cellsthroughout the agar. Then place the tube into a bucket of ice to solidify the medium. Repeat with theother three cultures. Incubate at 37 C for 48 hrs.

ii. Method 2. Thioglycolate broth.Obtain four screw-capped tubes of thioglycollate medium. The medium should be slightly pink or bluein the upper one-fourth of the tube due to the presence of resazurin or methylene blue respectively,

both redox indicators (they turn color when there is oxygen). Sodium thioglycolate, the active

ingredient, binds to O 2. Aseptically inoculate each of the tubes, taking care to stab to the bottom of thetube with your inoculating loop or needle - try not to introduce air. Dont shake or tighten the cap (you want air to get in the tube but not the medium). Incubate at 37C for 18-24 hrs (check Wed).

iii. Method 3. Gas-Pak Jar.Obtain two TSA plates from the lab instructor. Aseptically streak a single line of each of the fourorganisms on both of the plates and your unknowns. [Be sure to label each quadrant correctly].Immediately place one plate in the Gas-Pak Jar. It will be activated and placed at 37C for 48 hrs.Incubate the other plate at 37C in the air for 48 hours.

The Gas-Pak works as follows: To activate the system, water will be added to a reagent in the Gas-Pak to generate CO 2 and H 2. The jar contains a small amount of palladium that then catalyzes theconversion of O 2 and H 2 to H 2O. This removes sufficient O 2 to make an environment favorable foranaerobes. The jar also includes a redox indicator called methylene blue to check for anaerobiasis. Itis colorless when reduced and blue when oxidized.

D. Motility Work in Pairs.Motility will be tested by examining the distance cells migrate in semisolid medium. Remember motilitymay also be checked via a hanging drop or wet-mount method. Standard agar medium that we have beenusing is considered solid medium because it contains 1.5% w/v (1.5 g per 100 ml) agar. The semisolidmedium has only 0.5% w/v agar (swarming medium has even less, 0.3 % w/v agar ) and contains all thenutrients required for organisms to grow. The semisolid medium may also contain an indicator of cells withactive respiration called 2,3,5-tetraphenyltetrazolium chloride (TTC). This allows easier observation of wherethe organisms are growing since it turns red when reduced by an active electron transport chain (it iscolorless when oxidized).


6/11

MMBB255 Week 6 6

1. The bacterial cultures . Obtain these strains from your instructor. Escherichia coli Klebsiella pneumoniae Your unknownYour partners unknown

2. Inoculate by stabbing . Label each sectors, then aseptically stab eachorganism with an inoculating needle into the center of a quadrant ofsemisolid medium, see the diagram to the right.

3. Incubate. Incubate the plate upright (do not invert) at 30C and examinefor motility (movement away from the stab inside the agar not the surface)after 24 and then 48 hrs.

E. Growth curve - Finish1. Record your statistically valid plate counts (between 25-250), calculate the total viable cell

concentration, fill out the table below, and enter the calculated cfu/ml into the computer. time point

elapsed min.dilution

used # of coloniesx dilution

factor by volume

platedcalculated

cfu/ml

2. You will get graphing paper and all sections growth curve data next lab period. You should make agrowth curve of each growth condition and figure out the doubling time from the graph. Get help if youdo not know how to do this.

Thursdays Procedures:A. Carbohydrate fermentation and -galactosidase activity Work in Pairs.

1. Record Fermentation Data. After 24 hrs, record your results in the table below. Note: if for somereason you do not observe your cultures within about 24 hrs, make a note of the time and date below. TheFerm? heading in the table below is your conclusions that the organism does or does not ferment thatsugar- see the background.

Date that tubes were inoculated: _____________ time: ________________Date that observations were taken: _____________ time: ________________

Sample

Lactose Glucose SucroseGrowth

&Color

Gas Ferm?Growth

&Color

Gas Ferm?Growth &

ColorGas Ferm?

Neg. Control

E. aerogenes

C. freundii

E. faecalis

E. coli K. pneumoniae

P. mirabilis

P. aeruginosa

S. Typhimurium

Your Unknown


7/11

MMBB255 Week 6 7 2. Assay for -galactosidase activity. The enzyme ! -galactosidase breaks lactose into its two

monosaccharide components, glucose and galactose. Production of the enzyme -galactosidase isregulated by the presence of the substrate, lactose, and the absence of the more favorable substrate,glucose (you may want read about the Lac operon in your text). To demonstrate this, you will determinethe amount of ! -galactosidase produced by Escherichia coli grown in glucose vs. lactose.a. Mix your E. coli lactose and glucose PR tubes to disperse the cells. Label four 1.5 ml eppendorf (eppi)

tubes and fill them according to the table below. You do not need to be aseptic. Follow the flow chart below and note the footnotes about the various solutions:

Tube 1 Tube 2 Tube 3 (Blank) Tube 4 (Blank)Add 1.5 ml PR-glucose

cells to an eppi tubeAdd 1.5 ml PR-lactose cells

to an eppi tubeAdd 1.5 ml PR-glucose

cells to an eppi tubeAdd 1.5 ml PR-lactose cells

to an eppi tube

Centrifuge @ 12,000 RPMs for 4 minDiscard supernatant into originating PR tube.

Add 1 ml of 0.1% SDSMix to get all the cells resuspended and then transfer all the liquid to a spectrophotometer tube.

Add 2 ml Z buffer 2 and 1 ml ONPG 3

Add 2 ml Z bufferand 1 ml ONPG

Add 2 ml Z bufferand 1 ml H 2O

Add 2 ml Z bufferand 1 ml H 2O

Incubate at 37C for 5 min.

Add 1ml of 1M Na 2CO 34

Read Abs (OD) at 420 nm in the spectrophotometer.

b. Calculate the units of -galactosidase activity as follows: ! -gal Units = (A 420nm * 213)/Time (min.)

Units for glucose-grown cells:____________

Units for lactose-grown cells: _____________

What can you conclude from these results about the regulation of the gene ( lac Z) that encodes for

! -galactosidase?

B. More biochemical (enzyme) tests used to identify microbes.a. The catalase test. This test must be performed on fresh cultures (9, which stops the reaction and maximizes the absorbance at 420 nm.


8/11

MMBB255 Week 6 8

Results of the Catalase Test Positive Negative

Staphylococcus aureus

Micrococcus luteus

Streptococcus sp. Gp B or Enterococcus faecalis

your unknownb. The Oxidase DrySlide TM test. This test must be performed on fresh cultures (


9/11


10/11

MMBB255 Week 6 10 G. Aerobic and anaerobic growth Finished.

1. Results. Record your observations by noting where in the tube it grew and the quantity of growth. From yourobservations record your conclusions about the type of oxygen requirements. The definitions for the terms are inthe background reading material.

Conclusions*Agar Deeps Thioglycolate Gas-Pak

Escherichia coli Pseudomonas aeruginosa

Clostridium sporogenes

Streptococcus agalactiaeYour Unknown XXXXXXXXXXX XXXXXXXXXXX

*be sure to use the correct term: obligate aerobe, obligate anaerobe, microaerophile, aerotolerant, orfacultative anaerobe (see the background).

H. Motility Finished. 1. Record your results in the diagram. Observe in the agar itself

and not the surface; look from the side of the semi-solid agar plate. Did the organism move away from the stab line?

2. Which strains are motile?

Organism Non-Motile Motile

E. coli

K. pneumoniae

Your Unknown

I. Unknown Two Finish.

Agar Deeps Thioglycolate Gas-Pak vs. Air


11/11

MMBB255 Week 6 11

Study Questions:

1. Be able to determine what is positive and negative for each of the tests. Understand how each test works.

2. Oxidase is very useful to differentiate between which two groups of organisms? What is a positive reaction?

3. Catalase is very useful to differentiate between which two groups of organisms? What is the reactioncatalyzed by this enzyme and what do you see for a positive reaction?

4. What indicates that a sugar is fermented in the PR-sugar test? You may also have what byproduct as seen inthe small inverted test tube? What is that small test tube called?

5. What are the purposes of the various solutions in the ! -galactosidase test? Lactose is a disaccharidecomprised of which two monosaccharides? Be able to answer the question given at the end of thisexperiment.

6. Enzymes are named after the substrates they work on. Name the substrate for each enzyme. For exampleamylases substrate is amylose or starch.

7. Where does a microaerophile grow best in a thioglycollate tube? How about an obligate aerobe, obligateanaerobe, facultative anaerobe, and aerotolerant anaerobe?

8. What kinds of motility are there?

9. How does the Gas-Pak chamber work to give you anaerobic conditions? How do you know if it is working?

10. The first practicum is soon. Here are some typical questions:Practice #1:

Describe the cellular morphology and arrangement of this gram stain.

What gram reaction is it?

What is the difference between Gram positive eubacteria and Gram negative eubacteria?

Practice #2:Is this a positive or negative acid-fast stain?

Why do we have to use acid-fast stain for Mycobacteria?

Practice #3:Determine the O.D. at 420 nm of the yellow sample.

This was from the galactosidase assay we did using the ONPG substrate. What operon activity are wedetecting and is the above sample from the PR-Glucose or PR-Lactose medium?

biochemical tests.pdf

Documents