PowerPoint Presentation: WITCHES_OINTMENTS.Oct14
Milk is extremely perishable and many means have been developed to preserve it. The earliest one which has been used for many thousands of years is fermentation. Milk can be fermented by inoculating fresh milk with the appropriate bacteria and keeping it at a temperature which favors bacterial growth. As the bacteria grow, they convert milk sugar (lactose) to lactic acid. You can detect its presence by the tart or sour taste (sour is how we taste acid). The lowered pH caused by lactic acid preserves the milk by preventing the growth of putrefactive and/or pathogenic bacteria which do not grow well in acid conditions.
Fermentation is a means by which cells growing anaerobically can still generate a little ATP. Fermentation is defined biochemically as the catabolism of glucose (or other sugars) in which the terminal hydrogen acceptor is an organic molecule (carbon containing). During the breakdown of sugar, known as glycolysis, excess hydrogen atoms are generated and must be deposited somewhere. In lactic acid bacteria, they “dump” excess hydrogens on to pyruvic acid, the end product of glucose. This turns pyruvic acid into lactic acid.
Our muscles do the same thing, which causes the sting in over exercised muscles. In all fermentation, NADH gives up its hydrogen to produce NAD, which is required for further glycolysis. Yeast too performs fermentation, but with different terminal hydrogen acceptors (acetaldehyde) and products (CO2 and ethanol). You will note that alcoholic fermentation is also an anaerobic process. Since the terminal hydrogen acceptor in each of these microbiological processes is an organic molecule, they are, by definition, fermentation.
In contrast, respiration uses an inorganic terminal hydrogen acceptor (such as oxygen). If oxygen is the acceptor, then water is produced.
Casein, the predominant protein in milk, is soluble at a neutral pH, but insoluble in acid. Thus when milk sours, casein precipitates which thickens the product. Numerous strains of bacteria are capable of converting lactose to lactic acid. We will look at several fermented milk products to study their morphology and staining characteristics.
- Make a thin smear of each milk product well spaced on the same slide, labeling with a wax pencil Y, B and S. (see protocol Smear and Staining of Bacterial Specimens)
- Stain them according to the procedure for the Gram stain (see related protocol Gram Stain Protocol), or any simple stain such as methylene blue, should you only be interested in seeing bacterial morphology.
- View the stained smear at 400x to determine the characteristic features, select a field which is well spread and typically stained. Then switch to 1000x with oil. (The oil immersion lens is challenging to novices. Do not use this lens unless you have been instructed in its use.)
- Illustrate typical fields for each milk product showing all observed morphologies of bacteria. Label the morphologies and their probable identities according to the following type of bacteria expected in these fermented milk products:
Yogurt is produced by a mixed culture of two types of bacteria. Imbedded in particles of the protein casein, you will see chains of cocci or diplococci (Streptococcus thermophilus) and big rod-shaped bacilli (either Lactobacillus acidophilus or L. bulgaricus). If you do a Gram stain, the bacteria will be Gram positive (purple) and the protein will be pink. The illustrations at the top of the page are micrographs I took of a Gram stain of yogurt. The purple rods are Lactobacillus, the purple spheres are Streptococcus. The pink globs are casein, milk protein.
Buttermilk is the fermentation of milk by a culture lactic acid-producing Streptococcus lactis plus Leuconostoc citrovorum which converts lactic acid to aldehydes and ketones which gives it its flavor and aroma.
Sour cream is produced by the same bacteria as buttermilk, but the starting milk product is pasteurized light cream. Bacteria are less numerous than in buttermilk.
Protein Assay by Microbiuret: Standardization
From DBF’s Hopkins Notebooks, III, p. 102 & VI, p. 75.
Microbiuret reagent is an alkaline solution of copper ions which complex with the peptide bonds in protein to produce a blue-purple color (absorption max = 310 nm) . Following Beer’s Law, the color intensity of a small amount of protein mixed with this reagent should be proportional to protein concentration. Thus, this reagent can be used to assay soluble protein in unknown solutions. To do this, a “standard curve ” must be generated by assaying known amounts of protein . That is the purpose of this first exercise in this series.
Wear safety goggles and handle the microbiuret reagent with care since it is a dangerous caustic solution of 35% NaOH (lye). Any hint of slipperiness on your fingers should be rinsed off with a solution of 5% boric acid (in squirt bottles) followed by thorough hand washing.
FOR A TABLE OF TWO STUDENTS (perform experiment in pairs):
3 test tube racks: 2 for 13×100 mm, 1 for 16×150 tubes
12 13 x 100 mm test tubes
2 16×150 test tubes
1 5 mL pipet for water (in 16×150 test tube)
1 2 mL pipet for protein solution (in 16×150 test tube)
pipetman pipet bulb
spectrophotometer, warmed up
two cuvettes in test tube rack
8 mL microbiuret reagent 2 in Eppendorf pipettor or in 1 mL repipet
(front of room)
30-40 mL dH 2O in 125 mL flask
mL of mg/mL protein standard 3 in 13×100 test tube
Kimwipes, paper towel
STANDARDIZATION OF MICROBIURET REAGENT:
1) Write out an experiment table in your book (see Sample Layout of an Experiment)
2) Then set up 13×100 mm tubes for the standardization
3) Then add in sequence, accoding the to volumes in the following table
Here is the table for this standardization:
a) add water first (Always add the least expensive reagents first unless there is a compelling reason to do otherwise)
b) add the appropriate volume of protein.
c) add microbiuret reagent using a repeating pipetter.
Here is a picture illustrating the use of the Beckman Repeater
Here are the steps illustrated for setting up the standardization:
4) Vortex to mix well, let sit 15 min
5) Read tubes: use tube B as the blank, and read A325 in a spectrophotometer. Read at 310 nm if you have a UV capabilities.
6) Calculate the mg protein (or in each tube. (1 g = 10 3 mg = 10 6 ug).
7) Plot standardization curve (protein vs A 325) . Here is a typical standardization curve.
8) Determine conversion factor to convert from optical density at A 325 (OD) to mg protein: Determine the slope of the line where the curve is linear. The slope will be approximately 1.25 mg/OD unit at A 325 ).
Wash work areas well when finished to clean up any spilled caustic materials.
1 Our Spectronic 20s cannot measure in the UV range, but only measure absorbency down to 325 nm.
2 MICROBIURET REAGENT: (Safety glasses should be worn during this experiment since this solution is close to a 20% solution of NaOH. Handle with extreme caution.)
40 g NaOH (caution, caustic)
100 mL dH2O to dissolve NaOH with caution
400 mg CuSO4
40 mL water, agitate to dissolve
1) Q.s. To 150 mL with dH2O
2) Add solution B slowly to solution A with stirring.
Store in labeled bottle marked CAUTION : caustic
3 STANDARD PROTEIN, mg/mL: Prepare 1 mg/mL solution of standard protein (bovine serum albumin [BSA], or egg albumin) by adding 100.0 mg of powder to 80 mL dH2 O, thoroughly dissolve with stirring, avoiding foaming which denatures protein. If possible, let sit at 4C for a week to completely dissolve. Q.s. to 100.0 mL. Store at 4C. (Need ~7 mL/student).
For determination of protein in unknowns, see:
Sample Layout of an Experiment: Protein Concentration in Unknowns by Microbiuret
See Protein Assay by Microbiuret: Standardization for introduction, and how to standardize the microbiouret solution.
1. PLAN YOUR EXPERIMENT, WRITE OUT YOUR EXPERIMENT TABLE:
Calculate the dilutions of unknowns needed to bring their protein concentrations down to approximately 1-5 mg/mL. Plan two tubes per each diluted sample, one with 0.1 mL, the other with 1.0 mL. Calculate and record the amount of water required for each tube to q.s. to 2.0 mL. Create a table similar to one in the standardization with these nine columns:
Include a blank, as in the standardization procedure, and standardization tubes with 0.5 and 1.0 mg standard protein each.
2. PREPARE SAMPLE: DILUTE, SUSPEND OR DISSOLVE:
The final concentration of protein in the diluted samples should be between 1 to 5 mg/mL.
Solids: For a 1% suspension: weigh out 300-500 mg. Grind very fine in mortar and pestle. Add a few drops dH 2O, grind to paste, add few more drops, make slurry, wash grindings into graduated cylinder, q.s. to 100x weight with dH 2O (i.e., 30 mL for 300 mg solid).
Liquids: For concentrated fluids ( egg white or yolk, blood, milk, etc) make a 1:50 dilution : add 0.1 mL to 4.9 dH 2O. Collect saliva in 10 mL beaker, dilute 1:5 (0.4 mL + 1.6 mL dH 2O . For dilute biological fluids like urine, use undiluted as a first approximation. Dilute protein-rich materials 200x , saliva 5x. Vortex thoroughly after the diluting.
3. SET UP TUBES, ADD dH 2O TO TUBES AS IN YOUR TABLE:
Set up the appropriate number of labeled, clean 13 x 100 mm test tubes in a rack (2 tubes/sample). Add the dH2 O first [then the protein sample, finally the microbiuret reagent].
4. ADD PROTEIN ALIQUOTS TO THE SET OF TUBES:
Carefully following your protocol table, add the prescribed amounts of standard protein to the standardization tubes, and 0.1 and 1.0 mL aliquots of diluted specimens to their tubes. Samples should always be added just below the surface of the water.
5. ADD 1 mL OF MICROBIURET TO EACH TUBE.
Make a visual check to see that all tubes appear to have a identical final volume of 2 mL in them (water + sample). Then add 1.0 mL of microbiuret reagent by repipet, mix well by vortex, let sit for 15 min.
6. READ ABSORBENCY AT 325 nm:
Use the B tube (containing no protein) as the blank, determine the A 325 of each tube in succession. You may not need to wash the cuvette between samples, but drain it thoroughly, touching off the last drop from the cuvette on a paper towel prior to adding the next specimen to minimize cross contamination.
7. CALCULATE THE CONCENTRATION OF PROTEIN:
Calculate how much protein is in each tube using the conversion factor from the previous lab (A 325 of the specimen x conversion factor). Do the two standard tubes agree with the standardization? Calculate the concentration in the original sample:
Prepare a table of data of the results from the entire class.
Set up tables with prepared suspensions/dilutions of dog food, cat food, milk, etc.
Place pipeters with 10. And 0.1 volumes at each table, rotate around with tubes having appropriate volume of water in them.
Used Eppendorf Repipeter for distribution of MB.