Information

Why does ampicillin in solution turn yellow?

Why does ampicillin in solution turn yellow?


We are searching data for your request:

Forums and discussions:
Manuals and reference books:
Data from registers:
Wait the end of the search in all databases.
Upon completion, a link will appear to access the found materials.

I have a universal tube with 10 mg mL-1 ampicillin. When I got it, it was supposed to be sterile. It was opened for approximately 20 minutes for an experiment and has since been standing around sealed for a good month now.

Within the last couple weeks, it has gradually turned yellow. Right now the colour is faint, with a green-ish tint.

Why would it turn yellow? I know NADH is yellow, so that was my first guess. But I couldn't exaplain why ampicillin would cause NADH to accumulate, so I discarded that.

Side info: Ampicillin acts on bacterial cell walls, maybe that might help.


Why is NADH the first thing to come to mind? Time for some physical chemistry.

Beta-lactams has a shorter ring structure which give it a different absorbance (around 322 nm apparently (source needed)) which gives it a blueish hue. Typically Penicillin and Ampicillin start off as an off-white. As hinted by @Mad Scientist, the beta-lactam rings are unstable and will undergo acid-catalyzed hydrolysis which breaks the 4-membered ring.

The Penicillin core also near a ketone which can attack the ketone in the lactam (I'm pretty sure this qualifies as enol chemistry but it has been awhile). The cyclization will form a 5-membered oxazole ring. Based on my experience, unsaturated 5-membered rings will absorb at a higher wavelength tending to result in a yellowish color (570-590 nM) so I am going to suggest that is the source of your color.

Ampicillin degradation


How Is Lugol's Solution Typically Used As an Indicator in the Lab?

In a lab, Lugol's solution is typically used as an indicator for the presence of starch in a solution. Lugol's solution, also called Lugol's iodine, is a solution of elemental iodine and potassium iodide in water that generally causes a solution containing starch to turn deep blue.

In some cases, adding Lugol's iodine to a solution that contains no starch can cause a solution being tested to turn blue, such as when the Lugol's solution or the glassware being used is contaminated. Sometimes a control is used during testing, where Lugol's solution is also added to a solution known to contain starch (the positive control) and to a solution known not to contain starch (the negative control.) If the positive control turns blue, and the negative control does not turn blue, it helps confirm that the test is not defective.

Chemists do not fully understand how Lugol's solution causes a solution containing starch to turn blue. The chemical processes are complex. The mixture of elemental iodine and potassium iodide in Lugol's solution generates free iodine atoms that beta amylose, a starch, seems to force into a linear arrangement with energy level spacings conducive to the absorption of visible light such that the solution appears blue.


TEACHER PREPARATION:

Procedure:

A couple of days before the lab, start preparing the media for the activity. An alternative to the teacher preparing the media is to allow students to prepare their own.

Equipment per group of two to three students:

  • 3 - Empty sterile Petri dishes if students make their own plates, two for the gradient slant agar plates and one for the LB agar plate
  • 1 - LB agar plate with no ampicillin *
  • 4 - Large paper clips bent as spreaders (wrapped in aluminum foil and sterilized in a 350° F oven for 30 minutes)
  • 2 - Inoculating loops
  • 2- sterile transfer pipettes
  • 10 mL of sterile LB/nutrient broth for each student group *
  • 1- 50 mL culture tube
  • MM294 strain of E.coli *
  • 1 &ndash pencil
  • Ampicillin solution- 0.05 grams ampicillin salt dissolved in 1 ml of sterile distilled water
  • LB premix *
  • Agar *
  • Marking pen

* Ready-to-pour media and bacterial strains can be purchased from most biological supply companies.

Media Preparation To be completed two to three days before the activity.

LB broth solution:
Calculate the amount of nutrient broth that is to be supplied to the students and add extra for spillage and other factors. Weigh out 2.5 grams of LB premix and dissolve it in 100 ml of distilled water. For a larger amount, use multiples of the ingredients listed previously. Cap the bottle tightly and place it in a boiling water bath for at least 30 minutes.

Sterile Water:
Place 50 ml of distilled water in a screw top bottle. Tighten the cap and place the bottle in a boiling water bath for 30 minutes. Remove the bottle and allow it to cool.

Preparing Gradient Antibiotic LB Agar Plates:
Mix 2.5 grams of Luria broth and 1.5 grams of agar in 100 ml of distilled water in a Kimax or Pyrex bottle with a screw-on cap. Microwave the solution until it is clear (free of suspension). Caution: Never microwave any solution in a bottle with a tight cap or lid.

With the cap tightened down, place the bottle into a boiling water bath for at least 30 minutes. After 30 minutes, immediately pour enough of the agar into one sterile Petri dish to cover the bottom of each dish. Place the lid back on the plate and leave flat on the table top to cool and harden. Place the remaining agar in a 55°C water bath until you are ready to pour the gradient plates. If students are preparing their own plates, they can let the agar cool a short time on the table top and proceed to the next step. For a step-by-step tutorial on the preparation of the LB broth, LB agar plates and sterile water visit: www.biotech.iastate.edu/publications/ppt_presentations/default.html and find the section on &ldquoTransformation-Media Preparation.&rdquo

While the agar is cooling to 55°C in a water bath or on your tabletop, prepare two ampicillin gradient agar plates for each group. To start, rest one edge of each sterile Petri dish on a pencil.



When the agar is cool enough, pour the agar containing no antibiotic into the Petri dish until it is two-thirds of the way across the bottom of the Petri dish and cover. Repeat this with the second dish. Allow the agar to cool and harden. After drying, continue to the next step.


After the first layer has hardened, remove the pencil and lay the Petri dish flat on the table. If time allows, you can let the plates sit on the table top for several days to dry before proceeding to the next step. If you plan to complete the plates the same day, allow the remaining LB agar to cool until you can barely hold the warm flask in your hand and add 1 drop of ampicillin solution to the remaining LB agar. Pour the agar containing the antibiotic two-thirds of the way across the top of the first layer leaving the thickest edge of the first layer uncovered.

If you plan to complete the plates after several days of drying, prepare another bottle of LB agar exactly like the first day. Allow the LB agar to cool until you can barely hold the warm flask in your hand and add 2 drops of ampicillin solution per 100 ml of media. Pour the agar containing the antibiotic two-thirds of the way across the top of the first layer, leaving the thickest edge of the first layer uncovered.


Replace the lid of the Petri dish and allow the media to dry on the lab table until the condensation on the lid evaporates. The ampicillin will diffuse through the agars establishing a concentration gradient across the entire plate. The highest concentration of antibiotic is at the side with the thickest ampicillin agar and the lowest concentration is at the side of the thickest agar without ampicillin.


Safety and Clean Up:

It is important to follow your school district&rsquos lab safety rules and use established safe lab practices especially when the handling microorganisms.

All contaminated equipment can be made safe by soaking in a 10% Clorox solution for 15 minutes. Glassware can be washed with dish soap and water and plastic items disposed of in the regular trash.


DNA TRANSFORMATION OF BACTERIA-AMPICILLIN

Preparation for the DNA transformation experiment should begin at least 24 hours in advance of the laboratory period.

The following supplies can be provided to the class in groups of three students:

  • 2 microcentrifuge tubes (1.5 ml) containing 2 drops of sterile CaCl 2 and labeled "CaCl 2 ". The tubes can be put in the same ice container used to provide the DNA to the group of three students.
  • 1 aluminum foil packet containing 4 sterile toothpicks
  • 4 sterile plastic pipettes from the Office of Biotechnology
  • 1 aluminum foil packet containing 4 sterile paper clips that are large and smooth. The clips should be opened into a 90 o angle and the small end bent to close it.
  • 1 Sharpie marking pen
  • 1 glass test tube with a cap (provided by the Office of Biotechnology) containing 2 ml of sterile nutrient broth and labeled "Broth"
  • 2 petri dishes containing only nutrient agar and labeled "No Amp" on the bottom
  • 2 petri dishes containing nutrient agar and the antibiotic ampicillin. The dishes should be labeled "Amp" on the bottom. (Petri dishes provided by the Office of Biotechnology)
  • 2 copies of the laboratory instructions, one for each student

The following supplies can be shared by three students:

  • 1 petri dish containing colonies of E. coli (MM294)
  • 1 microcentrifuge tube (1.5 ml), labeled "P", containing 4 drops of plasmid DNA that is placed on ice to keep cold until used. The tube should be labeled "DNA".
  • 1 container for used toothpicks

The teacher should have available for the entire class:

  • 1 incubator for the petri dishes set at 37 o C or less. It is difficult to maintain the temperature precisely unless a research incubator is used. Prolonged temperatures above 40 o C will kill the bacteria. Temperatures lower than 37 o C will result in slower growth of the bacteria, but will not kill them.
  • 1 Sharpie marking pen
  • Containers for placing tubes on ice after DNA has been added, such as a styrofoam cup.
  • Containers for the 42 o C water bath, such as a styrofoam cup.

STERILIZATION OF SUPPLIES

  1. Sterilization of packets of toothpicks, and paper clips can be accomplished by wrapping each item in aluminum foil, labeling the contents with a marking pen, and

(a) baking them in an oven at 350 o F for 15 minutes

(b) putting them in a pressure cooker at 15 pounds for 15 minutes

(c) placing them in an autoclave for 15 minutes.

The pressure cooker and autoclave should be at the desired pressure for the 15-minute period. After the packets have cooled, they should be stored unopened at room temperature. The students should be instructed when opening the packets to touch only that part of the object that will not come in contact with the solutions or petri dishes.

(b) put in a pressure cooker at 15 pounds for 15 minutes

(c) placed in an autoclave for 15 minutes.

(a) boiling water for 30 minutes

(b) a pressure cooker at 15 pounds for 15 minutes

(c) an autoclave for 15 minutes.

Allow the bottle to cool until it is comfortable to hold, cap it tightly, and store in a refrigerator until used.

Before the class, put 2 ml of the LB into glass test tubes, leave the caps loose, and place them in an appropriate rack in boiling water for 30 minutes to sterilize them. After the 30 minute-period, remove the tube rack from the boiling water, let the tubes cool, then tighten the cap. Unused broth can be reboiled and stored in the refrigerator for future use.

"No Amp" plates: Prepare 3 "No Amp" plates for each group of 3 students one for preparation of the starter culture and 2 for each pair of students to use for transformation. It is best to prepare about 5 extra plates for the entire class in case contamination occurs in one or more of them. Place the required volume of distilled water in one or more glass bottles with caps. The bottles should not be more than half full. Add 25 mg of LB premix and 15 mg of agar /ml of distilled water. With the caps loose, sterilize the solution by one of the methods described for the calcium chloride (Item 3). After sterilization, the bottles should be swirled to mix the solution and cooled at room temperature to 55 o C, which is when the bottles can be held without an insulated glove. The petri dishes labeled "No Amp" should be poured immediately. The bottom of the dish should be covered with the agar. Agar begins to solidify at about 45 o C, therefore, it is important to pour the plates as rapidly as possible. If the "No Amp" agar does solidify, it can be reboiled and used again. Rinse the bottle with a large amount of tap water immediately after use so that the agar does not solidify in it or in the sink.

"Amp" plates: Prepare 2 "Amp" plates for each group of 3 students. Follow the same procedure as for the "No Amp" plates until the agar has cooled to 55 o C. Add 1 ml of the ampicillin solution (Item 4) per liter (1,000 ml) of solution, swirl to mix, and pour immediately the plates labeled "Amp". If the agar solidifies, it cannot be reheated because the ampicillin will be destroyed above 60 o C.

Allow the "No Amp" and "Amp" plates to harden for about 30 minutes or until the agar has a milky or opaque appearance, then turn the dishes upside down (lid down, agar up). If they are to be kept for more than 2 days, store them upside down in a refrigerator. The plates can be kept refrigerated for a month.

Note: People differ in their sensitivity to temperature and a teacher may prefer to measure the temperature of the agar to determine when 55 o C is reached, particularly for the solution to which ampicillin is added. It is not possible to put a thermometer into the heated agar solution because it will become contaminated. There are two alternatives that can be used.

(A) The bottle of agar can be put into a container with the same volume of cool tap water as the volume of the medium inside the bottle. When the temperature of the tap water reaches 55 o C, the contents inside the bottle should be at a similar temperature.

PREPARATION OF THE E. COLI STARTER PLATE

One petri dish containing live E. coli is needed for each group of four students. A strain of E. coli should be used that does not have resistance to ampicillin.

Use a sterilized transfer loop, a paper clip bent into a loop and sterilized, or a sterilized toothpick. Use the device to touch a colony of bacteria from a petri dish or test tube. Spread the bacteria on the plate in a zig-zag pattern to obtain individual colonies as the concentration of bacteria on the transfer device becomes less. Incubate the plates at 37 o C for 24-36 hours. Colonies should grow to the size of this 0 for use in the lab procedure.

CLEAN UP AFTER THE LABORATORY

Sterilize used toothpicks and 1.5 ml microcentrifuge tubes before placing them in the regular trash. Sterilize the pipettes before washing them. Sterilization can be achieved by placing them in boiling water for 30 minutes, autoclaving for 15 minutes, or putting them in a pressure cooker at 15 pounds for 15 minutes.

Wash glass bottles, pipettes, and paper clips for future use.

Petri dishes can be burned, if convenient. If not, freeze the plates overnight or allow them to dry out in the refrigerator for 1 month, then wrap them securely in a plastic bag and place them in the regular trash.

STUDENT INSTRUCTIONS

Genes control the traits that living organisms possess. Bacteria, such as E. coli , have genes on their chromosome and on a small circular piece of DNA called a plasmid. Genes can be transfered from one bacteria to another on the plasmid by a process known as transformation. In this experiment, a plasmid with a gene (DNA) for resistance to the antibiotic ampicillin will be used to transfer the resistance gene into a susceptible strain of the bacteria. The same technique is used to transfer genes (DNA) for production of insulin, growth hormones, and other proteins into bacteria. The transformed bacteria are used in fermentation to produce commercial quantities of the protein for treating diabetes, dwarfism, or other uses.

You will work with two other people in conducting this laboratory.

PreLab DAY 1

Step 1. Use a separate sterile toothpick to transfer a colony of E. coli about the size of this 0 into each of two tubes of calcium chloride. Use the toothpick to stir the cells vigorously and thoroughly into the solution. The solution should appear milky. Close the caps of both tubes and discard the toothpicks into the container provided for that purpose. One person in the pair should label one of the tubes "B1". The other person should label the other tube "B2".

Step 2. Place the tubes back in the ice and place the container of ice with tubes back in the refrigerator. (DO NOT FREEZE.) (The cold calcium chloride, in the tubes, conditions the surface of the bacteria for DNA uptake the following day.)

DAY 2

Step 1. Finger flick tube to resuspend cells.

Step 2. Open the tube labeled "B1" and with a sterile pipette add one drop of solution from the "P" tube. Close the tube. Do not add anything to the tube labeled "B2". (The plasmid DNA, from the "P"tube, added to the tube has a gene for resistance to ampicillin.)

Step 3. Place the tubes on ice for 15 minutes. (The cells are kept cold to prevent them from growing while the plasmids are being absorbed.)

Step 4. Remove the tubes from the ice and immediately hold them in a 42 o C water bath for 90 seconds. (The marked temperature change causes the cells to readily absorb the plasmid DNA).

Step 5. Use a sterile pipette to add 5 drops of sterile nutrient broth to each of the tubes. Close the tubes. Mix by tipping the tube and inverting it gently (The bacteria are provided nutrients to help them recover from the calcium chloride and heatshock treatments).

Note: For better results allow cell recovery at 37 o C for any amount of extra time, 20 minutes preferred.

Step 6. Label the underside of the four petri dishes with your name. On one "Amp" plate, print "B1" and on the other "Amp" plate print "B2". On one "No Amp" plate print "B1" and on the other "No Amp" plate print "B2".

Step 7. Use a fresh sterile pipette to place 3 drops of cell suspension from the tube labeled "B1" onto the center of a petri dish labeled "Amp"/"B1" and 3 drops to the center of a dish labeled "No Amp"/"DNA". Use another fresh sterile pipette to place 3 drops of cell suspension from the tube labeled "B2" onto the center of the dish labeled "Amp"/"B2" and 3 drops to the center of the dish labeled "No Amp"/"B2". Use a fresh sterile paper clip to spread the liquid evenly across the surface of each plate. Do not touch the part of the paper clip that comes in contact with the agar.

Step 8. Incubate the plates upside down for 24 hours at 37 o C.

Step 9. Analyze the results of the transformation by placing the two plates labeled "Amp" and the two plates labeled "No Amp" together. (The plate labeled "Amp"/"B2" should not have bacterial growth because the bacteria are killed because they did not have resistance to the antibiotic ampicillin. Bacterial growth on the "Amp"/"B1" plate is from cells that took up plasmids added in step 2 and that became resistant to ampicillin. There is extensive bacterial growth on both of the "No Amp" plates because the antibiotic was not present and both resistant and nonresistant bacteria could grow.)

Prepared by the Office of Biotechnology, Iowa State University revised 8/00


Unasyn

Unasyn (ampicillin sodium/sulbactam sodium) is a combination antibiotic indicated for the treatment of infections due to susceptible strains of microorganisms.

What are side effects of Unasyn?

Common side effects of Unasyn include:

  • fever, ,
  • headache,
  • rash,
  • diarrhea,
  • body aches,
  • nausea,
  • vomiting,
  • stomach pain,
  • bloating,
  • gas,
  • vaginal itching or discharge,
  • itching,
  • swollen/black/"hairy" tongue, (white patches inside your mouth or throat), or
  • pain, swelling, or irritation where the needle is placed.

To reduce the development of drug-resistant bacteria and maintain the effectiveness of UNASYN ® and other antibacterial drugs, UNASYN should be used only to treat infections that are proven or strongly suspected to be caused by bacteria.

DESCRIPTION

UNASYN is an injectable antibacterial combination consisting of the semisynthetic antibacterial ampicillin sodium and the beta-lactamase inhibitor sulbactam sodium for intravenous and intramuscular administration.

Ampicillin sodium is derived from the penicillin nucleus, 6-aminopenicillanic acid. Chemically, it is monosodium (2S, 5R, 6R)-6-[(R)-2-amino-2-phenylacetamido]-3, 3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2- carboxylate and has a molecular weight of 371.39. Its chemical formula is C16H18N3NaO4S. The structural formula is:

Sulbactam sodium is a derivative of the basic penicillin nucleus. Chemically, sulbactam sodium is sodium penicillinate sulfone sodium (2S, 5R)-3,3-dimethyl-7-oxo-4-thia- 1-azabicyclo [3.2.0] heptane-2-carboxylate 4,4- dioxide. Its chemical formula is C8H10NNaO5S with a molecular weight of 255.22. The structural formula is:

UNASYN, ampicillin sodium/sulbactam sodium parenteral combination, is available as a white to off-white dry powder for reconstitution. UNASYN dry powder is freely soluble in aqueous diluents to yield pale yellow to yellow solutions containing ampicillin sodium and sulbactam sodium equivalent to 250 mg ampicillin per mL and 125 mg sulbactam per mL. The pH of the solutions is between 8.0 and 10.0.

Dilute solutions (up to 30 mg ampicillin and 15 mg sulbactam per mL) are essentially colorless to pale yellow. The pH of dilute solutions remains the same.

1.5 g of UNASYN (1 g ampicillin as the sodium salt plus 0.5 g sulbactam as the sodium salt) parenteral contains approximately 115 mg (5 mEq) of sodium.

3 g of UNASYN (2 g ampicillin as the sodium salt plus 1 g sulbactam as the sodium salt) parenteral contains approximately 230 mg (10 mEq) of sodium.

INDICATIONS

UNASYN is indicated for the treatment of infections due to susceptible strains of the designated microorganisms in the conditions listed below.

Skin and Skin Structure Infections caused by beta-lactamase producing strains of Staphylococcus aureus, Escherichia coli,* Klebsiella spp.* (including K. pneumoniae*), Proteus mirabilis,* Bacteroides fragilis,* Enterobacter spp.,* and Acinetobacter calcoaceticus.*

NOTE: For information on use in pediatric patients (see PRECAUTIONSâ&euro&ldquoPediatric Use and Clinical Studies sections).

Intra-Abdominal Infections caused by beta-lactamase producing strains of Escherichia coli, Klebsiella spp. (including K. pneumoniae*), Bacteroides spp. (including B. fragilis), and Enterobacter spp.*

Gynecological Infections caused by beta-lactamase producing strains of Escherichia coli,* and Bacteroides spp.* (including B. fragilis*).

* Efficacy for this organism in this organ system was studied in fewer than 10 infections.

While UNASYN is indicated only for the conditions listed above, infections caused by ampicillin-susceptible organisms are also amenable to treatment with UNASYN due to its ampicillin content. Therefore, mixed infections caused by ampicillin-susceptible organisms and beta-lactamase producing organisms susceptible to UNASYN should not require the addition of another antibacterial.

Appropriate culture and susceptibility tests should be performed before treatment in order to isolate and identify the organisms causing infection and to determine their susceptibility to UNASYN.

Therapy may be instituted prior to obtaining the results from bacteriological and susceptibility studies when there is reason to believe the infection may involve any of the beta-lactamase producing organisms listed above in the indicated organ systems. Once the results are known, therapy should be adjusted if appropriate.

To reduce the development of drug-resistant bacteria and maintain effectiveness of UNASYN and other antibacterial drugs, UNASYN should be used only to treat infections that are proven or strongly suspected to be caused by susceptible bacteria. When culture and susceptibility information are available, they should be considered in selecting or modifying antibacterial therapy. In the absence of such data, local epidemiology and susceptibility patterns may contribute to the empiric selection of therapy.

QUESTION

DOSAGE AND ADMINISTRATION

UNASYN may be administered by either the IV or the IM routes.

For IV administration, the dose can be given by slow intravenous injection over at least 10â&euro&ldquo15 minutes or can also be delivered in greater dilutions with 50â&euro&ldquo100 mL of a compatible diluent as an intravenous infusion over 15â&euro&ldquo30 minutes.

UNASYN may be administered by deep intramuscular injection. (see DIRECTIONS FOR USE-Preparation for Intramuscular Injection section).

The recommended adult dosage of UNASYN is 1.5 g (1 g ampicillin as the sodium salt plus 0.5 g sulbactam as the sodium salt) to 3 g (2 g ampicillin as the sodium salt plus 1 g sulbactam as the sodium salt) every six hours. This 1.5 to 3 g range represents the total of ampicillin content plus the sulbactam content of UNASYN, and corresponds to a range of 1 g ampicillin/0.5 g sulbactam to 2 g ampicillin/1 g sulbactam. The total dose of sulbactam should not exceed 4 grams per day.

Pediatric Patients 1 Year Of Age Or Older

The recommended daily dose of UNASYN in pediatric patients is 300 mg per kg of body weight administered via intravenous infusion in equally divided doses every 6 hours. This 300 mg/kg/day dosage represents the total ampicillin content plus the sulbactam content of UNASYN, and corresponds to 200 mg ampicillin/100 mg sulbactam per kg per day. The safety and efficacy of UNASYN administered via intramuscular injection in pediatric patients have not been established. Pediatric patients weighing 40 kg or more should be dosed according to adult recommendations, and the total dose of sulbactam should not exceed 4 grams per day. The course of intravenous therapy should not routinely exceed 14 days. In clinical trials, most children received a course of oral antimicrobials following initial treatment with intravenous UNASYN. (see Clinical Studies section).

Impaired Renal Function

In patients with impairment of renal function the elimination kinetics of ampicillin and sulbactam are similarly affected, hence the ratio of one to the other will remain constant whatever the renal function. The dose of UNASYN in such patients should be administered less frequently in accordance with the usual practice for ampicillin and according to the following recommendations:

TABLE 3 : UNASYN Dosage Guide for Patients with Renal Impairment

Creatinine Clearance (mL/min/1.73m²)Ampicillin/Sulbactam Half-Life (Hours)Recommended UNASYN Dosage
&ge3011.5-3 g q 6h-q 8h
15-2951.5-3 g q 12h
5-1491.5-3 g q 24h

When only serum creatinine is available, the following formula (based on sex, weight, and age of the patient) may be used to convert this value into creatinine clearance. The serum creatinine should represent a steady state of renal function.

Females = 0.85 Ã&mdash above value

Compatibility, Reconstitution And Stability

UNASYN sterile powder is to be stored at or below 30°C (86°F) prior to reconstitution.

When concomitant therapy with aminoglycosides is indicated, UNASYN and aminoglycosides should be reconstituted and administered separately, due to the in vitro inactivation of aminoglycosides by any of the aminopenicillins.

Directions For Use

General Dissolution Procedures

UNASYN sterile powder for intravenous and intramuscular use may be reconstituted with any of the compatible diluents described in this insert. Solutions should be allowed to stand after dissolution to allow any foaming to dissipate in order to permit visual inspection for complete solubilization.

Preparation For Intravenous Use

1.5 g and 3.0 g Bottles: UNASYN sterile powder in piggyback units may be reconstituted directly to the desired concentrations using any of the following parenteral diluents. Reconstitution of UNASYN, at the specified concentrations, with these diluents provide stable solutions for the time periods indicated in the following table: (After the indicated time periods, any unused portions of solutions should be discarded).

DiluentTABLE 4 Maximum Concentration (mg/mL) UNASYN (Ampicillin/Sulbactam)Use Periods
Sterile Water for Injection45 (30/15)8 hrs at 25°C
45 (30/15)48 hrs at 4°C
30 (20/10)72 hrs at 4°C
0.9% Sodium Chloride Injection45 (30/15)8 hrs at 25°C
45 (30/15)48 hrs at 4°C
30 (20/10)72 hrs at 4°C
5% Dextrose Injection30 (20/10)2 hrs at 25°C
30 (20/10)4 hrs at 4°C
3 (2/1)4 hrs at 25°C
Lactated Ringer&rsquos Injection45 (30/15)8 hrs at 25°C
45 (30/15)24 hrs at 4°C
M/6 Sodium Lactate Injection45 (30/15)8 hrs at 25°C
45 (30/15)8 hrs at 4°C
5% Dextrose in 0.45% Saline3 (2/1)4 hrs at 25°C
15 (10/5)4 hrs at 4°C
10% Invert Sugar3 (2/1)4 hrs at 25°C
30 (20/10)3 hrs at 4°C

If piggyback bottles are unavailable, standard vials of UNASYN sterile powder may be used. Initially, the vials may be reconstituted with Sterile Water for Injection to yield solutions containing 375 mg UNASYN per mL (250 mg ampicillin/125 mg sulbactam per mL). An appropriate volume should then be immediately diluted with a suitable parenteral diluent to yield solutions containing 3 to 45 mg UNASYN per mL (2 to 30 mg ampicillin/1 to 15 mg sulbactam/per mL).

1.5 g ADD-Vantage® Vials: UNASYN in the ADD-Vantage® system is intended as a single dose for intravenous administration after dilution with the ADD-Vantage® Flexible Diluent Container containing 50 mL, 100 mL or 250 mL of 0.9% Sodium Chloride Injection, USP.

3 g ADD-Vantage® Vials: UNASYN in the ADD-Vantage® system is intended as a single dose for intravenous administration after dilution with the ADD-Vantage® Flexible Diluent Container containing 100 mL or 250 mL of 0.9% Sodium Chloride Injection, USP.

UNASYN in the ADD-Vantage® system is to be reconstituted with 0.9% Sodium Chloride Injection, USP only. See INSTRUCTIONS FOR USE OF THE ADD-Vantage® VIAL section. Reconstitution of UNASYN, at the specified concentration, with 0.9% Sodium Chloride Injection, USP provides stable solutions for the time period indicated below:

DiluentMaximum Concentration (mg/mL) UNASYN (Ampicillin/Sulbactam)Use Period
0.9% Sodium Chloride Injection (USP)30 (20/10)8 hrs at 25°C

In 0.9% Sodium Chloride Injection, USP

The final diluted solution of UNASYN should be completely administered within 8 hours in order to assure proper potency.

Preparation For Intramuscular Injection

1.5 g and 3.0 g Standard Vials: Vials for intramuscular use may be reconstituted with Sterile Water for Injection USP, 0.5% Lidocaine Hydrochloride Injection USP or 2% Lidocaine Hydrochloride Injection USP. Consult the following table for recommended volumes to be added to obtain solutions containing 375 mg UNASYN per mL (250 mg ampicillin/125 mg sulbactam per mL). Note: Use only freshly prepared solutions and administer within one hour after preparation.

UNASYN Vial SizeVolume of Diluent to be AddedWithdrawal Volume*
15 g3.2 mL4.0 mL
3.0 g6.4 mL8.0 mL
*There is sufficient excess present to allow withdrawal and administration of the stated volumes.

HOW SUPPLIED

UNASYN® (ampicillin sodium/sulbactam sodium) is supplied as a sterile off-white dry powder in glass vials and piggyback bottles. The following packages are available:

Vials containing 1.5 g (NDC 0049-0013-83) equivalent of UNASYN (1 g ampicillin as the sodium salt plus 0.5 g sulbactam as the sodium salt).

Vials containing 3 g (NDC 0049-0014-83) equivalent of UNASYN (2 g ampicillin as the sodium salt plus 1 g sulbactam as the sodium salt).

ADD-Vantage® package of 5 vials (NDC 0049-0031-02). Each vial containing 1.5 g (NDC 0049-0031-01) equivalent of UNASYN (1 g ampicillin as the sodium salt plus 0.5 g sulbactam as the sodium salt) are distributed by Pfizer Inc.

ADD-Vantage® package of 5 vials (NDC 0049-0032-02). Each vial containing 3 g (NDC 0049-0032-01) equivalent of UNASYN (2 g ampicillin as the sodium salt plus 1 g sulbactam as the sodium salt) are distributed by Pfizer Inc.

The 1.5 g UNASYN ADD-Vantage® vials are only to be used with the ADD-Vantage® Flexible Diluent Container containing 0.9% Sodium Chloride Injection, USP, 50 mL, 100 mL, or 250 mL sizes.

The 3 g UNASYN ADD-Vantage® vials are only to be used with the ADD-Vantage® Flexible Diluent Container containing 0.9% Sodium Chloride Injection, USP, 100 mL or 250 mL sizes.

Distributed by: Roerig Division of Pfizer Inc., New York, NY 10017

SIDE EFFECTS

Adult Patients

UNASYN is generally well tolerated. The following adverse reactions have been reported in clinical trials.

Local Adverse Reactions

Pain at IM injection site - 16%
Pain at IV injection site - 3%
Thrombophlebitis - 3% Phlebitis - 1.2%

Systemic Adverse Reactions

The most frequently reported adverse reactions were diarrhea in 3% of the patients and rash in less than 2% of the patients.

Additional systemic reactions reported in less than 1% of the patients were: itching, nausea, vomiting, candidiasis, fatigue, malaise, headache, chest pain, flatulence, abdominal distension, glossitis, urine retention, dysuria, edema, facial swelling, erythema, chills, tightness in throat, substernal pain, epistaxis and mucosal bleeding.

Pediatric Patients

Available safety data for pediatric patients treated with UNASYN demonstrate a similar adverse events profile to those observed in adult patients. Additionally, atypical lymphocytosis has been observed in one pediatric patient receiving UNASYN.

Adverse Laboratory Changes

Adverse laboratory changes without regard to drug relationship that were reported during clinical trials were:

Hepatic: Increased AST (SGOT), ALT (SGPT), alkaline phosphatase, and LDH.

Hematologic: Decreased hemoglobin, hematocrit, RBC, WBC, neutrophils, lymphocytes, platelets and increased lymphocytes, monocytes, basophils, eosinophils, and platelets.

Blood Chemistry: Decreased serum albumin and total proteins.

Renal: Increased BUN and creatinine.

Urinalysis: Presence of RBC&rsquos and hyaline casts in urine.

Postmarketing Experience

In addition to adverse reactions reported from clinical trials, the following have been identified during post-marketing use of ampicillin sodium/sulbactam sodium or other products containing ampicillin. Because they are reported voluntarily from a population of unknown size, estimates of frequency cannot be made. These events have been chosen for inclusion due to a combination of their seriousness, frequency, or potential causal connection to ampicillin sodium/sulbactam sodium.

Blood And Lymphatic System Disorders

Hemolytic anemia, thrombocytopenic purpura, and agranulocytosis have been reported. These reactions are usually reversible on discontinuation of therapy and are believed to be hypersensitivity phenomena. Some individuals have developed positive direct Coombs Tests during treatment with UNASYN, as with other beta-lactam antibacterials.

Gastrointestinal Disorders: Abdominal pain, cholestatic hepatitis, cholestasis, hyperbilirubinemia, jaundice, abnormal hepatic function, melena, gastritis, stomatitis, dyspepsia, black &ldquohairy&rdquo tongue, and Clostridium difficile associated diarrhea (see CONTRAINDICATIONS and WARNINGS sections).

General Disorders and Administration Site Conditions:Injection site reaction

Immune System Disorders: Serious and fatal hypersensitivity (anaphylactic) reactions (see WARNINGS section).

Nervous System Disorders: Convulsion and dizziness

Renal and Urinary Disorders:Tubulointerstitial nephritis

Respiratory, Thoracic and Mediastinal Disorders: Dyspnea

Skin and Subcutaneous Tissue Disorders:Toxic epidermal necrolysis, Stevens-Johnson syndrome, angioedema, Acute generalized exanthematous pustulosis (AGEP), erythema multiforme, exfoliative dermatitis, and urticaria (see CONTRAINDICATIONS and WARNINGS sections).

DRUG INTERACTIONS

Probenecid decreases the renal tubular secretion of ampicillin and sulbactam. Concurrent use of probenecid with UNASYN may result in increased and prolonged blood levels of ampicillin and sulbactam. The concurrent administration of allopurinol and ampicillin increases substantially the incidence of rashes in patients receiving both drugs as compared to patients receiving ampicillin alone. It is not known whether this potentiation of ampicillin rashes is due to allopurinol or the hyperuricemia present in these patients. There are no data with UNASYN and allopurinol administered concurrently. UNASYN and aminoglycosides should not be reconstituted together due to the in vitro inactivation of aminoglycosides by the ampicillin component of UNASYN.

SLIDESHOW

WARNINGS

Hypersensitivity

Serious and occasionally fatal hypersensitivity (anaphylactic) reactions have been reported in patients on penicillin therapy. These reactions are more apt to occur in individuals with a history of penicillin hypersensitivity and/or hypersensitivity reactions to multiple allergens. There have been reports of individuals with a history of penicillin hypersensitivity who have experienced severe reactions when treated with cephalosporins. Before therapy with a penicillin, careful inquiry should be made concerning previous hypersensitivity reactions to penicillins, cephalosporins, and other allergens. If an allergic reaction occurs, UNASYN should be discontinued and the appropriate therapy instituted.

Hepatotoxicity

Hepatic dysfunction, including hepatitis and cholestatic jaundice has been associated with the use of UNASYN. Hepatic toxicity is usually reversible however, deaths have been reported. Hepatic function should be monitored at regular intervals in patients with hepatic impairment.

Severe Cutaneous Adverse Reactions

UNASYN may cause severe skin reactions, such as toxic epidermal necrolysis (TEN), Stevens-Johnson syndrome (SJS), dermatitis exfoliative, erythema multiforme, and Acute generalized exanthematous pustulosis (AGEP). If patients develop a skin rash they should be monitored closely and UNASYN discontinued if lesions progress (see CONTRAINDICATIONS and ADVERSE REACTIONS sections).

Clostridium Dfficile - Associated Diarrhea

Clostridium difficile associated diarrhea (CDAD) has been reported with use of nearly all antibacterial agents, including UNASYN, and may range in severity from mild diarrhea to fatal colitis. Treatment with antibacterial agents alters the normal flora of the colon leading to overgrowth of C. difficile.

C. difficile produces toxins A and B which contribute to the development of CDAD. Hypertoxin producing strains of C. difficile cause increased morbidity and mortality, as these infections can be refractory to antimicrobial therapy and may require colectomy. CDAD must be considered in all patients who present with diarrhea following antibacterial drug use. Careful medical history is necessary since CDAD has been reported to occur over two months after the administration of antibacterial agents.

If CDAD is suspected or confirmed, ongoing antibacterial drug use not directed against  C. difficile may need to be discontinued. Appropriate fluid and electrolyte management, protein supplementation, antibacterial treatment of C. difficile, and surgical evaluation should be instituted as clinically indicated.

PRECAUTIONS

General

A high percentage of patients with mononucleosis who receive ampicillin develop a skin rash. Thus, ampicillin class antibacterial should not be administered to patients with mononucleosis. In patients treated with UNASYN the possibility of superinfections with mycotic or bacterial pathogens should be kept in mind during therapy. If superinfections occur (usually involving Pseudomonas or Candida), the drug should be discontinued and/or appropriate therapy instituted.

Prescribing UNASYN in the absence of proven or strongly suspected bacterial infection or a prophylactic indication is unlikely to provide benefit to the patient and increases the risk of the development of drug-resistant bacteria.

Patient Counseling Information

Patients should be counseled that antibacterial drugs including UNASYN should only be used to treat bacterial infections. They do not treat viral infections (e.g., the common cold). When UNASYN is prescribed to treat a bacterial infection, patients should be told that although it is common to feel better early in the course of therapy, the medication should be taken exactly as directed. Skipping doses or not completing the full course of therapy may (1) decrease the effectiveness of the immediate treatment and (2) increase the likelihood that bacteria will develop resistance and will not be treatable by UNASYN or other antibacterial drugs in the future.

Diarrhea is a common problem caused by antibacterial which usually ends when the antibacterial is discontinued. Sometimes after starting treatment with antibacterial, patients can develop watery and bloody stools (with or without stomach cramps and fever) even as late as two or more months after having taken the last dose of the antibacterial. If this occurs, patients should contact their physician as soon as possible.

Nonclinical Toxicology

Carcinogenesis, Mutagenesis, Impairment Of Fertility

Long-term studies in animals have not been performed to evaluate carcinogenic or mutagenic potential.

Pregnancy

Reproduction studies have been performed in mice, rats, and rabbits at doses up to ten (10) times the human dose and have revealed no evidence of impaired fertility or harm to the fetus due to UNASYN. There are, however, no adequate and well-controlled studies in pregnant women. Because animal reproduction studies are not always predictive of human response, this drug should be used during pregnancy only if clearly needed. (see â&euro&ldquoPRECAUTIONS, Drug/Laboratory Test Interactions section).

Use In Specific Populations

Labor And Delivery

Studies in guinea pigs have shown that intravenous administration of ampicillin decreased the uterine tone, frequency of contractions, height of contractions, and duration of contractions. However, it is not known whether the use of UNASYN in humans during labor or delivery has immediate or delayed adverse effects on the fetus, prolongs the duration of labor, or increases the likelihood that forceps delivery or other obstetrical intervention or resuscitation of the newborn will be necessary.

Nursing Mothers

Low concentrations of ampicillin and sulbactam are excreted in the milk therefore, caution should be exercised when UNASYN is administered to a nursing woman.

Pediatric Use

The safety and effectiveness of UNASYN have been established for pediatric patients one year of age and older for skin and skin structure infections as approved in adults. Use of UNASYN in pediatric patients is supported by evidence from adequate and well-controlled studies in adults with additional data from pediatric pharmacokinetic studies, a controlled clinical trial conducted in pediatric patients and post-marketing adverse events surveillance. (see CLINICAL PHARMACOLOGY, INDICATIONS AND USAGE, ADVERSE REACTIONS, DOSAGE AND ADMINISTRATION, and Clinical Studies sections).

The safety and effectiveness of UNASYN have not been established for pediatric patients for intra-abdominal infections.

OVERDOSE

Neurological adverse reactions, including convulsions, may occur with the attainment of high CSF levels of beta-lactams. Ampicillin may be removed from circulation by hemodialysis. The molecular weight, degree of protein binding and pharmacokinetics profile of sulbactam suggest that this compound may also be removed by hemodialysis.

Clinical Studies

Skin And Skin Structure Infections In Pediatric Patients

Data from a controlled clinical trial conducted in pediatric patients provided evidence supporting the safety and efficacy of UNASYN for the treatment of skin and skin structure infections. Of 99 pediatric patients evaluable for clinical efficacy, 60 patients received a regimen containing intravenous UNASYN, and 39 patients received a regimen containing intravenous cefuroxime. This trial demonstrated similar outcomes (assessed at an appropriate interval after discontinuation of all antimicrobial therapy) for UNASYN-and cefuroxime-treated patients:

Therapeutic RegimenClinical SuccessClinical Failure
UNASYN51/60 (85%)9/60 (15%)
Cefuroxime34/39 (87%)5/39 (13%)

Most patients received a course of oral antimicrobials following initial treatment with intravenous administration of parenteral antimicrobials. The study protocol required that the following three criteria be met prior to transition from intravenous to oral antimicrobial therapy: (1) receipt of a minimum of 72 hours of intravenous therapy (2) no documented fever for prior 24 hours and (3) improvement or resolution of the signs and symptoms of infection.

The choice of oral antimicrobial agent used in this trial was determined by susceptibility testing of the original pathogen, if isolated, to oral agents available. The course of oral antimicrobial therapy should not routinely exceed 14 days.

CONTRAINDICATIONS

The use of UNASYN is contraindicated in individuals with a history of serious hypersensitivity reactions (e.g., anaphylaxis or Stevens-Johnson syndrome) to ampicillin, sulbactam or to other beta-lactam antibacterial drugs (e.g., penicillins and cephalosporins).

UNASYN is contraindicated in patients with a previous history of cholestatic jaundice/hepatic dysfunction associated with UNASYN.

CLINICAL PHARMACOLOGY

General

Immediately after completion of a 15-minute intravenous infusion of UNASYN, peak serum concentrations of ampicillin and sulbactam are attained. Ampicillin serum levels are similar to those produced by the administration of equivalent amounts of ampicillin alone. Peak ampicillin serum levels ranging from 109 to 150 mcg/mL are attained after administration of 2000 mg of ampicillin plus 1000 mg sulbactam and 40 to 71 mcg/mL after administration of 1000 mg ampicillin plus 500 mg sulbactam. The corresponding mean peak serum levels for sulbactam range from 48 to 88 mcg/mL and 21 to 40 mcg/mL, respectively. After an intramuscular injection of 1000 mg ampicillin plus 500 mg sulbactam, peak ampicillin serum levels ranging from 8 to 37 mcg/mL and peak sulbactam serum levels ranging from 6 to 24 mcg/mL are attained.

The mean serum half-life of both drugs is approximately 1 hour in healthy volunteers.

Approximately 75 to 85% of both ampicillin and sulbactam are excreted unchanged in the urine during the first 8 hours after administration of UNASYN to individuals with normal renal function. Somewhat higher and more prolonged serum levels of ampicillin and sulbactam can be achieved with the concurrent administration of probenecid.

In patients with impaired renal function the elimination kinetics of ampicillin and sulbactam are similarly affected, hence the ratio of one to the other will remain constant whatever the renal function. The dose of UNASYN in such patients should be administered less frequently in accordance with the usual practice for ampicillin (see DOSAGE AND ADMINISTRATION section).

Ampicillin has been found to be approximately 28% reversibly bound to human serum protein and sulbactam approximately 38% reversibly bound.

The following average levels of ampicillin and sulbactam were measured in the tissues and fluids listed:

TABLE 1 : Concentration of UNASYN in Various Body Tissues and Fluids

Fluid or TissueDose (grams) Ampicillin/ SulbactamConcentration (mcg/mL or mcg/g) Ampicillin/ Sulbactam
Peritoneal Fluid0.5/0.5 IV7/14
Blister Fluid (Cantharides)0.5/0.5 IV8/20
Tissue Fluid1/0.5 IV8/4
Intestinal Mucosa0.5/0.5 IV11/18
Appendix2/1 IV3/40

Penetration of both ampicillin and sulbactam into cerebrospinal fluid in the presence of inflamed meninges has been demonstrated after IV administration of UNASYN.

The pharmacokinetics of ampicillin and sulbactam in pediatric patients receiving UNASYN are similar to those observed in adults. Immediately after a 15-minute infusion of 50 to 75 mg UNASYN/kg body weight, peak serum and plasma concentrations of 82 to 446 mcg ampicillin/mL and 44 to 203 mcg sulbactam/mL were obtained. Mean half-life values were approximately 1 hour.

Microbiology

Ampicillin is similar to benzyl penicillin in its bactericidal action against susceptible organisms during the stage of active multiplication. It acts through the inhibition of cell wall mucopeptide biosynthesis. Ampicillin has a broad spectrum of bactericidal activity against many gram-positive and gram-negative aerobic and anaerobic bacteria. (Ampicillin is, however, degraded by beta-lactamases and therefore the spectrum of activity does not normally include organisms which produce these enzymes).

A wide range of beta-lactamases found in microorganisms resistant to penicillins and cephalosporins have been shown in biochemical studies with cell free bacterial systems to be irreversibly inhibited by sulbactam. Although sulbactam alone possesses little useful antibacterial activity except against the Neisseriaceae, whole organism studies have shown that sulbactam restores ampicillin activity against beta-lactamase producing strains. In particular, sulbactam has good inhibitory activity against the clinically important plasmid mediated beta-lactamases most frequently responsible for transferred drug resistance. Sulbactam has no effect on the activity of ampicillin against ampicillin susceptible strains.

The presence of sulbactam in the UNASYN formulation effectively extends the antibacterial spectrum of ampicillin to include many bacteria normally resistant to it and to other beta-lactam antibacterials. Thus, UNASYN possesses the properties of a broad-spectrum antibacterial and a beta-lactamase inhibitor.

While in vitro studies have demonstrated the susceptibility of most strains of the following organisms, clinical efficacy for infections other than those included in the INDICATIONS and USAGE section has not been documented.

Gram-Positive Bacteria

Staphylococcus aureus (beta-lactamase and non-beta-lactamase producing), Staphylococcus epidermidis (beta-lactamase and non-beta-lactamase producing), Staphylococcus saprophyticus (beta-lactamase and non-beta-lactamase producing), Streptococcus faecalis&dagger (Enterococcus), Streptococcus pneumoniae&dagger (formerly D. pneumoniae), Streptococcus pyogenes&dagger, Streptococcus viridans&dagger.

Gram-Negative Bacteria

Hemophilus influenzae (beta-lactamase and non-beta-lactamase producing), Moraxella (Branhamella) catarrhalis (beta-lactamase and non-beta-lactamase producing), Escherichia coli (beta-lactamase and non-beta-lactamase producing), Klebsiella species (all known strains are beta-lactamase producing), Proteus mirabilis (beta-lactamase and non-beta-lactamase producing), Proteus vulgaris, Providencia rettgeri, Providencia stuartii, Morganella morganii, and Neisseria gonorrhoeae (beta-lactamase and non-beta-lactamase producing).

Anaerobes

Clostridium species,&dagger Peptococcus species,&dagger Peptostreptococcus species, Bacteroides species, including B. fragilis.

&dagger These are not beta-lactamase producing strains and, therefore, are susceptible to ampicillin alone.

Susceptibility Testing

For specific information regarding susceptibility test interpretive criteria and associated test methods and quality control standards recognized by FDA for this drug, please see: https://www.fda.gov/STIC.

Clinical Studies

Skin And Skin Structure Infections In Pediatric Patients

Data from a controlled clinical trial conducted in pediatric patients provided evidence supporting the safety and efficacy of UNASYN for the treatment of skin and skin structure infections. Of 99 pediatric patients evaluable for clinical efficacy, 60 patients received a regimen containing intravenous UNASYN, and 39 patients received a regimen containing intravenous cefuroxime. This trial demonstrated similar outcomes (assessed at an appropriate interval after discontinuation of all antimicrobial therapy) for UNASYN-and cefuroxime-treated patients:

Therapeutic RegimenClinical SuccessClinical Failure
UNASYN51/60 (85%)9/60 (15%)
Cefuroxime34/39 (87%)5/39 (13%)

Most patients received a course of oral antimicrobials following initial treatment with intravenous administration of parenteral antimicrobials. The study protocol required that the following three criteria be met prior to transition from intravenous to oral antimicrobial therapy: (1) receipt of a minimum of 72 hours of intravenous therapy (2) no documented fever for prior 24 hours and (3) improvement or resolution of the signs and symptoms of infection.

The choice of oral antimicrobial agent used in this trial was determined by susceptibility testing of the original pathogen, if isolated, to oral agents available. The course of oral antimicrobial therapy should not routinely exceed 14 days.

Animal Pharmacology

While reversible glycogenosis was observed in laboratory animals, this phenomenon was dose-and time-dependent and is not expected to develop at the therapeutic doses and corresponding plasma levels attained during the relatively short periods of combined ampicillin/sulbactam therapy in man.

PATIENT INFORMATION

Instructions for Use of the ADD-Vantage® VIAL

To Open Diluent Container: Peel overwrap from the corner and remove container. Some opacity of the plastic due to moisture absorption during the sterilization process may be observed. This is normal and does not affect the solution quality or safety. The opacity will diminish gradually.

To Assemble Vial and Flexible Diluent Container: (Use Aseptic Technique)

1. Remove the protective covers from the top of the vial and the vial port on the diluent container as follows:

a. To remove the breakaway vial cap, swing the pull ring over the top of the vial and pull down far enough to start the opening (see Figure 1), pull the ring approximately half way around the cap and then pull straight up to remove the cap (see Figure 2).

Note: Do not access vial with syringe.

b. To remove the vial port cover, grasp the tab on the pull ring, pull up to break the three tie strings, then pull back to remove the cover. (see Figure 3).

2. Screw the vial into the vial port until it will go no further. THE VIAL MUST BE SCREWED IN TIGHTLY TO ASSURE A SEAL. This occurs approximately ½ turn (180°) after the first audible click. (see Figure 4). The clicking sound does not assure a seal the vial must be turned as far as it will go. Note: Once vial is sealed, do not attempt to remove. (see Figure 4).

3. Recheck the vial to assure that it is tight by trying to turn it further in the direction of assembly.

1. Squeeze the bottom of the diluent container gently to inflate the portion of the container surrounding the end of the drug vial.

2. With the other hand, push the drug vial down into the container telescoping the walls of the container. Grasp the inner cap of the vial through the walls of the container. (see Figure 5).

3. Pull the inner cap from the drug vial. (see Figure 6). Verify that the rubber stopper has been pulled out, allowing the drug and diluent to mix.


NCERT Solutions for Class 11 Biology Chapter 13 Photosynthesis

These Solutions are part of NCERT Solutions for Class 11 Biology. Here we have given NCERT Solutions for Class 11 Biology Chapter 13 Photosynthesis.

Question 1.
By looking at a plant externally can you tell whether a plant is C3 or C4 ? Why and how?
Solution:
Plants that are adapted to dry tropical regions have the C4 pathway. They have a special type of leaf anatomy, they tolerate higher temperatures, they show a response to highlight intensities. Study vertical sections of leaves, one of a C3 plant and the other of a C4 plant.

Question 2.
By looking at which internal structure of a plant can you tell whether a plant is C3 or C4? Explain.
Solution:
In C4 plant internal structure of the leaf possess a special type of anatomy called ‘Kranz’ anatomy. ‘Kranz’ means ‘wreath’ and is a reflection of the arrangement of cells.

The bundle sheath cells may form several layers around the vascular bundles they are characterised by having large number of chloroplasts, thick walls impervious to gaseous exchange and no intercellular spaces.

While in C3 plants, there is no special type of leaf anatomy. There is only a single type of chloroplast inC3 i.e. granal, while in C4 chloroplasts are dimorphic, i.e, granite in the mesophyll cells and agranal in the bundle sheath cells.

Question 3.
Even though very few cells in a C4 plant carry out the biosynthetic-Calvin pathway, yet they are highly productive, can you discuss why?
Solution:
Though these plants have the C4 oxalacetic acid as the first CO2 fixation product they use the C3 pathway or the Calvin cycle as the main biosynthetic pathway.
In C4 plants photorespiration does not occur. This is because they have a mechanism that increases the concentration of CO2 at the enzyme site.
This takes place when the C4 acid from the mesophyll is broken down in the bundle cells to release CO2 this results in increasing the intracellular concentration of CO2 In turn, this ensures that the Rubisco functions as a carboxylase minimizing the oxygenase activity.

Now that you know that the C4 plants lack photorespiration, you probably can understand why productivity and yields are better in these plants. In addition, these plants show tolerance to higher temperatures.

Question 4.
RuBisCO is an enzyme that acts both as carboxylase and oxygenase. Why do you think RuBisCO carries out more carboxylation in C4 plants.
Solution:
RuBisCO or Ribulose bisphosphate carboxylase – oxygenase enzyme can bind to both C02 and O2. This binding is competitive. The relative concentration of C02 and 02 determines which one of the two will bind to the enzyme.

In C4 plants photorespiration does not occur. This is because they have a mechanism that increases the concentration of C02 at the enzyme site.

This takes place when oxaloacetic acid is broken down in the bundle sheath cells to release C02.

It results in increased intracellular concentration of C02. This ensures that the RuBisCO functions as a carboxylase and minimising the oxygenase activity.

Question 5.
Suppose there were plants that had a high concentration of chlorophyll b, but lacked chlorophyll a, would it carry out photosynthesis? Then why do plants have chlorophyll b and other accessory pigments?
Solution:
Though chlorophyll is the major pigment responsible for trapping light, other thylakoid pigments like chlorophyll b, xanthophylls, and carotenoids, which are called accessory pigments, also absorb light and transfer the energy to ‘chlorophyll a’.

Indeed, they not only enable a wider range of wavelengths of incoming light to be utilized for photosynthesis but also protect ‘chlorophyll a’ from photo-oxidation. Reaction centre chlorophyll-protein complexes are capable of directly absorbing light and performing charge separation events without other chlorophyll pigments but the absorption cross-section is small.

Question 6.
Why is the colour of a leaf kept in the dark frequently yellow, or pale green? Which pigment do you think is more stable?
Solution:
Chlorophyll is unable to absorb energy in the absence of light and loses its stability, giving the leaf a yellowish colour. This shows that xanthophyll is more stable.

Question 7.
Look at leaves of the same plant on the shady side and compare it with the leaves on the sunny side. Or, compare the potted plants kept in the sunlight with those in the shade. Which of them has leaves that are darker green? Why?
Solution:
Light is a limiting factor for photosynthesis Leaves get lesser light for photosynthesis when they are in shade. Therefore, the leaves or plants in shade perform lesser photosynthesis as compared to the leaves or plants kept in sunlight. In order to increase the rate of photosynthesis, the leaves present in shade have more chlorophyll pigments.

This increase in chlorophyll content increases the amount of light absorbed by the leaves, which in turn increases the rate of photosynthesis. Therefore, the leaves or plants in shade are greener than the leaves or plants kept in the sun.

Question 8.
The figure shows the effect of light on the rate of photosynthesis. Based on the graph, answer the following questions.
(a) At which point/s (A, B, or C) in the curve is light a limiting factor?
(b) What could be the Jimiting factor/s in region A?
(c) What do C and D represent on the curve?
Solution:

(a) In the region ‘A’ and half of ‘BTight is limiting factor because rate of photosynthesis is increasing with the intensity of light.
(b) All the other factors except light.
(c) C represents a region where a factor other than light is limiting, e.g., CO2. D represents the light intensity at which rate of photosynthesis is maximum under existing conditions (e.g., CO2).

Question 9.
Give a comparison between the following:
(a) C3 and C4 pathways
(b) Cyclic and non-cyclic photophosphorylation
(c) Anatomy of leaf in C3 and C4.
Solution:
(a) Differences between C3 and C4 pathway

(b) Differences between cyclic and non-cyclic photophosphorylation are

(c) Differences between the anatomy of leaf in C3 plants and anatomy of leaf in C4 plants are

VERY SHORT ANSWER QUESTIONS

Question 1.
Write one anatomical feature of C4 plants.
Solution:
Kranz anatomy in leaf.

Question 2.
Which of the following is not a useful function of the light reaction in photosynthesis?
(a) splitting water
(b) synthesis of NADPH
(c) converting light energy into chemical energy
(d) releasing oxygen for photorespiration
Solution:
(d) Releasing oxygen for photorespiration.

Question 3.
What is the starting substance in the CO2 fixation cycle? (Apr. 91)
Solution:
RuMP.

Question 4.
Where is PS II located in a chloroplast?
Solution:
PS II is located in the appressed regions of grana thylakoid

Question 5.
Name the reaction centre of PS I and PS II.
Solution:
P700 & P680

Question 6.
What type of light causes maximum photo-synthesis? (Oct. 1995)
Solution:
Red light

Question 7.
How many molecules of ATP and how many molecules of NADPH are spent to fix three molecules of CO2 in the Calvin cycle?
Solution:
9 ATP and 6 NADPH

Question 8.
Why do the stomata of CAM plants open during the night?
Solution:
As these plants grow in dry areas, they keep stomata close during the day to conserve water and open their stomata during the night for the diffusion of gases.

Question 9.
Mention one useful role of photorespiration in plants.
Solution:
It protects the plants from photooxidative damage.

Question 10.
Cyanobacteria and some other photosynthetic bacteria don’t have chloroplasts. How do they conduct photosynthesis?
Solution:
Cyanobacteria have bluish pigment phycocyanin, which they use to capture light for photosynthesis. Some green bacteria (cyanobacteria) are red or pink due to pigment phycoerythrin. Whatever the colour of cyanobacteria, they are photosynthetic and so can manufacture food.

Question 11.
What is phosphorylation? (M.Q.P.)
Solution:
Synthesis of ATP either with the help of light (during photosynthesis) or in presence of oxygen (during respiration) is called phosphorylation.

Question 12.
Name the organism Englemann used in his experiment.
Solution:
Cladophora.

Question 13.
Write the currently accepted equation of photosynthesis in plants.
Solution:

Question 14.
What is a pigment?
Solution:
A pigment is a substance that absorbs light of certain wavelength(s).

Question 15.
Write the full form of NADP
Solution:
NADP – Nicotinamide adenine dinucleotide Phosphate.

Question 16.
Expand RuBP
Solution:
Ribulose 1, 5 bisphosphates.

Question 17.
Give a reason for the following:
Some bacteria exhibit photosynthesis but they do not produce oxygen. (July 2006)
Solution:
Some photosynthetic bacteria do not use water as their source of hydrogen, hence do not liberate oxygen.

Question 18.
Mention two conditions where light can become a limiting factor.
Solution:
Conditions in which light can become a limiting factor:
(i) Plants in the shade.
(ii) Plants growing under the canopy in a dense forest.

Question 19.
What are antenna molecules?
Solution:
Antenna molecules are light-harvesting pigment molecules that occur on the outer side of a photosynthetic unit.

Question 20.
What is a quantasome? Where is it present?
Solution:
Quantasome means photosynthetic units. It is equivalent is 230 chlorophyll molecules. These are present in the grana lamellae.

SHORT ANSWER QUESTIONS

Question 1.
Specify how C4 photosynthetic pathway increases carbon dioxide concentration in bundle sheath cells of sugarcane?
Solution:
In C4 pathway of sugarcane, C02 from atmosphere enters through the stomata in the mesophyll cell and combines with phosphoenol pyruvate to form a 4-C compound oxaloacetic acid. The OAA is then transported to the bundle sheath where it is decarboxylatedto release C02 in bundle sheath.

Question 2.
Differentiate between absorption spectrum and action spectrum.
Solution:
The main differences between absorption spectrum and action spectrum are as follows.

Question 3.
What are quantasomes? (Oct. 94)
Solution:
Quantasome is a functional unit (Photo-synthetic unit) made of a group of pigment molecules required for carrying out a photochemical reaction. The Pigment molecules are embedded in the grana and differentiated as pigment system I (with chi 670, chi 680, P 700) and pigment system ll(with chi 670, chi 680, P 680, and Xanthophylls)

Question 4.
Distinguish between cyclic and non-cyclic photophosphorylation. (M.Q.P., March 2011)
Solution:
Non cyclic photophosphorylation (a) Cyclic photophosphorylation

  1. The path traversed by an electron is non-cyclic.
    (a) Path traversed by electron is cyclic.
  2. Both PSI and PSII are active.
    (b) Only PSI is active.
  3. It is accompanied by photolysis.
    (c) No photolysis.
  4. The major pathway that takes place.
    (d) Secondary pathway when additional ATP is needed.

Question 5.
What is Blackman’s law of limiting factors?
Solution:
F.F. Blackman (1905) extended a law to formulate the principle of limiting factors. “When a process is conditioned as to its rapidity by a number of separate factors, the rate of the process is limited by the pace of slowest factors.”

Question 6.
In the condition of water stress why the rate of photosynthesis declines?
Solution:
Due to water stress, stomata remain closed and so there is a decrease in CO2concentration and the leaf water potential is also reduced, decline the rate of photosynthesis.

Question 7.
What is a reaction centre? Give the reaction centres of PSI and PSII.
Solution:
Reaction centre is a chlorophyll component of the photosystem and it absorbs as well as accepts energy from other pigments and ejects an electron. The reaction centre of PSII is Chla680 or P680 and PSI is Chla700 or P700

Question 8.
Why is photorespiration considered a wasteful process?
Solution:
Photorespiration considered a wasteful process because
(i) 25% of photosynthetically fixed carbon is lost in the form of C02.
(ii) There is no energy-rich useful compound produced during this process.

Question 9.
Give two reasons as to why photosynthesis is important for sustaining life on earth.
Solution:
Photosynthesis is the most important process because
(i) it is the only natural process by which oxygen is liberated into the atmosphere.
(ii) it is the process by which food is manufactured for all living organisms.

Question 10.
Why does the rate of photosynthesis decrease at higher light intensities? What plays a protective role in such situations?
Solution:
Rate of photosynthesis decreases for two reasons :
(i) Other factors required for photosynthesis become limiting.
(ii) Destruction of chlorophyll by photo-oxidation.
Carotenoids play a protective role by:
(i) absorbing the excess light and
(ii) acting as an antioxidant to detoxify the effect of activated oxygen species.

Question 11.
What is C4 -pathway? Give an example. (March 2008)
Solution:
CA -pathway is an alternative photosynthetic pathway seen in plants like sugarcane/sorghum/ maize in which the stable compound is oxaloacetate a 4-C compound. It is called the Hatch-slack pathway.

Question 12.
What is kranz anatomy in plants?
Solution:
In Kranz Anatomy vascular bundles are surrounded by a layer of bundle sheath that contains a large number of chloroplasts in mesophyll cells and it is present in C4 plants e.g, Maize, Sugarcane, etc.

LONG ANSWER QUESTIONS

Question 1.
How is photosystem I different from photosystem II?
Solution:
The main differences between photosystem I and photosystem II are

Question 2.
Describe the factors that influence the rate of Photosynthesis. (Oct. 1989)
Solution:
The factors that affect photosynthesis may be both internal & External.

  • Chlorophyll: it is the light-absorbing pigment and only portions of the plant having chlorophyll can help in photosynthesis.
  • Protoplasmic factor: young seedlings when transferred from darkness to light show the presence of some factors which is believed to be enzymatic initiates photosynthesis and is called the protoplasmic factor.
  • Light: It is one of the most important factors which affects the process in 3 ways i.e. quantity, quality, and intensity. Quantity of light is otherwise duration and depends upon the photoperiod that is required by the plant quality refers to the wavelength, maximum photosynthesis occurs in red and blue light while minimum in green light. Intensity favours the process and low intensity decreases the rate of photosynthesis. Very high intensity brings about photooxidation of pigments which is called solarization.
  • CO2: An increase in CO2 concentration favours the process provided other factors are not limiting but very high concentrations are toxic and inhibit photosynthesis.
  • Temperature: Increase in temperature favour photosynthesis but above the optimum range the process decreases due to the denaturation of enzymes.

Question 3.
Explain the process of the biosynthetic phase of photosynthesis occurring in the chloroplasts.
Solution:
The biosynthetic phase of photosynthesis :

  • It occurs in the stroma of chloroplasts.
  • These reactions reduce the carbon dioxide into carbohydrates, making use of the ATP and NADPH2 produced in the photochemical reactions.
  • The reactions are also called as Calvin cycle.
  • The three phases of the Calvin cycle are as follows:

(i) Carboxylation
Six molecules of Ribulose 1,5 bisphosphate
react with six molecules of carbon dioxide to form six molecules of a short-lived 6C- compound.
The reaction is catalysed by RuBP carboxylase (RuBisCo).
The six molecules of the 6C-intermediate break into 12 molecules of 3- phosphoglyceric acid (3-PGA), an SC- compound.
It is through this step that carbon dioxide is fixed in the plant.

(ii) Reduction
12 molecules of 3-phosphoglyceric acid are converted into 12 molecules of 1, 3 diphosphate-glyceric acid, utilising 12 molecules of ATP and then reduced to 3- phosphoglyceraldehyde making use of 12 molecules of NADPH. Two molecules of phosphoglyceraldehyde react to form one molecule of glucose. It is in this step that there is an actual reduction of carbon dioxide leading to sugar formation.

(iii) Regeneration of RuBP
10 molecules of phosphoglyceraldehyde, by a series of complex enzyme-catalyzed reactions, are converted into six molecules of ribulose 1,5-bisphosphate six molecules of ATP are needed for this step. This step of ‘ regeneration of RuBP is important for the cycle to continue

We hope the NCERT Solutions for Class 11 Biology at Work Chapter 13 Photosynthesis, helps you. If you have any query regarding NCERT Solutions for Class 11 Biology at Work Chapter 13 Photosynthesis, drop a comment below and we will get back to you at the earliest.


Why does ampicillin in solution turn yellow? - Biology

Organic Molecules

These are complex, carbon-containing molecules associated with living organisms. Most also contain hydrogen and oxygen. There are five major types: carbohydrates, lipids, proteins, nucleic acids, and vitamins. We covered the first three types in lab. A review of our carbohydrate test data is provided on this page. Click the molecule types above to link to the associated review material.

Benedict's Reagent: A Test for Reducing Sugars

Carbohydrates are divided into two groups based on the complexity of their structure. Simple carbohydrates can form either a single ring structure (monosaccharides) or a double ring structure (disaccharides -- formed when a pair of monosaccharides bond). Simple carbohydrates include familiar sugars such the monosaccharides glucose (the basic fuel of cells) and fructose (found in fruits). Common disaccharides include sucrose (table sugar) and lactose (the sugar in milk).

Complex carbohydrates (polysaccharides) are chains of many bonded simple carbohydrates, and are often used for energy storage. These include starch, cellulose, and glycogen.

One test for the presence of many simple carbohydrates is to use Benedict's reagent. It turns from turquoise to yellow or orange when it reacts with reducing sugars. These are simple carbohydrates with unbound aldehyde or ketone groups. In lab, we used Benedict's reagent to test for one particular reducing sugar: glucose.

Interpreting Benedict's Reagent Results

Benedict's reagent starts out aqua-blue. As it is heated in the presence of reducing sugars, it turns yellow to orange. The "hotter" the final color of the reagent, the higher the concentration of reducing sugar. In general, blue to blue-green or yellow-green is negative, yellowish to bright yellow is a moderate positive, and bright orange is a very strong positive. (See below).

Terminology review : Controls

Water plus Benedict's reagent is a negative control for the sugar test. It demonstrates a negative test result (no sugar present). See tube 1 above.

Glucose plus Benedict's reagent is a positive control for the sugar test. It demonstrates what a strong positive result should look like. It also proves that our reagents haven't gone bad (they are capable of producing a positive result). See tube 4 above.

The point of controls is twofold. They give you standards to compare against, and they demonstrate that your reagents are working correctly.

Class Benedict's Reagent Results

Aside from our controls, we tested three solutions for glucose: starch, acid-treated starch, and amylase-treated starch. As starch is a polysaccharide, it is unsurprising that the starch solution tested negative for simple sugars.

We mixed HCl (an acid) into starch and re-tested for simple sugars. First, we had to adjust the pH of the solutions back to neutral before adding the Benedict's reagent. We used a pH indicator and NaOH (a base) for this. We then added the Benedict's reagent. We got moderately positive results (orangish color). This is because HCl breaks starch back down into its component monosaccharides (glucose, in this case).

Amylase is an enzyme that removes glucose molecules from starch. Both plants and animals use amylase when digesting starch. Unfortunately, amylase cannot break the beta-bonds which hold the glucose molecules together in cellulose. (If it could, we'd be able to eat hay). Based on this information, can you figure out what our results should be if we tested amylase-treated starch and amylase-treated cellulose solutions for reducing sugars?


Why does ampicillin in solution turn yellow? - Biology

Why do things change colour when heated

At some stage you would have seen a piece of metal change colour when heated. If you think you haven't look at a light bulb.

A light bulb contains a small strip of tungsten. When a current is sent through the metal, it heats up. This causes the tungsten to change colour from silver to bright white.

First thing first, what is light and what is it made of?

Light is made up of small particles called photons. These photons oscillates along a wave length. The more they oscillates the more intense the wave length.

Gamma rays are the most intense form of light energy, while radio waves are the weakest form.

Now that we know what light is, why is it that objects look the colour that they are.

The reasons objects look the colour that they are is because they reflect that colour back into our eyes and absorb every other colour.

White light is made up of every colour in the visible light spectrum. Meaning is an object looks white, outside, it is reflecting every colour back into our eyes.

If it looks blue, it is absorbing every colour but blue.

Throughout the electromagnetic spectrum, there is a small portion called visible light spectrum.

At this stage the photons are oscillates at the right wave length to produce the colours we see. Each colour has its own wave length.

Another reason some objects may look a specific colour is because they emit a specific wave length.

The sun dose not reflect or absorb different forms of light, but emits all wavelengths which is why it looks white.

So getting back to why things change colour when they heat up.

We know that everything is made up of atoms, and when we heat these atoms they begin to vibrate. When they vibrate they emit electromagnetic waves.

Objects which emit a blue colour are generally hotter than objects emitting a red/yellow colour (look back at the electromagnetic spectrum).

For example a star which burns blue has a surface temperature of 60,000 K, while a red star has surface temperature of only 3,500 K.

On a side note it is important to know that temperature and colour are not always linked.

If we change what we burn, we can change the colour of the flame without changing heat.

  • Strontium Chloride=RED
  • Sodium Carbonate=YELLOW
  • Cupric Sulfate=GREEN
  • Potassium Chloride=PURPLE
  • Cupric Chloride=BLUE

So why does metal change colour when its heated?

When we heat a piece of metal we are adding thermal energy to the atoms within the metal.

This causes them to start vibrating and then they begin to emit electromagnetic radiation.

When we start to heat a metal rod, you’ll notice it first glows red (lowest frequency). As we continue to heat it (giving more energy), it will eventually turn a bright white (all the visible spectrum).

This is because the atoms in the metal are vibrating so much that they emit a high electromagnetic frequency, which we see as different colours.


Qualitative and Quantitative Tests for Amino Acids and Proteins

There are six tests for the detection of functional groups in amino acids and proteins. The six tests are: (1) Ninhydrin Test (2) Biuret Test (3) Xanthoproteic Test (4) Millon’s Test (5) Hopkins-Cole Test and (6) Nitroprusside Test.

We divide the food we consume into three main classes: carbohydrates, the body’s most readily available energy source lipids, the body’s principal energy reserve and proteins, the body’s source of energy for growth and cellular maintenance.

Proteins also make up the second largest portion of cells, after water. They are large polymeric compounds that cells synthesize from various building blocks called amino acids.

The general structure for an amino acid is shown in the following figure:

Note that all amino acids contain carboxylic acid groups (—COOH), amino groups (—NH2), and substituent or replaceable side chains (—R). Twenty different amino acids, which differ only in the structures of their side chains, are used by human cells to build proteins. The side chain structure determines the class of the amino acid: nonpolar, neutral, basic, or acidic.

Human cells can synthesize most of the amino acids they need to build proteins. However, about 8 amino acids called essential amino acids cannot be synthesized by human cells and must be obtained from food. Amino acids incorporated into proteins are covalently linked by peptide bonds. Peptide bonds are amide bonds formed between the carboxylic acid group of one amino acid and the amino group of a second amino acid.

Equation 1 below shows a peptide linkage formation between two amino acids:

The peptide bond is indicated. Note that, because every amino acid contains at least one amino group and one carboxylic acid group, it is possible for a peptide bond to form in two different ways. For example, with glycine and valine, it is also possible for the peptide bond to form between the carboxylic acid group of valine and the amino group of glycine, producing valylglycine.

Pro­teins are composed of hundreds of amino acids linked by peptide bonds, forming a peptide chain. We define the direction in which the amino acids link by referring to the two ends of the chain as the N-terminus and the C-terminus. The N-terminus is the terminal amino acid in the chain that con­tains the only amino group not part of a peptide bond.

The C-terminus is the other terminal amino acid in the chain, containing the only carboxylic acid group not part of a peptide bond. Note that the N-terminus and the C-terminus are not determined by the side chains. The number of constitu­ent amino acids and the order, in which they are linked starting from the N-terminus, are referred to as the protein’s primary structure.

I. Amino Acid and Protein Solubility:

The physical properties of amino acids and proteins are mainly a result of their structure, both in the solid state and in various solutions. In this part of the experiment you will investigate the solubil­ity of selected amino acids and proteins in various solutions. Using your data you will compare amino acid and protein structural characteristics.

Solubility as a Function of Solution pH:

The presence of amino and carboxylic acid groups enables amino acids to accept protons from and donate protons to aqueous solution, and, therefore, to act as acids and bases. Because proteins contain both acidic and basic side-chains, they too can donate or accept protons. A molecule that functions simultaneously as an acid and a base is known as an amphoteric molecule.

In neutral aqueous solutions, amino acids act as amphoteric mol­ecules. For example, an amino acid with a neutral side chain contains two charges: one positive, due to the protonation of the amino group, and one negative, due to the dissociation of the carboxylic acid proton. This dou­ble ionic form of an amino acid is the zwitterionic form. Following figure shows an amino acid in the zwitterionic form.

Amino acids in zwitterionic form have many physical properties that are also associated with ionic salts. For example, zwitterionic amino acids are colourless, nonvolatile, crystalline solids with melting points above 200°C, usually melting with decomposition. They are soluble in water but not in nonpolar organic solvents such as cyclohexane.

Compared to organic amines and carboxylic acids of similar molecular weight, amino acids have much lower melting points and are highly soluble in polar organic solvents, but only slightly soluble in water. The amino and carboxylic acid groups of constituent amino acids, as well as the nature of various side-chains, allow proteins to possess some of these same properties. However, there are many other factors that must be con­sidered when discussing protein solubility.

The solubility of amino acids and proteins is largely dependent on the solution pH. The struc­tural changes in an amino acid or protein that take place at different pH values alter the relative solubility of the molecule. In acidic solutions, both amino and carboxylic groups are protonated. In basic solutions, both groups are un-protonated. Following figure shows an amino acid in acidic, neutral, and basic solutions.

The pH value at which the concentrations of anionic and cationic groups are equal is the isoelectric point for that amino acid or protein. Amino acids and proteins are least soluble at their isoelectric points. Most of the proteins found in human tissues and fluids have isoelectric points below pH 7.0 (below human body pH) and, therefore, exist mostly in their anionic forms.

II. Chemical Reactions of Amino Acid and Protein Functional Groups:

Certain functional groups in amino acids and proteins can react to produce characteristically coloured products. The colour intensity of the product formed by a particular group varies among proteins in proportion to the number of reacting functional or free groups present and their accessibility to the reagent. Now we will discuss various colour-producing reagents (dyes) to qualitatively detect the pres­ence of certain functional groups in amino acids and proteins.

Ninhydrin Test:

Amino acids contain a free amino group and a free carboxylic acid group that react together with ninhydrin to produce a coloured product. When an amino group is attached to the first, or alpha, carbon on the amino acid’s carbon chain, the amino group’s nitrogen atom is part of a blue-purple product, as shown in Equation 2. Proteins also contain free amino groups on the alpha carbon and can react with ninhydrin to produce a blue-purple product.

Amino acids that have secondary amino group attachments also react with ninhydrin. However, when the amino group is secondary, the condensation product is yellow. For example, the amino acid proline, which contains a secondary amino group, reacts with ninhydrin, as shown in Equation 3. Blue-purple and yellow reaction products positively identify free amino groups on amino acids and proteins.

Biuret Test:

The biuret test for proteins positively identifies the presence of proteins in solution with a deep violet colour. Biuret, H2NCONHCONH2, reacts with copper (II) ions in a basic solution to form a deep violet complex. The peptide linkages in proteins resemble those in biuret and also form deep violet complexes with basic copper (II) ions in solution. The general or biuret complex formed between the protein linkages and the copper (II) ion of the biuret test is shown in following figure.

Xanthoproteic Test:

Some amino acids contain aromatic groups that are derivatives of benzene. These aromatic groups can undergo reactions that are characteristic of benzene and benzene derivatives. One such reaction is the nitration of a benzene ring with nitric acid. The amino acids tyrosine and tryptophan contain activated benzene rings and readily undergo nitration.

The amino acid phenylalanine also contains a benzene ring, but the ring is not activated and, therefore, does not readily undergo nitration. This nitration reaction, when used to identify the presence of an activated benzene ring, is commonly known as the xanthoproteic test, because the product is yellow.

Xanthoproteic comes from the Greek word xanthos, which means yellow. The intensity of the yellow colour deepens when the reaction occurs in basic solution. This reaction is one of the reactions that occur if you spill a concentrated solution of nitric acid onto your skin. The proteins in skin contain tyrosine and tryptophan, which become nitrated and turn yellow.

Millon’s Test:

Millon’s test is a test specific for tyrosine, the only amino acid containing a phenol group, a hydroxyl group attached to a benzene ring. In Millon’s test, the phenol group of tyrosine is first nitrated by nitric acid in the test solution. Then the nitrated tyrosine complexes mercury (I) and mercury (II) ions in the solution to form a red precipitate or a red solution, both positive results. Proteins that contain tyrosine will, therefore, yield a positive result.

However, some proteins containing tyrosine initially forms a white precipitate that turns red when heated, while others form a red solution immediately. Both results are considered positive. Note that any compound with a phenol group will yield a positive test, so one should be certain that the sample that is to be tested does not contain any phenols other than those present in tyrosine.

Hopkins-Cole Test:

The Hopkins-Cole test is specific for tryptophan, the only amino acid containing an indole group. The indole ring reacts with glyoxylic acid in the presence of a strong acid to form a violet cyclic product. The Hopkins-Cole reagent only reacts with proteins containing tryptophan. The protein solution is hydrolyzed by the concentrated sulphuric acid at the solution interface. Once the tryp­tophan is free, it reacts with the glyoxylic acid to form the violet product.

Nitroprusside Test:

The nitroprusside test is specific for cysteine, the only amino acid containing a sulfhydryl group (—SH). The group reacts with nitroprusside in alkaline solution to yield a red complex.


Lack of proper nutrients

When yellow leaves happen where soil pH is ideal, a true nutrient deficiency may exist. Some nutrients are very mobile. Nitrogen, for example, moves through soil easily and leaches away. Unless soil nitrogen is regularly replaced through fertilizer applications, nitrogen deficiencies turn lawns and plant leaves yellow or pale green.

If you suspect nutrient deficiencies, proper fertilization and premium plant foods can help. Your local county extension agent can also shed light on the specific nutrients involved. Identifying which leaves turn yellow first and how the yellowing starts provides clues to common deficiencies 1 such as these:

  • Nitrogen deficiency shows up as a general yellowing. Older, inner leaves turn yellow first. As it progresses, yellowing moves outward, eventually reaching young leaves, too.
  • Potassium deficiency shows itself when leaf edges turn bright yellow, but the inner leaf stays green. Older leaves show symptoms first, and leaf edges soon turn brown.
  • Magnesium deficiency starts as yellow patches between leaf veins on older leaves. Veins stay green as yellow moves from the leaf center out. Leaf edges turn yellow last.
  • Iron deficiency also shows as yellowing between leaf veins, but it hits young leaves on plant tops and branch tips first.
  • Sulfur deficiency starts with the newest leaves, turning them yellow throughout.

Relationships between nutrients in soil and in plants are complex. For example, low potassium can make iron less available. 2 Yet excess potassium ties up calcium, magnesium and nitrogen, causing deficiencies of those nutrients. 1 That makes proper fertilization with trusted, premium plant foods crucial to keeping your plant free from yellow leaves.

Pennington UltraGreen All Purpose Plant Food 10-10-10 provides an ideal blend of essential primary nutrients plus the secondary and micronutrients that healthy, green plants need. For acid-loving plants, Pennington UltraGreen Azalea, Camellia and Rhododendron Plant Food 10-8-6 provides essential nutrients with extra micronutrients in forms that stay more available when soil pH isn't ideal. When magnesium or sulfur is lacking, Pennington Epsom Salt corrects yellow leaves. And when low iron's the issue, Ironite Plus 12-10-10 fights deficiencies and helps keep your plants green.

At Pennington, we understand how important healthy, vibrant green plants can be. We're committed to bringing you the finest in lawn and garden products — and helping you keep yellow leaves at bay. Let us help you learn and grow so you can enjoy the plants that brighten your day.

Always read product labels thoroughly and follow instructions.

Pennington is a registered trademark of Pennington Seed, Inc.

UltraGreen is a registered trademark of Central Garden & Pet Company.

1. University of Missouri Integrated Pest Management, "Diagnosing Nutrient Deficiencies."